Methods in Enzymology Vol 227: Metallobiochemistry Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environment in Metalloproteins

Preface The scope of metallobiochemistry has greatly expanded in recent years as ever more powerful techniques have been...

Author: John N. Abelson (Editor) | James F. Riordan (Editor) | Bert L. Vallee (Editor)

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Preface The scope of metallobiochemistry has greatly expanded in recent years as ever more powerful techniques have been brought to bear on the constituent elements that define and characterize the subject. Metallobiochemistry, Part A, Volume 158 of Methods in Enzymology, focused on progress in those areas in which early on there were major impediments to growth and development. Without the tools to measure metals with sufficient accuracy, precision, and sensitivity this scientific discipline could not have existed. In addition, unless it were possible to distinguish between the metals present in a biological sample that belonged there (because nature intended them to be) from those that merely appeared by accident (adventitious contamination), such metal analyses would have been meaningless. Technology overcame these hurdles, and Part A detailed the state of trace element analysis and the various approaches employed by the metallobiochemist to avoid artifacts and achieve the inorganic equivalent of microbiological sterility. It is a summary of the critical methods that have helped place the subject on a solid foundation. Metallobiochemistry, Part B, Volume 205 of this series, is devoted to metallothionein and related molecules. It is unusual for a Methods in Enzymology volume to feature a single molecule, but the surge of interest in metallothionein and its structural and possibly functional relationship to DNA-binding proteins suggested that such a volume would be timely and useful. It also seemed appropriate to stress that not all metal-containing biological molecules are metalloenzymes or electron-transport proteins. Parts C and D, Volumes 226 and 227, respectively, return to the theme of methods that have contributed to and are emerging as important factors in the advancement of the field. These methods embody concepts that had their origin about the time of the almost forgotten Sumner-Willst~itter controversy of the 1920s. Proteins, it was claimed, could hardly serve as specific biological catalysts if they were little more than nondescript colloids. Metal ions would prove to be the real actors on the enzymatic stage. Authority prevailed until the crystallization of urease seemingly dispatched the metal dogma to oblivion. Despite the extended protests of interest-vested diehards, protein chemistry became inextricably associated with enzymology and metals fell out of fashion. (Ironically, urease turned out to be a nickel enzyme.) Biochemists who had witnessed this metal-induced brouhaha were understandably reluctant to resurrect the idea that metals might have something to do with biological catalysis. Anyone wishing to make the case would have to have persistence along with persuasive and unassailix

X

PREFACE

able analytical data. Only through scrupulous attention to detail was it possible in those neonatal days of metallobiochemistry to gain the acceptance that allowed the field to grow and flourish. Despite its shortcomings the metal-cum-colloid view of catalysis did have one rather appealing feature: the metal would have unique properties among all the atoms of the protein and perhaps these could be exploited to gain important information. The metal could serve as a beacon to guide the investigator searching for an active site. It could also be a signal, either of the detailed steps of catalysis or of any other biological function with which the metal might be associated. Emission spectroscopy proved the significance of a metal-derived signal in principle, but was rather inconsiderate of the protein. Hence, attention shifted along with wavelength to absorption spectroscopy whereby it became possible to view the functional heart of a metalloenzyme directly. This window on the world of metallobiochemistry revealed unprecedented spectral features clearly indicative of an unusual coordination environment and likely characteristic of a catalytic site. Not all metals lend themselves to absorption spectroscopic investigation. Zinc, one of nature's most recurrent participants, is notoriously shy in this regard. Other metals are more expressive and revealing when viewed by alternative techniques. In these two volumes (226 and 227) we have assembled a broad representation of the physical and spectroscopic methods now available that can be useful for examining metals in biological systems and for probing their environments in metalloproteins and metalloenzymes. These approaches, while by no means all-inclusive, exemplify the wide variety of tools and the level of sophistication currently being applied to extract both the nuances and the general principles of metallobiochemistry pertinent to these systems. We are extremely grateful to our contributors for their willingness to participate in this endeavor. They have made a concerted effort to describe techniques in ways that would be most beneficial to the reader. The chapters differ from the more typical ones in this series in that they identify principles underlying a particular method, the kinds of questions that can be addressed, and the ways to interpret results. Step-by-step instructions were not practical in most cases, and generally the objective has been to provide a sense of what can be accomplished. It required more description than anticipated for most of the topics, and this necessitated two volumes instead of one. We appreciate the understanding of our colleagues at Academic Press and we thank them again as well as all the contributors for making this such a pleasant experience. JAMES F. RIORDAN BERT L. VALLEE

[1]

2D NMR OF PARAMAGNETICMETALLOPROTEINS

1

[1] T w o - D i m e n s i o n a l N u c l e a r M a g n e t i c R e s o n a n c e of Paramagnetic Metalloproteins

By ANT6NIO V. XAVIER, DAVID L. TURNER, and HELENA SANTOS Introduction Depending on the interactions between nuclear spins and unpaired electrons, the parameters of the nuclear magnetic resonance (NMR) spectra of paramagnetic molecules may be drastically different from those of diamagnetic onesl-5: the relaxation times, T~ and T2, may be shortened, and the chemical shifts, 8, can be changed. For nuclei that are more than a few bonds away from the paramagnetic center, their interaction with the unpaired electron is purely dipolar and depends on geometrical functions (including Curie relaxationS-7); thus, they provide important structural information. Although the relaxation induced by the unpaired electron can be such that the signals from resonating nuclei are so broad as to be undetectable by NMR, the paramagnetically induced shifts generally result in an increase in spectral resolution. Because the correlation time for the unpaired electron relaxation, zs, is the dominant parameter contributing to the relaxation of magnetic nuclei in a paramagnetic macromolecule, the application of NMR in the study of these molecules is limited to those cases in which zs is not too long. Several examples of the successful use of one-dimensional (ID) NMR in the study of paramagnetic molecules are available, 4 as well as examples of structural studies in which paramagnetic centers naturally present, extrinsically added, or introduced by isomorphous replacement of spectroscopically unsuitable ones are used to obtain geometrical information 8-I° or just to improve resolution and to assist in spectral assignments. i R. A. Dwek, R. J. P. Williams, and A. V. Xavier, in " M e t a l Ions in Biological S y s t e m s " (H. Sigel, ed.), p. 61. Dekker, N e w York, 1974. 2 G. N. L a Mar, in "Biological Applications of Magnetic R e s o n a n c e " (R. G. Shulman, ed.), p. 305. A c a d e m i c Press, N e w York, 1979. 3 j. D. Satterlee, Annu. Rep. N M R Spectrosc. 17, 79 (1986). 4 I. Bertini and C. Luchinat, " N M R of Paramagnetic Molecules in Biological S y s t e m s . " B e n j a m i n / C u m m i n g s , N e w York, 1986. 5 I. Bertini, L. Banci, and C. Luchinat, this series, Vol. 177, p. 246. 6 M. Gueron, J. Magn. Reson. 19, 58 (1975). 7 A. J. Vega and D. Fiat, Mol. Phys. 31, 347 (1976). 8 C. D. Barry, A. C. T. North, J. A. Glasel, R. J. P. Williams, and A. V. Xavier, Nature (London) 232, 236 (1971).

METHODS IN ENZYMOLOGY,VOL. 227

Copyright © 1993by AcademicPress, Inc. All rights of reproduction in any form reserved.

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PROBES OF METAL ION ENVIRONMENTS

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The advent of two-dimensional (2D) NMR techniques, which has greatly enhanced the range of potential applications of NMR spectroscopy to the study of macromolecules, 11 posed a further constraint in possible applications to the study of paramagnetic molecules. In fact, the 2D NMR technique can reveal scalar and dipolar (or chemical exchange) interactions, through the observation of connectivity signals (cross-peaks) between different nuclei (or the same nuclei but in different chemical environments) that develop during specially designed mixing and evolution periods. Because the frequency of labeling decays with the relaxation rate of the interacting nuclei, there is a stringent time scale for the useful application of 2D NMR to the study of paramagnetic molecules. Thus, specific conditions must be used in order to optimize the acquisition of the information required. The application of 2D NMR to paramagnetic proteins has grown exponentially, lagging just a few years behind the growth of applications to diamagnetic systems. For example, in 1986 Wuthrich 11 provided the first comprehensive account of 2D methods in protein NMR, Satterlee 3 mentioned that this technique should have application for paramagnetic systems in the future, and Bertini and LuchinaP mentioned just one example of a 2D NMR study of paramagnetic proteins. 12 The nuclear Overhauser effect spectroscopy (NOESY) 13 sequence was first applied to paramagnetic proteins in order to detect chemical exchange between two paramagnetic forms 12 (see Fig. 1) and between a diamagnetic and paramagnetic form [called exchange correlated spectroscopy (EXCTSY)14], making it possible to assign resonances in the paramagnetic (oxidized) spectrum from independently assigned resonances in the diamagnetic (reduced) one. Of course, those experiments also provided information about nuclear Overhauser effects (NOEs), but the technique was not widely used for paramagnetic proteins until 1988.15 Similarly, correlation spectroscopy ( C O S Y ) 16 w a s used to detect scalar couplings between aromatic protons 17 and between a-CH and NH

9 I. D. Campbell, C. M. Dobson, R. J. Williams, and A. V. Xavier, Annu. N. Y. Acad. Sci. 222, 163 (1973). ~0R. A. Dwek, " N M R in Biochemistry." Oxford Univ. Press (Clarendon), Oxford, 1973. H K. Wuthrich, " N M R of Proteins and Nucleic Acids." Wiley, New York, 1986. 12 H. Santos, D. L. Turner, A. V. Xavier, and J. LeGall, J. Magn. Reson. 59, 177 (1984). 13 j. Jeener, B. H. Meier, P. Bachmann, and R. R. Ernst, J. Chem. Phys. 71, 4546 (1979). 14 j. Boyd, G. R. Moore, and G. Williams, J. Magn. Reson. 58, 511 (1984). 15 S. J. McLachlan, G. N. La Mar, and K.-B. Lee, Biochim. Biophys. Acta 957, 430 (1988). 16 W. P. Aue, E. Bartholdi, and R. R. Ernst, J. Chem. Phys. 64, 2229 (1976). ~7G. Williams, G. R. Moore, R. Porteous, M. N. Robinson, N. Soffe, and R. J. P. Williams, J. Mol. Biol. 183, 409 (1985). 18 A. J. Wand, H. Roder, and S. W. Englander, Biochemistry 25, 1107 (1986),

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FIG. 1. NOESY spectra of partially oxidized Desulfooibrio oulgaris cytochrome c 3 showing cross-peaks resulting from intermolecular electron transfer. The sample was a 2 mM solution in 2H20 at 298 K. The spectrum was recorded on a Bruker (Karlsruhe, Germany) AMX500 (500 MHz) spectrometer using a mixing time of 25 msec with the transmitter frequency set at the position of the residual water line, which was suppressed by presaturation for 2 sec. Pure absorption peaks were obtained using time proportional phase incrementation (TPPI) with 2048 points in t2 and 512 increments in tl. The data were zero-filled to 2048 × 1024 points, and a window function for Lorentzian to Gaussian line shape transformation was applied in t2 and a cosine-square function in t~ prior to the 2D Fourier Transform (FT). A peak connecting resonances from molecules in which two and three hemes are oxidized is labeled with its specific assignment (c.f. Ref. 72).

protons ~8 far from the paramagnetic centers and later to assign protons close to an oxidized heme. 19 A variety of related methods including double quantum filter ( D Q F ) - C O S Y 2°,21 and 2D total correlation spectroscopy (TOCSY) 22 (see Fig. 2) were in use by 1990. 23-27 19 H. Santos and D. L. Turner, FEBS Lett. 226, 179 (1987). 2o M. W. Edwards and A. Bax, J. Am. Chem. Soc. 108, 918 (1986). 21 N. Muller, R. R. Ernst, and K. Wuthrich, J. Am. Chem. Soc. 108, 6487 0986). 22 L. Braunschweiler and R. R. Ernst, J. Magn. Reson. 53, 521 (1983). 23 y . Yamamoto, A. Osawa, Y. Inoue, R. Chuj6, and T. Suzuki, FEBS Lett. 247, 263 (1989). 24 y . Feng, H. Roder, and S. W. Englander, Biophys. J. 57, 15 (1990). 25 S. D. Emerson and G. N. La Mar, Biochemistry 29, 1545 (1990).

4

PROBES OF METAL ION ENVIRONMENTS

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FIG. 2. TOCSY spectrum of Methylophilus methylotrophus ferricytochrome c", 2 mM in 2H20 at 300 K. A 50 msec mixing time was used with a power level of 10 kHz and WALTZ modulation for spin locking. An exponential window function was used in t 2, and the remaining conditions for data recording and processing were the same as those given in the legend to Fig. 1. The complete spin systems of one of the axial histidine ligands (H) and one of the heme propionates (P) are indicated by boxes (c.f. Ref. 73).

Heteronuclear COSY was applied to paramagnetic systems earlier than homonuclear proton experiments. Correlation of 1~C and IH shifts of groups far from the paramagnetic center was used with 13C enrichment in 1983, z8 but correlations between 1H and 13C in natural abundance were used to assign methyl groups of an oxidized heme as early as 1986z9(Fig. 3). Any technique that can be applied to a diamagnetic protein can (and will) be used to study paramagnetic proteins. Indeed, large sections of the polypeptide chain that are remote from the paramagnetic center are 26 S. 27 L. 28 T. 29 H.

C. Busse, S. J. Moench, and J. D. Satterlee, Biophys. J. 58, 45 (1990). P. Yu, G. N. La Mar, and K. Rajarathnam, J. Am. Chem. Soc. 112, 9527 (1990). M. Chan and J. Markley, Biochemistry 22, 5996 (1983). Santos and D. L. Turner, FEBS Lett. 194~ 73 (1986).

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essentially indistinguishable from their equivalents in diamagnetic systems. We therefore concentrate on nuclei with large dipolar or Fermi contact shifts and/or relaxed by paramagnetic metal ions. For most purposes the nuclei outside a sphere or approximately 0.75 nm from the paramagnetic center can be considered as "diamagnetic." Before considering the information that may be obtained from two(three- or four-) dimensional experiments, it should be stressed that the same information can always be obtained from a series of one-dimensional experiments. These may be selective analogs of the 2D experiments or, in the case of the NOE, the development of a equilibrium with a resonance which is selectively and continuously saturated. Of course, this one-dimensional NOE experiment produces a larger effect than the dynamic process of relaxation of the complete spin system which occurs in NOESY, which is a dynamic NOE experiment. The effective sensitivity (signal-to-noise ratio, S/N) per unit time of a two-dimensional experiment may be expressed as 3°

which depends on the effective decay rate, T*2, and the length, 7"2, of the free induction decay sampled in each sequence as well as the decay rate, T#2, during the evolution period, which is incremented up to a maximum length 7"1. One-dimensional experiments only depend on the first term in the equation, as can be seen by letting 7"1 tend to zero, so they are less affected by more rapid relaxation. However, each selective one-dimensional experiment probes the interactions of a single nucleus, whereas a two-dimensional experiment provides information about all possible interactions simultaneously. It is therefore a balance between the second term of the equation and the number of interactions to be observed that governs the choice of approach. These may not be the only considerations because it is often convenient to have cross-peaks from known interactions present to provide a reference for the same sample and spectrometer conditions. It is clear, however, that if the interactions of a single resonance are sought, then a onedimensional experiment will be far more efficient.26,3° Experimental Considerations Having decided that the system under investigation is sufficiently complex to warrant the use of 2D methods, we may now consider which 3o D. L. Turner, J. Magn. Reson. 61, 28 (1985).

[1]

2D NMR OF PARAMAGNETICMETALLOPROTEINS

7

methods are the most useful. The simplest experiment for detecting scalar (J) couplings between nuclei is COSY, which may be applied to homonuclear or heteronuclear systems. The main advantage of COSY is that the magnetization transfer is effected almost instantaneously by a radio frequency pulse so that there is no loss of signal through relaxation. The main disadvantage is that the cross-peaks comprise several components of opposite phase which will cancel each other out if the intrinsic line width is large with respect to the coupling constant. A second complication, which is shared by most other 2D experiments, is that the spectrum correlates the frequency (with respect to the transmitter or reference frequency) of a resonance in F 2 with another in F~, together with the negative of its F~ frequency. If the spectral width in the second dimension (FI) is the same as that in F2, then the positive and negative frequency patterns will overlap. This may be resolved by canceling one set of signals by phase cycling (usually the positive Fl frequencies, called N-type selection), which is undesirable because information is being eliminated, or by separating the two patterns and then recombining them so that they are superimposed. The latter approach will also generate "pure phase" signals, that is, signals with simple absorption, or dispersion cross sections in both dimensions. A number of methods can be used to achieve this: States, 3~ TPPI, 32 and hypercomplex 33 procedures are most common. They share the same principles but differ in the routing of data such that various artifacts may appear in different places with respect to the true COSY spectrum. Most groups use absolute value spectra because of the cancellation of pure absorption cross-peaks that occurs when the lines a r e b r o a d . 27'34-36 This is unwise because all of the intensity at the center of the cross-peak comes form the dispersion mode (the absorption mode gives a zero signal on cross sections through the center, regardless of linewidth), so that the absolute value spectrum will degrade the signalto-noise ratio first by mixing the independent noise traces from the absorption and dispersion components and, second, by rendering it purely positive. The best procedure for broad lines is to use a matched filter, sin(TrJt) exp(-t/Tz), and to phase the cross-peaks correctly to be purely dispersive. This has the additional advantage of producing pure absorption and therefore narrow peaks on the diagonal. If the spectrum is poorly 31 D. J. States, R. A. Haberkorn, and D. J. Ruben, J. Magn. Reson. 48, 286 (1982). 32 D. Marion and K. Wuthrich, Biochem. Biophys. Res. Commun. 113, 967 (1983). 33 L. Muller and R. R. Ernst, Mol. Phys. 38, 963 (1979). 34 I. Bertini, F. Copozzi, C. Luchinat, and P. Turano, J. Magn. Reson. 95, 244 (1991). 35 j. D. Satterlee, D. J. Russell, and J. E. Erman, Biochemistry 30, 9072 (1991). 36 K. A. Keating, J. S. de Ropp, G. N. La Mar, A. L. Balch, F.-Y. Shiau, and K. M. Smith, lnorg. Chem. 30, 3258 (1991).

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PROBES OF METAL ION ENVIRONMENTS

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digitized, there may be some advantages in examining an absolute value plot of the spectrum as well. However, many pulse sequences for "magnitude" or "absolute value" COSY employ N- or P-type peak selection, and these should never be used because they lead to loss of half of the signal. Narrow diagonal peaks allow cross-peaks to be observed between resonances with small separations. This is the principal purpose of the double quantum filter in D Q F - C O S Y , together with the elimination of singlets that may arise from the solvent or from impurities. The elimination of singlets is not usually very important, but the narrow diagonal is achieved at the cost of one-half of the cross-peak intensity. A pure dispersion COSY is therefore preferable. The same applies to the " I S E C R - C O S Y ''37 experiment, which incorporates refocusing delays of the order of I / Z / f o r the purpose of bringing the components of the crosspeaks into the same phase: the signal is reduced by a further approximately 50 msec of relaxation for very little gain. Extended mixing periods can be useful, however, to find resonances that belong to the same spin system (a set of coupled nuclei within a single side-chain or prosthetic group) but do not couple directly with each other. Experiments of this type include relayed C O S Y 38 and T O C S Y . 22'39 The first involves a number of COSY-type magnetization transfer steps with refocusing delays that can be optimized for a particular series of coupling constants. The TOCSY experiment uses a somewhat different principle insofar as the radio frequency field is applied continuously during the mixing period and creates a series of cross-peaks to other nuclei of the same spin system that have all their components of the same phase. The transfer of magnetization between nuclei requires times of the order of 1/2J and is an oscillatory process, so some cross-peaks of the spin system may be weak for a single mixing time. In principle, a full set of crosspeaks between, for example, an amide proton and the shifts of all the protons in a particular residue may be observed, which may well allow a primary assignment to be made directly. There is a potential disadvantage in that correlations caused by cross-relaxation (NOEs) may then also occur, and these are of opposite sign to the cross-peaks generated by J coupling. Two protons may be coupled and also cross-relax, so that the cross-peak between them may be partially canceled. When short mixing times (<50 msec) are used to optimize an experiment by balancing the buildup of the cross-peaks against the loss of signal through relaxation, 37 S. Talburi and N. A. Scherage, J. Magn. Reson. 86, 1 (1990). 38 G. Eich, G. Bodenhausen, and R. R. Ernst, J. Am. Chem. Soc. 104, 3731 (1982). 39 A. Bax and D. G. Davis, J. Magn. Reson. 65, 355 (1985).

[1]

2D NMR OF PARAMAGNETICMETALLOPROTEINS

9

then the NOE may become the larger effect, and it is quite possible to see a negative NOE cross-peak rather than a positive J cross-peak connecting even geminal CH2 protons. Chemical exchange produces cross-peaks of the same sign as true TOCSY peaks, but these may be distinguished by varying either temperature or concentration. This has been used to help obtain cross-assignments between the oxidized and reduced forms of cytochrome c in half-reduced samples. 4° Although a simple continuous irradiation can be used in the TOCSY mixing period, the effect is very sensitive to the quality of the spin-locking field (which is a serious limitation since a strong spin-locking field may be needed to cover the larger spectral widths encountered in paramagnetic systems), so that self-compensating phase modulation schemes are usually preferred. The most common sequences are those used for broadband spin decoupling, such as MLEV 4j or WALTZ16. 42Many minor modifications of these sequences have been employed, but without obvious advantages. Citations for a variety of methods may be found in the articles referenced herein. We prefer to use dispersion mode COSY or TOCSY, according to the need to observe cross-peaks to directly coupled protons or to the whole of the spin system. With realistic sample concentrations it is usually possible to detect couplings that are of the order of one-tenth of the linewidth; it is certainly not necessary for multiplets to be resolved for these methods to work. Cross-peaks have been detected for signals with linewidths close to l kHz 43a but these are unlikely to be J cross-peaks. 43b Heteronuclear analogs of these experiments are identical in principle hut require simultaneous irradiation at the frequencies of the two isotopes. With COSY, the antiphase cross-peaks are then a bar to broadband decoupiing of the signal detected in t2. Fortunately, heteronuclear couplings are often large enough for refocusing methods of the type used in ISECR-COSY to be used to bring the components back into phase before decoupling, without requiring prohibitively long delays during which much of the signal may be lost through relaxation. The simple fact that the largest J3C-IH coupling constants are I0 times greater than proton-proton couplings is one of the reasons for the application of heteronuclear COSY to Fermi contact shifted resonances 29appearing several years before similar homonuclear experiments. 23 4o y . Feng, A. J. Wand, H. Roder, and S. W. Englander, Biophys. J. 59, 323 (1991). 4T M. H. Levitt, R. Freeman, and T. Frenkiel, J. Magn. Reson. 43, 502 (1981). 42 A. J. Shaka, J. Keeler, and R. Freeman, J. Magn. Reson. 53, 313 (1983). 43a I. Bertini, F. Capozzi, S. Ciurli, C. Luchinat, L. Messori, and M. Piccioli, J. Am. Chem. Soc. 114, 3332 0992). 43b I. Bertini, C. Luchinat, and D. Tarchy, Chem. Phys. Lett. 203, 445 (1993).

10

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The other main group of techniques is designed to detect NOEs (Fig. 4). The basic NOESY experiment is sufficient for most purposes. Because the cross-peaks develop at the longitudinal cross-relaxation rate but the signal decays with the shorter time constant T~ (which is usually dominated by paramagnetic relaxation), the cross-peak intensity n3'3°'35'44 first increases linearly as a function of mixing time, reaches a maximum for times of the order of T], and then decays away (NOESY cross-peaks have been detected for signals with T] = 2 msec25'43a'45-47). As noted above, the maximum effect is always less than that observed by continuous saturation 44 H. C h e n g , K. G r o h m a n n , a n d W. S w e e n e y , J. Biol. Chem. 267, 8073 (1992). 45 j. S. de R o p p and G. N. L a Mar, J. Am. Chem. Soc. 113, 4348 (1991). 46 L. Banci, I. Bertini, P. T u r a n o , a n d M. V. Oliver, Eur. J. Biochem. 2@4, 107 (1992). 47 L. Banci, I. Bertini, P. T u r a n o , J. C. Ferrer, and A. G. Mauk, lnorg. Chem. 3@, 4510 (1991).

[1]

2D NMR OF PARAMAGNETICMETALLOPROTEINS

11

in a one-dimensional experiment, but NOEs to a large number of protons may be detected simultaneously. Ideally, the linear region for the buildup of the cross-peaks may be used to measure internuclear distances, but in practice the peaks may be too small to measure accurately. Even so, it is possible to obtain approximate internuclear distances from experiments with longer mixing times despite the distortion caused by spin diffusion and variations in T~ .H One further advantage is that the presence of paramagnetic ions strongly diminishes spin diffusion effects, which otherwise might hamper distance calculations. 25'48 Also, the shorter T~ values allow a much faster repetition rate, thus increasing the signal-to-noise ratio obtainable in the time available for the experiment. Chemical exchange processes in macromolecules are formally equivalent to cross-relaxation, and so cross-peaks arising from chemical exchange may be observed in NOESY spectra. The experiment is sometimes referred to as EXCTSY or 2D exchange spectroscopy (EXSY) when it is deliberately applied to the measurement of chemical exchange. The main difference is that the rate of cross-relaxation is necessarily less than the total relaxation rate, but there is no such limit on the rate of chemical exchange. Under suitable conditions it is therefore possible to observe strong chemical exchange cross-peaks between resonances with T~ values much less than 10 m s e c . 49-51 The rotating-frame equivalent of NOESY, called ROESY, 52 might seem to have no advantage for macromolecules, as the maximum ROESY effect is smaller than for NOESY in the slow motion limit. Furthermore, the signal decays more rapidly, with the time constant T~p (~T2) during the mixing time, than with T~. There are two distinct advantages in practice, however: the most important is that chemical exchange cross-peaks are of opposite sign to those due to NOEs, and so the two effects are easily distinguished (Fig. 5). Second, in the slow motion limit that applies to macromolecules, the cross-relaxation rate in the rotating frame is twice the longitudinal rate. ROESY cross-peaks therefore build up at twice the rate of NOESY cross-peaks, and correspondingly shorter mixing times 48 y . Yamamoto, K. Iwafune, N. Nanai, A. Osawa, R. Chujo, and T. Suzuki, Eur. J. Biochem. 198, 299 (1991). 49 L. Banci, I. Bertini, F. Briganti, C. Luchinat, A. Scozzafava, and M. V. Oliver, lnorg. Chem. 30, 4517 (1991). 5o L. Skjeldal, W. M. Westler, B.-H. Oh, A. M. Krezel, H. M. Holden, B. L. Jacobson, I. Rayment, and J. L. Markley, Biochemistry 30, 7363 (1991). 51 I. Bertini, F. Briganti, C. Luchinat, L. Messori, R. Monnanni, A. Scozzafava, and G. Vallini, Eur. J. Biochem. 204, 831 (1992). 52 A. A. Bothner-By, R. L. Stephens, J. Lee, C. D. Warren, and R. J. Jeanloz, J. Magn. Reson. 106, 811 (1984).

12

PROBES OF METAL ION ENVIRONMENTS

[1]

10

-12

o

-14

0

0 -16 ppm

ppm '

I'4

I'2

lJO

FIG. 5. ROESY spectrum of partially oxidized Desulfovibrio uulgaris cytochrome c 3 showing both positive peaks (full lines) due to intermolecular electron transfer and negative peaks (dotted lines) due to intramolecular NOEs. A mixing time of 10 msec was used with a spin-lock power of 10 kHz. Other experimental details are given in the legend to Fig. 1.

can be used. In fact, it is possible to obtain ROESY cross-peaks of similar intensity to those in NOESY spectra, and, whereas the appearance of TOCSY-type cross-peaks in ROESY spectra of small molecules is a common problem, the reverse is true for macromolecules. As described earlier, in TOCSY spectra obtained with short mixing times, the positive crosspeaks between J-coupled nuclei may be canceled or overtaken by the negative cross-peaks of the ROESY effect, and they may also be confused with the positive cross-peaks caused by chemical exchange. In summary, it is often useful to perform all of these experiments. This approach is particularly important in paramagnetic systems because the hyperfine shifts often obscure the correlation between the observed chemical shifts and their functional group identity. 25 TOCSY can pick out all of the resonances belonging to a particular spin system, but COSY will show which nuclei have significant couplings and are therefore separated by just two or three bonds. NOESY can be used to obtain approximate internuclear distances or to cross-assign partners in chemical ex-

[1]

2D NMR OF PARAMAGNETICMETALLOPROTEINS

13

change. ROESY is then useful to separate the peaks resulting from NOEs from those due to chemical exchange (when present), and, finally, comparison with the TOCSY experiment may reveal peaks that could have been lost through cancellation. Two other practical aspects should also be taken into consideration: first the amount of data accumulated must be adequate for the experiment and the information required. Too little digital resolution may obscure important information) ° Second, special attention is needed for 2D data processing. The reader is referred to the comprehensive descriptions given in Refs. 27, 34, and 53. Survey of Achievements In the previous section, the application of 2D NMR experiments useful for obtaining structural information about the immediate environment of the paramagnetic center in proteins, by providing information about resonances which are hyperfine shifted and fast relaxing, was illustrated with spectra typical of data that can be obtained with state-of-art spectrometers and realistic protein concentrations. Now it is worth looking at specific achievements in the study ofparamagnetic proteins obtained using 2D NMR. Rather than trying to be exhaustive, we give special attention to examples of studies designed to understand the structural basis for the control of physicochemical parameters and thus for the functional mechanism of paramagnetic metal ion center. However, it should be stressed that many other 2D NMR experiments have been performed in the so-called "diamagnetic" region of the spectra of these proteins) 4-56 The presence of chemical exchange in a particular system can be used to give combined information derived from its effects in different types of 2D experiments in order to aid assignments as well as to resolve degeneracies.12'24'a3a'51 Parallel studies might be necessary to obtain confidence in the interpretation of such experiments (e.g., alteration of the chemical exchange rate by modification of the experimental solution conditions or alteration of the mixing time of the 2D experiment in order to identify different contributions for the cross-peak intensities24'57). In any case, the confidence of the assignments is dependent on obtaining internally selfconsistent networks. 24'4° The assignment of resonances of protons whose 53 j. S. de Ropp, L. P. Yu, and G. N. La Mar, J. Biomol. N M R 1, 175 (1991). 54 y . Feng, H. Roder, S. W. Englander, A. J. Wand, and D. L. Di Stefano, Biochemistry 28, 195 (1989). 55 B.-H. Oh and J. L. Markley, Biochemistry 29, 3993 (1990). 56 D. Whitford, Eur. J. Biochem. 203, 211 (1992). 57 F. A. Walker and U. Simonis, J. Am. Chem. Soc. 113, 8652 (1991).

14

PROBES OF METAL ION ENVIRONMENTS

[1]

chemical shift is dependent on the oxidation state of a nearby metal ion can be used to measure the difference between the midpoint redox potentials of different centers in a multiredox center protein. Such assignments have been performed using NOESY (EXCTSY) experiments in tetraheme proteins 12 as well as in 2(4Fe-4S) center ferredoxins. 5z A complete assignment of heme protons of the heme axial ligands 15,24,26,58 and of those of some nearby residues 25'27 is essential to determine the orientation of the magnetic axes of the heme iron.15'58 This information allows an extraction of the scalar contribution to the total paramagnetic shifts, thus providing important information regarding the extent of delocalization of unpaired electron density (electronic structure). 24,25,29Similarly, correlation between the structural orientation, as well as the protonation, of the axial ligands and the midpoint redox potential of the heme iron has been demonstrated. 59 Another important subject is that of the structural control of the heme redox potential. This has been probed by determining the orientation and mobility of the heme vinyl substituents in myoglobin 23'6° as well as the protonation state of a proximal/distal histidine residue in the redox equilibrium between low-spin ferricytochrome c" and high-spin ferrocytochrome c". 61 Proton exchange studies have also been used to probe structural fluctuations in the heme cavity. 24'62 The spin-state equilibrium in ferricytochrome b562 at high temperature has also been studied using the assignments obtained with a combination of COSY and NOESY experiments. 62 The assignments of the/3-CH 2 cysteinyl residues ligated to the 4Fe cluster of high-potential iron-sulfur proteins (HIPIP), obtained by 2D NMR experiments, 44'51'63'64 have been instrumental in cross-assigning the iron atoms to their position in the structure and in explaining their inequivalence in HIPIPs with different midpoint redox potentials. 44'5~Similar studies performed in 2(4Fe-4S) ferredoxins allowed important comparisons between the structures of oxidized and reduced proteins to be obtained.SJ'65

58 H. Santos and D. L. Turner, Eur. J. Biochem. 206, 721 (1992). 59 L. Banci, I. Bertini, P. Turano, M. Tien, and T. K. Kirk, Proc. Natl. Acad. Sci. U.S.A. 88, 6956 (1991). 6o y . Yamamoto, K. Iwafune, N. Nanai, R. Chujo, Y. Inoue, and T. Suzuki, Biochim. Biophys. Acta 1120, 173 (1992). 6J H. S. Costa, H. Santos, D. L. Turner, and A. V. Xavier, Eur. J. Biochem. 208, 427 (1992). 62 j. Wu, G. N. La Mar, L. P. Yu, K. Lee, F. A. Walker, M. L. Chiu, and S. G. Sligar, Biochemistry 30, 2156 (1991). 63 S. C. Busse, G. N. La Mar, and J. B. Howard, J. Biol. Chem. 266, 23714 (1991). 64 D. G. Nettesheim, S. R. Harder, B. A. Feinberg, and J. D. Otvos, Biochemistry 31, 1234 (1992). 65 I. Bertini, F. Briganti, C. Luchinat, L. Messori, R. Monnanni, A. Scozzafava, and G. Vallini, FEBS Lett. 289, 253 (1991).

[1]

2D NMR OF PARAMAGNETICMETALLOPROTE|NS

15

E v e n for a moderately large protein (but large from the viewpoint of N M R structural studies) like horseradish peroxidase (42 kDa), resonances with T~ as short as 2 msec and line widths greater than 500 Hz have been assigned. Estimates of distances of different nuclei to the heme iron have been obtained, providing information on the spatial arrangement around the heme cavity. 66'67 A comparison of this structure with that of other peroxidases, 47'67 as well as a comparison of the solution and crystal structure of proteins and/or the X-ray structure of proteins with that of homologous ones for which this type of information is not available, was performed for different myoglobins. 48 One situation where severe conditions are also present is that of the high-spin ferricytochrome c'. 46 With extremely large line widths (close to 1 kHz) and very short Tl values, COSY and N O E S Y spectra were obtained (an N O E was detected for a broad peak at 139 ppm with T~ of 0.19 msec). Here, the structural data were instrumental in rationalizing the influence of pH in the modification of the delocalization of unpaired electrons. One should of course not forget all the tricks that were developed earlier in order to facilitate the 1D NMR study of the more severe cases.~'8-~° An example worth mentioning is that used by Bertini e t al. 68 to study the oxidized form of Cu(II)2, Zn(II)2-superoxide dismutase. By isomorphous replacement of Zn(II) by Co(II), the NMR spectrum becomes manageable. Que and co-workers 69 applied this method to obtain N O E S Y spectra of uteroferrin (35 kDa) where the Fe(II) of the Fe(III)-Fe(II) dinuclear active center was replaced by Co(II). Clear N O E S Y connectivities were observed for nuclei with T~ between 20 and 80 msec and line widths well over 1 kHz. The assignments obtained provided the first definitive evidence for a coordinated carboxylate. Concluding Remarks Although the presence of paramagnetic centers in the active site of proteins may severely limit the use of NMR for structural studies, it may also contribute to important advantages in studying the physicochemical properties of the paramagnetic center as well as the structural arrangement around it (where the most important physiological actions take place). The increased resolution resulting from hyperfine shifts makes it possible to observe the resonances of nuclei close to the active site. The strong and precise geometrical dependence of the paramagnetic contribution to 66j. D. Satterlee and J. E. Erman, Biochemistry 30, 4398 (1991). 67j. S. de Ropp, G. N. La Mar, H. Wariishi, and M. H. Gold, J. Biol. Chem. 266, 15001(1991). 68I. Bertini, F. Capozzi, C. Luchinat, M. Piccioli, and M. S. Viezzoli, Eur. J. Biochem. 197, 691 (1991). 69R. C. Holz, L. Que, Jr., and L.-J. Ming, J. Am. Chem. Soc. 114, 4434 (1992).

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PROBES OF METAL

ION ENVIRONMENTS

[2]

the chemical shift and relaxation of these nuclei can give important information on the local architecture o f the active site even in the absence of a global structure determination. Thus, it can be useful even for proteins much larger than those diamagnetic proteins for which N M R has been successful in the determination of the full three-dimensional structure. Furthermore, the sensitivity of the paramagnetic perturbations makes it an extremely useful technique to probe even small structural changes in the protein active site caused by modifications of physicochemical properties, solution parameters (e.g., pH, 61 temperature, 58 and ionic strength62), as well as binding to substrates, inhibitors, or other proteins. In particular, the dispersion of 13C resonances adds a new and sensitive probe for very small changes. 58'7° Another useful and relatively fast application is for comparison of the active site architecture in solution for homologous native proteins or proteins subjected to site-specific mutagenesis. As shown in this chapter, 2D N M R can be used in essentially all the cases for which 1D N M R has proved to be an extremely useful structural technique. F u r t h e r m o r e , the use o f 2D experiments to study the immediate environment of the active site has obvious advantages over the 1D analogs for cases where the resonances are within the crowded regions. Indeed, in this case the ambiguities resulting from off-resonance effects 71 caused •I • • by the strong irradiation needed when saturating fast-relaxing signals are easily o v e r c o m e . 70R. Timkovich, Inorg. Chem. 30, 37 (1991). 71j. T. J. Lecomte, S. W. Unger, and G. N. La Mar, J. Magn. Reosn. 94, 112 (1991). 72C. A. Salgueiro, D. L. Turner, H. Santos, J. LeGall, and A. V. Xavier, FEBS Lett. 314, 155 (1992). 73H. Costa, H. Santos, and D. L.Turner, Eur. J. Biochem. (in press).

[2] C a d m i u m - 1 1 3 Nuclear Magnetic Resonance Applied to Metalloproteins B y JOSEPH E. COLEMAN

Introduction Among the naturally occurring isotopes of cadmium there are two, ll3Cd and lllCd, that have a nuclear spin of ½ and are useful as nuclear magnetic resonance (NMR) probes for cadmium-containing molecules. Both isotopes are present at a significant percent natural abundance, namely, 12.75 and 12.26% for lllCd and 113Cd, respectively. They are both

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

16

PROBES OF METAL

ION ENVIRONMENTS

[2]

the chemical shift and relaxation of these nuclei can give important information on the local architecture o f the active site even in the absence of a global structure determination. Thus, it can be useful even for proteins much larger than those diamagnetic proteins for which N M R has been successful in the determination of the full three-dimensional structure. Furthermore, the sensitivity of the paramagnetic perturbations makes it an extremely useful technique to probe even small structural changes in the protein active site caused by modifications of physicochemical properties, solution parameters (e.g., pH, 61 temperature, 58 and ionic strength62), as well as binding to substrates, inhibitors, or other proteins. In particular, the dispersion of 13C resonances adds a new and sensitive probe for very small changes. 58'7° Another useful and relatively fast application is for comparison of the active site architecture in solution for homologous native proteins or proteins subjected to site-specific mutagenesis. As shown in this chapter, 2D N M R can be used in essentially all the cases for which 1D N M R has proved to be an extremely useful structural technique. F u r t h e r m o r e , the use o f 2D experiments to study the immediate environment of the active site has obvious advantages over the 1D analogs for cases where the resonances are within the crowded regions. Indeed, in this case the ambiguities resulting from off-resonance effects 71 caused •I • • by the strong irradiation needed when saturating fast-relaxing signals are easily o v e r c o m e . 70R. Timkovich, Inorg. Chem. 30, 37 (1991). 71j. T. J. Lecomte, S. W. Unger, and G. N. La Mar, J. Magn. Reosn. 94, 112 (1991). 72C. A. Salgueiro, D. L. Turner, H. Santos, J. LeGall, and A. V. Xavier, FEBS Lett. 314, 155 (1992). 73H. Costa, H. Santos, and D. L.Turner, Eur. J. Biochem. (in press).

[2] C a d m i u m - 1 1 3 Nuclear Magnetic Resonance Applied to Metalloproteins B y JOSEPH E. COLEMAN

Introduction Among the naturally occurring isotopes of cadmium there are two, ll3Cd and lllCd, that have a nuclear spin of ½ and are useful as nuclear magnetic resonance (NMR) probes for cadmium-containing molecules. Both isotopes are present at a significant percent natural abundance, namely, 12.75 and 12.26% for lllCd and 113Cd, respectively. They are both

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

[2]

ll3Cd N M R APPLIED TO METALLOPROTEINS

17

obtainable in 96% purity. Although there is only one naturally occurring well-characterized protein, metallothionein, that accumulates cadmium, most metalloproteins containing the Zn z+ ion (5d ~°electron configuration) can be substituted with the heavier Cd z+ ion (6d~°). In the case of metalloenzymes, the Cd substitution has often led to a protein that maintains measurable catalytic activity. For a variety of Zn-containing DNA-binding proteins, which require Zn in order to maintain the fold required for DNA recognition and binding, the Cd-substituted proteins have retained DNA binding. Thus, from both structural and functional standpoints 113Cd (~11Cd) NMR can be used as a valuable probe of zinc metalloprotein structure and function. 1-3 The cadmium(II) ion has an ionic radius of 0.97 ,&, nearly the same as that of Ca 2+, 0.99 ,~ (Pauling radii). Thus, Cd 2+ can be substituted at most Ca 2+ binding sites found in proteins. 4 The Cd 2+ ion can also fit the sites occupied by the Cu + ion (5d J°) and thus can be used to probe the metal sites in copper electron transfer proteins in which copper alternates between the Cu + and Cu 2+ states. 5 Metal ion replacement employs anaerobic techniques under which the Cu ÷ ion is removed and replaced by 113Cd2+.5 This chapter describes the application of ll3Cd NMR methods to the study of metalloproteins. HaCd Nuclear Magnetic Resonance, Sensitivity, Chemical Shift, and Sample Preparation

Sensitivity of 11~CdNuclear Magnetic Resonance NMR detection of 113Cd has a sensitivity of approximately 1% that of 1H and about 70% that of 13C. Although the sensitivity and other NMR characteristics of lllCd and 113Cdare nearly identical, 113Cdhas been used almost exclusively in studies of biomolecules. As both spin ½isotopes of Cd are present at a natural abundance of approximately 12%, it would be possible to carry out NMR studies with cadmium of natural isotope abundance (7.5 times more sensitive than natural abundance ~3C NMR). Because the Cd ion is being substituted, however, it makes no practical sense to use the unenriched metal. Even with N3Cd of 96% abundance, most protein samples require I-2 ml of an approximately 1 mM protein I I. M. Armitage, A. J. M. Schoot Uiterkamp, J. F. Chlebowski, and J. E. Coleman, J. Magn. Reson. 29, 375 (1978). 2 I. M. Armitage and J. D. Otvos, in "Biological Magnetic Resonance" (L. J. Berliner and J. Reuben, eds.), Vol. 4, p. 79. Plenum, New York, 1982. 3 p. D. Ellis, Science 221, 1141 (1983). 4 S. Forsen, E. Thulin, and H. Lilja, FEBS Left. 104, 123 (1979). 5 H. R. Engeseth, D. R. McMillin, and J. D. Otvos, J. Biol. Chem. 259, 4822 (1984).

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PROBES OF METAL ION ENVIRONMENTS

[2]

solution for an adequate signal-to-noise ratio (S/N). Enhanced sensitivity at higher fields has under some conditions reduced this concentration by about 10-fold (see below).

113CdNuclear Magnetic Resonance Chemical Shifts The l laCd (lllCd) nucleus when coordinated to the mixtures of oxygen, nitrogen, and sulfur donor atoms found in proteins shows a chemical shift range of 800-900 ppm. The chemical shift zero is generally chosen as that of the resonance of 0.I M Cd(CIO4) 2. There is a significant contribution to the chemical shift tensor of the 113Cd nucleus from the large electron cloud surrounding the nucleus, the "paramagnetic" component. Thus, the chemical shift of the 113Cd nucleus is expected to show large changes as functions of ligands and coordination geometry. So far, the observations for H3Cd-substituted proteins show that the nature of the donor atoms, O, N, or S, and the number of donor atoms, rather than the gross symmetry of the complex, have the most dramatic effects on chemical shift. In the earliest 113Cd NMR studies of Cd 2+ in small molecules, 6-9 coordination complexes with oxygen donors were observed to be the most shielding, whereas sulfur donors were the most deshielding. One of the obstacles to the collection of ~13Cd NMR data in aqueous solution is the presence of facile exchange between multiple species of Cd 2÷ complexes formed with simple ligands, many of them hydrated. Thus, the observed NMR resonances often represent averaged chemical shifts reflecting fast chemical exchange between several species in solution, shifts which vary with concentration. Measurements in the solid state coupled with the application of magic angle spinning (MAS) techniques can circumvent this problem, ~°-~3 but the data necessary for an extensive correlation of chemical shifts with coordination geometry of ll3Cd complexes are not available as yet. It does appear that a tetrahedral arrange6 G. E. Maciel and M. Borzo, J. Chem. Soc., Chem. Commun., 394f (1973). 7 R. J. Kostelnick and A. A. Bothner-By, J. Magn. Reson. 14, 141 (1974). 8 A. D. Cardin, P. D. Ellis, J. D. Adom, and J. W. Howard, Jr., J. Am. Chem. Soc. 97, 1672 (1975). 9 R. A. Haberkorn, L. Que, Jr., W. O. Gillum, R. H. Holm, C. S. Liu, and R. C. Lord, lnorg. Chem. 15, 2408 (1976). l0 H. J. Jakobsen, P. D. Ellis, R. R. Inners, and C. F. Jensen, J. Am. Chem. Soc. 1114, 7442 (1982). ii j. j. H. Ackerman, R. V. Orr, V. J. Bartucka, and G. E. Maciel, J. Am. Chem. Soc. 101, 341 (1979). 12 T. T. P. Cheung, L. Worthington, P. DuBois Murphy, and B. C. Gerstein, J. Magn. Reson. 41, 158 (1980). 13 p. S. Marchetti, P. D. Ellis, and R. G. Bryant, J. Am. Chem. Soc. 1117, 8191 (1985).

[2]

ll3Cd N M R APPLIED TO METALLOPROTEINS

19

ment of ligands is generally more deshielding of the ll3Cd nucleus than an octahedral arrangement. Although protein complexes of H3Cd2÷ can be influenced by the exchange of monodentate ligands, CdE+-protein binding is generally stable. In fact, Cd 2÷ appears to bind remarkably well to a great variety of sites including those normally occupied by Mg2÷ as well as those normally occupied by Zn 2÷ and Ca z÷. Approximately 20 different metalloproteins (not including species or isozyme variants) have been examined using l l3Cd NMR as a probe of the immediate coordination environment of the metal ion. The range of 113Cdchemical shifts shown by ll3Cd2+ substituted in 20 different proteins is illustrated in Fig. 1. The donor atoms around the ~3Cd2 + ion are indicated as well. Several specific conclusions about structure can be made from these data. Oxygen is the most shielding ligand, and chemical shifts of about - 100 parts per million (ppm) characterize every 113Cd2+-substituted Ca 2+ site in which other structural data indicate an octahedron of oxygen atoms around the metal ion, calmodulin being the prototype. 113Cd2+ chemical shifts near - I00 ppm may be considered diagnostic of such octahedral oxygen sites in proteins, although some data suggest that a few of these sites may be 7- or 8-coordinate. Chemical shifts near 0 ppm can with some confidence be assigned as well to sites likely to contain all oxygen ligands.14 There is a range of chemical shifts between 40 and 320 ppm for proteins containing various mixtures of N and O donors at the Cd 2+ binding sites. The data do not allow precise statements of ligand number, but increasing numbers of nitrogen donors are associated with increasing downfield shifts. The Mn 2+ site ofconcanavalin A, the Zn 2+ site of carboxypeptidase A, and the Zn 2+ site of carbonate dehydratase contain one, two, and three imidazole nitrogens (in addition to oxygen) as ligands, respectively, and their H3Cd derivatives show increasing downfield shifts in that order, with values of 46, 132, and 210-270 ppm (Fig. 1). Sulfur donor atoms are the most deshielding, as predicted from model studies. 9'j5 Each sulfur donor present at a 113Cd2+ binding site is so deshielding that it is possible to use 113Cd NMR shifts to determine the number of sulfur donor atoms at an unknown site (Fig. 1). Although the boundary between $3 and $4 coordination may be considered narrow if cluster compounds like metallothionein or the GAL4 transcription factor with bridging -S- ligands are included, the correlation is reasonably accu14 p. Gettins and J. E. Coleman, J. Biol. Chem. 258, 396 (1983). 15 R. A. Santos, E. S. Gruff, S. A. Koch, and G. S. Harbison, J. Am. Chem. Soc. 112, 9257 (1990).

20

PROBES OF M E T A L ION E N V I R O N M E N T S

[2]

rllll --

i

m

m

o

m

Z m

Z

| o

_

o

i o z

m~ Z Z

a. (3-

z m

i

0 Z m

~Y

I

z I

IJ_~lIi i ~

~

.-

z n

[2]

ll3Cd N M R APPLIED TO METALLOPROTEINS

21

rate. 113Cd2+ coordinated to one thiolate and one thioether sulfur donor resonates in the range of 400 - 30 ppm (azurin, plastocyanin),5 whereas sites containing two thiolate ligands resonate near 500 ppm (the active site of alcohol dehydrogenase).16 Stellacyanin does not have a thioether ligand, but the second sulfur donor appears to be a cystine disulfide.5 ll3Cd 2+ bound to three thiolate ligands resonates between 630 and 670 ppm [gene 32 protein, human immunodeficiency virus (HIV) nucleocapsid protein]. 17,18 Isolated tetrathiolate sites examined thus far show ~13Cd2+ resonances from 704 to 751 ppm (the two Zn sites of the glucocorticoid receptor and the structural Zn site in alcohol dehydrogenase). ~6,~9 If the tetrathiolate sites that involve -S- ligands shared between two H3Cd2+ ions, as in the metal clusters found in metallothionein2,2°'2~ and the transcription factors GAL422'z3 and LAC9, 24 are included, then the chemical shift range moves moderately upfield from those for strictly tetrathiolate sites. Some of the resonances from such sites can overlap the chemical shifts shown by N S 3 coordination (Fig. 1).

Sample Preparation Although NMR spectrometers with multinuclear capabilities are now widely available, the low NMR sensitivity of the 1~3Cdnucleus has generJ6 B. R. Bobsein and R. J. Myers, J. Am. Chem. Soc. 102, 2454 (1980). 17 D. P. Giedroc, B. A. Johnson, I. M. Armitage, and J. E. Coleman, Biochemistry 28, 2410 (1989). 18 D. W. Fitzgerald and J. E. Coleman, Biochemistry 30, 5195 (1991). 19 T. Pan, L. P. Freedman, and J. E. Coleman, Biochemistry 29, 9218 (1990). 20 j. D. Otvos and I. M. Armitage, J. Am. Chem. Soc. 101, 7734 (1979). 2t j. D. Otvos and I. M. Armitage, Proc. Natl. Acad. Sci. U.S.A. 77, 7094 (1980). 22 T. Pan and J. E. Coleman, Proc. Natl. Acad. Sci. U.S.A. 86, 3145 (1989). 2~ K. H. Gardner, T. Pan, S. Narula, E. Rivera, and J. E. Coleman, Biochemistry 30, 11292 (1991). 24 T. Pan, Y. D. Halvorsen, R. C. Dickson, and J. E. Coleman, J. Biol. Chem. 265, 21427 (1990).

FIG. 1. Range of ll3Cd NMR chemical shifts, ~5,observed for H3Cd-substituted metalloproteins. A bar indicates that a range of 8 values have been observed for the same site under a variety of conditions (e.g., changes in pH or monodentate ligands). In the case of metallothionein, the bar represents the distribution of the s e v e n I13Cd signals observed for the threeand four-metal clusters in the mammalian HsCd7-substituted protein. Alkaline phosphatase, in the absence of phosphate, shows a well-defined signal for only one of the three 11sCd2+ sites owing to chemical exchange modulation of the signals when the other sites are occupied. The signal from the 06 site is not detected in the absence of the phosphate ligand. The [-]~denotes carbonate dehydratase with a C-coordinated C N - at the active site.

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PROBES OF METAL ION ENVIRONMENTS

[2]

ally required protein samples of 1-5 mM concentration. Most I J3Cd experiments have been done on instruments with ~H resonance frequencies of 90, 200, 250, and 400 MHz for which the ~3Cd resonance frequencies are 19.96, 44.4, 55.8, and 88.8 MHz, respectively. For reasonable data collection time, namely, less than 24 hr (10,000-I00,000 transients), a 10 mm diameter tube containing 1.8-2.0 ml of sample at 1-5 mM protein is required to obtain adequate signal-to-noise ratios. It has proved possible to obtain excellent 1~3Cd NMR spectra of proteins at 11.75 T (110.9 MHz for ll3Cd, 500 MHz for IH) without undue broadening from chemical shift anisotropy (see below). Enhanced sensitivity at the higher field has made it possible to detect a 113Cd resonance at a protein concentration of 0.1 mM in a 10 mm NMR tube. At the more usual concentrations of protein used for ~3Cd NMR (I-5 mM), it is possible at 110.9 MHz to use a 0.5 ml sample in a 5 mm NMR tube and detect a reasonable signal. Substitution of 113Cd2+ for the native metal ion has required the development of a variety of methods, reflecting the fact that a given method does not work for all proteins. A number of metalloproteins have proved to be stable in their apoprotein forms, so that the l l3Cd2+ c a n be added in stoichiometric fashion (e.g., carbonate dehydratase 25and alkaline phosphatase~4). At pH values above 5, Cd 2+ must be held in solution by a metal-complexing buffer. Tris-HCl or Tris-HCl-acetate at 10 mM are the most widely used, where the amino group of Tris forms a complex with Cd 2÷ stable enough to compete against the hydroxide ion but not to compete with most protein sites for the Cd 2+. Apoproteins can be prepared by removal of the native metal ions with chelating agents (often at pH values between 5 and 6; e.g., carbonate dehydratase 26) or with 1 M ammonium sulfate at pH 9 followed by dialysis against metal-free buffer (e.g., alkaline phosphatasel4). In the case of proteins containing sulfur ligands it is often possible to displace the Zn 2+ with organic mercurials and then dialyze against thiols to displace the mercurial, followed by metal-free buffer to remove the thiol (e.g., gene 32 protein27). For proteins containing thiolate ligands, it has proved advantageous to replace the metal by exchange rather than produce the unstable apoproreins as intermediates. Multiple thiol ligands may cross-link in the apoproreins, or Cd may form alternate ligand arrangements that then rearrange to the native conformation only slowly. It has often proved possible to 25 A. J. M. Schoot Uiterkamp, I. M. Armitage, and J. E. Coleman, J. Biol. Chem. 255, 3911 (1980). 26 j. B. Hunt, M. J. Rhee, and C. B. Storm, Anal. Biochem. 79, 614 (1977). 27 D. P. Giedroc, K. M. Keating, K. R. Williams, and J. E. Coleman, Biochemistry 26, 5251 (1987).

[2]

113Cd N M R APPLIED TO METALLOPROTEINS

23

exchange metal ions by adding 113Cd2+ in modest stoichiometric excess to the protein in a relatively small volume, incubating for 24 or 48 hr, and then dialyzing off the displaced metal, Zn 2+ in most cases. 17'23 Repeating the process will ensure complete replacement without using large amounts of expensive 113Cd2 +. Such exchanges should be done anaerobically under nitrogen and in the presence of 2-mercaptoethanol. The latter reagent, at a concentration of 1 mM, does not compete significantly for Cd 2+ with most thiolate-containing protein binding sites. For some proteins the presence of 2-mercaptoethanol appears to facilitate the exchange. Methods applied to the classic case of metallothionein are covered in Volume 205 of this series. 28 It is desirable to carry out the manipulations required to obtain the l~3Cd-substituted protein at protein concentrations of 0.1 mM or lower in order to avoid protein loss as dialysis bags are emptied or other surface contacts are made. The final sample can be prepared by concentration in an Amicon (Danvers, MA) or other ultrafiltration device once the protein is in the appropriate buffer. Standard ll3Cd NMR spectra can be run in 90% H20-10% D20. Proton decoupling is rarely employed (see below). Complete two-dimensional (2D) proton NMR spectra can be run on the same samples if the size of the protein is appropriate, but the protonated forms of Tris-HC1 and 2-mercaptoethanol must be removed. One can use deuterated buffers, but it is possible, once the protein contains stoichiometric Cd 2+ , to dialyze against H20, lyophilize, and redissolve the protein in pure H20. Careful monitoring of pH is required, but generally a water solution of a protein will not drift below pH 5.5. If the protein cannot be lyophilized, the protonated buffer can be exchanged for phosphate buffer as long as significant excess free Cd z+ is not present. The latter will precipitate as the phosphate or hydroxide complex. Because there are a limited number of ll3Cd r e s o n a n c e s from a single protein and multiple resonances, if present, are likely to be separated by A8 values significantly larger than the 113Cd line width, 113Cd NMR can be usefully applied to large proteins. Good signal resolution has been achieved for a protein, aspartate carbamoyltransferase (transcarbamylase), with a molecular weight of 310,000 (R. E. Cohen and H. K. Schachman, unpublished data). If one is interested in detecting coupling between Jl 3Cd and nonexchangeable protons on amino acid side chains by observing a 2D ~H correlation spectroscopy (COSY) spectrum, then it may be an advantage to use a sample in D20 solution to avoid the application of water suppression techniques. 28 j. F. Riordan and B. L. Vallee (eds.), this series, Vol. 205.

24

PROBES OF METAL ION ENVIRONMENTS

[2]

Relaxation of ll3Cd Nucleus Knowledge of the spin-lattice relaxation time (T I) and the nuclear Overhauser effect (NOE) applying to the l l3Cd nucleus when bound to protein sites is of great practical importance because both phenomena affect the NMR parameters one chooses for optimal signal observation. First and foremost, if one wishes to use the area under the observed mCd resonance peak to quantitate the l~3Cd giving rise to the observed signal by comparing it with the mCd resonance from a known standard, one must use a completely relaxed (maximum amplitude) signal. Because of the poor sensitivity of ll3Cd, it is often difficult to obtain enough data to determine Tl accurately, but it is important early in the study of a mCd-labeled protein to increase the delay time between pulses until the amplitude of the signal ceases to increase as the delay is further increased (i.e., the ll3Cd nucleus is allowed to fully relax between pulses). One can then determine how much of the ll3Cd added is giving a signal by comparison to the ll3Cd signal from the standard. It is important to know if one is observing all of the mCd bound to the protein or only a partially occupied or minor site (see section on chemical exchange below). Depending on the length of TI one may wish to compromise for routine spectra between complete relaxation and collecting more transients in a given time. Two major mechanisms of relaxation apply to the H3Cd nucleus, namely, dipolar relaxation from protons in the vicinity of the Cd 2+ ion and chemical shift anisotropy (CSA). Because solid-state l~3Cd spectra of model mCd2+ complexes show that mCd2+ complexes often have large CSA, it was predicted that in the case of the H3Cd nucleus bound to proteins the major relaxation mechanism would be CSA. In fact, there was concern that the effect of CSA on T2 relaxation of l l3Cd bound to large proteins would so broaden the l l3Cd signal that mCd NMR of proteins at high fields would be impossible. CSA has been shown to be the dominant relaxation mechanism for several l~3Cd-substituted proteins, 2,29,3°but line broadening has not been as severe as predicted. Because the protons on the amino acid side chains commonly found as ligands in most metalloproteins are more than 3.5 .~ distant from the metal ion, dipolar relaxation of "3Cd by adjacent protons has been thought unlikely to be a major relaxation mechanism. It is fortunate that the CSA relaxation mechanism is present; otherwise, pure dipolar relaxation from the distant protons in proteins would probably result in Tl values of at least 10 sec, and data collection would be very inefficient. z9 j. D. O t v o s and I. M. Armitage, Biochemistry 19, 4021 and 4031 (1980). to D. B. Bailey, P. D. Ellis, a n d J. A. Fee, Biochemistry 19, 591 (1980).

[2]

ll3Cd N M R APPLIED TO METALLOPROTEINS

25

Although these assumptions seem to be roughly correct, NOE (a dipolar process) measurements on the ll3Cd nucleus in proteins show that dipolar modulation processes can be present which are characterized by more effective relaxation frequencies than those provided by the overall protein reorientation (i.e., the rotational correlation time rR). Thus other, more rapid processes involving magnetic dipoles (most likely protons) must be present. One possibility is the presence of coordinated water molecules that rotate around their metal-oxygen bonds or exchange with bulk solvent either by breaking the metal-oxygen bond or exchanging one of the protons with bulk solvent. The frequencies for these processes can be estimated to be 10-8-10 -9 sec -1. A second source of dipolar interactions may be N - H backbone protons that form hydrogen bonds to the liganding thiolate sulfur atoms in a number of thiolate Zn and Cd complexes (e.g., metallothionein,3~ GAL4, 32 and aspartate carbamoyltransferase33). These N - H • •. S - - C d protons are closer to the metal ion than the 3.5 ,~ limit assumed for/3-CH 2 protons of cysteine or the CH protons of imidazole side chains. In the ideal case one would like to have enough information to make a complete analysis of relaxation for a given ll3Cd r e s o n a n c e from a protein, since such an analysis would provide interesting dynamic information. Unfortunately, the detailed structural information required to determine the nature of the dipolar interactions is usually not available, and the solid-state l l3Cd spectra of proteins required to determine the magnitude of the CSA are not obtained easily, although some are now being reported. ~3 From a practical standpoint, because dipolar relaxation and CSA relaxation are both frequency dependent, significant information about their contributions can be obtained if one measures the T~ and the NOE of a p r o t e i n - b o u n d 113Cd at two field strengths. An example of such an analysis is provided by the T~and NOE measurements on ll3Cd gene 32 protein obtained at 44.4 and 110.9 MHz [4.7 and 11.75 tesla (T)] (Table I). 17 There is a measureable negative NOE present at the lower field, "0 + I = 0.77, whereas no NOE is observed at the higher field "0 + 1 = 1.00. Thus significant dipolar interaction must contribute to relaxation at 4.7 T. In the absence of a measurement of CSA by solidstate NMR, one can assign a value of the CSA and calculate the T~ expected. If it is assumed that the T~ of 1.1 sec for the ll3Cd nucleus in 31 A. H. Robbins, D. E. McRee, M. Williamson, S. A. Collett, N. H. Xuong, W. F. Furey, B. C. Wang, and C. D. Stout, J. Mol. Biol. 221, 1269 (1991). 32 R. Marmorstein, M. Carey, M. Ptashne, and S. C. Harrison, Nature (London) 356, 408 (1992). 33 H. L. Monaco, J. L. Crawford, and W. N. Lipscomb, Proc. Natl. Acad. Sci. U.S.A. 75, 5276 (1978).

26

PROBES OF METAL ION ENVIRONMENTS

[2]

TABLE I SPIN-LATTICE RELAXATION TIME, TI, FOR ll3Cd NUCLEI AT PROTEIN BINDING SITES Protein

/'1 (sec)

Field strength (T)

Ref.

Alkaline phosphatase, Cd A, 170 ppm Alkaline phosphatase + Pi, CdB, 56 ppm Carbonate dehydratase Superoxide dismutase Gene 32 protein Gene 32 protein Azurin Plastocyanin Stellacyanin Metallothionein GAL4(62), 707 ppm GAL4(62), 669 ppm

5.2

4.7

29

6.2

4.7

29

1.6 1.2 0.5 1.1 0.15 0.15 0.12 -0.5 ~0.1 ~0.1

2.11 2.35 4.7 11.75 5.8 5.8 5.8 4.7 11.75 11.75

a 30 17 5 5 5 b 23 23

a I.M. Armitage and J.E. Coleman (unpublished). b I.M. Armitage (personal communication).

gene 32 protein at 11.75 T reflects primarily CSA, then a CSA of approximately 200 ppm is required to generate a T1 of about 1 sec for a ll3Cd nucleus held rigidly in a molecule with a rR of approximately 10 -8 s. ~7 One can combine the CSA calculation with the additional calculation of dipolar relaxation expected from one or more protons within 2.5 ,~ of the l l3Cd nucleus. Because of the way that Ho 2 and to2~-c2 enter the expression for CSA relaxation, 34 these terms have opposite and nonequivalent effects on the magnitude o f CSA relaxation as a function of field. Eventually at high enough field the Ho z term will dominate and CSA relaxation will increase with field strength. H o w e v e r , 4.7 and 11.75 T happen to be a pair o f fields at which the increase in the Ho 2 and 092 terms (for the ll3Cd resonant frequencies) approximately cancel out, and CSA relaxation is approximately the same at both fields. The dipolar contribution if present will be more effective at 4.7 T, and the TI may be shorter at the lower field, as is observed in the case of l~3Cd gene 32 protein (Table I). The limited T~ and N O E data available for mCd-substituted proteins are collected in Table I. Although detailed analysis or theoretical calculations are not possible at present, it is probably fair to assume that for those 34 T. C. Farrar and E. D. Becker, in "Pulse and Fourier Transform NMR," p. 59. Academic Press, New York, 1971.

[2]

27

ll3Cd N M R APPLIED TO METALLOPROTEINS

1.0

0.8

4.7 T f * " " f "

0,6 o., 0.2

o Ill

.11 T

-o.2

- 0.6 -0.8 - 1.0

- 1.4

IO-~l

I

I

I

I

I I Ill

10-1o

I

I

I

I

1 I I II

10-9

I

I

I

I

I I I I]

IO-a

I

I

I

I

1 I I I1

io-r

TR, Seconds FIG. 2. Plots of the 1H-113Cd NOE as a function of the molecular rotational correlation time, ~'R, in seconds. Two magnetic field strengths are shown, 2.11 and 4.7 T. The calculated curves are based on the isotropic rigid rotor model for dipolar relaxation from protons. (Data points are plotted from Ref. 2.)

proteins with relatively short ll3Cd T~ values (<0.5 sec), the CSA mechanism of relaxation is predominant. Concluding this discussion of ll3Cd relaxation, an NOE if present will be dependent on both the field strength and the motion, that is, effective rotational correlation time, TR, of the protein-bound 113Cd. If one uses the isotropic rigid rotor model and assumes that the appropriate motion is described by the overall tumbling of the macromolecule containing the ~13Cd, then one can construct theoretical curves of NOE versus TRfor the 1~3Cd nucleus as shown in Fig. 2. The curves assume that relaxation occurs entirely by the dipolar mechanism. Because of the negative magnetogyric ratio of the ll3Cd nucleus, NOE values, expressed as "O + 1, range from 1.25 for small cadmium complexes to a limiting value of 0.84 for a large macromolecular complex of ~'R = 10-7 sec. Depending on the field, there are intermediate values of TR where the NOE itself is - 1 and therefore r/ + 1 = 0. Thus, no signal would be observed if proton decoupling were applied. Note that this value of rR depends on the field strength at which the measurement is being made. These NOE considerations do not usually affect experiments with proteins because ~H-113Cd couplings are small and are obscured either by exchange averaging of the ln3Cd resonance or by the "natural" line width of the H3Cd signal. Thus, it is rarely necessary to employ proton decoupling. There have been exceptions. To resolve the l13Cd-l13Cd cou-

28

PROBES OF METAL ION ENVIRONMENTS

[2]

A It3Cd CPD

/&

B +,8- phenylpropionate

I

I ~llo

I

I ~6o

I

I ~4o

I

I izo

I

i ioo

I

PPM

C 100 --

J

1

lJ I

2

3

113Cd/A p DIMER

4

I so

[2]

ll3Cd N M R APPLIED TO METALLOPROTEINS

29

plings that are present in the ~13Cd cluster site of metallothionein, it was necessary to remove the small but significant ~H-l13Cd couplings.2'2° Resolution of the l l 3 C d - l l 3 C d coupling in metallothionein by decoupling the protons was first attempted at 2.1 T (90 MHz 1H), and the protein was discovered to have an NOE (,/ + 1) near zero at this field (see plot in Fig. 2). The experiment with proton decoupling was successful at a field of 4.7 T, where the z R at which r/ + 1 = 0 is shifted to significantly shorter values than that of the dipolar relaxation process occurring in metallothionein. The latter has an effective ZR of 1 0 - 8 - 1 0 -9 s e c and may reflect an exchanging H20 or an exchanging protein ligand at one of the metal sites. Despite the presence of a dipolar component, CSA remains the major relaxation mechanism in 113Cd-metallothionein.

Chemical Exchange Modulation of l l 3 C d Nuclear Magnetic Resonance Signals from Proteins The existence of two narrow NMR signals from two species in slow chemical exchange, a single narrow signal at an average chemical shift arising from two species in fast chemical exchange, or broadened single or double resonances from intermediate chemical exchange is familiar to t h e I H NMR spectroscopist. The low sensitivity of 113Cd NMR and the large chemical shift differences, A8 values, possible between two closely related H3Cd2+ complexes make chemical exchange modulation of l13Cd NMR signals in proteins an interesting but often undesirable complicating factor. Signal-to-noise ratios for the ll3Cd nucleus in a protein are low under the best of circumstances. Thus, employing high enough sample concentrations or accumulating enough data to observe ll3Cd signals broadened by intermediate chemical exchange is often impossible. Therefore, intermediate chemical exchange between two protein conformations generally causes the l l3Cd signal to disappear. Two classic examples are shown in Fig. 3. 113Cd-substituted carboxypeptidase A (1 M NaCl is required to solubilize the enzyme) does not show a detectable ll3Cd NMR

FIG. 3. Chemical exchange modulation of tt3fd NMR signals from lt3Cd-substituted carboxypeptidase A and alkaline phosphatase. The H3Cd-substituted carboxypeptidase A was studied in the absence (A) and presence (B) of the inhibitor fl-phenyl propionate. (C) 113Cd NMR signals observed during the titration of apoalkaline phosphatase with one to four tt3Cd ions. The narrow signal which titrates to maximum amplitude at two mCd2+/enzyme dimer has been assigned to the A site of the enzyme (see text). Spectra like those shown here have been published in Ref. 1 (A, B) and Ref. 14 (C).

30

PROBES OF METAL ION ENVIRONMENTS

[2]

signal. 35'36 On the other hand, if the tightly bound competitive inhibitor, /3-phenyl propionate, is added, a sharp 113Cd signal appears (Fig. 3). One has to conclude that the ll3Cd ion in the inhibitor-free protein was subject to a chemical exchange process, perhaps exchange of a coordinated CIor H20 at the active site. This exchange must give rise to at least two species whose chemical shift differences combined with the frequency of ligand exchange correspond to intermediate chemical exchange. The addition of the tightly bound inhibitor, which must be in slow exchange, then gives a single predominant species of Cd 2÷ complex. Although one can model the ll3Cd NMR signals of two exchanging ll3Cd species as a function of the exchange lifetime, 37 ~-, it has rarely been possible to observe the signals from two exchanging ll3Cd species in a protein. For example, if two exchanging ll3Cd species have signals separated by 100 ppm, then for a field strength of 4.7 T and an exchange lifetime, ~-, of 10 -4 sec, the signal as modeled is so broadened that it would not be likely to be detected because of the low sensitivity of ll3Cd NMR. 37 An exchange lifetime of 10 -3 to 10 -4 s e c would be fast chemical exchange for practically all IH NMR detection regimes. In contrast, the lower resonance frequencies for ll3Cd and the much larger A8 values characterizing mCd signals from different environments give rise to intermediate exchange frequencies that appear to match those of conformational changes found in 113Cd proteins (e.g., protein-ligand exchange, H20 or anion exchange). The failure to observe a 113Cd signal from a protein under initial conditions or the appearance of broadening of the signal when an additional modification of a protein is made has been such a common occurrence, that if one is attempting 113CdNMR on a new protein, chemical exchange modulation should be kept in mind. If one does not observe a signal initially, try other conditions or add ligands. The complexity of some of the chemical exchange modulations of 113Cd signals observed in proteins is illustrated by the second example (Fig. 3C). Addition of two H3Cd ions to alkaline phosphatase gives rise to a narrow 113Cd resonance at 168 ppm owing to the occupancy of a pair of identical sites (site A), one in each monomer of the symmetrical dimer. 14 Alkaline phosphatase possesses three closely spaced metal binding sites

35 I. M. Armitage, A. J. M. Schoot Uiterkamp, J. F. Chlebowski, and J. E. Coleman, J. Magn. Reson. 29, 375 (1978). 36 p. Gettins, J. Biol. Chem. 261, 15513 (1986). 37 j. E. Coleman, I. M. Armitage, J. F. Chlebowski, J. D. Otvos, and A. J. M. Schoot Uiterkamp, in "Biological Applications of Magnetic Resonance" (R. G. Shulman, ed.), p. 345. Academic Press, New York, 1979.

[2]

113Cd NMR APPLIEDTO METALLOPROTEINS

31

in each monomer. 38'39 There is a second metal binding site (site B) 3.9 away from A. Although sites A and B do not share any ligands, when the two B sites are titrated with l l3Cd, the original signal disappears (Fig. 3C). 14 A conformational modulation of the metal at site B must be transmitted to site A, which itself participates in an induced conformational modulation. Since the initial resonance disappears without broadening, the original species is not one of those participating in the exchange. The doubly occupied monomer must have at least two n e w 113Cd species that are exchanging at a frequency corresponding to intermediate chemical exchange. The basic process may relate to ligand or metal ion exchange at site B, which binds the ~3Cd2+ ion less tightly than site A. Unfortunately, the resonance positions of the species in chemical exchange cannot be observed, and thus one cannot determine the A8 values involved and make a more precise analysis of the frequency of the conformational processes responsible for the modulation. The complex exchange processes occurring in ll3CdA,H3CdB-alkaline phosphatase are completely damped out by addition of phosphate (a substrate as well as inhibitor) to the active site. Phosphate coordinates by bridging both A and B site l l 3 C d i o n s . 38,39 Two sharp ll3Cd NMR signals appear, one at 153 ppm from the two A site ~13Cd2+ and one at 70 ppm from the two B site 113Cd2+. As in carboxypeptidase A, ligand binding is observed to freeze out a chemical exchange process modulating the ~13Cdresonance in a protein.

l13Cd-113Cd Scalar Coupling in x13Cd-Substituted Metalloproteins When -S- ligands bridge between two 113Cd-~3Cd ions as in metal clusters, N3Cd-II3Cd J coupling is expected to be present. Only two examples of Cd 2÷ cluster sites occur in proteins, the four-Cd and three-Cd cluster sites found in metallothioneins and the binuclear Cd cluster found in the DNA-binding domains of yeast transcription factors like GAL4. In the presence of proton decoupling, the H3Cd-H3Cd J coupling has been observed directly in the case of metallothionein as fine structure appearing on the 113Cd resonances (Fig. 4). 20,40 With the good resolution shown in Fig. 4, decoupling can be used to distinguish which pairs of H3Cd signals represent J-coupled H3Cd ions. As indicated in Fig. 4, homonuclear decoupiing shows the crab metallothionein to contain two three-Cd clusters 3s E. E. Kim and H. W. Wyckoff, J. Mol. Biol. 218, 449 (1991). 39 j. E. Coleman, Annu. Rev. Biophys. Biomol. Struct. 21, 441 (1992). 4o j. D. Otvos and I. M. Armitage, in "Biochemical Structure Determination by NMR" (B. D. Sykes, J. Glickman, and A. A. Bothner-By, eds.), p. 65. Dekker, New York, 1981.

32

PROBES OF METAL ION ENVIRONMENTS

2

Cd

1

1

3

Ca

co

,T,z

6

Cd

[2]

5

0

Cd

2 3,4

6

B

660

650

640

I 630

,

I 620

PPM

FIG. 4. l l a C d NMR spectra (44.4 MHz) of native crab metallothionein 1 ( - 8 mM; l l 3 C d : Zn, 60 : 1) at pH 9.0. (A) Proton-decoupled spectrum of 23,000 transients. (B, C) Same as (A) but with homonuclear decoupling pulses applied at the frequencies indicated by the arrows. (From Ref. 40 with permission.)

represented by ~13Cd resonances 1, 2, 6 and 3, 4, 5. Two-dimensional ll3Cd-H3Cd COSY spectra have been collected on 113CdT-metallothionein, 4~'42 and most of the expected H3Cd-I13Cd couplings are represented by cross-peaks. Mammalian H3Cd7-metallothionein, containing a four-Cd 41 y. Boulanger, C. M. Goodman, C. P. Forte, S. W. Fesik, and I, M. Armitage, Proc. Natl. Acad. Sci. U.S.A. 80, 1501 (1983). 42 M. H. Frey, G. Wagner, M. Vasak, O. W. Sorensen, D. Neuhaus, E. W6rg6tter, J. H. R. K~igi, R. R. Ernst, and K. W0thrich, J. Am. Chem. Soc. 107, 6847 (1985).

[2]

ll3Cd N M R APPLIED TO METALLOPROTEINS

33

and a three-Cd cluster, may be a relatively unique example. The coupling between the two n3Cd ions of GAL4, connected by two bridging Cys -S-, has not been resolved either directly on the 1~3Cd signals or by a 2D l l3Cd-H3Cd COSY experiment. 43 Heteronuclear Nuclear Magnetic Resonance Techniques Applied to ll3Cd-Substituted Metalloproteins In contrast to the limited applicability of 1~3Cd-H3Cd coupling as a structural probe, J coupling of the n3Cd nucleus to nuclei that are part of the protein-ligand side chains or part of coordinated enzyme substrate analogs or inhibitors has proved to be useful in many situations. J coupling of U3Cd to IH, ~3C, 15N, and 31p have all been usefully employed as NMR spectroscopic probes of protein structure.

113Cd-IH Scalar Coupling The fl-CH 2 protons of all Cys residues that act as ligands to H3Cd show J coupling to the 113Cdnucleus when examined by proton NMR. 23'44-46The observed couplings range from 5 to 50 Hz and are most easily observed by examining the aft and tiff' proton cross-peaks in a 2D IH COSY spectrum of a protein substituted with ll3Cd. 44'46 If a 2D 1H COSY spectrum has been assigned by the standard techniques, then the Cys side chains coordinated to the metal ion can be identified. It is of course best to prepare a 1~2Cd(spin 0)-substituted protein as the control, as some changes in chemical shift of the protons on ligand side chains of a protein are likely to occur when substituting Cd for the native Zn ion. The characteristic patterns resulting from the splitting of the IH COSY cross-peaks by ll3Cd have been discussed in detail for metallothionein 44,45 and GAL423'46 and are not discussed further here. In theory, the pattern of IH-ll3Cd scalar coupling resolved on the ~H COSY cross-peaks can be used to detect coupling of the/3 protons of a given Cys ligand to two n3Cd ions, that is, the presence of a bridging Cys. 44'45In practice, the ~H-ll3Cd heteronuclear techniques described below are more definitive. ~H couplings to the second ~13Cd nucleus are often small (approximately the line width of the COSY cross-peaks) and the patterns of COSY cross-peaks arising from overlap of complex multiplets are often difficult to deconvolute. 23,44 43 T. Pan and J. E. Coleman, Biochemistry 29, 3023 (1990). 44 D. Neuhaus, G. Wagner, M. Vasak, J. H. R. K~igi, and K. Wiithrich, Eur. J. Biochem. 143, 659 (1984). 45 K. WiRhrich, " N M R of Proteins and Nucleic Acids," p. 158. Wiley (Interscience), New York, 1986. 46 T. Pan and J. E. Coleman, Proc. Natl. Acad. Sci. U.S.A. 87, 2077 (1990).

34

PROBES OF METAL ION ENVIRONMENTS

'/r/2) x

[2]

"/'r

1H

i |

hi iTM

T

,A ~'

tl

L, r'

7"

~J "1

t2

FIG. 5. Diagram of the pulse sequences for obtaining the lH-]I3Cd heteronuclear multiple quantum correlation (HMQC) spectrum described in the text and shown in Fig. 6A. The 113Cd spins are correlated with those of J-coupled protons, the /~-CH2, or their coupling partners, a-CH. The phase, 4), is cycled for the desired order, p, of H3Cd coherence (4) = kzr/p, k = 0, 1. . . . 2p - 1, with alternate addition and subtraction of the free induction decay, FID). The U3Cd-C~H relay peaks can be enhanced at the expense of the lt3Cd-Ct~H correlations by placing a single (~-/2)~ relay pulse at the position of the dashed line.

For relatively small proteins or protein fragments(under 100 amino acid residues) which bind H3Cd2+, there are two heteronuclear NMR techniques that detect 1H-H3Cd coupling and, when high-quality spectra are obtained, give unambiguous identification of H3Cd-coupled protons. These are a IH-113Cd heteronuclear multiple quantum correlation (HMQC) spectrum (or a IH-H3Cd heteronuclear COSY) and a ~13Cd-filtered 2D ~H COSY difference spectrum. The complete technical details are covered in Refs. 42, 44, and 47 for the HMQC method and in Refs. 48 and 49 for the H3Cd filter method. For ~H-JI3Cd HMQC spectroscopy, proton magnetization is begun with a 90 ° pulse, and when the initial coherence has evolved to antiphase magnetization with respect to l~3Cd during the delay time, ~-, a 90 ° 113Cd pulse is applied (Fig. 5). The 90 ° 113Cd pulse creates a heteronuclear two-spin coherence which is allowed to precess during the evolution period tl. In the middle of the t] period the application of a 180° ~H pulse refocuses the heteronuclear scalar interactions. Thus, only the ll3Cd chemical shifts and the homonuclear couplings will lead to modulations as a function of q. At the end of tl a second 90 ° llaCd pulse transfers the heteronuclear two-spin coherence back to proton coherence, which is then allowed to refocus during the second r delay and observed. A purging process using a phase-alternated 90 ° H3Cd pulse prior to acquisition eliminates proton coherence in antiphase with respect to heteronuclear 47 T. J. Norwood, J. Boyd, J. E. Heritage, N. Soffe, and LD. Campbell, J. Magn. Reson. 87, 488 (1990). 48 E. Worgotter, G. Wagner, and K. Wuthrich, J. Am. Chem. Soc. 108, 6162 (1986). 49 G. Otting and K. Wuthrich, Q. Rev. Biophys. 23, 39 (1990).

[2]

ll3Cd N M R APPLIED TO METALLOPROTEINS

35

couplings. A z filter [(Tr/2)x - ~'z - (~-/2)~] can be placed at the position of the dashed line in Fig. 5 before the collection of the free induction decay (FID) to remove the additional antiphase components of the proton signals caused by the homonuclear coupling. An example of the heteronuclear 1H-113Cd COSY technique applied to the binuclear ll3Cd cluster found in the DNA-binding domain of the transcription factor GAL4 is shown in Fig. 6A. The 1~3Cd chemical shifts are plotted on the ordinate. The signals for the two l|3Cd nuclei in GAL4 occur at 707 and 669 ppm. Each ll3Cd signal is associated with a group of/3 proton resonances whose ~H chemical shifts are plotted along the abscissa and represent protons to which the ll3Cd signal is scalar coupled. In this particular spectrum, the phase-alternated third rr/2 H3Cd pulse and preceding delay prior to acquisition were left out in order to improve signal-to-noise ratios; hence, the proton resonances show positive and negative phase patterns reflecting the heteronuclear coupling. The IH-1 t3Cd couplings mentioned below were determined independently from ~H COSY spectra. 46 Both purging processes should be applied if accurate coupling constants are to be measured directly from the HMQC spectra. The intensity of the proton resonances detected by the HMQC method depends on the magnitude of their coupling to the ll3Cd. The relative intensities of the coupled proton resonances vary with the delay time, r, which should be equal to 1/2J in order to obtain maximum intensity. Thus, with potential IH-ll3Cd J couplings of 5 to 50 Hz, there may not be a single value of ~- at which optimal intensity can be achieved for all the coupled protons. For the example of ~I3Cd2-GAL4 shown in Fig. 6A, J was assumed to be 10 Hz (T = 50 msec) in order to bring out couplings of 5-10 Hz. The intensities of the degenerate/3a, fib protons of C-21 (2.84 ppm), which have a coupling of 55 Hz to the 113Cdresonating at 669 ppm, are already washed out and weak with this relatively long r value. The /3a and/3b protons of C-28, however, have strong intensity associated with the 1~3Cd resonating at 707 ppm ( J = 30 and 10 Hz, respectively) and weaker but significant intensity associated with the 113Cd resonating at 669 ppm ( J ~ 5 Hz). The latter identify C-28 as a bridging ligand between the two lJ3Cd ions. In the case of bridging Cys ligands, coupling of the/3 protons to one of the 113Cdions is often significantly weaker than to the other. The other bridging C residue in the GAL4 binuclear complex is C-11, where the/3 a proton (2.96 ppm) is fairly strongly coupled to both ll3Cd ions ( J -~ 20 Hz). On the other hand, the /3b proton of C-11 (2.08 ppm) has a 12 Hz coupling to the 113Cdresonating at 669 ppm and a barely detectable coupling to the ~13Cd resonating at 707 ppm (~5 Hz). Thus, the two/3CH2 protons of a bridging Cys may have the same or different coupling constants to the second 113Cd.

36

PROBES OF METAL ION ENVIRONIVIENTS

[2]

A. 1H.113Cd COSY SPECTRUM c2a

b

c~s

•

c14

Lb

c1~ c~

•

b Cll

c14

Jlkm

E L 5"

I, ;"'

." 3,. ~°

3.4 cg, 3.2 , ~ 3 . o ~'

PPM 1H

2.8

2.6 a C38b

B. 113Cd.FILTERED2D 1H COSY DIFFERENCE SPECTRUM CI1 •

~"

2.0

2.5

3.5 ~m

4.0 4.5 4.0

3.5

3.0

D1 (ppm)

2.5

2.0

2.4

2.2

~12.0

707

[2]

ll3Cd N M R APPLIED TO METALLOPROTEINS

37

The application of a single ll3Cd filter to the acquisition of a 2D IH COSY difference spectrum for the Cd 2cluster formed by the N-terminal 62 residues of the transcription factor GAL4 is illustrated in Fig. 6B.23 In essence the method collects two 2D (~H-~H) COSY spectra, identical except for the phase of a ~r/2(U3Cd) editing pulse. A difference spectrum is then formed, and all proton signals subtract out except for those coupled to H3Cd, which respond to the phases of the editing pulse. In practice, this is accomplished by phase cycling of the ~'/2(H3Cd) pulse, and only the difference FID is finally recorded. 48 With a high sample concentration to maximize signal-to-noise ratios, the diagonal and associated cross-peaks for ll3Cd-coupled protons are the only features in the COSY difference spectrum (Fig. 6B). With this technique the intensity of the ~3Cd-coupled resonances does not depend on the magnitude of the coupling. A double HaCd filter can be generated by increasing the complexity of the phase cycling on the last U3Cd(zr/2) pulse. This method can detect bridging Cys ligands, since only the protons of bridging Cys will be coupled simultaneously to two ~13Cd nuclei. This has been applied successfully in the case of both 113Cd2-GAL423 and U3Cd7-metallothionein.48'49

lt3Cd-13C Scalar Coupling If carbon nuclei in the vicinity of the 113Cd nucleus in a protein can be 13C labeled, the observation of 13C-1~3Cd coupling can be a useful method of probing structure. One-bond ~3C-113Cd coupling is strong (-1000 Hz). On the other hand, three-bond ~3C-1~3Cd coupling (J = 20 Hz) is larger than two-bond ~3C-1~3Cd coupling. The coupling of ~3CN Ccoordinated to the U3Cd at the active site of H3Cd carbonate dehydratase (lj = 1040 Hz) is shown in Fig. 7A. 5° Three-bond 13CJI3Cd coupling to the y-~3C of the three His ligands at the active site of H3Cd6-alkaline phosphatase is illustrated in Fig. 7B. 5~ Each monomer of this enzyme, which forms a symmetrical dimer, contains 10 His residues. Isolation of 50 N. B.-H. Jonsson, L. A. E. Tibell, J. L. Evelhoch, S. J. Bell, and J, L. Sudmeier, Proc. Natl. Acad. Sci. U.S.A. 77, 3269 (1980).

FIG. 6. (A) Heteronuclear IH-11aCd COSY or HMQC spectrum of II3Cd2-GAL4(62). The pulse sequences are as described in Fig. 5 and by Norwood et al. 47 The delay time, 7, was set at 50 msec, corresponding to a IH-U3Cd coupling constant of l0 Hz. (B) U3Cd-filtered 2D 1H COSY difference spectrum of l13Cd2-GAL4(62) employing a single 113Cd filter (256 scans per experiment, 300 experiments, 5681.8 Hz sweep width, tl max = 26 msec). Protein samples for both spectra were 15 mM, pH 6, 35°. Heteronuclear correlation spectra of this type on I13Cd2-GAL4(62) have been published in Ref. 23.

38

PROBES OF METAL ION ENVIRONMENTS

[2]

A. 13C.113Cd Coupling ~I]l

l l l l l l l l l l l

lllll+l

IIII

IIIIII

HCAC + 13CN"

lll,lrll

rlrrl,,,,

400

,,,,r

l,,,,,t+,,

300

200

100

PPM B. 13C.113Cd Coupling

13C, 112CdAP --~3'

.

3,3' 4

13C, 113CdAP / i ,5 s

I

140

I

I

136

i

I

132

I

I

128

I

I

124

PPM FIG. 7. Heteronuclear coupling to the tl3Cd nucleus present in It3Cd-substituted Znmetalloenzymes. (A) 13C-I13Cd coupling observed on the ll3Cd NMR signal of the cyanide complex of ~3Cd human carbonate dehydratase C. Cyanide is C-coordinated to the active site Cd 2÷ . (Spectra reprinted from Ref. 50 with permission.) (B) t3CJl3Cd coupling to 3 of

[2]

ll3Cd NMR APPLIED TO METALLOPROTEINS

39

D. 31 p.113Cd Coupling E-P C. 15N-113Cd Coupling I I I I

III

I II

$1

Ili

I t

I1"

E.P

BCA + 15N-Neoprontosil I

"~ I'--

J = 190 Hz

BCA

I I l

I

I L I I I I I I L I I I L I L

400

300

J

13

8 PPM

PPM

FIG. 7 (continued) the 10 y-13C-labeledHis residues in E. coli alkaline phosphatase. The enzyme was labeled by growing a His auxotroph of E. coli on histidine labeled with 13C at the y-carbon (the fl-carbon was deuterated to avoid line broadening). Complete spectra of alkaline phosphatase at various llaCd stoichiometries are given in Ref. 51. (C) 15N-ll3Cd coupling observed on the 113CdNMR signal of tl3Cd bovine carbonate dehydratase A (BCA) complexed with the 15N-labeledsulfonamide neoprontosil. The sulfonamide, 15NH2--S(=O)E--R, is coordinated through the nitrogen, tI3Cd---NH--R. [Reprinted with permission from J. L. Evelhoch, D. F. Bocian, and J. L. Sudmeier, Biochemistry 20, 4951 (1981). Copyright 1981 American Chemical Society.] (D) 31p NMR spectra of the phosphoenzyme intermediates of E. coli tt3Cd6-alkaline phosphatase showing 31p_u3Cd coupling on the signal from the noncovalently bound phosphate, E. P. E-P is the phosphoseryl covalent intermediate. The 3tp spectra of E- P and E-P were recorded at different pH values to maximize formation of the intermediates by the Cd enzyme (pH 9 for E. P and pH 7 for E-P). Complete 113Cdand 3tp spectra of the phosphoenzymes can be found in Ref. 14. this e n z y m e p r o d u c e d f r o m a n E s c h e r i c h i a coli His a u x o t r o p h g r o w i n g o n y-13C-labeled h i s t i d i n e gives a p r o t e i n that s h o w s w e l l - r e s o l v e d laC r e s o n a n c e s for all 10 His r e s i d u e s (Fig. 7B). E a c h r e s o n a n c e c o r r e s p o n d s to t w o His, o n e in e a c h m o n o m e r . I n the 13C N M R s p e c t r u m of the 13Cdl a b e l e d l l3Cd6-alkaline p h o s p h a t a s e , t h r e e of the 13C r e s o n a n c e s (8, 9, a n d 10, Fig. 7B) s h o w 13C-U3Cd c o u p l i n g s (3j = 12-19 Hz). 51 T h u s , t h e r e are 51j. D. Otvos and I. M. Armitage, Biochemistry 19, 4021 (1980).

40

PROBES OF METAL ION ENVIRONMENTS

[2]

at least three His ligands distributed among the three n3Cd ions at the active center of alkaline phosphatase. Based on the work of Otvos and Browne 52 with y-13C-labeled His, resonances 8, 9, and 10 (Fig. 7B) with upfield shifts and resolved coupling could be assigned to the His whose imidazole side chains are coordinated to n3Cd through the N-3 nitrogen. The two 13C resonances, 1 and 2 (Fig. 7B), shifted downfield by 113Cd2+ binding, were thought to be ligands coordinated through N-1. 51 The lack of resolved coupling was explained by coordination through N-1 with the smaller two-bond coupling to 113Cd being unresolved. Thus, a total of five His ligands were thought to be present as ligands to the three Cd ions at the active center of alkaline phosphatase. 5~ The crystal structure of alkaline phosphatase initially suggested that of the three ll3Cd ions ligated at each active center, Cd A had three His ligands, CdB had one His ligand, and Cdc had none. 53 The 2.0 ,~ structure later showed, however, that Cd n has only two His ligands liganded through N-3. 38'39 An Asp-327, adjacent to His-372, was the third ligand rather than His-372 itself. CdB has a single His-370 as one of the ligands, again coordinated via N-3. Hence, the ~3C-ll3Cd coupling was signaling the correct number of His ligands, all coordinated through N-3. Unfortunately, y-~3C-labeled His has not been incorporated into a metalloprotein where the imidazole side chain of His is liganded through N-l, so we do not know if the weaker two-bond 13C-113Cd coupling can be resolved. It may be that 7J3C-labeled His will be a precise probe of both the number and ring nitrogen placement for imidazole side chain coordination in proteins. At present the probe can only be said to diagnose ll3Cd coordination to the N-3 of the imidazole side chain.

113Cd-15N Scalar Coupling 113Cd-15N coupling has been used on one occasion to confirm a structural feature for a ll3Cd-substituted enzymefl4 Sulfonamides are classic inhibitors of the enzyme carbonate dehydratase, and much indirect data suggest that the sulfonamide group itself, H2N--S(=O)2--R where R is a large heterocycle, coordinates the active site Znl ion. Such a coordination could be to the nitrogen or to one of the oxygens of the sulfonamide group. Even an X-ray structure, except at the very highest resolution, could not be interpreted unequivocally, ll3Cd NMR of n3Cd-carbonate dehydratase bound to an ~SN-labeled sulfonamide shows an 15N coupling (~J = 190 52 j. D. Otvos and D. T. Browne, Biochemistry 19, 4011 (1980). 53 H. W. Wyckoff, M. Handschumacher, H. M. K. Murthy, and J. M. Sowadski, Adv. Enzymol. Relat. Areas Mol. Biol. 55, 453 (1983). 54 j. L. Evelhoch, D. F. Bocian, and J. L. Sudmeier, Biochemistry 20, 4951 (1981).

[2]

ll3Cd N M R APPLIED TO METALLOPROTEINS

A

MAS, 1.6 KHz

41

B MAS, 1.0 KHz

C

I

I

I

I

I

I

.

I

I

I

I

200

.

[

I

I

I

100

[

I

0

I

!

-100

I

I

-200

I

~)

I

I

I

-300

.

I

I

I

I

I

I

I

-400

PPM

FIG. 8. Solid-state tl3Cd NMR spectra of partially rehydrated (020) lyophilized samples of mCd-substituted parvalbumin. (A, B) Magic angle spinning (MAS) spectra were recorded at two different frequencies of the sample which gives the static solid-state spectrum shown in (C). (Spectra adapted with permission from Ref. 13. Copyright 1985 American Chemical Society.) Hz) on the l l3Cd signal (Fig. 7C).54 Thus, the sulfonamide is N-coordinated to the Cd z+ ion at the active site of carbonate dehydratase.

113Cd-31p Scalar Coupling M a n y m e t a l l o e n z y m e s interact with phosphate-containing substrates, cosubstrates, or cofactors. Thus, 31P-O-113Cd coupling, e x p e c t e d to be around 30 H z b a s e d on model coordination c o m p o u n d s , can be a potential source of important structural information concerning p h o s p h a t e - m e t a l

42

PROBES OF METAL ION ENVIRONMENTS

[2]

ion interaction. This has been explored at some length for the phosphoenzyme intermediates formed by alkaline phosphatase.14,39 The 113Cd-substituted enzyme both binds phosphate in the noncovalent form and catalyzes the phosphorylation of the Ser residue at the active site. Both phosphoenzyme intermediates can be examined by 31p NMR. The 31p NMR signal of the noncovalent phosphate complex with the 113Cd enzyme (E. P) is a doublet showing a 30 Hz coupling, whereas the covalent phosphoenzyme complex (E-P) shows a singlet 31p signal (Fig. 7D). It is possible to interpret these findings as indicating that phosphate is initially coordinated to one of the 113Cd2+ ions (decoupling by irradiation of the ll3Cd resonances show this to be CdA). 14When the phosphoseryl residue is formed, the phosphate oxygens apparently move outside the coordination sphere of all the Cd 2+ ions, since no l l3Cd coupling is observed (Fig. 7D). However, a series of crystal structures of the phosphoenzyme intermediates of alkaline phosphatase are now available with resolutions from 2.0 to 2.8 A , 38'39 and the results suggest that, although 31p-o-I13Cd J couplings may be useful, they should be interpreted with some caution. The crystal structure of the noncovalent phosphate complex of the Zn-alkaline phosphatase shows that one phosphate oxygen is coordinated to ZnA with a typical P-O-ZnA bond angle of 120°, in agreement with the 31p-I13Cd J coupling observed for the Cd enzyme in solution. However, a second oxygen of the bound phosphate forms a coordination bridge to Zn R with a highly atypical P--O--ZnB bond angle of 175°. Unfortunately, the high pH (>9) required to form the E- P complex of the Cd-substituted enzyme prevents determination of the crystal structure of the E. P complex for the Cd enzyme. The structure of the covalent E - P complex formed at neutral pH by the Cd enzyme has been determined. 38 CdB appears to be coordinated to the ester oxygen of the E - P complex, yet no J coupling to 113Cd is observed on the solution 31p signal (Fig. 7D). Thus, the interpretation of the absence of 31p-113Cd coupling as indicating no coordination to phosphate may have to be treated with caution. Unusual P-O-I13Cd bonds formed in proteins may reduce coupling, or coordination with the ester oxygen of a covalently bound phosphate may not give rise to resolvable coupling. Conformational flux in a protein, perhaps not present in a crystal structure, may be an additional factor affecting the observation of 31p-o-I13Cd coupling. A 30 Hz 31p-o-113Cd coupling has also been observed on the 31p NMR signal of the inhibitor L-phenylalanine phosphoramidate phenyl ester bound to 113Cd-carboxypeptidase A, showing that the phosphate oxygen of the inhibitor coordinates the 113Cd ion at the active center. 36 Further work with phosphate-binding metalloenzymes will be needed to clarify the conclusions that can be made from the presence or absence of 31p-113Cd coupling.

[3]

L A N T H A N I D E SHIFT REAGENTS

43

Solid-State 113CdNuclear Magnetic Resonance of 113CdProteins Solid-state NMR spectra of mCd bound at protein binding sites, especially the comparison of a H3Cd static powder spectrum and the envelope of spinning side bands obtained by magic angle spinning (MAS), provide information about the ~13Cd chemical shift tensor. 13'15 This information can, in comparison to solid-state spectra of model l~3Cd compounds, lead to conclusions about the nature of the "3Cd complex not provided by the isotropic line from high-resolution solution NMR. The major hurdle has been the preparation of appropriate solid-state samples of n3Cd-substituted proteins. It was first thought that lyophilized powder samples would provide a ready means of accessing the solid-state spectra of most H3Cd proteins. Unfortunately, it has been shown that lyophilization to the point of significant dehydration of the protein leads to heterogeneity of the l~3Cd spectrum, apparently owing to heterogeneity in the precise conformation of the protein. This conformational heterogeneity is transferred to heterogeneity of the N3Cd complex such that multiple overlapping spectra of slightly different chemical shift determine the line shape. ~aThis was shown by the facts that MAS spectra failed to generate narrow l laCd lines and that controlled rehydration of the lyophilized protein powder with DzO resulted in narrowing of the MAS lines and the generation of a more typical solid-state mCd profile. Studies of this kind have been carried out on parvalbumin in which ll3Cd2+ was substituted for the single Ca 2+ and on concanavalin A in which "3Cd was substituted at both the Ca z+ and Mn 2+ binding sites (Fig. 8). Acknowledgment Original work on "3Cd NMR of metalloproteins carried out in the author's laboratory was supported by National Institutes of Health Grant DK09070.

[3] L a n t h a n i d e Shift R e a g e n t s

By CARLOS F. G. C. GERALDES Introduction The first report by Hinckley I more than 20 years ago of the use of lanthanide complexes to simplify unresolved proton resonances in IowI C. C. H i n c k l e y , J. Am. Chem. Soc. 91, 5160 (1969).

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

[3]

L A N T H A N I D E SHIFT REAGENTS

43

Solid-State 113CdNuclear Magnetic Resonance of 113CdProteins Solid-state NMR spectra of mCd bound at protein binding sites, especially the comparison of a H3Cd static powder spectrum and the envelope of spinning side bands obtained by magic angle spinning (MAS), provide information about the ~13Cd chemical shift tensor. 13'15 This information can, in comparison to solid-state spectra of model l~3Cd compounds, lead to conclusions about the nature of the "3Cd complex not provided by the isotropic line from high-resolution solution NMR. The major hurdle has been the preparation of appropriate solid-state samples of n3Cd-substituted proteins. It was first thought that lyophilized powder samples would provide a ready means of accessing the solid-state spectra of most H3Cd proteins. Unfortunately, it has been shown that lyophilization to the point of significant dehydration of the protein leads to heterogeneity of the l~3Cd spectrum, apparently owing to heterogeneity in the precise conformation of the protein. This conformational heterogeneity is transferred to heterogeneity of the N3Cd complex such that multiple overlapping spectra of slightly different chemical shift determine the line shape. ~aThis was shown by the facts that MAS spectra failed to generate narrow l laCd lines and that controlled rehydration of the lyophilized protein powder with DzO resulted in narrowing of the MAS lines and the generation of a more typical solid-state mCd profile. Studies of this kind have been carried out on parvalbumin in which ll3Cd2+ was substituted for the single Ca 2+ and on concanavalin A in which "3Cd was substituted at both the Ca z+ and Mn 2+ binding sites (Fig. 8). Acknowledgment Original work on "3Cd NMR of metalloproteins carried out in the author's laboratory was supported by National Institutes of Health Grant DK09070.

[3] L a n t h a n i d e Shift R e a g e n t s

By CARLOS F. G. C. GERALDES Introduction The first report by Hinckley I more than 20 years ago of the use of lanthanide complexes to simplify unresolved proton resonances in IowI C. C. H i n c k l e y , J. Am. Chem. Soc. 91, 5160 (1969).

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

44

PROBES OF METAL ION ENVIRONMENTS

[3]

field nuclear magnetic resonance (NMR) spectra marked the start of the application of the lanthanide-induced shift (LIS) method to a variety of NMR problems. One year later, Morallee e t al. 2 showed that the line broadenings induced by the gadolinium(III)-lysozyme complex in the proton resonances of fl-methyl-N-acetylglucosamine could be analyzed in terms of the absolute distance between the Gd(III) ion and various sugar protons. These observations stimulated much interest and activity in the use oflanthanides as NMR shift and relaxation reagents, in applications ranging from qualitative spectral simplification, proof of molecular stereochemistry, and quantitative analysis of dynamic solution structures to the most recent applications in NMR spectroscopy of perfused cells, organs, and intact animals and magnetic resonance imaging (MRI). All the applications are based on basic properties of the lanthanide cations, such as their Lewis acid behavior in the formation of complexes of high coordination numbers and their unpaired f electrons. When a Lewis base interacts with a lanthanide cation, any NMR-active nucleus within that base is influenced by the presence of the unpaired felectrons, leading to paramagnetic relaxation or broadening of that resonance and, in some cases, a shift to a different NMR frequency. If the interaction between the Lewis acid cation and the base is purely electrostatic, paramagnetic cations that have an anisotropic distribution o f f electrons originate a lanthanide-induced NMR dipolar (or pseudocontact) shift, which is the type of LIS useful in obtaining structural information. Gd(III) has 7 unpaired electrons distributed isotropically in its 4 f shell and, therefore, cannot produce a NMR dipolar shift. If, however, the Lewis acid-base interaction is partially covalent, a small amount of unpaired electron spin density can reach the molecular framework of the base and result in a second type of LIS, the contact (or scalar) shift. The goal of this chapter is to describe LIS methodologies and their application to study proteins in solution. The qualitative uses of lanthanides to simplify complex NMR spectra are not mentioned because highfield magnets and two- and three-dimensional methods have eliminated the need for LIS measurements for this purpose. The use of lanthanides as shift and relaxation reagents in organic solutions has been reviewed extensively, 3-9 as well as well as their applications as NMR structural 2 K. G. Morallee, E. Nieboer, F. J. C. Rossotti, R. J. P. Williams, and A. V. Xavier, Chem. Commun., 1132 (1970). 3 j. K. M. Saunders and D. H. Williams, Nature (London) 2411, 285 (1972). 4 j. Reuben, Prog. Nucl. Magn. Reson. Spectrosc. 9, 1 (1973). 5 W. de W. Horrocks, Jr., " N M R of Paramagnetic Molecules." Academic Press, New York, 1973. 6 R. E. Sievers (ed.), "NMR Shift Reagents." Academic Press, New York, 1973.

[3]

LANTHANIDE SHIFT REAGENTS

45

probes in aqueous solutions, particularly for biological systems including peptides and proteins. ~0-16The use of lanthanide shift reagents in conjunction with alkali metal NMR in biological systems is described in [4] of this volume. Lanthanide Ions as Structural Probes The use of metal ions as extrinsic structural probes, either by their specific binding to biochemical molecules that do not naturally contain metal ions or by substitution of those metal ions naturally present by others of similar chemical reactivity but possessing improved physical properties, has proved to be a versatile research tool. The trivalent lanthanide ions, which constitute a family with interesting electronic and magnetic properties and fairly similar chemical properties, constitute a set of potentially powerful probes. H They should, however, be used with caution, as various problems may arise. ~°'H'~5 First, the various lanthanide complexes of a particular ligand should be isostructural. Any variation in structure would jeopardize strategies for structure determination that involve, for example, the simultaneous analysis of paramagnetic shift or relaxation data obtained for complexes in which the central lanthanide ions are different [such as ytterbium(Ill) and Gd(III)]. For the cases where the question of isostructurality in solution has been examined in detail, contradictory conclusions have been reached. 17-19

7 M. R. Wilcott III and R. E. Davis, Science 19tl, 850 (1975). 8 j. Reuben and G. Elgavish, in "Handbook of Physics and Chemistry of Rare Earths" (K. A. Gschneider and L. Eyring, eds.), North Holland, New York, 1979. 9 T. C. Morrill (ed.), "Lanthanide Shift Reagents in Stereochemical Analysis." VCH Publ., New York, 1986. 10 R. A. Dwek, " N M R in Biochemistry." Oxford Univ. Press (Clarendon), Oxford, 1973. it E. Nieboer, Struct. Bonding (Berlin) 22, 1 (1975). 12 C. M. Dobson and B. A. Levine, " N e w Techniques in Biophysics and Cell Biology," Wiley, New York, 1976. ~3 L. Lee and B. D. Sykes, "Methods for Determining Metal-Ion Environments in Proteins: Structure and Function of Metalloproteins." Elsevier, North Holland, New York, 1980. 14 F. Inagaki and T. Miyazawa, Prog. Nucl. Magn. Reson. Spectrosc. 14, 67 (1981). 15 R. E. Lenkinski, in "Biological Magnetic Resonance" (L. J. Berliner and J. Reuben, eds.), Vol. 6, Chap. 1. Plenum, New York, 1984. 16 j. C. G. Biinzli and G. R. Choppin (eds.), "Lanthanide Probes in Life, Chemical and Earth Sciences." Elsevier, Amsterdam, 1989. 17 B. A. Levine and R. J. P. Williams, Proc. R. Soc. London A 345, 5 (1975). 18 A. D. Sherry and E. Pascual, J. Am. Chem. Soc. 99, 5871 (1977). 19 j. Reuben and G. A. Elgavish, J. Am. Chem. Soc. 100, 3617 (1978).

46

PROBES OF METAL ION ENVIRONMENTS

[3]

Second, the various lanthanide ions and their complexes should have very similar chemical properties. Although their coordination numbers are high (most commonly 8 and 9), their radii exhibit the well-known lanthanide ion contraction. Consequently, there is variation along the series of hydration numbers of the lanthanide aqueous ions and coordination numbers, as well as in the thermodynamic and kinetic properties of their complexes. 11,15.20 Third, the replacement of the naturally occurring ion by the probe should be isomorphic. The fact that the ionic radii of the lanthanides are very similar to the ionic radius of Ca 2+ (0.99 ,~) has led to many studies in which the former have been used as isomorphic replacements for Ca 2+. 11.21 However, only in a few cases, like thermolysin, has perfect isomorphous replacement actually been shown to occur. 22 Fourth, the replacement of the naturally occurring ion by the probe in a biological macromolecule (e.g., a metalloenzyme) should also be functional. In this respect, besides structural similarities, kinetic and equilibrium complexation similarities are also very important. 11 Lanthanide ions have been demonstrated to be good substitutes for Ca 2+ in a-amylase and in the trypsinogen to trypsin conversion, and for Mg 2÷ in isoleucyltRNA synthetase [L-isoleucine: t-RNA ligase (AMP)] and adenylylated glutamine synthetase (L-glutamate: ammonia ligate)." However, lanthanides act as competitive inhibitors for the Ca 2+ enzymes staphylococcal nuclease and concanavalin A, and for the Mg 2+ enzymes pyruvate kinase and yeast inorganic pyrophosphatase.l~ The extra charge and affinity for higher coordination numbers of lanthanides relative to divalent cations (e.g., Mg 2+ and Ca 2+) could cause them to distort the metal ion coordination polyhedron enough to destroy the catalytic ability of an enzyme. Lanthanide ions also have faster rates of inner sphere substitution than Mg 2+. Systematic studies of the functional replacement of lanthanides for Ca 2+ in a number of physiological processes have shown that the differences between their responses are as important as their similarities. 11,23,24 In spite of these limitations, lanthanide ions afford extensive applications as spectroscopic probes of the structure of biochemical macromolecules, such as proteins and nucleic acids. 11'25 Lanthanide ions can be used as (1) heavy atom isomorphous replacement probes for electron microscopy and in X-ray studies22,26; (2) luminescence probes of the 20 G. R. Choppin, Pure Appl. Chem. 27, 23 (1971). 21 R. B. Martin and F. S. Richardson, Q. Rev. Biophys. 12, 181 (1979). 22 p. W. Colman, L. H. Weaver, and B. W. Mathews, Biochemistry 13, 1719 (1974). 23 K. J. Ellis, lnorg. Perspect. Biol. Med. 1, 101 (1977). 24 C. H. Evans, Trends Biol. Sci., 445 (1983). 25 C. F. Meares and T. G. Wenzel, Acc. Chem. Res. 17, 202 (1984). 26 G. Nagahashi, W. W. Thompson, and R. T. Leonard, Science 183, 670 (1974).

[3]

LANTHANIDE SHIFT REAGENTS

47

structure of proteins and nucleic acids, namely, with europium(III) and terbium(III), owing to the enhanced fluorescence of Tb 3+ when bound to certain calcium- or iron-containing proteins (by factors of 104-105) 1~,16,21,27; (3) absorption spectroscopy and circular dichroism probes, as a consequence of the high sensitivity of certain f - f transitions to lanthanide chelation by the macromolecule¢6'28; (4) electron spin resonance (ESR) spectroscopy probes, as the Gd(III) ion can be easily observed in solution at room temperatureS6'29; (5) nuclear spin relaxation enhancement probes, particularly using the Gd(III) ion (proton relaxation enhancement studies and relaxometry studies are well documented)l°'11'3°'31; and (6) chemical shift probes for NMR spectroscopy, for example, with paramagnetic ions such as Eu(III) or Yb(III). The latter is the main subject of this chapter. Paramagnetic cationic shift reagents, such as Dy(PPP)27- [dysprosium (III)-bis-tripolyphosphate], can be used together with 43Ca2+ NMR to resolve resonances of Ca z+ ions weakly and tightly bound to proteins, such as a-lactalbumin, intestinal Ca2+-binding protein (ICaBP), or calmodulin, by adding the shift reagent to a solution containing the protein with excess Ca2+. 32-34 The diamagnetic lanthanides lanthanum(Ill) and lutetium(Ill) (La 3+ and Lu 3+) have also been used in competition binding studies of Ca2+-binding proteins. 32 For example, Ca2+/La 3+ competition has been investigated in Ca 2+ saturated ICaBP, together with 43Ca NMR, and CdZ+/Lu 3+ competition was studied in Cd2+-saturated parvalbumin together with 133Cd N M R . 33'35 Basic Theory of Nuclear Magnetic Resonance Effects of Paramagnetic Lanthanide Ions

Effects of Chemical Exchange Let us consider in this discussion exchange between two sites only that occurs on formation of 1 : 1 complexes, that is, chemical exchange between the ligand in a free state and in a metal complex: 27 W. de W. Horrocks and D. R. Sudnick, Acc. Chem. Res. 14, 384 (1981). 28 D. M. Dooley and J. H. Dawson, Coord. Chem. Rev. 60, 1 (1984). 29 G. H. Reed, R. D. Hershberg, and G. H. de Haas, in " N M R in Biochemistry" (S. J. Opella and P. Lu, eds.), p. 357. Dekker, New York, 1979. 3o D. R. Burton, S. Fors6n, G. Karlstr6m, and R. A. Dwek, Prog. Nucl. Magn. Reson. Spectrosc. 13, 1 (1979). 31 S. H. Koenig and R. D. Brown III, Prog. Nucl. Magn. Reson. Spectrosc. 22, 487 (1990). 32 H. J. Vogel and S. Fors6n, in "Biological Magnetic Resonance" (L. J. Berliner and J. Reuben, eds.), Vol. 7, Chap. 4. Plenum, New York, 1987. 33 H. J. Vogel and W. H. Braunlin, J. Magn. Reson. 62, 42 (1985). 34 H. J. Vogel, T. Andersson, W. Braunlin, T. Drakenberg, and S. Fors6n, Biochem. Biophys. Res. Commun. 122, 1350 (1984). 35 T. Drakenberg, M. Sward, A. Cav6, and J. Parello, Biochem. J. 227, 711 (1985).

48

PROBES OF METAL ION ENVIRONMENTS

[3]

M + L . k°n " ML ko~

(1)

where M, L, and ML represent the ligand, the metal ion, and the complex, respectively, and ko. and koff are the rates of formation and dissociation of the complex. Let us also consider a ligand nucleus exchanging between the two sites with different lifetimes rM and ~'L, different fractional populations PM and PL, different chemical shifts 8M and 8L [in parts per million (ppm)] or toM and toL (in hertz), and different relaxation times TiM, T2M, and Tin, TEL, respectively. The effects of chemical exchange on the observed NMR parameters depend on the exchange conditions relative to the two limits defined by the NMR chemical shift time scale, 2zr(toM - t o L ) . 36-39 In the fast exchange limit 2rr(tou - toL)~'M~ 1, a population-averaged chemical shift, 8obs, is given by ~obs = PM~M + PLt~L

(2)

Under the dilute species approximation (PL >> PM) and neglecting outersphere effects, the observed spin-lattice relaxation rate 1/T~obs, can be a p p r o x i m a t e d by 37'39 1/Tlobs

=

I / T l L + PM/(T1M + '/'M)

(3)

or 1/TIP = PM/(TIM + "/'M)

(4)

where 1/Tlp = 1/Tlobs - I/T1L is the paramagnetic contribution to the spin-lattice relaxation time. For cations that produce negligible shifts [e.g., Gd(III)], the observed paramagnetic contribution to the spin-spin relaxation rate obeys a similar equation, 1/T2p = PM/(T2M + z M)

(5)

where 1/T2p = 1/T2obs- 1/T2L If 7"M > TIM , T2M , Eqs. (4) and (5) reduce to 1~Tip ~ pM/I-M 36 T. J. Swift and R. E. Connick, J. Chem. Phys. 37, 307 (1962). 37 Z. Luz and S. Meiboom, J. Chem. Phys. 40, 2686 (1964). 38 j. Reuben and D. Fiat, J. Chem. Phys. 51, 4918 (1969). 39 j. S. Leigh, Jr., J. Magn. Reson. 4, 308 (1971).

(6)

[31

LANTHANIDE SHIFT REAGENTS

49

for i equals 1, 2, and all the resonances of the complex will have equal paramagnetic relaxation parameters governed by 1/ru. In the case of the lanthanides which induce shifts, 1/T2obs = PL/T2L + PM/T2M + PM(1 -- pM)2'rM[2'n'(OJM -- OJL)] 2

(7)

where the third term results from the chemical shift difference. In the slow exchange limit, 2"n'(toM -- (.OL)7 M >~> 1, the flee and complexed sites yield separate resonances with intensities proportional to P L and P M , respectively. The relaxation rates at each site are then given by l/Tiobs L = 1/TiL + 1 / r L 1/Tiobs M = 1~TiM + 1 / r M

(8) (9)

f o r / = 1,2.

Chemical Shifts The chemical shift of the lanthanide complex (lanthanide-induced shift, LIS) is given by the sum of three contributions4°-43: A M = Acf + A d + A c

(10)

where Acf is the diamagnetic complex formation shift, Ad is the dipolar shift, and Ac is the contact shift. The value of Acf, which arises from electrostatic interactions or metal ion-induced ligand conformational changes, is usually estimated from the effects of La(III) and Lu(III) binding to the ligand and are assumed to be constant through the lanthanide series. The dipolar or pseudocontact shift, Ad , results from a through-space interaction between the electron and the nuclear magnetic dipoles. Twelve of the fourteen available trivalent lanthanide cations have at least one unpaired electron, and eleven of these [excluding Gd(III)] form complexes with magnetic susceptibility anisotropy resulting from ligand field effects, which remove the spherical symmetry around the metal ion. Thus, the value of the dipolar magnetic field induced by the anisotropic electron magnetic moment is not averaged to zero by the fast rotational tumbling of the complex in solution, and therefore a dipolar shift arises, given by 1

Ad = 4--~r3 F(O, cb)

(ll)

4o R. M. Golding and M. P. Halton, Aust. J. Chem. 25, 2577 (1972). 41 B. Bleaney, J. Magn. Reson. 8, 91 (1972). 42 j. Reuben and G. A. Elgavish, in "Handbook of the Physics and Chemistry of Rare Earths" (K. A. Gschneider, Jr., and L. Eyring, eds.), North Holland, New York, 1979. 43 I. Bertini and C. Luchinat, " N M R of Paramagnetic Molecules in Biological Systems." Benjamin Cummings, Menlo Park, California, 1986.

50

PROBES OF METAL ION ENVIRONMENTS

[3]

where r is the distance from the paramagnetic center to the observed nucleus and F is the angular factor: F = (Xzz - X)(3 cos 2 0 - 1) + (X~x - Xyy) Sin2 0 COS 2~b

(12)

In Eq. (12), X~ (as = x x , yy, zz) are the principal components of the magnetic susceptibility tensor, ~, of the complex, with average value = Xxx + Xyy + Xzz)/3, whereas 0 and ~bare the nucleus polar coordinates. When Xxx = Xyy (axial symmetry), - 5X) t"3 cos 2 0 - 1 ) Ad-- (Xzz 4---~r

(13)

Thus, the dipolar shift could be calculated from ~ tensor components, which are given by the van Vleck equation. 43 In the case of the lanthanide(III) cations, the ~ tensor of the ground electronic state characterized by the quantum number J, is, to first approximation, isotropic, X~ = gj2fl2j(j + 1 ) / 3 K T , where the constants gj, fl, K, and T have the usual meanings, and thus causes no dipolar shift. The anisotropy of :~, which results from the ligand electrostatic field, has been shown by Bleaney41 in the case of a singly populated ground state J to be given by Xaa = _[gj2fl2j(j + 1)(2J - 1)(2J + 3)/30(KT)2]Da

(14)

: <JIl llJ> (r2)(A 2 - A ° ) <JIl llJ> ( r 2 ) ( - A ~ - A °) Dz -- <J/[o ll (2A °)

(15a) (15b) (15c)

where

Dy

:

are the ligand field parameters, ( r 2) is the average value of r 2 for the 4 f electrons, (Jl[~llJ) is a numerical coefficient characteristic of the lanthanide, and A ° and A22 are two crystal field coefficients. Therefore, Ad is • dependent on T-2: Ad =_[gjZfl2j(j + l ) ( 2 J - 1 ) ( 2 J + 3)/60(KT)2]r-3F'

(16)

where F ' = Dz(3 cos 2 0

--

I)

at- ( O x -

Dr)sin 2 0 cos 2 ~b

(17)

Terms of the electrostatic potential higher than second order have negligible contributions. 44 44 B. R. McGarvey,

J. Magn. Reson. 33, 445 (1979).

[3]

51

LANTHANIDE SHIFT REAGENTS TABLE I THEORETICAL DIPOLAR (Cj) AND CONTACT ((Sz)) VALUES

Lanthanide

Cja

( Sz ) b

Cerium (Ce) Praseodymium (Pr) Neodymium (Nd)

-6.3 - 11.0 -4.2 -0.7 4.0 0.0 -86 - 100 -39 33 53 22

-0.98 -2.97 -4.49 0.06 10.68 31.50 31.82 28.55 22.63 15.37 8.21 2.59

Samarium (Sm) Europium (Eu) Gadolinium (Gd) Terbium (Tb) Dysprosium (Dy) Holmium (Ho) Erbium (EL) Thulium (Tm) Ytterbium (Yb)

Reprinted with permission from Ref. 41. b Reprinted with permission from Ref. 40.

The dipolar shift can be rewritten as Ad =

Cj[fl2(r2)2A°(3 c o s 2 0 - 1) + f12(r2)2A2 s i n 2 0 c o s 2cb]/60(KT)r 3 (18)

where Cj = g j 2 ( j + 1)(2J -

1)(2J + 3)

(Jllo~lJ)

(19)

The value of Cj for each lanthanide listed in Table I includes contributions from excited state J levels that are populated at room temperature for some of the ions, for example, Samarium(Ill) and Eu(III).41 The numerical coefficient (Jl[allJ) changes sign for several of the ions, and this correctly predicts why some lanthanides induce shifts to high frequency and others to low frequency. The two crystal field coefficients, A ° and A 2, can also change sign with differing ligand fields. For a series of isostructural lanthanide-ligand complexes, where the values of r, 0, ~b, and the crystal field coefficients are independent of the lanthanide, the measured dipolar shift for any nucleus in that series of complexes should be proportional to Bleaney's Cj values. Thus, the lanthanide-independent constants may be combined with the Cj values to give

Ad:Ol(cos2°-a)r3"2(sin20c°82')"

(20)

52

PROBES OF METAL ION ENVIRONMENTS

[3]

where D1 and D 2 are temperature-dependent constants that depend on the individual lanthanides. If D 2 equals 0, axial symmetry occurs and the measured dipolar shifts b e c o m e proportional to Dl(3 cos a 0 - l)/r 3. Axial s y m m e t r y can o c c u r in two different ways. The lanthanide complex may contain a 3-fold or higher s y m m e t r y axis in solution, or there can be effective axial s y m m e t r y resulting from either rapid internal rotation about the lanthanide-ligand bond 45 or rapid interconversion of the geometrical isomers of the complex. 46 Such fluxional behavior has been proposed for many systems whose solid-state structures indicate nonaxial symmetry, although the paramagnetic shifts appear to possess axial s y m m e t r y in solution. It has been argued that the validity of using the axial symmetry assumption can be tested by examining shifts for different lanthanide complexes. If LIS values for a given complex are measured for a series of shift reagents containing different lanthanides, a plot of the measured LIS values versus Bleaney's Cj values is linear if the origin of the shift is purely dipolar. H o w e v e r , it should be noted that such linearity is not a test for axial s y m m e t r y since both D~ and D E should be constant for isostructural complexes. Deviations from linearity indicate that either the measured LIS values are not purely dipolar or the assumption of isostructurality is not valid. It has also been proposed that the constancy of internal shift ratios for different lanthanide complexes can be taken to indicate that the measured LIS values are contact free and conform to dipolar axial symmetry. 47-49 In that case, these shift ratios could be used with some confidence to derive geometrical information about the lanthanide complex. We note parenthetically that the measured LIS values could contain a contact c o m p o n e n t while the shift ratio, if indeed constant, should not. F o r example, a contact contribution could be present in each measured shift in the same ratio as the measured shift ratios. 16 Inspection o f Eq. (18) also suggests that constancy of shift ratios along the lanthanide series is not an absolute criterion of axial symmetry. It rather indicates that the complexes are either axially symmetric or strictly isostructural (A ° and A~ are constant for the entire series). It also has been shown that if the nuclei 45j. M. Briggs, G. P. Moss, E. W. Randall, and K. D. Sales, J. Chem. Soc. Chem. Commun., 1180 (1972). 46W. de W. Horrocks Jr., J. Am. Chem. Soc. 96, 3024 (1974). 47C. D. Barry, J. A. Glasel, A. C. T. North, R. J. P. Williams, and A. V. Xavier, Nature (London) 232, 236 (1971). 48I. D. Campbell, C. M. Dobson, R. J. P. Williams, and A. V. Xavier, Ann. N.Y. Acad. Sci. 222, 163 (1973). 49I. D. Campbell, C. M. Dobson, and R. J. P. Williams, Proc. R. Soc. London A 345, 41 (1975).

[3]

LANTHANIDE SHIFT REAGENTS

53

are in a particular spatial arrangement relative to the principal magnetic axis system they may have constant shift ratios, even though there are significant contributions of the nonaxial term in Eq. (20). 5°'51 Therefore, although it is likely that many lanthanide complexes achieve effective axial symmetry by some averaging process (as discussed above), routine assumption of axial symmetry based solely on constancy of shift ratios should be used with extreme caution. The Fermi contact contribution to the LIS, Ac , is much less prevalent for complexes of the lanthanides than for those of the transition metal ions, as the orbitals used by lanthanides in bonding have very little 4 f character. If a small fraction of unpaired electron spin density is delocalized into the orbitals of a ligand atom, that spin density may become polarized through the ligand bonds and produce an additional magnetic field at the nucleus being examined in the NMR experiment. This spin polarization mechanism usually causes a decrease of the contact shift as the number of bonds from the ligating atom increases. The magnitude of the contact shift depends on the hyperfine coupling constant, A, between the electron and the nuclear magnetic moments and the spin expectation value, (Sz), for a particular lanthanide: Ac = (A/h)(Sz)/(yflo/2rr)

(21)

Considering a singly populated ground J level, with Zeeman splittings less than KT, 52 (Sz) J =

--flgj(gj

--

B0 1)J(J + 1) 3K----T

(22)

and Ac is proportional to T -1. Values of (Sz) have been tabulated by Golding and Halton 4° for each lanthanide, which include excited electronic states that are thermally populated at room temperature, [e.g., from Sm(III) and Eu(III)] (Table I). These values can be either negative or positive owing to the large orbital contribution to the determination of the energy levels of the lanthanide ions. Once contact shift contributions to measured LIS values have been identified, they must be separated from the potentially structurally useful dipolar contributions. The approach introduced by Dobson et al. 53 involves 50 T. D. Marinetti, G. H. Snyder, and B. D. Sykes, Biochemistry 15, 4600 (1976). 51 j. W. M. de Boer, P. J. D. Sakkers, C. W. Hilbers, and G. de Boer, J. Magn. Reson. 25, 455 (1972). 52 W. B. Lewis, J. A. Jackson, J. F. Lemons, and H. Taube, J. Chem. Phys. 36, 694 (1962). 53 C. M. Dobson, J. P. Williams, and A. V. Xavier, J. Chem. Soc., Dalton Trans., 2662 (1973).

54

PROBES OF METAL ION ENVIRONMENTS

[3]

calculating the ratio of the shifts for one nucleus that contains contact and dipolar components (Aa+~) versus another nucleus in the same molecule that has only a dipolar shift (Ad) and plotting the ratio Ad+c/Aaversus (Sz)/A d for a series of lanthanides. However, the most general separation method was introduced by Reilley et al.,54 which relies on use of both theoretical Cj and (S z) values to separate those components. The total observed isotropic shift for a nucleus (after correcting for Acf effects) may be expressed as (23)

Aob s = A d + A c

Substituting reduced forms of Eqs. (18) and (21) into Eq. (23) gives Aobs = GCj + F(Sz)

(24)

where G is the complex geometrical term in Eq. (18) and F contains the hyperfine coupling constants and the remaining constants in Eq. (21). Equation (24) may be rearranged into two linear forms: (25a) (25b)

Aobs/(S z) = G ( q / ( S z ) ) + F Aobs/Cj = G + F((Sz)/C )

Equation (25a) should be used in linear regression analysis when AobS is dominated by dipolar shifts (calculated G / F >> 1) and Eq. (25b) when Aobs is dominated by contact effects (calculated G / F ~ 1). 54 Relaxation Rates

For paramagnetic lanthanide ions, the dipolar contribution to the nuclear relaxation rates (the contact contribution is usually negligible) can be expressed by the following terms of the Solomon-Bloembergen equations 10,55. 1 _ 2 yi2gjZJ(J + 1)/32 (

TIM

15

r6

3z¢ + 7r¢ ] \ 1 + O.)I2Tc2 1 + OksZTc2/

1 _ 1 "yi2gj2j(j + 1)/3 2 ( T2M

15

r6

3r c 4rc

+ 1 d'- ¢.OI2Tc2

~_ 13r~ '~ 1 + (.Os2Tc2/

(26) (27)

where the constants, YI, gi, J, and/3, have the usual meaning, to~ is the nuclear Larmor frequency, tos ~ 660toi, and rc is given by

1_!+I

1

"r~ zR

~-~l +--zi

54 C. N. Reilley, B. W. Good, and R. D. Allendoerfer, Anal. Chem. 48, 1446 (1976). 55 j. Reuben and D. Fiat, J. Chem. Phys. 51, 4918 (1969).

(28)

[3]

LANTHANIDE SHIFT REAGENTS

55

where TR is the rotational correlation time of the complex, Tie is the electron spin relaxation time, and ZM is the lifetime of the complex. When extreme narrowing conditions occur, tOsZc ~ 1, and l T1M

1 T2M

Cr-6Zc

(29)

where C = (4/3)ylZgj2j(j + 1)/32. Equation (29) can be used, under appropriate conditions, to obtain absolute values of the metal-to-nucleus distance r by specifying the values of zc . Data on the kinetics of lanthanide complex formation43 indicate that "rMexceeds 10 -7 sec. In the case of Gd(III), electron spin relaxation times Tie are rather long and can be estimated by using literature values for Tie obtained by EPR signal line widths and the frequency dependence of Tie1°:

1/Tje = Drv/(l + /360s2"rv2)

(30)

where "rv is the correlation time reflecting the rate at which solvent collisions modulate the zero-field splitting and/3 and D are constants for the particular spin system. At high fields (toH > 200 MHz), Tie is greater than or equal to 2 × 10 7 sec. Therefore, for proteins with rotational correlation times "rR no greater than 10-8 sec, the correlation time "rc of Eq. (28) is dominated by "rR, which can be obtained, for example, from analysis of protein ~3C relaxation times. Using this value in Eq. (29) leads to calculation of absolute Gd(III) nuclear distances from the analysis of Gd(III)induced relaxation enhancements. For the non-S-state lanthanides which have short Tie values ( ' ( 1 0 -12 sec), 56 "re is dominated by TI~ and very little nuclear paramagnetic relaxation occurs. Information on trends in T~ for a range of lanthanides have been obtained from T~ values of protons of lanthanide complexes with small ligands or proteins as a function of the effective magnetic moment of the c o m p l e x , 42,57 using Eq. (29). However, the non-S-state lanthanides have an additional nuclear relaxation mechanism, referred to as the Curie spin or magnetic susceptibility relaxation term. 58 This results from the interaction of the nuclear spins with the static magnetic moment related to (Sz) [see Eq. (22)], which is modulated by "re (but not by T~e): 16~/2/3o2gj4/34j2(J+l)2("rR) TlM× -- 5 (3KT)Zr 6 1 + ¢.,Ol2"rR2

(31)

56 B. M. Alsaadi, F. J. C. Rossotti, and R. J. P. Williams, J. Chem. Soc., Dalton Trans., 2147 (1980). s7 p. D. Burns and G. N. LaMar, J. Magn. Reson. 46, 61 (1982). 58 M. Gueron, J. Magn. Reson. 19, 58 (1975); A. J. Vega and D. Fiat, Mol. Phys. 31, 347 (1976).

56

PROBES OF METAL ION ENVIRONMENTS

1

1 "y2flo2gj4fl4j2(j + 1)2 (4rR + 3¢R (3KT)2r 6 _ 1 + £012TR2]

TZM× -- 5

[3]

(32)

where the subscript X refers to this term. The relative importance of this term increases with the square of the magnetic field. Lanthanide-induced shift and relaxation rate perturbations have been used to determine the conformations of molecules in solution. 14The relevant geometrical parameters in Eqs. (20) and (29) must be averaged by the fast molecular motions that take place in solution before fitting the observed perturbations to a structure or set of structures. Lanthanide-Induced Shift Studies of Biological Macromolecules Application of the LIS method to probe the aqueous structure of biological macromolecules has not developed as rapidly as for small molecules, mainly because of the experimental difficulties involved in such studies, such as poor spectral resolution, lack of resonance assignments, and multiple lanthanide ion binding sites. Although some LIS studies have been reported for tRNA, 59 most have been limited to low molecular weight proteins containing one or more selective lanthanide binding sites. Lanthanide ions are known to bind in normal Ca 2÷ binding sites on proteins, such as the amylases, thermolysin, calmodulin, or parvalbumin, or they may simply happen to occupy a relatively selective site where two or more carboxyl group side chains congregate, such as in concanavalin A or lysozyme. The Ln3+-ATP complexes have also been used as a substitute for Mg2+-ATP in kinases. 6° It is advantageous if the substitution of a Ln 3÷ for Ca 2÷ or Mg 2÷ is isomorphous and the system retains its normal activity, so that the information gained from LIS studies may be useful in understanding biological structure/function relationships. In practice, this has often not been observed; indeed, it has been suggested2~ that if a Ln3+-substituted protein is active, the Ca 2÷ (or Mg 2÷) plays a structural role, but if it is inactive, the ion has a catalytic role. We now describe a few typical applications of LIS studies to proteins. Mapping Active Site o f Nonmetalloenzyme: The Case o f Lysozyme

Hen egg white lysozyme is a small enzyme, having a single polypeptide chain of 129 amino acid residues and a molecular mass of approximately 14400 Da, that catalyzes the hydrolysis of fl-l,4-glycosidic linkages between residues in the polysaccharide components of bacterial cell walls. 59 C. R. J o n e s a n d D. R. K e a r n s , Proc. Natl. Acad. Sci. U.S.A. 71, 4237 (1974). 6o p. Transwell, E. W. W e s t h e a d , a n d R. J. P. Williams, FEBS Lett. 48, 60 (1974).

[3]

LANTHANIDE SHIFT REAGENTS

57

Trp 62

Tvr53

Thr

51

•

56 ~

Trp ~...-.~Ala 107

350 f" ~..~, Ala I10

FIG. 1. Schematic illustration of the active site region of lysozyme as determined by X-ray crystallography. (Reprinted with permission from Ref. 2.)

Its X-ray crystal structure has been determined to a resolution of 2.5 A, 61'62 the first enzyme to yield such an atomic resolution structure. It was also a very early test example for NMR lanthanide shift reagent applications by the Oxford enzyme g r o u p . 2'48'49 Lysozyme has no known metal ion requirements and binds lanthanide ions in solution only weakly (Kassoc ~ 103 M-l), with the primary site located in the enzyme active site near the catalytically active side-chain carboxyl groups of Glu-35 and Asp-52 (Fig. 1). 2'48'49 In solution, some other very weak (Kassoc -< 10 M -l) binding sites have been detected, resulting from binding to surfaceexposed carboxylic acid groups, but only the NMR effects arising from the strong binding site can be measured. 63Although the enzyme is inhibited by the lanthanide binding, the conformational perturbations resulting from this binding are small and confined to the immediate vicinity of the metal binding site, as implied by solution NMR studies 49'63 and finally shown by X-ray studies of the Gd(III)-lysozyme complex. 64 The ortho protons of Tyr-53 shift on addition of increasing amounts of a paramagnetic lanthanide other than Gd(III), so fast exchange conditions apply and the LIS values for various lanthanides were proportional to 61 C. C. F. Blake, L. N. Johnson, G. A. Mair, A. C. T. North, D. C. Phillips, and V. R. Sarma, Proc. R. Soc. L o n d o n B 167, 378 (1967). 62 T. Imoto, L. N. Johnson, A. C. T. North, D. C. Phillips, and J. A. Rupley, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 3, p. 666. Academic Press, New York, 1972. 63 C. M. Dobson and R. J. P. Williams, in "Metal-Ligand Interactions in Organic Chemistry and Biochemistry" (B. Pullman and N. Goldblum, eds.), Part 1, p. 255. Reidel, Dordrecht, The Netherlands, 1977. 64 K. Kurachi, L. C. Sieker, and L. H. Jensen, J. Biol. Chem. 250, 7663 (1975).

58

PROBES OF METAL ION ENVIRONMENTS

[3]

Bleaney's Cj values (Table 1). 49 Although several proton resonances shift when a lanthanide is titrated into the protein, many could not be resolved because of overlap with other unshifted resonances in the spectra, a frequent problem with protein spectra, particularly in the early studies at 270 MHz proton frequency. Campbell e t a t . 48'65 introduced an ingenious solution to this problem by adding incremental amounts of Gd 3+ and generating an NMR difference spectrum after each addition. Because Gd 3+ induces line broadening only in those resonances nearest the lanthanide binding site, the resonances that appear in each new difference spectrum reflect nuclei further removed from the Gd 3+ binding site. Ions that produce a paramagnetic shift are then added in combination with Gd 3+ to obtain LIS values (Fig. 2) for a number of resonances near the lanthanide binding region which could not normally be resolved. Some of the reported 49 LIS values and relaxation rate enhancements of a number of CH resonances of hen egg white lysozyme induced by the binding of various paramagnetic lanthanides are summarized in Table II. A semiquantitative comparison of the structure of the protein in solution and in the crystal was then attempted. As the relative relaxation of resonances induced by Gd 3+ is simply proportional to the relative values of 1/r 6 for the different nuclei [see Eq. (29)], where r is the distance between the bound lanthanide and the nucleus in question, a plot of observed relative distances against the calculated relative distances from the X-ray structure can be made 49 (see Fig. 3A). Allowance has been made for rotation of methyl groups and for flipping of tyrosine residues. The correlation of the NMR data with the X-ray structure is generally good, except for the Val-109 resonances and also for the resonances at longer distances (r -> 14 ,~), where the relaxation from the major binding site is small and difficult to measure, and where relaxation from the weaker binding sites is more important. The proton resonances were assigned to specific amino acid protons in the primary sequence of the enzyme using a comparison of experimental NMR data and the X-ray crystal structure. The LIS values induced by the other paramagnetic lanthanides are proportional to 1/r 3 and to functions related to angles made between the vector joining the metal to the nucleus and the axis of magnetic susceptibility determined by the ligand field effects, and which depend on its symmetry. As this symmetry cannot be determined directly, Eq. (20) with D 2 ~ 0 or D 2 = 0, corresponding, respectively, to rhombic and axial symmetry, could be used to fit the experimental LIS data. It was considered by the authors 49 (see Table II for some of the data) that the observed LIS ratios were reasonably independent of the nature of lanthanide (with the notable 65 I. D. Campbell, C. M. Dobson, and R. J. P. Williams, J. Magn. Reson. 11, 172 (1973).

[3]

59

LANTHANIDE SHIFT REAGENTS

Lysozyme (5 mM) +Eu (111)(3.3 mM)

'\\ i

Lysozyme(5 raM) w I!

Lysozyme(5 raM) + Pr(lll) (10 mM) I

2

I

1

I

0

~/ppm FIG. 2. G d 3÷ differencespectra of the methyl region of lysozymein the absence and presence of shift probes. (Reprintedwith permissionfrom Ref. 48.) exception of Tm3+), suggesting that the shifts may be described by the axial symmetry equation [Eq. (20), with D2 = 0]. Given the coordinates of the assigned nuclei and the metal ion binding site from the crystal structure, the ratios of (3 cos 2 0 - 1)/r 3 for each nucleus were calculated for all possible directions of the magnetic symmetry axis (which defines 0). For a very limited range of directions, the observed and calculated ratios were found to be close (see Table II and Fig. 3B). The agreement for many resonances was found to be excellent, indicating general accord between the crystal structure of lysozyme and the solution structure as determined by the LIS method; moreover, it shows that the conformation of the enzyme near the metal binding site (also the active site) is unique and well defined. However, the assumption of axial symmetry of the LIS values and therefore the possibility of obtaining a more detailed structure of the

60

PROBES O F M E T A L I O N E N V I R O N M E N T S

[3]

T A B L E II PROTON S H I F T AND RELAXATION DATA FOR LANTHANIDE(III) IONS IN H E N EGG WHITE LYSOZYME a

Shift ratios G d 3+

Observed Resonance

Observed broadening ratio

r b (/~)

P r 3+

N d 3+

Calculated

V a l - 1 0 9 CZdH3 V a l - 1 0 9 C~2H3 Ala-110 CH 3 T r p - 108 CVH Trp-108 NH Ala-31 CH 3 Thr-51 CH 3 o-Tyr-53 m-Tyr-53 L e u - 5 6 CvlH3 L e u - 5 6 C~'2H3 I1e-98 CY2H3 Met-105 CH 3 Met-12 CH3 L e u - 1 7 C~IH3 L e u - 1 7 C~2H3 o-Tyr-20 o-Tyr-23 T r p - 6 3 CVH Ala-107

2300 2300 1750 1200 -163 140 100 100 95 95 75 35 25 25 25 -----

8.81 6.02 6.26 6.87 -10.44 9.61 10.90 9.87 11.85 9.60 13.3 12.6 14.7 17.2 15.8 -----

73 - 109 59 -117 -45 -151 100 160 0 -9 -32 - 14 -2 --0 - 14 53 -68

-110 -380 64 -55 --69 100 100 -12 --11 5 7 12 ------

65 175 -80 -137 -21 -lll 100 144 - 1 - 19 -23 -25 -6 ---3 - 10 42 -49

Reprinted with permission from R e f . 49. b Crystal structure.

a

protein in solution are much more doubtful. Some differences in shift ratios were indeed observed for the different lanthanides (see Table II), indicating that the assumption of axial symmetry is not strictly correct. In fact, Agresti e t al. 66 have reanalyzed the LIS data and statistically tested the validity of such an assumption. Both Nd 3÷ and C e 3÷ w e r e found to exhibit considerable nonaxial contributions to the dipolar LIS values. The assumption of axial symmetry was statistically rejected with 97.5% confidence. Lenkinski e t al. 67 also rejected the assumption of axial symmetry for the LIS shifts observed for the Co 2÷ derivative of lysozyme. Using the shift perturbations produced by Co 2÷ and the broadenings induced by 66 D . G . A g r e s t i , R . E . L e n k i n s k i , a n d J. D . G l i c k s o n , B i o c h e m . B i o p h y s . R e s . Commun. 76, 711 (1977). 67 R . E . L e n k i n s k i , D . G . A g r e s t i , D . M . Chen, and J. D . Glickson, Biochemistry 17, 1463 (1978).

[3]

LANTHANIDE SHIFT REAGENTS

61

A 1.0

I 0 0.5

Oo

i

!

i

i

i

i

i

Ii1.01

= I

Calculated PP" Shift Ratios

B "0

/

o o 1.0

J I

I

-1.0

/~

I

A

i

Observed |

I

1.0

o

.0

FIG. 3. Correlation of calculated and observed (A) relative distances from the Gd 3+ ion in lysozyme and (B) relative shifts induced by the Pr 3+ ion in lysozyme. (Reprinted with permission from Ref. 48.)

Gd 3+, the assignment of the signals from the indole N H protons of the tryptophan residues in lysozyme were cross-checked by using both metals, in association with the crystal structure. 68 ~3C NMR spectroscopy was also used to investigate the effect of chemical modification of Trp-108 on 68 R. E. Lenkinski, J. L. Dallas, and J. D. Glikson, J. Am. Chem, Soc. 101, 3071 (1979).

62

PROBES OF METAL ION ENVIRONMENTS

[3]

the binding of lanthanide ions, which is weakened by a factor of more than 20. 69 Weak and less specific binding of lanthanide ions was also observed to the surface of various enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, 7° horse ferricytochrome c, 71 and basic pancreatic trypsin inhibitor (BPTI). 72 The observed shifts were used to help assignments, a task of less importance now with the present high-field instruments and multidimensional techniques. For proteins that lack a single, well-defined lanthanide binding site, formation of a specific nitrotyrosine derivative by chemical modification was proposed as a binding site for lanthanides as protein NMR structural probes. 73 This approach was successfully applied to a study of the Gd 3+induced proton relaxation effects of dinitro-BPTI, where Gd 3+ interacts with nitrotyrosine-21.74 However, the interpretation of the Pr 3+- and Eu 3÷induced LIS values was found to be complex, owing to the presence of nonaxial symmetry contributions. 75 The general applicability of this approach has also not been demonstrated.

Probing Active Site of Mg2+-ATP-Dependent Phosphoglycerate Kinase Yeast 3-phosphoglycerate kinase (PGK), a glycolytic enzyme that catalyzes the reversible phosphorylation of 3-phosphoglycerate (PGA) by ATP, is a monomer with a molecular mass about 45 kDa. Its schematic high-resolution X-ray crystal structure 76'77 (see Fig. 4) shows that the polypeptide chain is organized into two structurally independent domains that are joined by a short helical hinge composed of two chains. More than 50% of the residues are organized in two large/3 sheets that, between them, contain 14 strands (A-N) and 13 a helices (I-XIII). The catalytic mode of Mg2÷-ATP (or Mg2+-ADP) binding to PGK, illustrated in Fig. 4, is to the C-terminal domain, where a hydrophobic depression binds the adenine group. The phosphate chain of the cofactor points away from the C-terminal domain toward the N-terminal domain. The active site of the 69 K. Dill and A. Allerhand, Biochemistry 16, 5711 (1977). 7o R. A. Dwek, H. R. Levy, G. K. Radda, and P. J. Seeley, Biochim. Biophys. Acta 377, 26 (1975). 71 C. M. Dobson, G. R. Moore, and R. J. P. Williams, FEBS Lett. 51, 60 (1975). 72 S. J. Perkins and K. Wiithrich, Biochim. Biophys. Acta 536, 406 (1978). 73 T. D. Marinetti, G. H. Snyder, and B. D. Sykes, J. Am. Chem. Soc. 97, 6562 (1975). 74 T. D. Marinetti, G. H. Snyder, and B. D. Sykes, Biochemistry 15, 4600 (1976). 75 T. O. Marinetti, G. H. Snyder, and B. D. Sykes, Biochemistry 16, 647 (1977). 76 R. D. Banks, C. C. F. Blake, P. R. Evans, R. Haser, D. W. Rice, G. W. Handy, M. Merrett, and A. W. Philips, Nature (London) 279, 773 (1979). 77 H. C. Watson and N. P. C. Walter, EMBO J. 1, 1635 (1982).

[3]

~ / ~

LANTHANIDE SHIFT REAGENTS

63

M2° g

leu

FIG. 4. Schematic drawing of the active-site cleft of PGK as determined from X-ray crystallography. The a-helical segments are denoted by cylinders and the/3-sheet strands by arrows. The residues associated with the basic-patch region of the N-terminal domain (His-62, His-167, and His-170 and Arg-38, Arg-65, and Arg-168) are also shown. Inset shows the bound substrates Mg2+-ATP and PGA. (Reprinted with permission from Refs. 76 and 77.)

enzyme is probably divided between the two domains, with the C-domain carrying the cofactor and the N-domain the PGA substrate and the catalytic site. Nucleotide binding to PGK in solution has been studied using and proton 6°'8°-82 NMR techniques. Conformational changes were moni-

31p78,79

78 B. D. Nageswara Rao, M. Cohn, and R. K. Scopes, J. Biol. Chem. 253, 8056 (1978). 79 B. D. Ray and B. D. Nageswara Rao, Biochemistry 27, 5574 (1988). 80 W. J. Fairbrother, D. Bowen, L. Hall, and R. J. P. Williams, Eur. J. Biochem. 184, 617 (1989). 81 W. J. Fairbrother, H. C. Graham, and R. J. P. Williams, Eur. J. Biochem. 190, 161 and 407 (1990).

64

PROBES OF METAL ION ENVIRONMENTS

[3]

toted on binding of ATP and ADP both with and without Mg 2÷. These studies clearly show the presence of two ATP binding sites on PGK. Its primary binding site involves electrostatic interactions between the nucleotide triphosphate chain and arginines in the basic-patch region (located in the N-domain, see Fig. 4), identified as the general anion binding site. The PGA substrate binds close to Arg-168. As the Mg 2÷ concentration is increased relative to ATP, hydrophobic binding of the adenosine moiety of ATP occurs to PGK at a secondary site, which is equivalent to the catalytic site at the C-domain observed by X-ray crystal studies. The affinity of the catalytic site is increased relative to the primary electrostatic site with increasing Mg 2÷ concentration. Binding to the catalytic site causes a conformational change of His-167. These conclusions are consistent with a kinetic study 83 which has shown ATP 4- to be an activator of PGK at low concentrations and an inhibitor at higher concentrations. The inhibition was shown to be competitive with respect to both substrates (MgE+-ATP and PGA). The activating site is the PGA binding site. A kinetic and proton NMR study 6° at neutral pH showed that MgZ÷-ATP has an apparent K m that is dependent on the PGA concentration ( K m = 0.073 mM at [PGA] = 5 mM) and that L n a + - A T P (Ln = La, Eu, Pr, and Yb) is a competitive inhibitor of PGK, with apparent KI = 0.04 raM. Mg2+-ATP binds at the catalytic site with Kd = 0.15 mM, whereas La3+-ATP shows much tighter binding at the same site. These observations validate the use of Ln3+-ATP complexes as shift and relaxation probes of the resonances of nuclei in the active site region of PGK, and of other ATP phosphotransferases. In fact, data from this technique have been obtained that enabled the mapping of the geometry of the metal/ ATP/PGK active site complex, s3-85 Using paramagnetic difference spectroscopy, the line-broadening inhibitor Gd3+-ATP and the substrate Mn2+-ATP were found to induce identical perturbation of the enzyme resonances, a4,85 These perturbations are quite specific, corresponding to various residues located at the basic patch of the N-domain [His-62 (peak 3 in Fig 5A), His-167 (peaks 4, 15), His-170 (peak 5)] and others located near the ATP binding site at the C-domain [Tyr-193 (peak 14) and Phe-342 (peak 12)] (see Fig. 4 for the residue locations and Fig. 5A for the spectrum and signal numberings). The effects of the shift probes Ln3+-ATP (Ln = Pr, Eu 3+) (see Fig. 6) are in agreement with the broadening data: the largest LIS values are 82 M. Larsson-Raznikiewicz and R. Schierbeck, Biochim. Biophys. Acta 481, 283 (1977). s3 p. Tanswell, E. W. Westhead, and R. J. P. Williams, Biochem. Soc. Trans. 1, 79 (1974). 84 p. Tanswell, E. W. Westhead, and R. J. P. Williams, Ear. J. Biochem. 63,,249 (1976). 85 H. R. Wilson, R. J. P. Williams, J. A. Littlechild, and H. C. Watson, Ear. J. Biochem. 170, 529 (1988).

[3]

65

LANTHANIDE SHIFT REAGENTS A

H-2

H-all 34 5

110

I

9

I

H-I'

I

I

8 7 6 Chemical Shift (ppm)

B 3

I

I

I

7 6 Chemical Shift (ppm)

5

C

His 62 I,t'tis1~

9.0

8.0

His His

7.0

6.0

Chemical Shift (ppm)

FIG.5. Paramagneticdifferencespectraofthe PGKaromaticprotonregionin the presence of (A) 20 g,MGd3+-ATP;(B) 40/xMGd3+-P2; (C) 2/~M[Cr(CN)6]3-. (Adaptedwith permission from Refs. 81 and 84.)

observed for His-167 and (opposite sign) His-62 and His-170. The broadening probe Gd3+-P2 (P2 = pyrophosphate) (see Fig. 5B), and the corresponding shift probe Eu3+-P2, cause very similar effects to the Lna+-ATP chelates, 84 whereas the anionic broadening probe [Cr(CN)6] 3- (Fig. 5C) and the corresponding shift probe [Fe(CN)6] 3- affect only the histidine residues in the basic patch region of the N-domain. 81 This binding site

66

PROBES OF M E T A L ION E N V I R O N M E N T S -50

[3]

-

-4O -30 ~" -1-

-20

.=,-2_ -10

09

i

,

I

i

I

I

I

I

I

~

I

1.0

ao 20

ss

S

lO [Pr • ATP]/[enzyme] (mol/mol) |

'

I

I

I

0.5

I

I

I

I

110

-10' -20 -30 -401 FIG. 6. LIS for PGK histidine resonances as a functionof the concentrationof the shift probes Eua+-ATP and pr3+-ATP. (©) His-62; (0) His-167; (I) His-170. (Adapted with permission from Ref. 84.) corresponds to the general anionic site for PGK, made up of a cluster of positively charged side chains of His-62, His-167, His-170, Arg-21, Arg65, and Arg-168. 81 The broadening and shift effects produced by the Ln3+-ATP probes were then used to map geometrically the enzyme active site, including the conformation of the ATP, the position of various enzyme side-chain residues, and an approximate position for the substrate PGA 84 (Fig. 7). Because the X-ray structure (Fig. 4) indicates that the basic-patch histidine residues are at least 12 ,~ from the ATP binding site, it follows from the above observations that the enzyme as seen by NMR must differ from that seen in crystals. The observed proximity of the two binding sites in the two domains supports a hinge-bending movement hypothesis postulated to explain the catalytic mechanism of this enzyme, 76 whereby the open or substrate-binding form with the two domains apart transforms into the closed and catalytically active form, where the active site cleft and the

[3]

LANTHAN1DE SHIFT REAGENTS

67

site of the transferable phosphate group come into close proximity. Thus, this example illustrates the utility of selective paramagnetic perturbations even for a large protein.

Studies of Ca2+-Binding Proteins: Parvalbumin Another protein that has been studied extensively using the LIS method illustrates a much more complex yet potentially more informative situation. This is the work of Lee and Sykes 86'87 on the CaZ+-binding muscle protein parvalbumin. This protein, having a molecular mass around 11 kDa and a known X-ray crystal structure,88 binds 2 equivalents of Ca z+ in two distinct binding domains called the "CD and EF hands." Each of these consists of a short a-helical structure, a loop around the Ca 2+ site which contains regularly spaced carboxyl, carbonyl, and hydroxyl sidechain ligands for the metal ion, followed by a second a-helical region. Unlike lysozyme, these binding domains have a very high affinity for Ca z+ (Kd -~ 10-9 M) and an even higher affinity for the Ln 3+ cations. It has been shown that the CD domain has a significantly higher affinity for the larger Ln 3+ ions, whereas the EF domain is less selective [e.g., for Yb 3+, K d ~ (4-7) × 10-l° M for CD, K d ~- (2-6) × 10-l° M for EF]. This fact allows, in the presence of Ca z+ and given the preference of Ca z+ for the CD site, preferential loading of the EF site by the smaller ion, Yb 3+ (paramagnetic) or Lu 3+ (diamagnetic), at low Ln 3+/protein ratios, followed by loading of the other site at a higher ratio. The NMR spectra shown in Fig. 8 illustrate the complexity of this LIS study, as compared to the lysozyme study. As incremental amounts of Yb 3÷ are titrated into the protein, several proton resonances disappear from their diamagnetic positions and several new resonances appear both upfield and downfield from their normal diamagnetic positions, between - 2 0 and +36 ppm. Some resonances result from Yb 3+ binding in the EF site, whereas others arise as a result of Yb 3÷ binding in the CD site, in a sequential loading scheme (Fig. 9). This is typical of a slow-exchange situation and is more difficult to interpret since the correspondence between the diamagnetic and paramagnetic resonances is not known. The first problem is then the proper assignment of the many shifted resonances observed. The approach taken by Lee and Sykes was to measure the paramagnetic shifts and broadenings induced by Yb 3+ binding in the proton NMR spectrum and to use the known X-ray structure of the 86 L. Lee and B. D. Sykes, Biochemistry 19, 3208 (1980); L. Lee and B. D. Sykes, Biochemistry 211, 1156 (1981); L. Lee and B. D. Sykes, Biochemistry 22, 4366 (1983). 87 T. C. Williams, D. C. Corson, and B. D. Sykes, J. Am. Chem. Soc. 106, 5698 (1984). 88 R. H. Kretsinger and C. E. Nuckolds, J. Biol. Chem. 248, 3313 (1973).

68

PROBES OF METAL ION ENVIRONMENTS \

[3]

/ \

/ \

®~,®"

/

~-~

@,,, @

\

/ \

/ \

/

@ --(+)-,, -

~

5'

~--

,43'

\

2'

', @

/

\

•

2 (--)

± +

@ T "'~

@

J" j.

j. f j f J@ J

(--)

2'

(J~)~/[ " "

.,.

,. " "

(--)

%,

",

® 0"(~.. %.

@ J. +.L I

~-J

[3]

LANTHANIDE SHIFT REAGENTS

69

protein to determine the unknown parameters required to interpret those effects in terms of the structure of the protein. First, the line broadening effects of the shifted proton resonances were analyzed as a method for the determination of metal-proton distances in the EF site of parvalbumin. 87 The spin-lattice relaxation times and the line widths of several peaks of the NMR spectrum for a yb3+/protein ratio of 0.8 (Fig. 8C) were measured at three different frequencies namely, 200,270, and 400 MHz. Plots of those line widths as a function of the square of the frequency, o~2, were found to be linear. From Eqs. (26), (27), (31), and (32), it is clear that only the susceptibility relaxation term is field dependent. Thus, the intercepts of those plots gave the contribution to the line width governed by the nonsusceptibility contribution, which probably arises from proton dipole-dispole interactions in the protein. For toi2 ~ 0, Lee and Sykes, using relevant values for the constants of Eqs. (27) and (32), showed that for the yb3+-parvalbumin complex at 270 MHz (1/Tzx)/(1/T2s) ~ 16, and thus the susceptibility relaxation term dominates the normal Solomon term. It was similarly shown that the T~ values of the resonances are dominated by the electronic dipolar relaxation mechanism. The metal-proton distances given in Table III were obtained by calculating the susceptibility contribution to the line width, (1/T2x), and analyzing them in terms of Eq. (32), using relevant constants. These distances are therefore an aid to the assignment of the shifted resonances, by comparison of the experimental distances with those calculated from the crystal structure of the protein. The analysis of the dipolar shifts poses a second problem, related to locating the direction and magnitude of the magnetic susceptibility tensors, which define the values of D~ and D2 in Eq. (20). Axial symmetry could not be assumed, as LIS data were available only for Yb 3÷. LIS data for other ions could perhaps have been obtained, but, as indicated above, the selectivity of the EF site over the CD site changes with increasing cation size, and this precludes taking LIS measurements with a sufficient number of other Ln 3÷ ions to test for axial symmetry. Thus, the analysis of the LIS values requires the specification of eight parameters in Eq. (20): three coordinates for the metal ion, three Euler angles that relate the principal symmetry axis of the magnetic susceptibility tensor of Yb 3÷

FIG. 7. Schematic of the active site of PGK showing the relative coordination of the enzyme and ATP protons to the lanthanide (designated M). The protons are located spatially on the basis of the NMR data. The dashed lines represent the dipolar cone of the LIS, and the plus signs (+) define the symmetry axis (PGA, 3-phosphoglycerate; 3, His-62; 4, 15, His-167; 5, His-170; 12, Phe-342; 14, Tyr-193). (Adapted with permission from Ref. 84.)

70

PROBES OF METAL ION ENVIRONMENTS

[3]

A

B

|

30

1

i

i

i

18

J

i

6

i

i

-6

i

- 18

1

|

-30

ppm 8

c

35

30

25

20

15

10

54 55 59 53 ~7 ^

15~2I g ~ -~

30

s2

-;s

-~o

ppm Fro. 8. Proton spectrum (270 MHz) of (A) CaZ+-saturated carp parvalbumin and (B) parva]bumin with 0.8 equivalents of yb3÷; (C) shows spectrum (B) with numbered resonances. [Adapted with permission from L. Lee and B. D. Sykes, Biochemistry 20, 1156

0981). Copyright 1981 American Chemical Society.]

[3]

LANTHANIDE SHIFT REAGENTS

71

2.0 1.4

0.6 ~ ~J~] 34 30 26 22 18 14 & ppm

8ootB ILl

600I

rr

400! 2O0

m

2 3 4 YbJPo F ~ . 9. (A) Proton spectrum (270 MHz) of carp parvalbumin (positive shift portion) at Yb3*/protein ratios of 0.6, 1.1, 1.4, and 2.0. (B) Areas of three different resonances as a function of Yb3+/protein ratio (&, peak 3; Ira, peak 8; Q, peak next to 8, see Fig. 8C). [Adapted with permission from L. Lee and B. D. Sykes, Biochemistry 20, 1156 (1981). Copyright 1981 American Chemical Society.]

to the axis system of the crystal, and Dl and D z . The coordinates obtained by X-ray crystallography for Ca2+ in the EF site of parvalbumin were taken as the coordinates for Yb 3÷. Only five of the shifted resonances (three proton, one l~3Cd, and one 13C) could be assigned with certainty,

72

PROBES OF METAL ION ENVIRONMENTS

[3]

T A B L E III SUSCEPTIBILITY CONTRIBUTION TO SPIN--SPIN RELAXATION OF PROTON N M R RESONANCES OF yb3+-PARVALBUMIN AND MEASURED METAL-PROTON DISTANCES a

Resonance b

Measured r (,~)

1/T2x (sec -I)

3 4 5 6 7 8 10 51 56 61 65 66

5.8 5.9 6.2 6.5 6.2 7.9 8.8 7.7 6.2 7.0 6.6 5.6

141 139 102 78 101 24 12 26 102 46 67 179

Reprinted with permission from Ref. 86. Spectroscopy conducted at 270 MHz. b See Fig. 8C for peak assignments.

so these were used to locate the direction and the magnitude of the Y b 3÷ susceptibility tensors using coordinates for these five nuclei from the X-ray structure. With these parameters determined (see the best-fit calculated versus observed LIS values in Table IV), LIS values for all other nuclei in the protein could be predicted, and several resonances were assigned on this basis. 86 It was noted that the calculated LIS values for protons T A B L E IV COMPARISON OF CALCULATED AND OBSERVED LANTHANIDEINDUCED SHIFTS IN PARVALBUMIN a

ap Nucleus

Observed

Calculated

r b (~)

~H His-26C2H lH His-26C2H IH N - a c e t y l - C H 3 U3Cd C D metal site 13C Arg-75 ~:-carbon

0.485 0.343 0.033 -0.270 0.318

0.475 0.332 0.082 -0.286 0.351

13.6 15. l 20.0 11.9 23.2

Reprinted with permission from Ref. 86. b F r o m E F site.

[3]

LANTHANIDE SHIFT REAGENTS

73

close to the EF binding site (5-10 ,~) were generally larger than the observed shifts, suggesting that the solution structure in this region is less compact than that predicted from the crystal structure. This illustrates the power of the LIS method in emphasizing relatively small structural differences between the solution and solid state. Ln 3+ ions were later used to replace Ca 2+ and assign various ~3C resonances of parvalbumin. 89 The approach described above provides a useful methodology that can be applied to analyze the spectral perturbations induced by the trivalent lanthanide ions in the slow exchange limit. Because of the great degree of sequence similarity between parvalbumin and many other CaZ+-binding proteins of the EF family, such as calmodulin (CAM), troponin C (TnC), the myosin light chains, and the intestinal calcium-binding proteins (ICaBP), and the fact that some of these proteins already have known X-ray crystal structures, an approach involving LIS is, in principle, applicable to them. Some preliminary studies have been undertaken, such as in troponin C fragments 9°'91 and in ICaBP. 92 Studies of non-EF-type Ca 2+binding proteins, such as elastase, have also been reported. 93 Probing Surface o f Proteins

The processes whereby a protein surface recognizes another surface are of fundamental importance in a wide range of biochemical systems. Recognition, which is rather selective, is largely dependent on general electrostatic interactions. The potential energy surfaces of the interacting proteins depend on the polarity size, shape, and flexibility of their surfaces, which are determined by the geometric distribution of their surface residues. 94 The surface cationic groups (guanidinium from arginines or ammonium from lysines) or anionic groups (carboxylates from aspartates and glutamates) may be found concentrated in certain regions, yielding cationic and anionic patches which constitute mountains and wells in such a potential energy surface. Hydrophobic patches may also be found. Studies of the binding of small charged cationic and anionic species to protein surfaces constitute a powerful method to study this problem. 89 D. J. Nelson, lnorg. Chim. Acta 27, L71 (1978). 90 L. Lee, B. D. Sykes, and E. R. Birnbaum, FEBS Lett. 98, 169 (1979).

91j. Garirpy, L. E. Kay, I. D. Kuntz, B. D. Sykes, and R. S. Hodges, Biochemistry 24, 544 (1985). 92W. J. Bridsall, D. C. Dalgano, B. A. Levine, R. J. P. Williams, C. S. Fullmer, and R. H. Wasserman, in "Calcium BindingProteins: Structure and Function" (F. L. Siegel, E. Carafoli, R. H. Kretsinger, D. H. MacLennam, and R. H. Wasserman, eds.), p. 405. Elsevier, New York, 1980. 93j. L. Dimicoli and J. Bieth, Biochemistry 16, 5532 (1977). 94S. C. Tam and R. J. P. Williams,Struct. Bonding (Berlin) 63, 103 (1985).

74

PROBES OF METAL ION ENVIRONMENTS

[3]

These studies have been systematically carried out for ferricytochrome c, 95-97 using small complexes of differing size, shape, and charge that are paramagnetic N M R shift and/or relaxation agents. Analysis of the location of their binding sites was possible through observation of NMR effects on specific proton resonances, which function as reporter groups (Fig. 10). For example, binding studies of the spherical relaxation agents [Cr(CN)6] 3÷, [Gd(dipicolinate)3] 3-, and [Cr(NH3)6] 3+ yield, respectively, the location of positive, hydrophobic, and negatively charged surface regions. Cylindrical probes like [Fe3+(EDTA4-)] - will find dipolar regions. All those surface regions are quite mobile. Such an approach has also been applied to detect histidine-, lysine-, and arginine-containing cationic patches in PGK 81 and myosin subfragment S1, 98 using [MIII(CN)6] 3probes, and hydrophobic patches in S1, 98 using [Gd(DOTA)]-. A different LIS approach has been used to assign 13C resonances in the bacteriophage fd ssDNA-binding gene 5 protein (G5P). This protein, which in solution is a dimer with a molecular mass of about 20 kDa, may be reductively methylated to introduce 13C-enriched methyl groups into all six lysyl residues without significantly disrupting its ability to bind ssDNA. 99 This derivative has been studied by ~3C NMR, and three of the modified lysine resonances are affected by the binding of oligonucleotides. The problem encountered in this protein, which does not normally bind metal ions, was how to assign the resonances to the proper lysyl residues in the protein sequence. In this case, the assignment was accomplished by titrating the protein with negatively charged Ln 3÷ tetraazamacrocylic phosphate extrinsic probes, Tb(DOTP) 5- as a LIS probe and Gd(DOTP) 5as a relaxation probe. The spectrum of the 13C-enriched protein before and after the addition of Tb(DOTP) 5- is shown in Fig. 11. Resonances 1, 2, and 5 shifted to higher frequency, and resonances 3 and 4 shifted to lower frequency throughout a titration until 1 equivalent of chelate per protein dimer had been added. Further additions of the probe did not affect the spectrum. Addition of Gd(DOTP) 5- specifically broadened resonance 2 (and 1 to some extent). Competition experiments between the shift probe 95 C. G. S. Eley, G. R. Moore, G. Williams, and R. J. P. Williams, Eur. J. Biochern. 124, 249 (1976). 96 G. Williams, C. G. S. Eley, G. R. Moore, M. N. Robinson, and R. J. P. Williams, FEBS Lett. 150, 295 (1982). 97 C. O. Arean, G. R. Moore, G. Williams, and R. J. P. Williams, Eur. J. Biochem. 173, 607 (1988). D. C. Dalgarno, H. P. Prince, B. A. Levine, and I. P. Trayer, Biochim. Biophys. Acta 707, 81 (1982). R. Dick, C. F. G. C. Geraldes, A. D. Sherry, C. W. Gray, and D. M. Gray, Biochemistry 28, 7896 (1989).

[3]

75

LANTHANIDE SHIFT REAGENTS

K5

126 I K oo

~'-~1

~~'E~

9

C terminis

V3 4-13:1

~ / ~ ~ K E 8 2 7 1

Top D93 02 K5 I

--5 6 ~ . ~ E69 K99~ ~ , , , - . ~ ~M65 Clerminus--~, C'~3"~6, ~ ' ~ ~ 1 rEK73 T1tO2~3~/~j~:~60~~6, / E66 ( )H33{ -~"~'-~L .-'~L-X==,,J, / 7

DSO Back FIG. 10. Binding sites for [Cr(CN)6]3- on cytochrome c. Filled regions are positively charged, and striped regionsare negativelycharged.Symbolsare single-letternotationsfor amino acids. The square grids denote the six anion binding sites. (Reprinted with permission from Ref. 94.)

and a hexanucleotide show that the binding is mutually exclusive, which suggests that the binding site for the phosphonate chelate overlaps the DNA binding sites. A comparison of the experimental and calculated (based on the protein crystal structure ~°°) shift and broadening effects 10oG. D. Brayer and A. McPherson, J. Mol. Biol. 169, 565 (1983); G. D. Brayer and A. McPherson, Biochemistry 23, 340 (1984).

76

PROBES OF METAL ION ENVIRONMENTS

[3]

4

5 6

A

3,4

5

•

l

. . . .

I

45

. . . .

J

. . . .

I

. . . .

i

. . . .

44

6

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43

. . . .

~

. . . .

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42

(ppm) FIG. 11. 13C NMR spectrum (50.1 MHz) of [methyl-13C]G5P (A) before and (B) after the addition of [Tb(DOTP)] 5-. Resonances 1-6 correspond to the six dimethyllysyl residues, whereas resonance 7 is the partially modified N-terminal dimethylmethionyl residue. (From Ref. 99.)

allowed location of the probe at the protein surface and assignment of resonances 1-6. Resonance 2 corresponds to Lys-24, whose large perturbation can be explained by a substantial movement of the flexible DNAbinding loops containing this residue on binding of the chelate. This is an example of a system that has relatively specific lanthanide chelate binding, which may be saturated at the concentrations used in the NMR experiment while maintaining rapid chemical exchange conditions. This advantage allows determination of the stoichiometry plus a direct evaluation of the diamagnetic chemical shift of each resonance. One further advantage of this system is that the Ln(DOTP) 5- structures are by definition axially symmetric, l°l so the LIS data may be fit to the axial symmetry model without the usual laborious tests for axial symmetry and without making assumptions concerning ligand field averaging. ~01A. D. Sherry, C. F. G. C. Geraldes, and W. P. Cacheris, Inorg. Chim. Acta 139, 137 (1987).

[3]

LANTHANIDE SHIFT REAGENTS

77

The interaction of Gd(DOTP) 5- with very similar IKe and M13 G5P proteins has been studied by proton NMR as a function of pH.~°2 This study showed that these probes bind to the protein at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there exists one high-affinity binding site for the probe per G5P monomer, which coincides with the ssDNA-binding domain of a phosphate-binding electropositive cluster at the protein surface. At pH 5, a second lowaffinity probe binding site became apparent. Soluble spin labels, such as the nitroxide tempol, approach protein surfaces randomly and "bleach" their protons to NMR measurements because of paramagnetic relaxation effects. The simplification of twodimensional nuclear Overhauser effect spectroscopy (NOESY) spectral cross-peaks observed for amide protons in the presence of nitroxides and in DzO, which depend on proton exposure to the nitroxides and to the solvent, reflects the native folding pattern of the protein. A correlation of the spectral simplifications with the known tertiary structure of peptides and proteins, such as gramicidin S, lysozyme, and BPTI, has been explored to provide useful information about protein conformation and dynamics, such as the location of specific amide groups.l°3'~°4 Conclusions and Future Prospects Despite much effort, the prospect of obtaining the quantitative structure of a protein in solution using lanthanides as NMR shift and relaxation probes independently of its X-ray crystal structure has never materialized. However, at least for small proteins, this structural goal has been reached using a combination of the nuclear Overhauser effect and multidimensional NMR. 1°5 Nevertheless, useful qualitative or semiquantitative structural information can still be obtained using the lanthanide probe method, even for medium-sized proteins. Enzymes with no metal requirements are the least amenable to the use of lanthanides as probes, owing to the difficulty of, in general, finding a single, specific metal binding site. MgZ+-ATP-dependent kinases and Ca2+-binding proteins may, on the other hand, constitute the most promising systems to explore, through the use of isomorphous substitution. Axial 102j. p. M. van Duynhoven,I. M. A. Nooren, D. W. Swinkels,P. J. M. Folkers, B. J. M. Harmsen, R. N. H. Konings,G. L. Tesser, and C. W. Hilbers,Biochemistry, submitted. 103N. Niccolai, A. Bonci, M. Rustici, M. Scarselli, P. Neff, G. Esposito, P. Mascagni, A. Motta, and H. Molinari,J. Chem. Soc., Perkin Trans. 2, 1453(1991). 104G. Esposito, A. M. Lesk, H. Molinari, A. Motta, N. Niccolai, and A. Pastore, J. Mol. Biol. 224, 659 (1992). 105K. Wtithrich,Science 243, 45 (1989); K. Wiithffch,Acc. Chem. Res. 22, 36 (1989).

78

PROBES OF METAL ION ENVIRONMENTS

[4]

symmetry of the experimental LIS values should not be assumed as a rule, but rather considered the exception. Ideally, axial symmetry should be proved by methods independent of LIS measurements, if possible through a study of the magnetic susceptibility tensor for the Ln3÷-protein deriv~ttives in single crystals. Examples of calcium-binding proteins that are amenable to LIS studies and that have already been studied by luminescence or ESR spectroscopic methods using lanthanide ions include calmodulin, a-lactalbumin, phosp h o l i p a s e A 2 , and C a 2 + - A T P a s e . 16'1°6'1°7 Lanthanides are also sometimes good probes at Fe 3÷ sites, as demonstrated in ESR studies of Gda÷-trans ferrin derivatives. 108,109In fact, proton LIS studies have been reported for Ln3+-transferrins. 1~° It is hoped that many other applications of lanthanides as NMR probes of proteins will arise in the future. Acknowledgments The authorthanks Prof. R. J. P. Williamsfor inspirationand JuntaNacionalde Investigaqho Cientificae Tecnol6gia(JNICT), Portugalfor financialsupport. 106 E. M. Stephens and C. M. Grisham, Biochemistry 18, 4876 (1979). 107G. H. Reed, R. D. Hershberg, and G. H. de Haas, in " N M R in Biochemistry" (S. Opella and P. Lu, eds.), p. 361. Dekker, New York, 1979. i08 p. B. O'Hara and S. H. Koenig, Biochemistry 25, 1445 (1986). 109 O. Zak and P. Aisen, Biochemistry 27, 1075 (1988). ll0 L. Messori and M. Piccioli, J. Inorg. Biochem. 42, 185 (1991).

[4] A l k a l i M e t a l N u c l e a r M a g n e t i c R e s o n a n c e By DUARTE MOTA DE FREITAS Introduction The alkali metal ions Na t and K ÷ are abundant in biological systems, generally in the concentration range of 1.0-I000 mM. ~ In contrast, the concentrations of Li ÷, Rb ÷, and Cs ÷ in most biological systems are negligible. ~Li ÷, however, is present at appreciable concentrations (in the range of 0.2-5.0 mM) in tissues of manic-depressive patients receiving lithium carbonate treatment. 2 i C. A. Pasternak (ed.), "Monovalent Cations in Biological Systems." CRC Press, Boca Raton, Florida, 1990. 2 M. Schou, in "Lithium and the Cell: Pharmacology and Biochemistry" (N. J. Birch, ed.), p. 1. Academic Press, San Diego, 1991.

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

78

PROBES OF METAL ION ENVIRONMENTS

[4]

symmetry of the experimental LIS values should not be assumed as a rule, but rather considered the exception. Ideally, axial symmetry should be proved by methods independent of LIS measurements, if possible through a study of the magnetic susceptibility tensor for the Ln3÷-protein deriv~ttives in single crystals. Examples of calcium-binding proteins that are amenable to LIS studies and that have already been studied by luminescence or ESR spectroscopic methods using lanthanide ions include calmodulin, a-lactalbumin, phosp h o l i p a s e A 2 , and C a 2 + - A T P a s e . 16'1°6'1°7 Lanthanides are also sometimes good probes at Fe 3÷ sites, as demonstrated in ESR studies of Gda÷-trans ferrin derivatives. 108,109In fact, proton LIS studies have been reported for Ln3+-transferrins. 1~° It is hoped that many other applications of lanthanides as NMR probes of proteins will arise in the future. Acknowledgments The authorthanks Prof. R. J. P. Williamsfor inspirationand JuntaNacionalde Investigaqho Cientificae Tecnol6gia(JNICT), Portugalfor financialsupport. 106 E. M. Stephens and C. M. Grisham, Biochemistry 18, 4876 (1979). 107G. H. Reed, R. D. Hershberg, and G. H. de Haas, in " N M R in Biochemistry" (S. Opella and P. Lu, eds.), p. 361. Dekker, New York, 1979. i08 p. B. O'Hara and S. H. Koenig, Biochemistry 25, 1445 (1986). 109 O. Zak and P. Aisen, Biochemistry 27, 1075 (1988). ll0 L. Messori and M. Piccioli, J. Inorg. Biochem. 42, 185 (1991).

[4] A l k a l i M e t a l N u c l e a r M a g n e t i c R e s o n a n c e By DUARTE MOTA DE FREITAS Introduction The alkali metal ions Na t and K ÷ are abundant in biological systems, generally in the concentration range of 1.0-I000 mM. ~ In contrast, the concentrations of Li ÷, Rb ÷, and Cs ÷ in most biological systems are negligible. ~Li ÷, however, is present at appreciable concentrations (in the range of 0.2-5.0 mM) in tissues of manic-depressive patients receiving lithium carbonate treatment. 2 i C. A. Pasternak (ed.), "Monovalent Cations in Biological Systems." CRC Press, Boca Raton, Florida, 1990. 2 M. Schou, in "Lithium and the Cell: Pharmacology and Biochemistry" (N. J. Birch, ed.), p. 1. Academic Press, San Diego, 1991.

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

[4]

ALKALI METAL N M R

79

Many enzymes are activated by K + alone (e.g., pyruvate kinase, phosphofructokinase, and aldehyde dehydrogenase), a few enzymes are activated by Na + alone (oxaloacetate decarboxylase), and one membranebound enzyme (Na +,K*-ATPase) is activated by both K + and Na ÷ ions. J Whereas the intracellular K + concentrations are generally high for most cell types, the extracellular K ÷ concentrations are low. In contrast, the intracellular Na + concentrations are typically lower than the Na + concentrations in extracellular fluids. The Na + and K + gradients, which result from active and passive ion transport mediated by membrane-bound proteins, and the corresponding transmembrane potential difference play an important role in nerve transmission arid muscle contraction. 1 The Li + ion has pharmacological importance in the treatment of manic-depressive psychosis2; Rb + and Cs + ions are sometimes used in physiological studies as tracers of K + transport. Despite the physiological and pharmacological importance of alkali metal ions and the rather high concentrations in biological systems, the understanding of the environment of alkali metal ions in biomolecules is scant compared to that available for transition metal ions. Alkali metal ions are diamagnetic, and their complexes with biological ligands are colorless, ruling out the application of common methods, such as electron spin resonance and optical spectroscopy. Total alkali metal ion concentrations in metalloproteins or concentrations during ion transport generally can be determined by conventional methods, such as atomic absorption (AA) spectrophotometry or the use of radioisotopes; however, the determination of the environment of alkali metal ions in biomolecules is not amenable to these conventional methods. The determination of ion concentrations during transport experiments performed with invasive methods, such as AA or radioisotopes, requires the separation of cells from the suspension medium, followed by cell lysis, prior to analysis. 3 The radioisotope method is not applicable to Li + transport because lithium radioisotopes have extremely short half-lives. If an alkali metal cation is present in two different environments in a biological sample, in either the free hydrated form or bound to a cytoplasmic protein or a membrane component, only alkali metal nuclear magnetic resonance (NMR) spectroscopy would be suitable for probing these different environments. A molecular understanding of the binding and transport properties of alkali metal ions can be obtained from alkali metal NMR methods; magnetically or chemically nonequivalent pools of alkali metal cations have different relaxation and sometimes different chemical shift properties. 3 D. Mota de Freitas, M. T. Espanol, and E. Dorus, in "Lithium Therapy Monographs" (V. S. Gallicchio, ed.), Vol. 4, p. 96. Karger, Basel, 1991.

80

PROBES OF METAL ION ENVIRONMENTS

[4]

Several reviews of alkali metal N M R and its applications to biological s y s t e m s h a v e appeared4-11; these should be consulted for earlier accounts o f alkali metal N M R s p e c t r o s c o p y and for details on the N M R properties of alkali metal nuclides. This chapter provides a practical description of the w a y in which N M R s p e c t r o s c o p y can be used for determining the e n v i r o n m e n t of alkali metal ions in m e t a l l o e n z y m e s and metalloproteins as well as for studying alkali metal ion distribution and transport across cell m e m b r a n e s . Because of recent d e v e l o p m e n t s in N M R applications to t r a n s m e m b r a n e transport of alkali metal ions in cell suspensions and perfused organs, this physiologically important area of alkali metal N M R applications is described here in depth. The principles that form the basis o f the N M R transport m e t h o d s illustrated here with h u m a n red blood cell (RBC) suspensions are also applicable to perfused organs. 9-N Details on the w a y in which the interpretation of alkali metal N M R data is obtained and the special precautions to be taken w h e n this technique is used are also given.

Special Features of Alkali M e t a l Nuclear Magnetic Resonance Spectra The basic principles and applications of N M R s p e c t r o s c o p y are described elsewhereJ2; in this section we describe unique aspects of alkali metal N M R spectra. With the advent of Fourier transform nuclear magnetic r e s o n a n c e ( F T - N M R ) s p e c t r o m e t e r s , the introduction of superconducting magnets, and i m p r o v e m e n t s in p r o b e design, new information on the m e t a l l o b i o c h e m i s t r y of alkali metal ions has been obtained f r o m alkali metal N M R s p e c t r o s c o p y . T h e N M R properties of the biologically important alkali metal nuclides are shown in Table I. F o r comparison, the N M R properties of the m o r e c o m m o n nuclides IH and 13C are also listed in Table I. With the exception o f 6Li, all alkali metal nuclides have a relatively high natural abundance. The N M R receptivity of a nuclide provides a 4 j. Mason (ed.), "Multinuclear NMR," p. 625. Plenum, New York, 1987. 5 C. Detellier, in "NMR of Newly Accessible Nuclei: Chemically and Biochemically Important Elements" (P. Laszlo, ed.), Vol. 2, p. 105. Academic Press, New York, 1983. 6 F. W..Wehrli, in "Annual Reports on NMR Spectroscopy" (G. A. Webb, ed.), Vol. 9, p. 125. Academic Press, New York, 1979. 7 S. Fors6n and B. Lindman, Methods Biochem. Anal. 27, 289 (1981). s j. j. Dechter, in "Progress in Inorganic Chemistry" (S. J. Lippard, ed.), Vol. 29, p. 285, Wiley, New York, 1982. 9 C. S. Springer, Jr., Annu. Rev. Biophys. Chem. 16, 375 (1987). l0 R. K. Gupta, in "NMR Spectroscopy of Cells and Organisms" (R. K. Gupta, ed.), Vol. 2, p. 1. CRC Press, Boca Raton, Florida, 1987. II K. Kirk, N M R Biomed. 3, 1 (1990). t2 j. j. Villafranca, this series, Vol. 177, p. 403.

~

+

..g a

_

~ ~ 1 1

xx~x~Xll %

iI

.,.¢ ,-I

N m

Z >

Z ©

Iga

~xx~xxxg

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,-] i~1 <

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Z

82

PROBES OF METAL ION ENVIRONMENTS

[4]

measure of how strong the NMR signal should be; this is a useful NMR parameter because, in addition to the natural abundance of the nuclide, it also takes into account the intrinsic sensitivity of a nuclide, which is a function of the gyromagnetic ratio, y, and the nuclear spin, I. The receptivities of the 7Li, 23Na, 87Rb, and 133Cs a r e considerably higher than that of 13C, and, therefore, 7Li, 23Na, 87Rb, and 133Cs NMR spectra of biological samples can be obtained quickly. Although the 39K nuclide is highly abundant, its detection by NMR is hampered by acoustic ringing problems associated with a low gyromagnetic ratio, and by a correspondingly low observation frequency) The low receptivity of the 6Li nuclide is generally circumvented in biological applications by the use of lithium salts enriched in the 6Li isotope. 13,14The 6Li isotope is a by-product of nuclear reactions, and lithium salts enriched in 6Li can be obtained inexpensively from the Oak Ridge National Laboratory (Oak Ridge, TN). The chemical shifts of 6Li+, 7Li÷, 23Na+, 39K+, and 87Rb+ cations are virtually insensitive to solvation or complexation by biomolecules. 4'5 Because of the large size of the 133Cs÷ cation, the chemical shift of this nuclide is very sensitive to binding in biological systems. T M Therefore, whereas one can use chemical shift information to determine the environment of the 133Cs+ cation in biological systems, line width or relaxation data are generally used for probing the environment of the other alkali metal nuclides. All alkali metal nuclides of biological interest have a nuclear spin I greater than or equal to I, possessing a finite quadrupole moment. The major relaxation mechanism for 23Na, 39K, and 87Rb nuclides is quadrupolar, resulting in broad NMR lines. The 87Rb÷ NMR lines are particularly broad; this effect is thought to be associated with the high value of the Sternheimer antishielding factor (see Table I), a cation property that describes the magnification of the electric field gradient at the nucleus by the surrounding electrons. For the 6Li, 7Li, and 133Csnuclides, the quadrupole moments are very small, giving rise to narrow NMR lines. The dipolar relaxation mechanism predominates in the case of 6Li and is also important in the case of 7Li.5 When the correlation time 7c, a parameter that describes molecular motion, is much shorter than the N M R observation frequency co, the extreme narrowing condition applies (co2r2 ~ 1). Under this condition,

13 R. Ramasamy, D. Mota de Freitas, C. F. G. C. Geraldes, and J. A. Peters, lnorg. Chem. 30, 3188 (1991). 14 A. Abraha, E. Dorus, and D. Mota de Freitas, Lithium 2, 118 (1991). is D. G. Davis, E. Murphy, and R. E. London, Biochemistry 27, 3547 (1988). ~6L. Wittenkeller, D. Mota de Freitas, C. F. G. C. Geraldes, and A. J. R. Tom6, lnorg. Chem. 31, 1135 (1992).

[4]

ALKALI METALNMR

83

the spin-lattice (l/T0 and spin-spin (l/T2) relaxation rates are equal and reflect a single exponential decay given by I/T 1 = I/T z = 3(2I + 3)X2Tc/4012(21 -- 1)

(1)

where X is the product of the quadrupolar coupling constant, eZqQ/h, and the asymmetry factor, 1 + 7/2/3 (eq is the electric field gradient, eQ is the electric quadrupole moment, and h is Planck's constant). In a homogeneous magnetic field, T2 is related to the line width at half-intensity of the signal, AVl/2, by

1/T2 = 'B'AI-~I/2

(2)

In most biological applications, the alkali metal nuclide is bound to a high molecular weight, slowly moving protein or intracellular component; the extreme narrowing condition no longer applies, resulting in a T~ value that is greater than the T2 value. For I = 1 (e.g., 6Li) the relaxation decay is still monoexponential, whereas for I = ½ (e.g., 133Cs) there are four components for the relaxation decay. 4 When I = ~ (which is the case of 7Li, 23Na, 39K, and 87Rb) and the relaxation and exchange times in the bound state are much shorter than those in the free state, the relaxation decay is biexponential. 4'5 The time dependences of the longitudinal, Mz(t), and transverse, MT(t), magnetizations for I = ~ nuclides can then be solved analytically and are given by Eqs. (3) and (4), respectively, 4'5'17 Mz(t) = Mz(0)[0.2 e x p ( - t / T l ' ) + 0.8 exp(-t/Tl")] MT(t) = MT(0)[0.6 exp(-t/T2') + 0.4 exp(-t/T2")]

(3) (4)

where the single- and double-primed symbols denote the relaxation times for the two components. T~', Tl", T2', and T2" are given by

1/T 1' = 1~Tie + PbX2/lO [7c/(1 + o32'/'c2)] 1/T1" = 1/Tlf + PbX2/lO [%/(1 + 4t02"rc2)]

(5) (6)

I / T 2' = 1/T2f + PbXZ'rc/20 [1 + 1/(1 + o~z~'c2)] 1/T2" = 1/T2f + PbXZ'rc/20 [1/(1 + 4o927"c2) + 1/(1 + ¢..o2"1-c2)]

(8)

(7)

where the subscripts f and b refer to the free and bound states. Equations (5)-(8) hold only when Pb, the mole fraction of bound ions, is very small compared to pf, the mole fraction of free ions in solution. Details of the limitations of the fast exchange two-site model for fitting relaxation data of nuclei with I = ~ have been described; an asymmetric continuous distribution of correlation times for the fluctuating electric field gradients 17D. W. Urry, T. L. Trapane, C. M. Venkatachalam, and R. B. McMichens, this series, Vol. 171,286.

84

PROBES OF METAL ION ENVIRONMENTS

[4]

experienced by the alkali metal nuclides in biological samples provides a better description of the relaxation behavior of I = ~ nuclei) 8'~9 A Lorentzian line shape is characterized by a Avl/8/Avv2 ratio of (7) 1/2, which is observed in the extreme narrowing condition. Equation (4) indicates that, in most biological samples in which it is observed, a nonLorentzian line shape can be deconvoluted into a narrow Lorentzian curve, owing to the slow relaxation component and accounting for 40% of the total signal intensity, and a broad Lorentzian curve, originating from the fast relaxation component and responsible for the remaining 60% of the total signal intensity.17 In some biological samples, the broad component may be indistinguishable from the baseline, resulting in only 40% visibility for the NMR resonance. The narrow component is associated with the +½ to -½ transition, whereas the broad component is due to the +~ to +½ and the -½ to -~ transitions. Unlike solution samples for which only one NMR resonance is generally observed, in micelle suspensions and in the solid state the ionic motion is anisotropic, and three transitions are sometimes observed for I = ~ nuclides.18

Use of Nuclear Magnetic Resonance Spectroscopy for Probing Alkali Metal Ion Environments in Metalloenzymes and Metalloproteins In addition to probing of the binding of alkali metal ions to membrane components 17 and DNA, 2° alkali metal NMR spectroscopy has also been used extensively for probing the environment of alkali metal ions in K ÷activated enzymes, such as pyruvate kinase 21 and Na ÷,K+-ATPase. 22 Although K ÷ can be determined directly by 39K NMR spectroscopy, when K ÷ is bound to a protein its observation is difficult because of short relaxation times. In contrast, because of the small quadrupole moments and the high receptivities of the 7Li and 133Cs nuclei, readily observable and narrow 7Li or 133Cs NMR resonances can be obtained by substitution of K ÷ by Li ÷ or Cs ÷ in K+-activated enzymes. The environment of the alkali metal ion and the competition with K ÷ in K÷-activated enzymes can be probed by means of 7Li or 133Cs (and sometimes 23Na) NMR relaxation measurements. 18 W. D. Rooney and C. S. Springer, Jr., N M R Biomed. 4, 209 (1991). 19 W. D. Rooney and C. S. Springer, Jr., N M R Biomed. 4, 227 (1991). 20 S. Padmanabhan, M. Paulsen, C. F. Anderson, and M. T. Record, Jr., in "Monovalent Cations in Biological Systems" (C. A. Pasternak, ed.), p. 321. CRC Press, Boca Raton, Florida, 1990. 21 j. M. Van Divender and C. M. Grisham, J. Biol. Chem. 260, 14060 (1985). 22 C. M. Grisham and W. C. Hutton, Biochem. Biophys. Res. Commun. 81, 1406 (1978).

[4]

ALKALI METAL N M R

85

22

20

18

o

16

v'-

i--

14

al

12

t

°

t A

13,

10

i

i

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i

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i

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i

2.5

Mg2+/mM FIG. 1.7Li T1values for Li-ATP (open symbols) and Li-ADP complexes (filled symbols) in the presence of increasing concentrations of Mgz÷. We obtained TI measurements by using the inversion recovery pulse sequence (D-180°-z-90°-AQ),, where D was at least 5T l , r is the interpulse delay which was arrayed between 0.3 and 20 sec in seven increments, n (4) is the number of scans, and AQ is the time (1 sec) during which the emitted radio frequency signals were collected. Nucleotide were as follows: circles, 3 mM; squares, 5 mM; and triangles, 7 mM. The Li + concentration was 5 mM in all samples. Each TI value is an average of two readings made on separately prepared samples. (Reprinted by permission of the publisher from Ref. 23. Copyright 1991 by Elsevier Science Publishing Co., Inc.)

Figure 1 illustrates how alkali metal NMR relaxation measurements can be used for probing alkali metal ion binding and competition with other metal ions for binding sites in biomolecules. Figure 1 shows the T~ values for 7Li in solutions of ATP or ADP containing both Li + and Mg 2ions. 23 Li ÷ ions are in fast exchange on the 7Li N M R time scale. The 23 A. Abraha, D. Mota de Freitas, M. M. C. A. Castro, and C. F. G. C. Geraldes, J. lnorg. Biochem. 42, 191 (1991).

86

PROBES OF METAL ION ENVIRONMENTS

[4]

observed 7Li chemical shift and T1values therefore represent the weighted average of free and bound Li + ions. Because the 7Li+ nucleus has a narrow chemical shift range, 5 the 7Li NMR chemical shifts are not sensitive to Li + binding to nucleotides. In contrast, 7Li T1relaxation times are sensitive to motion. Free Li + ions have large T1 values, whereas those that are tightly bound, if visible by 7Li NMR spectroscopy, have relatively small TI values. The T1 values for 7Li decreased in the presence of increasing concentrations of ATP and ADP (Fig. 1), confirming that 7Li T1 values are dependent on Li ÷ binding to adenine nucleotides. In the presence of increasing concentrations of Mg 2÷, the 7Li T1 values increased because of displacement of Li + by Mg 2+ from ATP- and ADP-binding sites. If simultaneous binding of Li + and Mg 2+ to ATP and ADP had occurred, the 7Li TI values in the presence of Mg 2+ would be the same as in its absence, but this was not observed. The example given in Fig. 1 is for a small biological ligand. In the case of large biomolecules, such as metalloproteins and metalloenzymes, the contribution of rr, the reorientation correlation time, toward ~c may complicate the analysis of NMR relaxation data. 17 With the exception of 133Cschemical shifts, alkali metal NMR chemical shifts are not very sensitive to changes in ligation, as mentioned above. Because the alkali metal ion is generally in fast exchange between the free and bound states, the observed chemical shift is a weighted average of the limiting chemical shifts for the free and bound forms. The low sensitivity of the NMR experiment requires that a large excess of the free hydrated metal ion be present. A comparison of the alkali metal NMR chemical shift data for protein samples with those obtained for aqueous samples of low molecular weight model compounds generally does not provide an unambiguous assignment of the alkali metal ion environment in metalloenzymes and metalloproteins. As for all spectroscopic studies using metal-substituted enzymes, it is also important to investigate, with independent biochemical methods, what is the influence of the metal replacement on the structure and function of K+-activated enzymes. Some Ca 2+- and Mg2+-activated proteins, such as calmodulin, 24 Ca2÷-ATPase, 25 parvalbumin, 26and phosphoglucomutase, 27were also investigated by 23Na NMR 24'26or 7Li NMR 25'27methods with the use of Na +- or Li+-substituted protein derivatives, respectively. 24 A. Delville, J. Grandjean, P. Laszlo, C. Gerday, H. Brzeska, and W. Drabikowski, Eur. J. Biochem. 109, 515 (1980). 25 E. M. Stephens and C. M. Grisham, Biochemistry 18, 4876 (1979). 26 C. Gerday, J. Grandjean, and P. Laszlo, FEBS Lett. 105, 3.84 (1979). 27 G. Rhyu, W. J. Ray, Jr., and J. L. Markley, Biochemistry 24, 2536 (1985).

[4l

ALKALIMETALNMR

87

Most K+-activated enzymes also contain Mg2÷ binding sites. Replacement of the diamagnetic Mg 2÷ ion by the paramagnetic Mn 2+ ion generally leaves the activity of the metaUoenzyme unchanged. Alkali metal NMR spectroscopy has therefore been used for measurements of the distances between the monovalent and divalent cations, and thus for mapping the active site structure of metalloenzymes.25,28'29The distance r between the alkali metal ion and the paramagnetic Mn 2+ center is calculated from the dipolar contribution to the relaxation process, which is given by the Solomon-Bloembergen equation: r 6 = 6 / 1 5 72g2f12S(S +

1)T1Mrc/(1 +

co2rc2)

(9)

where y is the gyromagnetic ratio, g is the electronic factor, fl is the Bohr magneton, S is the electronic spin of Mn 2÷, T1M is the paramagnetic contribution of the protein-Mn 2+ complex to the TI value of the alkali metal ion being investigated by NMR spectroscopy, r c is the correlation time, and co is the nuclear Larmor precession frequency. The values of the product of the constants in Eq. (9) for Mn2+-substituted proteins containing 6Li, 7Li, 23Na, 39K, 87Rb, or 133Cs are 1287, 1779, 1563, 876, 1680, and 1239, respectively. 29 TIM is calculated from

p(1/TIM ) = l/Tl(P-Mn ) - 1/TI(P_Mg ) - 1/T1Mn

(10)

where p is the mole fraction of alkali metal ion bound to the protein-Mn 2+ complex, 1/T1(P-Mn) and 1/TI(P-Mg) are the observed spin-lattice alkali metal NMR relaxation rates in the presence of protein-Mn 2+ and protein-Mg 2+ complex, respectively, and 1/Tli n is the contribution from free Mn 2+ in solution, which is calculated from the difference in T1 relaxation rates between control samples of alkali metal ions with and without Mn 2+ in the absence of protein. 29 The correlation time Tc can be estimated conveniently, with a single NMR spectrometer, from the ratio of T1M values for the two naturally occurring isotopes of lithium, 6Li and 7Li.28'29 Many K*-activated enzymes also require ATP as a substrate. The paramagnetic, kinetically inert analog Crm(ATP) has also been employed for measuring the distances between the alkali metal cation and the substrate binding site. 22 Detailed accounts of the methods available for measuring distances between monovalent and divalent cations in metalloenzymes and metalloproteins are available. 3° 2s F. M. Raushel and J. J. Villafranca, J. Am. Chem. Soc. 11}2, 6618 (1980). 29 F. M. R a u s h e l and J. J. Villafranca, Biochemistry 19, 5481 (1980). 30 j. j. Villafranca and F. M. Raushel, Annu. Rev. Biophys. Bioeng. 9, 363 (1980).

88

PROBES OF METAL ION ENVIRONMENTS

[4]

U s e of N u c l e a r M a g n e t i c R e s o n a n c e Spectroscopy for S t u d y i n g Alkali

Metal Ion Distribution anti Transport in Cell Suspensions and Perfused Organs For obtaining physiologically important information on the distribution and transport properties of alkali metal cations in cell suspensions or perfused organs by alkali metal NMR spectroscopy, a significant transmembrane difference in either the chemical shift or the relaxation rate of the nuclide of interest is required. Whereas a transmembrane difference in chemical shift can be achieved for the 6Li, 7Li, 23Na, 39K, and 87Rb nuclides only by use of a shift reagent (SR), a transmembrane mCs chemical shift difference can be observed even in the absence of SRs. Transmembrane differences in relaxation rates for all alkali metal nuclides are, however, present for most cell suspensions and perfused organs. The transport of an alkali metal cation across a cell membrane may be either slow or fast relative to the NMR time scale. Unlike the transmembrane differences in chemical shift that can be used only for monitoring of slow transport processes, the transmembrane difference in relaxation rates can be used for monitoring of both slow and fast transport processes. A practical description of the alkali NMR methods available for monitoring of slow and fast transport processes in tissue samples is given below.

Shift Reagent Method Negatively charged lanthanide SRs are insoluble in hydrophobic cell membranes and are repelled by the negatively charged head groups of phospholipids at the surface of cell membranes. Unless decomposition of the shift reagent occurs or the cell membrane is leaky, SRs remain in the extracellular compartment or suspension medium during the course of an NMR experiment. Because of the presence of a paramagnetic lanthanide ion in SRs, the extracellular alkali metal NMR resonance is subject to a pseudocontact shift and thus is separated from the intracellular signal. Details concerning the origin of pseudocontact shifts afforded by lanthanide SRs are given in a separate chapter of this volume. 31 The most popular lanthanide SRs used in conjunction with alkali metal NMR spectroscopy are Dy(TTHA) 3-, Tm(DOTP) 5-, and Dy(PPP)27-, where D y 3+ and T m 3+ denote dysprosium and thulium ions and ppps-, TTHA 6-, and DOTP 8- represent the ligands triphosphate, triethylenetetraminehexaacetate, and 1,4,7,10-tetrazacyclododecane-N,N',N",N"-tetrakis(methane phosphonate), respectively. The methods of preparation are given below. 31 C. F. G. C. Geraldes, this volume [3].

[4]

ALKALI METALNMR

89

Preparation of Dy(TTHA) 3-. For preparation of 5 ml of a 0.125 M stock solution of the Na ÷ form of Dy(TTHA) 3-, 0.2356 g of dysprosium chloride (Aldrich, Milwaukee, WI) is dissolved in 3 ml of water. Triethylenetetraminehexaacetic acid (H6TTHA; Aldrich) in powder form (0.3090 g) is added to the dysprosium chloride solution, which is then titrated during stirring with a total of 1.5 ml of a 2.5 M NaOH solution added in 30-/xl aliquots. 32'33 The pH must be maintained at less than 8.0 to avoid the formation of insoluble dysprosium hydroxide. For preparation of the tetramethylammonium, (CH3)4N +, form of the Dy(TTHA) 3- shift reagent, tetramethylammonium hydroxide instead of NaOH is used in the titration step. Preparation of Tm(DOTP)5-. After synthesizing the DOTP 8- ligand according to a published procedure, 34 the Tm 3+ complex is prepared by mixing a 10-30 mM solution of DOTP 8-, whose pH has been adjusted to the range 8-9 with ammonium hydroxide, with 1 equivalent of thulium acetate (Aldrich) at 80°. The mixture is dried on a rotary evaporator, and the ammonium acetate impurity in the white solid product is removed by using vacuum sublimation. 35 Preparation ofDy(PPP)27- . Sodium triphosphate (Aldrich) is recrystallized three times from 40% (v/v) ethanol. A 30 mM stock solution of the Na* form of the Dy(PPP)27- shift reagent is prepared from dysprosium chloride (Aldrich) and purified sodium triphosphate in a ratio of 1 : 2.5. Purified sodium triphosphate (75 raM) is added dropwise to a 30 mM dysprosium chloride solution until complete formation of the complex is achieved, as indicated by the clarity of the solution. It is important to use a ligand-to-lanthanide ratio larger than 2 to ensure complete complex formation. 36 The (CH3)4N+ and K + forms of this SR are useful for certain ion transport studies; the bulky tetramethylammonium cation minimizes the competition with the alkali metal cations being studied. The tetramethylammonium and K + forms o f sodium triphosphate are obtained by passage of sodium triphosphate through a column packed with Dowex 50W (Sigma, St. Louis, MO), which was presaturated with (CH3)4NC1 or KC1, respectively. The preparation of the (CH3)4N+ and K + forms of the 32 S. C. Chu, M. M. Pike, E. T. Fossel, T. W. Smith, J. A. Balshi, and C. S. Springer, Jr., J. Magn. Reson. 56, 33 (1984). 33 M. M. Pike, D. M. Yarrnuch, J. A. Balshi, R. E. Lenkinski, and C. S. Springer, Jr., lnorg. Chem. 22, 2388 (1983). 34 A. D. Sherry, C. R. Malloy, F. M. H. Jeffrey, W. P. Cacheris, and C. F. G. C. Geraldes, J. Magn. Reson. 56, 33 (1984). 35 D. C. Buster, M. M. C. A. Castro, C. F. G. C. Geraldes, C. R. Malloy, A. D. Sherry, and T. C. Siemers, Magn. Reson. Med. 15, 25 (1990). 36 p. j. Brophy, M. K. Hayer, and F. G. Riddell, Biochem. J. 210, 961 (1983).

90

PROBES OF METAL ION ENVIRONMENTS

[4]

Dy(PPP)z 7- SR is the same as for the Na + form of the SR, as mentioned above. The residual amount of Na + in the tetramethylammonium and K + forms of the Dy(PPP)2 7- SR is determined by AA spectrophotometry. The use of the SR method for investigating Li + and Cs + distribution and transport in biological samples is illustrated in Figs. 2 and 3 for Li ÷loaded and Cs+-loaded RBC suspensions. Figure 2A shows that the intraand extracellular 7Li+NMR resonances are not resolved in an RBC suspension that does not contain a shift reagent. This observation is consistent with Li + existing mostly as free hydrated ions in both cellular compartments, and with the 7Li nucleus in ionic environments having a narrow chemical shift range that is insensitive to changes in ligation. Figure 2B, C shows that discrimination between the intra- and extracellular 7Li+ NMR resonances in RBC suspensions can, however, be accomplished by incorporation in the suspension medium of a cell membrane-impermeable SR, such as Dy(PPP)27- or Dy(TTHA)3-. 37'38 Because of differences in overall negative charge, the shift induced by Dy(PPP)27- is larger than that induced by Dy(TTHA) 3- at the same concentration.3S Whereas Dy(PPP)Jinduces an upfield pseudocontact shift of the extracellular 7Li+ NMR resonance, Dy(TTHA) 3- induces a downfield shift. 38 The opposite signs of the pseudocontact 7Li + shifts induced by Dy(PPP)27- and Dy(TTHA) 3are due to the different locations of the Li + cation relative to the cones around the effective magnetic axes of these SRs. 13 SRs have also been used for discriminating between the chemical shifts ofintra- and extracellular 6Li, 23Na, 39K, and 87RbNMR resonances in RBC suspensions. 10,14,36,39 In contrast to the requirement for SRs in the medium or perfusate to obtain chemical shift separation of intra- and extracellular 6Li+, 7Li ÷, 23Na+, 39K+,and S9Rb+ NMR resonances, Fig. 3A shows that SRs are not needed for separation of the chemical shifts of intra- and extracellular 133Cs+ NMR resonances in Cs+-loaded RBC suspensions. J5,16The physical basis for the resolution of intra- and extracellular 133Cs+ NMR resonances arises from Cs + binding to intracellular phosphates, in particular 2,3diphosphoglycerate, and from the nonideality of intracellular water induced by hemoglobin. 16The positions of the intra- and extracellular mCs + NMR resonances in the presence of SRs (Figs. 3B-D) were inverted relative to the positions when no SR was used (Fig. 3A). The mCs + shift induced by Tm(DOTP) 5- (Fig. 3B) was much larger than those for 37 M. T. Espanol and D. Mota de Freitas, lnorg. Chem. 26, 4356 (1987). 38 R, Ramasamy, M. T. Espanol, K. M. Long, D. Mota de Freitas, and C. F. G. C. Geraldes, Inorg. Chim. Acta 163, 41 (1989). 39 j. L. Allis, R. M. Dixon, A. M. Till, and G. K. Radda, J. Magn. Reson. 85, 524 (1989).

[4]

ALKALI METAL N M R

91

A

B

C

IIII[|IHIH H ] l H l l I H l [ H I q t I l l ] H H I H l l [ H I q l l l l [ l l t l l l l l l l H HH t Hilt HIIHC[ Hlqt HI]lH q

6

4

2

0

-2

-4

-6

-8

-10

-12

ppm FIG. 2. (A) 7Li NMR (116.5 MHz, 37°) spectrum of packed RBCs suspended in a medium containing 3.5 mM LiCI, 140 mM NaC1, 5 mM KCI, 10 mM glucose, and 50 mM HEPES, pH 7.4. Packed RBCs were incubated with 150 mM LiCI at 37° for 12 hr prior to NMR measurements. D20 (17%) was present in the medium for frequency lock. The hematocrit was 13%. (B) 7Li NMR spectrum of the same RBC suspension as in (A), except that 3 mM NaTDy(PPP)2" 3NaCI was present in the medium instead of 30 mM NaCI. (C) 7Li NMR spectrum of the same RBC suspension as in (A), except that 7 mM Na3Dy(TTHA) was present in the medium instead of 21 mM NaCI. The 7Li NMR spectra were obtained with a single pulse sequence, (D-P-AQ),, where D [6.5 sec, except for (A) where it was 50 sec] is the delay between successive signal acquisitions, P is the radio frequency excitation pulse (45°), AQ is the time (1.0 sec) during which the emitted radio frequency signals were collected, and n (8) is the total number o f scans taken for each spectrum. The total accumulation time was approximately 1 min except for (A), which took 7 min.

92

PROBES OF METAL ION ENVIRONMENTS

[4]

A

C

l's ....

o

1'o . . . .

~ ....

6 ....

-~"

:io . . . .

ppm FIG. 3. 133CsNMR (39.4 MHz, 37°) spectra. (A) Cs+-loaded carbon monoxygenatedRBCs suspended at 33% hematocrit in an isotonic medium containing 10 mM CsCI, 140 mM NaCI, 5 mM glucose, and 5 mM HEPES, pH 7.4. (B) RBC suspensions similar to that in (A) at 40% hematocrit, except that 5.0 mM Tm(DOTP) 5- replaced 25 mM NaCI in the medium. (C) RBC suspension similar to that in (A) at 33% hematocrit, except that 5.0 mM Dy(TTHA) 3replaced 15 mM NaCI in the medium. (D) RBC suspension similar to that in (A) at 45% hematocrit, except that 5.0 mM Dy(PPP)27- replaced 50 mM in the medium. The symbols i and o denote intra- and extracellular mCs+ NMR resonances. The 133CsNMR spectra were obtained with a single pulse sequence, (D-P-AQ),,, where D (25 sec) is the delay between successive signal acquisitions, P is the radio frequency pulse (54°), AQ is the time (1.0 sec) during which the emitted radio frequency signals were acquired, and n (1000) is the total number of transients. (Reprinted with permission from Ref. 16. Copyright 1992 American Chemical Society.)

[4]

ALKALI METAL

NMR

93

TABLE II INTRA- AND EXTRACELLULARTI VALUES IN RED BLOOD CELL SUSPENSIONSWITHOUT SHIFT REAGENT Nucleus

Tt(in)

Tl(out)

[M+]in (mM)

[M+]out (mM)

Ref.

6Li 7Li 23Na 39K 87Rb mCs

20 sec 5.4 sec 35 msec 24 msec 0.9 msec 4.5 sec

120 sec 16.5 sec 69 msec 75 msec 3 msec 13.6 sec

10 1.0 10 130 1-8 0.3-3.0

100 3.5 75 75 10 10

a 40 41 41 39 15

D. Mota de Freitas and A. Abraha, unpublished observations (1991).

Dy(TTHA) 3- (Fig. 3C) and Dy(PPP)27- (Fig. 2D) at the same concentration. The negative sign of the 133Cs+ shift induced by Dy(PPP)27- is the opposite of that observed for 6Li +, 7Li ÷, 23Na+, 39K+, and 87Rb+ NMR resonances and is associated with the large size of the Cs + ion and its location in the equatorial region formed by the cone around the effective magnetic axis of the Dy(PPP)z7- SR. 16

Modified Inuersion Recovery Method Table II shows the intra- and extracellular alkali metal NMR T~ values for RBC suspensions not containing a shift reagent. Because of increased intracellular viscosity and binding of alkali metal ions to intracellular components, the intracellular T~ values of alkali metal NMR resonances are, in general, lower than the corresponding extracellular T1 values in cell suspensions or perfused organs in the absence of an SR. The transmembrane difference in T~ values is the basis for the application of the modified inversion recovery (MIR) pulse sequence, (DI-180°-Dz60°-AQ),, where Dl and D 2 are delays between successive pulses, AQ is the time during which emitted radio frequencies were acquired, and n is the total number of scans, for eliminating the extracellular alkali NMR resonance and selectively observing the intracellular NMR resonance. 40,42 40 D. Mota de Freitas, M. T. Espanol, R. Ramasamy, and R. J. Labotka, lnorg. Chem. 29, 3972 (1990). 41 O. Burstein and E. T Fossel, J. Magn. Reson. 73, 150 (1987). 42 y . Seo, M. Murakami, E. Suzuki, and H. Watari, J. Magn. Reson. 75, 529 (1987).

94

PROBES OF METAL ION ENVIRONMENTS

[4]

A

13

D

"l~m,~l

40

JmJml

35

30

J,,

,I

25

Jm,~l

J~,+l~,,~In,,,

20

15

10

I,,~,

I

5

0

ppm FIG. 4. (A) 7Li NMR (116.5 MHz, 37°) spectrum of Li+-loaded RBCs ([Li+]in = 1.0 mM) suspended at 13% hematocrit in a medium containing 3.5 mM LiCI, 5 mM KCI, 140 mM NaC1, 10 mM glucose, and 50 mM HEPES, pH 7.5. A single pulse sequence, (D-60°-AQ), was used, consisting of a repetition rate D of 60 sec and a flip angle of 60° before spectral acquisition AQ of 1 sec. The spectrum represents the two overlapped intra- and extracellular 7Li÷ NMR resonances. (B) 7Li NMR spectrum of the same RBC suspension as in (A), except that an MIR pulse sequence, (D+-180°-D2-AQ), was used. The values of DI and D2 were 60 and 11.5 sec respectively. The spectrum represents the intracellular 7Li+ NMR resonance. (C) Computer-generated difference (C = A - B) spectrum, which represents the extracellular 7Li+ NMR resonance. (D) 7Li NMR spectrum of the Li+-contalning suspension medium (no RBCs), using the same MIR method as in (B). A total of 8 scans were taken for each spectrum with a total accumulation time of approximately 9.7 min, except for spectrum (A), which took 8 min. (Reprinted with permission from Ref. 40. Copyright 1990 American Chemical Society.)

Figure 4 illustrates the use of the MIR method for the study of the t r a n s m e m b r a n e d i s t r i b u t i o n o f L i + in L i + - l o a d e d R13C s u s p e n s i o n s in t h e a b s e n c e o f a n S R . T h e s t a n d a r d o n e - d i m e n s i o n a l 7Li N M R s p e c t r u m o f t h e R B C s u s p e n s i o n s h o w n in F i g . 4 A c o n t a i n s o n l y o n e s i g n a l , w h i c h

[4]

ALKALI METAL N M R

95

100

[]

0

ooc~oo

O

O A

0

.z, 50

&

A

A

0

c 0

0 a On

.~_ ¢-

A

100'

O

r-

0 a

-

A &

O []

.3

& 50

A., .8

<> o <>

0-

0

<> 0

<> a

A

0

oo ° 0 0

o []

O<>a

A &&A

A

<> &

-50

,

0

,

2

,

,

4

,

,

6

,

,

8

,

,

,

•

,

,

,

10 12 14 16

-50 0

20 ,

40 ,

d0

1O0 '

120 ,

D2 Delay (see) FIG. 5. Plot of 7Li+ NMR resonance of Li÷-ioaded RBCs in a Li+-free medium (<>), Li ÷free RBCs suspended in a Li÷-containing medium (A), and Li+-loaded RBCs in a Li +containing medium ([]) as a function of the delay time D 2, with the MIR pulse sequence. The other 7Li NMR parameters were the same as for Fig. 4B. The Li+-free medium contained 112.5 mM choline chloride, 10 mM glucose, 85 mM sucrose, 0.1 mM ouabain, and 10 mM Tris-C1, pH 7.5, and the Li+-containing medium was made up of 3.5 mM LiCI, 5 mM KCI, 140 mM NaCI, 10 mM glucose, and 50 mM HEPES, pH 7.5. (Reprinted with permission from Ref. 40. Copyright 1990 American Chemical Society.)

represents the overlapped intra- and extracellular 7Li+ NMR resonances, and which was recorded with the same pulse sequence as that shown in Fig. 2A. Figure 4B shows the 7Li NMR spectrum of the intraceUular 7Li ÷ NMR resonance obtained by the MIR method. Figure 5 shows the effect

96

PROBES OF METAL ION ENVIRONMENTS

[4]

of varying D2 on the 7Li+ NMR resonance intensities of Li÷-free RBCs in a Li+-containing medium, Li+-loaded RBCs in a Li+-free medium, and Li+-loaded RBCs in a Li+-containing medium. When D2 was I 1.5 sec, the extracellular 7Li+ signal for Li+-free RBCs in a Li+-containing medium (triangles, Fig. 5) is nulled, whereas the intracellular 7Li÷ signal for Li +loaded RBCs suspended in a Li+-free medium (diamonds, Fig. 5) has reached 81% of its maximum intensity. Therefore, the extracellular 7Li+ NMR resonance in Li+-loaded RBCs suspended in a Li+-containing medium (squares, Fig. 5) was nulled in the MIR experiment when D z was 11.5 sec (Fig. 4B). The 7Li+ NMR resonance observed with the MIR pulse sequence in Fig. 4B was therefore due to intracellular Li + only, and not to a combination of intra- and extracellular 7Li+ NMR resonances. For a D2 value of 11.5 sec and after a 60° pulse was applied, the magnetization component of extracellular 7Li÷ along the axis of signal detection is negligible, and its signal disappears. This conclusion was confirmed by the absence of a 7Li NMR signal for a Li+-containing medium (no RBCs) with the MIR pulse sequence (Fig. 4D). The computer-generated difference spectrum shown in Fig. 4C represents the extracellular 7Li+ NMR resonance. The MIR method has also been applied for discriminating between the intra- and extracellular 39K+NMR resonances in perfused salivary glands in the absence of an SR. 42 It should be possible to extend the MIR method to other cell suspensions and perfused organs in which a transmembrane difference in T1 values (see Table II) exists, by setting the D 2 delay to a value equal to In 2 × Tl(out). H The experimentally determined value of D2 (11.5 sec) that resulted in nulling of the extracellular 7Li+ NMR resonance was in excellent agreement with the theoretical value (11.4 sec). Magnetization Transfer Method For slow alkali metal ion transport processes in which the rate of metal ion transport is much slower than the alkali metal NMR relaxation times (e.g., Li ÷ uptake or Li+in-Na+out exchange in human RBC suspensions), 14'4° the rates of metal ion transport can be calculated from the timedependent intensity changes of the alkali metal NMR resonances measured by either the SR or the MIR method. However, for alkali metal ion transport processes that take place on a time scale that is comparable to the alkali metal NMR relaxation times (e.g., ionophore-induced Li ÷ or Na ÷ transport in vesicle suspensions), 43,44 one can calculate the rates of metal 43 F. G. Riddell, S. Arumugam, and B. G. Cox, J. Chem. Soc., Chem. Commun., 1890 (1987). 44 F. G. Riddeil and S. Arumugam, Biochim. Biophys. Acta 945, 65 (1988).

[4]

ALKALIMETALNMR

97

ion transport by using the magnetization transfer method.45 After inversion of the extracellular alkali metal NMR resonance, which was shifted by d Hz from the intracellular signal by use of an SR, the inverted magnetization is transferred during a variable interpulse delay z via chemical exchange to the intracellular alkali metal NMR resonance, resulting in a decrease of its intensity. The pulse sequence 90°-q-90°-z-90°-AQ-D~ is used with the transmitter set at 2d and d Hz, respectively, downfield from the extraand intracellular alkali metal NMR resonances, with tl set at 1/2d and D~ equal to 5Tl(in). 43

Other Methods For some ionophore-induced alkali metal ion transport processes, the time scale of metal ion transport is faster than the alkali metal NMR relaxation times,46 and the rates of metal ion transport can be calculated from the broadening of the intracellular alkali metal NMR resonance, which has been shifted from the corresponding extracellular resonance by use of an SR. A double-quantum coherence transfer pulse sequence has also been proposed for the selective observation of intracellular alkali metal NMR s i g n a l s f The pulse sequence is 90°-r/2-180 °r/2-90°-8-90°-AQ, where z is the preparation time and 8 is the evolution time during which a 64-step phase cycle is employed for selecting for double-quantum coherence. This sequence is selective for nuclei subject to biexponential T2 relaxation, which is the case of most alkali metal nuclides in intracellular compartments. The usefulness of this method for monitoring alkali metal ion transport processes is limited by its poor sensitivity and by the nonlinear relationship between the double-quantum signal intensities and intracellular alkali metal ion concentrations. H Information Obtained from Alkali Metal Nuclear Magnetic Resonance Spectra Alkali metal T~relaxation measurements can be used for the calculation of binding constants of an alkali metal cation to a metalloprotein or metalloenzyme. J7 The calculation of the binding constant, Kb, of an alkali metal cation M + to a protein or enzyme assumes a two-state model (free and bound metal ions) undergoing fast exchange and a total alkali metal ion concentration, [M+], that is large with respect to the binding site concentration, [B]. Under these conditions, Eq. (11) is valid: 45 G. A. Morris and R. Freeman, J. Magn. Reson. 29, 433 (1978). 46 F. G. Riddell and M. K. Hayer, Biochirn. Biophys. Acta 817, 313 (1985). 47 j. Pekar, P. F. Renshaw, and J. S. Leigh, J. Magn. Reson. 72, 159 (1987).

98

PROBES OF METAL ION ENVIRONMENTS

14]

AR-I = (Rob s -- Rfree)-l = Kb{[B](Rbou,d _ Rfr¢¢)}_1 + [M+]{[B](Rboun ~ _ Rfre~)}_l

(11)

where Robs , Rfr~e, and Rbound are the reciprocals of Tl(obs), Tl(free) , and Tl~bound), respectively. One can obtain K b from a linear plot of AR -1 versus [M +] by taking either the ratio of the slope by the y-intercept or the reciprocal of the x-intercept. When one strong and one weak binding site are present in the metalloprotein, the plot of AR -1 versus [M ÷] is a curve; the slope at low [M +] concentration is used for calculating the strong binding constant, whereas the limiting slope at high [M +] concentration is used for determining the weak binding constant. Methods of analysis for interpretation o f alkali metal N M R relaxation data involving multiple binding sites in metalloproteins are also available. 17 Because ~33Cs N M R chemical shifts are also sensitive to changes in ligation, the binding constant characterizing the interaction between Cs + in fast exchange with a metalloprotein or metalloenzyme can also be obtained from Eq. (12): l/A8 = I/(8obs -

8free) =

I/{Kb[CS+](Sbound - -

8free)} +

l/(Sbound - -

8ohs = XfreeSfree+ XboundSbound

8free)

(12) (13)

where Xfree and Xbound are the mole fractions of free and bound Cs + ions, respectively, a n d 8obs, 8fre,, and 8bound are the chemical shifts of the observed, free, and limiting bound 133Cs+ N M R resonances. Equation (12) can also be used for any other alkali metal nuclide for which appreciable changes in chemical shift a c c o m p a n y metal ion binding to a metalloprotein or metalloenzyme. Alkali metal T l measurements can also be used for calculating the distances between the alkali metal cation and a paramagnetic metal ion or a paramagnetic substrate analog in a metalloprotein or metalloenzyme from Eqs. (9) and (10). F r o m the correlation time ¢c, the rates of association and dissociation of an alkali metal ion to a biomolecule can also be calculated, as described previously. 17 Intra- and extracellular alkali metal ion concentrations, [M+]in and [M+]out, expressed in millimoles o f M + per liter of cells or tissue, in cell suspensions and perfused organs containing an SR in the medium can be calculated from Eqs. (14) and (15): [M+]in

[M+]out =

=

Ain[M+]s/(AsCtu)

Aout[M+]s/{As(1

-

Ct)}

(14) (15)

where Ain and Aout are the peak areas under the intra- and extracellular alkali metal N M R resonances which are obtained by means of the integration routines included in the software provided by the manufacturers o f

[4]

ALKALI METALNMR

99

all commercial NMR spectrometers, [M+]s and As are the known concentration and peak area of a standard alkali metal ion solution which is measured separately, Ct is the cell volume which can be measured with a Coulter counter (Model ZM, Coulter Electronics, Hialeah, FL) or, in the special case of red blood cells, with a microhematocrit unit (Model IEC, MB, Needham Heights, MA), and v is the visibility factor. The visibility factor for the intracellular alkali NMR resonances may sometimes be less than 1.0 and is generally obtained by comparison of the concentrations measured by NMR spectroscopy with those measured with an independent technique, such as atomic absorption. 4° Multiple-pulse NMR experiments can also be used for determining the visibility factor. J8.J9 Alternatively, the intracellular water content can be determined by 23Na and 2H NMR methods. I° Intracellular alkali metal ion concentrations, expressed in millimoles of M ÷ per liter of cell water, are obtained by dividing the concentrations obtained from Eq. (14) by fw, the ratio of the volume of intracellular water to the total cell volume. Because the MIR-derived intracellular NMR resonances have undergone only partial relaxation, a correction factor must be introduced in the denominator of Eq. (14) for calculation of MIR-derived intracellular alkali metal ion concentrations. In the case of 7Li MIR spectra of Li+-loaded RBC suspensions (Fig. 4B), the correction factor is 0.81. The correction factor, c, to be used in the calculation of MIR-derived intracellular alkali metal ion concentrations can, in general, be obtained from Eq. (16): c = 1 - 2 exp(-~'/T 1)

(16)

where ~- is the sum of the interpulse delay and acquisition time used in the MIR pulse sequence to null the extracellular NMR resonance, and T~ is the spin-lattice relaxation time of the intracellular NMR resonance of the alkali metal cation under investigation. In addition to obtaining information on the transmembrane distribution of alkali metal ions in cell suspensions and perfused organs, one can also use alkali metal NMR spectroscopy to calculate rates and rate constants for transmembrane ion transport. 9-11'14'4° The use of 7Li and 23Na NMR spectroscopy for monitoring ionophore-induced alkali metal ion transport in human RBC suspensions is shown in Fig. 6. 48 Whereas the peak areas of the intracellular 7Li + and 23Na+ NMR resonances increased appreciably within 75 min in the presence of the ionophore dibenzyl-14-crown-4 (DBI4C4), there were no significant changes in its absence. Alkali metal ion transport induced by DB 14C4 is first-order with respect to both metal ion and ionophore concentrations and is therefore second-order overall. 48 48 A. A b r a h a and D. Mota de Freitas, Lithium 3, 203 (1992).

100

PROBES OF METAL ION ENVIRONMENTS

[4]

A 67.5

..

.37.5 7.5 ,

2

. . . . .

. . . . ,

_ _ ~ .

0

.

.

.

,

. . . . . . . . .

-2

,

. . . . . . . .

-4 ppm

,

.

-6

.

.

.

.

.

.

.

4

2

0

-2

-4

-6

-8

-10 -12

ppm

FIG. 6. 7Li (A) and 23Na (B) NMR spectra of human RBC suspensions at 37° in the presence of the Li÷-selective ionophore dibenzyl-14-crown-4 (at 0.30 mM concentration) at 7.5, 37.5, and 67.5 min and in its absence at 67.5 min (the three lower spectra are for samples containing ionophore, whereas the top spectrum is for a sample without ionophore). RBCs were suspended at 26% hematocrit in 140 mM NaCl (SR contribution included), 5.0 mM LiCl, 5.0 mM Na7Dy(PPP)2, 0.1 mM ouabain, 0.1 mM 4,4'-diisothioeyanostilbene-2,2'disulfonate (DIDS), 10 mM glucose, and 10 mM HEPES, pH 7.4. The transport inhibitors ouabain and DIDS were present in the suspension medium to prevent Na ÷ and K ÷ transport via Na÷,K+-ATPase and Cl- transport via the anion-exchange protein. The osmolarity of the medium was maintained at 295 -+ 5 mOsM. The intracellular 7Li+ and 23Na NMR resonances are at 0 ppm, whereas the corresponding extracellular signals are upfield. 7Li and 23Na single-pulse NMR spectra were obtained after 56 and 900 scans, respectively, each requiring a total of 15 min. Each spectrum represents the midpoint for each 15 rain of accumulation time extending over 75 min. The radio frequency excitation pulses were 60°, the acquisition time was l sec for both 7Li and 23Na NMR spectra, and the interpulse delay was 16 sec for the 7Li NMR spectra. No delay was used for the 23NaNMR spectra. (Reprinted with permission from Ref. 48.) I n Fig. 6, the D B 1 4 C 4 c o n c e n t r a t i o n w a s k e p t c o n s t a n t , a n d t h u s the i o n o p h o r e - i n d u c e d alkali m e t a l i o n t r a n s p o r t follows p s e u d o - f i r s t - o r d e r k i n e t i c s . T h e p s e u d o - f i r s t - o r d e r rate c o n s t a n t s , k, w e r e d e t e r m i n e d f r o m the slopes o f the plots o f ln([M+]t/[M+]o) v e r s u s time b y l i n e a r r e g r e s s i o n a n a l y s i s . T h e k v a l u e s for D B l 4 C 4 - i n d u c e d t r a n s p o r t o f Li ÷ a n d N a ÷ in h u m a n R B C s u s p e n s i o n s w e r e 0.15 a n d 0.12 hr -1, with a L i ÷ / N a ÷ selectivity r a t i o o f 1.2. 48 B y u s i n g alkali m e t a l N M R s p e c t r o s c o p y , , w e h a v e t h e r e f o r e s h o w n t h a t the i o n o p h o r e D B 1 4 C 4 is a n efficient c a r r i e r for Li ÷ a c r o s s h u m a n R B C m e m b r a n e s e v e n in the p r e s e n c e o f a large e x c e s s o f N a ÷ i o n s , the s i t u a t i o n t h a t is p r e s e n t in sera o f m a n i c - d e p r e s s i v e p a t i e n t s receiving lithium carbonate.

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ALKALI METAL N M R

101

The ionophore-induced alkali metal ion transport shown in Fig. 6 was slow relative to the NMR time scale, and therefore rate constants could be obtained conveniently by monitoring the time dependence of the peak areas of the alkali metal NMR resonances. When the rate of transmembrane ion exchange is comparable to the alkali metal NMR relaxation rates, however, the magnetization transfer method must be used. The time dependence of the decrease in intensity of the noninverted intraceUular NMR signal is used for calculation of the rate of alkali metal ion transport, as described in detail elsewhere. 45 In contrast, when the rate of transmembrane alkali metal ion exchange is much faster than the alkali metal NMR relaxation rates, the rate constants k are given by k = rrLB

(17)

where L B is the line broadening induced by fast alkali metal ion exchange. This method was used for calculation of the k values of monensin-induced Na + efflux in phosphatidylcholine vesicle suspensions by 23Na NMR spectroscopy .46 Alkali metal NMR T1 and T2values have also been used for investigating the binding of alkali metal ions to cell membranes. 17'49-51 For example, in Li+-loaded RBCs ([Li+]i, = 1.5 raM), the 7Li T2 values, 90 +- 10 msec, were significantly shorter than the T1 values, 5.6 -+ 0.6 s e c . 49'50 Whereas the difference between intracellular RBC 7Li + Tl and T2 values was large, no such difference was found with a 1.5 mM LiC1 aqueous solution, the viscosity of which was adjusted to that of intracellular RBC with glycerol ( T l = 1.0 --- 0.1 sec; T 2 = 0.8 --- 0.1 s e c ) . 49 In contrast, Li+-loaded RBC ghosts with 1.5 mM Li + concentration also have longer Tl (12.1 -+ 1.1 sec) than T2 values (0.16 - 0.01 sec). 5° No other large differences between T1 and T2 values were observed for the other major anionic RBC components, the spectrin-actin matrix, hemoglobin, or the ATP-Mg z- complex. Slow motions contribute only to T2 values, whereas components of motion at the resonance frequency contribute to both 7"1 and T2 values. Thus, the high intracellular viscosity decreased both 7Li* TI and Tz values but was not responsible for the large difference between T1 and T2 values observed in packed Li+-loaded RBCs. Because the same relaxation behavior was observed in RBC ghosts and intact RBCs, the large difference between intracellular 7Li ÷ Tl and T2 values was attributed to the binding of Li ÷ to 49 j. W. Pettegrew, J. F. M. Post, K. Panchalingam, G. Withers, and D. E. Woessner, J. Magn. Reson. 71, 504 (1987). 50 D. Mota de Freitas, A. Abraha, Q. Rong, S. Mo, and L. Wittenkeller, J. lnorg. Biochem. 43, 386 (1991). 51 D. W. Urry, T. L. Trapane, S. K. Andrews, M. M. Long, H. W. Overbeck, and S. Oparil, Biochem. Biophys. Res. Commun. 96, 514 (1980).

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PROBES OF METAL ION ENVIRONMENTS

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the RBC membrane. 5° Therefore, 7Li NMR relaxation times constitute a good probe for obtaining information on Li ÷ interactions with the RBC membrane; this may prove to be useful in elucidating the abnormal Li + transport properties in RBCs, and possibly other tissues, of manic-depressive patients. 23Na NMR relaxation times have also been used successfully for probing of the abnormal binding of Na + to RBC membranes of hypertensive patients. 5~

Precautions When Using Alkali Metal Nuclear Magnetic Resonance Spectroscopy in Biological Applications The NMR relaxation times are much shorter when an alkali metal nuclide binds strongly to a metalloprotein or a metalloenzyme, resulting in a very broad or even an NMR-invisible bound component. ~7 Free and bound alkali metal ions are generally in the fast exchange domain relative to the NMR time scale, and a large excess of metal ion to protein is used to enhance the sensitivity of alkali metal NMR experiments; the alkali metal NMR resonance observed under these conditions is narrow because of the large contribution of free metal ions. Measurements of T~, as opposed to line width, are generally preferred for calculating binding constants of alkali metal ions to proteins or for the determination of distances between diamagnetic monovalent and paramagnetic divalent cations at the active sites of metalloenzymes because they are not subject to magnetic field inhomogeneity effects or exchange broadening. 3° Because Na + and K + ions are naturally present at relatively high concentrations in cells or organs, no tissue Na + or K + loading is necessary for obtaining Z3Na or 39K NMR spectra. The sensitivity of the 7Li nucleus is such that Li + levels of tissues from manic-depressive patients receiving lithium carbonate (which are normally in the 0.2-0.5 mM range) can also be monitored by 7Li NMR spectroscopyfl2 Rb + and Cs + are present only in trace amounts; therefore, they can be investigated by 87Rb or ~33Cs NMR spectroscopy only if the tissue is first loaded to approximately 0.1 mM. 15,16,39Because of the relatively poor sensitivity of NMR spectroscopy, high cell densities are needed. Cell or organ viability during the course of the NMR experiment is maintained by use of a perfusion system that provides nutrients and oxygen and also removes waste products. Details of methods for obtaining high-density cell cultures and perfusion systems suitable for NMR experiments are available. 53 5z D. Mota de Freitas, J. Silberberg, M. T. Espanol, E. Donas, A. Abraha, W. Donas, E. Elenz, and W. Whang, Biol. Psychiatry 28, 175 (1990). 53 B. S. Szwergold, Annu. Rev. Physiol. 54, 775 (1992).

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103

Metal competition and pH studies 32'35'38have shown that Dy(TTHA) 3and Tm(DOTP) 5- are the most promising shift reagents for 7Li, 23Na, and 39K NMR ion transport investigations in that they interact weakly with the alkali metal cation and yet induce relatively large pseudocontact shifts that are independent o f p H in the physiological pH range. Also, these SRs are, in general, less susceptible to competition from other physiologically relevant monovalent and divalent cations. In the case of Tm(DOTP) 5-, however, Ca 2÷ can appreciably bind to the SR, resulting in a decrease in the alkali metal NMR pseudocontact shift. Because Ca 2+ is essential for the viability of perfused organs, extra Ca 2+ must be added to a peffusate containing Tm(DOTP) 5- so that the free Ca/+ concentration is maintained. 35 Because of the high negative charges of SRs, the amounts of extracellular alkali metal ions bound to a given SR may be appreciable and may result in SR-dependent changes in the transmembrane alkali metal ion distribution in cell suspensions and perfused organs. By comparing the SR method with the MIR and AA methods, which do not require SRs, we found that the transmembrane Li + and Na + ratios in Li÷-loaded RBC suspensions measured in the presence of Dy(PPP)27- were different from those measured in the presence of Dy(TTHA) 3- or in the absence of SR. 4° We also found that the rates of Li+~n-Na+out exchange in RBC suspensions measured by 7Li NMR spectroscopy in the presence of Dy(PPP)J- were significantly higher than the rates measured in the absence of SRs by the MIR or AA methods or by 7Li NMR spectroscopy in the presence of Dy(TTHA) 3- or Dy(DOTP) 5 .54 Maintenance of the potential across the cell membrane during the course of an NMR experiment is essential for ensuring cell or tissue viability. 31p and ~9F NMR methods exist for measuring the membrane potential. 55'56 By using both of these NMR methods, we found that, in Li+-free RBC suspensions, the membrane potentials measured in the presence of either 5 mM Dy(PPP)27- or 5 mM Dy(DOTP) 5- were significantly less negative than those measured in the presence of 7 mM Dy(TTHA) 3 or in the absence of SR. 54 The Dy(PPP)27- SR was shown to undergo decomposition in perfused rat muscle; the ligand triphosphate is believed to be hydrolyzed by membrane phosphatases. 57 Free Dy 3÷

54 R. Ramasamy, D. Mota de Freitas, W. Jones, F. Wezeman, R. Labotka, and C. F. G. C. Geraldes, Inorg. Chem. 29, 3979 (1990). 55 K. Kirk, P. W. Kuchel, and R. J. Labotka, Biophys. J. 54, 241 (1988). 56 R. E. London and S. A. Gabel, Biochemistry 28, 2378 (1989). 57 N. A. Matwiyoff, C. Gasparovic, R. Wenk, J. D. Wicks, and A. Rath, Magn. Reson. Med. 3, 164 (1986).

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PROBES OF METAL ION ENVIRONMENTS

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formed from the decomposition of SRs was found to diffuse into cells. 58 The apparent size and shape of Li+-free RBCs, studied by scanning electron microscopy and Coulter counter methods, were, however, unchanged by the presence of SRs in the suspension medium at concentrations lower than 10 mM. 54Effects of Dy(PPP)27- and Dy(TTHA) 3- on the cell structure stability of kidney cortical tubules and hepatocytes have also been reported. 58 The effect of SRs on the energy metabolism of a cell suspension or a perfused organ can be checked easily by 31p NMR spectroscopy; a nontoxic SR should have no effect on the peak areas of the 31p NMR resonances of ATP, inorganic phosphate, and phosphocreatine. 59 Control experiments testing the effect of the SR, at the desirable concentration, on the transmembrane alkali metal ion distribution and transport in the cell suspension or perfused organ under investigation, as well as on the membrane potential, cell structure stability, and energy metabolism, should therefore be conducted. If a concentration of SR cannot be found that provides clear discrimination between intraand extracellular alkali metal NMR resonances, and that has no side effects on ion distribution and transport, membrane potential, cell structure stability, and energy metabolism, the MIR method should be employed. Although the MIR method does not require an SR, its applicability is dependent on the existence of a significant transmembrane difference in T~ values, which may not always be present in cell suspensions or perfused organs. When the SR method is employed, the extracellular alkali metal NMR resonance has a T1 value that is considerably lower than the intracellular TI value because of the proximity of the paramagnetic lanthanide ion to extracellular ions. For obtaining quantitative measurements with the SR method, it is therefore recommended to use 90° radio frequency pulses and a delay between the successive radio frequency pulses that is at least five times the value of the intracellular alkali metal T~ relaxation time; otherwise, the intracellular alkali metal NMR resonance is only partially relaxed and has not reached its full intensity. The erroneous conclusion that the 7Li NMR-determined intracellular Li + concentrations in Li +loaded RBCs suspended in a medium containing Dy(PPP)27- were lower than those measured by AA spectroscopy probably originated from setting the delay between successive pulses to a value that did not allow full relaxation of the intracellular 7Li+ NMR resonance. 6° 58 y . Boulanger, A. Fleser, R. Amarouche, H. Ammann, M. Bergeron, and P. Vinay, Biophys. J. 5, 1 (1992). 59 T. Ogino, G. I. Shulman, M. J. Avison, S. R. Gullans, J. A. Den Hollander, and R. G. Shulman, Proc. Natl. Acad. Sci. U.S.A. 82, 1099 (1985). 6o M. S. Hughes, K. J. Flavell, and N. J. Birch, Biochem. Soc. Trans. 16, 827 (1988).

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ALKALIMETALNMR

105

With the MIR method, one should set the interpulse delay D z (Fig. 4) by first performing a calibration as shown in Fig. 5; an estimate of the ideal D2 value can, however, be obtained quickly from the expression In 2 x Tl(out), as mentioned above. In all NMR transport experiments, it is essential to confirm that the suspension medium is isotonic by measuring it directly with, for example, a Wescor vapor pressure osmometer (Wescor Inc., Logan, UT). When an SR is employed, its contribution toward the osmolarity of the isotonic suspension medium should be taken into account by use of a lesser amount of alkali metal salts. 4° A recent report 6~indicated that there was no transmembrane difference in 7Li+ T1 values in Li +loaded RBC suspensions, which would preclude the application of the MIR method. We found, however, that the inability to observe a transmembrane difference in 7Li+ T~ values in Li+-loaded RBC suspensions may be associated with the use of a hypotonic suspension medium and consequent cell lysis, Li ÷ redistribution across the RBC membrane during the course of a long TI relaxation measurement, and Li +in-Na ÷outexchange induced by the presence of Na + in the suspension medium. 6z Under these unsatisfactory experimental conditions, 6~ the measured T~ values were due not to intracellular 7Li+ ions alone, but to a combination of intra- and extracellular ions. Magnetic susceptibility can also contribute to the observed chemical shifts and line widths of alkali metal NMR resonances from cell suspensions and perfused organs.16'63 Because the geometry of most cells and organs is nonspherical, the alkali metal nuclei inside and outside the tissue experience different magnetic fields, resulting in an additional, albeit small, chemical shift difference between the intra- and extracellular compartments. The contribution of the bulk magnetic susceptibility (BMS) to the observed chemical shift is dependent on the tissue morphology, the volume susceptibility of the media inside and outside the tissue, and the orientation of the NMR sample relative to the applied magnetic f i e l d . 63 For NMR experiments performed with superconducting magnets, which is often the case, paramagnetic substances afford a positive susceptibility, resulting in a downfield shift. In contrast, diamagnetic substances afford a negative susceptibility, which originates an upfield shift. Because the orientations of the magnetic moments of alkali metal nuclei relative to the applied magnetic field are different in superconducting magnets and electromagnets, the sign and magnitude of the BMS shifts are opposite in these 61 R. P. Gullapalli, R. M. Hawk, and R. A. Komoroski, Magn. Reson. Med. 20, 240 (1991). 62 D. Mota de Freitas, Q. Rong, and S. Mo, Magn. Reson. Med. 29, 256 (1993). 63 S. C.-K. Chu, Y. Xu, J. A. Balschi, and C. S. Springer, Jr., Magn. Reson. Med. 13, 239 (1990).

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PROBES OF METAL ION ENVIRONMENTS

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two types of NMR spectrometers. 63 Magnetic susceptibility should, in principle, change the chemical shifts and line widths of all alkali metal nuclei equally. Because significant inhomogeneity components are present in most tissue samples, a contribution of the gyromagnetic ratio to the observed BMS shift is also present. 63 Alkali metal nuclei with a lower resonance frequency therefore generally show smaller shifting and linebroadening effects. 16.63Magnetic susceptibility effects should therefore be taken into account in the interpretation of alkali metal NMR spectra from cell suspensions and perfused organs. Future Outlook Alkali metal NMR spectroscopy will most likely continue to be used for probing the binding and environment of alkali metal ions in metalloproteins and metalloenzymes, despite its inherent problems of broad lines and visibility of the NMR resonance. Because these problems are less pronounced with 7Li and 133Cs NMR spectroscopy, these two alkali metal nuclides are the probes of choice for gaining information on the active site of alkali metal-activated enzymes, as well as that of Ca/+- and Mg 2÷containing proteins. Continued activity in applications of alkali NMR spectroscopy to cell suspensions and perfused organs is also anticipated; this research should lead to novel information at a molecular level on the mechanisms of alkali metal ion transport in biological systems, a problem of major physiological and medical interest. Alkali metal NMR relaxation measurements should prove useful in the understanding of diseases associated with abnormalities of ion transport, in particular manic depression and hypertension. ~o,52 Acknowledgments Work on 7Li, 23Na, and 133CsNMR in our laboratory was supported in part by U.S. Public Health Service Grant MH-45926. I am grateful to Prof. Carlos Geraldes(University of Coimbra, Portugal) and Dr. RavichandranRamasamy(Universityof Texas, Dallas) for helpful suggestions.

[5l

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107

[5] C a l c i u m N u c l e a r M a g n e t i c R e s o n a n c e B y S. FORSI~N, C. JOHANSSON,

and S. LINSE

Introduction A survey of the nuclear properties of naturally occurring isotopes of all elements in the periodic table reveals that nearly all of the 30 or so essential elements in biological systems have at least one potentially valuable isotope amenable for nuclear magnetic resonance (NMR) studies. Calcium has one isotope, 43Ca, with a nuclear spin I of ½, meaning that the nucleus also has an electric quadrupole moment. The nuclear parameter that determines the frequency at which NMR signals may be observed at a given magnetic field, namely, the magnetogyric ratio, is such that at a field of 11.7 tesla (T) the 43Ca NMR signals will be observable at 33.5 MHz. At this field ~H NMR signals would occur at 500 MHz. The low NMR frequency for 43Ca also implies low relative spectroscopic sensitivity; if the sensitivity of ~H nuclei is put as 1.00, the sensitivity for an equal amount of 43Ca nuclei would only be 0.0064. We must also consider the low natural abundance of 43Ca of only 0.145%. In view of this and other • factors to be discussed below, it is not surprising that a3Ca NMR was slow to develop; in fact, the first observation of 43Ca NMR signals from Ca 2+ ions bound to a protein molecule at millimolar concentrations was made in the early 1980s. L2 In this chapter we outline how 43Ca NMR can be used to obtain unique information about biological systems: to identify and characterize Ca 2+ binding sites, to study compartmentalization and transport processes, to determine Ca 2+ binding constants, and to obtain rates of chemical exchange of Ca 2+ ions from a macromolecule under equilibrium conditions. 3 We also briefly describe the experimental requirements for observation of 43Ca signals and how to extract biochemically relevant information from the observed NMR spectra. A general account by Sanders and Tsai on the practical aspects of NMR studies of quadrupolar nuclei like 43Ca appears in an earlier volume of this series .4 1 T. Andersson, T. Drakenberg, S. Forsrn, E. Thulin, and M. Sward, J. Am. Chem. Soc. 104, 576 (1982). z S. Forsrn, T. Andersson, T. Drakenberg, E. Thulin, and M. Swgrd, Fed. Proc. 41, 2981 (1982). 3 S. Fors6n, T. Drakenberg, and H. Wennerstr6m, Q. Reu. Biophys. 19, 83 (1983). 4 C. R. Sanders II and M.-D. Tsai, this series, Vol. 177, p. 317.

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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PROBES OF METAL ION ENVIRONMENTS

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TABLE I SELECTED PARAMETERS OF 43Ca

Nuclear spin (I)

Natural abundance (%)

NMR frequency at 11.7 T (MHz)

Electric quadrupole moment (10 -28 m 2)

IH = 1.000

13C = 1.000

{

0.145

33.5

- 0.05

0.0064

0.4

Relative sensitivity"

" Equal n u m b e r o f nuclei.

Experimental Aspects It is practical to summarize some of the relevant atomic and nuclear properties of 4 3 C a (Table I). We may infer from Table I that even for a calcium sample containing exclusively the isotope 43Cathe NMR sensitivity is only 0.6% of that of IH and comparable to that of ~3C at its natural abundance (1. I%). As a consequence, it is advisable that 43CaNMR studies be performed on isotope-enriched samples. Most 43Ca NMR studies in laboratories interested in biological applications have employed 40-60% isotope-enriched 43Ca.

Nuclear Magnetic Resonance Spectroscopic Behavior o f 43Ca: Effects Due to Electric Quadrupole Moment Before we continue our discussion on how to obtain biochemically relevant information from 43Ca NMR spectra we must briefly summarize the characteristic NMR spectroscopic properties of 43Ca. NMR signals of nuclei with spin I = ½, like ~H, 15N, ~3C, etc., are generally characterized by two relaxation rates: R 1 (= 1/TI) and R z (= 1/T2). R 1 represents the decay rates of the longitudinal and R2 the transverse nuclear magnetizations owing to the interaction of the nuclei with their environment. When we write Rx = 1/Tx we imply that the relaxation is exponential and may be characterized by a single time constant, Tx. A peculiarity of magnetic nuclei with spin I > ½is that their relaxation under certain conditions may be nonexponential. 5 Furthermore, because nuclei with spin I > ½ always possess a nuclear electric quadrupole moment, the most effective relaxation mechanism is usually due to the interaction of this nuclear quadrupole with fluctuating electric field gradients produced by the external environment. Quadrupolar interactions may also result in an apparent shift of the resonance frequency, so called second5 p. H u b b a r d , J. Chem. Phys. 53, 985 (1970).

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CALCIUM N M R

109

order dynamic frequency shifts .6,7 The magnitude of these shifts depends on the square of the coupling strength of the nuclear quadrupole with the external field gradients (the quadrupole coupling constant, X) and is inversely proportional to the field at which the NMR spectrum is recorded.8 With NMR spectrometers equipped with high-field superconducting magnets (B0 -> 10 T) and in most biological applications considered here, the effect may be neglected. When the rate of tumbling of a molecular system containing a quadrupolar nucleus, such a s 43Ca, is very slow and/or when the tumbling is nonisotropic, then the electric field gradient set up by the atoms surrounding the quadrupolar nucleus is asymmetric. Therefore, the NMR spectra will consist of multiple resonances separated by"quadrupolar splittings."9 Such effects are observed on solid samples and also in the presence of biological membranes and in liquid crystalline systems.

Chemical Shift Range No detailed study of 43Ca chemical shifts in different types of complexes have been performed, but available information indicates that the range is of the order of +-30 parts per million (ppm) from the 43CaNMR signal in a dilute aqueous solution of CaCI 2. Studies on Ca 2÷ complexed to proteins show that the shift is dependent on the nature and disposition of the protein ligands. So-called EF-hand sites, found in proteins of the calmodulin superfamily, are characterized by shifts at roughly + 10 ppm.

Relaxation Rates in Absence of Chemical Exchange Because one of the most useful applications of43CaNMR is the determination of chemical exchange rates, for example, of Ca 2+ ions bound to a biological macromolecule in dynamic equilibrium with "free" Ca z+ ions in solution, it is useful to first consider situations in which no chemical exchange takes place. Relaxation behavior of magnetic nuclei is usually discussed for two well-defined experimental situations: (i) rapid isotropic motion or "extreme narrowing," where tOo2rc 2 < < 1, and (ii) slow isotropic motion or"non-extreme narrowing," where tOo2~'c2 -> 1. Here Zc is a characteristic tumbling rate of the molecular system containing the 43Ca nucleus and oJo is the NMR frequency (Larmor frequency) of the 43Ca nuclei. 6 R. Poupko, A. Baram, and Z. Luz, Mol. Phys. 27, 1345 (1974). 7 L. G. Werbelow and A. G. Marshall, J. Magn. Resort. 43, 443 (1981). 8 P.-O. Westlund and H. WennerstrOm, J. Magn. Reson. 50, 451 (1982). 9 C. A. Fyfe, "Solid State NMR for Chemists." CRC Press, Guelph, Ontario, Canada, 1983.

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PROBES OF METAL ION ENVIRONMENTS

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In the case of rapid isotropic motion, as would be expected to prevail for 43Ca2+ ions binding to a small organic molecule, a peptide, or perhaps even a small protein, the relaxation rates R, and R2 will be identical and governed by a single relaxation time constant, T, according to 1° 27r2 2 R l = R 2 = (l/T) = - ~ - X ~'~

(1)

where X is the quadrupole coupling constant (in Hz) characterizing the interaction of the nuclear electric field gradient with the external electric field gradient. The value of X will typically be in the range 105 to 106 Hz. In Eq. (1) we have neglected any asymmetry in the external electric field gradient, which is reasonable since this term is in most cases close to unity. Equation (1) means that the relaxation time, T, for a 43Ca2+ ion binding to a molecule with ~'c = 1 nsec and with X = 1 MHz equals 2.5 msec, corresponding to a line width of the observed N M R signal of about 140 Hz. The use of Eq. (1) is valid if too ( = 27rVo) -< 1 × 108 radians/sec. It should be noted that in the rapid isotropic motion situation the NMR line shape will be Lorenzian. In view of this it may come as a surprise that the line width of the 43Ca2+ NMR signal in a 100 m M aqueous solution of an inorganic calcium salt is less than 1 Hz.I1 In the case of slow isotropic motion the relaxation of a nucleus like 43Ca with spin I > ½, will in general not be a simple exponential decay. In the case of 43Ca (I = ½) the transverse and longitudinal nuclear magnetizations will decay as a weighted sum of four exponentials. 5 This means that the line shape of a 43Ca NMR signal will no longer be a Simple Lorenzian but more complex. However, in a considerable number of biological applications the experimental conditions are such, or can sometimes be made such, that OSorc is in the proximity of unity, a situation one might call "intermediate isotropic motion." In this case the transverse and longitudinal relaxation rates will often appear singly exponential though different. Halle and Wennerstr6m have used perturbation theory to derive approximate analytical expressions for R, and R212 [Eq. (2)]: 10 A. Abragam, in "The Principles of Nuclear Magnetism." Oxford Univ. Press (Clarendon), London and New York, 1961. it S. Fors6n and B. Lindman, Methods Biochem. Anal. 27, 289 (1981). 12 B. Halle and H. Wennerstr6m, J. Magn. Reson. 44, 89 (1981).

[5]

CALCIUM N M R

0.2rc 0.8r c 27r2 2 1 + Worc RI = "~-X 2 2 + 1 -¥-Z-2 2 + 4¢Oo~'c/ R2

=

27r2 2 ( 0.5~'c 0.2~-c - - ~tOo~-c 2 + 1 + 4¢Oo~'c/ 22 ~ - - X 0.3~'c + 1 +

111

(2a) (2b)

Equations (2a) and (2b) are valid approximations for experimental conditions such that Worc -< 1.5. The apparent complexity of the above experimental situation is in fact beneficial. Because the observed relaxation rates Rj and R 2 are unequal, it follows that if we have determined both these rates we may independently calculate rc and X!

Effects of Chemical Exchange When a magnetic nucleus like 43Ca is being rapidly transferred between two environments characterized by different interactions with the nucleus this constitutes a mechanism for relaxation. The appearance of a 43Ca NMR spectrum will be dependent on the rate of chemical exchange in relation to other NMR parameters. We begin to assume that a 43Ca nucleus is present in two different environments, say, A and B, characterized by different chemical shifts and different intrinsic relaxation rates. In the absence of chemical exchange we would then observe two separate 43Ca NMR signals. Typically environment A could be uncomplexed, "free," 43Ca2+ ions in aqueous solution, and B could be 43Ca2+ ions (strongly) binding to a biological macromolecule, for simplicity assumed to undergo rapid isotropic motion. The NMR signal from site A would then be sharp and narrow, whereas that from site B would be broad owing to efficient relaxation in this site (long re, high X). If we gradually could turn on chemical exchange of the 43Ca2+ nucleus, the first effect to be discerned would be an additional broadening of the two NMR signals. The additional broadening, AAvl/2, is given by AA/-"I/2 =

1/~7"ex

(3)

where rex represents the average lifetime of a 43Ca2+ ion in the site. If the populations of the two sites are PA and PB, it then follows that PA/PB = A B rexh'ex. If PA is not much larger than PB, the additional line broadening may be most easily detected on the narrow A signal. The "turning on" of chemical exchange can often in practice be accomplished by raising the sample temperature. Equation (3) may be used to determine slow exchange rates even if the population of 43Ca2+ ions in the fast relaxing site, PB, is so small that no NMR signal from the B site is observable.

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PROBES OF METAL ION ENVIRONMENTS

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We now turn to the other extreme experimental situation, that is, when the rate o f chemical exchange of the 43Ca2+ ion between the two different environments is much faster than both the inverse of the chemical shift difference ANAB ( = NA -- NB) and the relaxation rates (R1 and R2) at both sites. In this case the 43Ca N M R spectrum of our model system would consist of a single signal. The N M R properties of this signal will be a weighted average of the properties at sites A and B. Thus, the L a r m o r f r e q u e n c y of the observed signal Noss = PANA + PBNB and the relaxation rate R °Bs = R °Bs = R °ss = PARA + PBRs. In this experimental situation the exchange rate may not be determined from the N M R spectrum. This situation is, however, very useful if one is primarily interested in determining association constants for Ca 2+ interacting with a biological macromolecule. The relaxation rate, and accordingly the N M R signal line width, of 43Ca2+ at the macromolecular binding sites is very much larger than for " f r e e " 43Ca2+. This means that even if only a small fraction of the 43Ca2+ ions is bound to the macromolecular site the averaged 43Ca2+ N M R signal will be considerably broadened. Let us consider Ca z+ binding to a single binding site on a protein molecule, P. We may write for the equilibrium constant Ka: C a 2+ + P .

Ka--

kon

, Ca2+ . p ko~

(4)

kon _ [ C a 2+ . p ]

kon

[Ca2+][P]

The fraction o f Ca 2+ bound to the protein, PB, is [Ca 2+ • P]/[Ca2+]tot, where [Ca2+]tot is the total Ca z+ concentration. If the experiment is conducted at conditions such that Ca z÷ ions are in large excess over the protein, the population of " f r e e " Ca 2+ , P r , will be close to unity. It then follows from Eq. (4) that P s = ga[P]tot/(1 + ga[Ca]tot)- At a given set o f Ca 2+ N M R relaxation rates, RoB s will be Ka[PltotRs

Ross = PFRF + PsRB = PFRF + 1 + Ka[Ca2+]tot

(5)

We may consider only the observed relaxation rate in excess over the value for " f r e e " Ca 2+, REX, and since PF ~ 1 we have ga[P]totRs

REX = Ross - RF = 1 + Ka[Ca2+]to t

(6)

It is apparent that the relation in Eq. (6) may be used to determine Ca 2+ binding constants by following the dependence of REX, that is

[5]

CALCIUM N M R

113

7rA/~l/2, on the total Ca 2+ concentration. The upper limit of K a values that can be determined is dependent on the lowest Ca 2+ concentrations at which 43Ca NMR signals can be reliably detected. Ka values of up to 104 M- l may certainly be detected with a good experimental setup. For higher K a values the assumption of fast chemical exchange may also not be valid. A good test if the fast exchange criterion is met is to raise the temperature of the sample. The observed 43Ca NMR line width should then decrease slightly owing to the faster tumbling of the protein molecule and the correspondingly slower relaxation of the bonded 43Ca2+ ion. The NMRaccessible range of Ka values, from approximately 1 to 104 M -l, is difficult to study by other physical techniques. If the Ca 2+ chemical exchange rate is neither very slow nor very fast, but comparable to the rate of relaxation at the macromolecular binding site, the appearance of the 43Ca NMR spectrum will depend in a complex way on the exchange rate, the Ca 2+ binding constant, and the relaxation rate. The extraction of these physical parameters from the experimental spectrum requires a detailed analysis of Ca 2+ to protein concentration ratios. A procedure that has been used for a number of years in our department is described in the literature) 3,14 The procedure depends on an analysis of the total band shape of the 43Ca NMR spectrum. For systems where it has been possible to compare rate constants obtained from the NMR method with rate constants obtained from stopped-flow measurements, the rates are found to agree within the errors of measurements. Under favorable circumstances the rate constants that may be determined using 43Ca NMR range from less than 10 to 104 sec-1. Although a total band-shape analysis of the 43Ca NMR spectrum is always advisable, it is possible to use a simple analytical expression for the excess line width, REX, in systems where the concentration of uncomplexed, " f r e e , " Ca 2+ is much larger than the concentration of the Ca2+-protein complex (PF > > PB)- Then we have

Ri,EX -

PB

(7)

(Ri,B) 1 + 'rex

where i = 1 (longitudinal relax) or 2 (transverse relax) and R;,B is the relaxation rate at the protein site with the Ca z+ exchange lifetime %x. Equation (7) is valid as written when the 43Ca NMR chemical shift difference between free and bound states is small. Equation (7) will not result in very accurate values of the Ca 2+ exchange rate but may nevertheless be useful in many situations. 13 T. Drakenberg, S. Fors6n, and H. Lilja, J. Magn. Reson. 53, 412 (1983). 14 M.-D. Tsai, T. Drakenberg, E. Thulin, and S. Fors6n, Biochemistry 2 ~ 3635 (1987).

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[5]

Special Experimental Problems of 43Ca Nuclear Magnetic Resonance NMR spectroscopy of quadrupolar nuclei like 43Ca NMR differs in many respects from NMR of spin I = ~ nuclei. We briefly comment on some of the most pertinent factors. Sample Preparation. As mentioned in the introduction, 43Ca NMR on problems of biological interest generally necessitates the use of isotopeenriched 43Ca. An enrichment of 40 to 60% may be sufficient. With a reasonably sensitive NMR spectrometer (see below) this should allow studies of samples with millimolar or even submillimolar Ca z+ concentrations. EDTA, EGTA, or other organic chelators that may have been used in the preparation of the macromolecular sample should be removed. ~H NMR is the best way of checking this. Control of pH of the sample is also important. H + will compete with Ca 2+ , and below pH 5 carboxylate side chains in proteins will gradually become protonated and lose the ability to strongly ligand to Ca 2+. Spectrometers. NMR probes suitable for 43Ca studies are usually not standard on modern commercial NMR spectrometers and may have to be acquired separately. Good NMR probes and spectrometer systems should have the characteristics of high sensitivity and low acoustic ringing. The sensitivity should always be tested on standard samples using experimental conditions (number of pulses, total acquisition time, a reasonably large 43Ca NMR line width, e.g., 100-200 Hz) similar to those expected in the biological applications planned. Acoustic ringing is a phenomenon that in particular affects NMR spectra of nuclei at low frequencies. Mechanical, "acoustic," waves are generated in the probe as a result of the radio frequency pulses used to excite the magnetic nuclei under study and will result in a "rolling" baseline. These effects can largely be eliminated by pulse cycling but can also be suppressed through careful probe designs. 1,~1,15 Acquisition of 43CaNuclear Magnetic Resonance Spectra. One of the benefits of the high 43Ca relaxation rates usually encountered in biological applications is that high rates of radio frequency pulsing may be used to obtain the NMR spectrum. High pulse rates do, however, put high demands on the spectrometer: high power output at the low-frequency band where 43Ca signals appear so as to make the 90° pulses short, high dynamic range, control of the preacquisition delay time, etc. These and other requirements have been discussed at some length by Sanders and T s a i . 4 It should be emphasized here that the line shape of a 43Ca NMR signal may be complex and non-Lorentzian. To truly reproduce the NMR specI5 I. P. Gerothanassis, Prog. NMR Spectrosc. 19, 267 (1987).

[5]

CALCIUMNMR

115

trum and avoid artificial broadening it is necessary to sample the free induction decay (FID) after a radio frequency pulse for a time TS that satisfies the condition T, > ~T2, where T2 is the longest time constant that characterizes the decay of the FID.

Experimental Applications A comprehensive review of essentially all 43Ca NMR studies of calcium-binding proteins has been published.16 In this chapter we highlight a few applications aimed at determining different biophysical parameters.

Chemical Shift 43Ca ions bound to proteins frequently give broad (400-800 Hz) signals within a small shift range (+-30 ppm). It is therefore often difficult to discriminate signals from calcium ions in different environments, although band-shape analysis can help to identify partially overlapping components 17 as illustrated in Fig. 1. The problem can also be overcome by addition of various lanthanide shift reagents [e.g., Dy(PPP)2 v- , where Dy is dysprosium(III) ion and ppps- is triphosphate] to the protein solution, whereby the signal from free calcium ions experiences a shift change of between 60 and -125 ppm. 18'19 A drawback is that some shift reagents broaden the signals beyond detection.

Ca2+-Binding Constants The first determination of the calcium affinity of a protein using 43Ca NMR concerned the single Ca 2+ site in prophospholipase A2, for which the binding constant was calculated as K a = 2.5 x 103 M -1 (Refs. 13, 20, and 21). This initial work has been followed by several other studies of calcium sites as diverse as that in factor XIII of the blood coagulation cascade (K a = 400 M - l ) , 22 bone y-carboxyglutamic acid protein (Ka = 16 C. Johansson and T. Drakenberg, Annu. Rep. NMR Spectrosc. 22, 1 (1989). 17 H. J. Vogel, T. Drakenberg, and S. Fors6n, Biochemistry 24, 3870 (1985). 18 H. J. Vogel, T. Andersson, W. Braunlin, T. Drakenberg, and S. Forsfn, Biochem. Biophys. Res. Commun. 122, 1350 (1984). 19 H. J. Vogel and W. Braunlin, J. Magn. Res. 62, 42 (1985). 20 T. Andersson, T. Drakenberg, S. Fors6n, T. Wieloch, and M. Lindstr6m, FEBS Lett. 123, 115 (1981). 21 T. Drakenberg, T. Andersson, T. S. Fors6n, and T. Wieloch, Biochemistry 23, 2387 (1984). 22 M. M. Sarasua, K. A. Koehler, C. Skrzynia, and J. M. McDonagh, J. Biol. Chem. 257, 14102 (1982).

116

PROBES

'

'

'

I

1000

OF

'

METAL

'

'

'

ION

I

0

ENVIRONMENTS

'

'

'

'

[5]

I

'

"

'

-1000

Hz

FIG. 1.43Ca NMR spectrum at 24.34 MHz from a solution of 2.2 mM total calcium and 1 mM of the calbindin D9k mutant Y13F. (Top) Experimental spectrum; (middle) calculated spectrum; (bottom) individual Lorentzian lines. The observed band can be resolved into three Lorentzian signals, one for free calcium and two broader lines of approximately equal intensity for calcium in the two bindings sites in the protein. [Redrawn with permission after S, Linse, P. Brodin, T. Drakenberg, E. Thulin, P. Sellers, K. Elmd6n, T. GrundstrSm, and S. Fors6n, Biochemistry 26, 6723 (1987).]

5 × 103 to 1 × 105 M - l ) , 23 a n d the p o l y p e n t a p e p t i d e o f e l a s t i n (K a = 7 o r 35 M-1).24 Interaction between Macromolecules 43Ca N M R c a n also b e u s e d to s t u d y the i n t e r a c t i o n b e t w e e n m a c r o m o l e c u l e s if at l e a s t o n e o f t h e m b i n d s c a l c i u m . I n the c a s e o f c a l c i u m b o u n d to p r o t h r o m b i n f r a g m e n t 1, B o u h o u t s o s - B r o w n et al. z5 h a v e f o u n d that the 43Ca N M R line w i d t h is s e n s i t i v e to b o t h p r o t e i n a s s o c i a t i o n a n d to the i n t e r a c t i o n o f the p r o t e i n with a p h o s p h o l i p i d m e m b r a n e . 23 M. Sv~ird,T. Drakenberg, T. Andersson, and P. Fernlund, Eur. J. Biochem. 158, 373 (1986). 24D. W. Urry, T. L. Trapane, and C. M. Venkatachalam, Calcif. Tissue Int. 34, $41 (1982). 25E. Bouhoutsos-Brown, C, H. Pletcher, G. L. Nelsestuen, and R. G. Bryant, J. lnorg. Biochem. 21, 337 (1984).

[5]

CALCIUM NMR &v

12

/Hz

1 17

×

o

200

o

150

100

50

i

270

320

370 ~T/K

FIG. 2. Temperature dependence of the 43CaNMR linewidth (aVl/2) of the Lorentzian signal from free calcium in the presence of three different calbindin D9k mutants: (©) (A14A + AI5D) with kon = 515 sec-l; (×) (AI5D + P20G) with koff = 92 sec-~; and (11) (A142x + A15D + P20G + N21A) with koff = 6700 sec -t. Note that the calcium/protein ratios are different for the three plots. [Redrawn with permission after C. Johansson, P. Brodin, T. Grundstr6m, S. Fors6n, and T. Drakenberg, Eur.J. Biochem. 202, 1283(1991).]

Calcium Exchange Rate, ko#, Quadrupole Coupling Constant, X, and Correlation Time, ~',. As described earlier, the t e m p e r a t u r e d e p e n d e n c e of the 43Caline width gives direct information of the exchange rate, koff, for the calcium ions, as depicted in Fig. 2. F u r t h e r m o r e , from the t e m p e r a t u r e dependence the quadrupole coupling constant, X, and correlation time, %, for calcium in the binding site can be obtained. This gives information on the environment of the site, and f r o m a total band-shape analysis of the Lorentzian signals as a function of t e m p e r a t u r e information on the t h e r m o d y n a m i c s of calcium binding can be obtained. S o m e proteins belonging to the calmodulin superfamily of calciumbinding proteins have been extensively studied by 43Ca NMR: troponin C, parvalbumin, calmodulin, and calbindin D9k. F o r calmodulin (CAM), with a correlation time of 8.2 nsec and a quadrupole coupling constant of 1.15 M H z , studies of both the intact protein and its two tryptic fragments, each comprising one globular domain with two Ca 2+ sites, led to the identification of the two C-terminal Ca 2+ sites as the high-affinity sites with slow Ca 2+ exchange (kofr --~ 10 sec-~) and the N-terminal sites as the

118

PROBES OF METAL ION ENVIRONMENTS

[6]

low-affinity c a l c i u m / m a g n e s i u m sites with an intermediate exchange rate of about 500 s e c - J (Refs. 1, 26, and 27). Finally one might raise the question whether the d e v e l o p m e n t o f multidimensional high-resolution ~H N M R methods will m a k e 43Ca N M R studies redundant. It is, h o w e v e r , evident from the a b o v e presentation that 43Ca N M R provides unique information about Ca 2÷ ions interacting with m a c r o m o l e c u l a r sites. A striking e x a m p l e is provided in the case of calbindin D9k, which has been subject to detailed engineering and structural (~H N M R and X-ray) studies in our laboratory. Several mutant f o r m s of the protein have been extensively studied by 43Ca N M R , w h e r e b y , for instance, different factors that affect the calcium exchange rate in the Nterminal site have b e e n identified. J7,28-32 In addition, a study of calciumbinding l y s o z y m e s and a-lactalbumins 33 illustrates that 43Ca N M R will continue to be an indispensable m e t h o d for the study of calcium binding proteins. 26S. Fors6n, A. Andersson, T. Drakenberg, O. Teleman, E. Thulin, and H. J. Vogel, in "Calcium Binding Proteins" (B. de Bernard, G. L. Soltocasa, G. Sandri, E. Carafoli, A. N. Taylor, T. C. Vanaman, and R. J. P. Williams, eds.), p. 121. Elsevier, Amsterdam, 1983. 27A. Teleman, T. Drakenberg, and S. Fors6n, Biochim Biophys. Acta 873, 204 (1986). 28S. Linse, P. Brodin, T. Drakenberg, E. Thulin, P. Sellers, K. Elmd~n, T. Grundstr6m, and S. Fors6n, Biochemistry 26, 6723 (1987). 29S. R. Martin, S. Linse, C. Johansson, P. M. Bayley, and S. Fors6n, Biochemistry 29, 4188 (1990). 30C. Johansson, P. Brodin, T. Grundstr6m, E. Thulin, S. Fors6n, and T. Drakenberg, Eur. J. Biochem 187, 455 (1990). 31p, Brodin, C. Johansson, S. Fors6n, T. Drakenberg, and T. Grundstr6m, J. Biol. Chem. 265, 11125 (1990). 32C. Johansson, P. Brodin, T. Grundstr6m, S. Fors6n, and T. Drakenberg, Eur. J. Biochem. 202, 1283 (1991). 33j. M. Aramini, T. Drakenberg, T. Hiraoki, Y. Ke, K. Nitta, and H. J. Vogel, Biochemistry 31, 6761 (1992).

[6] P u l s e d E l e c t r o n N u c l e a r M u l t i p l e R e s o n a n c e Spectroscopic Methods for Metalloproteins and Metalloenzymes B y HANS THOMANN and MARCELINO BERNARDO

1. Introduction Pulsed electron nuclear multiple r e s o n a n c e ( P E N M R ) s p e c t r o s c o p y refers to a b r o a d class o f techniques in which solid-state pulsed nuclear

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All fights of reproduction in any form reserved.

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PROBES OF METAL ION ENVIRONMENTS

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low-affinity c a l c i u m / m a g n e s i u m sites with an intermediate exchange rate of about 500 s e c - J (Refs. 1, 26, and 27). Finally one might raise the question whether the d e v e l o p m e n t o f multidimensional high-resolution ~H N M R methods will m a k e 43Ca N M R studies redundant. It is, h o w e v e r , evident from the a b o v e presentation that 43Ca N M R provides unique information about Ca 2÷ ions interacting with m a c r o m o l e c u l a r sites. A striking e x a m p l e is provided in the case of calbindin D9k, which has been subject to detailed engineering and structural (~H N M R and X-ray) studies in our laboratory. Several mutant f o r m s of the protein have been extensively studied by 43Ca N M R , w h e r e b y , for instance, different factors that affect the calcium exchange rate in the Nterminal site have b e e n identified. J7,28-32 In addition, a study of calciumbinding l y s o z y m e s and a-lactalbumins 33 illustrates that 43Ca N M R will continue to be an indispensable m e t h o d for the study of calcium binding proteins. 26S. Fors6n, A. Andersson, T. Drakenberg, O. Teleman, E. Thulin, and H. J. Vogel, in "Calcium Binding Proteins" (B. de Bernard, G. L. Soltocasa, G. Sandri, E. Carafoli, A. N. Taylor, T. C. Vanaman, and R. J. P. Williams, eds.), p. 121. Elsevier, Amsterdam, 1983. 27A. Teleman, T. Drakenberg, and S. Fors6n, Biochim Biophys. Acta 873, 204 (1986). 28S. Linse, P. Brodin, T. Drakenberg, E. Thulin, P. Sellers, K. Elmd~n, T. Grundstr6m, and S. Fors6n, Biochemistry 26, 6723 (1987). 29S. R. Martin, S. Linse, C. Johansson, P. M. Bayley, and S. Fors6n, Biochemistry 29, 4188 (1990). 30C. Johansson, P. Brodin, T. Grundstr6m, E. Thulin, S. Fors6n, and T. Drakenberg, Eur. J. Biochem 187, 455 (1990). 31p, Brodin, C. Johansson, S. Fors6n, T. Drakenberg, and T. Grundstr6m, J. Biol. Chem. 265, 11125 (1990). 32C. Johansson, P. Brodin, T. Grundstr6m, S. Fors6n, and T. Drakenberg, Eur. J. Biochem. 202, 1283 (1991). 33j. M. Aramini, T. Drakenberg, T. Hiraoki, Y. Ke, K. Nitta, and H. J. Vogel, Biochemistry 31, 6761 (1992).

[6] P u l s e d E l e c t r o n N u c l e a r M u l t i p l e R e s o n a n c e Spectroscopic Methods for Metalloproteins and Metalloenzymes B y HANS THOMANN and MARCELINO BERNARDO

1. Introduction Pulsed electron nuclear multiple r e s o n a n c e ( P E N M R ) s p e c t r o s c o p y refers to a b r o a d class o f techniques in which solid-state pulsed nuclear

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All fights of reproduction in any form reserved.

[6]

PULSED ELECTRON NUCLEAR MULTIPLE RESONANCE

l 19

magnetic resonance (NMR) methods are combined with pulsed electron paramagnetic resonance (EPR) methods for measuring the magnetic interactions of paramagnetically coupled NMR-active nuclei. In metalloproteins and metalloenzymes, the paramagnetism typically originates from the active site such as the transition metal ion or cluster of ions or from an organic cofactor. Examples of frequently encountered transition metal sites in metalloenzymes and proteins include mononuclear copper, iron, and molybdenum centers, iron-sulfur clusters, oxo-bridged copper and iron clusters, manganese clusters, and heme centers. The combination of the NMR with EPR techniques provides the advantage of increased spectral resolution as well as significantly higher sensitivity than would be obtained in the NMR experiment alone. Continuous wave irradiation techniques have generally been used to excite both the NMR and EPR transitions in most multiple resonance experiments. The additional advantages of employing pulsed excitation techniques are presented and demonstrated in this chapter. Detailed chemical and electronic structure information on the active site in the protein or enzyme can be derived from the analysis of the NMR frequencies of paramagnetically coupled nuclei even when single crystals are not available. High-resolution data on the electronic structure derived from spectroscopic methods are particularly useful if the coordination structure is known from the crystal structure. The combination of crystallographic and spectroscopic data offers the best potential for understanding chemical functionality such as electron transfer, redox potentials, and substrate specificity and reactivity. In magnetic resonance experiments, structural information is derived from the magnetic interactions between the unpaired electron spin on the metal sites and the paramagnetically coupled nuclei of the central metal atoms and ligand nuclei. This interaction can in some cases be observed as line splittings, known as the hyperfine splittings, in an EPR spectrum. In studies of metalloproteins it is frequently the case that the hyperfine splittings from ligand nuclei and even from the central metal nuclei are not resolved in the EPR spectra. This low spectral resolution arises from many line broadening mechanisms, particularly the anisotropy of the g factor as well as the overlap of hyperfine splittings from the many nuclei in the coordination sphere of the transition metal site. EPR spectra broadened by these line broadening mechanisms are referred to as inhomogeneously broadened lines. Inhomogeneous line broadening obscures the structural information otherwise provided in the analysis of the hyperfine splittings. In principle, greater spectral resolution could be obtained in the NMR spectrum of the paramagnetically coupled nuclei. In a system with one

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electron coupled to many nuclei, each electron spin state can be coupled to many combinations of nuclear spin states. As a result there are many nuclear hyperfine line splittings in the EPR spectrum, which ultimately results in an inhomogeneously broadened line shape. On the other hand, the electron spin can be in only one of two spin states, so the NMR line for each nucleus can only be split into two lines. The increase in spectral resolution can be qualitatively understood by considering the average spectral density of lines in an EPR spectrum compared to the lines in the NMR spectrum. The relative number of lines in the EPR and NMR spectra can be compared using the spectral density functions introduced by Hyde.~ The average spectral density for an EPR spectrum is K

II (2Nklk + 1) k=l PEPR =

(1)

K

Z 2AkNA

k=l

The numerator in Eq. (1) corresponds to the number of EPR lines observed that are due to the hyperfine splitting, Ak, assuming no degeneracy, with K groups of N k equivalent nuclei with nuclear spin I k . The denominator describes the total width of the EPR spectrum. For simplicity, only isotropic first-order hyperfine interactions are considered. The corresponding average spectral density for the NMR spectrum of paramagnetically coupled nuclei is 2K PNMR -- Amax

(2)

where Areax determines the width of the spectrum. Because we have used the simplifying assumption that only the first-order hyperfine interaction generates line splittings, only two NMR lines are observed for each group of K equivalent nuclei. Comparing Eq. (1) to Eq. (2) it is clear that the EPR spectral density increases much faster than the NMR spectral density. Under appropriate conditions, nuclei coupled to an unpaired electron can be observed directly in the NMR experiment. 2,3 The paramagnetic interaction shifts the NMR resonance frequency and can broaden the line. An essential criterion for direct NMR detection is that the electron J. S. Hyde, "Magnetic Resonance in Biological Systems." Pergamon, London, 1967. 2 G. N. LaMar, W. D. J. Horrocks, et al., "NMR of Paramagnetic Molecules." Academic Press, New York, 1973. 3 I. Bertini and C. Luchinat, " N M R of Paramagnetic Molecules in Biological Systems" Benjamin Cummings, Menlo Park (1986).

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spin-lattice relaxation time, Tie, or electron correlation time, re, is on the order of 10- ~1sec or less if well-resolved NMR lines are to be observed. The short electron spin relaxation time assures that the anisotropic g factor and hyperfine interactions are averaged to a small value, resulting in narrow NMR lines. The paramagnetic shift is then determined by the isotropic hypertine interaction arising from the unpaired spin density on the nucleus. The pulsed techniques described in this chapter for the EPR detection of NMR transitions are well suited to those situations where the direct NMR detection of paramagnetically coupled nuclei fails. If the electron spin relaxation times are not favorable, the NMR lines are broadened beyond detection. The low sensitivity of the NMR experiment is another limiting factor. As an example, the NMR spectra of the central metal nuclei in paramagnetic transition metal complexes can be readily observed by indirect detection using pulsed excitation techniques but cannot be observed using direct NMR detection. Within the broad class of electron nuclear multiple resonance methods, electron nuclear double-resonance (ENDOR) spectroscopy is the most established. 4-v Traditionally the ENDOR experiment has been performed by continuously irradiating both the EPR and NMR transitions simultaneously. NMR transitions are detected indirectly via the change in EPR signal intensity caused by the nuclear spin flip. ENDOR transitions are therefore NMR transitions detected indirectly via an EPR transition. The ENDOR signals are often referred to as " E N D O R enhancements" because the signal corresponds to an increase in the EPR signal intensity. Because the radio frequency (rf) field irradiating the NMR transition and the microwave field irradiating the EPR transition are continuously on during the experiment, this version of the ENDOR experiment is known as continuous wave (CW)-ENDOR. In the pulsed electron nuclear multiple resonance techniques discussed in this chapter, both the NMR and EPR transitions are irradiated using pulsed techniques. The first pulsed ENDOR experiment was demonstrated by Mims in 1965, 8 less than 10 years after Feher first reported the 4 M. M. Dorio and J. H. Freed (eds.), "Multiple Electron Resonance Spectroscopy." Plenum, New York, 1979. 5 L. Kevan and L. D. Kispert (eds.), "Electron Spin Double Resonance Spectroscopy." Wiley (Interscience), New York, 1979. 6 H. Kurreck, B. Kirste, et al., " E N D O R Spectroscopy of Radicals in Solution." VCH Publ., New York, 1988. 7 A. J. Hoff (ed.), "Advanced EPR: Applications in Biology and Biochemistry." Elsevier, Amsterdam, 1989. 8 W. B. Mims, Proc. R. Soc. L o n d o n 283, 452 (1965).

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CW-ENDOR experiment. 9 Subsequently, several pulsed ENDOR studies of organi~ and inorganic radicals were reported. ~°-13 The first pulsed ENDOR experiments on a metalloprotein were reported in 1988.14 There are several factors motivating the development of pulsed methodology in electron nuclear multiple resonance experiments. 8'13'15-19 Detecting the E N D O R enhancement by pulsed excitation techniques eliminates the sensitivity of the ENDOR signal to the detailed balance between the electron and nuclear spin relaxation rates. This sensitivity is well known in CW-ENDOR experiments 4'5 and has two important consequences. First, it often precludes the observation of ENDOR signals except over a very limited temperature range. Second, the relaxation rates can determine and limit the E N D O R enhancement and the ENDOR line shapes, thereby affecting the sensitivity and resolution. In contrast, in the pulsed E N D O R experiment, the only requirements for observing an ENDOR enhancement are that (1) the electron spin phase memory time is sufficiently long to observe a free induction decay (FID) or a spin echo and (2) the time scale in which the electron spin polarization decays is comparable to or longer than the time required to flip the nuclear spin. If these conditions are satisfied, a pulsed ENDOR experiment will succeed, whereas the CW-ENDOR experiment may or may not succeed depending on the spin relaxation rates and other experimental conditions. As will become apparent in this chapter, all CW-ENDOR experiments have direct analogs in pulsed E N D O R versions. A second important motivation for developing pulsed methodology is that pulsed methods significantly increase the ability to manipulate the spin system. As has been amply demonstrated in NMR spectroscopy, this ability greatly extends the information that can be derived from a 9 G. Feher, Phys. Rev, 103, 834 (1956). l0 I. M. Brown, D. J. Sloop, et al., Phys. Rev. Lett. 22, 324 (1969). 1i p. F. Liao and S. R. Hartmann, Phys. Rev. B 8, 69 (1973). 12 W. A. J. A. v. d. Poel, D. J. Singel, et al., Mol. Phys. 49, 1017 (1983). 13 M. Mehring, P. Hofer, et al., Bet. Bunsen-Ges. Phys. Chem. 91, 1132 (1987). 14 H. Thomann, M. Bernardo, et al., "Time Domain ENDOR Studies of Disordered Solids." 29th Experimental NMR Conference, Rochester, New York, 1988. 15 A. Grupp and M. Mehring, "Modern Pulse and Continuous Wave Electron Spin Resonance Spectroscopy." Wiley, New York, 1990. J6 H. Thomann and M. Bernardo, Spectrosc. Int. J. 8, 119 (1990). 17 C. Gemperle and A. Schweiger, Chem. Rev. 91, 1481 (1991). ts H. Thomann and M. Bernardo, "Advances in Chemistry," Vol. 229. ACS Books, Washington, D.C., 1992. i9 H. Thomann and W. B. Mims, "Pulsed Magnetic Resonance: NMR, ESR, and Optics," p. 362. Oxford Univ. Press (Clarendon), Oxford, 1992.

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spectrum. 2° Examples of pulsed methods which would be extremely difficult or impossible to implement as CW-ENDOR analogs include two dimensional (2D) E N D O R ] ~'22coherence transfer ENDOR,10,23 and multiple quantum (MQ) ENDOR. 24'25 We proceed in the next section by first describing the spectrum of NMR transition frequencies typically observed for paramagnetically coupled nuclei. This is followed by sections describing the basic physical principles for pulsed techniques in electron nuclear spectroscopy. Next, we follow with a section on the experimental and instrumental considerations, providing the nonspecialist with an overview of the experimental methodology. Discussion of specific PENMR experiments with examples of applications to metalloenzymes and metalloproteins is given in subsequent sections.

2. Electron Nuclear Double Resonance Energy Levels and Transition Frequencies When the electron spin relaxation time is not sufficiently short to average the anisotropic magnetic interactions to their isotropic values, the NMR transitions for paramagnetic nuclei are spread over a wide spectral bandwidth. 2'3 This is generally the situation encountered at low temperatures (near liquid helium) where most pulsed EPR experiments on metalloenzymes must be performed. The distribution of NMR frequencies arises from the anisotropies of both the electronic g factor and the hyperfine interaction. Additional broadening can arise for quadrupolar nuclei or if the electron spin multiplicity is greater than S = ½. In the latter case the zero field splitting can lead to line broadening. 26 The interaction energy between a single electron with S -- ½ and a nucleus with the applied magnetic field and between the electron and nucleus are formally described by the spin Hamiltonian26: ~

= f l S ' g . H o - fingnI ' H o + S ' A ' I

+ I'P'I

(3)

20 R. R. Ernst, G. Bodenhausen, et al., "Principles of NMR in One and Two Dimensions." Oxford Univ. Press (Clarendon), Oxford, 1987. 21 C. Buhlmann, A. Schweiger, et al., Chem. Phys. Lett. 154, 285 (1989). 22 H. Thomann and M. Bernardo, Chem. Phys. Lett. 169, 5 (1990). 23 p. Hofer, A. Grupp, et al., Phys. Rev. A 33, 3519 (1986). 24 M. Mehring, P. Hofer, et al., Europhys. Lett. 6, 463 (1988). 25 H. Thomann and M. Bernardo, lsr. J. Chem. 32, 323 (1992). 26 A. Abragam and B. Bleaney, "Electron Paramagnetic Resonance of Transition Metal Ions." Oxford Univ. Press (Clarendon), Oxford, 1970.

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where S and I are electron and nuclear spin angular momentum operators. The properties of this spin Hamiltonian and the spectra that it defines have been discussed in several general texts on EPR spectroscopy. 26-3° The first and second terms in Eq. (3) describe the interaction of the electron and nuclear spin, respectively, with the applied static magnetic field, H 0. These terms are known as the electron and nuclear Zeeman interactions, respectively. The nuclear Larmor frequency is given by vn = gnflnHo/h, where gn is the nuclear g factor which depends on the chemical identity of the nucleus, fin is a fundamental constant known as the nuclear Bohr magneton which depends on the charge-to-mass ratio of the nucleus, and h is Planck's constant. The electron Larmor frequency is defined by a similar expression except that the electron g factor for a metal typically has a large anisotropic component so that the product of g and H 0 is expressed through a matrix equation as indicated in Eq. (3). The anisotropy of the g factor is a consequence of the fact that the spin-orbit coupling imparts an orientation dependence to the quantisation axis of the electron spin. The third term in Eq. (3) describes the magnetic interaction between the electron and a nucleus. The unpaired electron generates a magnetic field at the nucleus which vectorially adds to the applied static field to produce an effective local magnetic field. The local magnetic field can comprise an isotropic component arising from the finite unpaired electron spin density at the nucleus. This is also known as a Fermi contact interaction. A finite Fermi contact interaction indicates that a covalent interaction exists between the atom hosting the unpaired electron and the nucleus. This covalent coupling need not, however, be through a direct bond. It could also arise through an indirect bonding pathway because of electron correlation effects. Because of this ambiguity the isotropic interaction is sometimes referred to as a transferred hyperfine interaction. The hyperfine coupling is also composed of an anisotropic interaction which can comprise two contributions. One is the classic through-space dipolar coupling between the electron and nucleus. The second is a quantum mechanical effect arising from the distribution of the unpaired spin density in the molecular wave function as the electron in a transition metal complex is generally not well represented as a point dipole. This anisotropy is expressed by a matrix coupling for the interaction between the electron and 27 N. M. Atherton, "Electron Spin Resonance: Theory and Applications." Halsted Press (Wiley), New York, 1973. z8 W. Gordy, "Theory and Applications of Electron Spin Resonance." Wiley, New York, 1980. 29 j. E. Wertz and J. R. Bolton, "Electron Spin Resonance." Chapman & Hall, London, 1986. 3o j. R. Pilbrow, " E P R of Transition Metal Ions." Oxford (1991).

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nucleus. The energies of these hyperfine interactions can vary from a few kilohertz to tens of megahertz for ligand nuclei and to several hundred megahertz for the central metal nuclei. The hyperfine interaction with a nucleus of spin I causes each of the eigenvalues m s of S z to be split into (21 + 1) spin states labeled according to the eigenvalues m I . The allowed eigenvalues for the projection of Iz along the z axis are - I < mi < + I, where the mx differ by unity. In favorable cases, the hypeffine interaction can be observed as line splittings in the EPR spectrum. Allowed EPR transitions obey the selection rule lares] = 1 so that the splitting of each ms state into (2I + 1) levels will result in (21 + l) EPR transitions. ENDOR transitions are NMR transitions detected via an EPR transition. The hyperfine interaction generates a local magnetic field which produces a shift of the NMR resonance frequency with respect to the Larmor frequency. The frequency shifts depend on the relative orientation of the applied magnetic field with respect to the principal axis system in which the hyperfine matrix is diagonal. Expressions for the orientation-dependent ENDOR frequencies in randomly oriented transition metal complexes have been d e r i v e d . 26'31-33 In the coordinate system in which the g matrix is diagonal, the nuclear spin Hamiltonian can be written a s 33 3 ~(~n = ~ [( s ' ° A ) i i=1

(gnflnHohi)]Ii

(4)

where the nuclear spin operator I will have three components, I;, where the index i = 1, 2, 3 refers to the x, y, and z axes, respectively. The h i are unit vectors for the directions of the magnetic field expressed in spherical polar coordinates: ( h 1 , h 2 , h 3) = ( c o s qb

sin 0,

sin ~b sin 0,

cos 0)

(5)

where (0, ~b) are the polar and azimuthal angles that relate the magnetic field vector to the coordinate system in which the g-matrix is diagonal. The prime on S' indicates that S is expressed in the coordinate system in which the g matrix is diagonal. The term S' •A defines the local magnetic field arising from the hyperfine interaction. This local field will either add or subtract to the applied magnetic field depending on the eigenvalue, ms,, of S'. Expressing this orientation dependency by an orientationsl A. Schweiger, F. Graf, et al., Chem. Phys. 17, 155 (1976). 32 B. M. Hoffman, J. Martinsen, et al., J. Magn. Reson. 59, 110 (1984). s3 G. C. Hurst, T. A. Henderson, et al., J. Am. Chem. Soc. 1M, 7294 (1985).

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dependent hyperfine coupling, the nuclear frequencies for S = ½ are given by t,+ = t'n ± 4 [ a ( o , 6)1

v± = 41A(O,ch)[+ t,.

(2Vn>lal;I= 4) (2~. < Ia[; I = 4)

(6)

where the subscripts refer to the NMR frequencies in the two electron spin manifolds. The expressions in Eq. (6) indicate that for a nucleus with I = 4, two ENDOR lines will be observed, one for each of the two electron spin manifolds. These lines will be displaced either about the nuclear Larmor frequency or about one-half of the hyperfine coupling depending on the relative magnitudes of these terms as indicated in Eq. (6). For the simplified case of axial symmetry, the orientation dependence of the hyperfine coupling is given by

A(O, ~b) = [i=~l(j~=lAjigj) ] 1/2

(7)

where the indices i, j = 1, 2, 3 refer to the coordinate axes x, y, and z. In most cases the microwave pulses excite only a small region of the EPR spectrum centered about the magnetic field selected. 34This magnetic field position selects a subset of molecular orientations defined by the resonance condition: hv = gflH o where the orientation dependence of the anisotropic g factor is given by

g(O' dP) = [ ~ (gihi)2]

(8)

The number of molecular orientations which contribute to the ENDOR spectrum is minimum for values of (0, ~b) that correspond to a principal axes of the g matrix. This was first reported by Rist and Hyde 35 and is referred to as g factor orientation selectivity. Higher spectral resolution is observed in ENDOR spectra recorded at magnetic field values corresponding to those values of (0, ~b) which correspond to these principal axes. These improved spectral resolution is only realized, however, if the principal axes of the g and A matrices are collinear or if the largest components of the two principal axes systems coincide. In ENDOR spectra of noncrystalline materials, a single pair of lines will only be observed if the principal axes of the g and hyperfine matrices are collinear. Otherwise, a powder pattern line shape arising from the angular dependence indicated in Eq. (7) will be obtained. According to Eqs. (4) and (8), the 34 W. B. Mires, "Electron Paramagnetic Resonance," p. 263. Plenum, New York, 1972. 35 G. H. Rist and J. S. Hyde, 52, 4633 (1970).

J. Chem.Phys.

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powder pattern will also depend on the magnetic field position within the EPR spectrum at which the ENDOR spectrum is recorded. An additional complication arises if the nuclear spin angular momentum I > ½.Then the fourth term in Eq. (3) describing the nuclear quadrupole interaction is nonzero. The nuclear quadrupole interaction describes the interaction of a nucleus with I > ½with the electric field gradients at the nucleus. These electric field gradients arise from a nonspherical charge distribution caused by the atoms in the vicinity of the nucleus. The magnitude and orientation of these gradients are determined by the electronegativity and the geometric positions of the atoms surrounding the nucleus. 36 The nuclear quadrupole interaction is described by a nuclear spin selfcoupling. The anisotropy of the quadrupole interaction is described by the quadrupole tensor, P, which is traceless so that it is fully characterized by five independent components. These are usually taken as the magnitude of the quadrupole coupling, e2qQ/h, the quadrupole asymmetry, ~, and the three Euler angles, (a, /3, y), that describe the orientation of the quadrupole tensor with respect to the principal axes of the g matrix. The quadrupole coupling constant and asymmetry parameter are related to the tensor elements by

e2qQ = 2•(2• - l)Pzz (9) exx - eyy

"0 =

Pzz

The elements Pxx, eyy, and Pzz are the principal values of the quadrupole tensor P and are defined so that Iezzl >- Ieyyl >- Iexxl. The quadrupole interaction results in additional splittings of the ENDOR lines. If the magnetic field, H0, is oriented parallel to one of the principal axes and the g, A, and P tensors are collinear, the first-order ENDOR frequencies are given by VENDOR= IAil 2 - + v "+- 3 Ieil(2m ~ + 1)

(IAI > 2v, > 3leil) (lO)

Iail b'ENDOR =

2

+ 31eil(2m I + 1) + Vn

- - Z.

(]AI > 31e,I > 2v.)

where the A i and Pi denote the principal values of the A and P tensors along the axis i. The nuclear transitions in Eq. (lO) are assumed to increase in the quantum number m I, that is, m t --~ m I + 1. 36 E. A. C. Lucken, "Nuclear Quadrupole Coupling Constants." Academic Press, London, 1969.

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For an I = 1 nucleus such a s 1 4 N , four ENDOR transitions are expected. However, if the hyperfine splitting is resolved in the EPR spectrum, each mi state can be selectively excited. In this case, less than the 4 ENDOR transitions expected may be observed, depending on the mi levels involved in the EPR transition. A two-line ENDOR spectrum can be observed if the EPR transition connects the mi = 0 to mi = 0 states, whereas a four-line spectrum can be observed if the EPR transition connects either of the m I ----- - I states. A reduced number of ENDOR lines than the 41 expected can also be observed for nuclei with I > 1 if the individual EPR transitions can be irradiated. 3. Basic Principles of Sublevel Polarization Transfer A distinguishing feature of the pulsed electron nuclear multiple resonance experiments discussed in this chapter is that no Mx,y component of the electron spin magnetization is present when the rf pulses are active. We consider the effect of the rf pulses on the NMR transitions only as far as these NMR transitions affect the electron spin polarization M z . It is also possible to observe an ENDOR signal ifMx,y magnetization is finite during this time. 1°'13 Such experiments are known as coherence transfer pulsed ENDOR experiments. Interesting aspects of the quantum mechanical properties of magnetic dipole transitions have been observed by coherence transfer pulsed ENDOR. 13 However, coherence transfer ENDOR studies are experimentally more demanding than sublevel polarization transfer ENDOR. The line widths in coherence transfer are also generally broader than observed in polarization transfer experiments because of the short electron spin phase memory times. For these and other reasons, only polarization transfer ENDOR experiments have been applied in metalloprotein studies. We now describe the details of pulsed ENDOR experiments where the transverse components of the electron spin magnetization Mx,y -- 0 during the polarization transfer step of the pulse sequence. A simple four-level system will suffice to describe the basic principles of sublevel polarization transfer techniques. The sublevel polarization transfer principles described in this section are the basis for all pulsed ENDOR experiments. These are the pulsed analogs of the CW-ENDOR experiment. The principles of sublevel polarization transfer also form the basis for a wide variety of more complex pulsed electron nuclear multiple resonance techniques. 15-19,21-25 The two most commonly used pulse sequences for the sublevel polarization transfer experiment, shown in Fig. 1, were proposed by Mims s and Davies. 37 In both cases a single if pulse is applied to excite NMR 37 E. R. Davies, Phys. Lett. A 47, 1 (1974).

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I I I

tTw-q H-

i m NMR

b t~w--I b--

+gml

mA

b-~-q

NMR I

'+ Preparation I

"I

I

Mixing

~t

Detection

FIG. 1. Davies ENDOR (a) and Mims ENDOR (b) pulse sequences.

transitions. As shown below, this NMR transition corresponds to a transfer of spin polarization between two spectral positions in the EPR spectrum. In the pulse sequence proposed by Mires, the nuclear spin flip interferes with the refocusing of a stimulated electron spin echo. In the pulse sequence proposed by Davies, the nuclear spin flip modifies the intensity of an electron spin echo. In both cases, the reduction of the primary or stimulated electron spin echo is a consequence of sublevel polarization transfer. It is useful to divide the pulses into conceptual periods, as indicated in Fig. 1, similar to the procedure used in modern pulsed NMR spectroscopy. 2° In the preparation period, sublevel polarization is created from the electron spin longitudinal polarization. In the mixing period, this polarization is transferred between two positions in the EPR spectrum which are split by the hyperfine coupling. This time period could therefore more accurately be referred to as the polarization transfer period, but we retain the term mixing in order to be consistent with the NMR literature. The purpose of the detection period is self-explanatory; it is to observe the effects of the pulses on the spin system in the periods preceding the detection period. It is assumed in the discussion below that the reader is familiar with certain concepts in magnetic resonance, namely, (1) the concept of the rotating frame, (2) the response of the spins to pulsed excitation, (3) the

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concept of spin polarization, and (4) the concept of spin coherence. A comprehensive discussion of these topics can be found in any general text on magnetic resonance. 3.1. P r e p a r a t i o n P e r i o d : S u b l e v e l P o l a r i z a t i o n T r a n s f e r

In all the pulsed ENDOR experiments we consider in this chapter, one or two microwave pulses are applied in the preparation period to create a sublevel nuclear spin polarization. This sublevel nuclear spin polarization is created from the electron spin polarization owing to the equilibrium distribution of spins among the electron Zeeman spin energy levels. Because geflHo >>g n f l H o , the dominant contribution to the electron spin polarization is from the electron Zeeman interaction, AE = hv = g d 3 H o . At the magnetic field strengths at which ENDOR experiments are performed, the polarization arising from the nuclear Zeeman and hyperfine interactions is negligible compared to the electron polarization and is ignored. The ratio of spins in the upper, N u , and lower level, Nl, electron Zeeman spin energy levels is given by the Boltzmann relation: 1 - (gBHo/kT) = 1 - 8

Nu = N l e x p ( - g ~ H o / k T ) - - ~

(11)

where the total number of spins N = Nl + Nu. When the electron is coupled to nuclei, the polarization is distributed over the hyperfine sublevels in each electron spin manifold. Referring to the four-level system in Fig. 2, at the start of the pulse sequence the electron spin polarization is given by AP14 + AP23 = 8/2 + 8/2 = 8. Each box in Fig. 2 represents the electron spin polarization corresponding to 8/2.

a

E4~

b

c

\

E3

E2 E1 Preparation

Mixing

Detection

FIG. 2. Energy level diagram for the interaction of one-electron with one I = ½nucleus showing the transfer of spin populations during the preparation, mixing, andMetection periods of the Davies ENDOR sequence.

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Sublevel polarization will be created if at the end of the preparation period not all EPR transitions have been equally excited. In the case of single-pulse excitation, this is accomplished by selectively inverting the spin populations on a subset of EPR transitions. In our example of the fourlevel system, let us suppose that a microwave pulse selectively excites the EPR transition E1 ~ E4 in Fig. 2. For the present purposes, we can treat this transition as if it were an independent two-level system. If the excitation pulse applied to the EPR transition E~ ~ E4 is a rr pulse, the electron spin polarization for this effective two-level system will change sign. The polarization of the four-level system immediately after the selective microwave excitation pulse is shown in Fig. 2b. Notice that now API4 --k- ~ 2 3 = - 8 / 2 71- 8/2 = 0. An EPR spectrum recorded immediately following the selective excitation pulse would have one emission peak and one normal absorption peak. For the analysis of the ENDOR experiment, we examined the spin polarization in the hyperfine sublevels. Before the microwave preparation pulse, the spin polarization in both hyperfine sublevels is: AP~2 = 0, AP34 = 0. After the pulse, A P I z = - 8 / 2 and AP34 -- - 8 / 2 . The selective excitation of the microwave preparation pulse has resulted in a transfer of the electron spin polarization to sublevel spin polarization. We refer to sublevel spin polarization to distinguish from nuclear spin polarization. Sublevel spin polarization is more precisely described as longitudinal electron nuclear two-spin order. In contrast, nuclear spin polarization is not a function of the electron spin states. 17'2° The sublevel spin polarization has been referred to as nuclear spin alignment in order to avoid confusion with nuclear spin polarization. 13,15 Sublevel polarization can also be created using two microwave pulses in the preparation period, as shown in the pulse sequence of Fig. lb and first demonstrated by Mims. 8 One advantage of using a two-pulse excitation is that the microwave pulses need no longer correspond to selective EPR excitations. After a first thought it might appear that nonselective pulses could not produce sublevel polarization. In fact, no sublevel polarization is present immediately following the first pulse. However, if the pulses are 7r/2 pulses, then during the time z following the first pulse, the local magnetic field arising from the hyperfine coupling will cause the Mx,y components of the magnetization corresponding to the two spin packets (of the two EPR transitions) to accumulate different phase factors. After a time ~"this accumulated phase is stored along the z axis by applying the second ¢r/2 pulse. After this second pulse, the electron spin polarization, [APl4 + AP23] is modulated by the terms cos(A~'/2) cos(f~:'), whereas the periodicity of sublevel polarization, [API2 + AP34] is now given by sin(A~'/2) sin(f~:-). Note that the creation of sublevel polarization also requires a resonance offset, denoted by f~s. Note also that no sublevel

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polarization exists if the product Az/2 = mr, where n = 0, I, 2, etc. 8 These are known as blind spots in the Mims ENDOR experiment. 8 Their significance will become apparent below.

3.2 Mixing Period: Sublevel Polarization Transfer After the sublevel polarization has been created in the preparation period, an rfpulse is applied in order to transfer this sublevel polarization to another hyperfine sublevel. This transfer is accomplished by inducing a nuclear spin flip, that is, an NMR transition. We can treat the magnetization from the two sublevels, E 3 and E 4 , and E 1 and E 2 , as pairs of isolated two-level systems. The sublevel population difference AP34and AP~2 created in the preparation period then corresponds to longitudinal sublevel magnetization, Mz(34) and Mz~12) , for these sublevels. For the example in Fig. 2, we have selected the frequency of the rf mixing pulse to be on resonance with the sublevel transition E 3 ~ E 4. The rf pulse will transform the longitudinal component, Mz(34 ) of this sublevel magnetization to Mz~34) cos OR. The nutation angle, OR equals yneH2tp, where Yn is the nuclear gyromagnetic ratio,/-/2 is the rf magnetic field intensity, and tp is the rf pulse length. Note that the sign of the nutation angle can be positive or negative since ~n can be positive or negative. The factor e takes into account the enhancement of the nuclear transition rate owing to the electronic magnetic field at the nucleus. This electronic field arises from the hyperfine interaction, z6 When OR = 7r, Mz(34 ) is transformed from - M z ( 3 4 ) t o +Mz(34 ) . This corresponds to the transformation of (AP34) to -(AP34) as indicated in Fig. 2c. After an rf 7r pulse, the electron spin polarization is now AP~4 + AP23 = 0, while the net sublevel polarization is AP12 + z ~ 3 4 = 0. In this simple four-level system, this nuclear spin flip corresponds to the transfer of electron polarization from one EPR line (the transition El ~ E4) to the second EPR line (the transition E: ~ E3). An EPR absorption spectrum collected after the rf rr pulse is applied would show no absorption from either transition. In fact, this polarization transfer could in principle be detected by sweeping (with the microwave frequency) through the EPR absorption spectrum. In practice, it is easier to detect it from the change in amplitude of an electron spin echo.

3.3. Detection Period The transfer of polarization can in principle be detected by any method for observing the EPR transitions. This includes sweeping the EPR absorption such as in conventional EPR, observing the free induction signal using a single microwave pulse, observing the electron spin echo intensity,

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or directly reading the longitudinal magnetization. Direct detection of the longitudinal polarization requires special instrumentation. 38'39 Likewise, detection using low-power continuous wave excitation requires a more complex instrumental setup capable of performing both pulse and continuous wave EPR experiments. The most common method for reading out the electron polarization is to observe the amplitude of a primary electron spin e c h o 37 o r of a stimulated echo. 8 It is also possible to detect the FID following single-pulse excitation. 4° However, for samples with extremely broad inhomogeneous EPR lines such as is usually observed for transition metals in proteins and enzymes, the FID decays within the dead time of the receiver.

4. Amplitudes in Pulsed Electron Nuclear Double Resonance Spectra The amplitudes in the ENDOR spectra are determined by the transition probabilities that depend on the spin Hamiltonian parameters and also on experimental parameters that depend on the details of the experiment. In this section we describe how the various experimental parameters of the pulse sequence affect the amplitudes in the pulsed ENDOR spectrum. The experimental parameters selected for the preparation, mixing, and detection periods must each be considered for a quantitative analysis of the ENDOR amplitudes. In most cases the effects of the experimental conditions on the ENDOR amplitudes, EA(v+), can be considered separately for each pulse period so that the final amplitudes can be expressed by the following product relation: EA(v±) = P(A, Ap) M(~) D(A, Ap, z)

(12)

where P, M, and D represent the functions that determine the ENDOR amplitudes in the preparation, mixing, and detection periods, respectively. The arguments of the functions in each case indicate the dominant contributions to the ENDOR signal amplitude in each period. A more detailed analysis of each of these periods is discussed below, but first we must consider the more complex spectra usually encountered for frozen solutions of transition metals in enzymes and proteins. In this case, the discussion of the simple four-level model must be expanded to include the effects of inhomogeneous line broadening.

38 G. Whitfield and A. G. Redfield, Phys. Rev. 106, 918 (1957). 39 A. Schweiger and R. R. Ernst, J. Magn. Reson. 77, 512 (1988). 4o T. Wacker and A. Schweiger, Chem. Phys. Lett. 191, 136 (1992).

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4.1. lnhomogeneously Broadened Electron Paramagnetic Resonance Lines: Hole Burning One way to envision an inhomogeneously broadened spectral line is to consider the overlap of many pairs of lines, each arising from the EPR transitions in a four-level system, but where each four-level system is characterized by a slightly different hyperfine interaction. If a sufficient number of transitions overlap in the ESR spectrum, no individual lines will be resolved, and the overall line shape will approach a single line with a Gaussian line shape profile. In inhomogeneously broadened lines a saturation hole can be "burned" into the line shape by the microwave excitation pulse(s). The width, tp, of the microwave pulse determines its bandwidth in the frequency domain. For a square pulse, this frequency is roughly given by Ap ~- 1/tp. A hole will be burned into the line if Ap is less than the line width. The ability to burn a hole into an absorption line in magnetic resonance and in optical spectroscopy is in fact often taken as an indication that the absorption line is arising from inhomogeneous rather than homogeneous broadening mechanisms. Homogeneous broadening arises from lifetime broadening, as opposed to static (time-independent) broadening mechanisms which contribute to inhomogeneous line broadening. An example of a saturation hole burned into an inhomogeneously broadened ER line shape is illustrated in Fig. 3a. The spin packets at the center of the saturation hole are on-resonance and in the example of Fig. 3a are shown to have an inverted electron spin polarization following the excitation pulse. Those spin packets far away from the center of the saturation hole, that is, far away from the resonance condition, are not affected by the excitation pulse. The profile of the saturation hole describing the connection between the on-resonance spin packets whose polarization is inverted and the off-resonance spin packets whose polarization is unaffected by the pulse is determined by several factors. In practice, the transmitter pulses generally do not have ideal rectangular pulse shapes. The finite probe Q tends to broaden the sharp transitions of a rectangular pulse. Furthermore, spin dynamics mechanisms, such as spectral and instantaneous diffusion, also broaden the excitation profile. Although such spin dynamics mechanisms may not be significant on the short time scale of the microwave excitation pulse in the preparation period, they usually become significant over the longer time scale during which the rf pulse is applied. As result of these complications, the magnetization profile excited by a microwave pulse can to a first approximation be described by a Lorentzian function41: 4J W. B. Mims, K. Nassau, et al., Phys. Rev, 123, 2059 (1961).

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a

b

FIG. 3. EPR spectra of an inhomogeneously broadened line showing the saturation/ inversion " h o l e " after the preparation period in the Davies E N D O R (a) and the Mims E N D O R (b) pulse sequences.

P(A, Ap) = 4(v_+ -

Vn)2/[4(1.'+ -- Vn)2 + (2Ap) 2]

(13)

where P(A, Ap) represents the sublevel spin polarization and dip is the width of the Lorentzian hole. Under limiting conditions in which ideal ~r/2 and ~r pulses are assumed, the width can be related to the microwave magnetic field intensity: Ap = gefleHl/h, where H~ is the magnitude of the microwave magnetic field intensity. A hole can also be burned into the inhomogeneous absorption line using two microwave pulses in the preparation period, as shown in the pulse sequence of Fig. lb. The pattern of electron polarization created by the pulses is shown in Fig. 3b. For the two-pulse excitation, the polarization described by Eq. (13) [or Eq. (14)] is multiplied by the function cos(2zrAff) where Ai is the hyperfine coupling of the jth nucleus. 8 This creates a sawtooth pattern of electron spin polarization that is superimposed onto the saturation hole burned into the inhomogeneously broadened EPR.

4.2. Preparation Period: Hyperfine Contrast Selectivity In the case of the inhomogeneously broadened EPR line, the NMR transition in the mixing period corresponds to the transfer of polarization from within the hole burned into the EPR line in the preparation period

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to another part of the EPR spectrum. Thus, the sublevel transition, i.e., the polarization transfer, corresponds to a displacement, Ad , of the polarization within the EPR spectrum. If the displacement remains entirely within the saturation hole, no change in EPR signal intensity can be observed. For a preparation consisting of single-pulse excitation, this means that the displacement of the electron spin polarization must be greater than Ap/2. Of course polarization can only be transferred if the rf matches a hyperfine sublevel transition. This establishes the important criterion that A > Ap/2 in order to observe an ENDOR signal in the Davies E N D O R experiment. Nuclei with hyperfine couplings A ~ Ap/2 will not contribute to the E N D O R spectrum.16'37'48 Because the width Ap of the saturation hole determines the minimum hyperfine coupling that can be observed, the choice of the preparation pulse conditions imposes a selectivity on the magnitude of the hyperfine couplings that will be observed in the ENDOR spectrum. This is known as hyperfine contrast selectivity and can be used to suppress selectively E N D O R amplitudes for nuclei with small hyperfine couplings. As shown in Section 5, this can be useful if ENDOR lines from nuclei with smaller and larger hyperfine couplings overlap in the ENDOR spectrum. The effect of the hyperfine contrast selectivity mechanism on the amplitude of the Davies E N D O R lines can be placed on a more semiquantitative basis by substituting the expressions for the hyperfine coupling (see Section 2) into Eq. (13). Substituting into Eq. (13) we have

P(A, Av) = A2/[A2 + (2Ap)2]

(14)

According to Eq. (14), P(A, Ap) decreases continuously from unity when Ap = 0 to P(A, Ap) = 1 when Ap ~ ~. The ENDOR signal intensity is determined not only by the excitation conditions in the preparation period but also by the excitation conditions in the detection period. The ENDOR signal intensity can be expressed by the product of P(A, Ap) with D(A, Ap) where D (A, Ap) represents the detection period. 47 While the explicit expressions for D(A, Ap) depend on the details of the detection pulses used, the functional form for D(A, Ap) is similar to P(A, Ap) but with an additional Ap in the numerator. For the present discussion, it is sufficient to note that D(A, Ap) increases from 0 when Ap = 0 to D(A, Ap) ~ 1 when Ap = ~ ~. The maximum ENDOR signal is obtained when the product P(A, Ap) D(A, Ap) reaches a maximum. 42'47'48 This maximum occurs at the crossover point between the decreasing function P(A, Ap) and the increasing function D (A, Ap) and is obtained when A = 2A~. The microwave excitation pulse intensity can therefore be adjusted to enhance or suppress E N D O R lines selectively depending on the magnitude of the hyperfine coupling. 42'47'48

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Note that because A = 2(v_+ - v,), the hyperfine contrast selectivity mechanism is not dependent on the type of nucleus, that is, on gn, but only on the difference between the ENDOR frequencies and the nuclear Larmor frequency. The mechanism for hyperfine contrast selectivity is therefore neither a homonuclear nor a heteronuclear suppression effect as has been suggested in the literature. 42 It is therefore not correctly described as a proton suppression technique. This is evident in the Davies ENDOR spectra of stellacyanin (see Section 6), where the proton ENDOR signals from the methylene protons on the thiolate ligand which have large A values are in fact enhanced and not suppressed under preparation pulse conditions that suppress the protons with small A values. The two-pulse excitation imposes an additional constraint for observing ENDOR signals. For a preparation period consisting of two-pulse excitation, the condition on the polarization displacement i.e., the maximum ENDOR effect, is obtained for z = (2n + 1)~r/A and is zero for r = 2 n r t / A where n = 0, 1, 2, etc. s The latter correspond to the blind spots in the Mims ENDOR experiment mentioned above. We see that for two-pulse excitation, the condition that A > AJ2 is relaxed, but at the expense of introducing blind spots at periodic intervals in the hole burned into the EPR line. The Mims ENDOR experiment offers excellent sensitivity for detecting ENDOR lines from nuclei with small hyperfine interactions. In the Mims ENDOR experiment, the interpulse delay time z determines the relative amplitudes of the ENDOR transitions. The amplitudes of weakly coupled nuclei are suppressed the least by choosing large values of z because this creates a sawtooth pattern where the "teeth" are more closely spaced. A direct comparison between the ENDOR amplitudes in the Davies and Mims experiments recorded under similar microwave excitation pulse conditions is shown in Fig. 9 for a blue copper protein, stellacyanin (see Section 6. I). The Mires ENDOR spectrum (Fig. 9a) was recorded using microwave excitation pulses of 0.05/zsec and r = 0.30/zsec. This Mires spectrum may be contrasted with the Davies ENDOR spectrum (Fig. 9b), which was recorded with a similar microwave excitation pulse width. Note that the weakly coupled protons dominate the Mims ENDOR spectrum, whereas the ENDOR signals from these protons are completely suppressed in the Davies ENDOR spectrum. The Mims ENDOR experiment is also more effective in detecting ENDOR signals from nuclei that have both small g. and small hyperfine coupling values. For example, an 14N nucleus with a very small hypertine coupling such that A ¢ 2Ap can be more readily detected by Mims ENDOR 42 p. E. Doan, C. Fan, et al., J. Magn. Reson. 95, 196 (1991).

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because the Davies experiment would require a very long preparation pulse length. In the Mims experiment short pulse widths can be used by choosing a long interpulse delay time, r. Weakly coupled laN nuclei have been detected in Mims ENDOR studies of model copper complexes 43 and for nitrogen ligands coupled to an iron-sulfur center in the enzyme hydrogenase. 44 A direct comparison of the NMR transitions detected by electron spin echo envelope modulation (ESEEM) and stimulated echo ENDOR spectroscopies has been reported 43'44 and is discussed in Section 8. One complication of the Mims ENDOR experiment involves the blind spots where no E N D O R effect is observed. On the other hand, these blind spots can aid in the assignment of hyperfine coupling values. The n = 1 blind spots are indicated by the arrows in Fig. 9a. The blind spots can be easily identified by recording several spectra using different r values. The upper bound on r will be determined by the electron spin phase memory time, which (at liquid helium temperature) is usually on the order of 2-3 /zsec for metalloproteins. The lower bound on the usable ~"is determined by the spectrometer dead time.

4.3. Mixing Period: Hyperfine Enhancement Factor The dependence of the ENDOR amplitudes on the polarization transfer step in the mixing period can be expressed by

M(v-+) = ½[1 - COS(0R_+)]

(15)

where OR_+ = yeffB2t~ is the Rabi nutation angle, 45 yeff is the effective nuclear gyromagnetic ratio in which the hyperfine enhancement factor is taken into account, B2 is the rf magnetic field intensity, and trf is the rf pulse length. The qualifier --- on R in OR_+refers to the two electron spin manifolds corresponding to the eigenvalues, ms. For simplicity, we drop this qualifier in subsequent discussions below. The effective nuclear gyromagnetic ratio is related to the nuclear gyromagnetic ratio by the hyperfine enhancement factor, e. For an isotropic hyperfine interaction, A, the enhancement factor is given by 26 e = I1 + msA/val

(16)

where v, is the nuclear Larmor frequency. For proton A values typically encountered in metalloenzyme ENDOR studies, the enhancement factor 43 E. J. Reijerse, N. A. J. M. v a n Earle, eI al., J. Magn. Reson. 67, 114 (1986). H. T h o m a n n , M. Bernardo, et ai., J. A m . Chem. Soc. 113, 5911 (1991). 45 I. I. Rabi, Phys. Rev. 51, 652 (1957).

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t~ Us) FIG. 4. Transient nutation patterns showing the Rabi oscillation frequencies at two positions in the ENDOR spectrum of stellacyanin at g = 2.10 and r = 0.23 tzsec. Other experimental conditions are as in Fig. 8b. The ordinate axis, AX, is the difference in EPR signal intensity with and without the rf pulse.

is in the range 0 < e < 2. Nitrogen nuclei directly bound to a metal ion can have large hyperfine interactions so that the enhancement is typically in the range 2 < e < 20. As discussed below, significant effects on the ENDOR amplitudes will be observed if e ~ I. The nutation of the sublevel magnetization can be observed using either the Davies or Mims ENDOR pulse sequences by either incrementing the rf magnetic field intensity in a stepwise manner or by incrementing the rf pulse length on successive pulse sequence iterations) 3'46'47 If the pulse length is incremented, the nutation of the nuclear magnetization will be superimposed on the time-dependent signal arising from the loss of the electron spin polarization. The latter is a consequence of the electron spin-lattice relaxation or spectral diffusion mechanisms. The effect of electron polarization decay during the mixing time can be removed by digital filtering or by subtracting the ENDOR signal obtained by repeating the Davies or Mims pulse sequence with the rf power set to zero on alternate pulse sequence iterations. 46 C. Gemperle, A. Schweiger, et al., Chem. Phys. Lett. 145, 1 (1988). 47 p. Hofer, "Development of Pulsed ENDOR and Applications to Polyacetylene." Ph.D. Thesis. Stuttgart, 1988.

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Exam[ales of transient nutation patterns showing the Rabi oscillation frequencies at two positions in the E N D O R spectrum of the blue copper protein stellacyanin are shown in Fig. 4. The first maximum in each waveform corresponds to the maximum polarization transfer, which is obtained when OR = rr. The sudden decrease in the signal intensity at the end of each waveform is a direct measurement of the signal baseline. When the baseline is directly measured, the advantage of measuring the transient nuclear nutation by subtracting the signal with rf power on from the signal with no rf power is that the absolute ENDOR enhancement can be measured. From Fig. 4 it is evident that the polarization transfer, and therefore the ENDOR amplitude, is reduced for OR < 7r and for OR > zr. However, the loss in signal amplitude is very small for OR > 7r. Note also that the Rabi oscillations are rapidly dampened for OR> ~'. This damping can arise from a combination of effects including nuclear spin relaxation during the rf pulse, rf magnetic field inhomogeneity, and dephasing caused by the destructive interference between signals arising from a distribution of E N D O R frequencies. The latter is expected if the ENDOR line is inhomogeneously broadened. Multiple Rabi oscillation periods can be observed if the E N D O R line is narrow and therefore sustains little or no inhomogeneous broadening. 16-18,48 Davies and Mims E N D O R spectra are typically recorded using a fixed rf power level and rf pulse length. ENDOR spectra are often recorded over a wide frequency range encompassing many different types of nuclei with a wide range of hyperfine coupling magnitudes. The frequency-dependent nuclear nutation angle arising from the hyperfine enhancement is then directly manifest in the relative ENDOR amplitudes. This becomes evident by considering the effective flip angles, 0Reft, for protons and nitrogen nuclei. Suppose the proton and nitrogen nuclear Larmor frequencies are 13 and 1 MHz, respectively. If OR = ~r at 13 MHz, then for a proton hyperfine coupling of 10 MHz, ORelf is equal to 0.627r and 1.387r in the two electron spin manifolds. Assuming the same rf pulse length and/42 intensity for the nitrogen as for protons, the flip angles for nitrogen are multiplied by TN/3'H ~ 0.07. For a nitrogen hyperfine coupling of 36 MHz, OReff is 1.237r and 1.37w, whereas for a nitrogen coupling of 18 MHz, ORelf is 0.58zr and 0.72zr. This is in good agreement with the approximately 2:1 intensity ratio observed for the two nitrogen E N D O R lines in the spectrum of stellacyanin shown in Fig. 8b. The intensity of the proton ENDOR lines for the 48 H. Thomann and M. Bernardo, "Pulsed ENDOR Methods for Metalloproteins and Enzymes." Annual Rocky Mountain Conference on Applied Spectroscopy, Denver, Colorado, 1990.

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thiolate methylene protons are lower than expected because of hyperfine anisotropy. The nitrogen and proton ENDOR lines are approximately equal in intensity for Davies ENDOR spectra recorded at gy as shown in Fig. 12b. 4.4. D e t e c t i o n P e r i o d

Although in principle a variety of schemes could be used to detect the electron spin polarization, in practice spin echo or stimulated echo detection is normally used for metalloprotein studies. The echo intensity depends on several factors, which can be expressed as D(A, Ap, ~-) = [Em(,r)][Emod(~-)]

(17)

4.4.1. Electron C o h e r e n c e Effects. The first term, Em(z), in Eq. (17) expresses the fact that the echo is detected at finite interpulse delay times, ~', during which the echo intensity will be reduced by irreversible dephasing mechanisms. The reduction in echo intensity is described by the phase memory decay time, Tm, which is defined as the time for the echo signal to fall to roughly 37% (e- 1) of its initial value at z = 0. In most metalloproteins, Tm is on the order of 2 to 3/zsec. The explicit consideration of the phase memory effects becomes important if Tm is not constant for all ENDOR lines in the spectrum. This could arise, for example, if ENDOR lines from more than one electron radical site contribute to the ENDOR spectrum and the radical sites do not have the same Tm values. 4.4.2. E l e c t r o n N u c l e a r C o h e r e n c e Effects. The second term, Emod(Z), in Eq. (17) expresses the fact that, if nuclear modulation is present, the spin echo envelope intensity may be a periodic function of the interpulse delay time, z. The observations of nuclear modulation of the echo envelope intensity requires the coherent excitation of both semiforbidden and allowed EPR transitions. 49 The echo envelope will be modulated with the periodicity of the v+, u_ ENDOR frequencies as well as the sum and difference of these frequencies. As the simplest example, we consider an electron coupled to a nucleus with I = ½. The echo envelope intensity is then described by 49

Emod('r) -=- 1 -- k/2 - k/2{cos(27rv+ ~') + cos(2~'v_ r) - ½cos[(27rv+ + 2~'v_)r] - ½cos[(27rv+ - 2Try )~']} (18) where v+ and v_ are the hyperfine frequencies in the two electron spin manifolds. The modulation depth factor, k, is given by k = [27ru,B/(21rv+)(27rv_)] 1/2

where B = (1/h)(gegd3d3,)(3 cos 0 sin O)/r 3. 49W. B. Mims, Phys. Rev. B 5, 2409 (1972).

(19)

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If the nuclei are not coupled to each other, the modulation for more than one nucleus is given simply by the product OfEmodfor the individual nuclei: Em~

= [Ij[Emod(/j)]

(20)

which contains the modulation frequencies for the individual nuclei as well as combination frequencies from the product. A quantitative description of the nuclear modulation of the electron spin echo envelope usually requires consideration of additional interactions such as the nuclear quadrupole interaction for nuclei with I > ½and must include orientational averaging and orientation selectivity effects. 5° Our focus in this chapter is not, however, a quantitative description of the nuclear modulation phenomenon but rather the manifestation of the nuclear modulation phenomenon in the pulsed ENDOR spectrum. 4.4.2.1. Hyperfine correlation. The most trivial situation that can be encountered if nuclear modulation is present is if all ENDOR lines in the ENDOR spectrum are equally modulated in amplitude for ENDOR spectra recorded for different ~" values of the spin echo delay in the detection period. A somewhat less trivial but important situation arises if only a subset of ENDOR lines are amplitude modulated by the electron spin echo envelope modulation function. 5~ This situation can be encountered, for example, if ENDOR lines from two different electron radical sites contribute to the ENDOR spectrum but only one of the radical sites has an additional nucleus giving rise to the nuclear modulation of the electron spin echo envelope. The nuclear modulation effect can in fact be used as a spectral editing technique as discussed in Section 9 on ESEEMedited ENDOR. 4.4.2.2. Partial excitation. In some cases it is possible that the microwave pulses in the detection period coherently excite both the allowed and semiforbidden EPR transitions associated with an NMR transition in one electron manifold while the ENDOR transition is detected for the NMR transition from the same nucleus in the second electron spin manifold. This situation can be encountered for protons, where the hyperfine interaction is large and of the order of A ~ 2vn . It is then likely that the one sublevel will give rise to electron spin echo envelope modulation (ESEEM), whereas the other sublevel will only be observed as an ENDOR transition. For example, the amplitude of the v+ ENDOR line in Davies ENDOR spectra recorded at several values of ~" will then be amplitude modulated with periodicity cos[2zr(v_)7]. If the modulation arises from more than one nucleus, then the relative amplitudes in the ENDOR spectrum can depend on the periodicity of the ESEEM frequencies for the 50 S. Dikanov, "Electron Spin Echo Spectroscopy." CRC Press, Boca Raton, Florida, 1992. 51 H. Thomann and M. Bernardo, Chem. Phys. Lett. submitted (1993).

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individual nuclei as well as on the combination frequencies that arise from the product function in Emoa-

4.4.3. Orientation Selectivity by Electron Spin Echo Envelope Modulation. The nuclear modulation effect can also influence the pulsed ENDOR spectrum by another mechanism. The spin echo modulation depth and frequency are functions of the anisotropic hyperfine interaction and of the nuclear quadrupole interaction i f / > ½. Usually the nucleus responsible for the nuclear modulation signal is not the same nucleus that is responsible for the ENDOR signal. Both nuclei can of course be part of the same paramagnetic molecule, with each nucleus described by a hyperfine and quadrupole interaction tensor. However, the orientation and magnitudes of the hyperfine and quadrupole tensors for the nucleus responsible for the nuclear modulation will in general not be the same as for the nucleus responsible for the ENDOR signal. As an example consider the simplified case that the electron-nuclear hyperfine coupling is purely dipolar. The ENDOR frequencies are then given by 27rv+ = [ ( B / 2 ) 2 + (Aft2 + 27rVn)2] 1/2 2try_ = [ ( B / 2 ) 2 + (Aal2 27"gl)n)2]1/2 -

(21a) (21b)

where Aa = (1/h)(g~ gj3d3n)(3 cos 2 0 - 1)/r3, r being the distance between the electron and nucleus and 0 the angle between the magnetic field H0 and the vector pointing from the electron to the nucleus, and the parameter B was defined in Eq. (19). Because the ESEEM spectrum and the echo envelope waveform are Fourier transform pairs, selecting a particular r value in the spin echo readout period corresponds to the selection of an orientation or range of orientations in the ESEEM spectrum. This selects a corresponding set of orientations for the nuclei contributing to the ENDOR spectrum. The orientation selected for each nucleus depends on the anisotropy of the hyperfine interaction and on the relative orientations of the hyperfine and quadrupole tensors.

5. Experimental and Instrumental Considerations In this section we describe some practical considerations concerning the performance of pulsed electron nuclear experiments. When properly designed, a spectrometer used for pulsed ENDOR experiments can also be utilized for electron spin echo experiments, particularly ESEEM spectroscopy. Experimental considerations for electron spin echo spectroscopy have been discussed by Mims. 33,s2,s3As we illustrate in this chapter, s2 W. B. Mims and J. Peisach, "Biological Magnetic Resonance." Plenum, New York, 1981. 53 W. B. Mims and J. Peisach, "Advanced EPR Techniques, Applications in Biology and Biochemistry." Elsevier, Amsterdam, 1989.

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tlator ~

~

~ero~

FIG. 5. Block diagram of a pulsed electron nuclear multiple resonance spectrometer.

several new experiments require the capability for performing both ESEEM and pulsed ENDOR experiments. A block diagram of a pulsed multiple resonance spectrometer capable of performing the experiments described in this chapter is shown in Fig. 5. Important experimental and instrumental considerations are described below.

5.1. Time Scales

It is essential that the EPR and NMR transitions occur on a time scale short compared to the electron and nuclear spin relaxation times, respectively. This criterion dictates the conditions for the time scales of the microwave (mw) and rfpulses. As discussed further below, this condition also affects the temperature at which experiments can be performed. The microwave pulses must be short compared to the electron spin phase memory time, Tm. Tm characterizes the decay of the transverse magnetization. Mx,y. Spin packets that have dephased during the time the pulse is applied will not contribute to the detected signal at the end of the pulse sequence. This has the effect of reducing the overall signal-to-noise ratio in the experiment. At liquid helium temperatures (4 K), the electron spin phase memory time, Tin, for transition metal ions with one unpaired electron (i.e., spin ½) in frozen solutions of metalloenzymes is determined by the random

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nuclear spin flip-flops of the abundant protons in the protein. This random field is fairly consistent from one protein to another so that the T~n value observed is consistently on the order of 2 to 3/xsec. The phase memory times rapidly decrease with increasing temperature. In most cases, the electron spin echo signal can only be observed up to about 25 K. For electron spin echoes to be observed, the microwave pulses should be on the order of a few hundred nanoseconds. To adjust the bandwidth of the hole burned into the EPR line by the preparation pulse, the microwave pulses should also be independently adjustable in time increments of 0.05 /zsec or less. This fine tuning of the width of the microwave pulses provides flexibility in experimental design. An example of where such pulse width adjustments are necessary arises when taking advantage of hyperfine contrast selectivity mechanisms as a spectral simplification method, as discussed in Sections 4 and 6 of this chapter. High resolution for the timing increments during the mixing period is not so essential for pulsed polarization transfer ENDOR experiments. Timing increments of 0.1/zsec are certainly adequate and are easily obtainable with, commercially available programmable timing sources. Shorter timing increment adjustments between the microwave pulses are, however, essential if the same spectrometer is to be used for ESEEM experiments or for some of the new combined ESEEM/ENDOR experiments described in this chapter.

5.2. Temperature The temperature is an important experimental parameter in all electron nuclear multiple resonance experiments. This is true whether pulsed or CW excitation techniques are employed. In both cases the EPR signal intensity is larger at lower temperature. At low temperature both a larger electron spin polarization is obtained and the spin relaxation rates are longer. However, the effect of temperature on the pulsed ENDOR and the CW-ENDOR signals is manifest in quite different forms. IN CW-ENDOR, the ENDOR signal intensity is determined by the ratio of electron and nuclear spin relaxation rates as well as on the electron-nuclear cross-relaxation rates. 4-6 The latter are transitions in which both the electron and nucleus change their spin state. The sensitivity to the details of the electron and nuclear spin relaxation rates in CW-ENDOR has two practical consequences. First, the intensity of the C W - E N D O R signal is a sensitive function of temperature. Because the electron and nuclear relaxation rates generally do not follow the same functional dependence with temperature, the observation of the

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CW-ENDOR signal is often restricted to a narrow temperature window. Furthermore, the relative intensities of lines within a CW-ENDOR spectrum are not simply related to the number of nuclei that contribute to the line. As already discussed earlier in this chapter, one of the primary advantages of employing pulsed excitation in ENDOR experiments is that the ENDOR enhancement is not dependent on this relationship between the electron and nuclear spin relaxation rates. However, the success of the pulsed ENDOR experiment does require electron spin phase memory times that are sufficiently long to observe a spin echo. It is also essential that the electron spin polarization created in the preparation period does not decay by spin-lattice relaxation or spectral diffusion mechanisms before sublevel polarization transfer can be detected. During the time interval between the preparation period and the detection period, the electron longitudinal spin polarization created in the preparation period will decay back toward thermal equilibrium. The rate of this decay is characterized by the electron spin-lattice relaxation time, Tie. Tie characterizes the lifetime of the spin in the excited spin state. The loss of electron longitudinal polarization by spin-lattice relaxation will reduce the EPR signal intensity observed in the detection period. This loss in signal intensity competes directly with change in the EPR signal intensity arising from the displacement of the longitudinal electron spin polarization due to sublevel polarization transfer. The loss of electron spin polarization during the mixing period results in a reduced signal-to-noise ratio in the ENDOR spectrum. For transition metal ions, Tie values decrease rapidly with increasing temperature. T~ values can range over many orders of magnitude in time, from less than nanoseconds at high temperature to minutes at low temperatures. The magnitude and explicit temperature dependence of Tie depend on many factors including the spin multiplicity of the ion, the electronic orbital degeneracy, and the ligand field of the coordination complex. Pulsed ENDOR experiments of transition metal ions are generally performed in the liquid helium temperature range, where T~e values are generally on the order of milliseconds or longer.

5.3. Sample Volume, Concentration, and Sensitivity In general, higher sample concentrations give higher signal intensities, but there are two factors that set the upper limit on the usable concentration levels. First, it may simply be difficult to concentrate the protein beyond a certain limit. Second, at higher concentrations

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the spin-spin interactions between metal centers may shorten the electron spin phase relaxation time so that electron spin echoes are more difficult to detect. The concentration at which these spin-spin interactions between metal ions can be observed depends on whether the metal ions are buried within a protein or are situated near the protein surface. Shorter Tm values can often be observed when concentrations of the metal ions exceed the range of a few millimolar. It is important to recognize that the shortening of the T~ values will be observed at concentrations far below those required to observe broadening effects in the EPR spectrum. Attention should also be paid to the possibility of aggregation on freezing. Aggregation can increase the possibility of spin-spin interactions. Sample volumes depend on the probe design. For most designs, sample volumes in the range of 100 to 300 t~l are optimum. We have found that it is particularly convenient if the probe accommodates samples in the standard 4-mm EPR sample tubes. The sensitivity of the pulsed ENDOR experiment depends in large measure on the width of the inhomogeneous EPR absorption spectrum, the electron spin relaxation rates, and the efficiency of the probe design. The width of the EPR line is important because only a small portion of the overall resonance line is sampled by the pulses at a given magnetic field setting. The excitation bandwidth of the microwave pulses samples roughly 0.5-10 G out of an EPR line which may be several hundred gauss or wider. The sensitivity that can be expected for metal ions or metal clusters with spin multiplicites greater than ½ is lower than for spin ½ samples because the EPR signal is spread over a greater magnetic field range. The effect of short electron spin relaxation rates has already been discussed. Short phase memory times reduce the electron spin echo intensity, resulting in a lower signal-to-noise ratio of the detection signal. The ENDOR signal intensity is a measure of the change in the echo intensity due to sublevel polarization transfer and is not a measure of the absolute echo intensity. Of course, if the echo is weak because of a short Tin, changes in its intensity will be more difficult to detect. Short T~e times compete directly with the sublevel polarization transfer to result in a reduction of the echo intensity. This results in a direct reduction in the ENDOR signal intensity. In practice, the ENDOR enhancement observed for copper proteins and copper enzymes, iron-sulfur proteins and enzymes, and hemes are in the range of 1 to 10% of the EPR signal intensity. This applies to experiments performed at roughly 2 K, with sample concentrations of roughly 1 mM and sample volumes of about 200/~1.

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5.4. Microwave Transmitter The short phase memory time observed for frozen solutions ofmetalloproteins and enzymes dictates many aspects of the microwave transmitter design. It is essential that microwave magnetic field strengths on the order of 10 G (in the rotating frame) be available at the sample. This is necessary so that the spins on-resonance can be rotated through a sufficiently large angle in a time short compared to Tm. As a guiding rule, the spins should be rotated by a ~" flip on a time scale roughly 10 times shorter than Tm. This sets the criterion Hi -> zr/YTm for the required microwave magnetic field strength. Probes used in electron spin echo and pulsed ENDOR must have a low Q value (see discussion of probes below), so the ability to deliver these high field strengths requires the use of high-power microwave transmitters. In most modern spectrometers this is achieved by amplifying a low-level microwave source signal to power levels between 100 and 1000 W using a traveling wave tube amplifier (TWTA). The configuration of the TWTA in the microwave transmitter is shown in the block diagram of Fig. 5. Alternate (and less expensive) methods of achieving the high microwave power levels such as the use of magnetrons is also possible. However, these alternatives are less desirable because of the poor pulseto-pulse stability and difficulty in generating phase coherence between microwave pulses. A second microwave source must be added for hyperfine selective ENDOR experiments. 2z Alternatively, the hyperfine selective ENDOR experiments may be performed using one microwave source and jumping the magnetic field between the preparation and detection periods. 21 Besides the ability to generate the appropriate microwave power levels, other important aspects of the microwave transmitter are the capability to control the phase of the pulse and the ability to generate pulses over a broad frequency range. Control of the phase provides the capability to phase cycle the pulses in a pulse sequence, The use of phase cycling techniques helps eliminate the contribution of spurious signal responses to the desired signal. At minimum it is desirable to have the capability to shift the phase by ~', but solid-state microwave components are readily available to also allow generation of ~-/2 phase shifts. The latter are essential if the spectrometer is also to be used for Fourier transform EPR experiments but are not critical for ESEEM or pulsed ENDOR experiments. 5.5. Probes The probe is one of the most critical components in the spectrometer. In pulsed EPR experiments, the probe must convert the power microwave

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delivered by the transmitter to magnetic field at the sample. In pulsed ENDOR and related multiple resonance experiments, the probe must also convert the high power rf pulses delivered from the transmitter to a magnetic field at the sample. The probe must have resonance modes in which the magnetic and electric fields are separated and must accommodate the sample in a region of maximum magnetic field and minimum electric field. The conversion of microwave power to magnetic field can be accomplished by using probes with high-Q resonant modes or by confining the magnetic field to a small volume at the sample. The first approach is commonly used in CW-EPR spectroscopy, where cavity structures with Q values of several thousand are used. High-Q structures cannot be used in pulsed EPR spectroscopy. High-Q structures will not admit the bandwidth of frequencies contained in the short microwave transmitter pulses. In addition, the microwave transmitter power in the probe must decay to levels below the signal level before the EPR signal can be observed. This decay time, known as the probe ring down time, must be short compared to the phase memory time, Tm. The time during which ring down occurs is known as the dead time of the spectrometer. The microwave power level in the probe decays as exp(-oJt/Q), where oJ is the microwave frequency. Mims has estimated that a decay of order 140 dB is necessary for the transmitter power to be reduced to the thermal noise power level. 34 This value can be used to estimate the spectrometer dead time resulting from the probe ring down time: t(ringing) ~ 14[ln(10)]Q/to ~ 32Q/to. For probes with Q values of the order of 100, t(ringing) is approximately 60 nsec, which is roughly a factor of 2 shorter than the actual dead time observed experimentally for many spectrometers. The microwave magnetic field at the sample is directly proportional to the product of the probe Q and the filling factor. The filling factor, ,/, is approximately given by the ratio of the sample volume to the total volume of the probe in which the magnetic field is finite. By the reciprocity relationship, the sensitivity is also determined by this relationship. The use of low-Q probes demanded for fast ring down can be compensated by increasing the filling factor. This is accomplished by confining the magnetic field to a small volume of space at the sample. Several probe designs with low Q values and large filling factors have been described in the literature. Mims designed a transmission cavity in which the sample is placed directly on the center conductor of the microwave transmission line. 8 It has the advantage of having the highest filling factor but is inconvenient to use, especially if the sample is air and temperature sensitive. Probe designs which accommodate standard 4- or 5-mm

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EPR sample tubes include the slotted tube resonator 47.54and the loop gap 55 and bridged loop gap resonators. 56 A modified slotted tube resonator was used in all experiments described in this chapter. An additional complexity introduced in pulsed multiple resonance experiments is that an rf circuit must also be present to deliver the rf magnetic field. Ideally, the rf circuit should not interfere with the performance of the microwave circuit. In practice, this is difficult to achieve. The presence of the rf elements usually degrades the performance of the microwave circuit. This is often manifest by slightly longer ring down times than would be obtained in the absence of the rf circuit. The coil can be wound on the outside or inside of the microwave shield. The former design has the advantage that it minimizes the interference with the microwave circuit but has the disadvantage that a lower rf field is obtained for a given transmitter power. The latter offers the highest power-to-field conversion factor but is more difficult to design without compromising the microwave circuit. The design of the rf circuit is in many respects much more simple than that required for NMR experiments. This is because the rf circuit only needs to deliver the rf power to the sample. Because the NMR signal is not detected, the Q value of the rf circuit is not critical. Indeed, the Q value must by necessity be extremely low to accommodate the extremely wide frequency range of the ENDOR spectrum. The rf circuit is in fact better described as a low-pass rf filter rather than by a Q-factor value. In practice some compromise must be made between the efficiency of converting rf power to rf magnetic field and the usable frequency range. Circuits with higher induction values generate higher rf magnetic fields but start to attenuate the rf input power at lower frequencies. At X-band EPR frequencies, the ENDOR of ligand nuclei, especially protons and nitrogen atoms, are observed in the range from 1 to 30 MHz. However, as is demonstrated in Section 6, the sensitivity in pulsed ENDOR experiments is sufficient so that ENDOR spectra from 1 to 200 MHz can be recorded. The ENDOR from central metal nuclei with large hyperfine splittings are observed in the higher frequency range.

5.6. Radio Frequency Transmitter The specifications for the rf transmitter are very similar to those of a typical high-power pulsed NMR transmitter for studies of solids. The detailed specifications for the rf transmitter will depend on the type of 54 M. Mehring and F. Freysoldt, J. Phys. E: Sci. Instrum. 13, 894 (1980). 55 W. Froncisz and J. S. Hyde, J. Magn. Reson. 47, 515 (1982). 56 S. Pfenninger, J. Forrer, et al., Rev. Sci. Instrum. 59, 752 (1988).

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multiple resonance capabilities desired. As a minimum for pulsed ENDOR experiments, the transmitter should be capable of delivering rf pulses of sufficient magnitude to rotate the nuclear spins in a time short compared to the time scale over which the electron spin polarization decays after the preparation pulse. The electron spin-lattice relaxation time, Tie, can be taken as a measure of this decay. It should be recognized, however, that the spectral diffusion of energy across the inhomogeneous EPR resonance line width can enhance the polarization decay by several orders of magnitude. As a conservative estimate, the NMR transition should occur in a time an order of magnitude shorter than T~e. This assumes that the nuclear phase memory relaxation time, T2n, is longer than 10T~e. Little direct experimental data o n T2n for paramagnetically coupled nuclei are available. Preliminary experimental data suggest that the T2n values for nuclei shifted away from the nuclear Larmor frequency by the hyperfine interaction are longer than for nuclei at their Larmor frequency. 16'~8 In the absence of other data, we assume for the current discussion that T2, is not the limiting criterion for the time scale in which the NMR transition must occur. If this is not the case, then the condition tp < 10T2, should replace the condition tp < 10Tie in the discussion that follows. Recalling that the nutation angle 0 is equal to ey,H2tp, it is clear that the rf power requirements will vary depending on the value of the hyperfine enhancement factor, e, as well as on the rf circuit design parameters. We can obtain an estimate of the required rf magnetic field at the sample by using our rule of thumb that tp -< 10TI~and a 7r pulse for maximum ENDOR enhancement, H 2 = 107r/eYnTle. Protons and nitrogen are two nuclei commonly observed in metalloenzyme studies. Assuming Tie = 10 msec, we find that for protons H2 = 0.738/e G, whereas for ~4N//2 = 10.2/e G. For protons with small hyperfine couplings, the H2 required is in fact rather modest, as fields of 1 G in the rotating frame are easily achieved. The inverse linear relationship between H2, TI~, and e means that the irradiating magnetic fields required will vary significantly with the hyperfine coupling and the TI~ value. In metalloenzyme studies at X-band microwave frequencies, the proton hyperfine coupling A is typically no larger than 2v~. Thus e is no greater than 2 for the NMR transition in one of the rns electron spin manifolds but no less than 0 for the NMR transition in the other electron spin manifold. If A is approximately equal to 2v, so that e = 0.01, an H 2 of about 75 G may be required for arr pulse. For a nucleus with a low gyromagnetic ratio, such as the ~4N nucleus, much larger H2 fields are required. 14N nuclei that coordinate metal ions typically have hyperfine couplings in the range 5 < A < 40 MHz depending on the type of metal ion and the details of the coordination structure. As

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a result, enhancements of up to e = 20 can be observed. The enhancement factor results in larger effective gyromagnetic ratios. This results in a significantly lower rf magnetic field requirement. In practice, magnetic fields of the order of 50 G can be easily achieved using a 500- to 1000-W rf amplifier. Larger field strengths can be achieved if the rf circuit is tuned to a narrower frequency range. Using a more modest rf power amplifier with an output in the range of 100 to 500 W, protons with small hyperfine couplings, A < 2v., are easily observable, whereas nuclei with low y values are easily observable if the hyperfine enhancement is sufficiently large. Proton ENDOR lines with A ~ 2v n can exhibit a pronounced amplitude asymmetry between the ENDOR transitions in the two electron spin manifolds. This is because the hyperfine enhancement reduces the effective y for the nucleus to an extremely small value. This amplitude asymmetry will also be observed in ENDOR spectra recorded at spectrometer microwave operating frequencies other than X-band. Of course, the relevant A value will depend on the value of 2v,, which is in turn determined by the spectrometer microwave operating frequency.

5.7. Choice of Radio Frequency and Microwave Operating Frequency Range It is highly desirable that the ENDOR spectrometer be capable of operation at several microwave frequencies. This is most useful to observe how the ENDOR frequencies shift between two magnetic field values but at constant g value. This information can be very useful for identifying the nucleus associated with given ENDOR lines in cases where many ENDOR lines overlap. The overlap between nitrogen with large hyperfine couplings (i.e., A > 2Vn) and protons with small hyperfine couplings (A < 2Vn) is effectively eliminated in ENDOR spectra recorded at Q-band microwave frequency. This overlap can also be eliminated in spectra recorded at X-band microwave frequency by suppressing the ENDOR signal for nuclei with small A values using hyperfine contrast selectivity (see Sections 4 and 6). However, this will also suppress the ENDOR signals from nuclei with small A values other than protons. Operation at other spectrometer frequencies is less effective as a method for identifying ENDOR lines from nuclei with small 3' values and large A values such as, for example, in distinguishing between 57Fe and 14N. In that case, another recently developed ENDOR method, hyperfine selective (HS)-ENDOR, described in Section 11.1, can be helpful in assigning overlapping ENDOR lines. The HS-ENDOR method is particularly useful for resolving overlapping lines from nuclei centered at A/2, that is, nuclei with hyperfine couplings A exceeding 2v,. Because

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the hyperfine coupling does not shift with applied magnetic field, operation at another microwave frequency will not be effective in removing the overlap. For the greatest versatility, the rf transmitter should be very broad banded, operating from 1 to 200 MHz. ENDOR from ligand nuclei are observed in the range from less than 1 to roughly 30 MHz at X-band frequencies, whereas proton ENDOR can be observed up to 70 or 80 MHz at Q-band frequencies. The ENDOR from the central metal nuclei can be observed at up to several hundred megahertz. The Cu ENDOR for a blue copper protein is an example discussed in Section 6. 6. Mims and Davies Electron Nuclear Double Resonance Studies of Metalloproteins The blue copper protein stellacyanin serves as a good illustrative model for demonstrating the pulsed ENDOR methodology discussed above. Copper ions in blue copper proteins are typically coordinated in a distorted tetrahedral geometry by two imidazole nitrogens from histidine residues, a thiolate sulfur from a cysteine residue, and a thioether sulfur from a methionine. 57-6° Stellacyanin is unusual among the blue copper proteins because the polypeptide is known to contain no methionine residues. 6~ Stellacyanin also exhibits several unusual properties for blue copper proteins, including the lowest redox potential among the blue copper proteins and a more rhombic EPR spectrum in contrast to the usual axial spectrum. Moreover, the protein exists in a reversible perturbed blue form between pH 8 and 1 1.5 that has slightly different properties but is still a blue copper protein. 61'62 The protein ligands in the high-pH form (pH > 8) of stellacyanin have been identified using pulsed ENDOR techniques.44 A spectrosopic model for the copper coordination structure of the high-pH form of stellacyanin is shown in Fig. 6. The electron spin echo detected EPR (ESE-EPR) spectrum of stellacyanin recorded at pH 11 is shown in Fig. 7a. ESEEPR spectra are recorded by plotting the ESE intensity while incrementing the magnetic field in a stepwide manner on successive pulse sequence iterations. Recording the EPR spectrum in this manner yields the absorption spectrum rather than the derivative (more correctly the first harmonic of the absorption) that is normally recorded on the conventional EPR 57 j. A. Fee, Struct. Bonding (Berlin) 23, 1 (1975). 58 E. T. Adman, R. E. Stenkamp, et al., J. Mol. Biol. 123, 35 (1978). 59 p. M. Coleman, H. C. Freeman, et al., Nature (London) 272, 319 (1978). 60 H. B. Gray and E. I. Solomon, "Copper Proteins." Wiley (Interscience), New York, 1981. 61 j. Peisach, W. G. Levine, et al., J. Biol. Chem. 242, 2847 (1967). 62 B. G. Malmstrom, B. Reinhammer, et al., Biochim. Biophys. Acta 205, 48 (1970).

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.•,(18.2) 'k

c

Rb ~4

N / ~,,.CIl

/~""N3('9.o)

,o,7

/, H

~.

S

1~1(42) /

(21.4) ~,'~

a R4

~

a J

II

./,------NS

(4.62)H,2

TM

/a

(, 8,

~H

FIG. 6. Spectroscopic model for the copper coordination structure in the high-pH form of stellacyanin. Numbers in parentheses are the hyperfine couplings in megahertz.

spectrometer. For easy comparison with the conventional EPR spectrum, the digital derivative of the ESE-EPR spectrum is shown in Fig. 7b. When the nuclear modulation of the electron spin echo envelope is deep, amplitude distortions in the ESE-EPR spectrum can be observed. In favorable cases, such amplitude distortions can be minimized by using microwave pulses with weak magnetic field intensities or by superimposing ESE-EPR spectra recorded at several values of the interpulse delay time. In spite of these potential complications, the ESE-EPR spectrum serves as a useful guide for identifying the position in the EPR spectrum at which the E N D O R spectrum is recorded. The pulse sequences for the Davies and Mims pulsed ENDOR experiments are shown in Fig. la,b, respectively. In both the Mims and Davies experiments, the E N D O R spectrum is recorded by stepping the rf frequency on successive pulse sequence iterations. This is necessary because the excitation bandwidth of the rf pulse is small compared with the width of the ENDOR spectrum.

6.1 Pulsed Electron Nuclear Double Resonance Spectroscopy of Proton and Nitrogen Ligand Nuclei The two Davies ENDOR spectra of the blue copper protein stellacyanin shown in Fig. 8 were recorded at the magnetic field position corresponding to g = 2.20 (approximately gz) in the EPR spectrum. The markedly different appearance of the spectra in Fig. 8a arises from the suppression of E N D O R lines from nuclei with small hyperfine couplings by the hyperfine contrast selectivity mechanism described in Section 4. The spectrum in Fig. 8a was recorded using a preparation pulse with a bandwidth ofroughly

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a

I

I

2600

2800

I

I

3000 3200 Magnetic FiNd (G)

I

3400

FIG. 7. ESE-EPR spectrum at 1.6 K of the high-pH form of stellacyanin (a) and its first derivative (b). Experimental conditions: Umw = 8.884 GHz, tmw = 0.02 and 0.04/~sec, r = 0.57/zsec.

2.5 MHz, whereas the bandwidth was 25 MHz in Fig. 8b. The ENDOR lines from protons with small hyperfine couplings, which are the stronger peaks centered around the proton Larmor frequency identified by Un in Fig. 8a, are not observed in Fig. 8b. In ENDOR spectra of transition metal ion complexes recorded at Xband microwave excitation frequencies, the ENDOR lines from protons with small hyperfine couplings often overlap with the ENDOR lines from other nuclei, particularly 14N.63 Protons with small hyperfine couplings include those on the ligand pendant groups coordinating the metal ion, on the solvent molecules, on the polypeptide backbone near the metal ion, and possibly on substrates coordinated to the metal ion. Other nuclei that may have an ENDOR line overlapping with these weakly coupled proton ENDOR lines must have large hyperfine couplings. The overlap of ENDOR lines from protons with small hyperfine interactions with ENDOR lines from nuclei with large hyperfine couplings can be eliminated by suitable choice of the preparation conditions as discussed 63 j. E. Roberts, J. F. Cline, et al., J. A m . Chem. Soc. 106, 5324 (1984).

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©

I

I

I

I

I

I

5

10

15

20

25

30

rf(Mnz) FIG. 8. Davies E N D O R spectra o f stellacyanin at g = 2.20 recorded under microwave inversion and detection pulse widths,/row, of 0.40, 0.20, 0.40/xsec (a) and 0.04, 0.02, 0.04 /~sec (b) illustrate hyperfine contrast selectivity (see text). Other experimental conditions: Vmw = 8.843 GHz, r = 0.52/zsec (a) and 0.51 /zsec (b), t~ = 5.50/xsec.

in Section 4.42,48E N D O R lines from protons with small hyperfine couplings can be suppressed if a narrow microwave pulse width is used in the preparation period. This suppression effect is illustrated in the Davies E N D O R spectrum of Fig. 8b. In the Davies ENDOR experiment, the necessity of using a finite preparation pulse width means that the amplitude for protons with very small hyperfine couplings will always be suppressed by the hyperfine contrast selectivity mechanism. In the Mims ENDOR experiment, the interpulse delay time r instead of the microwave pulse width(s) is the relevant factor that determines the relative amplitudes of the ENDOR transitions. ENDOR amplitudes from nuclei with very small hyperfine couplings can be observed by choosing large values of r in the preparation period. This creates a sawtooth pattern of electron spin polarization where the "teeth" are more closely spaced. The gradient of the polarization as a function of offset from the resonance center is then very large, so that small couplings can be detected without amplitude suppression. A direct comparison between the Davies and Mims ENDOR spectra recorded using similar microwave excitation pulse widths is shown

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I

I

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I

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5

l0

15

20

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30

rf(MUz) FIG. 9. M i m s E N D O R s p e c t r u m of stellacyanin obtained with tp = 0.05 ~ s e c and r = 0.03 ~ s e c (a) c o m p a r e d with the s a m e Davies E N D O R s p e c t r u m s h o w n in Fig. 8b (b). Other experimental conditions are identical.

in Fig. 9. The Mims ENDOR spectrum in Fig. 9a was recorded using microwave excitation pulses of 0.05/xsec and r = 0.30/xsec. This Mims spectrum may be contrasted with the Davies ENDOR spectrum in Fig. 9b, which was recorded with a similar microwave excitation pulse width. Note that the weakly coupled protons dominate the Mims ENDOR spectrum, whereas the ENDOR signals from these protons are virtually completely suppressed in the Davies ENDOR spectrum. One complication of the Mires ENDOR experiment are the blind spots expected at A i r = n, where no ENDOR effect is observed. On the other hand, these blind spots can aid in the assignment of hyperfine coupling values. The n = 1 blind spots are indicated by the arrows in Fig. 9a. The blind spots can be easily identified by recording several spectra using different r values. The upper bound on r will be determined by the electron spin phase memory time, which is usually on the order of 2-3/xsec for metalloproteins. The lower bound on the usable r is determined by the spectrometer dead time. The suppression of the proton ENDOR lines centered near the proton Larmor frequency reveals two broad lines centered at roughly 8.5 and

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17.5 MHz. and two somewhat narrower lines at 22.6 and 24.8 MHz. Based on the similarity of these ENDOR lines to those observed for other blue copper proteins, 63 the lines centered at 8.5 and 17.5 MHz are assigned to the imino nitrogens, denoted by N~ and N b in Fig. 6, on the imidazole ligands of protein histidine residues that coordinate the copper ion. For these nitrogen atoms, IAI -> 2Vn SO the ENDOR line is centered at A/2. The line splittings expected from the nitrogen Larmor frequency and from the quadrupole interaction (see Section 2) are not well resolved and serve only as line broadening mechanisms. The nitrogen Larmor splitting can, however, be observed under other experimental conditions (see below). At 9.5 MHz the laN ENDOR line also overlaps part of the proton ENDOR spectrum whose amplitudes have not been completely suppressed by the hyperfine contrast selectivity mechanism. The weak broad intensity between 12.5 and 14.5 MHz arises from the v+ branch of this residual part of the proton ENDOR spectrum. The v branch of the residual proton ENDOR spectrum overlaps the 14N ENDOR line centered at 8.5 MHz to give the asymmetric line observed. Based on comparative studies of blue copper proteins 63 and on isotope labeling studies, 64 the ENDOR lines at 22.6 and 24.8 MHz are assigned to the two-proton ENDOR transitions for the two methylene protons on a thiolate ligand. The thiolate methylene protons are identified as H~ and H~2 in Fig. 6. This assignment yields A = 18.2 MHz for H~1 and A = 21.4 MHz for H~2. The two ENDOR lines from the second electron spin manifold are expected at 3.0 and 1.3 MHz for H~1 and H~2, respectively. The intensity of these lower frequency ENDOR transitions are too small to be detected under the experimental conditions used to record the spectra shown in Fig. 8. The low intensity is a result of the small hyperfine enhancement factor (see discussion on amplitudes below). The amplitudes for both the ~4N and ~H ENDOR lines are also determined by electron nuclear coherence effects as discussed in Section 4. These effects are illustrated in Fig. 10, where two Davies ENDOR spectra of stellacyanin are shown. These spectra were recorded using identical experimental conditions except for the spin echo delay time in the detection period. The spin echo envelope modulation waveform recorded using the same microwave pulse conditions that were used in the detection period of the Davies ENDOR experiment is shown as an inset in Fig. 10. One pronounced difference between the two spectra in Fig. l0 is that the relative ENDOR amplitudes for the two methylene proton lines at 21.4 and 23.1 MHz are observed to depend on ~-. The low-frequency partners for these proton ENDOR lines are expected at 3.0 and 1.3 MHz, T. H. Stevens, C. T. Martin, et al., J. Biol. Chem. 257, 12106 (1982).

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~

159

4

5'

~ 10

1'5 rf (MHz)

' 20

2'5

30

FIG. 10. Davies ENDOR spectra of stellacyanin at g = 2.20 recorded at a two-pulse modulation minimum at r = 0.40 tzsec (a) and at a maximum at r = 0.51 tzsec (b) indicated in the inset. Other experimental conditions: T = 1.3 K, Vmw= 9.236 GHz,tmw= 0.03,0.05, 0.03 /xsec, trf = 2.00 ~sec.

respectively. F o r this small splitting, the m i c r o w a v e pulses used in the detection period have sufficient bandwidth to excite coherently the allowed and semiforbidden E P R transitions. If the nuclear modulation arises from the 1.3 M H z line, the minima and m a x i m a for the intensities of the 23.1 M H z line would be separated by roughly 0.38/zsec, which is consistent with the o b s e r v e d results. Another possibility is that the r - d e p e n d e n t amplitudes shown in Fig. 9 arise f r o m the excitation of semiforbidden E P R transitions associated with the 14N nucleus f r o m the remote amino nitrogen of the imidazole ligand. The shape of the 14N E N D O R line centered at 17.5 M H z (Fig. 10) is also a function of the echo delay time r in the detection period. This serves as an example of the E S E E M orientational selectivity effect discussed in Section 4. The 14N E N D O R line has been assigned to the histidine imidazole imino nitrogen, N] in Fig. 6. the dominant contribution to the modulation of the echo w a v e f o r m is f r o m the amino nitrogen, N~ in Fig. 6, on the same histidine imidazole ligand. The E S E E M orientation selectivity i m p r o v e s the resolution of the E N D O R line since a smaller n u m b e r of

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0.4-

0.2-

0.0"--- 1.52 MHz -"

-0.2 O

-0.4 -

-0.6-

-0.8-

_/ 10

•

2.38 MHz

-

4.62 MHz

I

I

I

I

I

I

11

12

13

14

15

16

rf (MI-Iz) FIG. 1 1. Davies ENDOR spectrum at g = 2.10 of stellacyanin centered around the proton Larmor frequency obtained by reducing the rf step size from that used in Fig. 8b.

nuclei corresponding to a subset of the hyperfine (and quadrupolar) orientations contribute to a given spectral range in the ENDOR spectrum. The protons with smaller hyperfine couplings can also be useful in identifying the protein residues that ligate the transition metal ionY A high-resolution Davies ENDOR spectrum of stellacyanin centered at the proton Larmor frequency recorded at the gy position in the EPR spectrum is shown in Fig. 11. The hyperfine splittings for the protons with small hyperfine couplings can be measured with high accuracy in pulsed ENDOR spectra. This is a direct benefit of the fact that ENDOR lines in spectra recorded using pulsed techniques are not susceptible to the line broadening or frequency shift effects frequently encountered in spectra recorded using CW excitation techniques. The spectral assignments of the four proton couplings of 4.62, 1.52, 2.38, and 0.87 MHz are shown in Fig. 11. Using the measured, proton 65 H. Thomann, M. Bernardo, et al., J. A m . Chem. Soc. submitted (1993).

17

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hyperfine couplings for the methine protons on the imidazole ring of Cu(II)[(imid)4] 66 a s a reference, the hyperfine couplings measured for stellacyanin can be assigned to the protons on the imidazole methine carbons in the histidine imidazole ring that is coordinated to the copper i o n . 67 This assignment procedure assumes that the mechanism for the delocalization of the unpaired electron spin density from the copper atom on to the methine carbon protons in imidazole ring is similar in the model compound and the copper protein. The unpaired spin density on the nitrogen is delocalized onto the imidazole ring through the tr-Tr interaction on the nitrogen. The magnitude of the hyperfine coupling for the nitrogen directly bound to the Cu(II) ion is then a measure of the unpaired electron spin density in the imidazole ring. The ~4N hyperfine couplings for the imidazole imino nitrogens in stellacyanin can then be used as a scaling parameter to calculate the expected proton hyperfine couplings for the methine protons in the imidazole ring. The measured values for the imino imidazole nitrogen hyperfine coupling in Cu(II)[(imid)4] is A = 40 MHz, whereas the proton couplings for the C-2 and C-5 protons are 5.4 and 1.7 MHz, respectively. Using these references values, the proton couplings calculated for the C2 and C-5 protons for the imidazole ring with the imino nitrogen A = 35 MHz in stellacyanin are 4.73 and 1.40 MHz, respectively. For the imidazole ring with the imino nitrogen A = 17 MHz, the calculated couplings for the C-2 and C-5 protons are 2.30 and 0.72 MHz, respectively. These values are within the experimental error of the measured proton couplings shown in Fig. 11. This good agreement between the assignment of the proton and nitrogen hyperfine couplings serves as an internal serf-consistency check for the assignment of the proton and nitrogen ENDOR lines to an imidazole ligand. 6.2. Pulsed Electron Nuclear Double Resonance Spectroscopy of Central Metal Nuclei In favorable cases, the hyperfine splittings from the central metal nucleus can be observed directly in the EPR spectrum. However, this is the exception rather than the rule for metals coordinated in proteins and enzymes. Furthermore, the selection rules for the EPR spectrum generally prevent the detection of the quadrupole interaction for central metal nuclei with spin I > ½. The advantage of the ENDOR technique is that both the hyperfine and quadrupole interaction for the central metal nucleus can be determined with high precision. This is possible even when the hyperfine 66 H. L. van Camp, R. H. Sands, et al., J. Chem. Phys. 75, 2098 (1981). 67 H. Thomann and M. Bernardo, in Biological Magnetic Resonance,Vol. 13. Plenum Press, N e w York (1993).

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splitting from the central metal nucleus is not resolved in the EPR spectrum. Pulsed techniques offer specific advantages over traditional continuous wave irradiation techniques in ENDOR studies of the central metal nuclei. These advantages are more readily appreciated when considering the general characteristics of ENDOR spectra for a central metal nucleus with spin I > ½. The large values of the hyperfine and quadrupole interactions spread the E N D O R lines over a wide frequency range in the ENDOR spectrum. In most cases, the ENDOR lines also tend to be broad. These characteristics of broad lines spread over a wide frequency range require high sensitivity and minimum spectral distortion for accurate measurement. These criteria are more readily fulfilled using pulsed techniques. The decoupling of the E N D O R signal from the detailed balance between the electron and nuclear relaxation rates results in spectra that are not frequency shifted or broadened by rapid passage effects. 69 In addition, the sensitivity for the detection of ENDOR lines with large hyperfine interactions is optimized by using preparation pulses with large excitation bandwidths (see Section 4). Significantly, these large excitation bandwidths in the preparation period do not introduce line distortion effects. Thus, pulsed methods are particularly well suited for studies of the central metal E N D O R especially if the hyperfine couplings are large such as for Cu(II) ions. Cu E N D O R spectra of stellacyanin recorded using the Davies pulse sequence are shown in Fig. 12. The wide ENDOR frequency sweep range, from 1 to 200 MHz, shown in Fig. 12 encompasses the proton and nitrogen as well as the Cu E N D O R signals. For the broad bandwidth excitation pulse used in the preparation period, the proton ENDOR lines for the imidazole ring protons do not contribute to the spectrum. However, the E N D O R lines for the two imino (i.e., directly coordinated to the cupric ion) nitrogens of the two histidine imidazole ligands and the proton ENDOR from the methylene carbon of the thiolate ligand have large hyperfine couplings and do contribute to the spectrum. These are the narrow lines (relative to the Cu ENDOR lines!) observed below 25 MHz in Fig. 12. The intensities of the nitrogen ENDOR lines for the imino imidazole nitrogen with A ~ 19 MHz in Fig. 12 are small owing to the short rf pulse widths used to record the spectra. The changes in intensity observed for the methylene protons in Fig. 12 is attributed to a combination of two effects. One is the anisotropy of the hypertine interaction. This anisotropy is manifest in the dependence of the amplitudes on the g value at which the E N D O R spectrum was recorded. Electron-nuclear coherence is the second phenomenon which can affect the relative intensities of the methylene proton E N D O R lines in Fig. 12. The electron-nuclear coherence is

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PULSED ELECTRON NUCLEAR MULTIPLE RESONANCE

I

I

I

50

100

150

163

200

rf(Mnz) F1G. 12. Cu E N D O R spectra of stellacyanin recorded using the Davies pulse sequence near gx at g = 1.967 (a), near gy at g = 2.088 (b), and near gz at g = 2.25 (c). Other experimental conditions: T < 1.3 K, Umw = 8.884 GHz, tmw = 0.04, 0.02, 0.04/zsec, t~f = 2.00/zsec, r = 0.55/~sec.

manifest as the nuclear modulation of the electron spin echo envelope in the detection period (see Section 4). The broad lines at frequencies above 30 MHz in the Davies ENDOR spectra shown in Fig. 12 are assigned to the ENDOR transitions from the Cu nucleus. This assignment is consistent with previous CW-ENDOR studies of stellacyanin.68 However, in contrast to the CW-ENDOR spectra, in the pulsed ENDOR spectra all ENDOR lines have the same phase, consistent with the assertion that rapid passage effects 69 do not affect the pulsed ENDOR spectrum. This is significant because rapid passage results in line broadening and frequency shifts that can yield inaccuracies in the hyperfine and quadrupole couplings deduced from the CW-ENDOR spectra. Recording of the 1H, IaN, and 63'65CuENDOR transitions in one widefrequency spectrum has the advantage of making possible a more direct comparison of the relative transition intensities for these nuclei. The Cu 68 j. E. Roberts, T. G. Brown, et al., J. A m . Chem. Soc. 102, 825 (1980).

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E N D O R transitions are very intense, actually comparable to the amplitudes of 'the strongly coupled nitrogen and methylene proton ENDOR transitions. As discussed in Section 4, a quantitative comparison of ENDOR amplitudes for the different nuclei must take into account the hyperfine contrast selectivity, the frequency dependence of the hyperfine enhancement factor, and the anisotropy of the g and hyperfine interactions on the ENDOR. For the cupric ion, the 63Cu and 65Cu nuclei, both with I = g, are expected to give rise to three ENDOR transitions in each of the two electron spin manifolds, so that up to six lines could in principle be observed in the E N D O R spectrum. The lines are rather broad, however, so that the ENDOR lines from the 63Cu and 65Cu nuclei are not separately resolved. At magnetic field strengths typical in X-band ENDOR studies, the hyperfine interaction is significantly larger than the nuclear Zeeman and quadrupole interactions. The Cu ENDOR transitions are therefore centered at [A[/2. Additional line splittings are expected from the nuclear Zeeman and quadrupole couplings (see Section 2). For I = ~, 41 = 6 lines are expected in the E N D O R spectrum. However, as seen in Fig. 12a, only two lines are resolved in the ENDOR spectra recorded at g = 1.967 (gx = 2.03). The value [A~[ = 176 MHz is obtained by assigning the center of the doublet to [A[/2. This is consistent with the hyperfine splitting observed in the EPR spectrum near gx. The splitting of the doublet is much larger than twice 2v c'' (which is approximately 7.30 MHz for 63Cu and 7.81 MHz for 65Cu at this magnetic field). This splitting is therefore assigned to the Cu quadrupole interaction which gives [Pxl = 5.8 MHz. The unresolved Cu Larmor frequency splitting results in the broadening of the two ENDOR lines centered at [A[/2 - 3[P[. Using a similar analysis, we find that [Az[ is 130 MHz and IPzl is 4,3 MHz for the ENDOR spectrum in Fig. 12c recorded at g = 2.25 (near gz)7. Comparison between Continuous Wave and Pulsed Electron Nuclear Double Resonance Studies The Davies and Mims ENDOR experiments are the pulsed analogs of the conventional CW-ENDOR experiment. In this section, we contrast the relative sensitivity of the two experimental approaches and discuss how the spectra obtained by the two methods depend on the experimental parameters and conditions. From the discussion in Sections 4 and 6, it would appear that spectra acquired using pulsed techniques are inherently more complicated because of the sensitivity to the detailed experimental parameters such as the microwave and rf pulse and timing conditions.

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These experimental conditions primarily determine the relative signal amplitudes in the ENDOR spectrum. In the CW-ENDOR experiment, an alternate set of experimental parameters determines the relative signal amplitudes. These include the magnetic field and rf modulation frequencies and amplitudes and the rf and microwave power levels. In both the pulsed and CW experiments, these parameters can be adjusted in order to define experimental conditions that enhance or suppress selected ENDOR lines. For example, in the CW-ENDOR experiment the use of large magnetic field modulation amplitudes can be used to suppress the ENDOR signals from nuclei with small hyperfine couplings. By far the major contrast between the pulsed and CW-ENDOR experimental approaches is a consequence of how the ENDOR signals, particularly the ENDOR amplitudes, depend on the electron and nuclear spin relaxation rates. The sensitivity of the CW-ENDOR signal to the detailed balance between electron and nuclear spin relaxation rates has been well documented. 5 This sensitivity has the consequence that the amplitudes in the CW-ENDOR experiment can in general not be related to the number of nuclei contributing to the ENDOR line. In some cases it has the consequence that the CW-ENDOR signal can only be observed over a narrow temperature range. Another important consequence is that the ENDOR frequencies, line shapes, and signal phases will be shifted if the rf sweep rate is comparable or greater than the spin relaxation rates. 69Rapid sweep rates are often used to enhance the signal-to-noise ratio since the sweep rate determines the signal acquired per unit time interval. 68 In the pulsed experiments, the process of electron and nuclear spin excitation and detection occurs on time scales short compared to the spin relaxation times. For coherent pulsed excitation of the EPR and NMR transitions, it is necessary that to~-2 ~ TleT2e and 092 - 2 <~ TlnTzn. Because the time scales are determined by the spin relaxation rates, these conditions require the use of intense microwave and rf magnetic fields. The power requirements for the microwave and rftransmitters in pulsed experiments have been discussed in Section 5. The use of these short, intense excitation pulses has the important consequence of decoupling the ENDOR signal from the detailed balance between the electron and nuclear spin relaxation rates. The essential criterion required to achieve this decoupling is the same as the criterion for coherent pulsed excitations. The use of pulsed excitations short compared to the spin relaxation times is also essential for separating the operations of creating polarization, polarization transfer, and detection. 69 M. Weger, Bell Syst. Tech. J. 39, 1013 (1960).

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The decoupling of the E N D O R signal intensity from the ratio of electron and nuclear spin relaxation rates has several important consequences. If the effects of hyperfine contrast selectivity, hyperfine enhancement, and nuclear modulation phenomena in the detection period are properly taken into account, the amplitudes in the pulsed ENDOR experiment can be related to the number of nuclei that contribute to the ENDOR line. Second, in the pulsed experiment, the phase of all ENDOR signals is always the same regardless of whether the central metal or ligand nuclei are observed. A third important practical consequence of this decoupling is that the experimental conditions which result in intense ENDOR amplitudes from central metal nuclei, such as from 63Cu, 65Cu, 55 Mn, or 57Fe, do not frequency shift or broaden the ENDOR lines. The latter two consequences are evidence of the reduced sensitivity to the rapid passage effects that are frequently encountered in CW-ENDOR studies of metalloenzymes and proteins. Unlike NMR, pulsed techniques in ENDOR do not, however, necessarily supersede CW techniques. It is not always possible to observe an FID or electron spin echo. This is the case if the electron phase relaxation time is short compared to the dead time of the spectrometer. In this case polarization transfer pulsed ENDOR experiments are not possible. This situation is typically observed for free radicals in the liquid state. It may then be possible to observe ENDOR signals using coherence transfer pulse techniques, 7° but the ENDOR lines acquired by coherence transfer techniques are severely broadened compared to the linewidths typically observed in CW-ENDOR of liquid state samples. Thus, CW and pulsed E N D O R techniques are more appropriately described as complementary rather than competitive techniques.

7.1. Continuous Wave Electron Nuclear Double Resonance Amplitudes In the limiting case of negligible relaxation effects, the amplitudes of the CW-ENDOR lines have been shown to increase with the square of the E N D O R frequency. 71 This result can be easily derived from the hyperfine enhancement factor in the limit of small sublevel Rabi nutation angles. The use of a low rf power level corresponds to a small Rabi nutation angle. The E N D O R amplitude, EA, is proportional to COS(0R). For small 0R_~, COS(0R_+)can be expanded in a power series

EA(vrf) = ½[1 - CoS(OR_+)] (22)

~--~0R_+2/4 ~ - ( m s A ) 2 / ( 2 V n ) 2 = {ms2/Vn2}(v+_ -

Vn)2

70 j. Forrer, S. Pfenninger, et al., Rev. Sci. lnstrum. 61, 3360 (1990). 71 L. R. Dalton and A. L. Kwiram, J. Chem. Phys. 57, 1132 (1972).

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The last equality explicitly indicates that the amplitude of the two ENDOR lines at v+ and v for a given I = ½nucleus are related by the square of the rf frequency.

8. C o m p a r i s o n b e t w e e n E l e c t r o n S p i n E c h o E n v e l o p e M o d u l a t i o n a n d Pulsed Electron Nuclear Double Resonance Spectroscopies

ESEEM and pulsed ENDOR are for the most part complementary techniques. In general, when one technique works well the other usually does not. As a consequence of this complementary nature, studies in which both ESEEM and pulsed ENDOR spectroscopies are utilized provide excellent opportunities for corroborating and cross-checking spectral line assignments. In particular, combination frequencies observed in the ESEEM spectrum are not observed in the ENDOR spectrum. The observation of electron spin echo envelope modulation requires that both allowed and semiforbidden EPR transitions are coherently excited. 49 The microwave power available at the sample to excite the EPR transitions is relatively small (of the order of 5-10 G in the rotating frame), so that semiforbidden transition probabilities are low when the magnitude of the hyperfine interaction exceeds roughly 30 MHz. 49 The maximum modulation depth in the ESEEM experiment is obtained under the conditions that the hyperfine and quadrupole interactions are of comparable magnitude and the hyperfine interaction cancels the nuclear Zeeman interaction in one of the electron spin manifolds. 72'73 Under these conditions the hyperfine anisotropy only broadens the ESEEM lines in this "canceled manifold" to second-order, resulting in narrow ESEEM lines. TM In contrast, these are in fact the conditions under which ENDOR is most difficult to observe. If the nuclear Zeeman field is exactly canceled by the hyperfine interaction, the NMR transition probability is zero, so that no ENDOR signal is observed. Furthermore, whereas the excitation of semiforbidden EPR transitions in pulsed ENDOR offers new experimental methodologies as demonstrated in this chapter, it is also a complication because only allowed transitions are necessary for the ENDOR experiment. ENDOR signals are in fact progressively more easily observed at higher radio frequencies. Thus, the techniques of ESEEM and ENDOR are truly complementary.

72 W. B. Mims and J. Peisach, J. Chem. Phys. 69, 4921 (1978). 73 H. L. Flanagan and D. J. Singel, J. Chem. Phys. 87, 5606 (1987). 74 E. J. Reijerse and C. P. Keijzers, J. Magn. Resort. 71, 83 (1987).

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The power of combining the methods of ESEEM and pulsed ENDOR spectroscopies is demonstrated 82 in a study of the oxidized hydrogenase enzyme. 75-77 Hydrogenase catalyzes the activation of molecular hydrogen according to the following reaction: H2 ~ 2H + + 2e-.75-77 The proposed site for the Hz oxidation and production in the Fe-only hydrogenase isolated from the anaerobic bacterium Clostridium pasteurianum is a novel FeS center that has been labeled the hydrogenase or H cluster. The rhombic EPR spectrum with g values 2.00, 2.04, and 2.10 and consistent with spin multiplicity S --- ½ observed for the purified oxidized hydrogenase enzyme has been assigned to the H cluster. The ESEEM spectrum obtained from the cosine Fourier transform of the three-pulse stimulated echo envelope recorded at the low-field extrema (gmax = 2.10) of the EPR spectrum is shown in Fig. 13B. The spectrum was recorded at the low-field extrema in the EPR spectrum to take advantage of the resolution enhancement afforded by g matrix orientation selectivity. 32'33'35'43'78'79 The line assignments shown in Fig. 13 were based on the analysis of the ESEEM spectra recorded at several microwave excitation frequencies. The ESEEM lines at 0.58, 3.37, and 3.95 MHz in Fig. 13 are assigned to the v0, v_, and v+ nuclear quadrupolar relaxation (NQR) transitions for a n 14N nucleus, N1, with nitrogen quadrupole parameters K = 1.21 MHz, ~ = 0.21. The magnetic field at which this ESEEM spectrum was recorded is close to the field at which the nuclear Zeeman field is canceled by the hyperfine field. At the cancellation condition, the three ESEEM transitions in this manifold are the pure 14N NQR transitions, v0, v_, and v+, from which the quadrupole coupling parameters can be directly calculated. The 14N quadrupole coupling constants are related to the ESEEM frequencies by v+ = K(3 + "0), v_ = K(3 - ~7), and v0 = 2KT/, where e2qQ/h = 4 K . 36 The exact cancellation is recognized in multifrequency ESEEM studies by the fact that the v+ ESEEM frequency reaches its minimum value. Near cancellation, the value of the quadrupole coupling deduced from the ESEEM spectrum will be accurate to within 10% for v, - ]AI/2 < 0.25v,, where ~'n = g n f l n n o / h ' 7 4 The peaks at 2.4 and 5.4 MHz in Fig. 13 arise from the second electron spin manifold in which the hyperfine field adds to the Zeeman field. The 75 J.-S. Chen, L. E. Mortenson, et al., "Iron and Copper Proteins," p. 68. Plenum, New York, 1976. 76 C. van Dijk, H. J. Grande, et al., Eur. J. Biochem. 1117, 251 (1980). 77 W. R. Hagen, A. van Berkel-Arts, et al., F E B S Lett. 201, 158 (1986). 7s H.-Y. Jin, "The Electron Spin Echo Studies of Metalloproteins." Ph.D.Thesis. City Univ. of New York, New York, 1989. 79 j. B. Cornelius, J. McCracken, et al., J. Phys. Chem. 94, 6977 (1990).

[6]

PULSED

ELECTRON

NUCLEAR

MULTIPLE

169

RESONANCE

ENDOR Frequency(MHz) 4

0.00

A

v(NI) I I

(D

-0.02

IiiI 11

-0.04

II

<0 I

I I I

~L

E

,<

I

IIV.+(N1) II II II II

-0.06 I vain =z(N1)

E

< nO a Z Lll

W W UJ

/.,

0

F 2

+'+if+" 1 4

~70

T 6

8

ESEEM Frequency (MHz) FIG. 13. M i m s E N D O R s p e c t r u m o f the oxidized form of hydrogenase I (A) and corresponding three-pulse ESEEM spectrum (B) close to the low-field edge at g = 2.09. Numerical simulation of the ESEEM spectrum from one of the nitrogens is shown in (C). Mims E N D O R conditions: T = 1.6 K , Umw = 9 . 0 7 6 G H z , tmw = 0.05 p, s e c , trf = 6 5 . 0 0 p,sec.

broader 2.4 MHz peak is assigned to the overlapping (u0, v_) ESEEM lines. In most cases, these ESEEM lines are usually broad and are not detected. In the present case they are most likely observed because of g value orientation selectivity. The ESEEM line at 5.36 MHz is assigned to the u+ or "Am~ = 2 NMR transition" from this second electron spin manifold. 8° The ESEEM line assignments for this nitrogen, N1, were further confirmed by numerical simulations in which g matrix orientation selectivity is explicitly incorporated. Using the quadrupole parameters obtained directly from the experimental spectrum, the simulated ESEEM spectrum, shown in Fig. 13c, quantitatively reproduces both the ESEEM frequencies and intensities. The ESEEM line at 3.87 MHz, identified as v'(N2) in Fig. 13, is tentatively assigned to a second nitrogen, N2, for which v, - IAI/2 ~> 80 The quotes are used to indicate that the ESEEM line does not actually arise from a A m ! = 2 N M R transition although it is formally equivalent to one.

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PROBES OF METAL ION ENVIRONMENTS

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0.25Vn SO that pure NQR frequencies cannot be observed. This assignment is based Qn the observed shift of 0.44 kHz/G, which is close to the gyromagnetic value for the nitrogen nucleus (0.31 kHz/G). The additional ESEEM line at 4.45 MHz is assigned to a sum (v' + v0) combination frequency between N1 and N2. This assignment was corroborated by the Mims ENDOR spectrum. The Mims ENDOR spectrum recorded at the same microwave excitation frequency and magnetic field position as the ESEEM spectrum is shown in Fig. 13A. The Mims E N D O R spectrum could be observed because at this magnetic field value the hyperfine coupling does not exactly cancel the nitrogen Zeeman field. The nitrogen ENDOR lines were not observed by conventional CW-ENDOR 81or in the Davies ENDOR experiment. As discussed in Section 6, the Mims ENDOR experiment is better suited for detecting the ENDOR signals from nuclei with such small hyperfine couplings. The spectral line assignments in the ESEEM spectrum can be corroborated using the Mims ENDOR experiment. The direct comparison between the two spectra in Fig. 13A,B also graphically illustrate the complementary nature of the two techniques. ENDOR amplitudes for ~4N nuclei with small hyperfine couplings are strongly attenuated owing to the low hyperfine enhancement factor. In contrast, the ESEEM intensity is largest when the hyperfine coupling cancels the nuclear Zeeman coupling so that the nucleus is effectively in zero magnetic field. This has the consequence (given other conditions equal) that ESEEM lines are more intense at the lower frequency range of the spectrum, whereas ENDOR lines are more intense at higher rf frequencies. Thus, the "Ami = 2 transition" at 5.4 MHz is much weaker in the ESEEM spectrum than in the ENDOR spectrum. The width of this peak is also determined by the hyperfine anisotropy and the orientation of the quadrupole tensor principal taxes with respect to the g tensor. The peak at 4.45 MHz in the ESEEM spectrum (Fig. 13B) was assigned above to the combination frequency v'(N1) + v0(N2). The presence of this peak in the ESEEM spectrum but not in the Mims ENDOR spectrum is consistent with its assignment as a combination frequency between the two nitrogens N1 and N2. If the conditions are favorable, the ESEEM experiment may have a distinct advantage of sensitivity compared to the ENDOR experiment. This may occur if the hyperfine and quadrupole interactions and the magnetic field conditions result in large amplitude modulation depths of the echo envelope. Under favorable conditions, the modulation depth can approach 100% of the echo intensity. In contrast, the ENDOR signal is sl j. Telser, M. J. B e n e c k y , et al., J. Biol. Chem. 262, 6589 (1987).

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usually less than 5% of the EPR signal intensity. For ligand nuclei with small gyromagnetic ratios and small hyperfine couplings, even smaller ENDOR enhancement will be observed. 9. Electron Spin Echo Envelope Modulation-Edited Electron Nuclear Double Resonance Spectroscopy The electron spin echo envelope modulation observed in the detection period of a Davies ENDOR experiment offers a new method for editing the pulsed ENDOR spectrum. The basic principles underlying this ESEEM editing of the ENDOR spectrum are the electron-nuclear coherence effects described in Section 4.4.2. In this section we demonstrate the utility of this new technique in a study of the iron-sulfur cluster active site in the enzyme hydrogenase. Some properties of this enzyme have been described in Section 8. The Davies ENDOR spectra for the oxidized enzyme enriched with 57Fe and recorded using two different ~" values in the spin echo readout are shown in Fig. 14a,b. The ENDOR line at 17.5 MHz observed in both spectra is assigned to the central metal 57Fe hyperfine interaction based on the observed splitting of 2v, for 57Fe. The ENDOR line at 4.5 MHz observed in Fig. 14b also has the 2v, splitting characteristic for 57Fe. However, this ENDOR line is not readily identified in Fig. 14a. The spin echo envelope waveform recorded at the same g value and under identical microwave excitation pulse conditions is shown as an inset in Fig. 14. The low-frequency modulation has been assigned to a protein nitrogen covalently coordinated to the H cluster as discussed in Section 8. The Davies ENDOR spectra in Fig. 14a,b were recorded for z values corresponding to the first minima and first maxima, respectively, in the echo envelope modulation pattern. The difference between these Davies ENDOR spectra is shown in Fig. 14c. The 57Fe line at 17.5 MHz vanishes in the difference spectrum, indicating that the intensity of this ENDOR line was identical to the Davies ENDOR spectra recorded at the two z values. In contrast, the 57Fe line at 4.5 MHz is observed in Fig. 14b and in the difference spectrum. A second line at 8.5 MHz with the 2v, splitting characteristic for 57Fe is also observed in the difference spectrum. This ENDOR line is not apparent in the spectra of Fig. 14a,b. One interpretation consistent with these results is that the 57Fe ENDOR lines at 4.5 and 8.5 MHz and the line at 17.5 MHz arise from two different FeS centers with overlapping EPR spectra. The intensity of the ENDOR lines for the two 57Fe nuclei with hyperfine coupling values A = 9.0 and 82 H. T h o m a n n , M. Bernardo, et al., J. A m . Chem. Soc. 113, 7044 (1991).

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PROBES OF METAL ION ENVIRONMENTS

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a

5

lO

15

20

rf (MHz) FIG. 14. Davies E N D O R spectra at g = 2.00 of the oxidized h y d r o g e n a s e e n z y m e enriched with 57Fe recorded with T = 0.39 p~sec (a) and 0.49 p~sec (b) in the spin echo readout

and their difference (c). The inset shows the positions in the two-pulse modulation waveform where the Davies ENDOR spectra were taken. Other experimental conditions: T = 1.3 K, Vmw= 9.085 GHz, tmw 0.10, 0.05, 0.10 p.sec, Trf = 17.00/zsec. =

17.0 M H z are o b s e r v e d to be modulated by the E S E E M frequencies arising f r o m an I4N nucleus coordinated to the FeS cluster. In contrast, the intensity of the E N D O R line for the 57Fe nucleus with hyperfine coupling value A = 35.0 M H z is not a function of these 14N E S E E M frequencies. It therefore a p p e a r s that one o f the two distinct FeS clusters are o b s e r v e d to be modulated b y the E S E E M frequencies arising from an 14N nucleus coordinated to the FeS cluster. Direct evidence corroborating this interpretation is in fact provided by pulsed E N D O R - i n d u c e d E P R spectra presented in Section 13 of this chapter.

10. Electron Nuclear Double Resonance-Edited Electron Spin Echo Envelope Modulation Spectroscopy The E N D O R - e d i t e d E S E E M experiment is a new method for assigning peaks in a complex E S E E M spectrum and for correlating the lines in

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ENDOR and ESEEM spectra. In the ENDOR-edited ESEEM spectrum, an rf mixing pulse in the Davies ENDOR experiment preselects the ESEEM lines that will contribute to the ESEEM spectrum. The technique can be particularly useful when ESEEM lines originate from overlapping EPR signals. The ENDOR editing of the ESEEM spectrum then provides a method for correlating the nuclei that separately contribute to the ESEEM and ENDOR spectra from the same paramagnetic molecule. The ENDOR-edited ESEEM spectrum is the Fourier transform of the difference between the echo envelope waveforms recorded in the detection period of the Davies ENDOR experiment. The difference waveform is obtained from the waveform recorded with rf power applied minus the waveform recorded with no rf power applied in the mixing period. The frequency of the rf pulse preselects the nucleus that will contribute to the ESEEM spectrum. The mechanism for this selection can be understood by considering the effect of the preparation and mixing pulses in the Davies ENDOR experiment on the spin population transfer in a four-level system comprising for example, a spin ½nucleus coupled to an electron. At the end of the mixing period, a hyperfine sublevel in which the longitudinal component of the sublevel magnetization has been transferred by an rf pulse will be of opposite polarity compared to the start of the mixing period. The mixing pulse therefore has the effect of changing the relative amplitudes of the allowed and semiforbidden EPR transitions arising from these hyperfine sublevels. This change in relative amplitudes is detected in the echo envelope modulation depth for the echo envelope waveform recorded in the detection period. If neither of the hyperfine sublevels in the two electron spin manifolds is on-resonance with the mixing pulse, the relative amplitudes of the allowed and semiforbidden EPR transitions associated with these sublevels will not be affected during the mixing period. Thus, only the ESEEM frequencies from the hyperfine sublevels affected by the NMR transition in the mixing period will be observed when taking the difference with rf power on and off on alternate pulse sequence iterations. This provides a convenient method of correlating ESEEM lines and ENDOR lines. An application of ENDOR-edited ESEEM experiments is shown in Fig. 15, where the two-pulse ESEEM time domain waveforms of the oxidized hydrogenase enzyme purified from Clostridium pasteurianum are shown. All three of the two-pulse ESEEM waveforms in Fig. 15 were collected with the microwave excitation frequency and magnetic field set to the high-field extrema of the EPR spectrum. The low-frequency modulation arises from an 14N nucleus coupled to the FeS cluster, known as the H cluster, as discussed in Section 8. The 57Fe Davies ENDOR

174

[6]

PROBES OF METAL ION ENVIRONMENTS

--3_._ II~

0.0

b

T

0.5

r

1.0

T

1.5 x ~s)

I"

2.0

T

2.5

FIG. 15. Two-pulse E S E E M waveform (a) and corresponding ENDOR-edited E S E E M waveforms with va = 5.10 MHz (b) and 18.0 MHz (c) for the oxidized hydrogenase enzyme enriched with 57Fe. Other experimental conditions: T = 1.3 K, g = 2.008, Vmw = 9.242 GHz, trnw = 0.10, 0.05, 0.10 /xsec, tff = 5.00 /xsec.

spectrum of the H cluster recorded at the high-field extrema of the EPR spectrum has been discussed in Section 9.57Fe ENDOR lines originating from three inequivalent iron sites are observed. The laN ESEEM lines can be correlated with the 57Fe ENDOR lines using the ENDOR-edited ESEEM experiment. In Fig. 15a the two-pulse spin echo envelope modulation waveform observed with no rf power applied is shown. This waveform is collected by recording the spin echo intensity as a function of the spin echo delay time, r, in the detection period. To compare directly the echo envelope amplitudes, all waveforms

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PULSED ELECTRON NUCLEAR MULTIPLE RESONANCE

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were recorded using a Davies pulse sequence with the same mixing period delay regardless of whether rf power was actually applied. In Fig. 15b, the difference waveform for the two-pulse spin echo waveform recorded with and without rf power applied in the mixing period and with the rf frequency set to 5.10 MHz is shown. The difference waveform is collected by gating the rf power off on alternative pulse sequence iterations. Note that the 14N modulation is clearly observed in the difference waveform. This indicates that the 14Nand 57Fenuclei must have a hyperfine sublevel in common. These 14N and 57Fe nuclei must therefore represent an EPR signal arising from the same FeS cluster. If this were not the case, then the 14N modulation depth would not be affected by the 57Fe NMR transition, and the difference spectrum would not show nuclear modulation. The difference waveform observed with the rf frequency set to 18.0 MHz is shown in Fig. 15c. The ESEEM frequency arising from the InN nuclear modulation is not observed in the difference waveform when the rf mixing pulse frequency is set to the 57Fe ENDOR line at 18 MHz. This means that the modulation depth is the same with and without the rf mixing pulse, so that the nuclear modulation vanishes in the difference waveform. This implies that the laN and 57Fenuclei do not share common hyperfine sublevels that can give rise to both the ESEEM and ENDOR frequencies. One interpretation consistent with these results is that the STFe nuclei associated with the two ENDOR lines arise from two distinct FeS clusters which have overlapping EPR spectra at the g value at which the ENDOR and ESEEM spectra where recorded. Evidence to support this interpretation is derived from the larger modulation depth in Fig. 15b compared to the depth in Fig. 15a. One method to compare the modulation depths between two echo envelope waveforms is to take the ratio of the relative minima, Imin, to maxima, /max, as defined in Fig. 15. The dotted horizontal line in each under each ESEEM waveform is the true baseline of the echo envelope waveform. The baseline is measured by stepping the receiver gating off of the electron spin echo signal. This can be identified by the abrupt step in the signal at the long time limit of the echo envelope waveform. Notice that the ratio Imin/Ima x is larger in the ENDOR-edited ESEEM waveform than in the ESEEM waveform recorded with no rf power applied. If the EPR signals from the two FeS clusters overlap at the magnetic field at which the ESEEM waveforms and ENDOR spectra were recorded, the normal two-pulse ESEEM waveform in Fig. 15a will have intensity from both FeS clusters, whereas the ENDOR-edited ESEEM waveform in Fig. 15b will only have intensity from the FeS cluster that also has the 14N nuclear modulation.

176

PROBES OF METAL ION ENVIRONMENTS

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An alternative interpretation of the change in modulation depth observed in the ENDOR-edited ESEEM waveform is also possible. The change in ESEEM modulation depth could in principle also arise from orientation selectivity effects. As discussed in Section 2, the echo envelope modulation depth vanishes for certain orientations (0 = 0, 7r/2) of the anisotropic hyperfine tensor. In principle, it is possible that the 57Fe NMR transition frequency at particular positions in the EPR spectrum accidentally coincides with an orientation of the ~4N hyperfine tensor for which the modulation depth vanishes. Whether this is the case can be established by recording the ENDOR-edited ESEEM waveforms at other magnetic field positions in the EPR spectrum. In the present case, however, the interpretation of the ENDOR-edited ESEEM data as arising from two distinct FeS clusters is also consistent with pulsed ENDOR-induced EPR spectra as discussed in Section 13 of this chapter. 1 1. Electron Nuclear Electron Triple Resonance: Two-Dimensional Electron Nuclear Double Resonance Studies A common feature of ENDOR spectra for metalloproteins is that the ENDOR lines are broad with significant line shape overlap. A variety of factors contribute to this broadening and overlap, including the anisotropies of the g factor, hyperfine, and quadrupole interactions, the number of different nuclei that contribute to the ENDOR spectrum, and the possible distribution of hyperfine and quadrupole couplings that may arise if the molecular sites are not magnetically equivalent. The latter is manifest as g strain and A strain and represents the distribution of the g and A values among the different molecular sites. 34 This broadening and overlap have the consequences that the ENDOR lines are often difficult to assign to particular nuclei and the hyperfine coupling values deduced from the ENDOR lines are less precisely defined. Under these circumstances the two-dimensional (2D) ENDOR techniques for generating hyperfine selective (HS) ENDOR and EPR subspectra can be used to great advantage. 2~'22In the HS-ENDOR experiment, an ENDOR spectrum is obtained for those nuclei with a preselected hyperfine coupling. In the EPR subspectra, the hyperfine coupling for a selected E N D O R line is directly determined without the need to first assign the ENDOR line to a particular nucleus. The 2D ENDOR experiments can be understood using the concepts of sublevel polarization transfer introduced in Section 3. The pulse sequence for the 2D ENDOR experiment is shown in Fig. 16. The preparation and mixing periods for the 2D ENDOR experiment are identical to the Davies E N D O R experiment. The microwave preparation pulse burns a

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PULSED ELECTRON NUCLEAR MULTIPLE RESONANCE

177

i

L

EPRI

m mA

EPR2

I- T -I NMR i

I

I I I

I I I

Preparation I

Mixing

I,

Detection

FIG. 16. Electron nuclear electron triple resonance pulse sequence used for 2D ENDOR experiments.

hole into the inhomogeneously broadened EPR line at positions Bp in the EPR spectrum, as shown in Fig. 17. The mixing pulse transfers the polarization at Bp to another position in the EPR spectrum indicated by Bo in Fig. 17. These two positions in the EPR spectrum are connected by a hyperfine coupling. In the Davies (or Mims) experiment, the polarization transfer is detected by measuring the change in electron spin polarization at Bp. In the 2D ENDOR experiment, the polarization transfer is detected by measuring the change in electron spin polarization at the new position in the EPR line denoted by Bd in Fig. 17. This can be accomplished by shifting the microwave frequency in the detection period from that used in the prepara-

FIG. 17. EPR spectra of an inhomogeneously broadened line showing the inversion " h o l e " at Bp after the preparation period and the position B d of the detection pulses.

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PROBES OF METAL ION ENVIRONMENTS

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tion period by an amount A as shown in Fig. 17. 22 Alternatively, it can be achieved by jumping the magnetic field between the preparation and detection periods. 2~The combination of exciting two separate EPR transitions and one nuclear hyperfine transition constitutes a triple resonance experiment or effectively a combined electron electron double resonance (ELDOR) and ENDOR experiment (i.e., an ELDOR-ENDOR experiment).

11.l. Hyperfine Selective Electron Nuclear Double Resonance Studies A polarization transfer from positions Bp to Bd in the EPR spectrum of Fig. 17 will only be observed if the frequency shift, A, matches the hyperfine coupling that separates the two EPR transitions. In a series of pulsed ENDOR spectra, each recorded using different A values, a hyperfine selective ENDOR spectrum will be observed whenever A -- A. When more than one nucleus contributes to the ENDOR spectrum, each HSENDOR spectrum comprises transitions from those nuclei with a preselected hyperfine coupling determined by A = Ai, where A i is the hyperfine coupling for the ith nucleus. Each HS-ENDOR spectrum is therefore a slice o f a 2D ENDOR spectrum where one axis corresponds to the sublevel mixing frequency, as in the standard ENDOR experiment, whereas the other axis corresponds to the difference frequency, A, between the preparation and detection frequencies. The spectral resolution along the A axis is determined by the excitation bandwidths selected for the microwave pulses in the preparation and detection periods. In the 2D ENDOR plot, the conventional ENDOR spectrum corresponds to the spectrum along the A -- 0 axis. It is the "inverted" image of the projection onto the A = 0 axis of all HS-ENDOR spectra recorded for A > 0. The HS-ENDOR spectrum is recorded in a manner analogous to the Davies ENDOR experiment except that the microwave frequency in the detection period is offset from the preparation period by A. Each HSENDOR spectrum, corresponding to a given A, is then recorded by incrementing in a stepwise manner the rf frequency of the sublevel mixing pulse on successive pulse sequence iterations. A sublevel polarization transfer is only detected for those nuclei for which A = A. Because the mixing pulse transfers "negative" electron spin polarization from position Bp to BO, the polarization transfer will be observed as a decrease in the EPR susceptibility at B 0. Transitions in the HS-ENDOR spectrum therefore have the appearance of an "emission" line rather than an "absorption" line as observed in the Davies ENDOR spectrum. The ENDOR spectra for the high-pH perturbed form of the blue copper protein stellacyanin serve as a good example of the power of the HS-

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PULSED ELECTRON NUCLEAR MULTIPLE RESONANCE

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a

b

C

d

5

I

I

I

I

I

10

15

20

25

30

,f(Mltz) FIG. 18. Davies ENDOR spectra for the native (pH 7) form (a) and the high-pH perturbed (pH 11) form (b) of stellacyanin. HS-ENDOR spectra with A = 19.0 MHz (c), 42.0 MHz (d), and 36.0 MHz (e) were used to assign the feature at 21 MHz in the spectra of the highpH form to a nitrogen. Other experimental conditions: T = 1.65 K, g = 2.07,/"mw 9.080 GHz (detection), tmw = 0.10, 0.05, 0.10/zsec, t~r = 8.00/.~sec. Reproduced with permission from reference 44. Copyright 1991 by the American Chemical Society. =

ENDOR technique. 44 The Davies ENDOR spectra (A = 0) for the native (pH 7) protein and for the high-pH perturbed ( p H I 1) form are shown in Fig. 18a,b, respectively. The two Davies spectra are identical except for the broad peak at 21 MHz observed in the high-pH form. This new ENDOR line is difficult to assign because of the strong overlap with the nitrogen line at 18 MHz and the proton line at 24 MHz. If the new line arises from a new proton cut'piing it would correspond to A = 20 MHz. If it arises

180

PROBES OF METAL ION ENVIRONMENTS

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f r o m a new nitrogen coupling it would c o r r e s p o n d to A = 42 M H z . The new E N D O R line at 21 M H z is not o b s e r v e d in the H S - E N D O R s p e c t r u m with A = 19 M H z , s h o w n in Fig. 18c, but it is o b s e r v e d when A = 42 M H z , as shown in Fig. 18d, indicating that it arises from a nitrogen with A -~ 42 M H z . The imino imidazole nitrogen with A -- 35 M H z is o b s e r v e d in the H S - E N D O R spectrum, shown in Fig. 18e, obtained for A = 36 M H z . B e c a u s e the two imidazole imino nitrogens and cysteinyl methylene protons are o b s e r v e d with identical frequencies and amplitudes at p H 7 and 11, the r e s o n a n c e at 21 M H z m u s t arise from a third nitrogen (i.e., a fourth ligand). A second illustration o f the 2D E N D O R e x p e r i m e n t is shown in Fig. 19. The Davies E N D O R s p e c t r u m for the 57Fe-enriched oxidized h y d r o g e n a s e e n z y m e isolated f r o m the bacterium Clostridium pasteurianum is shown in Fig. 19a. Properties of this e n z y m e h a v e been discussed in Section 8. The two lines in the Davies E N D O R spectrum, shown in Fig. 19a, at 4.5 and 17.5 M H z are assigned to 57Fe E N D O R lines based on the characteristic 2v, splitting observed. T h e s e c o r r e s p o n d to 57Fe nuclei with central

!

!

I

I

5

10

15

20

rf (MHz) FIG. 19. HS-ENDOR spectra with A = 0.0 MHz (a), 30.0 MHz (b), and 34.4 MHz (c) for the oxidized hydrogenase enzyme enriched with 57Fe. Other experimental conditions: T = 1.6 K, Vmw= 9.085 GHz, tmw = 0.10, 0.05, 0.10/~sec, trf = 17.00/~sec, ~ = 0.49 p.sec.

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metal hyperfine couplings of A = 9.0 and 35.0 MHz. A broad weak line centered at 8.5 MHz in Fig. 19a is also assigned to an 57Fe ENDOR transition. As discussed in Section 9, the intensity of this line depends on the echo delay time used in the detection period. The 57Fe ENDOR line at 17.5 MHz is partially obscured by overlap with the proton ENDOR lines. Furthermore, the amplitude asymmetry of the proton ENDOR lines on the high-frequency side of the proton Larmor frequency (vu -~ 13 MHz) suggests that an additional 57FeENDOR line may be overlapping with these proton lines. This is in fact confirmed in the HS-ENDOR spectrum with A = 30.0 MHz shown in Fig. 19b. A fourth 57Fe ENDOR line, identified by the characteristic 2v n splitting for the 57Fe nucleus, is observed in the HS-ENDOR spectrum shown in Fig. 19b. The complete line shape for the 57Fe ENDOR line at 17.5 MHz, which is partially obscured by the proton ENDOR lines in the Davies ENDOR spectrum in Fig. 19a, is completely resolved in the HS-ENDOR spectrum with A = 34.4 MHz shown in Fig. 19c. Thus, using a combination of the Davies, ESEEM-edite~l ENDOR, and HS-ENDOR experiments, a total of four magnetically inequivalent 57Fe ENDOR lines are identified for the active FeS cluster in the hydrogen-activating enzyme hydrogenase isolated from the bacterium Clostridium pasteurianum. It is interesting to contrast the HS-ENDOR experiment with other methods for reducing the overlap of ENDOR lines. In the discussion on hyperfine contrast selectivity above, we have already demonstrated how the ENDOR amplitudes from the weakly coupled proton ENDOR lines can be suppressed by the appropriate selection of the microwave preparation pulse width. Another approach is to record the ENDOR spectra at several microwave excitation frequencies. 83-85 Recording the ENDOR spectrum at Q-band EPR is an effective method for eliminating the overlap of nitrogen and proton ENDOR lines, as demonstrated for copper proteins by Werst et al. 85 The weakly coupled protons will shift to the higher ENDOR frequency centered about the proton Larmor frequency, whereas the strongly coupled nitrogen nuclei will remain centered at A/2. The greatest advantage of the 2D ENDOR experiment is in cases where nuclei with large hyperfine couplings overlap. In pulsed ENDOR experiments the hyperfine contrast selectivity mechanism is not effective in eliminating the overlap of strongly coupled nuclei whose ENDOR fre83 O. Burghaus, A. Toth-Kischkat, et al., J. Magn. Reson. 80, 383 (1988). 84 H. C. Box, "Radiation Effects: ESR and ENDOR Analysis." Academic Press, New York, 1977. 85 M. M. Werst, C. E. Davoust, et al., J. A m . Chem. Soc. 113, 1533 (1991).

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quencies are centered at A/2. Recording ENDOR spectra at higher microwave excitation frequencies is also not likely to reduce the spectral overlap since the nuclei with large hyperfine couplings will remain centered at A/2. Although a direct comparison between the pulsed 2D ENDOR and high-frequency E N D O R experiments has not yet been reported, it is likely that no one technique is the panacea for all spectral simplification problems. Because the 2D experiment is a pulsed experiment, it will have the advantage of being insensitive to the details of the electron and nuclear spin relaxation rates. The sensitivity of each HS-ENDOR spectrum is comparable to that obtained for the Davies experiment. However, the total experiment time required will be longer for the 2D experiment since it will be necessary to record HS-ENDOR spectra at multiple values of A. Another consideration is that implementation of the 2D experiment requires a relatively minor modification of the pulsed ENDOR spectrometer. However, the construction of a pulsed ENDOR spectrometer must be balanced against constructing a separate microwave probe and transmitter/receiver system for operation (either CW or pulsed) at the second microwave frequency. When the many parameters are taken into consideration, it is apparent that no one technique is the cure-all for all spectral simplification problems.

12. Pulsed Electron. Nuclear Nuclear Triple Resonance: Pulsed Double Electron Nuclear Double Resonance Studies Electron nuclear nuclear triple resonance experiments are ENDOR experiments in which a second rf irradiation field is applied. Two types of electron nuclear nuclear triple resonance experiments have been demonstrated in the continuous wave irradiation mode. 86 In one mode of the experiment, the two NMR transitions corresponding to the hyperfine sublevels in the two electron spin manifolds connected by the EPR transition are simultaneously irradiated by the two rf fields. This experiment, introduced by MObius et al.,86 who coined the term Special Triple resonance, primarily has the advantage of enhancing the sensitivity in the CW-ENDOR experiment. It also reduces the sensitivity of the CWENDOR amplitudes to the detailed balance of the spin relaxation rates. In the second type of electron nuclear nuclear triple resonance experiment, known as General Triple resonance, two nonequivalent nuclei are irradiated at their respective NMR transition frequencies. The experiment is performed by observing the intensity change for one ENDOR line 86 K. MObius and R. Biehl, "Multiple Electron Resonance Spectroscopy," p. 475. Plenum, New York, 1979.

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i I

t~w.,.I fl_

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EPR

i

V

NMR1

NMR 2 I I

Preparation I

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F16. 20. Pulsed implementation of double ENDOR.

while saturating the NMR transition of the second nucleus. There are two important advantages of this experiment: (1) the relative signs of nonequivalent nuclei can be determined, and (2) the connectivity of the nuclear sublevels can be determined. General Triple resonance was first demonstrated by Cook and Whiffen, who originally coined the term double ENDOR to describe the experiment. 87 Here we adopt the original nomenclature (double ENDOR) in order to avoid confusion with the electron nuclear electron triple resonance technique described in this chapter. The pulsed implementation of double ENDOR, shown in Fig. 20, was first demonstrated on an organic radical by Mehring et al. 13The frequency for one of the rf pulses Oil, identified as NMR 1in Fig. 20, is set to irradiate a selected ENDOR line. The frequency of the second rf pulse %2, identified as NMR 2 in Fig. 20, is then incremented on successive pulse iterations analogous to the experimental procedure in the Davies ENDOR experiment. Intensity changes in the ENDOR spectrum that may result from the irradiation at COl2are more easily identified by taking the difference spectrum, that is, the double ENDOR difference spectrum, between the ENDOR and double ENDOR spectrum. In principle, the ordering of the two rf pulses in the pulse sequence shown in Fig. 20 is not significant; the pulse with fixed frequency can precede or follow the pulse that is swept, or the two can be applied concurrently. This assumes, however, that relaxation rates are negligible or that the relaxation rates effect the longitudinal polarization of all hyperfine sublevels equally. 87 R. J, Cook and D. H. Whiffen, Proc. R. Soc. London 84, 845 (1964).

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ENDOR lines observed in the double ENDOR difference spectrum must have sublevels in common that are connected by the EPR and two NMR transitions. Thus, the observation of a peak in the double ENDOR difference spectrum indicates that the two ENDOR lines irradiated by the two rf fields must arise from two inequivalent nuclei coupled to the same electron. The relative sign of two hyperfine coupling constants, Ai and Aj, associated with these two inequivalent nuclei can be also be deduced from the double ENDOR difference spectrum. An increase in the ENDOR intensity, observed as an absorption line in the double ENDOR difference spectrum, indicates that A i and A~ are of opposite sign. A decrease in the ENDOR intensity, observed as an emission line in the double ENDOR difference spectrum, indicates that Ai and Aj have the same sign. An illustration of the pulsed double ENDOR experiment is shown in Fig. 21. The Davies ENDOR spectrum of the hydrogenase H cluster (see Section 8 for a discussion of the H cluster) is shown in Fig. 21a. The ENDOR lines centered at 4.5, 9.5, 14.5, and 17.5 MHz are assigned to 57Fe. As discussed in Sections 9 and 10, this assignment is based on the

I

I

I

I

5

IO

15

20

rf(Ml-lz) FIG. 21. Davies E N D O R s p e c t r u m for the oxidized h y d r o g e n a s e e n z y m e enriched with 57Fe (a) a n d double E N D O R difference s p e c t r u m with a second rf pulse at 17.8 M4-1z (b). O t h e r experimental conditions: T = 1.3 K, g = 2.008, Vmw = 9.214 G H z , tmw = 0.05, 0.03, 0.05/xsec, trfl = 10.00 g.sec, tn2 = 4.00/xsec.

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characteristic 2/,n splitting observed in spectra recorded in the Davies, HS-ENDOR, and ESEEM-edited ENDOR experiments. The ENDOR spectrum shown in Fig. 21a was recorded at the high-field extrema of the EPR spectrum. The pulsed double ENDOR difference spectrum observed when setting the fixed rf frequency to 17.80 MHz is shown in Fig. 2lb. When the two rf pulses in the mixing period of the pulse sequence in Fig. 20 are set to irradiate the same NMR transition, a decrease in the ENDOR signal intensity is observed because the second rf pulse effectively reverses the polarization transfer resulting from the first pulse. In this case the second rf pulse reduces the ENDOR signal intensity, which is observed as an emission line in the double ENDOR difference spectrum. This accounts for the negative peak at 17.80 MHz in Fig. 2lb. When the second rf pulse irradiates an NMR transition arising from the same nucleus but in the other electron spin manifold, the ENDOR signal intensity increases the analogy to the increase observed in the Special Triple experiment. This is observed as an absorption line in the double ENDOR difference spectrum and accounts for the positive peak at 16.85 MHz. Note that this peak is offset by 2vn from the peak at 17.80 MHz as expected if the two ENDOR lines arise from the same 57Fe nucleus. An additional weak and very broad negative peak is observed centered at 14.6 MHz. The observation of this broad peak at 14.6 MHZ suggests that the ENDOR line at 17.8 MHz has a level in common with the ENDOR line at 14.6 MHz. This implies that the two 57Fe nuclei must be coupled to the same electron and must therefore arise from the same FeS cluster. No change in the ENDOR intensities for the 57Fe lines at 4.5 and 9.0 MHz is evident in the double ENDOR difference spectrum. This suggests that the two 57Fe ENDOR lines do not share common hyperfine sublevels with the 57Fe line at 17.8 MHz. We conclude based on these results that the two low-frequency 57Fe ENDOR lines and the two high-frequency lines arise from two different FeS clusters whose EPR signals overlap at the magnetic field at which the ENDOR spectra are recorded. Further evidence for two overlapping EPR spectra is obtained in pulsed ENDORinduced EPR spectra as demonstrated in Section 13. 13. Pulsed Electron Nuclear Double Resonance-Induced Electron Paramagnetic Resonance Studies The ENDOR-induced EPR (EI-EPR) experiment, first demonstrated by Hyde, 88 generates an EPR spectrum by plotting an ENDOR transition 88 j. S. H y d e , J. Chem. Phys. 43, 1806 (1965).

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as a function of the applied magnetic field. It is a particularly useful technique for separating overlapping EPR spectra arising from different radicals or different molecular conformations of a radical. The technique takes advantage of the fact that the magnetic sites which generate the overlapping EPR spectra may have nonoverlapping lines in the ENDOR spectrum. Tracking the amplitude for one of the ENDOR lines as a function of magnetic field will generate the EPR spectrum for only that magnetic site. Using CW-ENDOR excitation, the EI-EPR spectrum has been recorded using one of two methods. One approach is to take the difference between the normal EPR spectrum and the spectrum recorded with an rf field set to an E N D O R frequency. 88 This requires the use of Zeeman field modulation and either amplitude or frequency modulation of the rf field. An alternative approach is to use a frequency-modulated rf field without Zeeman field modulation. 89 Using this single modulation encoding, the EI-EPR spectrum is displayed directly as an absorption spectrum. Pulsed excitation methods offer a new experimental approach for recording EI-EPR spectra. Because the EPR spectrum is recorded using spin echoes, we identify these spectra as EI-SE/EPR spectra to distinguish them from experiments in which the EPR signal is recorded using CW excitation techniques. The EI-SE/EPR spectrum can be recorded using either the Davies or Mims pulsed ENDOR schemes by tracking the amplitude of the E N D O R line selected by the radio frequency as a function of the magnetic field. In the EI-SE/EPR experiment, the EPR absorption spectrum is recorded directly without taking the difference of two spectra and without using any modulation encoding methods. An example of EI-SE/EPR spectroscopy is shown in Fig. 22. In Fig. 22a the SE/EPR spectrum for the hydrogenase H cluster is shown. The SE/EPR spectrum is recorded by tracking the electron spin echo intensity as a function of magnetic field. This generates directly an absorption display as opposed to the derivative display observed by phase detection when using magnetic field modulation encoding. If nuclear modulation effects and spin relaxation effects do not significantly affect the spin echo amplitude, the digital derivative of the SE/EPR spectrum corresponds to the standard EPR spectrum. The SE/EPR spectrum of the hydrogenase H cluster shown in Fig. 22a is qualitatively consistent with the rhombic spectrum with g values of 2.00, 2.04, and 2.10 observed by conventional CW-EPR. However, the SE/EPR line shape is not quantitatively consistent with a single rhombic 89 R. J. Cook, J. Sci. lnstrum. 43, 548 (1966).

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3100

3150

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3200 3250 Magnetic Field (G)

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187

Fie. 22. Spin echo EPR spectrum for the oxidized hydrogenase enzyme enriched with STFe(a) and corresponding ENDOR-induced SE/EPR (EI-SE/EPR) spectra with vre = 5.10 MHz and trf = 10.00/zsec (b), Vrf = 18.00 MHz and trf = 3.00/xsec (c), and Vrf = 24.00 MHz and ta = 3.00/~sec (d). Other experimental conditions: T = 1.3 K, Vmw= 9.214 GHz, tmw = 0.10, 0.05, 0.10 p, sec.

p o w d e r p a t t e r n . T h i s is m o s t e v i d e n t f r o m t h e a m p l i t u d e a s y m m e t r y in t h e high-field r e g i o n o f t h e s p e c t r u m . T h i s a s y m m e t r y c o u l d a r i s e f r o m n u c l e a r m o d u l a t i o n o r s p i n r e l a x a t i o n e f f e c t s . A l t e r n a t i v e l y , it c o u l d a r i s e f r o m t h e o v e r l a p o f a s e c o n d E P R signal. T h e s e p o s s i b i l i t i e s c a n be d i s t i n guished using EI-SE/EPR spectroscopy. The Davies ENDOR spectra of the hydrogenase H cluster recorded at t h e high-field e x t r e m a o f t h e E P R s p e c t r u m h a s b e e n d i s c u s s e d in

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Sections 9-12. Four magnetically inequivalent 57Fe sites with hyperfine coupling values of approximately 9.0, 17.0, 28, and 32 MHz can be identified using a combination of the Davies ENDOR, HS-ENDOR, ESEEMedited E N D O R experiments. The EI-SE/EPR experiment can be used to determine whether these four magnetically inequivalent iron sites arise from the same radical site or from more than one radical site. The EI-SE/EPR spectra shown in Fig. 22b-d were recorded using the Davies pulsed ENDOR sequence. The spectrum shown in Fig. 22b was recorded with the rf mixing pulse frequency set to irradiate the 57Fe ENDOR line at 5.10 MHz. Note that the amplitude asymmetry observed at the high-field region in the SE-EPR spectrum shown in Fig. 22a is not observed in the EI-SE/EPR spectrum shown in Fig. 22b. The EI-SE/EPR spectrum in Fig. 22b displays the powder absorption pattern expected for a rhombic EPR signal arising from a single radical site with electron spin S=½. A completely different line shape is observed in the EI-SE/EPR spectrum, shown in Fig. 22c, obtained when the rf mixing pulse frequency is set to irradiate the 57Fe ENDOR line at 18 MHz. The line shape of the EI-SE/EPR spectrum in Fig. 22c has axial rather than rhombic symmetry. The most plausible interpretation of these results is that the two 57Fe E N D O R lines at 5. I and 18.0 MHz arise from two different radical species which have overlapping EPR spectra. This interpretation is also consistent with the results from the several other pulsed multiple resonance experiments discussed throughout this chapter. The E N D O R frequencies are not constant as the magnetic field is swept in an EI-EPR or EI-SE/EPR experiment. The nuclear Zeeman frequency is a function of the magnetic field. The ENDOR frequencies are also field dependent if the g factor or hyperfine interactions are anisotropic. Because the ENDOR frequency is field dependent, the radio frequency cannot match the NMR transition across the entire EPR spectrum. If the radio frequency is not adjusted during the field sweep, pronounced amplitude effects can be observed in the EI-EPR or EI-SE/EPR spectra. For nuclei with a large gyromagnetic ratio such as ~H or 19F, significant amplitude effects will be observed for even narrow field sweep widths. For nuclei with a small gyromagnetic ratio such as ~4N or 57Fe, these effects become significant only if the magnetic field sweep range exceeds 100 G. This is apparently the case for one of the iron sites with hyperfine coupling A = 9 MHz that was irradiated to generate the rhombic powder pattern observed in the EI-SE/EPR spectrum shown in Fig. 22b. If the hyperfine anisotropy is large, significant amplitude variations can be observed even over a narrow field sweep range. This is apparently the case for the second iron site with hyperfine coupling A -- 35 MHz

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that was irradiated to generate the axial powder pattern observed in the EI-SE/EPR spectrum shown in Fig. 22c. Additional evidence for the greater magnetic field dependence of this 5VFe ENDOR line is obtained by recording the EI-SE/EPR spectrum with the rf mixing pulse frequency set to 24 MHz. Because no 57Fe ENDOR line is observed at 24 MHz in the ENDOR spectrum recorded at g = 2.00, no intensity is observed at the high-field edge in the EI-SE/EPR spectrum. However, an ENDOR signal is observed at 24 MHz for g > 2. The decrease of the EI-SE/EPR signal intensity with decreasing magnetic field observed in the spectrum of Fig. 22d originates from the frequency shift and broadening of the 57Fe ENDOR line observed with increasing g values. This suggests that the hyperfine interaction for this 57Fe nucleus is considerably more anisotropic than observed for the two 57Fe nuclei with smaller hyperfine coupling. This anisotropy is also evident in a series of Davies ENDOR spectra recorded at several magnetic field positions across the EPR spectrum. 14. Concluding Remarks The combination of the modern pulsed techniques in EPR spectroscopy with modern pulse techniques in solid-state NMR spectroscopy have already created a variety of new experimental methodologies for metaUoenzyme and metalloprotein studies. The impact that these new experimental methodologies will have in addressing problems in the biological sciences will certainly continue to expand as new laboratories enter the field. At the same time, new techniques and methodologies are currently under development that offer even greater and exciting potentials for structure and function elucidation. A potential limitation of both the crystallographic and spectrosopic methods discussed in this chapter are that the proteins must be in the solid state. For crystallographic studies, the protein must also obviously be in a crystalline form. For the spectroscopic studies frozen solutions can be studied and crystals are not necessary, but most experiments must be performed at low temperature. Structural data deduced from physical methods must be interpreted with the caveat that the data on the solid state must be related to the physiological state. Such a comparison can in fact provide insight into relevant physiological function. For example, a distribution of conformational states of the peptide side chains that coordinate a metal in the frozen solution state may reflect the conformational states dynamically accessed in the high temperature solution state.

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[7] C o n t i n u o u s W a v e E l e c t r o n N u c l e a r D o u b l e Resonance Spectroscopy

By CHRISTOPHER J. BENDER and PHILIP AISEN Introduction and History The development of the transistor in the 1950s generated intense investigation into the properties of semiconductors. In the early years of electron paramagnetic resonance (EPR) spectroscopy considerable interest therefore focused on silicon crystals doped with donor atoms from Group Va of the periodic table. The doping atom, phosphorus, arsenic, or antimony, brought with it an unpaired electron and a resulting EPR spectrum. Because the natural abundance of silicon-29 (29Si) is 4.7%, each unpaired electron sampled the nuclear moments of many silicon atoms, both nearby and remote. Resulting hyperfine interactions were often too small, compared to the EPR line widths, to be resolved and analyzed by conventional EPR spectroscopy. This difficulty was circumvented by Feher L2 in the seminal work introducing ENDOR (electron nuclear double resonance) spectroscopy. The great insight of Feher was that nuclear magnetic resonance (NMR) transitions, inaccessible to conventional NMR spectroscopy, could be detected by EPR. 1.2 ENDOR spectroscopy soon made its own transition from solid-state physics to biology with studies of a photoinduced free radical in N A D P H dehydrogenase 3 and the copper protein stellacyanin. 4 Although X-band EPR showed no interpretable features in the radical signal, the ENDOR spectrum clearly revealed a characteristic "matrix line" centered at the free proton frequency (near 14.5 MHz), with poorly resolved structure suggestive of electron coupling to ring and aliphatic protons. A strong line at 19 MHz could be attributed, by comparison to spectra of free ravin radicals, to protons of the freely rotating 8-methyl group of the ravin ring. Origin of the radical in the ravin cofactor rather than the protein fabric of the enzyme was thereby established. In stellacyanin, well-resolved lines between 16 and 20 MHz were found. Because of their relative invariance to 10% shifts in observing field, these lines were attributed to coordinated J G. 2 G. 3 A. 4 G.

Feher, Phys. Rev. 103, 834 (1956). Feher, Phys. Reo. 114, 1219 (1959). Ehrenberg, L. E. G. Eriksson, and J. S. Hyde, Biochim. Biophys. Acta 167, 482 (1968). H. Rist, J. S. Hyde, and T. Vf.nng~u'd, Proc. Natl. Acad. Sci. U.S.A. 67, 79 (1970).

METHODS IN ENZYMOLOGY.VOL. 227

Copyright © 1993by Academic Press, Inc. All rights of reproduction in any form reserved.

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nitrogen. This early inference has been supported by spectroscopic and modeling studies with comparisons to blue copper proteins of known structure, 5 although it has not yet been verified by X-ray diffraction because of persistent difficulties in crystallizing the protein. With the application of ENDOR spectroscopy to the study of heine proteins 6 and iron-sulfur proteins 7 the power of the method in revealing hyperfine interactions invisible to conventional EPR spectroscopy was clearly demonstrated.

Instrumentation and Technique General

Feher 8'9 proposed a polarization phenomenon based on the Overhauser 1° description of coupled electron and nuclear spin states. This phenomenon, which became known as ENDOR, was initially verified by employing an EPR technique, adiabatic rapid passage, 1~ commonly used to monitor spin-lattice relaxation rates. An adiabatic rapid passage experiment entails forward and reverse magnetic field sweeps through an EPR line, so that on successive sweeps the spin population is alternately inverted so long as the sweep rate per field cycle exceeds the spin relaxation rate. In establishing the ENDOR effect, Feher used a modified superheterodyne EPR spectrometer that was equipped with a rectangular TEl01 cavity, around which was wound a radio frequency (rf) c o i l . 12'13 The radio frequency circuit consisted of an oscillator, power amplifier, the coil, and a 50 ohm (fl) termination. The dispersive EPR signal was detected during adiabatic rapid passage without modulation, and spin population dynamics were determined from the relative intensities of the EPR line on successive sweeps. A pictorial representation of the experiment, adapted from Feher, 9

5 S. Wherland, O. Farver, and I. Pecht, J. Mol. Biol. 2114, 407 (1988). 6 C. P. Scholes, R. A. Isaacson, T. Yonetani, and G. Feher, Biochim. Biophys. Acta 322, 457 (1973). 7 j. Fritz, R. Anderson, J. Fee, G. Palmer, R. H. Sands, J. C. M. Tsibris, I. C. Gunsalus, W. H. Orme-Johnson, and H. Beinert, Biochim. Biophys. Acta 253, 110 (1971). 8 G. Feher, Phys. Rev. 103, 500 (1956). 9 G. Feher and E. A. Gere, Phys. Rev. 103, 501 (1956). l0 A. Overhauser, Phys. Rev. 92, 411 (1953). It F. Bloch, Phys. Reo. 70, 460 (1946). 12 G. Feher, Bell Syst. Tech. J. 26, 449 (1957). 13 G. Feher and E. A. Gere, Phys. Rev. 114, 1245 (1959).

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Hi

T~

_/ £ =::=b'

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Fl~. 1. Diagrammatic representation of Feher's original ENDOR experiment based on adiabatic rapid passage. H represents the swept dc magnetic field, dX '/dH is the EPR signal intensity, and ]msml) is a representation of the state diagram, where a, b, a', and b' represent the states of a system with S = ½and I = ½and filled boxes indicate most heavily populated energy levels. (Left) Conventional adiabatic rapid passage experiment; (Right) experiment varied by an NMR transition between states a' and b' before the second field sweep. (After Ref. 9.) describes the relationship b e t w e e n the spin dynamics and the o b s e r v e d signal (Fig. 1). In a later paper, F e h e r 13 described detection o f E N D O R f r o m its effect on the E P R signal o f a saturated portion of an inhomogeneously b r o a d e n e d r e s o n a n c e line. This m a n n e r o f performing E N D O R s p e c t r o s c o p y is now used almost exclusively, dynamical detection schemes being relegated to what is n o w k n o w n as dynamical nuclear polarization (DNP). The premise o f the E N D O R detection technique is that a portion of the E P R line is saturated, and hence the detected signal, which depends on the population difference b e t w e e n energy levels in resonance, is near zero b e c a u s e of spin population equilibration. A swept radio f r e q u e n c y field, by exciting transitions in nuclei coupled to the o b s e r v e d electron, depopulates one o f the states corresponding to the saturated E P R transition. This nuclear r e s o n a n c e is detected as an increase in the E P R signal intensity. The E N D O R s p e c t r u m is therefore r e c o r d e d as the change in E P R signal intensity (of a saturated line) as a function of the imposed radio frequency. F o r this r e a s o n E N D O R m a y be regarded as an E P R - d e t e c t e d N M R experiment. Modulation

Ordinarily, an E P R s p e c t r o m e t e r operates with phase-sensitive detection locked to a f r e q u e n c y that modulates the dc magnetic field. Poole 14 14c. P. Poole, "Electron Spin Resonance, A Comprehensive Treatise," 2nd Ed. Wiley (Interscienee), New York, 1983.

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describes the field modulation technique in detail, and some of the salient features are repeated here. Under conditions of field modulation the detector of the EPR spectrometer receives a signal input from a small band of the spectrum. The detected signal oscillates from that observed at H o - H mto that seen at H o + H m, where H o is the dc field and H m is the modulation amplitude. When the field is off-resonance, the front end of the diode detector oscillates between two equal power levels, which is therefore transparent to the phase-sensitive components of the remainder of the detector circuit. As the dc field is swept, a region of the spectrum is entered where microwave power is absorbed. In this region the power level detected at the diode varies during the modulation cycle, and this modulated power is detected by the phasesensitive circuitry. The signal level of the phase-sensitive detector increases to a maximum at the inflection of the absorption peak, then decreases to pass through zero at the midpoint of the line, reaches a minimum at the inflection point of the falling edge of the absorption peak, and finally returns to baseline as the traversal of the EPR line is completed. An analogous analysis of the dispersion mode of detecting an EPR resonance detects maximum modulated power at the midpoint of the EPR line. In an ENDOR experiment observations are made on a saturated line that is "collapsed" by equilibration of the ground and excited spin states. If field modulation is used as part of the phase-sensitive detection scheme, a change in signal level is seen as the rf sweep through the NMR transition depopulates one of the electron spin states and lifts the condition of EPR line saturation. The resulting enhancement usually amounts to 5-10% of the amplitude of the unsaturated EPR line. A 10% change in signal intensity at the midpoint of the EPR line is a larger quantity in dispersion than in absorption mode. For this reason the EPR signal is usually detected in dispersion mode during an ENDOR experiment in which field modulation is used. The present-day commercial standard, in the implementation by Bruker Instruments, Inc., is frequency modulation (FM) of the rf source prior to amplification. A frequency-modulated rf source undergoes a timedependent variation of its output frequency; such a source is defined by its carrier wave, fc, a frequency deviation or modulation depth, Af, and a modulation frequency, fro- The parameters are defined pictorially in Fig. 2A. As an example, a 10 MHz carrier frequency that is modulated with a +-100 kHz frequency deviation at a fixed modulation frequency of 12.5 kHz (as on the Bruker ENDOR accessory) will vary in output between 9.90 and I0.10 MHz during an 80/~sec cycle. Its spectrum will feature side bands at frequencies above and below the carrier frequency. These

194

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PROBES OF METAL ION ENVIRONMENTS

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side bands o c c u r as an infinite set (f~ -+ nfm[n = 1, 2, 3 . . . . ), although only a few will h a v e significant amplitude. The n u m b e r of significant side bands, and hence the bandwidth of the signal, depends on the modulation amplitude (i.e., deviation) and frequency. Bandwidth increases as f r e q u e n c y deviation increases or modulation f r e q u e n c y decreases, in a relationship often e x p r e s s e d as a modulation index, A f / f m. Line width distortions can result f r o m an i m p r o p e r f r e q u e n c y deviation (modulation amplitude or depth) or modulation frequency, m u c h as line width distortions can o c c u r in a conventional E P R

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experiment when the field modulation is improperly used. 14 One source of line shape distortion unique to FM is the derivation of sideband amplitude (i.e., power) from the carrier frequency. Excessive modulation amplitude or inadequate modulation frequency will burn a hole in the output band of the rf source, leaving only side bands. Sweeping through the ENDOR transitions with such a distorted rf source will, in turn, distort the observed line shape. Distortion of the ENDOR line often occurs prior to achieving the maximum signal intensity by varying Af. For this reason FM was rejected in favor of other modulation schemes in early ENDOR spectrometers. Amplitude modulation (AM) of the rf, described in the early literature,15'16 is more often used in conjunction with a second field modulation. ~7Phase modulation of the microwave field in conjunction with AM has been employed for powder ENDOR.18 As the name suggests, the amplitude of the carrier frequency is modulated by an imposed waveform, usually an audio frequency. The audio frequency is heterodyned with the rf carrier to give the modulated waveform (Fig. 2B), with percent modulation determined from the~ratio of the peak-to-peak amplitude of the modulation and carrier signals: percent modulation = 100 × Vp.p(audio)/Vp.p(rf). A modulation percentage in excess of 100 (overmodulation) will lead to gaps in the signal. Side bands corresponding to the sum and difference of modulation and carrier frequencies are produced during amplitude modulation. Because only two AM side bands are generated, an AM signal has an inherently narrower bandwidth than an FM signal with the same modulation frequency. A second difference between AM and FM is the power distribution over the signal band. With AM the power is divided among the carrier and two sidebands, and the power distribution is dependent on the percent modulation. At 100% modulation the sideband powers are maximal, each delivering one-sixth of the total output power. This differs from FM, in which overmodulation enhances side bands at the expense of carrier. Amplitude modulation tends to be used in ENDOR only as a secondary modulation in conjunction with field modulation because the double modulation permits measurement of the ENDOR signal despite events such as drift that might otherwise cause artifacts. Rejection of AM as a sole modulation technique can be rationalized on the following basis. With AM the amplitude of the signal varies sinusoidally, and at 100% modulation nuclear transitions are switched on and off at the modulation frequency. 15 W. T. Doyle, Rev. Sci. Instrum. 33, 118 (1962). 16 H. Seidel, Z. Phys. 163, 218 (1963). t7 D. S. Leniart, in "Multiple Electron Spin Resonance Spectroscopy " (M. M. Dorio and J. H. Freed, eds.) p. 5. Plenum, New York, 1979. is j. S. Hyde, T. Astlind, L. E. Goran Eriksson, and A. Ehrenberg, Rev. Sci. lnstrum. 41, 1598 (1970).

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The EPR detector level would then be oscillating between the power level of the saturated signal and the corresponding power of the normal EPR line when the condition of saturation is removed by the NMR transition. This, in theory, is a good feature for ENDOR, but the condition (27rfm) - l > Tl must still be met. For example, the Bruker ENDOR accessory operates with a modulation frequency of 12.5 kHz, so that AM ENDOR is constrained to spin samples with T1 shorter than 13 /zsec. The experimental limitation imposed by the audio frequency must be considered in studying the temperature dependence of an ENDOR line, since variations of T~ with temperature may result in T1 approachingfm -1. Amplitude modulation of an rf source in an ENDOR experiment may also result in baseline distortions. These distortions are presumably due to mechanical effects (e.g., microphonics) as the rf power is pulsed at high powers. For these reasons a double modulation scheme involving an audio AM and a low-frequency field or phase modulation is used; the second low-frequency modulation serves as a filter. A drawback in the use of Zeeman modulation for ENDOR is that the signal-to-noise ratio (S/N) is lost because signal is observed on only half of the cycle. Most test instruments and radio components are designed for operation at a fixed frequency. The ENDOR experiment, however, requires a modulated swept frequency. To achieve this, rf oscillators operating over discrete bands can be combined to cover a broad frequency range; a 0-100 MHz source might require several bands. The ENDOR spectral baseline may then distort as the rf source crosses two bands. The Illinois EPR Research Center (Urbana, IL) has devised a modified FM modulator based on a surface acoustic wave device that eliminates this distortion. 19 The modulator is compatible with standard frequency synthesizers and is available from the Illinois EPR Research Center.

Sensitivity Enhancement at Low Modulation Frequencies As already indicated, ENDOR experiments are usually performed with low-frequency modulation. Receiver noise figures of heterodyne and homodyne spectrometers are comparable at modulation frequencies of 100 kHz, but at lower frequencies a so-called flicker noise in homodyne receivers can overwhelm the normal diode noise. Flicker noise also arises from detector diodes in a manner inversely proportional to the modulation frequency. 18,20Noise in a homodyne receiver operating at 10 kHz may be four times greater than noise at 100 kHz. 2°

t9 R. B. Clarkson, R. L. Belford, and C. Reiner, Rev. Sci. Instrum. 61, 3356 (1990). s0 C. Hoentzsch, J. R. Niklis, and J. M. Spaeth, Rev. Sci. Instrum. 49, 1100 (1978).

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The problem of sensitivity loss owing to the low modulation frequency can be remedied by making a modification to the microwave bridge. 2°'21 This modification consists of inserting a low-noise preamplifier prior to the receiver. Normally, the microwave radiation from the klystron is fed via a circulator to the cavity and then on to the detector diode (i.e., receiver). A circulator is a three- (or sometimes four-) port ferrite device acting as a one-way distributor of microwave radiation. Radiation from the klystron enters the circulator at port 1. At port 2, radiation leaves and is reflected back from the cavity, with the reflected microwave radiation from the cavity exiting the circulator (bound for the receiver) at port 3. The amplifier should be placed between port 3 of the circulator and the input of the receiver. An isolator between the amplifier and the receiver is recommended to halt microwave power reflected by the receiver. The theory of the sensitivity enhancement gained as a result of this modification is described in detail by Hoentzsch et al., 2° but the gist of the idea is to boost the signal amplitude prior to detection by the diode with little penalty in added noise. The flicker noise is then favorably scaled relative to the amplified signal (with respect to spectrometer performance) and a sensitivity enhancement, typically a factor of four, may be gained. Low-noise amplifiers are available from LORAL/NARDA (Hauppauge, NY) or Miteq (Hauppauge, NY). Bruker will install amplifiers on their current spectrometers on request. Resonators and Electron N u c l e a r Double R e s o n a n c e Coil

In principle, the sole design requirement of a continuous wave (CW)ENDOR cavity is that it be possible to impose a sample simultaneous dc, microwave, and rf magnetic fields along mutually orthogonal axes. 21aThis condition is difficult to implement in practice because the engineering requirements of two high-frequency circuit components, namely, a tuned microwave resonator and an untuned NMR probe, must be reconciled. As discussed above, ENDOR detection is made as a measure of net change in EPR signal intensity during microwave power saturation. Any factor that causes a loss in EPR signal-to-noise ratios will, in turn, have deleterious effects on ENDOR sensitivity. Desirable attributes of a resonant cavity for EPR spectroscopy are reviewed by Poole. 14 Of these, the one most often affected by making cavity modifications for ENDOR is the Q or quality factor. For example, dielectric losses from the metal of an NMR probe inserted in a cavity will decrease the cavity Q factor with 21 G. Grampf, Rev. Sci. lnstrum. 56, 2050 (1985). 21a Despite the general rule regarding orthogonality of Hi and H2, Newton and Hyde describe S-band ENDOR studies using a loop-gap resonator and ff coil arrangement in which both fields are parallel [M. E. Newton and J. S. Hyde, J. Magn. Resort. 95, 80 (1991)].

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a corresponding loss of signal intensity. Such a spoiled cavity may be useless for all but the most concentrated samples. The crux of the design problem, therefore, is to introduce the NMR probe with minimal perturbation to the electric field component of the resonant microwaves. Two strategies for modifying the microwave cavity without significantly spoiling Q can be devised: (1) an NMR probe is inserted within the resonant field in such a way that the electric field is minimally perturbed; (2) the NMR probe is positioned outside the resonant field, either as an integral component of the resonator itself or as an independent external structure. Each of these strategies is discussed in turn. Most commonly used ENDOR cavities (including commercial designs) are of the first type. In general, with this strategy a cavity with a very high Q factor is chosen, so that spoiling by the coil will be relatively unimportant. Widely used cavities that feature high Q factors are the rectangular TE~o2 and cylindrical TEo11, with resonant modes illustrated in Fig. 3. To minimize perturbation of the electrical component of the microwave field the metal wire of the probe must lie very nearly perpendicular to the electric field lines. For the two cavities illustrated in Fig. 3, this means the NMR probe is best fashioned as vertical posts parallel to the sample axis. The posts can be joined either externally or internally to form the loops supporting the rf field.

TElo 2

• •

XxX XxX

TEol I

Flo. 3. Mode diagrams and schematic drawings of two TE cavities used for ENDOR spectroscopy. Magnetic field lines are indicated by the dashed lines in the plane of the page; electric field lines are represented by the conventional dots and crosses which indicate vector lines perpendicular to the page. The four centrally located " p o s t s " in the accompanying schematics mark the position of the NMR probe described in the text.

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The simplest example of the parallel post design is a single loop of magnet wire that is cemented onto a quartz support (single wall tube or Dewar flask). The free leads of the magnet wire loop are connected to the remainder of the rf circuit, and the entire assembly is inserted into either of the two cavities mentioned above. This design can be elaborated by adding two more parallel posts and fashioning a Helmholtz coil; the posts/coil may be affixed to the side of a tubular support or permanently mounted within the cavity. ENDOR cavities so produced tend to have very high Q factors (10,000) owing to the inherently high Q of the starting cavity. The loop form of the rf probe or coil, however, is not efficient for generating an rf field because of its a large surface-to-current ratio. It is normally used for samples for which low rf field strengths (< 1 G) are adequate to drive the spin system. As the spin-lattice relaxation time of the sample decreases, higher rf current is required (see the following section for discussion of ENDOR enhancement and relaxation rates). Higher power can be attained using a coil wound with multistranded fine wire (e.g., Litz wire) to increase current capacity. As the number of wire strands increases, however, more metal is introduced into the cavity and further Q spoiling is risked. Powers greater than 100 W with such a coil system can generate higher rf fields but at the threat of melting the coil. The difference between analogous coils used in CW-ENDOR as opposed to NMR experiments is the manner in which they are used. An NMR Helmholtz coil is part of a tuned circuit that operates at a fixed frequency; power delivery is therefore efficient (and in modern spectrometers, pulsed), and heat losses are low. In a CW-ENDOR spectrometer the coil is not tuned and must be swept through a broad frequency range. An unmatched circuit (see below) tends to be inefficient in power handling, with resultant heating that can lead to catastrophic failure in coils of fine-gauge wire. A helical coil (solenoid) is a far better structure for generating a strong rf field at moderate power levels (100-300 W) but is not compatible with either of the two cavities discussed above. Biehl et al. z2 have incorporated a helical coil into a cylindrical T M l l o cavity so that the wire helix does not interfere with the electric field lines. This cavity/coil combination is ideal for general ENDOR spectroscopy because the coil may be elongated along its axis to provide a strong rf field over the entire active region of the microwave magnetic field of the cavity, that is, along the entire length of its central axis. It will accept a sample in a capillary tube, as well as an insert Dewar vessel and standard EPR quartz sample tube. z2 R. Biehl, W. Lubitz, K. M6bius, and M. Plato, J. Chem. Phys. 66, 2074 (1977).

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The Q factor of the cylindrical TMHocavity is moderate (the unloaded Q of some commercial cavities were measured in the range of 5000 to 6000) and is spoiled to some degree by the finite pitch of the wire helix. Fanatical attention to detail in constructing helices for a Bruker 250ENB cavity is typically rewarded with loaded Q values of 1200-2000, depending on the quartz insert (C. J. Bender, unpublished observations, 1988); details of coil construction are outlined below. One useful feature of cylindrical cavities resonating in the TMHomode is that the resonant frequency is independent of the axial length, whereas the Q factor increases commensurately (up to a point). This feature allows optimization of cavity dimensions and Q for samples of a given volume. The Bruker ENDOR accessory is now the most commonly used commercial E N D O R unit. It employs a cylindrical TMHocavity, and a coil that can be wound and cemented onto a quartz Dewar flask or be a freestanding design. In the latter version the coil is wound on a Teflon template mandrel and held rigid with heat-shrink tubing. ~3 Once fabricated, the coil may be slid off the template and onto a cavity insert Dewar. A great advantage of the free-standing coil is that the design leaves the coil uncommitted to a single Dewar, thereby facilitating its handling, testing, and use. The typical coil consists of 16 turns of AWG 12 silver wire with an inside diameter of 10.0 mm (compatible with commercial Dewar inserts from Oxford Instruments and Wilmad Glass Co.) and a length (spacing between the brass end pieces) of 4.0 c m . 24 As the Bruker cavity has, for the most part, become an "industry standard," guidelines for preparing compatible coils follow. 1. The brass end pieces are generally fabricated first to ensure compatibility with both the cavity stacks and the Dewar insert. The inner diameter should be a close fit to the precision bore quartz tubing of the Dewar; the outer diameter must be adequate to make contact at the points where the BNC (or type N) spring contacts screw into the cavity stack. The remainder of the brass tube (i.e., below the region of electrical contact) is turned down to approximately the thickness of the wire that will be used to make the coil. 2. The template on which the coil will be wound is constructed of 0.25 inch (i.d.) by 0.50 inch (o.d.) Teflon tubing through which is forced a 0.250 inch (o.d.) brass rod. The brass core provides rigidity and prevents sagging during heating as the shrink tubing is applied (see Step 6). The template is then cooled at - 7 8 ° and turned on the lathe until the brass end pieces just slide onto the trimmed template when it is cold (several trimming 23 G. Hurst, K. Kraft, R. Schultz, and R. Kreilick, J. Magn. Reson. 49, 159 (1982). 24 C. J. Bender and G. T. Babcock, Rev. Sci. Instrum. 63, 3523 (1992).

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steps may be required). The idea is to have the template of such a diameter that the coil slides onto it when cold but is held tight when at room temperature. 3. With the brass end pieces positioned on the template, the coil is wound directly onto the template. Cowinding the wire with a heavy thread ensures a uniform spacing of the coil turns; optimal coil performance is achieved when the spacing between the individual turns is 1 to 2 times the wire diameter. The wire gauge selected will therefore depend on the number of turns in the coil. 4. Wire selected for coil construction may vary in composition and shape according to personal preferences. Round or square magnet wire may be used as described above; round wire may be flattened in a roller mill in order to minimize its tendency to creep. Phosphorbronze ribbon may also be used in place of wire; the pitch of a helix fashioned from the ribbon tends to be dictated by the width of the ribbon, so that interwinding spacing is best judged by eye. Current at radio frequencies is carried at a layer near the surface of the conductor (see standard tables of skin depth), and the composition of the wire can be selected from any number of metals, although economic constraints usually limit the choices to silver or copper. Because of the skin depth effect, it is important that the wire or ribbon used be free of surface imperfections and kinks. Long-term buildup of oxide also tends to limit lifetime of a coil, which may be extended by storing coils with an antitarnish pad ordinarily used for silverware. 5. The number of turns to an ENDOR coil depends on the frequency range that will be swept; 30-35 turns of narrow wire is adequate for frequencies below 6 MHz and 16-20 turns of larger wire above 5 MHz. Ribbon wire works well for constructing broad band coils because of its inherently low impedance, but it should be matched (see below) or operated in a manner to minimize heat radiation (the large relative surface area renders ribbon helices very good radiators of heat that will destabilize the tuned cavity). 6. Once wound, the coil leads are soldered to the brass end pieces. The coil can be held rigid during winding and soldering by overwrapping with Teflon tape. Best results have been achieved when the two ends are affixed at opposite sides of the coil. The thread spacer is then removed and heat-shrink tubing is applied. In the original description of free-standing coils, 23 the authors specify Kynar or Teflon heat-shrink tubing, but a word of caution is advised. Heat-shrinkable TFE Teflon does not flow well when heated and the individual turns of the coil may not be rigidly held in place. Kynar will flow well and grab the wire, but most grades of Kynar have dielectric constants too high for EPR applications. AIN Plas-

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tics (Mount Vernon, NY) offers Kynar tubing suitable for use in the E N D O R cavity. SPC Technology manufactures a heat-shrinkable twolayer tubing consisting of an FEP Teflon inner tube that flows readily to encase the wire coil and an outer TFE Teflon layer that provides rigidity. The tubing is relatively thick, however, and may require turning on the lathe to make it conform to the ENDOR cavity. 7. Once the shrink tubing is in place, the coil and template are returned to the freezer, and the coil is removed for testing and use after the template has shrunk. Because the coil is prone to damage by rough handling, especially if pushed or pulled from the brass end pieces (the coil should always be mounted onto a Dewar flask by using a rotating motion, as though it were being screwed on), we recommend that the shrink tube be cemented to the brass end pieces. AIN Plastics sells a kit for chemically etching and cementing Teflon components; satisfactory results may also be obtained by abrading the edges of the Teflon heat-shrink tubing with a file and cementing the parts with cyanoacrylate. Other methods for modifying conventional EPR cavities for E N D O R spectroscopy or fabricating dedicated ENDOR cavities have been described.Z5, ~6 Last, the loop-gap resonator (LGR) has recently become popular in EPR studies, particularly for solution samples of limited size. z7 Although applications of the LGR to E N D O R were predicted, there have been few examples of a LGR-based ENDOR resonator, and these have been applied to pulsed experiments where the orthogonality requirement is lifted, zs'z9 Newton and Hyde have used the LGR design at S-band for CW-ENDOR studies, 3° but no X-band CW-ENDOR experiment that uses a LGR has been reported.

Impedance Matching of Electron Nuclear Double Resonance Coil The rf circuit that is used for ENDOR spectroscopy consists of a modulated source, an amplifier, and the coil. The source and amplifier are typically constructed to operate with a 50 ~ load, although most modern amplifiers (e.g., those made by ENI, Rochester, NY, and Amplifier Research, Souderton, PA) are built to accommodate any load imped25 j. S. Hyde, J. Chem. Phys. 43, 1806 (1965). 26 D. Schmalbein, A. Witte, R. R6der, and G. Laukein, Rev. Sci. Instrum. 43, 1664 (1972). 27 j. S. Hyde and W. Froncisz, in "Advanced EPR: Applications in Biology and Biochemistry" (A. J. Hoff, ed.), p. 277. Elsevier, Amsterdam, 1989. 28 j. Forrer, S. Pfenninger, J. Eisenegger, and A. Schweiger, Rev. Sci. lnstrum. 61, 3360 (1990). S. Pfenninger, J. Forrer, A. Schweiger, and Th. Weiland, Rev. Sci. lnstrum. 59, 752 (1988). M. E. Newton and J. S. Hyde, J. Magn. Reson. 95, 80 (1991).

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ance without failure. It is therefore common to operate an ENDOR spectrometer with an unmatched rf coil that is terminated with a 50 O dummy load. The impedance of the coil is typically lower than the nominal 50 fl value, but terminating with the load, in effect, clamps the total circuit impedance to some value near 50 fl. Because the load and line impedances are not exactly matched, the amplifier must be protected from rf power reflected from the coil. In a typical ENDOR circuit configuration a 50 lq line is connected to a coil of low impedance (-12 f~) that is then terminated with another section of 50 II line and the dummy load. Power reflections will occur owing to the mismatch in the cavity/coil. If inefficient power delivery were the only manifestation of operation in this manner, there would be little reason to match the ENDOR circuit since power can be simply increased. What is often overlooked, however, is the fact that the coil is a reactive circuit element, and its behavior may vary over the frequency range selected for a sweep of the ENDOR spectrum. At high frequencies inductive and capacitative circuit elements exhibit a frequency-dependent impedance. The characteristic impedance of an ideal capacitor or inductor will vary inversely or directly with applied frequency. Real circuit components, however, each possess resistive, capacitative, and inductive properties, and therefore may resonate at a specific frequency. At this resonant frequency the impedance characteristics of a circuit element will dramatically change; a capacitor will begin to behave as though it were an inductor, and vice versa. The dimensions of the rf coil used in ENDOR studies usually preclude self-resonance, but other parasitic reactances may lead to a spurious resonance condition. For example, the cavity wall is incorporated into the rf circuit when the coil is terminated with a 50 O load. The cavity wall is at ground relative to the coil, and a parasitic capacitance develops between the coil and ground. Measurements of this parasitic capacitance in a TM~o cavity indicate that the capacitance is on the order of 100 pF at 10 MHz, and a characteristic impedance plot of the ENDOR circuit reveals a resonance in the region of 30 MHz that can be attributed to the parasitic capacitance. 24 The spurious resonance can be associated with baseline artifacts that commonly appear at the same frequency. An impedance matching network can eliminate the problem of spurious circuit resonances while making rf power delivery more efficient. A servodriven impedance matching circuit (Fig. 4C) that continuously adjusts a trimmer capacitor in response to a phase measurement has been described. 3~ A broadband match can also be achieved with transformers or 31 j. Forrer, A. Schweiger, and H. H. Giinthard, J. Phys. E: Sci. lnstrum. 10, 470 (1976).

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i

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13

D

FIG. 4. N e t w o r k s u s e d to m a t c h E N D O R coils to 50 f~ lines. See text for details and references.

a wideband (i.e., low Q) matching network. Ruthroff 32 and Sevick 33 offer expositions on the construction and use of transformers as matching components. The simplest example is a 1 : 4 transformer that consists of several turns of magnet wire wound on an iron-powder toroidal core (Micrometals, Inc., Annaheim, CA). A 1:4 ratio is used because the coil typically has an impedance of 12 ~ . Alternatively, a broadband matching network may be used to match the coil to the 50 f~ line. Peri6 and Dulcic 34 describe a circuit of two parallel branches, the first branch consisting of the coil and a 50 ~ resistor and the second of a variable capacitor and another 50 f/resistor. The two branches supply inductive and capacitive reactance, the resistors serving to clamp the impedance at or below 50 ~. Parasitic reactances of the coil/ cavity ensemble are, however, neglected. To eliminate these resonances, a matching network was devised by adding a capacitor in series and an inductive shunt to the circuit. 24 The resultant circuit makes up what is known as a zr network and showed none of the self-resonance properties found in the coil/cavity combination alone. Figure 4 illustrates four networks that describe the ENDOR coil and impedance matching circuit elements. The network of Fig. 4D is an adaptation based on an antenna tuner described by Brumbaugh 35 and provides a quick means of general matching to a nonspecific coil. The same multiple 32 C. L. Ruthroff, Proc. 1. R. E., 1337 (1959). 33 j. Sevick, " T r a n s m i s s i o n Line T r a n s f o r m e r s , " 2nd Ed. A m a t e u r Radio League, Newington, Connecticut, 1990. 34 M. Peri6 and A. Dulcic, J. Phys. E: Sci. Instrum. 14, 700 (1981). t5 j. F. B r u m b a u g h , Amat. Radio Today, 46 (1991).

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C

D

GSOURCE

RL FIG. 5. Circuit representation of the magic tee coupler, and its use for matching an ENDOR probe in situ.

tapped inductor (Radiokit) can be used in the matching network described by Bender and Babcock 24 (4D). The 50 12 resistors depicted in Fig. 4B,D correspond to the high power loads; in the Peric and Dulcic network (Fig. 4A), these are 50 12 noninductive wire-wound resistors. High-wattage wire-wound resistors are difficult to find but may be substituted by three 150 12 resistors in parallel. The measurement of impedance and the reactance parameters (L, C) of a component is not crucial to the design of an effective ENDOR coil and matching circuit. A simple method of matching a circuit has been described by Fukushima and Roeder. 36 Two transmission lines are balanced using a magic tee, a four-port device that splits an rf signal with phase coherence on all but one port, which is shifted by 180° (Fig. 5). A low-power 50 12 termination is attached to port A (Fig. 5); the frequency synthesizer is attached to port C, an oscilloscope to port D, and the ENDOR circuit to port B. The rf signal at port C is delivered at ports A and B with relative phase shifts of 180°. Because ports A and B (Fig. 5) are terminated, the rf signals on each arm are reflected back to the magic tee and recombined at the output, port D. If the impedance of both arms is the same, there will be no further phase shift of the signal and the two 36 E. Fukushima and S. B. W. Roeder, "Experimental Pulsed NMR: A Nuts and Bolts Approach." Addison-Wesley, Reading, Massachusetts, 1981.

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signals combined at D should null each other because of the 180° shift imposed by the magic tee. Matching of the ENDOR network is therefore achieved by adjusting circuit components until the signal detected on the scope is minimized.

Triple Resonance Double ENDOR, or Triple resonance, spectroscopy is a technique often used to determine the relative signs of hyperfine coupling constants and establish the number of equivalent nuclei contributing to a given ENDOR transition. 37 In applying this technique, one or more ENDOR transitions are saturated; and the enhancement of other ENDOR lines is observed. There are two types of Triple resonance schemes, General and Special. During a General Triple experiment a single transition is saturated with a " p u m p " rf field as the ENDOR spectrum is swept. In Special Triple both components of an ENDOR pair are simultaneously driven while detecting the saturated EPR signal. Details of the Triple resonance phenomenon are covered in the theory section (see below). Instrumental requirements for Triple resonance studies include a second rf source and a device for combining two rf signals. A power combiner may be used for General Triple; a double balanced mixer is required for Special Triple. Modulation is usually applied to the scanning rf field, although sometimes the pump field is modulated. A Wavetek (Model 3000446, San Diego, CA) modulated rf source and a second programmed test source (PTS-160; PTS, Inc., Littleton, MA) source with a Bruker ER250 ENDOR accessory are used at Michigan State University (East Lansing, MI). For General Triple experiments the rf output of both rf sources is fed to a power combiner prior to amplification. Because the power meter cannot discriminate by frequency, the lowest power level (i.e., the sweep) is set first. Special Triple experiments are performed in a similar manner. The PTS source is programmed for the nuclear Larmor frequency37a and fed to the local oscillator (LO) port of a mixer. The modulated rf sweeper is then programmed to sweep upward from 200 kHz, and this output is fed to the rf (R) port of the mixer. At the X port of the mixer modulated rf fields of frequenciesfzrs -+ fsweepare obtained, and this port is connected to the input of the power amplifier. Care must be taken in selecting a mixer for Special Triple experiments because their specifications vary by a considerable amount. Triple requires an output of rf signals at frequencies fl -+ f2, free of harmonics (fl --+ nf2) and leaks (f~ and f2)- These requirements translate to a high isolation, 37 K. M6bius, M. Plato, and W. Lubitz, Phys. Rep. 87, 171 (1982). 37a In solid systems where g is anisotropic, the ENDOR spectrum is often asymmetric about the nuclear Larmor frequency, so that appropriate corrections should be made.

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40-60 dB, between the X port and the R and L O ports. Two r e c o m m e n d e d mixers are the double-balanced Anzac 37(Burlington, MA) and the HewlettPackard (Cupertino, CA) Model 10514 or 10534, which perform well at low input frequencies. Unfortunately, the Hewlett-Packard mixers are now discontinued items, but they can often be found on the used equipment market. Triple resonance has been most successful in spin systems with E N D O R lines that are homogeneously broadened and easily saturated, particularly radicals in solution 37'38 and some crystalline solids. 39-42 E N D O R spectra of p o w d e r samples are inherently broadened inhomogeneously owing to the random orientation of individual spins relative to Ho. When a General Triple experiment is conducted on such a sample, the pump rf field will burn a hole in the spectral line rather than saturate it. The theory of inhomogeneous line broadening suggests certain properties of spin-spin relaxation p h e n o m e n a 43 that, in principle, may facilitate adjustment of experimental parameters to alleviate the so-called triple problem. An easier approach is to broaden the spectrum of the rf pump. This can be done by applying FM to the pump, pulse modulating the pump, or "clipping" the sine waves of the rf signal. The latter two methods broaden the spectrum of the rf signal by introducing additional Fourier components. The same effect can be achieved by mixing the rf carrier with a noise source to enhance sensitivity in powder E N D O R studies. Electron Nuclear Double Resonance Experiment: T h e o r y and Application In a static magnetic field, the population difference between electron energy levels in a paramagnetic system at thermal equilibrium is given by the Boltzmann distribution. When the paramagnetic system is irradiated with high microwave power at its resonant (Larmor) frequency in that field, the population difference between EPR levels specified by the Boltzmann distribution is reduced or obliterated if power is absorbed faster than it can be dissipated by nonradiative relaxation mechanisms. The spin system is then said to be saturated, and one observes a concomitant loss in EPR signal intensity. 38H. Kurreck, B. Kirste, and W. Lubitz, "Electron Nuclear Double Resonance Spectroscopy of Radicals in Solution." VCH, Weinheim, 1988. 39j. R. Niklas, R. U. Bauer, and J.-M. Spaeth, Phys. Status Solidi 171, 1196 (1983). 40N. S. Dalai and C. A. McDowell, Chem. Phys. Lett. 6, 617 (1970). 41W. Kolbe and N. Edelstein, Phys. Rev. B 4, 2869 (1971). 42D. A. Hampton and G. C. Moulton, J. Chem. Phys. 63, 1078 (1975). 43C. P. Poole and H. A. Farach, "Relaxation in Magnetic Resonance." Academic Press, New York, 1971.

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In the CW-ENDOR experiment, the static magnetic field is fixed at or near the center of the EPR line of interest while the electron resonance is microwave power saturated. Should the electron spin be coupled to a nuclear spin, however, the loss of intensity resulting from saturation may be restored in part by inducing transitions between nuclear spin states. Such transitions open new relaxation pathways for the electron, thereby returning some of the lost population difference. In practice, the sample is swept over a continuous range of radio frequency fields encompassing nuclear transitions of interest. As the radio frequency field sweeps through a nuclear transition, the change in EPR intensity is detected by the EPR spectrometer, thereby generating the ENDOR spectrum. The E N D O R phenomenon is best explained by using a four-level energy diagram that represents a S = ½, I = ½spin system (Fig. 6). The lefthand side of Fig. 6 illustrates the effect of Zeeman splitting, and this pattern of four energy levels is spatially modified in the right-hand side of Fig. 6 in order to facilitate the discussion. Allowed radiation-induced transitions are denoted by arrows; dashed lines indicate relaxation pathways. In the absence of saturating microwave or radio frequency fields, the relative population of the four spin states is determined by the Boltzmann distribution. Ignoring, for the time being, relaxation pathways of Fig. 6, either of two EPR transitions can be saturated, and the corresponding nuclear sublevels will become equally populated. A subsequent sweep of the NMR spectrum will induce two transitions: I - - } ~ I - +} and I+ + } ~ I + - ) . These NMR transitions will depopulate one or the other

I+-> ,,_

fill

!r,o ,

i

/

\ /

,,_

?,

l]ir, o \

Jl.;

/ITx/~\ v,

I---->

,

~ONIC ZEEMAN

NUCLEAR ZEEMAN

I

I-÷>

FIG. 6. Electron a n d nuclear Z e e m a n splitting (left) a n d spectroscopic description o f the E N D O R p h e n o m e n o n in a s y s t e m with S = ½, 1 = ½ (right). D a s h e d lines indicated by T t e r m s are the relaxation p a t h w a y s that c o m p e t e with radiation-induced transitions (arrows).

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CONTINUOUS WAVEENDOR SPECTROSCOPY

209

energy level associated with the saturated EPR transition, and there will be an enhancement of the detected EPR signal. The conceptual nature of the experiment is unchanged by taking into account the various relaxation routes. What now becomes important is the relative magnitude of the spin relaxation rates and the rate of the radiation-induced transitions. In short, the success of an ENDOR experiment depends to a great extent on balancing the kinetics of energy level transitions. The probability of a radiation-induced transition is proportional to the intensity of the radiation field, so that a necessary condition for a successful ENDOR experiment is that the NMR transition rate be greater than the spin relaxation rate, 44,44a l/Tle ( o r We). This means that factors such as the applied rfpower and temperature will be experimentally controllable variables that affect the success of observing an ENDOR enhancement. Somewhat less influential are the cross-relaxation pathways indicated in Fig. 6 by Tx and Txx. In general, cross-relaxation occurs because of the finite width of spectral lines, and when these lines overlap, mutual spin flips are possible. Txx is a process that only becomes relevant when the hyperfine interactions are anisotropic45; under such conditions the transition I+ + ) ~ I- - ) becomes partially allowed. Relaxation by these routes is relatively slow for most spin systems, hence their diminished role in determining whether an ENDOR experiment will succeed. Usually, the effect of cross-relaxation is seen in the intensity of a line corresponding to a single nuclear sublevel. For example, when one of the two transitions of an ENDOR spectrum are missing, cross-relaxation is typically blamed. 6 The effects of cross-relaxation on the intensity of ENDOR lines and experiments to verify this mechanism are covered in several texts and reviews.43, 45-48

One of the lessons of this relaxation-dependent analysis of the ENDOR enhancement is that the signal intensity of an ENDOR experiment will be strongly influenced by those factors that govern the relaxation rates 44 L. Kevan and L. D. Kispert, "Electron Spin Double Resonance Spectroscopy." Wiley, New York, 1976. 44a Spin lattice relaxation is a nonradiative process that returns the spin system to thermal equilibrium. 45 A. Abragam and B. Bleaney, in "Electron Paramagnetic Resonance of Transition Ions." Oxford Univ. Press (Clarendon), Oxford, 1970. 46 N. M. Atherton, in "Multiple Electron Spin Resonance Spectroscopy" (M. M. Dorio and J. H. Freed, eds.), p. 387. Plenum, New York, 1979. 47 j. H. Freed, in "Electron Spin Resonance in Liquids" (L. T. Muus and P. W. Atkins, eds.), p. 387. Plenum, New York, 1972. 48 j. Owen and E. A. Harris, in "Electron Paramagnetic Resonance" (S. Geschwind, ed.), p. 427. Plenum, New York, 1972.

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PROBES OF M E T A L ION E N V I R O N M E N T S

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of the spin system. The signal obtained in a steady-state ENDOR experiment is not analogous to the signal measured in a swept field NMR experiment, and line intensities are not simply proportional to the relative numbers of contributing nuclei. The relative intensity of the ENDOR lines corresponding to each of the nuclei will depend on the nuclear spin relaxation times. If these relaxation rates differ by a substantial amount, integrated line intensities will not reflect the number of contributing nuclei. For this reason, it is a risky practice to use spectral subtraction as a means to resolve hidden ENDOR lines. A further difficulty lies in guaranteeing a reproducible rf field from sample to sample; Triple resonance is required for reliable quantitative analysis. Experimental factors affecting the relaxation parameters of an electron-nuclear spin system will, in turn, influence the quality of the ENDOR spectrum. This means working with spin concentrations sufficiently small to eliminate Heisenberg exchange effects. Sometimes deoxygenating the sample improves ENDOR spectral quality. The secondary structure of a globular protein usually provides insulation that prevents spin-spin interactions between whatever paramagnetic species is located at its " c o r e . " It is often advantageous to concoct a solvent system that forms an isotropic, homogeneous glass on cooling. With protein samples this can be achieved with a supporting buffer solution that is 50% by volume in either glycerol or ethylene glycol, or 2.5-3.0 M in sodium perchlorate. Optimal conditions must be found by trial and error. The principal benefits of ENDOR over conventional EPR spectroscopy are the simplicity and greater resolution of the hyperfine spectrum. The number of lines in an EPR spectrum subject to nuclear spin-spin coupling are determined by a multiplicative law; this law is additive for the ENDOR experiment. Two ENDOR lines are observed for each set of equivalent coupled nuclei, and these pairs of ENDOR lines are typically denoted v_ and v+. The additive nature of ENDOR lines greatly facilitates the determination of hyperfine coupling constants, as can be seen by comparing the EPR and ENDOR solution spectra of the 1,4-naphthosemiquinone radical anion (Fig. 7). In the ENDOR spectrum, the three distinct proton groups (i.e., at ring positions 2,3; 5,8; and 6,7) yield three pairs of ENDOR spectral lines that are designated by letter in Fig. 7; labels a and a' denote paired E N D O R transitions v_ and v+, respectively. In contrast, these six protons would generate 64 EPR lines, not all of which would necessarily be resolved. The much greater resolving power of ENDOR can also be appreciated in Fig. 7. First, the widths of the ENDOR lines are of the order of 10 kHz, which is considerably narrower than those of an EPR spectrum. Second, the weak couplings of the protons at ring positions 5-8 are clearly

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CONTINUOUS WAVE ENDOR SPECTROSCOPY

211

A FIc. 7. EPR and ENDOR spectra of the 1,4-naphthosemiquinoneanion radical. The much simpler ENDOR spectrum clearly resolves the hyperfine coupling constants of the three nonequivalent proton types. resolved in the E N D O R spectrum (labeled a and b, Fig. 7). Such small couplings (<2 MHz) would not ordinarily be resolved in an EPR spectrum but might be gleaned from fine adjustments in spectral simulations. Many EPR studies of biological systems require samples that are frozen or sufficiently viscous, such that a lack of fast motional averaging results in an inhomogeneously broadened E P R line. Under such conditions hyperfine splittings may be lost in the EPR line widths. The increased resolution of E N D O R allows retrieval of this lost information, with the added advantage of allowing the principal components and orientation of the hyperfine tensor to be determined from p o w d e r E N D O R spectra. 49-52 A p p r o a c h to Spin H a m i l t o n i a n

The analysis of free radical E N D O R spectra can usually be achieved without a detailed knowledge of the quantum mechanical description of spin energetics. When dealing with transition metal complexes, however, additional energetic terms b e c o m e important in the analysis, and very many scenarios lead to as many different analyses (see Abragam and Bleaney 45 for an extensive catalog of case studies). As a result, it is necessary in these cases to fall back on the spin Hamiltonian description of the paramagnetic species and independently calculate the relevant energies. 49j. s. Hyde, G. H. Rist, and I. R. Eriksson, J. Phys. Chem. 72, 4269 (1968). 5oL. R. Dalton and A. L. Kwiram, J. Chem. Phys. 57, 173 (1972). 51A. L. Kwiram, J. Chem. Phys. 57, !132 (1972). 52p. j. O'Malley and G. T. Babcock, J. Am. Chem. Soc. 108, 3995 (1986).

212

PROBES OF METAL ION ENVIRONMENTS

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The Hamiltonian contains all the energetic contributions for a mechanical system, both kinetic and static. An electron in a molecule will be described in terms of its motion and various interactions (e.g., Coulombic). The diverse contributions to the total energy are taken to be additive, which allows the Hamiltonian to be written as a sum of terms each of which may be individually computed. Thus, to a first approximation the Hamiltonian of a molecule subjected to an EPR experiment may be set down as = ~[~electronic+ ~spin

This approximation allows separation of a spin Hamiltonian, ~spi,, from the total Hamiltonian, and thereby permits one to calculate parameters describing the results of a magnetic resonance experiment. If, for the time being, we reconsider the case of a simple organic free radical (S = ½, I = ½) and the level diagram illustrated in Fig. 7, the spin Hamiltonian can be written as the sum of the electronic and nuclear Zeeman energies, and the hyperfine interaction energy, ~spi. = ~Ze + ~Zn + ~HF

thus further decomposing its terms. If one now operates with this Hamiltonian on a state function that describes the unpaired electron in terms of its spin and angular momentum, ~(L,S,I), the energy levels of that state, depicted in Fig. 7, are obtained. Additional terms are added to the above spin Hamiltonian as needed. For example, a quadrupole term is added when I --- 1 nuclei are included as part of the spin interaction scheme. A transition metal complex is described by a spin Hamiltonian that contains many terms: ~spin = ~Ze + ~Zn + ~HF + ~Q + ~CF + ~SS + ~SO

where the subscripts Q, CF, SS, and SO designate quadrupole, crystal field, spin-spin interactions, and spin-orbit interactions, respectively. Individual terms of the spin Hamiltonian generally involve tensor operations, so that in calculating the energy for a given Hamiltonian term the relative orientation of the laboratory reference frame and the g, hyperfine, and quadrupole tensor coordinate axes must be taken into account. Theoretical analysis of the various spin Hamiltonian terms and their interplay is best offered in several texts devoted to the subject, to which we refer the interested reader. 45'53-56 53j. E. Wertz and J. R. Bolton, "Electron Spin Resonance: Elementary Theory and Practical Applications." Chapman & Hall, New York, 1986. 54j. R. Pilbrow, "Transition Ion Electron Paramagnetic Resonance." Oxford Univ. Press (Clarendon), Oxford, 1990. 55j. W. Orton, "Electron Paramagnetic Resonance." Gordon & Breach, New York, 1969. 56j. S. Griffith, "The Theory of Transition Metal Ions." Cambridge Univ. Press. Cambridge, 1961.

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CONTINUOUS WAVEENDOR SPECTROSCOPY

213

Spin Hamiltonian Terms from Electron Nuclear Double Resonance Spectrum In metalloproteins IH, 2H, 13C, 170, 338, and various metal nuclear couplings have been detected and analyzed by ENDOR spectroscopy. Occasionally a metalloprotein may be of interest because of an associated radical species (e.g., ribonucleotidexdiphosphate reductase); however, most interest in metalloprotein ENDOR is devoted to the ligand structure of the metal center in the resting or functioning state of the host protein. Nitrogen couplings arise from the amino acids coordinated to the metal in the protein; if sufficient spin density is delocalized onto the amino acid ligands, strongly coupled proton lines will also appear in the ENDOR spectrum. Protons weakly dipole-coupled to the paramagnetic species appear as a matrix line centered at the proton Larmor frequency. 44 The nuclear Zeeman energy also can be determined from the line splittings in an ENDOR spectrum and used to identify the nuclear origins of a given set of lines. Furthermore, the appearance or absence of lines corresponding to weakly coupled nuclei may permit determining whether a solvent or other molecule is accessible to the site of the paramagnetic ion (or radical). The ENDOR spectrum of an immobilized sample often features an intense broad line centered at the Larmor frequency of those nuclei close enough to interact with the paramagnet. For example, the ENDOR spectrum of a transition metal ion in a protein will usually show an intense line at the proton Larmor frequency ( - 14 MHz at X-band) that is due to very weak (note: Vn --+ ½A in the limit of A ~ 0 is Vn) dipolar interactions between the transition metal ion and the nonbonded protons of the surrounding protein milieu. This so-called matrix ENDOR line acts as a probe of the environment of the paramagnet within a 5-10 A radius, depending on the nature of the spin interaction. 44 If we consider as a case study the problem of accessibility of water to the active site of a metalloprotein, we would design our experiment as a comparison of the ENDOR spectra taken in H20- and D20-containing solvent (i.e., buffer). If water is accessible, then following D20 exchange the proton Larmor line should collapse somewhat, and there should be seen a deuterium matrix line. Similar analyses can be performed with other nuclei as labels on other cofactors. The lines in an ENDOR spectrum are usually dispersed according to a simple rule that reflects the relative magnitude of the nuclear Larmor frequency and the hyperfine coupling constant. A pair of ENDOR lines 1 for a set of equivalent nuclei will be observed at v+ = IVn + ~A] if v, > 1½A[ or at v_+ = [½A -+ b,.l if vn ~- I½AI. If quadrupole interactions are a factor, an additional term is added (see below).

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PROBES OF METAL ION ENVIRONMENTS

[7]

In the absence of quadrupole interactions the hyperfine coupling is read directly from the ENDOR spectrum according to the aforementioned rule. One can then compute the spin density of that nucleus by using a McConnell relation Aiso = Qp

(1)

where p denotes the spin density at the nucleus. The proportionality factor is called the Q value (distinct from the cavity Q value and quadrupole constant) and will vary among the types nuclei studied. Strongly coupled protons are classified by their proximity to the carbon bearing the unpaired spin density. One denotes these protons directly bound to that carbon as an a proton. Protons bound to a carbon that is itself bound to a carbon bearing unpaired spin density are denoted /3 protons, and so forth. For most cases, only o~ and/3 protons contribute to the hyperfine structure of an electron magnetic resonance solution spectrum. The value of Q in the McConnell relation for ot protons, Aiso = Qp, varies for the type of C - H fragment. The Q value represents an overlap of sorts between the molecular orbitals of the adjacent p and s orbitals57and for this analysis approximates - 7 0 MHz. 53 The/3 proton couplings are subject to an analysis similar to that of protons except that the Q value is dependent on the rotational angle about the C~-C~ bond axis. Neglecting a small constant factor, the isotropic coupling constant for/3 protons is Aiso =

Blp

COS 2 0

(2)

where 0 is the dihedral angle that is defined by the axis of the Pz orbital on which the spin density resides and the C~-H~ bond, 58 and B 1 is on the order of 160 MHz. 59The contribution for a rotationally averaged/3 proton would be Aiso = ½Blp. Analysis of spectra obtained from solid samples is complicated by the contributions of anisotropic terms. In the solid state the dipolar interactions between the unpaired spin and atomic nuclei are no longer motionally averaged out, and the resultant ENDOR powder spectrum contains the principal (i.e., diagonal) terms of the hyperfine tensor as the turning points of the powder pattern. In other words, the powder ENDOR line may be axial or rhombic and subject to analysis as with the analogous EPR line shape. 49 For ot protons, the dipolar contribution to the hyperfine tensor leads to a rhombic ENDOR line with turning points at approximately 57 H. M. M c C o n n e l l and D. B. C h e s t n u t , J. Chem. Phys. 28, 107 (1958), 58 W. Derbyshire, Mol. Phys. 5, 225 (1962). 59 R. W. F e s s e n d e n and R. H. Schuler, J. Chem. Phys. 39, 2147 (1963).

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215

1Also, Also, and ~z' o3A Xis .49'60A/3 proton appears as an axial line, and in certain cases, such as a methyl group, this axial line will further split into three axial lines as rotational averaging about the C~-Ct3 bond ceases. The highly resolved ENDOR spectrum now becomes very useful because it permits further analysis of the hyperfine tensor of a molecule to derive structural or electronic information based on the two models of dipolar interaction. The simplest analysis follows from the point dipole approximation, which applies to interactions between a nucleus and an electron in a spherical orbital or when the interaction distance is large (>3-4 ,~). Using this approximation, one obtains an axial dipolar contribution to the hyperfine tensor with one term

A.± =-pg/3/3nlzI-lR

3

(3)

where the subscript ~ denotes a dipolar hyperfine coupling component, /3 terms are the Bohr magneton of the electron and nucleus, R is the distance over which the interaction occurs,/x is the magnetic moment of the nucleus, and I is its spin. In units that are convenient for the analysis of ENDOR spectra (i.e., megahertz), A~± = - 14.1ptzI-1R -3

(4)

It follows that the second term of the axial dipolar interaction is A~,IL= - 2 A , ± . At shorter interaction distances (e.g., the proton adjacent to the p orbital in the C~-H~ fragment) the point dipole model must be replaced by one that corrects for changes in symmetry and effective distance of interaction. 61 In a molecular system the unpaired electron spin is often delocalized over more than one atomic center, and the dipolar interaction for a given nucleus is not limited to its nearest neighbor. For a delocalized spin system a method described by Heller and Cole 62 can be used. This procedure entails the independent computation of all dipolar interactions for a given nucleus, followed by rotation of each of the tensors onto a common axis. The coincident tensors are then diagonalized and summed to give the total dipolar hyperfine contribution. 5z,63 The utility of this procedure is that

6o H. M. McConnell, C. Heller, T. Cole, and R. W. Fessenden, J. Am. Chem. Soc. 82, 766 (1960). 61 W. Gordy, "Theory and Applications of Electron Spin Resonance." Wiley, New York, 1980. 62 C. Heller and T. Cole, J. Chem. Phys. 37, 243 (1962). 63 C. J. Bender, M. Sahlin, G. T. Babcock, B. A. Barry, T. K. Chandreshekar, S. P. Salowe, J. Stubbe, Lindstr6m, L. Petersson, A. Ehrenberg, and B.-M. Sj6berg, J. Am. Chem. Soc. 111, 8079 (1989).

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PROBES OF METAL ION ENVIRONMENTS

[7]

it permits one to fine-tune the spin density distribution and therefore (indirectly) the Q value of an electron-nuclear interaction if one is provided with a good estimate of the molecular structure. It conversely allows one to predict a structure if one is given an accurate estimate of the spin density distribution. A detailed description of the procedure as applied to the first scenario has been given during the analysis of the ENDOR spectrum of the tyrosyl radical of ribonucleotide reductase. 63 The hyperfine coupling constant of nitrogen is subject to a similar McConnell-like analysis. The nuclear spin of ~4N, I = 1, leads to a triplet splitting of each electronic Zeeman level. As part of a r r system the unpaired electron spin is associated with a p orbital on the nitrogen, whose symmetry imparts an axial line shape to the anisotropic hyperfine tensor. The resultant spectral features occur at All = Aiso + 2A~, and A± = Ais o A~. The isotropic coupling constant is often related to the spin density on nitrogen in a manner similar to that of a proton, that is, A~soN = QNON, where the Q value of 14N is taken to be approximately 56 M H z . 61 The orbitals of atoms such as nitrogen and carbon hybridize in their molecular structures and hence are not described by pure hydrogenlike atomic orbital functions. The total spin density contribution may possess some s-orbital character. Spin polarization of the tr bonds and lone pair electrons will also affect the hyperfine coupling of an ~4N nucleus in an extended ~- system. The sign of the hyperfine interactions due to the polarization effects may differ from that of the principal contribution (i.e., the p orbital), which is why in many cases the simple McConneU relation given in the previous paragraph tends to overestimate measured nitrogen couplings. The analysis that corrects for the various inductive effects was first derived for the analogous problem of relating hyperfine coupling constants to the spin density of 13C. The concept is quite simple and entails a decomposition of the total isotropic coupling constant into a sum of individual McConnell-like relations; the first analysis of l a N hyperfine coupling constants 64 contains an extra constant, but in its simplified form the corrected McConnell relation for nitrogen couplings

is 61

Ai~o = QIp= + Qz ~ Pi

(5)

where the summation is taken over the various secondary interactions arising from spin polarization, hybridization, etc. The relevance of this decomposition scheme to the topic of the chapter is that the ENDOR 64 E. W. Stone and A. H. Maki, J. Chem. Phys. 39, 1635 (1963).

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CONTINUOUSWAVEENDOR SPECTROSCOPY

217

technique greatly facilitates the analysis of ~4N couplings because anisotropic terms of nitrogen and proton hyperfine interactions can be picked out, and the latter used as a "check." 65 Single crystals of metalloproteins suitable for study by EPR and ENDOR are rarely available. Frozen solution spectra reflect a powder pattern of all molecular orientations relative to the applied magnetic field. Because the spin Hamiltonian terms are tensors, the spectroscopic parameters of the system are strongly dependent on orientation, often confounding unambiguous interpretation. Single-crystal-like ENDOR spectra may be obtained, however, by saturating the so-called turning points of the EPR powder spectrum,66-68 thereby selecting for resonance a small population of the sample in which spins are distributed along a narrow solid angle with a comparatively discrete orientation of the hyperfine tensor to the applied magnetic field. Under such conditions relatively narrow angleselected lines are obtained, which greatly facilitates the interpretations of hyperfine couplings. As demonstrated by Rist and Hyde, 67-69 singlecrystal-like ENDOR spectra of a nitrogen nucleus yields four lines with energies of v = 1½A -+ v. - Q'I, where v. is the nuclear Larmor frequency and Q' is the quadrupole interaction. Axially symmetric paramagnetic metal complexes yield a second case of powder ENDOR spectra amenable to simplified analysis. 53'69If hyperfine and quadrupole reference frames are colinear and corresponding tensors have axial symmetry, then the ENDOR resonant frequencies are given by v = I½A~cos 2 0 + ½A2 sin 2 0 -+ Q'(3 cos 2 0

--

1) ± vnl

(6)

where 0 represents the angle between the applied magnetic field and the z axis of the hyperfine and quadrupole tensor. 69 From the analysis of Eq. (6) by Rist and Hyde, 69 integral averaging at EPR turning points yield maxima from which it is possible to obtain the quadrupole, Zeeman, and hyperfine terms. A case study is provided in the analysis by Rist and Hyde of powder ENDOR spectra of planar copper complexes. 69 The Triple resonance technique can be used to glean further infor/'nation from the electron-nuclear hyperfine interactions. As described previously, there are two distinct experimental techniques: (1) General Triple, in which a single NMR transition is saturated while the ENDOR spectrum 65 W. H. Nelson, F. M. Atwater, and W. Gordy, J. Chem. Phys. 61, 4726 (1974). 66 T. Doyle, Phys. Rev. 126, 1421 (1962). 67 G. H. Rist and J. S. Hyde, J. Chem. Phys. 49, 2449 (1968). 68 G. H. Rist and J. S. Hyde, J. Chem. Phys. 50, 4532 (1969). 69 G. H. Rist and J. S. Hyde, J. Chem. Phys. 52, 4633 (1970).

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VROaES OF METAL ION ENVIRONMENTS

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is recorded, and (2) Special Triple, during which in the course of the rf sweep both the upper and lower NMR transitions of a given nucleus are driven simultaneously.37'7° General Triple is used to determine the relative signs of the hyperfine coupling constant. Although the sign of a given hyperfine coupling constant follows from the McConnell relation, there are times when experimentally determining the sign will aid in an assignment. For example, when the magnitude of the dipolar hyperfine terms exceeds the isotropic component one gets a mix of positive and negative principal hyperfine terms. The theory of Triple is simply explained for an S = ½ system with coupled nuclei. Energy levels are divided into an upper and lower spin manifold (m S = + ½, and m s I, respectively). If A > 0 for a given nucleus, then its low-frequency ENDOR line v_ comes from an NMR transition in the upper manifold; v+ for this nucleus arisies from the NMR transition in the lower spin manifold. The converse holds for nuclei with A<0. If one of the NMR transitions in either of the electron spin manifolds (i.e., v+ or v_) is now saturated, the spin populations of all levels in that manifold are approximately equalized. To some extent, populations of the sublevels in the other electron spin manifold are augmented because of the induced EPR transition and electron spin-lattice relaxation processes. If the ENDOR spectrum is now swept, line intensities corresponding to the NMR transitions of the equilibrated spin manifold have "collapsed," whereas those of the opposing spin manifold have been enhanced. The rule of General Triple is that for couplings of identical sign the enhancements coincide with regard to the symmetry about the Larmor frequency. It follows that the General Triple method provides information on the relative signs of hyperfine coupling constants. When both NMR transitions, v_ and v+, are driven simultaneously, as in a Special Triple experiment, the signal intensity of the NMR transition becomes independent of the rf field i n t e n s i t y 37'7°'7°a and is solely a function of those spin states involved in the transition. The ratio of intensities for any pair of Special Triple lines then corresponds to the ratio of the number of nuclear spins responsible for the lines in question. In other words, the line intensities of a Special Triple spectrum can be quantitatively analyzed in the same manner as in a conventional NMR spectrum.

7o K. P. Dinse, R. Biehi, and K. M6bius, J. Chem. Phys~ 61, 4335 (1974). 70a Freed and co-workers have developed a theory of ENDOR based on stochastic dynamical arguments. The theory is detailed in the volume edited by Dorio and Freed (see Ref. 46).

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219

Electron Nuclear Double Resonance Spectroscopy at Q or S Band Traditionally, most ENDOR spectra of metalloproteins have been taken at X-band microwave frequencies, probably because of the availability of commercial instruments and their relative ease of use. Q-band spectroscopy, however, may offer advantages in particular situations. When sample is limited, the enhanced sensitivity at higher frequency may make possible experiments that would not be feasible at lower frequency. Proton resonances overlapping with resonances of other nuclei will be shifted to higher frequencies where they are unlikely to mingle with and confound interpretation of resonances from other nuclei, so that mutually obscuring effects are no longer likely to be problematic. The higher magnetic field of Q-band spectroscopy results in a correspondingly higher Larmor frequency for nuclei other than protons as well. This effect, shifting resonances to higher frequencies more amenable to detection than those obtaining at X-band, has been put to advantage in recent studies of iron-sulfur proteins.7~ For nuclear spins greater than 1, mixing of nuclear states by the nuclear quadruple operator can give rise to Ami = 2 transitions observable at Q-band frequencies but likely to be lost at X-band (so-called distant ENDOR signals, observed when internuclear magnetic interactions are stronger than the hyperfine coupling of nuclei to electrons of the paramagnetic center). 72 Relaxation pathways are then provided by polarization transfer from the nucleus under observation to the paramagnetic electron via intervening spins. Distant ENDOR resonances may be better resolved at higher microwave frequencies. They are typically centered at nuclear Larmor frequencies and require lower rf powers for detection than do local ENDOR resonances. Because of complex cross-relaxation effects, the amplitudes of distant ENDOR signals may be surprisingly large, 72 so that nuclei in small concentrations, such as naturally abundant ~3C, may be observed. Distant ENDOR has been observed from naturally abundant 13C as well as from 14N in Q-band ENDOR spectra of ironsulfur proteins. 71 S-band (2-4 GHz) ENDOR studies of radical centers in diamond and y-irradiated sucrose have been reported using a loop-gap resonator. 3° In addition to the increased resolution associated with cancellation of A and g strain effects at low microwave frequencies, S-band ENDOR provides the advantage of exploiting the hyperfine enhancement effect (see below) as the reduction in microwave frequency substantially increases nuclear transition probabilities compared to those observed at X-band frequencies. 71 A. L. P. Houseman, B.-H. Oh, M. C. Kennedy, C. Fan, M. M. Werst, H. Beinert, J. L. Markley, and B. M. Hoffman, Biochemistry 31, 2073 (1992). 72 E. Boroske and K. M6bius, J. Magn. Reson. 28, 325 (1977).

220

PROBES OF METAL ION ENVIRONMENTS

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Applications of S-band E N D O R spectroscopy to paramagnetic metal centers in biological systems have not yet been described.

Simulations o f Electron Nuclear Double Resonance Spectra

Perturbation and matrix diagonalization methods have both been used to simulate E N D O R spectra, generally following methods successfully used for the simulation of E P R spectra. 73-76 Perturbation methods have the great advantages of simplicity, since they are based on closed analytic expressions, and o f providing physical insights not readily attainable from numerical procedures. E x a c t diagonalization of the matrix of the parameterized spin Hamiltonian o v e r all molecular orientations contributing to the E P R spectrum at the observing field, and using a basis function set spanning the electron spin manifold and all nuclear manifolds of relevance, offers the most general and powerful approach to simulation of E N D O R spectra, 7v but it is expensive in c o m p u t e r and experimenter time. It has the great advantage in principle, however, of applicability to systems with S > ½ without simplifying assumptions, although to our knowledge simulations by direct diagonalization of E N D O R spectra from metalloproteins with S > ½ have not yet been reported. F o r systems with S = ½, the matrix diagonalization approach is relatively simple and powerful for extracting hyperfine coupling matrices from experimental spectra. TM Probably the most useful and used strategy for simulating and interpreting E N D O R spectra for systems with S = ½, or that can be represented with an effective spin S' = ½, is that advanced by Hoffman and colleagues. 79'8° At the heart of the method is the perturbation expression presented by Rist and H y d e 69 v+ = Ia/2 +- VNI

(7)

where vN is the nuclear L a r m o r frequency. Since A in Eq. (7) may not be constant along orientations of constant g, the task in simulation is to calculate the appropriate value of the hyperfine coupling, A. 73 F. K. Kneub0hl, J. Chem. Phys. 33, 1074 (1960). 74 R. Aasa and T. Vanngard, in "Paramagnetic Resonance" (W. Low, ed.), Vol. 2, p. 509.

Academic Press, New York, 1963. 75A. S. Yang and B. J. Gaffney, Biophys. J. 51, 55 (1987). 76C. P. Keijzers, E. J. Reijerse, P. Stam, M. F. Dumont, and M. C. M. Gribnau, J. Chem. Soc., Faraday Trans. 1 83, 3493 (1987). 77m. Kreiter and J. Htittermann, J. Magn. Reson. 93, 12 (1991). 78G. J. Baker and J. B. Raynor, J. Chem. Soc., Faraday Trans. 1 84, 4267 (1988). 79B. M. Hoffman, J. Martinsen, and R. A. Venters, J. Magn. Reson. 59, 110 ~1984). 8oB. M. Hoffman, R. A. Venters, and J. Martinsen, J. Magn. Reson. 62, 537 (1985).

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221

The initial step is to calculate the curve of points, S, as a function of the polar orientation angles 0 and ~b, corresponding to the value of g selected in the EPR spectrum by the fixed observing field. At any given frequency z,, an orientation along S will contribute to the ENDOR intensity if the hyperfine coupling at that orientation satisfies the resonance condition, u = /"-+ENDOR"The expected ENDOR intensity at frequency u will thus reflect the sum of probabilities that the hyperfine couplings taken over all points along S satisfy Eq. (4). In analogy to methods commonly used for the simulation of EPR spectra, 74'81 a shape function is defined for the probability that the hyperfine coupling assumes a particular value of A at the observing field defined by g: N(A+_)g =

[(SS/SA. )e[

(8)

The ENDOR intensity at frequency ~ is then the combined contributions from N ( A + ) g and N ( A _ ) g . The problem now is to calculate the shape functions N ( A _ )g. It is convenient to take the frame in which the g matrix is diagonal as the reference coordinate system. The orientation of the observing magnetic field must be specified in this frame, and the frame in which the hyperfine matrix is diagonal must be transformed to the reference coordinate system. 45'79 The principal values of the A matrix in its own frame, and the rotation angles, are then adjustable parameters in the simulation program. When the g and A matrices are coaxial, calculations are straightforward: ~ 2)1x 2 + (gy2 _ gz2)ly2 ( g 2 _ gz 2) = (gx 2 -- gz (A 2 -

A z 2)

(Ax 2

Az2)lx 2 + ( A y 2 - Az2)ly 2

(9) (10)

The polar angles 0 and th (or, equivalently, direction cosines) are obtained as functions o f g x , gy , gz , and g, and A x , Ay , A z , and A from the simultaneous Eqs. (9) and (10). Because S, the locus of points at constant g, also depends on 0 and qb, 1(SS/SA)gl becomes a calculable quantity. If the A and g tensors are not coaxial, the A tensor is rotated from the coordinate system in which it is diagonal to the g reference frame. The Euler angles involved in the transformation are then additional adjustable parameters in the simulation. Calculation of A, and of I(6S/3A)g I, becomes more tedious but is still tractable. In either case, the ENDOR spectrum is obtained by stepping through the range of A of experimental interest. The intensity calculated at each value of A is multiplied by a hyperfine or ff enhancement factor, z,/vN , reflecting the increase in effective radio frequency field perceived by the 81 A. D. Toy, S. H. H. C h a s t o n , J. R. Pilbrow, and T. D. Smith, lnorg. Chem. 10, 2219 (1971).

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PROBES OF METAL ION ENVIRONMENTS

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nucleus owing to its hyperfine interaction with the electron spin. 82 A persistent difficulty in simulating ENDOR spectra is the inability to compute transition probabilities reliably, as these depend on both EPR and NMR transitions, TM as well as on the physical characteristics of the ENDOR coil used for rf irradiation of the sample. The usual, and usually successful, approximation is to set all ENDOR transition probabilities to unity by using a constant line shape function in the convolution of 8 function spectra. An implicit assumption in the foregoing is that the components of EPR and ENDOR spectra are 8 functions arrayed over fields or frequencies in the simulations. To achieve a more realistic and useful simulation these restrictions must be relaxed. First, an ENDOR line shape function is used to convolve the calculated 8 function frequencies arising over the curve of constant g. A second convolution introduces broadening effects of finite EPR line widths. The net result is an overall broadening of the double-8 function simulation, although important details in simulated spectra usually survive such broadening. A difficulty in the first-order perturbation approach of Eq. (4) may arise when the hyperfine interaction of interest is dominated by dipolar coupling, rather than contact interaction, so that the A tensor is highly anisotropic and even asymmetric, although still diagonalizable with real eigenvalues, 83 In such instances electron and nuclear spins may be quantized along different directions, and the ENDOR frequencies may not be symmetric about the nuclear Larmor frequency at all g values represented in the EPR spectrum. This potential difficulty, which may be of greater theoretical than practical consequence, is circumvented when spectra are simulated by diagonalization of the matrix of the full spin Hamiltonian. 76'78 The general approach is to calculate ENDOR frequencies over the range of azimuthal angles 0 and ~b determined by the value of g at the observing field. Principal values of the A matrix are approximated from ENDOR at turning points of the EPR spectrum, then varied to optimize the fit of the simulated to experimental spectrum. The orientation of A and g tensors with respect to one another is similarly varied; the entire fitting procedure is essentially an interactive, iterative process. When a satisfactory fit is achieved, the 8 function (stick) spectrum may be convolved with a line shape function, usually a Gaussian. The result is a simulated ENDOR powder spectrum that takes on the characteristics of a single-crystal 82 S. Geschwind, in "Hyperfine Interactions" (A. J. Freeman and Frankel, R. B. eds.), p. 225. Academic Press, New York, 1967. 83 G. C. Hurst, T. A. Henderson, and R. W. Kreilick, J. A m . Chem. Soc. 1071 7294 (1985).

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CONTINUOUSWAVEENDOR SPECTROSCOPY

223

ENDOR spectrum at appropriate turning points in the EPR spectrum. A general program for simulating EPR, ENDOR, and ESEEM (electron spin echo envelope modulation) spectra has been developed, 76 but it is so computationally intensive as to require a fast mainframe computer to run in reasonable times. In contrast, the perturbation approach taken by Hoffman and colleagues can be implemented easily on a personal computer. Lines predicted in simulation programs are not always evident in experimental spectra. Sometimes, the difficulty is experimental, when microwave or radio frequency power is not optimized to achieve proper saturation of transitions under study. Occasionally, the problem is one of cross-relaxation: two lines at the same frequency may broaden one another beyond detectability of either. 6 In such case, a judicious choice in experimental microwave frequency may relieve the disparity between experiment and prediction. 6

I l l u s t r a t i v e E x a m p l e s : E l e c t r o n N u c l e a r D o u b l e R e s o n a n c e Studies of M e t a l l o p r o t e i n s with S -- ½

Iron-Sulfur Proteins As already indicated, two-iron ferredoxins from a variety of sources, enriched with 57Fe, were among the first metalloproteins studied by ENDOR spectroscopy. EPR spectra of these proteins revealed g tensors of nearly axial to rhombic symmetry, with little or no resolved hyperfine structure apparent in spectra. TM Hyperfine interactions, however, were strikingly evident in X-band ENDOR spectra of the reduced state of the proteins and could be explained taking an S = ~ ground state, as expected of one high-spin Fe(III) antiferromagnetically coupled to a high-spin Fe(II). Difference ENDOR spectroscopy of proteins freed of iron and reconstituted with either 57Fe or 56Fe essentially restricted features to those generated by hyperfine interactions of the unpaired electron with the magnetic iron nucleus. Proof that observed ENDOR lines actually arose from 57Fe hyperfine couplings came from the invariance of line frequencies with 10,% shifts in EPR frequencies and corresponding observing fields, as well as from line splittings with twice the nuclear Zeeman frequency of 57Fe. In contrast, proton spectra, because of their dominant nuclear Zeeman terms, 84 y. I. Shethna, P. W. Wilson, R. E. Hansen, and H. Beinert, Proc. Natl. Acad. Sci. U.S.A. 52, 1263 (1964).

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scaled with external field as microwave frequency was altered. Two distinct sets of 57Fe E N D O R lines, as expected from the two types of iron atoms in the spin-coupled Fe(II)-Fe(III) system, could thus be distinguished. Taking advantage of angle selection effects afforded by the anisotropic EPR spectra made it possible to extract components and the relative orientation of one of the 57Fehyperfine tensors. In all, the study provided a clear example of how detailed information may be extracted from ENDOR spectra. Extension of ENDOR spectroscopy to four-Fe clusters in iron-sulfur proteins was soon accomplished. 85 The high-potential protein from Chromatium showed two types of iron atoms, each with a nearly isotropic hyperfine tensor as expected of the relatively high symmetry in the cubanelike structure of the iron-sulfur cluster. A distinctly lower symmetry was found in the eight-iron protein of Clostridium pasteuranium, possibly because of intercluster spin-spin couplings. An unexpected and interesting finding was that the hyperfine couplings were appreciably smaller than those of the two-Fe protein, suggesting a greater electron delocalization onto the iron ligands. Advantages of ENDOR spectroscopy at Q-band microwave frequencies over that at traditional X-band frequencies were subsequently exploited to characterize hyperfine couplings in the [4Fe-4S] cluster of beef heart mitochondrial aconitase. 86 In addition to the obvious improvement in sensitivity accruing with higher frequency, Q-band spectroscopy offers two further advantages in delineating hyperfine interactions to ligand nuclei. First, confounding proton lines are shifted to higher frequencies, well apart from those of ligands (see above). At X-band frequencies, the proton Zeeman frequency is near 14 MHz, whereas at Q-band it is close to 40 MHz. Most ligand resonances, centered at frequencies largely determined by field-insensitive hyperfine energies, would be expected in a lower frequency range. Studies of solvent-metal interactions, using deuterated water, are facilitated at higher observing fields. Deuteron resonances at X-band typically occur between 0.5 and 3.5 MHz, where detection of E N D O R signals is difficult, but at Q-band extend from 6.5 to 9 MHz, where signals are easily recorded. The studies of aconitase reported by Hoffman and colleagues 86 represent a substantial achievement in the application of ENDOR spectroscopy to the study of active site structure and mechanisms in metalloenzymes. 85 R. E. Anderson, G. Anger, L. Petersson, A. Ehrenberg, R. Cammack, D. O. Hall, R. MuUinger, and K. K. Rao, Biochim. Biophys. Acta 376, 63 (1975). 86 M. M. Werst, M. C. Kennedy, H. Beinert, and B. M. Hoffman, Biochemistry 29, 10526 (1990).

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CONTINUOUSWAVE ENDOR SPECTROSCOPY

225

The species examined included reduced active enzyme, enzyme-substrate complex, enzyme-[170]carboxyl-labeled substrate complex, enzyme-perdeuterated substrate complex, several enzyme-substrate analog and enzyme-inhibitor complexes, and 57Fe- and 33S-substituted iron-sulfur clusters. Angle selection and isotope substitution effects were correlated to extract principal values and relative orientations of hyperfine tensors in the free enzyme and substrate-enzyme complex. Perhaps most interesting, and of greatest relevance to the enzymatic mechanism, was the demonstration of solvent binding (as OH-) to a particular iron atom of the four-Fe cluster and its protonation on binding of a substrate carboxylate ligand to the cluster. Nitrogen ENDOR signals remained virtually unchanged on binding of substrate, substrate analogs, or inhibitors to the enzyme. Enzyme-catalyzed interconversion of citrate and isocitrate therefore does not appear to involve displacement of an endogenous ligand, but rather addition of substrate ligands to the F e 4 - S 4 cluster and a change in the protonation state of bound solvent. A detailed discussion of the catalytic mechanism of aconitase, and the insights gleaned from ENDOR spectroscopy, has been presented. 87

Copper Proteins Copper is among the most tractable metals for ENDOR spectroscopy. The anisotropic EPR spectrum of Cu(II), with its axial g tensor and relatively narrow EPR lines, lends itself well to exploitation of angle selection effects; use of isotopically pure 63Cu and 65Cu (each with a nuclear spin of ~) simplifies interpretation of copper hyperfine splittings; electron relaxation times of Cu(II) are favorable for the saturation demanded by ENDOR spectroscopy; and hyperfine couplings of copper to ligand nuclei are generally in a satisfactory radio frequency range for ENDOR detection. Cu(II) has therefore been widely used as a probe of metal-binding sites in proteins, and enzymes dependent on copper have been favorite subjects for ENDOR experiments. The most important information elicitable from ENDOR spectroscopy of a copper-bearing protein usually pertains to the ligands and solvent accessibility of the bound copper species. Studies with model Cu 2+ complexes demonstrated the potential of ENDOR in revealing superhyperfine splittings not resolved in EPR spectra, 88 in particular those attributable to the remote (nonligated) nitrogen nuclei of imidazole ligands first demonstrated in ESEEM spectroscopy. 89 Investigations of the blue copper pro87 H. Lauble, M. C. Kennedy, H. Beinert, and C. D. Stout, Biochemistry 31, 2735 (1992). 88 H. L. Van Camp, R. H. Sands, and J. A. Fee, J. Chem. Phys. 75, 2098 (1981). 89 W. B. Mims and J. Peisach, Biochemistry 15, 3863 (1976).

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tein stellacyanin expanded work already cited 4 in demonstrating coordination of at least two nitrogen ligands to the active copper, although the amino acid source of the nitrogen could not be ascertained. 9°'9~The occurrence of two nitrogen ENDOR lines in spectra obtained at EPR turning points, originally thought to arise from two equivalent planar nitrogens with tensor orientations perpendicular to each other, was subsequently attributed to two inequivalent nitrogen ligands, each with a nearly isotropic h y p e r f i n e t e n s o r . 92"93 Such isotropy is said to be characteristic of copper coordination by histidyl nitrogen. 92 (It might be noted, in passing, that an anisotropic A tensor may produce a powder ENDOR pattern at a fixed field within the EPR powder pattern if many orientations are represented in this field. 94'95) For the first time, copper hyperfine splittings could be observed in the ENDOR spectrum of a copper protein; anisotropy of the copper hyperfine tensor was ascribed to substantial departures of the copper ligand field from tetragonal symmetry. Cytochrome-c oxidase has been the subject of copper ENDOR studies in a number of laboratories. The cytochrome oxidase molecule, of still uncertain polypeptide structure and molecular mass, 96 bears four distinct metal centers, arranged in two pairs. One pair, where dioxygen is bound and reduced during the catalytic cycle, consists of a binuclear cluster of a heme (heme a3) and a copper ion, CUB, and is inaccessible to EPR-ENDOR spectroscopy. The other pair, heme a and Cu A, participates in the flow of electrons from reduced cytochrome c to the dioxygen-binding binuclear center. Early work demonstrated an indeterminate number of weakly coupled protons with anisotropic hyperfine interactions and at least one nitrogen ligand to the EPR-visible "intrinsic" CUA of the enzyme. 97 Only a single pair of lines attributable to nitrogen could be identified, possibly because the partner pair was lost in the broad lump of matrix proton resonances. Final identification of the nitrogen resonances came

9o M. M. Werst, C. E. Davoust, and B. M. Hoffman, J. Am. Chem. Soc. 113, 1533 (1991). 91 j. E. Roberts, T. G. Brown, B. M. Hoffman, and J. Peisach, J. Am. Chem. Soc. 102, 825 (1980). 92 H. Yokoi, Biochem. Biophys. Res. Commun. 108, 1278 (1982). 93 j. E. Roberts, J. F. Cline, V. Lum, H. Freeman, H. B. Gray, J. Peisach, B. Reinhammar, and B. M. Hoffman, J. Am. Chem. Soc. 106, 5324 (1984). 94 S. P. Greiner and M. Baumgarten, J. Magn. Reson. 83, 630 (1989). 95 G. A. Rottman, K. Doi, O. Zak, R. Aasa, and P. Aisen, J. Am. Chem. Soc. 111, 8613 (1989). 96 S. I. Chan and P. M. Li, Biochemistry 29, 1 (1990). 97 H. L. Van Camp, Y. H. Wei, C. P. Scholes, and T. E. King, Biochim. Biophys. Acta 537, 238 (1978).

[7]

CONTINUOUS WAVEENDOR SPECTROSCOPY

227

from use of enzyme from yeast grown with 15N-substituted histidine98; at least one histidine was demonstrated to be a ligand of CuA. Another study identified copper hyperfine signals, thus establishing the participation of Cu(II), rather than the alternative possibility of a thiyl radical (R-S.) coordinated to Cu(I), in the heme a--CUA site of the enzyme. 99 The small copper hyperfine energies found, 68-90 MHz, were taken to indicate a high degree of covalency of the Cu(II) ion. During enzymatic turnover, the ordinarily invisible CuB generates an EPR signal which can be distinguished from that given by Cu A.~00ENDOR spectroscopy of this copper shows resonances attributed to three nitrogen ligands, of which at least one appears to be from a histidine residue. Studies of proton resonances of cytochrome-c oxidase have also been revealing. Comparative studies of native yeast cytochrome oxidase with enzyme in which deuterium was substituted for protons at the/3 carbon atoms of cysteine made possible assignment of specific lines to/3 protons of cysteine ligated to CUA.J°I On going from fully oxidized (a 3+" CUA2+" a33 +" CUB2+) or two-electron reduced CO-ligated enzyme (a 3+ • CUAz+ • aa2+ • CO" CuB+) to a more completely reduced species, the ENDOR splittings of the/3 protons was substantially reduced, suggesting that the methylene protons sense a reductive event remote from the CuA site. The value of copper as an ENDOR probe of metal-binding sites in proteins was shown in a study of 65Cu-substituted transferrin, l°z the iron transport protein of blood plasma. The transferrin molecule consists of a single polypeptide chain arranged in two lobes, each lobe bearing a metalbinding site that can accommodate a large variety of metal ions in addition to Fe(III). 1°3 Spectra obtained with the protein loaded with two Cu(II) ions were qualitatively identical to the spectrum from the protein selectively labeled at the A site in the C-terminal lobe, thus indicating similarity in the ligand structures of the sites. Nearly isotropic hyperfine splittings from a single nitrogen nucleus were identified and assigned to a ligand imidazole. The small principal values of the coupling tensor, 30.8-31.5 98 T. H. Stevens, C. T. Martin, H. Wang, G. W. Brudvig, C. P. Scholes, and S. I. Chan, J. Biol. Chem. 257, 12106 (1982). 99 B. M. Hoffman, J. E. Roberts, M. Swanson, S. H. Speck, and E. Margoliash, Proc. Natl. Acad. Sci. U.S.A. 77, 1452 (1980). 100j. Cline, B. Reinhammar, P. Jensen, R. Venters, and B. M. Hoffman, J. Biol. Chem. 258, 5124 (1983). 101 C. Fan, J. F. Bank, R. G. Dorr, and C. P. Scholes, J. Biol. Chem. 263, 3588 (1988). J0~ j. E. Roberts, T. G. Brown, B. F. Hoffman, and P. Aisen, Biochim. Biophys. Acta 747, 49 (1983). 103 D. C. Harris and P. Aisen, in "Iron Carriers and Iron Proteins" (T. M. Loehr, ed.), VCH, Weinheim, 1989.

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MHz, were attributed to electron withdrawal by a coordinated tyrosine. A resolved copper hyperfine line, observed at the g± extremum of the EPR line, provided a value for the perpendicular component of the axial copper hyperfine tensor, which could not be obtained from the EPR spectrum itself. The ligand structure of the sites, revealed by X-ray crystallography, TM subsequently corroborated inferences about histidine ligation from ENDOR spectroscopy. Electron Nuclear Double Resonance Studies of Metalloproteins with S > ½ For paramagnetic metal ions with S > ½, the effects of zero-field (finestructure) terms in the spin Hamiltonian must be taken into account in interpreting ENDOR spectra. Two cases should be distinguished, depending on whether the zero-field tensor has near-axial or lower symmetry.

S > ~, Axial Symmetry In the first instance, the analysis of experimental spectra is usually a relatively straightforward extension of the analysis of spectra from systems with S = ½. The hyperfine Hamiltonian can be transformed from the true spin representation to an effective spin representation with S' = -.~105 Perturbation expressions applicable to S = ½ systems must be appropriately modified in the S' = ½representation, so that the ENDOR frequency is specified by I"ENDOR --

g'A g 2

---+ P i

(1 1)

where g' is the effective or apparent g value corresponding to the observing field, g is the free-electron g value which may be taken as 2, and A is the hyperfine coupling energy. As in the case of S = ½systems, the A tensor must be transformed to the appropriate reference frame, usually that in which the g tensor is diagonal. For systems with S > ½the presence of a zero-field splitting presents both difficulty and opportunity in the interpretation of ENDOR spectra. The interaction of energy levels from which resonance transitions are observed to close-lying but otherwise "silent" states can generate a large anisotropic "pseudonuclear Zeeman effect" (PNZE), present in addition to the true nuclear Zeeman interaction. Splittings produced by the pseudo104 C. A. Smith, B. F. A n d e r s o n , H. M. Baker, and E. N. Baker, Biochemistry 31, 4527 (1992). 105 A. E. True, M. J. N e l s o n , R. A. Venters, W. H. O r m e - J o h n s o n , and B. M. H o f f m a n , J. Am. Chem. Soc. 1111, 1935 (1988).

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nuclear Zeeman effect can be much larger than the scalar splitting of the nuclear Zeeman interaction. 45 The origin of the pseudonuclear Zeeman effect can be seen in the second-order perturbation expression obtained when the fine structure splitting energies are substantially greater than energies from electronic Zeeman and hyperfine terms. The eigenfunctions of the fine structure term are taken as the zero-order wave functions in the perturbation treatment, with the electronic Zeeman and hyperfine terms combined as a perturbation. Terms in ~2 and [2 then arising make no or little contribution to the ENDOR spectrum and may be ignored. Cross-terms involving the interaction of the electronic Zeeman term with the hyperfine term, however, give rise to the PNZE, which takes the form PNZE = ~ ((0IA(S" hli)(ilglz,(B" s)10) +

(0[g~a" (B. S)[i)(i4A(S • i)10)~ WoC~// }

(12)

Thus, if the zero-field separations of Kramers doublets (W0 - Wi) are sufficiently small, a splitting of ENDOR frequencies results that can be much larger than the splittings caused by the nuclear Zeeman term itself. The equation for the PNZE simplifies to PNZE -- CxB~[~ + CyBy[y + CzBz[ z

(13)

in which the Ci terms represent appropriate constants in an expression that has the form of an anisotropic nuclear Zeeman interaction. Because the denominators in the full expression for the PNZE [Eq. (12)] are the zero-field splittings, it becomes possible to estimate the components of the fine-structure term in the spin Hamiltonian from the observed ENDOR splittings. 95,1°5 This is the opportunity accompanying the PNZE. Information to be gleaned from ENDOR spectroscopy of metalloproteins with S > ½is illustrated in a study of the molybdenum-iron cofactor of Azotobacter vinelandii nitrogenase enriched with 57Fe.105Still uncertain when the study was undertaken, the EPR-active Mo-Fe-S cluster is now known from X-ray crystallography to have the overall composition MoFe7S81°6 with a total spin S = ~ and an EPR signal of near-axial symmetric (h = 0.053). The PNZE made it possible to estimate the zero-field separation between the two Kramers doublets with high precision, a value of 12.2 cm-~ being obtained. 107Because of the spread in ENDOR frequencies I06 j. Kim and D. C. Rees, Science 257, 1677 (1992). 107 R. A. Venters, M. J. Nelson, P. A. McLean, A. E. True, M. A. Levy, B. M. Hoffman, and W. H. Orme-Johnson, J. A m . Chem. Soc. 108, 3487 (1986).

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brought about by the effect described in Eq. (I), it was possible to distinguish and characterize the 57Fe hyperfine tensors in five of the seven iron atoms of the cofactor cluster in a striking example of multisite polycrystalline ENDOR spectroscopy. Each of the resolved Fe sites was found to differ from its partners in hyperfine tensor components and orientation despite the strong electron delocalization within the cluster, indicating a remarkable complexity in electronic configuration yet to be related to functional activity. The possibility that the unseen iron atoms have hyperfine parameters identical to those of the observed iron atoms must now be considered in view of the seven-Fe structure of the Mo-Fe cofactors, but other possibilities are not excluded.

S > ~, Orthorhombic Symmetry In the second instance, when the fine structure tensor of the spin Hamiltonian has lower than axial symmetry, the simple eigenfunctions of Sz are no longer eigenfunctions of the full spin Hamiltonian. It is therefore necessary to diagonalize the matrix of the fine structure tensor in order to obtain the correct zero-order wave functions for perturbation calculations. (The shortcomings of the g tensor approach in the analysis of EPR spectra has long been appreciated.~°8) In one protein studied, transferrin, iron is bound to each of the two similar but not identical sites of the protein as high-spin (S = ~) Fe(III). The features of the EPR spectrum of each site are best described with a spin Hamiltonian dominated by the fine-structure term. Because the fine-structure tensor is almost completely rhombic (E/DI ~ 0.3 15), the EPR spectrum shows a prominent and nearly isotropic feature at g' = 4.3, arising from the virtually isotropic middle Kramers doublet of the S = ~ sextet. All orientations of the paramagnetic Fe(III) centers contribute to the g' = 4.3 EPR signal, and therefore to the ENDOR spectrum. Energies of the uppermost and lowermost Kramers doublets are orientation-dependent, however, so that the PNZE leads to an anisotropic powder-type ENDOR spectrum from the g' = 4.3 EPR line, even though that line is almost isotropic. Numerical diagonalization over all orientations of the matrix of the full spin Hamiltonian is then required for calculation of ENDOR frequencies. Zero-field splittings can then be obtained from the parameters of the spin Hamiltonian that give the best match of calculated to observed frequencies. A slight difference in D, distinguishing the two sites of transferrin, was thereby detected in the 57Fe ENDOR spectra of the protein selectively occupied at each site. Differences between the 57Fe ENDOR spectra of the two sites of transferrin were much ~06W. E. Blumberg, in "Magnetic Resonance in Biological Systems" (A. Ehrenberg, B. G. Malmstr6m, and T. V~inng~rd, eds.), p. 119. Pergamon, Oxford, 1967.

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more striking than differences between EPR spectra, but a further analysis of the ENDOR differences was not undertaken. Concluding Comments The rapidly accelerating progress in the biological applications of ENDOR spectroscopy seen since the 1970s reflects a combination of instrumental and conceptual advances. Structural information, and its functional implications, has been wrested from ENDOR studies of almost all magnetic nuclei coupled to the paramagnetic centers encountered in biological molecules. ENDOR spectroscopy, with the detailed insight it offers into the local structure of a paramagnetic centers in solution and the alterations of such structure during biological function, can often complement the tremendous power of X-ray crystallography in studies of metalloproteins. Interpretation of ENDOR spectra merges intuition with analytic rigor, with the limitations of the one often surmounted by the power of the other. Sensitivity problems persist but are yielding to studies at ever higher frequencies. Fruitful exploitation of Triple resonance in studying metalloproteins has yet to be accomplished. The wide availability of the personal computer, with its dramatically increasing speed and power, has enormously facilitated processing and interpretation of spectroscopic data. A persistent difficulty is to achieve understanding of the relaxation processes and pathways governing ENDOR spectroscopy; such understanding has yet to attain the peaks achieved by other, perhaps simpler, spectoscopies. In the end, the ingenuity and resourcefulness of experimenters remain the most powerful tools in the armamentarium of ENDOR spectroscopy. Acknowledgments Preparation of this chapter was supported in part by Grants GM-40168 and RR-02583 (J. Peisach), and DK-15056(P. Aisen)from the NationalInstitutes of Health, U.S. Public Health Service.

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ION ENVIRONMENTS

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[8] V a n a d y l ( I V ) E l e c t r o n N u c l e a r D o u b l e Resonance/Electron Spin Echo Envelope Modulation Spin Probes B y N. DENNIS CHASTEEN

Introduction The oxycation vanadyl(IV), VO 2+, has been historically used as an electron paramagnetic resonance (EPR) spin probe of metal binding sites in proteins. 1 More recently, VO 2+ has been employed as an E N D O R (electron nuclear double resonance) and E S E E M (electron spin echo envelope modulation) spin probe. These higher resolution resonance techniques provide more detailed information about the metal binding site than is possible with E P R spectroscopy alone. In particular, magnetic nuclei with couplings less than the natural width of 5-15 G of the EPR line can be identified and studied by E N D O R or E S E E M . In favorable cases, one can obtain information about the geometrical arrangement of magnetic nuclei in the vicinity of the electron spin as well. In this chapter we briefly review some of the salient features of the E P R spectra of vanadyl(IV)-protein complexes and then give a synopsis of some recent E N D O R and E S E E M work. The principles o f E P R , E N D O R , and E S E E M spectroscopies are discussed elsewhere in this volume and are not reiterated here. Some aspects of E N D O R and E S E E M spectroscopy of v a n a d y l - p r o t e i n complexes and small chelates have been recently reviewed. 2

Electron Paramagnetic Resonance Spectroscopy VO 1÷ is an S = ½paramagnet with a 3d 1 outer electron configuration. When complexed, VO 2÷ has an orbitally nondegenerate ground state with no excited states nearby in energy. These are the requirements for observing electron resonance at room temperature with solution samples as well as at low temperatures with samples in the frozen state, a situation c o m m o n l y encountered with biological EPR studies. i N. D. Chasteen, in "Biological Magnetic Resonance" (L. Berliner and J. Reuben, eds.), Vol. 3, p. 53. Plenum, New York, 1981; N. D. Chasteen, Struct. Bonding (Berlin) 53, 107 (1983). z S. S. Eaton and G. R. Eaton, in "Vanadium in Biological Systems" ~N. D. Chasteen, ed.), p. 199. Kluwer Academic Publ., Dordrecht, The Netherlands, 1990. METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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233

Parallel Lines -1/2

+100"

I

,

""'A'

I

r-I

1/

t-i E

0

//a

IT I1. LIJ

i/2 ~"+ 3/2 ,~

5•3, 2

Perpandicul

-100

I

s

i

2320

i

3320

Field

I

4320

{Gauss)

FIG. 1. F r o z e n solution spectrum (77 K) of VO 2-- complexed with apoferritin. Parallel and perpendicular hypertine lines are indicated. Two forms of VO 2+ binding, a and fl, are apparent.

The large moment of the 99.75% abundant I = ½ 51V nucleus causes EPR spectra of frozen solutions to be composed of overlapping patterns of eight lines. Such a spectrum is shown in Fig. 1 for the VO 2+ ion complexed with apoferritin, the iron storage protein. 3 To a good approximation, VO 2+ EPR spectra can be analyzed using the following spin Hamiltonian: '~ : fl(gxxHxSx + gyyHySy + gzzHzSz) - g.fln(H,:ix + nyiy + H J z ) + AVxx,~:,iv+ A vyySyI ^ ~vy Jr mVzzSziV z "~ ~ S . A L. i L

(1)

L

The g factor terms represent the electron Zeeman interaction, and the terms in AVx,:, AVyy, and AV~x denote the vanadium nuclear hyperfine interaction. The sum term includes all ligand nuclear superhyperfine interactions. Here x, y, and z represent the coordinate system for the g matrix, which is assumed to be coincident with that of the vanadium nuclear 3 N. D. Chasteen and E. C. Theil, J. Biol. Chem. 257, 7672 (1982).

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hyperfine matrix. The coordinate systems for the nuclear superhyperfine interactions of the various ligands, Y~S • A L • i L, are not generally coincident with the g matrix coordinate system. VO 2÷ complexes typically show axial or pseudoaxial EPR spectra such that gzz = gll, gxx = gyy -- g±, AVzz = AVlland AVx~ = AVyy -- AV±. Thus, to a first approximation, the frozen solution EPR spectrum is a superposition of two sets of eight hyperfine lines with vanadium nuclear hyperfine couplings AVlland AV± (Fig. 1). The parallel and perpendicular lines in Fig. 1 arise from those molecules in the polycrystalline sample having their VO bond axis parallel or perpendicular to the direction of the applied magnetic field. The labeling of the hyperfine lines in Fig. I according to the m I values of the 51V nucleus assumes a negative coupling constant. Values for AvH range from (-)138 × 10 - 4 to (-)183 × 10 - 4 cm -1 (150 to 203 G) and AV± from (-)47 × 10 - 4 t o (-)71 × 10-4 cm -1 (51 to 77 G) in going from strong to weak ligand fields. 4 Similarly gll changes from 1.972 to 1.932 and g± from 1.985 to 1.972.1'4 The spin Hamiltonian parameters gll, g±, AVll, and AV± reflect the ligand environment of the metal. Multiple binding sites are often evident in EPR spectra of VO2+-protein complexes. In the case of VO2+-apoferritin, two forms of VO 2+ binding, labeled a and/3, are evident in the EPR spectrum of Fig. 1 and are attributed to two states of hydrolysis of the metal (see below). The identity of the coordinating ligands can be inferred from the values of the spin Hamiltonian parameters using additivity relationships1; however, a better approach is to observe ligand nuclei directly using E N D O R or ESEEM spectroscopy. Ligand nuclear superhyperfine couplings are not often observed in VO 2+ EPR spectra owing to the fact that the unpaired electron resides in a molecular orbital that is largely dxy in character and nonbonding, the x and y axes being approximately along the equatorial ligand-metal bonds. Ligand superhyperfine couplings typically less than the peak-to-peak EPR line widths, while not observable by EPR, can be measured by ENDOR and ESEEM. They generally fall in the range 2 MHz (e.g., 13C)to 16 MHz (e.g., 1H).5-7 Table I summarizes the proteins studied to date by EPR, ENDOR, or ESEEM spectroscopy and the nuclear spin couplings observed. 4 N. D. C h a s t e e n , R. J. D e K o c h , B. L. Rogers, and M. W. H a n n a , J. Am. Chem. Soc. 95, 1301 (1973). 5 p. A. Tipton, J. M c C r a c k e n , J. B. Cornelius, and J. Peisach, Biochemistry 28, 5720 (1989). 6 D. Mustafi and M. W. Makinen, Inorg. Chem. 27, 3360 (1988). 7 D. Mustafi, J. Telser, and M. W. Makinen, J. Am. Chem. Soc. 114, 6219 (1992); D. Attanasio, J. Phys. Chem. 90, 4952 (1986).

TABLE I PROTEINS STUDIED BY VO 2+ ELECTRON SPIN PROBES Protein

Resonance method(s)

Nuclei observed

Bromoperoxidase Calmodulin Carbonate dehydratase Carboxypeptidase A Collagen Ferritin Insulin Lactoferrin Ovotransferrin Pyruvate kinase

EPR~/ESEEM b EPR C EPR d EPR e EPR f EPRg/ENDORh/ESEEM i EPRJ EPRk/ESEEM l EPRm,n EPR°/ESEEM p

ATP : L-methionine s-adenosyltransferase Serum albumin Testicular S-100-1ike protein Transferrin Xylose isomerase

EPR q:

IH, 2H, 14N, 5Iv 51V 51V 5IV 51V IH, 2H, 14N, 51V 51V 1H, 2H, I3C, 14N, 5Iv 51V 13C, 14N, 170, 23Na, 51V, 31p, 133Cs 170, 31p, 51V, 203,205T1

EPR s't EPR" EPR"/ENDORh/ESEEM ~ EPR°/ENDOR °

5IV 5~V IH, 2H, 13C, 14N, 5iV IH, 14N, 51V

a E. de Boer, K. Boon, and R. Wever, Biochemistry 27, 1629 (1988). E. de Boer, C. P. Keijzers, A. A. K. Klaassen, E. J. Reijerse, D. Collison, C. D. Garner, and R. Wever, FEBS Lett. 235, 93 (1988). c R. H. Ahmed, J. Nieves, L. Kim, L. Echegoyen, and D. Puett, J. Protein Chem. 6, 431 (1987). d j. j. Fitzgerald and N. D. Chasteen, Biochemistry 13, 4338 (1974). e R. J. DeKoch, D. J. West, J. C. Cannon, and N. D. Chasteen, Biochemistry 13, 4347 (1974). f R. P. Ferrari, lnorg. Chim. Acta 176, 83 (1990). g N. D. Chasteen and E. C. Theil, J. Biol. Chem. 257, 7672 (1982). h p. M. Hanna, N. D. Chasteen, G. A. Rottman, and P. Aisen, Biochemistry 30, 9210 (1991). i G. J. Geffen, P. M. Hanna, N. D. Chasteen, and D. J. Singel, J. Am. Chem. Soc. 113, 9513 (1991). J N. D. Chasteen, R. J. DeKoch, B. L. Rogers, and M. W. Hanna, J. Am. Chem. Soc. 95, 1301 (1973). k A. Carmichael and J. S. Vincent, FEBS Lett. 105, 349 (1979). t S. S. Eaton, J. Dubach, K. M. More, G. R. Eaton, G. Thurman, and D. R. Ambruso, J. Biol. Chem. 264, 4776 (1989). m D. Casey and N. D. Chasteen, J. Inorg. Biochem. 13, l l l (1980). n D. Casey and N. D. Chasteen, J. lnorg. Biochem. 13, 127 (1980). o K. A. Lord and G. H. Reed, Arch. Biochem. Biophys. 281, 124 (1990). P P. A. Tipton, J. McCracken, J. B. Cornelius, and J. Peisach, Biochemistry 28, 5720 (1989). q G. D. Markham, Biochemistry 2,3, 470 (1984). r G. D. Markham and T. S. Leyh, J. Am. Chem. Soc. 109~ 600 (1987). s N. D. Chasteen and J. F. Francavilla, J. Phys. Chem. 80, 897 (1976). t N. D. Chasteen, J. K. Grady, and C. E. Holloway, Inorg. Chem. 25, 2754 (1986). u j. C. Cannon and N. D. Chasteen, Biochemistry 14, 4573 (1975). v R. Bogumil, J. Hiittermann, R. Kappl, R. Stabler, C. Sudfeldt, and H. Witzel, Eur. J. Biochem. 196, 305 (1991).

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Electron Nuclear Double Resonance Spectroscopy VO 2÷ complexes have been investigated by ENDOR since the early 1980s. Some of the earliest and most definitive work was carried out by van Willigen and co-workers on model vanadyl complexes, s-ll One advantage of using VO 2÷ as an ENDOR spin probe is that its electron spin energy levels are relatively easy to saturate. Measurements can be carried out at I00 K with microwave powers of the order of 20 mW and radio frequency powers of 70-200 W. 6-11 However, stronger ENDOR signals are obtained around 5 K, the temperature where most protein work has been carried out. Concentrations of VO2+-protein complexes generally employed fall in the range 1-2 mM in order to minimize the amount of signal averaging required. A second advantage of VO 2÷ ENDOR spectroscopy is that the anisotropic EPR spectrum affords the opportunity of doing angle-selective ENDOR spectroscopy. The same is also true for VO z÷ ESEEM measurements. By placing the static magnetic field at "parallel" or "perpendicular" features of the EPR spectrum, one is able to make ENDOR measurements on a subset of molecules in the sample having their VO bond axes (the axis of gzz and Azz) approximately parallel or perpendicular to the direction of the applied magnetic field. Spectra gathered at the extreme ends of the spectrum, namely, at the ml = -½[[ and +½[[ lines (Fig. 1), represent true "single-crystal spectra" with the field along the VO bond axis of the complex. Spectra gathered at other parallel features, namely, at the m I = -----½[[, ±za[[, and ±~[[ lines, also have contributions from intermediate field orientations from neighboring hyperfine components; therefore, these spectra do not correspond to " p u r e " single-crystal spectra. Nevertheless, resonances from the subpopulation of parallel molecules corresponding to the m I = ± 11 field positions generally dominate the ENDOR spectrum. The ml = - 11 line rather than the mi = -Ill line is often used in ENDOR measurements because of its greater intensity (Fig. 1). In the case of perpendicular spectra, the m I = + ½ / and mi = +~J_ lines have fewer contributions from neighboring hyperfine components, but other lines have been used as well. Perpendicular lines, in addition to having contributions from intermediate orientations from neighboring hyperfine manifolds, contain signals from a range of in-plane orientations, 8 C. 9 B. 1o C. 11 H.

F. Mulks, B. Kirste, and H. van Willigen, J. Am. Chem. Soc. 104, 5906 (1982). Kirste and H. v a n Willigen, J. Phys. Chem. 86, 2743 (1982). F. M u l k s a n d H. v a n Willigen, J. Phys. Chem. 85, 1220 (1981). v a n Willigen, J. Magn. Reson. 39, 37 (1980).

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a fact that can complicate the analysis of the ENDOR spectrum, particularly in the case of 14N ENDOR (see below). The most intense feature in the EPR spectrum is due to near coincidence of parallel and perpendicular lines (Fig. 1). This feature corresponds to either the m I = +½ o r --½ hyperfine component, depending on the EPR spin Hamiltonian parameters of the complex. ENDOR measurements at this position have contributions from all orientations; however, the ENDOR spectrum in these cases is dominated by perpendicular features since there is statistically a higher population of perpendicularly oriented molecules in the sample. ENDOR spectra of VO2+-protein complexes have been limited to studies of ~H and 14N couplings, although 3Jp couplings have been observed in nucleotide complexes. 7 In the case of ~H ENDOR, the nuclear Zeeman energy at magnetic fields of X-band EPR is usually greater than the hyperfine coupling. In this case the ENDOR lines are given to a first order by VENDO R = I V . ±

Aii/21

(2)

where i equals x, y, and z. Equation (2) predicts a pair of proton ENDOR lines in the ENDOR spectrum centered about the free proton precessional frequency VH and separated by the hyperfine coupling constant Aii. Figure 2 shows the ENDOR spectra of VO2+-substituted D-xylose isomerase, an Mg 2+ enzyme that catalyzes the reversible isomerization of a-D-xylose to o~-Dxylulose/2 The ENDOR spectrum obtained with the magnetic field set on the m I = - 11 line is illustrated in Fig. 2a. Here differences from the free precessional frequency are plotted, and, therefore, VH corresponds to 0 MHz in Fig. 2a. Two pairs of IH lines with couplings under 1.5 MHz are apparent. In addition, there is a larger 3.5 MHz coupling labeled AA' (Fig. 2a). In Fig. 2b the ENDOR spectrum gathered at the ml = -~l[ EPR line is shown. The feature corresponding to AA' in the ml = - 11 spectrum is also observed in the ml = -~ll spectrum but is flanked by two other lines, presumably from orientations arising from the overlapping m~ = -½ hyperfine manifold. The presence of these additional lines as well as the asymmetry in intensity distribution in the two halves of the spectrum, the high frequency side of the spectrum being less prominent, suggest that the AA' coupling arises from a proton neither on the axis of the VO bond

12R. Bogumil, J. Hfittermann, R, Kappl, R. Stabler, C. Sudfeldt, and H. Witzel, Eur. J. Biochem. 196, 305 (1991).

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PROBES OF METAL ION ENVIRONMENTS

°

Jk I/

--A -3

[8]

mr-"21.

//--A

-2 -1 0 .1 *2 Frequency (MHz]

I *3

-c -3

I/-'°2°'c,

-2 -1 0 *1 *2 Frequency (MHz)

+3

FIG. 2. IH E N D O R spectra o f four-VO2+-substituted D-xylose i s o m e r a s e (B site) in frozen solution at a b o u t 5 K. (a) Magnetic field set on the mi = -½11E P R line; (b) rn I = -~11 E P R line; (c) ml = - ~ ± E P R line; (d) m I = +½ll, ± E P R line; (e) m~ = +½11,± E P R line in D20. ( F r o m Bogumil et al. 12)

or perpendicular to it. ~2The range of couplings observed is designated by the A and A' bands in Fig. 2b. The identity of the proton is unknown, but presumably the signal is due to a proton residing on the protein. If the maximum observed coupling of 4 MHz corresponds to Arl of the proton and is attributed solely to an electron-proton magnetic point dipole interaction, then a vanadiumproton distance of approximately 3.4 A is estimated, a value expected for a proton located in the second coordination sphere. A rigorous analysis of the proton E N D O R of protein complexes is usually not possible because sufficient information on the origin of the resonances is not normally available. Protein samples with selectively deuterated amino acids are needed to assist in the analysis. In principle one can obtain detailed structural information from proton ENDOR coupling constants provided that the principal values of the superhyperfine coupling tensor are known in magnitude and sign. An analysis

[8]

VANADYL(IV) E N D O R / E S E E M

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239

of the rich IH ENDOR spectrum of the V O 2+ ion in methanol solution has been carried out within the point dipole framework to elucidate the structure of the vanadyl-methanol complex. 6 This method has also been used in structural studies of vanadyl-nucleotide and vanadyl-Schiff base complexes: ENDOR spectra of the VO2+-substituted D-xylose isomerase complex obtained at the m I = - ~ ± and +½_1_field positions are shown in Fig. 2c-e. Comparison of the m I = + 1 1 spectra in HzO and D20 (Fig. 2d,e) show an exchangeable proton denoted BB' with a coupling of 1.7 MHz assigned to the N H proton of imidazole of histidine. 12This value is higher than the value of approximately 1 MHz reported for the VO(imidazole)42+ complex 8 and for histidine coordination in VOZ+-apoferritin.13 The larger coupling with D-xylose isomerase is in keeping with the larger 14N coupling observed with this protein (see below). In the case of 14N ENDOR, the nuclear Zeeman energy at magnetic fields of X-band EPR is generally smaller than the hyperfine coupling. In this case the ENDOR lines are given to a first order by I"ENDOR :

IAii/2 +- VN +- ~Qiil

(3)

where i equals x, y, and z. Four lines centered about Aii/2 are predicted. Figure 3 shows the 14N region of the ENDOR spectra of voE+-D-xylose isomerase measured at the m I = +½_l_ and m I = -~H EPR lines. Figure 3b shows a tentative assignment of the parallel spectrum from which values of the hyperfine coupling Azz = 13.2 MHz and quadrupole coupling Qzz = 0.72 MHz are obtained using Eq. (3). The z axis in this instance refers to the principal axis of the g matrix. The 2vN separation between the two doublets aids in assignment of the spectrum (Fig. 3b). The values of Azz and Qzz for xylose isomerase and other vanadyl protein and small chelate complexes are summarized in Table II. The large value of Azz = 13.2 MHz for xylose isomerase relative to those of the other complexes has been attributed to imidazole coordination in a nonequatorial fashion. 12 The presence of at least two inequivalent 14N nuclei is evident in the ENDOR spectra of VO 2+ complexes with apoferritin, 13imidazole, and carnosine s and are assigned to the coordinating N-1 and remote N-3 nitrogens of the imidazole ring. The presence of a second 14N is not evident in the spectrum of xylose isomerase (Fig. 3b). Assignment of the perpendicular spectrum (Fig. 3a) is not straightforward. The analysis is complicated by the broad lines encountered in 14N ENDOR studies of proteins in comparison to those of small chelates, perhaps reflecting significant "A strain" from local disorder in the metal 13 p. M. Hanna, N. D. Chasteen, G. A. Rottman, and P. Aisen, Biochemistry 30, 9210 (1991).

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PROBES OF METAL ION ENVIRONMENTS

[8]

l a

m z = +1/2

perpendicular

m r = -5/2 parallel L

~

I

2

3

4

5 6 7 Frequency (MHz)

8

T

9

10

FIG. 3. 14N ENDOR spectra of four-VO2÷-substituted D-xylose isomerase (B site) in frozen solution at about 5 K. (a) Magnetic field set on the mI = +½11,1EPR line; (b) m[ = -~I[ EPR line. The four-line pattern from a single 14Ncoupling is indicated. (From Bogumil et al) 2)

site of the frozen sample.13 Also, the marked variation in intensity of the E N D O R lines makes weak lines next to strong ones difficult to discern.13 The perpendicular spectrum is further complicated by the possibility of up to eight resonances, four from each of the x and y components described by Eq. (3). Difficulties in assigning perpendicular 14N E N D O R spectra are also e n c o u n t e r e d with small chelates. 9 Given the present level of sophistication of analysis, the principal value of 14N E N D O R lies in simply identifying nitrogen coordination in protein complexes. E N D O R measurements at more than one microwave frequency (e.g., X-band and Q-band), 14 should aid considerably in more rigorous assignment of E N D O R spectra in the future. Electron Spin Echo Envelope Modulation Spectroscopy A n u m b e r of properties of VO 2+ make it well suited for E S E E M measurements. The ability to obtain orientation information by selectively 14 M. M. W e r s t , C. E. D a v o u s t , and B. M. H o f f m a n , J. A m . Chem. Soc. 113, 1533 (1991).

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TABLE II 14N ELECTRON NUCLEAR DOUBLE RESONANCE PARAMETERS FOR H o PARALLEL TO V ~ O BOND Ligand ApoferritinC D-Xylose isomerase d I midazole e Histidine e Carnosinee Pyridine f Ammonia f

Nitrogen a

Azz (MHz)

Qzz (MHz) b

N- 1 N-3 N N- 1 * N-3 N-3* N- 1" N-3 N* N*

7.14 6.36 13.2

0.24 0.85 0.72 0.23 0.80 0.76 0.24 0.79 0.85 0.51

7.40

6.64 6.00 7.10 6.67 6.50 5.63

N-1 and N-3 refer to the sp 3 and sp 2 nitrogens, respectively, of the imidazole ring, and the asterisk denotes the coordinating nitrogen. b Qzz is the quadrupole coupling where z is taken as the V ~ O bond direction and does not necessarily correspond the principal z axis of the quadrupole coupling tensor, which usually lies along the V - N bond direction [see C. I. H. Ashby, C. P. Cheng, and T. L. Brown, J. A m . Chem. Soe. 100, 6057 (1978)]. From P. M. Hanna, N. D. Chasteen, G. A. Rottman, and P. Aisen, Biochemistry 30, 9210 (1991). From R. Bogumil, J. Hiittermann, R. Kappl, R. Stabler, C. Sudfeldt, and H. Witzel, Eur. J. Biochem. 196, 305 (1991). e From C. F. Mulks, B. Kirste, and H. van Willigen, J. A m . Chem. Soc. 104, 5906 (1982). f B. Kirste and H. van Willigen, J. Phys. Chem. 86, 2743 (1982).

pulsing with the static field set on parallel and perpendicular features in the continuous wave (CW) spectrum has already been mentioned within the context of ENDOR measurements. Because the ground state is largely nonbonding in nature, 14N couplings from directly bonded nitrogen can be observed, 5'~5unlike the situation with Cu E+ where the remote uncoordinated nitrogen of imidazole is seen in the ESEEM.~6 The depth of modulation is greatest when the hyperfine coupling is comparable to the nuclear Zeeman energy, ~v a situation which is observed with VO 2+. Generally good modulation depths are obtained with a variety of nuclei. 5 Moreover, 15 G. J. Gerfen, P. M. Hanna, N. D. Chasteen, and D. J. Singel, J. A m . Chem. Soc. 113, 9513 (1991). 16 W. B. Mims and J. Peisach, in "Advanced EPR: Applications in Biology and Biochemistry" (A. J. Hoff, ed.), p. I. Elsevier, Amsterdam, 1989, and references therein. 17 W. B. Mims and J. Peisach, in "Biological Magnetic Resonance" (L. Berliner and J. Reuben, eds.), Vol. 3, p. 213. Plenum, New York, 1981, and references therein.

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PROBES OF METAL ION ENVIRONMENTS

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the spectra of this relatively simple S = ½system are usually quite amenable to analysis, but measurements at more than one microwave frequency are needed to avoid erroneous assignments. 5,15 Optimum concentrations for ESEEM measurements are in the 1-2 mM VO 2+ concentration range as for ENDOR. Both two- and three-pulse spin echoes are generally collected depending on the magnitude of the couplings and the modulation decay times of the observed nuclei. As a brief illustration, Fig. 4 shows the two-pulse ESEEM pattern and the corresponding frequency spectrum for VO 2+ complexed to apoferritin. ~5 An expanded portion of the low-frequency region is shown in Fig. 5. The collection of peaks below 10 MHz are assigned to laN. By a combination of frequency tracking, where the ESEEM measurement is made at more than one microwave frequency, and field sampling within the EPR spectrum, the ESEEM frequency spectrum can be assigned in some detail. For example, the broad weak feature near 11.5 MHz and above is assigned to the sum combination of the fundamental doublequantum frequencies of the upper and lower I = 1 manifolds of the Ms = ---½spin states. The peak at approximately 9 MHz is assigned to the higher frequency partner double-quantum transition and the negativephase peak at 6.7 MHz to the sum combination of the single-quantum

0.0

1.0

2.0

3.0

4.0

30.0

40.0

~sec

0.0

10.0

20.0 MHz

FIG. 4. Two-pulse E S E E M pattern (top) a n d corresponding f r e q u e n c y s p e c t r u m (bottom) o f VO2*-apoferritin ct c o m p l e x at 4.2 K with the field set on the m 1 = +½11line. (From Gerfen et al. 15)

[8]

VANADYL(IV) E N D O R / E S E E M i

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243

w

t"03

05

0.0

I

I

I

I

I

2.5

5.0

7.5

10.0

12.5

MHz 14

FIG. 5. Expanded view of the N region of the ESEEM spectrum of the VO 2+ -apoferritin a complex shown in Fig. 4. (From Gerfen et al. tS)

fundamentals. The feature at approximately 5 MHz comprises a number of overlapping peaks, namely, the lower frequency partner of the doublequantum transition, the double-quantum difference combination, and the higher frequency single-quantum fundamental. The lower frequency partner single-quantum transition appears at around 2 MHz. These assignments correspond to an 14N hyperfine coupling of around 6.7 MHz, a value typical of coordinated histidine cis to the vanadyl oxo group 8'~3,~5 and comparable to the value of approximately 7 MHz obtained from ENDOR studies of VO2+-apoferritin. 13 The ESEEM spectrum in Fig. 3 is very similar to that observed for vanadyl-transferrin, where histidine is a ligand. 18 The 14N quadrupole coupling constant Qzz is too small to be readily extracted from these spectra but has been obtained from ENDOR spectra. Normally ~H couplings are not observed in ESEEM spectra, and only a peak from "matrix" protons is observed at the Larmor frequency (16.3 MHz in Fig. 4). However, the feasibility of using the negative-phase dipolar-shifted 1H sum combination peak near 33 MHz in Fig. 4 for probing first-coordination sphere water protons has been demonstrated.15 In this study, it was shown that the differences between the a and/3 forms of VO z÷ bound to apoferritin (Fig. 1) is due to deprotonation of a first coordination sphere water molecule. ~5Analysis of the shifted 1H sum combina~s S. S. Eaton, J. Dubach, K. M. More, G. R. Eaton, G. ThurnSan, and D. R. Ambruso, J. Biol. Chem. 2,64, 4776 (1989).

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tion peak should find application in the study of other S = ½ systems as well. The sum combination peaks of the aqua-VO 2+ complex has been analyzed in detail and should serve as a model for future work with proteins. 19 In general, assignment of ESEEM spectra of vanadyl complexes proceeds along similar lines as for other S = ½metal ions. The reader should consult the papers listed in Table I for additional examples of ENDOR and ESEEM assignments and references to the original literature. The ESEEM study of the vanadyl-pyruvate kinase complex shows examples of ESEEM spectra with couplings from a diversity of nuclei (Table I); the spectra of this protein complex are particularly rich in information. 5 i9 A. M. Tyryshkin, S. A. Dikanov, R. G. Evelo, and A. J. Hoff, J. Chem. Phys. 97, 42 (1992).

[9] M u l t i d i m e n s i o n a l N u c l e a r M a g n e t i c R e s o n a n c e M e t h o d s to P r o b e M e t a l E n v i r o n m e n t s in P r o t e i n s By G E R A R D W . C A N T E R S , CORNELIS W . H1LBERS, M A R T VAN DE K A M P , a n d SYBREN S. W I J M E N G A

1. Introduction This chapter deals with methods to obtain information about metal sites in proteins by means of nuclear magnetic resonance (NMR) techniques. NMR spectroscopy has developed into a powerful method to study structures both of metal environments in proteins and of proteins in general. Because its application in chemistry and biochemistry has been dealt with already in detail elsewhere, we refrain from introducing the basic principles of NMR; for the interested reader a number of excellent textbooks and up-to-date review articles are available (see Section 2). When considering metal sites in proteins, attention is given not only to metals that are naturally found in metal binding sites. It can be illuminating to replace the naturally occurring metal by another metal with better NMR properties. For example, calcium (Ca)- and zinc (Zn)-binding proteins can be advantageously studied by replacing the metal with cadmium (Cd). However, one should be prepared for possible deformations the replacement may cause at the metal site. Further, study of metals that do not naturally have a physiological function may be of interest, for instance, when these metals have therapeutic value, like platinum (Pt) in the wellknown Pt antitumor drugs. We also note that nuclei for which no physiologMETHODS IN ENZYMOLOGY,VOL. 227

Copyright © 1993by AcademicPress, Inc. All rights of reproductionin any form reserved.

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PROBES OF METAL ION ENVIRONMENTS

[9]

tion peak should find application in the study of other S = ½ systems as well. The sum combination peaks of the aqua-VO 2+ complex has been analyzed in detail and should serve as a model for future work with proteins. 19 In general, assignment of ESEEM spectra of vanadyl complexes proceeds along similar lines as for other S = ½metal ions. The reader should consult the papers listed in Table I for additional examples of ENDOR and ESEEM assignments and references to the original literature. The ESEEM study of the vanadyl-pyruvate kinase complex shows examples of ESEEM spectra with couplings from a diversity of nuclei (Table I); the spectra of this protein complex are particularly rich in information. 5 i9 A. M. Tyryshkin, S. A. Dikanov, R. G. Evelo, and A. J. Hoff, J. Chem. Phys. 97, 42 (1992).

[9] M u l t i d i m e n s i o n a l N u c l e a r M a g n e t i c R e s o n a n c e M e t h o d s to P r o b e M e t a l E n v i r o n m e n t s in P r o t e i n s By G E R A R D W . C A N T E R S , CORNELIS W . H1LBERS, M A R T VAN DE K A M P , a n d SYBREN S. W I J M E N G A

1. Introduction This chapter deals with methods to obtain information about metal sites in proteins by means of nuclear magnetic resonance (NMR) techniques. NMR spectroscopy has developed into a powerful method to study structures both of metal environments in proteins and of proteins in general. Because its application in chemistry and biochemistry has been dealt with already in detail elsewhere, we refrain from introducing the basic principles of NMR; for the interested reader a number of excellent textbooks and up-to-date review articles are available (see Section 2). When considering metal sites in proteins, attention is given not only to metals that are naturally found in metal binding sites. It can be illuminating to replace the naturally occurring metal by another metal with better NMR properties. For example, calcium (Ca)- and zinc (Zn)-binding proteins can be advantageously studied by replacing the metal with cadmium (Cd). However, one should be prepared for possible deformations the replacement may cause at the metal site. Further, study of metals that do not naturally have a physiological function may be of interest, for instance, when these metals have therapeutic value, like platinum (Pt) in the wellknown Pt antitumor drugs. We also note that nuclei for which no physiologMETHODS IN ENZYMOLOGY,VOL. 227

Copyright © 1993by AcademicPress, Inc. All rights of reproductionin any form reserved.

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ical role is known may occur in proteins like the metallothioneins, which sequester hazardous metals such as mercury (Hg), Cd, or silver (Ag). The study of the polypeptide environment of these metals can also be of interest. Time and again the merits and drawbacks of the NMR technique for the study of structure and function of proteins have been contrasted with those of the X-ray diffraction technique. Suffice it to say that for detailed structural studies, NMR spectroscopy, in contrast to the latter method, is limited to proteins with a molecular weight of approximately less than 20,000, although advances in the technique [three- (3D) and four-dimensional (4D) NMR, isotope labeling] may shift the limit of its applicability up to molecular weights of 30,000 or higher. However, when considering only the metal and its immediate surroundings, this limitation is much less restrictive. Metal sites in proteins with molecular weights much higher than 30,000 have been studied to good effect by NMR spectroscopy. Moreover, the NMR method is well suited to study conformational equilibria and time-dependent processes. Finally, chemical shifts may provide information about electronic distributions, and thus reactivity, in the active sites of metalloproteins. In this chapter the following topics are dealt with. Section 2 contains an introduction to general NMR methodology used for protein structure determination. Section 3 is dedicated to the structure of metal environments in proteins. It starts with the use of pH variations to identify metal ligands. Subsequently, techniques to probe the environment of a metal inside a protein are discussed first for paramagnetic, then for diamagnetic metal ions. The last part of Section 3 is devoted to the study of extrinsic ligands. Section 4 deals with time-dependent phenomena, namely, conformational equilibria, N H exchange rates, and NMR relaxation times. Section 5 focuses on probing the oxidation state of a metal site and changes thereof. Section 6, finally, deals with the topic of modeling metal sites in proteins. 2. Nuclear Magnetic Resonance and Protein Structure Nowadays one- (1D), two- (2D), and three-dimensional (3D) NMR spectroscopy techniques are indispensable tools in the study of structure and function of biomacromolecules. The popularity of multidimensional NMR spectroscopy started by the end of the 1970s with the development of 2D NMR techniques, followed by the advent of 3D NMR spectroscopy and even 4D NMR spectroscopy in the late 1980s. Although the pulse schemes and multidimensional NMR spectra have become quite complex and involved, the determination of a protein structure by NMR spectros-

246

PROBES OF METAL ION ENVIRONMENTS

[9]

copy is still based on only two main types of information, namely, J coupling constants and nuclear Overhauser enhancement (NOE) intensities. When a paramagnetic metal is present, additional structure information can be obtained from resonance line widths and chemical shifts (see below). The J coupling between two nuclei provides information about their through-bond connectivity. In particular vicinal coupling constants, 3j, are of interest as they may yield torsion angles via the well-known Karplus equations.l'2 NOEs provide for distance information. 3 The NOE denotes the phenomenon that the intensity of the NMR signal of a nucleus is affected when the magnetization of a nearby nucleus is perturbed, for example, by selective irradiation. This intensity change is due to transfer of magnetization from the irradiated nucleus to the nucleus under observation. The magnetization transfer does not take place through covalent bonds, but rather directly through space, and is mediated by the nuclear dipole-dipole coupling; its effectiveness falls off with the sixth power of the distance between the nuclei. NOEs are routinely determined nowadays from so-called NOESY (2D NOE spectroscopy), although the 1D NOE variant is sometimes used in the case ofparamagnetic proteins (see below). NOEs thus give valuable structural information about a protein. To probe the two types of NMR information, two main classes of NMR experiments are in use. J coupling information can be obtained from correlated spectroscopy, for example, 2D correlation spectroscopy (COSY) and 2D total correlation spectroscopy (TOCSY) [or homonuclear Hartmann-Hahn spectroscopy (HOHAHA)], whereas NOEs can be effectively studied by NOESY. The theoretical basis of 2D NMR spectroscopy has been discussed in detail in the book by Ernst e t al. 4 Comprehensive reviews have been given of the application of 2D NMR techniques to study protein structures, 5-7 and the book by Wfithrich I gives a good introduction to this subject. Methodological aspects of 2D NMR spectroscopy have been described in two recent volumes of this series .8 Several exciting developments since the late 1980s promise to extend the NMR methodolI K. Wiithrich, " N M R of Proteins and Nucleic Acids." Wiley, New York, 1986. 2 D. Neff, G. Otting, and K. Wiithrich, J. Am. Chem. Soc. 112, 3663 (1990). 3 D. Neuhaus and M. Williamson, "The Nuclear Overhauser Effect in Structural and Conformational Analysis." VCH, New York, 1989. 4 R. R. Ernst, G. Bodenhausen, and A. Wokaun, "Principles of Nuclear Magnetic Resonance in One and Two Dimensions." Oxford Univ. Press (Clarendon), Oxford, 1987. 5 A. Bax, Annu. Rev. Biochem. 58, 223 (1989). 6 G. M. Clore and A. M. Gronenborn, Protein Eng. 1, 275 (1987). 7 K. Wfithrich, Acc. Chem. Res. 22, 36 (1989). 8 N. J. Oppenheimer and T. L. James (eds.), this series, Vols. 176 and 177.

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

247

ogy with respect to both the size of the proteins that can be studied as well as the accuracy and precision of the structure determinations. These developments entail the extension of 2D NMR to 3D and 4D NMR spectroscopy, applied in a homonuclear fashion or used in conjunction with uniform 15N and ~3C labeling of proteins. The theoretical basis and early use of 3D NMR spectroscopy has been reviewed by Griesinger et al. 9 The utility of homonuclear 3D NMR methods has been demonstrated for several proteins. 1°-~3 The application of 3D and 4D NMR methods to structure determination has been reviewed by Clore and Gronenborn.14 The basic strategy for determining a protein structure by means of these types of NMR experiments is relatively straightforward and can be divided into three stages: (1) sequential assignment of main-chain and side-chain resonances; (2) derivation of a secondary structure based on a limited set of observed NOEs, coupling constants, and N H exchange rates; and (3) calculation of the 3D structure based on distance and torsion angle constraints derived from NOE intensities and the magnitude of J coupling constants, respectively, possibly supplemented with hydrogen bond constraints from NH exchange measurements. Stereospecific assignments greatly improve the quality of the distance and angle constraints. The first two stages of this strategy as applied to the blue copper protein azurin from P s e u d o m o n a s a e r u g i n o s a 13't5'16 are illustrated in Fig. 1, whereas two exemplary 3D solution NMR structures of metalloproteins, namely, P s e u d o m o n a s a e r u g i n o s a cytochrome c55~~7 and French bean plastocyanin, TMshown in Figs 2 and 3, illustrate the results of the final stage. The application of multidimensional NMR, possibly with 15N and/or 13C labeling, greatly improves the structure determinination procedure. 9 C. Griesinger, O. W. SCrensen, and R. R. Ernst, J. Magn. Reson. 84, 14 (1989). 10 H. Oschkinat, C. Cieslar, and C. Griesinger, J. Magn. Reson. 86, 453 (1990). 11 G. W. Vuister, R. Boelens, A. Padilla, G. J. Kleywegt, and R. Kaptein, Biochemistry 29, 1829 (1990). 12 S. S. Wijmenga and C. P. M. van Mierlo, Eur. J. Biochem. 195, 807 (1991). 13 M. van de Kamp, G. W. Canters, S. S. Wijmenga, A. Lommen, C. W. Hilbers, H. Nar, A. Messerschmidt, and R. Huber, Biochemistry 31, 10194 (1992). 14 G. M. Clore and A. M. Gronenborn, Annu. Rev. Biophys. Biophys. Chem. 20, 29 (1991). 15 H. Nar, A. Messerschmidt, R. Huber, M. van de Kamp, and G. W. Canters, J. Mol. Biol. 218, 427 (1991). 16 H. Nar, A. Messerschmidt, R. Huber, M. van de Kamp, and G. W. Canters, J. Mol. Biol. 221, 765 (1991). 17 D. J. Detlefsen, V. Thanabal, V. L. Pecoraro, and G. Wagner, Biochemistry 30, 9040 (1991). 18 j. M. Moore, C. A. Lepre, G. P. Gippert, W. J. Chazin, D. A. Case, and P. E. Wright, J. Mol. Biol. 221, 533 (1991).

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FIG. 2. Solution NMR structure of Fe(II) cytochrome c551 from Pseudomonas aeruginosa. 17 Stereoviews are given of the best-fit superposition of the main-chain atoms of 10 calculated structures (top) and of an expansion of the heine environment, showing mainchain atoms for residues 10-20 and 55-65 and side-chain atoms for the axi~tl Fe ligands His-16 and Met-61 (bottom). (Reprinted with permission from Detlefsen et al/7 Copyright 1991 American Chemical Society.)

First, an increased number of assigned NOE contacts can be obtained, and 3j(HN-H~) couplings can be obtained with higher accuracy. 2'19,2° In addition, an increased number of main-chain J couplings can be determined. In particular, heterocouplings between protons and other nuclei

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS IO

M e t 92

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251 10

Met 92

FIG. 3. Solution NMR structure of French bean Cu(I)-plastocyanin. ~8 Stereoviews are given of the best-fit superposition of the main-chain atoms of 16 calculated structures with the position of the Cu atom indicated by a circle (top) and of an expansion of the Cu site, showing the main-chain and side-chain atoms of the iigand residues of the best 10 structures (bottom). (Reprinted with permission from Moore et al. 18)

19 L. E. Kay and A. Bax, J. Magn. Reson. 86, 110 (1990). 2o p. Schmieder, V. Thanabal, L. P. McIntosh, F. W. Dahlquist, and G. Wagner, J. Am. Chem. Soc. 113, 6323 (1991).

252

PROBES OF METAL ION ENVIRONMENTS

[9]

(15N, 13C), 3 j ( H N - C ~ ) , 3 j ( H N - C ' ) , 3j(Haf--C'i_l), 20a can be informative as they provide together with the 3j(HN-H~) couplings more accurate 05 main-chain torsion angle constraintsZ~ ; also 3J(H'~Ni+1)couplings depend on the tO main-chain torsion angle although no simple cyclic relationship exists between them. 22a'b Side-chain torsion angles XI can be obtained from 3j(N-H~) couplings.23 Improved stereospecific assignments are facilitated by the improved resolution of 3D and 4D spectra. Also, relaxation parameters of ~3C and ~SN nuclei can be measured; such data may be of value in obtaining better defined spectral density functions and may, thus, help in better characterizing the internal and the overall motion of the protein. 24-29 Finally, better resolution furthers the detection of hydrogen exchange between amide NHs and bulk water, which provides a handle on the detection of hydrogen bonds. 3°-35 Because internal water molecules and water molecules in the hydration sphere of a protein may be considered to be intimately connected to both the structure and function of the protein, it is often worthwhile to investigate the structural aspects of these water molecules in addition to the protein structure itself. A number of special NMR techniques are available for this study. Although they are fairly new, they are mentioned here because they may turn out to be of use for the study of metal sites (see below). By using X-ray diffraction of crystals, both surface-bound and internal water molecules can be detected, provided the X-ray data 20a Expressions of the form nJ(Ai-Bj) indicate the n-bond spin-spin coupling constant between nuclei A and B of residues i and j, respectively. zl p. Schmieder and H. Kessler, Biopolymers 32, 435 (1992). 22a G. Wider, D. Neff, G. Otting, and K. Wiithrich, J. Magn. Reson. 85, 426 (1989). 22b A. Demarco, M. Llin~s, and K. W~ithrich, Biopolymers 17, 2727 0978). 23 G. T. Montelione, M. E. Winkler, P. Rauenbuehler, and G. Wagner, J. Magn. Reson. 82, 198 (1989). 24 L. E. Kay, D. A. Torchia, and A. Bax, Biochemistry 28, 8972 (1989). 25 L. E. Kay and D. A. Torchia, J. Magn. Reson. 95, 536 (1991). 26 L. E. Kay, L. K. Nicholson, F. Delaglio, A. Bax, and D. A. Torchia, J. Magn. Reson. 97, 359 (1992). 27 j. W. Peng, V. Thanabal, and G. Wagner, J. Magn. Reson. 95, 421 (1991). 28 G. Wagner, V. Thanabal, B. J. Stockman, J. Peng, N. R. Nirmala, S. G. Hyberts, M. S. Goldberg, D. J. Detlefsen, R. T. Clubb, and M. Adler, Biopolymers 32, 381 0992). 29 j. W. Peng and G. Wagner, J. Magn. Reson. 98, 308 (1992). 30 G. Wagner and K. WiRhrich, J. Mol. Biol. 1611, 343 (1982). 31 W. J. Chazin and P. E. Wright, J. Mol. Biol. 202, 623 (1988). 32 D. Marion, M. Ikura, R. Tschudin, and A. Bax, J. Magn. Reson. 85, 393 (1989). 33 S. Spera, M. Ikura, and A. Bax, J. Biomol. N M R 1, 155 (1991). 34 p. R. Gooley, D. Zhao, and N. E. MacKenzie, J. Biomol. N M R 1, 145 (1991). 35 j. D. Forman-Kay, A. M. Gronenborn, P. T. Wingfield, and G. M. Clore, J. Mol. Biol. 220, 209 (1991).

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are of sufficient resolution, the mobility of the water molecules is low, and water binding sites show sufficient occupancies. 1H NMR spectroscopy offers the advantage that, besides the structural aspects, the dynamic aspects of water molecules can be studied, too. However, it suffers from the drawback that signals from these waters must be distinguished against the huge background signal of the bulk water. Several schemes have been developed to overcome this problem. Because internally bound and surface-bound water molecules have different residence times, different techniques have been developed for their study. In the detailed investigations of Otting et a/. 36-39 o n BPTI (bovine pancreatic trypsin inhibitor) bound internal water molecules could be determined via NOESY experiments using no water presaturation. The presence of these water molecules showed up at the F1 frequency of water in NOESY and ROESY (rotating frame NOESY) spectra, where NOE connectivities were observed between protein resonances and the water signal. A comparison with the BPTI X-ray structure showed that all four bound internal waters detected by X-ray techniques in the crystal are also present in solution. The advent of homo- and heteronuclear 3D NMR has also meant an important step forward in this area. In heteronuclear 3D 1H{15N} N O E S Y - H M Q C and R O E S Y - H M Q C experiments (HMQC is heteronuclear correlation spectroscopy via heteronuclear multiple quantum coherence; here, ~H is the observed and ~SN the detected nucleus), internally bound water molecules can be detected more easily owing to the reduced overlap, the relevant protein-water cross-peaks now being spread out in the F 2 F 3 15N-~H plane at the F1 IH frequency of water 4°'41 (see also Fig. 10). In the past, water molecules of the hydration shell of a protein usually escaped detection when 2D NMR methods were applied because of their extremely short residence times (generally <1 nsec, but see Clore and Gronenborn41). Otting et a1.,38'39 however, observed a plethora of NOE contacts between aliphatic surface residues and water molecules in the hydration shell in an elegant 3D homonuclear NOESY-TOCSY experiment. Owing to the high mobility of the interaction vector connecting the protons of a particular water molecule with those of a surface, residue, the NOE has a negative sign and is of relatively low intensity. 36 G. Otting and K. Wfithrich, J. Am. Chem. Soc. 111, 1871 (1989). 37 G. Otting, E. Liepinsh, and K. Wiithrich, J. Am. Chem. Soc. 113, 4363 (1991). 38 G. Oning, E. Liepinsh, B. T. Farmer II, and K. Wfithrich, J. Biomol. N M R 1, 209 (1991). 39 G. Otting, E. Liepinsh, and K. Wiithrich, Science 2,54, 974 (1991). 4o G. M. Clore, A. Bax, P. T. Wingfield, and A. M. Gronenborn, Biochemistry 29, 5671 (1990). 41 G. M. Clore and A. M. Gronenborn, J. Mol. Biol. 223, 853 (1992).

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PROBES OF METAL ION ENVIRONMENTS

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3. Metal Environments

3.1. pH Effects Two types of information about the protein environment of a metal can be distinguished. For some purposes a mere knowledge of which types of residues coordinate to the metal is sufficient. More often, however, one also would like to know the specific structure of the metal site and the manner in which the ligands are arranged around the metal. One way to identify ligands is to study the titration behavior of the protein by means of NMR spectroscopy. Residues that coordinate metals in metalloproteins often are amino acids that can be (de)protonated (e.g., His, Asp, Glu, Cys, and Tyr42-46). The titration behavior of these amino acids will usually be affected by metal binding in the sense that these residues exhibit abnormal PKa values or do not titrate at all. PKa values of titrating amino acids can be determined by measuring the chemical shift of one or more protons of the amino acid as a function of pH. It should be kept in mind, however, that unusual pK a values can also be the result of internal hydrogen bonding or the apolar hydrophobic environment in which the side chain is situated. Therefore, pH titration experiments directed at the identification ofligand residues should ideally be performed by comparing the holo- and apoproteins (Fig. 4). 47-49 If the protein under investigation is not too large (< 100 residues), 1D 1H N M R spectroscopy (preferably in D20) can already be informative. When searching for signals of titratable residues one should look in the following regions of the spectrum. In the aromatic region of the spectrum [around 7 parts per million (ppm)], His C~H and C~H and Trp CSH resonances can be recognized as singlets, and Tyr C~H resonances as doublets. The C~H resonances of Asp and Cys and the CVH resonances of Glu should be searched for in the aliphatic region (between 2 and 4 ppm). By using this approach it was shown, for example, that at least one histidine coordinates to the copper in the blue copper protein azurin, this histidine 42 j. p. Glusker, Adv. Protein Chem. 42, 1 (1991). 43 p. Chakrabarti, Biochemistry 28, 6081 (1989). 44 p. Chakrabarti, Biochemistry 29, 651 (1990). 45 p. Chakrabarti, Protein Eng. 4, 49 (1990). 46 p. Chakrabarti, Protein Eng. 4, 57 (1990). 47 H. A. O. Hill, J. C. Leer, B. E. Smith, C. B. Storm, and R. P. Ambler, Biochem. Biophys. Res. Commun. 70, 331 (1976). 48 K. Ugurbil and R. Bersohn, Biochemistry 16, 3016 (1977). 49 M. van de Kamp, F. C. Hali, N. Rosato, A. Finazzi Agro, and G. W. Canters, Biochim. Biophys. Acta 1019, 283 (1990).

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

CU(I)-HOLOAZUBIN

APOAZURIN HISlt7

255

+ .

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FIG. 4. Comparison of the pH dependence of the 300 MHz 1D IH NMR spectrum of Pseudomonas aeruginosa apoazurin and Cu(I)-holoazurin in 2H20.47-49 The singlet resonances in the apoazurin spectrum denoted by a + sign and which shift as a function pH are assigned to a Cu ligand residue, His-117, that titrates between pH 6 and 9 in the apo- but not in the holoprotein. b e i n g n o n t i t r a t a b l e in t h e h o l o p r o t e i n a n d t i t r a t i n g n o r m a l l y in t h e a p o p r o t e i n (Fig. 4). 47-49] W h e n o v e r l a p p r e c l u d e s o b s e r v a t i o n o f t i t r a t a b l e signals, 2D p r o t o n spectroscopy often can be of help (COSY, TOCSY, NOESY). A disadvant a g e is t h a t t h e a c q u i s i t i o n o f t h e s e s p e c t r a t a k e s m u c h m o r e t i m e . T h i s

256

PROBES OF METAL ION ENVIRONMENTS

[9]

problem can be alleviated by using 2D heteronuclear techniques, preferably 2D ~H{15N}HSQC spectroscopy (i.e., heteronuclear correlation spectroscopy via heteronuclear single quantum coherence); spectra can be recorded within 30 min, provided the protein is labeled. The application of this technique for the investigation of the titration behavior of various groups throughout a protein is illustrated by Forman-Kay et al. for thioredoxin. 5° 3.2. P a r a m a g n e t i c M e t a l Ions

A paramagnetic metal ion derives its paramagnetism from one or more unpaired electron spins, in which cases the total spin quantum number S amounts to S = ½ or S > ½, respectively. When more than one energy level is thermally accessible for the unpaired electron(s), as in the case of the ferredoxins, the effective electron spin is a thermal average of the electron spin values of the individual states. The magnetic moment associated with the electronic spin causes a dipolar magnetic field around the metal site, which, close to the metal, can be sizable. In the vicinity of the metal the regular motion of the nuclear spins in the externally applied field will therefore be perturbed, which leads to shifts and line broadenings of the corresponding NMR signals. Because the strength of the dipolar field falls off with r -3 and its effect on the nuclear relaxation falls off with r -6, the paramagnetic effect is localized within a relatively small volume of space around the metal. This conclusion still holds when delocalization of the electron spin through covalent bonds is taken into consideration as well. Thus, the paramagnetism of a metal site in principle provides a tool to focus attention to the immediate environment of the site under consideration, not only for smaller proteins (e.g., ferricytochrome c and ferredoxin), but also for much larger proteins, like peroxidase. Once again, it should be stressed that a paramagnetic metal may be incorporated into a protein by replacement of the naturally occurring diamagnetic ion. Conversely, if the natural form of the protein is paramagnetic, it may be useful to study a diamagnetic form of the protein, in order to discriminate between the effects that are and those that are not due to paramagnetism. Several reviews have appeared that deal with the use of paramagnetic effects in the study of metal centers in metalloproteins (see, e.g., Bertini et al. 51 and Villafranca 52 and references therein). Also, the use of paramagnetic effects in combination with NMR spectroscopy is 5o j. D. Forman-Kay, G. M. Clore, and A. M. G r o n e n b o m , Biochemistry 31, 3442 (1992). 5~ I. Bertini, L. Banci, and C. Luchinat, this series, Vol. 177, p. 246. 52 j. j. Villafranca, this series, Vol. 177, p. 403.

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discussed elsewhere in this volume. 53'54Therefore, here only limited attention is given to the subject, mainly focusing on aspects connected with the application of multidimensional NMR methods to the study of paramagnetic metal centers. There are two ways in which electronic paramagnetism can be transmitted to nuclei. First, spin density may be delocalized through covalent bonds. Unless the ligand contains a system of conjugated ~r bonds, as in the case of the heme group, for instance, delocalization of spin density becomes negligible beyond two or three tr bonds. Spin density delocalized into orbitals of a particular nucleus gives rise to a magnetic, so-called Fermi contact (Fc) interaction between electronic spin and nucleus. The principal characteristic of the Fc interaction is that its magnitude and modulation in time depend only on the motion of the electronic spin and not on the orientation of the molecule with respect to the external magnetic field. In other words, the interaction is isotropic. To understand the effect unpaired spin density has on a nuclear resonance signal, it is important to realize that in an external magnetic field the electronic spin may assume different orientations. For instance, when S = ½the electronic spin may be oriented up or down. The field exerted on a nucleus by the electronic spin through the Fc term will be of opposite sign for these two orientations. The net field will thus be proportional to the difference in population of the up and down states, and the resonance of the nucleus will experience a shift that is proportional to the spin density at the nucleus and a Boltzmann factor. This so-called Fc shift can be an important diagnostic tool for probing the electron spin distribution over the metal ligands. Moreover, the Fc shift may give a clue as to the distance of the nucleus to the metal in terms of the number of covalent bonds. The second way the paramagnetism of the metal can influence a nuclear resonance is by means of the through-space dipolar interaction between electron spin and nuclear spin. In contrast to the Fc interaction, the dipole-dipole interaction is anisotropic, that is, its magnitude and sign depend on the orientation of the vector connecting nucleus and metal with respect to the external magnetic field. Averaged over all orientations in space the net effect of the dipole-dipole interaction on the nuclear resonance averages to zero. In solution, where fast tumbling occurs, the dipole-dipole interaction does not, therefore, produce shifts of the NMR signals. There is, however, one important exception encountered in various paramagnetic metalloproteins, namely, when the magnetic moment associated with the electronic spin carries a contribution from the orbital 53 A. V. Xavier, D. L. Turner, and H. Santos, this volume [1]. 54 C. F. G. C. Geraldes, this volume [3].

258

PROBES OF METAL ION ENVIRONMENTS

[9]

motion of the electron(s). It is said that the g tensor of the electronic spin is anisotropic. In that case the dipole-dipole field does not average to zero when the molecule tumbles, and the nuclear resonance experiences what is called a pseudocontact shift. The magnitude of the pseudocontact shift depends on the magnitude of the g tensor anisotropy, the magnitude of the electronic magnetic moment, the distance from the paramagnetic metal, and the orientation of the metal-nucleus vector with respect to the principal axis system of the g tensor. For detailed expressions the reader is referred to the literature: 5-58 Apart from their position, the width of the NMR signals may also be affected. The amount of broadening depends on the magnitude of the field (Fc and/or dipole-dipole) exerted by the metal at the position of the nucleus and on the rate by which this interaction is modulated. Roughly speaking, one can say that the NMR signals will be sharper the faster the modulation. To observe NMR signals from nuclei close to the metal one needs correlation times on the order of 10-H sec or shorter. For most cases only two types of modulation need to be considered: (1) rotational motion of the protein, which modulates the dipole-dipole interaction, and (2) longitudinal and transverse motion of the electronic spin, which modulates the Fc as well as the dipole-dipole interaction. A very approximate estimate of the rotational correlation time, ~'c, can be obtained from the relation 3 zc "~ lO-12Mr, which shows that a protein with an M r of 10,000 already has a correlation time of about 10 nsec. Rotational motion in the case of proteins will therefore in most cases be too slow to yield resonance signals that are sharp enough to be observable. Only in the case where the longitudinal and transverse motion of the electron is fast enough is there a chance to observe signals from nuclei in the close vicinity of the metal. Because the transverse relaxation time is always shorter than the longitudinal relaxation time, the critical parameter to look at is the longitudinal relaxation time, zs, of the electronic spin S, and the experimental approach differs depending on the magnitude of this parameter. M e t a l I o n s w i t h S h o r t 7 s V a l u e s (10-11-10 -13 sec). Paramagnetic metal ions with short ~'s include V(III), Cr(II), low-spin Fe(III), high-spin Fe(II), high-spin Co(II), Ni(II), and Ru(III). 5~ Proteins that contain more than one metal ion may also fall in this class, provided at least one of the ions belongs to the fast relaxing category and provided the exchange coupling 55 y . Feng, H. Roder, S. W. Englander, A. J. Wand, and D. L. Di Stefano, Biochemistry 28, 195 (1989). 56 y . Feng, H. Roder, and S. W. Englander, Biochemistry 29, 3494 (1990). 57 y . Gao, J. Boyd, G. J. Pielak, and R. J. P. Williams, Biochemistry 30, 1928 (1991). 5s y . Gao, J. Boyd, G. J. Pielak, and R. J. P. Williams, Biochemistry 30, 7033 (1991).

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MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

259

between the metal ions is strong enough. A case in point is Cu(II)Co(II)superoxide dismutase. As argued above, resonance signals from protons close to the metal will be shifted (often over distances of tens of parts per million) outside the main envelope of the NMR spectrum of the protein. The paradoxical situation then occurs that, although the paramagnetically shifted resonances are broadened, their resolution improves since they are few in number, their (pseudo-)contact shifts disperse them over a broad region, and overlap with signals from the rest of the protein is reduced, in many cases even to zero. This is also the reason why, as far as their metal site is concerned, proteins can be studied up to a much higher molecular weight (>40,000) than usual. It has been shown that not only 1D NOE but also NOESY and COSY techniques can successfully be applied to study these signals 55-69 (Fig. 5). Analysis of the NOESY and COSY spectra provides for side-chain assignments and spatial information about the metal ligands. For example, in this way the spin systems of catalytically important residues in the heme pocket of horseradish peroxidase could be identified. 6~'62Sometimes these experiments can be advantageously complemented by isotope (H/D) exchange experiments, preferably performed on the apo- and the holoprotein. 5~ Line widths provide information about the distances of protons to the metal. Thus, replacement of Cu in the blue copper protein stellacyanin by Co(II) has been used by Dahlin e t al. 7° in an attempt to elucidate the structure of the metal site (but see Fields e t a / . 71) When more than one oxidation state of the metal is experimentally accessible, as in the case of the heme-containing proteins, the assignment of the resonances in one oxidation state can be used to help assigning resonances in the other oxidation state by means of saturation transfer 59 L. P. Yu, G. N. La Mar, and H. Mizukami, Biochemistry 29, 2578 (1990). 60 L. P. Yu, G. N. La Mar, and K. Rajarathnam, J. Am. Chem. Soc. 112, 9527 (1990). 61 j. S. de Ropp and G. N. La Mar, J. Am. Chem. Soc. 113, 4348 (1991). 62 j. S. de Ropp, L. P. Yu, and G. N. La Mar, J. Biomol. N M R 1, 175 (1991). 63 j. D. Satterlee and J. E. Erman, Biochemistry 30, 4398 (1991). 64 j. D. Satterlee, D. J. Russell, and J. E. Erman, Biochemistry 30, 9072 (1991.). 65 B.-H. Oh and J. L. Markley, Biochemistry 29, 3993 (1990). 66 B.-H. Oh, E. S. Mooberry, and J. L. Markley, Biochemistry 29, 4004 (1990). 67 B.-H. Oh and J. L. Markley, Biochemistry 29, 4012 (1990). 68 L. Skjeldal, W. M. Westler, B. H. Oh, A. M. Krezel, H. M. Holden, B. L. Jacobson, I. Rayment, and J. L. Markley, Biochemistry 30, 7363 (1991). 69 I. Bertini, F. Capozzi, S. Curli, C. Luchinat, L. Messori, and M. Piccioli, J. Am. Chem. Soc. 114, 3332 (1992). 70 S. Dahlin, B. Reinhammar, and J. ,~ngstr6m, Biochemistry 28, 7224 (1989). 7J B. A. Fields, J. M. Guss, and H. C. Freeman, J. Mol. Biol. 222, 1053 (1991).

260

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spectroscopy, or its 2D variant, 2D exchange spectroscopy (EXSY) ( F i g . 6). 55,56,68,72,73

When the g tensor of the metal ion is anisotropic and its components are known, very precise information about minor structural variations in solution can be obtained, provided the crystal structure of the protein is available. The information is derived from a comparison of the observed pseudocontact shifts with the calculated values. For instance, a quantitative evaluation of the shifts of the proton resonances of ferricytochrome c facilitated the detection of small localized, redox statedependent variations in structure. 55-58 Conversely, when the structure of the protein is known, observed pseudocontact shifts allow the determination of the g tensor elements. 55-58 Fc shifts provide information about the distribution of spin density over the metal ligands and, thus, about the ground state electronic properties, as, for instance, for the ferredoxins. TM Study of the temperature dependence of the contact and pseudocontact shifts (Curie and anti-Curie behavior) can sometimes also be helpful in this respect. 5~ M e t a l Ions with Long 7"s Values (10 8-10-9 sec). Paramagnetic metal ions with long r S include Cu(II), Mn(II), VO(IV), Gd(II), V(II), and Cr(III). 51 The major effect these metal ions have on the NMR spectrum of the protein is that signals from protons close to the metal are broadened beyond detection. For instance, with ~-s = 10 9 sec and at a spectrometer frequency of 300 MHz, nuclei within a sphere with a radius of about 10 _A around the metal become invisible in the NMR spectrum. When it is possible to compare the paramagnetic and diamagnetic forms of the protein, for example, for a Cu(I) and Cu(II) protein, these resonances can be observed in a difference spectrum, but it is clear that the information will usually not be very specific. An exception is the case where electron 72 j. Boyd, G. R. Moore, and G. Williams, J. Magn. Reson. 58, 511 (1984). 73 y . Feng, A. J. Wand, H. Roder, and S. W. Englander, Biophys. J. 59, 323 (1991). 74 S. C. Busse, G. N. La Mar, and J. B. Howard, J. Biol. Chem. 266, 23714 (1991).

FIG. 5. Two-dimensional NMR spectra of the strongly paramagnetic protein horseradish peroxidase (42 kDa) recorded at 300 MHz. 61 (A, A') ID JH NMR reference traces. (B) NOESY map collected with 10-msec mixing time [ 1280 transients/free induction decay (FID); phase-sensitive acquisition in IK memory space with 512 increments of t I ; repetition rate 11 s-I; total experiment time 16 hr]. (B') Split diagonal portion of a magnitude-COSY map (4480 transients/FID; acquisition in 1K memory space with 256 t I increments; repetition rate 23 s -1; total experiment time 14 hr). (C') Split diagonal portion of a NOESY map as in (B) but recorded with a 3-msec mixing time. (D) Structure and labeling of the heme group. (Reprinted with permission from de Ropp and La Mar. 61 Copyright 1991 American Chemical Society.)

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F1 FIG. 6. EXSY spectrum at 470 M H z o f an equimolar mixture of tuna Fe(II)- and Fe(III)cytochrome c. 72 To optimize cross-peak intensity arising from chemical exchange and to reduce intensity arising from N O E transfer, the apparent unimolecular electron exchange rate, kuni = 40 s -l, was chosen to be larger than the reciprocal mixing time, zM-I = 10 s -I, which in turn was larger than the estimated cross-relaxation rate, o-ij = - 1 . 4 sec -~. Offdiagonal cross-peaks correlate resonances o f the diamagnetic ferro- with those of the paramagnetic ferricytochrome. (Reprinted with permission from Boyd eta/. 72)

exchange between the diamagnetic and paramagnetic forms in solution is fast. When a solution of the diamagnetic protein contains a slight amount of the oxidized form (0.1-10%) the signals of the ligand protons are selectively broadened owing to the electron self-exchange (ESE) reaction, and they can be picked up fairly easy in a difference spectrum. The method works well with ESE rates of the order of 104-106 M -z sec -l (see below).

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

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Examples are provided by studies of the blue copper proteins azurin and amicyanin (Fig. 7).13'75'76 3.3. Diamagnetic Metal Ions Instead of a paramagnetic ion, the active site of a protein or an enzyme may contain a diamagnetic ion. Diamagnetic ions also often occur at positions in the polypeptide framework where they have a structural function like increasing the thermodynamic stability of the protein. A brief overview of available NMR techniques is given below, but for detailed information the reader is referred to recent reviews 77-81and to other contributions to this volume. 82-84 One-Dimensional NMR Spectroscopy. Metal ions in proteins that have been studied directly by metal NMR include (spin in parentheses) the alkali metals 7Li (~), 23Na (a), 39K (~), 87Rb (~), and 133Cs (½), the alkali earth metals 25Mg (~) and 43Ca (½), the transition metals 67Zn (~), ll3Cd (1) (Fig. 8), 85 5IV (½), 57Fe (½), 59C0 (½), 95M0 (½), l°9Ag (½), 195pt (½), and 199Hg (½), and finally 2°5T1 (½).77,80,81(V, Fe, So, and Mo in this list were studied in their diamagnetic oxidation states.) Important parameters to consider in designing NMR experiments on metal nuclei are natural abundance, sensitivity, and relaxation properties. Detailed information on a number of these parameters can be found in Mason. 77 When the natural abundance of the chosen nucleus is low, use of isotopically enriched samples should be considered. This requires either growth of the organism from which the protein is isolated on isotopically enriched medium (i.e., enriched in the metal isotope) or removal of the metal from the protein and reconstitution of the site with the desired metal isotope. The latter procedure often is the less expensive of the two, and when the isotope to be inserted is different from the naturally occurring element it, of course, represents the only choice. 75 G. W. Canters, H. A. O. Hill, N. A. Kitchen, and E. T. Adman, J. Magn. Reson. 57, 1 (1984). 76 A. Lommen, S, S. Wijmenga, C. W. Hilbers, and G. W. Canters, Eur. J. Biochem. 201, 695 (1991). 77 j. Mason (ed.), "Multinuclear NMR." Plenum, New York, 1987. 78 S. Fors6n, T. Drakenberg, and H. Wennerstr6m, Q. Rev. Biophys. 19, 83 (1987). 79 M. F. Summers, Coord. Chem. Rev. 86, 43 (1988). 8o C. Johansson and T. Drakenberg, Annu. Rep. N M R Spectrosc. 22, l (1989). 81 C. R. Sanders II and M.-D. Tsai, this series, Vol. 177, p. 317. 82 j. E. Coleman, this volume [2]. 83 D. Mota de Freitas, this volume [4]. 84 S. Fors6n, C. Johansson, and S. Linse, this volume [5]. 85 H. R. Engeseth, D. R. McMillin, and J. D. Otvos, J. Biol. Chem. 259, 4822 (1984).

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FIG. 7. Comparison of portions of double quantum filtered (DFQ)-COSY spectra recorded of a diamagnetic, completely reduced [100% Cu(I)] Pseudomonas ae~ ~ginosa azurin sample (top) and of a partially oxidized sample containing 10% paramagnetic Cu(II)-azurin (bottom) (kcx ~ l06 M-I sec-i).13 The assigned peaks i n t h e top spectrum s h o w e x t e n s i v e p a r a m a g n e t i c line broadening i n t h e bottom spectrum, owing to the proximity of the corresponding residues to the Cu (Cu ligand residue numbers are 45, 46, 112, 117, and 121).

At a fixed magnetic field, the signal-to-noise ratio (S/N) in an N M R experiment is proportional to a factor (yN) 5/z, in which YN represents the gyromagnetic ratio of the nucleus. 4 A low gyromagnetic ratio means a low N M R sensitivity, and many metal isotopes fall into this category. However, it appears possible to circumvent the problem of low sensitivity if the metal (X) nucleus has a J coupling to a nucleus with a better sensitivity, for instance, ill. In that case use can be made of polarization transfer

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Chemicalshift (ppm) FIG. 8. Proton-decoupled II3Cd NMR spectra at 55.8 MHz of 113Cd-substituted blue copper proteins, in which the metal ion is strongly coordinated by two histidines and a cysteine thiolate.85 The H3Cd chemical shift spans a wide range from 750 to -200 ppm and reflects the nature and the number of coordinating ligands. Ps., Pseudomonas; AI., Alcaligenes. (Reprinted with permission from Engeseth et al. 85)

between the 1H and X nuclei. As the proton has a high gyromagnetic ratio, its Zeeman levels carry a relatively high Boltzmann polarization corresponding to a large net magnetic moment. Various pulse schemes have been developed by which this reservoir of proton spin polarization can be converted to polarization of the low-sensitivity nucleus. The two more c o m m o n pulse schemes in use for this purpose are insensitive nuclei enhancement by polarization transfer ( I N E P T ) and distortionless enhancement by polarization transfer (DEPT). 4 It should be noted that transfer of polarization occurs in a time equal to 1/2J during which T2 relaxation

266

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occurs (T2 is of the order of the fastest relaxing nucleus, 86-88 which is generally ~H). This gives a lower limit for the X-~H coupling constant, which should be of the order of or larger than 1/I"2. Assuming that no T2 relaxation occurs, the S/N ratio of a polarization transfer experiment (INEPT, DEPT) is given b y 4

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where ./obs and ./exc are the gyromagnetic ratios of the observed nucleus and the excited nucleus, respectively, T is the duration of a single sweep experiment, and T~ is the longitudinal relaxation time of the observed nucleus. For low--/ nuclei coupled to ~H, a gain in sensitivity is thus obtained with respect to direct X-nucleus detection of roughly -/H/-/x ; for example, for 113Cd a gain of 4.5 can be obtained. When a nuclear spin exhibits short longitudinal or transverse relaxation times, the corresponding resonance line is broadened: the faster the relaxation, the broader the line and the more difficult the resonance signal is to detect. The relaxation properties of the nucleus are therefore an important parameter to consider. The main relaxation mechanism for protons in a diamagnetic environment is the modulation in time of the nuclear dipolar interaction. For a metal at least two additional sources of relaxation come into play, the quadrupole interaction and the chemical shift anisotropy (CSA). Quadrupole relaxation may play a role for nuclei with I > ½, which possess a nonzero quadrupole moment, eQ (eQ = 0 f o r / = ½nuclei). 77,78,s°,sl More precisely, the quadrupole moment is a second-rank tensor that interacts with the local electric field gradient. This field gradient is a molecular property and is anchored to the molecular framework. When the protein tumbles, the gradient tumbles and drags the nuclear quadrupole with it, causing the nuclear spin to reorient and enhancing relaxation. Depending on the magnitude of eQ, the nuclear quadrupole-induced relaxation can be considerable. For instance, compared to 15N (I = ½), ~4N (I = 1) has unfavorable properties in this respect. Metal nuclei like 133Csand 6Li have favorably low quadrupole moments compared to 23Na o r 87Rb. Next to the quadrupole interaction, the chemical shift anisotropy may be a source of enhanced relaxation. The chemical shielding of a nucleus critically depends on the distribution of the electrons in the covalent bonds around s6 T. J. Norwood, J. Boyd, J. E. Heritage, N. Soffe, and I. D. Campbell, J. Magn. Reson. 87, 488 (1990). s7 A. Bax, M. Ikura, L. E. Kay, D. A. Torchia, and R. Tschudin, J. Magn. Reson. 86, 3O4 (1990). ss A. G. Palmer III, J. Cavanagh, P. E. Wright, and M. Rance, J. Magn. Reson. 93, 151 (1991).

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

267

the nucleus. In general this distribution is not isotropic, and one expects the shielding to depend on the direction of the magnetic field with respect to the molecular framework. The chemical shift is thus expected to be anisotropic. For protons this anisotropy is practically zero. For a metal like Hg, however, the CSA tensor can exhibit large anisotropies. This means that when the molecule tumbles the chemical shift is modulated in time, which again causes relaxation. The presence of a metal in a polypeptide can be detected by NMR spectroscopy through the presence of fine structure on resonance signals from nuclei close to the metal (1H, ~3C, 15N), in other words, through heteronuclear J coupling. 77 For instance, when the proton NMR spectrum of a protein has been assigned, the observation of fine structure from a metal nucleus on one or more proton signals immediately identifies the ligands of the metal. Metal-proton J couplings have been observed in the range of 20-40 Hz for 199Hg, for instance; similarly, J13Cd-1H couplings varying from 10 to 90 Hz have been observed in Cd-loaded metallothioneins. Conversely, splitting may be observed directly in the NMR spectrum of the metal. As stated above, for the performance of metal NMR spectroscopy, an enormous gain in sensitivity can be obtained by coupling the metal spin bath to that of the protons. The INEPT and DEPT pulse sequences used for this purpose require a nonzero J coupling between metal and protons on one or more nearby residues. J couplings may provide information about the type and conformation of the ligands that bind to the metal. Chemical shifts in the metal NMR spectrum may tell something about the character of the ligands. Line widths in the metal NMR spectrum may provide information about the field gradient at the position of the nucleus and thus about the electronic distribution around the metal. When a solution of the apoprotein is titrated with a solution of the metal ion, NMR spectroscopy of the metal or the protein may reveal the magnitude of the association constant as well as the stoichiometry of the metal binding. For a detailed discussion of these aspects, the reader is referred to the literature. 77-8~ Two-Dimensional NMR Spectroscopy. The presence of a heteronuclear J coupling between metal and protons in principle allows for the use of heteronuclear 2D NMR correlation spectroscopy techniques to probe the metal environment. Two different types of experiments may be considered, namely, 1H{X} HMQC and IH{X} HSQC type experiments, which make use of DEPT-like and INEPT-like magnetization transfer, respectively. 86-88 Various schemes have been tried with l°9Ag and 113Cd. The fact that the 1H and X nuclei resonate at largely different frequencies thereby provides for attractive possibilities to perform spin gymnastics at the two nuclei separately. For instance, by means of a special pulse

268

PROBES OF METAL ION ENVIRONMENTS

[9]

sequence (a so-called X filter), it is possible to obtain a COSY spectrum in which signals occur only from protons that couple directly to the X nucleus. Similarly, by use of a double X filter, protons can be singled out that couple to two metal nuclei. 89'9° These techniques are powerful tools to identify and assign the ligands that bind to the metal(s) in the protein. When the protein contains one or more clusters of metal ions, their presence and configuration can be investigated by homonuclear metal-metal correlation spectroscopy. The structure determination of metallothionein nicely demonstrates this approach. Metallothionein is a small protein of 62 residues that contains 20 cysteines and seven metal ions that are all bound to cysteine sulfur atoms. Reconstitution of the protein with NMR-active 113Cd and NMRinactive 1~2Cd enabled the identification of Cys C ' H proton resonances in the IH COSY spectra. Four different NMR techniques were applied to determine unequivocally the metal-Cys arrangement in metallothionein. (1) First, a ~H-~H COSY spectrum was acquired. The presence of a scalar coupling between ll3Cd and the C ' H of a Cys manifests itself in the fine structure of the ~H-~H COSY C~H-C~H cross-peaks of the cysteine. "Incomplete" or exclusive COSY (E.COSY)-Iike crosspeak structures are obtained in homonuclear 2D IH NMR spectra when correlated protons have nonvanishing J couplings with the same heteronucleus. 9~ Ct~H-C~H cross-peaks acquire an E.COSY-Iike appearance when both/3 protons are connected to the same Cd, whereas an additional splitting arising from J ( H ' - C d ) is observed when only one Ct3H is bound. (2) In addition, an X-filter technique 89'9° was used to simplify the proton spectra either by selecting or removing the proton signals from metal-bound residues. (3) Further, the presence of more than one metal was established by acquiring 1H{ll3Cd} HMQC spectra. 92 (4) Finally, the occurrence of J coupling between different Cd ions was probed by means of 113Cd-l13Cd COSY spectroscopy. In this way, it could be shown that metallothionein contains two metal clusters, one with three and the other with four Cd 2÷ ions, and each Cd ion appeared to be coordinated to four cysteine residues. 92-94 89 E. W6rg6tter, G. Wagner, and K. Wiithrich, J. Am. Chem. Soc. 108, 6162 (1986). 90 G. Otting and K. Wfithrich, Q. Rev. Biophys. 23, 39 (1990). 91 D. Neuhaus, G. Wagner, M. Vasfik, J. H. R. K~igi, and K. Wfithrich, Eur. J. Biochem. 143, 659 (1984). 92 M. H. Frey, G. Wagner, M. Vastik, O. W. SCrensen, D. Neuhaus, E. W6rg6tter, J. H. R. Kfigi, R. R. Ernst, and K. Wiithrich, J. Am. Chem. Soc. 107, 6847 (1985). 93 K. Wiithrich, this series, Vol. 205, p. 502. 94 B. A. Messerle, A. Schaffer, M. Vasfik, J. H. R. Kfigi, and K. W0thrich, J. 34ol. Biol. 225, 433 (1992).

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

269

These techniques have been applied to study the metal coordination in the GAL4 transcription factor (a zinc finger). By replacing the naturally occurring Zn with 113Cd, the metal-Cys cluster structure and the topology of the metal site could be determined, 95'96 and this helped in establishing the three-dimensional structure of the protein. 97'98 It is foreseeable that the experiments mentioned here will find a wider application for the study of the environments of other metals in proteins, especially S = ½metals.

3.4. Extrinsic Ligands So far in this chapter attention has focused on the environment of metals as shaped by the intrinsic components of the protein. In many cases, however, extrinsic ligands also form an essential part of a metal site. Examples are metal-containing proteases (e.g., carboxypeptidase and thermolysin, the external iigand being the substrate which donates a carbonyl oxygen to the metal), the transport proteins myo- and hemoglobin (which have 02 or CO coordinated to the metal), and metalloenzymes where water molecules may coordinate temporarily or permanently to the metal, as in some iron-containing nonheme proteins, some single or multicopper-containing oxidases and oxygen transport proteins, or proteins that contain a catalytic Zn site. 44'45 Extrinsic ligands may also be formed by substrate molecules, product molecules, or inhibitors that bind to a catalytically active metal site. In all these cases ligands not provided by the protein may form part of the metal environment, and there are several ways to establish their presence and to study their properties. Substrates.When a substrate (or inhibitor) binds tightly to a metal site in a metalloenzyme and its signals can be observed in the NMR spectrum of the protein-substrate complex, the position and conformation of the substrate is open for scrutiny by the multidimensional NMR techniques described above. NOESY investigations in particular can be helpful in this respect. When it is possible to label the substrate with a paramagnetic label, its position in the complex with the protein can be determined by looking at the protein signals that are affected on binding of the labeled compound. Types of spin labels that can be used are paramagnetic metal ions or ion 95 K.H. Gardner, T. Pan, S. Narula, E. Rivera, and J. E. Coleman, Biochemistry 30, 11292 (1991). 96 T. Pan and J. E. Coleman, Biochemistry 30, 4212 (1991). 97 p. j. Kraulis, A. R. C. Raine, P. L. Gadhavi, and E. D. Laue, Nature (London) 356, 449 (1992). 98 j. D. Baleja, R. Marmorstein, S. C. Harrison, and G. Wagner, Nature (London) 356, 451 (1992).

270

PROBES OF METAL ION ENVIRONMENTS

[9]

complexes, well-known examples being Cr(III) and Gd(II) and, to a lesser extent, Cu(II) and Co(II), and nitroxide free radical-containing compounds. 52'99 Conversely, paramagnetism of a metal in the protein metal site to which the substrate binds may be used to study the substrate binding mode. For an understanding of the paramagnetic effect that is exerted either by the labeled substrate on the protein or by a paramagnetic metal ion in the protein metal site on the substrate, the reader is referred to Section 3.2 and the references therein. A substrate can also be isotopically labeled with, for example, ~SN or 13C, to make it distinguishable from the signals deriving from the protein. The use of heteronuclear X-filtering then offers the possibility of obtaining information on the individual partners in the complex and their interactions. 9° When the substrate binds weakly, the conformation of the substrate in its bound state can be studied by 1D and 2D transfer-NOE experiments. The method works well when the substrate is small and binds to a large metalloenzyme, because then the long correlation time (~'c) of the complex facilitates the detection of NOEs. ~00 Small Molecules. Water molecules often form an intrinsic part of the active site of a protein or enzyme, as is also the case for metalloproteins. 43-46 Examples are provided by proteins that contain a catalytic Zn site. Experiments reported to be useful for the study of water molecules that either are internally bound or are part of the hydration sphere of a protein are IH ROESY, 3D 1H TOCSY-ROESY, and 3D 1H{15N}R O E S Y - H S Q C , when combined with water suppression techniques that do not make use of presaturation of the w a t e r signal. 36-41 Rotating frame NOEs (ROEs) yield residence times and facilitate the determination of the positions of the water molecule relative to the protein. A different technique, called NMRD (NMR dispersion; also known as relaxometry and proton relaxation enhancement, PRE), has been worked out by Koenig, Bertini, and others. 5~'52'1°1 When a metal-bound water molecule exchanges with the bulk of the surrounding solvent, the relaxation properties of the bulk water signal are affected, provided the exchange is sufficiently fast. Thus, by measuring the relaxation properties of the bulk water as a function of protein concentration, for instance, information is obtained about the presence of water in the coordination 99 p. A. Kosen, this series, Vol. 177, p. 86. t00 p. R. Rosevear and A. S. Mildvan, this series, Vol. 177, p. 333. t01 D. M. Dooley, M. A. McGuirl, C. E. Cote, P. F. Knowles, I. Singh, M. Spiller, R. D. Brown III, and S. H. Koenig, J. A m . Chem. Soc. 113, 754 (1991).

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

271

shell of the metal. The method works especially well when the metal under consideration is paramagnetic, because the relaxation properties of the water molecules that are bound to the metal will be strongly affected by the paramagnetism of the metal. Under favorable circumstances not only the presence of water molecules in the coordination sphere of the metal but also their residence time can be determined. Finally, small molecules, like H20, CO, and 0 2 , can be studied by ID 170 NMR. 102 Metal Site Accessibility. It can sometimes be of importance to know how accessible a metal site is from the outside. When the site is paramagnetic, its accessibility for a particular compound can be probed directly by observing the resonances of the compound as outlined earlier in this section for substrates and small molecules. When the site is diamagnetic, its accessibility can be probed with a paramagnetic compound by looking at broadening of signals from residues in the vicinity of the metal site. By varying the charge and/or the aromatic character of the paramagnetic compound, the nature of the interactions that govern the access to the metal site can be probed. For instance, with Cr(III) compounds like Cr(phen)33+, Cr(NH3)63÷, Cr(EDTA)-, and Cr(ox)33-, but also with small paramagnetic proteins like ferricytochrome c, the surface charge around the metal site and the hydrophobic character of the protein surface close to the metal site can be investigated.l°3-1°6 Spin-labeled substrate analogs can be used for similar purposes. 52'99 4. Mobility of Metal Environments The mobility of the protein matrix around a metal site often has a direct bearing on the activity of the protein. A study of this mobility can, therefore, be of crucial importance for an understanding of the mode of operation of the protein. The fortunate characteristic of NMR spectroscopy with regard to protein studies is that the time scales probed by this technique cover the time scales at which many biologically important (inter)actions take place, such as enzyme catalysis and channel opening. Dynamic phenomena related to protein metal sites that can be studied by NMR spectroscopy include the following. t02 B. Halle, T. Andersson, S. Fors6n, and B. Lindman, J. Am. Chem. Soc. 103, 500 (1981). 103 p. C. Driscoll, H. A. O. Hill, and C. Redfield, Eur. J. Biochem. 170, 279 (1987). i04 S. Bagby, P. C. Driscoll, K. G. Goodall, C. Redfield, and H. A. O. Hill, Eur. J. Biochem. 158, 413 (1990). 10s D. Whitford, Eur. J. Biochem. 203, 211 (1992). i06 G. Esposito, A. M. Lesk, H. Molinari, A. Motta, N. Niccolai, and A. Pastore, J. Mol. Biol. 224, 659 (1992).

272

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PROBES OF METAL ION ENVIRONMENTS

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0.8

0.0

kl[D*] D÷*A

k_ 1

0.8 1.0 FHisD ÷

k2 B

k-~

C

FIG. 9. (A) Half-width at half-height (in radians/sec) of the C2H signal of the Cu ligand His-96 of reduced amicyanin from T h i o b a c i l l u s v e r s u t u s as observed in 300 (circles) and 600 (triangles) MHz IH NMR spectra as a function of the deuteronated fraction, f a i s o + . (Notice that the pD decreases from left to right.) Experimental conditions: 1.4 mM reduced amicyanin, 20 mM phosphate in D20; temperatures as indicated. The solid lines are computer simulations on the basis of the three-site exchange model depicted at the bottom. At high pD (faisn÷ "~ 1) the line width is governed by the (relatively fast) transformation between A and B; at low pD (fHisD÷ ~ 1) the linewidth is governed by the (relatively slow) transformation between species B and C. (B) The data are compatible with a structural transformation as observed in plastocyanin.lll At high pH (or pD) the Cu site is coordinated by four ligands (His-54, Cys-93, His-96, and Met-99). This state might correspond with species A (see above). On lowering the pH His-96 becomes protonated (species B) and subsequently undergoes a conformational switch (species C). The NMR data so far have not been analyzed for the details of the conformational switch, and the transition from B to C has been represented here only by way of an example as a rotation around the Ca-C y bond. (Reprinted with permission from Lommen and Canters. m)

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

273

I-I+

H? s

FIG. 9. (continued)

First, the situation may arise that the coordination shell of the metal occurs in different conformations, which are in dynamic equilibrium. Usually this is connected with a titratable ligand residue, the different conformations corresponding to different states of protonation of the residue. Examples can be found among the Zn-finger peptides 1°7-H° and the blue copper proteins H1-1~3 (Fig. 9). The metal site may also be affected by 107 G. M. Clore, J. G. Omichinski, and A. M. Gronenborn, J. Am. Chem. Soc. 113, 4350 0991). t08 B. A. Krizek, B. T. Amann, V. J. Kilfoil, D. L. Merkle, and J. M. Berg, J. Am. Chem. Soc. 113, 4518 (1991). 109 R. X. Xu, S. J. Horvath, and R. E. Klevit, Biochemistry 30, 3365 (1991). H0 M. Kochoyan, T. F. Havel, D. T. Nguyen, C. E. Dahl, H. T. Keutmann, and M. A. Weiss, Biochemistry 30, 3371 (1991). m j. M. Guss, P. R. Harrowell, M. Murata, V. A. Norris, and H. C. Freeman, J. Mol. Biol. 192, 361 (1986). 112 A. Lommen and G. W. Canters, J. Biol. Chem. 265, 2768 (1990). tl3 A. Lommen, K. I. Pandya, D. C. Koningsberger, and G. W. Canters, Biochim. Biophys. Acta 1076, 439 (1991).

274

PROBES OF METAL ION ENVIRONMENTS

[9]

conformational changes in a more remote part of the protein, with the effect of the change in protein structure at one point being transmitted over a shorter or longer distance to the immediate environment of the metal. Allosteric cooperative effects as encountered in hemoglobin are well-known examples; one could also think of changes in conformation of, for example, the loops on which metal ligands are often located. Substrate binding also sometimes induces conformational changes at the metal site that may be worthwhile to study in detail. Second, one sometimes is interested in the properties of the empty metal site. Naturally, the residues in the metal site often exhibit a much greater conformational freedom in the apoprotein than in the holoprotein, and it can be of importance to study the effect of metal binding on this flexibility. Comparative studies have been reported, for example, for the a p o , (Ca2+)2, and (CdZ+)l forms of calbindin D9k .114'115 Also, the proteinbound metal can be involved in a dynamic exchange with metal ions that are free in solution, and, apart from the metal-binding constant, one might like to know kinetic details of this equilibrium. Finally, the dynamic aspects of the protein structure around the metal site can be of interest. Different parts of a protein may have different mobilities, and the presence of a metal can have a strong bearing on the dynamics of the polypeptide framework in the immediate neighborhood of the metal. Various techniques are at the disposal of the NMR spectroscopist, depending on the aspect of the dynamics of the protein to be investigated. Conformational equilibria can be investigated by the methods usually applied to study chemical exchange processes. 116,117For the study of internal protein mobility, the measurement of NH exchange rates 118 and of NMR relaxation times 119 is of relevance.

4.1. Conformational Equilibria Conformational exchange in proteins is generally relatively slow (millisecond time scale). It can be effectively studied with the help of straightforward 1D NMR techniques by looking at shifts and widths of the signals from nuclei that are affected by the exchange. These techniques have the advantage that they require relatively unsophisticated instrumentation, 114 S. Linse, O. Teleman, and T. Drakenberg, Biochemistry 29, 5925 (1990). i15 N. J. Skelton, J. K6rdel, M. Akke, and W. J. Chazin, J. Mol. Biol. 227, ll00 (1992). ll6 B. D. N. Rao, this series, Vol. 176, p. 279. ll7 j. j. Led, H. Gesmar, and F. Abildgaard, this series, Vol. 176, p. 311. lib p. R6sch, this series, Vol. 176, p. 342. ii9 R. E. London, this series, Vol. 176, p. 358.

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275

they are easy to implement, and that there is extensive literature available on the theoretical and practical aspects. 116'117 Variation in temperature, pH, and concentration of exchanging species may help in establishing which lines to look at. A good example is provided by the study of the blue copper protein amicyanin. One of the Cu ligand histidines in this protein has been found to titrate with pH, and this titration leads to a conformational change at the Cu site. 113 The process has been followed by ~H NMR spectroscopy as function of pH, temperature, and magnetic field, and its kinetics have been determined by line shape analysis, n2 In the case of amicyanin it was found that for a correct description exchange between at least three different species had to be invoked (Fig. 9). A three-site exchange model was also used by Clore et al. for the analysis of the conformational dynamics induced by protonation of a ligand histidine at the metal coordination site of a Zn finger. 1°7 This exchange problem was examined by using 2D magnetization transfer (EXSY/ NOESY) experiments as a function of the mixing time. 4.2. N H Exchange

The rate by which backbone amide protons exchange with protons from the surrounding water molecules depends on the accessibility of the backbone amides to water molecules and on the stability of the protein, especially the stability of the hydrogen bonds in which the backbone amides participate. 118'12°'121Currently, two models are in use to describe the exchangeability of NH protons. 34'12°According to one model, localized unfolding of the protein is necessary for the water molecules to gain access to and to enhance exchange with a particular amide proton. In the alternative scheme, exchange is mediated by water diffusion through channels in the protein structure. No consensus about the correct description seems to have been reached yet. At the moment it is not easily possible to translate backbone proton exchange rates into general statements about protein dynamics. However, exchange rates do provide useful information for making comparisons. For instance, when a particular point mutation enhances the N H exchange rates around the site of the mutation, it may be concluded that the mutation has a destabilizing effect on the structure. Similarly, when binding of a substrate analog to the active site of an enzyme decreases the N H exchange rates of particular surface residues, it may be concluded that the substrate hampers access of the bulk water to these residues or that the local flexibility leading to proton exchange is diminished. An example of the first phenomenon is provided by the n0 S. W. Englander a n d N. R. Kallenbach, Q. Rev. Biophys. 16, 521 (1984). 12l R. J. P. Williams, Eur. J. Biochem. 183, 479 (1989).

276

PROBES OF METAL ION ENVIRONMENTS

[9]

Ca2+-containing protein calmodulin, where clear differences have been observed in the N H exchange rates between free calmodulin and calmodulin complexed with a substrate. 33 The measurement of N H exchange rates can also be used to study the influence of metal binding on protein folding. 114,115 The rates by which amide protons exchange may vary over many orders of magnitude. The slowly exchanging amide protons in a protein are commonly defined as the amide hydrogens that can be observed after the protein has been dissolved in D20. Because the signals in the amide region of the 1D ~H NMR spectrum of a protein normally show extensive overlap, it is preferable to use a 2D scheme to resolve the resonances. 3°'32 Marion et al. 32 present methods to record 2D NMR spectra (e.g., NOESY or IH{~SN} HMQC) in 5 min or less, which puts a lower limit of at least several minutes on the exchange time for an amide proton to be observable and to be classified as a slow exchanger. The amide exchange rate depends strongly on pH, 1A2° because in the range pH 4-9 amide hydrogen exchange is catalyzed by O H - ions. If the experiments can be performed at a low pH around 4, most of the amide hydrogens can still be observed 5 min after the protein is dissolved in D20. However, for proteins that cannot be handled at such a low pH, exchange rates can only be obtained for a much smaller fraction of the hydrogens. At pH 7, for example, most of the amide protons exchange more rapidly, and their exchange rates cannot be measured by dissolving the protein in D20 and looking at the disappearance of the proton signals. When the D20 exchange method is not practicable, the exchange rates can be established from the intensity of the cross-peaks at the FI frequency of water (the so-called tol-H20 line) in NOESY spectra, using water suppression techniques that do not involve water saturation. 122,123The to~-H20 line, however, is often too crowded to assign reliably the resonances on this line. Then recourse has to be taken to 3D NMR techniques. In 3D ~H{15N}NOESY-HMQC spectra, recorded without water presaturation, the F 2 F 3 ~SN-~H plane at the F1 ~H frequency of water contains all the now well-separated exchange cross-peaks with water (Fig. 10). A complication is the presence, in this plane, of cross-peaks arising from NOE magnetization transfer to amide protons from (a) C~H protons that resonate close to the water frequency and (b) bound water. The latter cross-peaks, (a) and (b), may be distinguished from exchange peaks in 122 B. A. Messerle, G. Wagner, G. Otting, C. Weber, and K. Wiithrich, J. Magn. Reson. 85, 608 (1989). u3 p. j. Hore, this series, Vol. 176, p. 64.

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

A

L

o O

,, Hl17 H48 N¢2H

+

1--47

.a

11o

' O

-9"*

j' 'o f-| O

,

120

• io+12+,,,.+ 119 (t~

If

q 114--I

~

B

,

130 ~m

'~

t oe

,

,0

,

o o ,je,_2, gO

0

: ~t~P 00~0110AL )~0 |O 121_~ftp

, 0--47

j

~17"~: ~ g" t

114--0

101.........

g

n

I

~h"

o

l l;, 2,

O

F2 15N

11o 45--0 120

,,

0

"1 |'119

':i; °,, ': 91.........

,

OA ~AV~A O~II

o oo,J.IJt' o'lj O

F3'1't'~ '''111 .........

d 116

,o

1

o ,

118--0

113-o Hl17 H46 N~2H

15N

'+-'

, lip

121|1/•

F2

e

,o'

118__t 113--1

277

81.........

130

ppm

?1''" ...... 6'''pprn

FIG. 10. (A) The F2F3 =SN-IH plane at the F1 IH frequency of H20 (-4.65 ppm) of a 600 MHz 3D tH{XSN}NOESY-HSQC spectrum (100-msec mixing time) recorded without water presaturation for a 1.5 mM uniformly 15N-labeled Cu(I)-azurin sample from Pseudomohas aeruginosa? 3 (B) F2F3 ISN-IH projection of the NOESY-HSQC spectrum, showing all the ISN-IH one-bond correlations. Assignments refer to residue numbers, residues 45, 46, 112, 117, and 121 being Cu ligands. The projection in (B) is equivalent to the familiar 2D IH{]SN} HSQC spectrum of the protein. In the F2F3 plane in (A), cross-peaks show up owing to NH exchange with H20, the cross-peak intensity being proportional to the exchange rate; some cross-peaks are also present that are due to NOE magnetization transfer to the amide protons from C~H protons that resonate close to the water frequency. Examples of the latter type of NOE cross-peaks are the peaks that are assigned to residues 47, 113, 114, and 121. Whereas the main-chain NH exchange is relatively slow (<0.1 sec -1) for residues 45, 46, and 112, faster exchange (in the range 1-100 sec -1) is observed for the main-chain NH protons of residues 117, ll8, and ll9 and for the side-chain N~2H protons of His-46 and His-117. The latter two resonances are boxed because they are folded into the 120 ppm region of the spectrum.

278

PROBES OF METAL ION ENVIRONMENTS

[9]

a 3D ROESY-HM(S)QC experiment) 5 A second approach, which also involves the use of a 15N-enriched sample, has been worked out by Spera et al.33 In this approach NOE cross-peaks are separated from the exchangerelated cross-peaks by repeating the experiment at a number o f p H values. Because the exchange is strongly pH dependent and the NOEs are not, these two effects can be separated. The advantage of the heteronuclear methods over homonuclear 2D schemes is that they provide a means to determine the NH exchange rates more completely and over many orders of magnitude in an accurate fashion. The usefulness of a comprehensive determination of amide proton exchange rates is exemplified by the aforementioned study of free calmodulin and calmodulin complexed with a 19-residue myosin light chain kinase peptide. 33 In the complex the usual trends in NH exhange are observed, that is, toward the ends of the a helices one sees a strong increase in amide proton exchange rates, indicating increased fraying. However, a very slow exchanging amide proton was also found, which is not involved in the hydrogen-bonding network of the protein, indicating that apart from hydrogen bonding solvent accessibility is also an important parameter determining the exchange rate. Another aspect is that this type of measurement focuses on the rapidly exchanging amides, which are mostly located at the surface, and some clear differences are observed when comparing calmodulin and calmodulin complexed with peptide. These differences were not found when only slowly exchanging amides were measured. A similar approach was used in the detailed study of amide proton exchange of calbindin D9k. 114,115It was found that addition of Ca 2+ or Cd 2+ retarded the exchange of many amide protons observed to be in a hydrogen-bonding network, with the largest changes found in the ion-binding loops, consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops. Another example of the study of amide exchange in metalloproteins is the study of cytochrome c2 in the reduced and oxidized state by Gooley et al. 34 The authors present a detailed analysis of the factors that may influence the exchange rate. Because very small (I ,~) differences between the structures of the two states may cause variations in the NH exchange rate of up to a factor of 100, lack of detailed knowledge of the structure of these states makes it difficult to draw definite conclusions from the available data.

4.3. N u c l e a r Relaxation and Cross-Relaxation

NMR relaxation is caused by the modulation in time of magnetic interactions, primarily (when interaction is restricted to proteins) magnetic

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

279

dipole-dipole interactions between n u c l e i . 4A24 The time-dependent modulation of the interaction may be considered as a time-dependent perturbation that on one hand induces transitions between the stationary Zeeman levels of the nuclei (relaxation time T1) and on the other hand destroys coherence of the motion of the nuclear spins (relaxation time Tz). When the structure of the protein is known, the magnitude of the magnetic dipole-dipole interaction can be calculated to a high degree of precision. A study of the relaxation behavior of the NMR signals can give information about the time dependence of this interaction and thus about the internal protein dynamics. Similar considerations apply to the nuclear Overhauser effect. 3 This effect is due to the transfer of magnetization from one nuclear spin to another as mediated by the nuclear dipole-dipole interaction. The magnitude of the NOE depends, as in the case of the longitudinal (Tj) and transverse (/'2) relaxation times, on the magnitude of the dipole-dipole interaction and its modulation in time. Mathematically the expressions for the relaxation times (T1 and T2) and NOEs are conveniently formulated in terms of spectral density functions. The spectral density function represents the frequency distribution of the time modulation of the magnetic dipole-dipole interaction. It has a simple mathematical form in the case of a rigid spherical molecule and is characterized by a limited set of parameters, the most important one being the correlation time, r. When the interaction is modulated by more than one type of motion (e.g., overall tumbling of the protein and internal mobility), more than one correlation time is needed to characterize the overall modulation of the interaction. The aim of relaxation and crossrelaxation measurements is to extract information about these correlation times or, more generally, about the spectral density function. In fact, the T~ and T2 relaxation times and the NOE each probe certain points on the spectral density function. The model mostly used to analyze relaxation data is the one put forward by Lipari and Szabo,125 in which internal motion is taken into account in a model-free manner as an order parameter S. A good example of the application of this model to the analysis of relaxation data is a study by Kay et al. on S-Nase, 24 in which 15N T l a n d T 2 values as well as 15N to IH NOEs were measured. We note that some controversy exists concerning the correct method of measuring 15N and 13C T2 values because different experiments produced slightly different (mainly too short) T2 values. 27 124 A. Abragam, "The Principles of Nuclear Magnetism," Oxford Univ. Press (Clarendon), Oxford, 1983. 125 G. Lipari and A. Szabo, J. Am. Chem. Soc. 104, 4546 (1982).

280

PROBES OF METAL ION ENVIRONMENTS

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A very interesting approach has been proposed by Wagner et al.,27-29 who measured six different relaxation rates, related to amide proton and amide nitrogen coherenes, that probe the relevant spectral density function at five different frequencies. From these values the spectral density function can be calculated without the need to introduce any motional model. The relation between this approach and that of Lipari and Szabo is that the latter authors claim that any type of internal motion can be described by a three-parameter spectral density function, the three parameters being two time constants and an order parameter. For an overview of 1D NMR methods used to measure Tj and T2 relaxation rates we refer to London.119 In addition, we mention a number of novel approaches that have been explored with the advent ~SN- and/or ~3C-labeled material. Labeling enables the accurate measurement of the relaxation of heteronuclei ~3C and ~SN directly bonded t o p r o t o n s . 24-29 This is particularly useful for obtaining motional information, since the relaxation of these nuclei is governed predominantly by dipolar interactions with these directly bonded protons. Moreover, the interpretation of the relaxation data requires only that the ~H-X bond length be known and, in general, does not require a knowledge of the overall structure of the molecule in question.

5. Oxidation State Changes This volume deals with the study of environments of metals in metalloproteins, and strictly speaking the oxidation state of the metal is not a property of the metal environment. Yet oxidation state is a crucial parameter that determines in part the physicochemical properties of the metal site, and its study is particularly important when more than one oxidation state is accessible to the metal, as in redox proteins. The redox activity of a metalloprotein is often studied by kinetic techniques (rapid flow/ rapid freeze, stopped flow, flash photolysis, pulse radiolysis). Electron paramagnetic resonance (EPR) spectroscopy is also frequently applied since usually at least one of the oxidation states of a redox protein is paramagnetic. However, for the study of the dynamic equilibrium between two redox forms of the same protein, NMR spectroscopy has some advantages over the other techniques, which is why the subject is briefly discussed here. Metals commonly occurring in redox-active proteins comprise V (e.g., in the vanadium chloro- and bromoperoxidases126), Mn (e.g., in the water 126 R. Wever and M. G. M. Tromp, J. lnorg. Chem. 43, 404 (1991).

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MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

281

splitting enzyme of the photosynthetic reaction center~27), Mo and W (in some hydrogenases and nitrogenases127), and especially Cu and Fe, which can be found pervasively throughout nature in a great variety of oxidases, oxygenases, and O2-transporting proteins. During enzyme turnover V in the peroxidases does not seem to change its oxidation state, and for Mo and W the situation is not clear in this respect; however, for Mn and especially Cu and Fe many examples are known of proteins in which the metal changes its oxidation state during turnover. When two redox forms of a metalloprotein are in dynamic equilibrium it means that they can exchange electrons during an encounter. In a number of cases NMR spectroscopy appears exceptionally suitable to measure the rate of this so-called electron self-exchange (ESE) reaction. The study of this type of reaction has gained popularity for various reasons. First, although in most cases the ESE reaction is probably not of physiological significance, its study may provide insight into the redox mechanism of a protein and into its dependence on parameters as temperature, ionic strength, and pH. Second, in the Marcus theory, which has been widely used to analyze electron transfer reactions in solution, the ESE rate figures prominently) 28 NMR methods that are applicable to the study of ESE reactions comprise saturation transfer and chemical exchange related techniques. They have successfully been applied to determine ESE rates in the range of 102-107 M - l s e c - I .

5.1. Exchange Studied by Saturation Transfer A necessary condition for a 1D NMR saturation transfer experiment to be feasible is that the NMR signal of at least one nucleus must occur at different positions in the NMR spectra of the two exchanging species. The experiment starts with the application of a strong radio frequency (rf) pulse at the resonance position of this nucleus in, say, species 1. The result is a partial or complete loss of signal intensity, depending on the strength and length of the rf pulse. When the protein changes its redox state as a result of an electron transfer event, the (partial) saturation is carried over to the other redox species (species 2), and the corresponding NMR signal also drops in intensity. The magnitude of the intensity loss depends on the amount of saturation brought about by the rf pulse, the longitudinal relaxation time of the nucleus in species 2, and the individual lifetimes of the two redox species. The amount of saturation and the T~ of species 2 can be measured experimentally. Analysis of the saturation 127 j. j. R. Frausto da Silva and R. J. P. Williams, "The Biological Chemistry of the Elements." Oxford Univ. Press, Oxford, 1991. 128 R. A. Marcus and N. Sutin, Biochim. Biophys. Acta 811, 265 (1985).

282

PROBES OF METAL ION ENVIRONMENTS

[9]

transfer data then provides for the lifetimes of the reacting species and, thus, for the second-order ESE rate when the concentrations of the reacting species are taken into account. In the case of complete rf saturation, the relation between the various parameters is given by I/I o = T1-1/(TI - l + k)

(2)

in which I and I 0 are the intensities of the resonance of species 2 in the presence and absence of rf irradiation, and k is the inverse lifetime of s p e c i e s 2.129 When complete saturation is not attainable, the experiment is still feasible, albeit a more elaborate procedure has to be followed by which the experiment is performed as a function of the time of irradiation and the amount of saturating power applied.~29 As can be seen from Eq. (2), the method works best if k is of the order of magnitude of T~-1. Because for proteins the latter often amounts to about 1 s e c -1 for protons, this means with protein concentrations of 1 mM that ESE rates of 102-104 M -~ sec -1 are within reach of the method. Heme proteins often are amenable to this type of study because the resonance signals of a number of protons occur at positions that differ widely for the reduced and the oxidized form of the protein and many of them are well resolved. ~30The experiment is often performed by irradiating a peak of the oxidized protein. These peaks can be fairly broad, and this can make it difficult to judge whether the irradiated peak has been truely saturated by the rf field. The risk exists that the peak is considered saturated while in reality some intensity is still present in the background, which may lead to errors in the ESE rate. Whether saturation has been achieved can be checked by repeating the experiment with progressively longer irradiating times and/or irradiating power. Eventually, the observed I/I o ratio should become independent of these two parameters. 5.2. E x c h a n g e Studied by T 1 and T2 M e a s u r e m e n t s

When a nucleus resonates at different positions in the NMR spectra of the oxidized and the reduced form, the interconversion of the two species can be analyzed by NMR techniques that are normally used to study chemical exchange processes. The difference in resonance position may be due to paramagnetic effects, as in the case of the heme proteins, but it may also be due to small conformational changes induced by the change in oxidation state. Thus, in plastocyanin, azurin, and amicyanin rE9 O. Jardetzky and L. C. K. Roberts, " N M R in Molecular Biology." Academic Press, New York, 1981. 130D. W. Concar, D. Whitford, G. J. Pielak, and R. J. P. Williams, J. Am. Chem. Soc. 113, 2401 (1991).

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283

some upfield-shifted resonances undergo small changes in resonance position on oxidation of the protein, whereas the corresponding protons are far removed from the metal site. 75A31'132 The shifts are caused by small changes in conformation that are transmitted over a long distance from the site of oxidation to remote parts of the protein. Small shifts can also be introduced by changes in the pKa of titratable residues that may occur on oxidation or reduction of the protein. Such a change in PKa of, for instance, a histidine may change the average state of protonation of this residue and thus the average resonance positions of its ring protons. 133 The customary procedure to study chemical exchange by NMR spectroscopy starts with the determination of the T1 1 and/or T 2 I relaxation rates of a signal that is clearly visible in the NMR spectrum of the reduced as well as the oxidized form. Subsequently the change in T1-1 and/or T2-l caused by the chemical exchange is measured for a solution containing a mixture of the two species. From this change the interconversion rate can be determined. The analysis of two-site and to some extent of threesite exchange processes by means of NMR studies has been thoroughly documented in the literature. 75'112'129A32'134-139 When the effects on TI -~ and T2-~ owing to ESE are small, as expected when the exchange rate is low (103 M -l sec-1), it is usually advantageous to study the T1-1 relaxation rate. In proteins the longitudinal relaxation rate of protons, for instance, can be appreciably longer than the transverse relaxation rate. A small change in the relaxation rates can therefore be determined with better precision for T1-1 than for T2-1.140'141 The chemical exchange method has successfully been applied to study ESE rates in the range of 103-106 M -1 sec -1. Under favorable conditions this range can be expanded to 102-107 M-I sec-~. A chemical exchange analysis may also be feasible when a resonance signal of a particular nucleus can only be observed in the NMR spectrum of the diamagnetic species. This applies when in the paramagnetic species 131 F. A. Armstrong, P. C. Driscoll, and H. A. O. Hill FEBS Lett. 190, 242 (1986). 132 A. Lommen, G. W. Canters, and J. van Beeumen, F~ur. J. Biochem. 176, 213 (1988). 133 A. Romero, C. W. G. Hoitink, H. Nar, R. Huber, A. Messerschmidt, and G. W. Canters, J. Mol. Biol. 229, 1007-1021 (1993). i34 A. Patterson, Jr., and R. Ettinger, Z. Elektrochem. 64, 98 (1960). m j. S. Leigh, Jr., J. Magn. Reson. 4, 308 (1971). 136 A. C. McLaughlin and J. S. Leigh, Jr., J. Magn. Reson. 9, 296 (1973). 137 C. M. Groeneveld and G. W. Canters, J. Biol. Chem. 263, 167 (1988). 138 C. M. Groeneveld and G. W. Canters, Eur. J. Biochem. 153, 559 0985). 139 E. R. Johnston and D. M. Grant, J. Magn. Reson. 47, 282 (1982). 140 D. W. Dixon, X. Hong, S. E. Woehler, A. G. Mauk, and B. P. Sishta, J. Am. Chem. Soc. 112, 1082 (1990). i41 D. W. Dixon, X. Hong, and S. E. Woehler, Biophys. J. 56, 339 (1989).

284

PROBESOF METALION ENVIRONMENTS

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the NMR signal is broadened beyond detection. The exchange reaction can then be studied by observing the NMR spectrum of a solution of the diamagnetic species while small amounts of the paramagnetic species are added in successive steps. A gradual broadening of the signals deriving from nuclei close to the metal is observed that is proportional to the amount of added paramagnetic protein. The simplest description of the phenomenon is that most of the time the NMR spectrometer "observes" the nuclei while they are in a diamagnetic environment. This is because the fraction of paramagnetic species is small. Every now and then, as the result of an electron exchange event, the protein becomes paramagnetic and the nuclei experience a short and strong magnetic pulse from the unpaired electron(s) at the metal nucleus. This destroys the regular motion of the nuclei under the influence of the external magnetic field. As a result the NMR spectrometer observes the nuclei in an unperturbed fashion only during short time spans of length 70 . "TOrepresents the average lifetime of the diamagnetic species and is equal to kESE[P], kEsE being the secondorder ESE rate and [P] the concentration of paramagnetic species. The observed change in T1-1 and T1-2 is given by AT -l = kESE[P]

(3)

When [P] is known, the ESE rate can then be determined immediately from Eq. (3). An illustration is presented in Fig. 11. A necessary condition for this method to be feasible is that the paramagnetic pulse experienced by the nucleus during the time the protein is in the paramagnetic form must be sufficiently strong to destroy the magnetization and the coherence in the motion of the nucleus under study. This condition has been analyzed in detail for the case of the blue copper proteins and model compounds. 137A42Generally speaking, the closer the nucleus is to the metal, the easier this condition is fulfilled. The best signals to choose for this type of study are the ones corresponding to nuclei of the metal ligands. When the "strong pulse" condition is not fulfilled, for instance, because the signal under consideration corresponds to a nucleus that is at some distance from the metal, the method is still feasible, but the analysis becomes more complicated. 137,138Also, the "paramagnetic pulse" must be short enough, that is, the fraction ofparamagnetic species must be small ( < - 5 % ) . If this fraction becomes larger (10-20%), a more elaborate equation than Eq. (3) must be used. 135-139 The method has successfully been applied for the determination of ESE rates in the range of 104-106 M -1 sec -1. 142C. M. Groeneveld,J. van Rijn, J. Reedijk, and G. W. Canters, J. 4893 (1988).

A m . Chem. Soc.

U0,

[9]

MULTIDIMENSIONAL

NMR

fp =0.008

T:

294 K / L/~.

OF M E T A L SITES IN PROTEINS

285

T= 310 K

fp =0.0

T=302 K

fp =0.004

T=32O K ,

fp =0.012 l~xj ~ 6.0 5.5 ppm

ppm

FIG. 11. Region of the 300 MHz proton NMR spectrum of azurin, showing the signal (5.88 ppm) of the C~2Hproton of the Cu |igand His-46 that was used for the line broadening experiments. Conditions: 20 mM phosphate buffer, pH 9.0, and 2 mM azurin. (Left) Broadening increases as the temperature is raised owing to the increase in the ESE rate, kEsE [see Eq. (3)]. (Right) Broadening increases from top to bottom owing to the increase in concentration of the paramagnetic particles, [P] [see Eq. (3)]. (Reprinted from Groeneveld and Canters. 138) 6. Metal Force Fields The d y n a m i c properties of a protein can be studied not only by N M R techniques but b y computational techniques as well. To this end, molecular dynamics (MD) simulations are usually employed. The ingredients required f o r an M D simulation are (1) the a t o m coordinates of the protein, as obtained, for instance, f r o m an N M R or an X-ray diffraction study, and (2) the forces acting on each atom, as obtained f r o m the gradient of the potential energy of each atom. The potential energy is usually considered as the sum of a C o u l o m b term, a van der Waals term, and various covalent bonding terms. To characterize the latter a knowledge of the force constants of the various types of motion of each nucleus is sufficient, provided the harmonic approximation is adequate. The collection of parameters describing the potential energy function of a nucleus m a y be considered as the force field of that nucleus. F o r the force fields of nonmetal atoms, good approximations exist that have b e e n tried out and refined in MD simulations of m a n y proteins. The exceptions are the force fields of metal atoms, for which no reliable data sets were available until relatively recently. This provided problems w h e n

286

PROBES OF METAL ION ENVIRONMENTS

[9]

simulations had to be performed on metalloproteins. The topic is receiving attention now, because MD simulations are increasingly used to analyze the dynamic behavior of proteins or protein domains, including metalloproteins. Moreover, MD simulations are often used in the final stages of an NMR structure determination to relieve possible strains remaining after a distance geometry calculation. The need for reliable force fields thus is equally strongly felt for non-metal-containing proteins as for metalloproteins. For this reason the topic of force fields of metals in proteins is briefly addressed here. One of the problems encountered when designing a force field for a metal is that little or no information is available on force constants in model compounds, although there has been some progress in this direction.143's44 An analysis of the infrared (IR) or resonance Raman enhancement (RRE) spectrum of a metalloprotein with the aim to extract the metal-dependent vibrations is also of little avail, since the analysis of the frequency spectrum would require a knowledge of the vibrational modes of metal plus protein and so far no feasible way seems to have been designed to tackle a problem of such formidable complexity. A second problem is that of uncompensated charge. Because the metal ion is often inside the protein, the charge on the metal may grossly distort the protein structure during an MD run simply because of electrostatic effects. This is because Coulomb interactions between the metal and charges elsewhere in the protein are propagated in a medium that has a low effective dielectric constant and because they are of long-range character (1/r dependence). An added complication arises when the metal may occur in more than one oxidation state. The force field of the metal should be able to mimic a change in oxidation state without destroying the stability of the protein. Third, many metalloproteins are able to exchange the metal in their active site with metal ions in solution. Ideally, the force field should allow metal-protein dissociation and association at a realistic rate. However, state-of-the-art methodology at present cannot be expected to mimic this aspect of the dynamics of a metal site. Metal force fields up to now have been designed to model a situation in which the metal is permanently bound to the protein. The problem of charge effects can be alleviated simply by reducing the charge on the metal or by partly redistributing the charge of the metal over the ligands. The latter method is preferable, because some or all of the ligands will usually carry a charge opposite to that of the metal. By 143 V. S. Allured, C. M. Kelly, and C. R. Landis, J. A m . Chem. Soc. 113, 1 (1991). 144 A. Vedani and D. W. Huhta, J. A m . Chem. Soc. 112, 4759 (1990).

[9]

MULTIDIMENSIONAL N M R OF METAL SITES IN PROTEINS

287

smearing the charge of the metal over the ligands, the net charge of the metal site is conserved, while at the same time the Coulomb interactions are attenuated. The practial question is to what extent the charge neutralization has to be carried through. Another obvious way during an MD run to prevent the metal from wandering through the protein structure under the influence of long-range electrostatic forces is to tie it to the ligands by means of bonds with large force constants. This method might serve the purpose of immobilizing the metal, provided the protein has enough stability of its own to resist the distortive Coulomb forces exerted by the metal, but it would create a more or less solidified hard kernel inside the protein that would distort the dynamics of the metal surroundings. The best way, so far, to design an acceptable metal force field seems to be by trial and error. Criteria by which the quality of a metal force field can be judged include the overall stability of the protein structure during an MD run. Energy and root mean square (rms) deviation from the start structure should level out after a starting up period of, say, 50 psec. The stability should be judged in runs of a few hundred picoseconds at the least. A second criterion is the geometry of the metal site. Preferably this should on average (when considered over a few hundred picoseconds) deviate by not more than one- or two-tenths of an angstrom from the experimental structure. A third criterion is the metal-ligand vibrational frequencies. If there is a way to identify experimentally these vibrations, then it is worthwhile to see if they can be found in the frequency spectrum of the metalplus-ligand motions in the MD run. For instance, the 600 nm RRE spectra of blue copper proteins closely reflect the vibrational activity of the Cu-S(Cys) bond. A frequency analysis of the Cu-S(Cys) motion in the MD run should match at least part of the RRE spectrum of the protein. A number of force fields appear to have been used with some success in MD simulations of proteins containing Ca, Zn, or Cu. Equilibrium values of metal-ligand distances are usually taken from crystallographic or model compound data. Points that ought to be but at the moment are not always addressed comprise the charge distribution on metal plus ligands, the force constants for stretching, bending, and improper dihedral modes, and the values of Lennard-Jones parameters of the metal. Ahlstr6m et al. in an MD simulation of calbindin Dgk incorporated the Ca 2+ ion by taking into account only Coulomb and van der Waals interactions. The Ca ligands exclusively consist of carbonyl and carboxyl groups, and the stability of the resulting structure is probably due to the large negative charge present on the Ca surroundings. In the MD simulation of various ZnE÷-containing enzymes by Hoops

288

PROBES OF METAL ION ENVIRONMENTS

[9]

TABLE I FORCE FIELD PARAMETERS FOR ZINC SITES IN PROTEINS a Bonds

Ro (fit)

K R (kcal/mol A 2)

O-H Zn-N Zn-OH Zn-OH 2

0.957 2.05 1.80 2.05

553 40 94 40

Angles

O 0 (°)

K o (kcal/mol rad 2)

C-N-Zn H-O-Zn N-Zn-N N-Zn-O

126 126 109.5 109.5

20 100 20 20

F r o m Ref. 145.

et al., 145 the formal charge on the metal had to be reduced to values in the range of +0.69 to +1.03 in order to obtain stable structures. An extensive quantum chemical study was undertaken by these authors to establish the effect that the charge distribution on the Zn ligands may have on the properties of the metal site during an MD run. They report some measure of success in optimizing the charge distribution. The parameters they used to mimic the covalent bonds and the angular motions involving the metal are summarized in Table I. ~45 They were designed within the framework of the general Amber force field. Wright and co-workers in their restrained MD simulations ofplastocyanin ~8 used a limited set of parameters to constrain the position of the Cu within the protein framework. For this purpose it appeared sufficient to implement Cu-ligand stretch modes and bending modes centered on the Cu and the ligands. They do not report the use of formal charges on the Cu or the adjustment of ligand charges. The force field used by Freeman and co-workers to model the Cu-containing stellacyanin on the basis of the three-dimensional structure of cucumber basic protein was more elaborate. 71 As in the case of the Zn proteins, it was found that the formal charge on the metal had to be reduced, in this case from +2 to +0.5, in order to get stable structures in the MD simulations. The values for the force constants of the metal-ligand bonds, the bending modes and the improper dihedral modes are collected in Table 11. 71 An extensive set of simulations was performed on azurin [G. W. Canters, W. F. van Gunsteren, and H. J. C. Berendsen, unpublished re145 S. C. H o o p s , K. W. A n d e r s o n , and K. M. Merz, Jr., J. A m . Chem. Soc. 113, 8262 (1991).

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289

TABLE II FORCE FIELD PARAMETERSFOR COPPER SITE IN PLASTOCYANINa

Bonds

K R (kcal/mol ~2)

Cu-N(His) Cu-S(Cys) Cu-S(Met)

100 90 60

Angles

K o (kcal/mol rad 2)

X-Cu-Y Cu-X-Y

30 50

Improper dihedrals

K E (kcal/mol rad 2)

Cu-CV-NS-C~(His) Cu-S~-C~-Ca(Cys)

50 20

From Ref. 71.

suits, 1992] in order to design within the GROMOS framework a proper set of parameters for the Cu. For the oxidized protein (Cu 2÷ form) best results were obtained with a net charge of +0.58 on Cu plus ligands, with a charge of +0.40 on the Cu, +0.23 on the ligand histidines, and -0.28 on the cysteine. For the reduced protein these numbers are 0.0, +0.18, +0.14, and -0.46, respectively. A slight polarization of the Met-121-S ~ had to be introduced as well, and the charge on the carbonyl of Gly-45 had to be reduced from -0.38 to -0.20 in order to prevent the Cu from being pulled over to the Gly-45 carbonyl. No metal-ligand bonds were TABLE III FORCE FIELD PARAMETERSFOR THE COPPER SITE IN AZURINa Angles

Oo (o)

Ko (kcal/mol tad 2)

C-N-Cu(His) C-S-Cu(Cys)

126 109.5

20 80

Improper dihedrals

-=0 (°)

K.- (kcal/mol rad 2)

(His)NS-CV-Ne-Cu

0

40

a From G. W. Canters, W. F. van Gunsteren, and H. J. C. Berendsen, unpublished results, 1992.

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PROBES OF M E T A L ION E N V I R O N M E N T S

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needed to obtain a stable Cu site, but nonzero force constants for a number of angle modes and improper dihedrals appeared necessary to keep the metal fixed in the proper position (see Table III). Analysis of the Cu-ligand motions over a period of several hundred picoseconds showed the presence of frequencies that occur in the same range as observed in the RRE spectrum. The efforts discussed above represent first attempts at mimicking the properties of metal sites in proteins. It is clear, however, that further work has to be invested in optimizing metal force fields for MD simulations.

[10] O p t i c a l l y D e t e c t e d M a g n e t i c R e s o n a n c e o f T r i p l e t S t a t e s in P r o t e i n s

By

ARNOLD

J. HOFF

1. Introduction Triplet states are with few exceptions (notably dioxygen) (photo)excited metastable states of usually aromatic, organic molecules. These types of molecules abound in all biological material, and the triplet state is an important probe of the molecular structure. Triplets have certain physical properties that make them eminently useful for identifying reactants in photoreactions, for probing structural environments, binding, etc., and for serving as gentle perturbers. Studying the response of the system then gives important clues to its structure and function. In this chapter I first introduce the physical characteristics of the triplet state and then discuss a variant of the technique of electron paramagnetic resonance (EPR), namely, optically detected magnetic resonance (ODMR) in zero magnetic field, which is one of the most important spectroscopic tools to study the triplet state. Most attention is devoted to the absorbance-detected magnetic resonance (ADMR) technique, the use of which has greatly expanded since the early 1980s. By ADMR it is possible to monitor low-temperature triplet absorbance difference spectra with unparalleled accuracy and sensitivity. For coupled pigment systems such as encountered in photosynthetic membranes, this has opened up a new field for the study of pigment interaction, interpretation of optical spectra, etc. The applications of ODMR in protein research are subsequently discussed, with emphasis on pigment-protein complexes in photosynthesis. Finally, the possibilities of ODMR spectroscopy of metalloproteins are examined. In writing this overview of theory and techniques of ODMR METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, lnc. All rights of reproduction in any form reserved.

290

PROBES OF M E T A L ION E N V I R O N M E N T S

[10]

needed to obtain a stable Cu site, but nonzero force constants for a number of angle modes and improper dihedrals appeared necessary to keep the metal fixed in the proper position (see Table III). Analysis of the Cu-ligand motions over a period of several hundred picoseconds showed the presence of frequencies that occur in the same range as observed in the RRE spectrum. The efforts discussed above represent first attempts at mimicking the properties of metal sites in proteins. It is clear, however, that further work has to be invested in optimizing metal force fields for MD simulations.

[10] O p t i c a l l y D e t e c t e d M a g n e t i c R e s o n a n c e o f T r i p l e t S t a t e s in P r o t e i n s

By

ARNOLD

J. HOFF

1. Introduction Triplet states are with few exceptions (notably dioxygen) (photo)excited metastable states of usually aromatic, organic molecules. These types of molecules abound in all biological material, and the triplet state is an important probe of the molecular structure. Triplets have certain physical properties that make them eminently useful for identifying reactants in photoreactions, for probing structural environments, binding, etc., and for serving as gentle perturbers. Studying the response of the system then gives important clues to its structure and function. In this chapter I first introduce the physical characteristics of the triplet state and then discuss a variant of the technique of electron paramagnetic resonance (EPR), namely, optically detected magnetic resonance (ODMR) in zero magnetic field, which is one of the most important spectroscopic tools to study the triplet state. Most attention is devoted to the absorbance-detected magnetic resonance (ADMR) technique, the use of which has greatly expanded since the early 1980s. By ADMR it is possible to monitor low-temperature triplet absorbance difference spectra with unparalleled accuracy and sensitivity. For coupled pigment systems such as encountered in photosynthetic membranes, this has opened up a new field for the study of pigment interaction, interpretation of optical spectra, etc. The applications of ODMR in protein research are subsequently discussed, with emphasis on pigment-protein complexes in photosynthesis. Finally, the possibilities of ODMR spectroscopy of metalloproteins are examined. In writing this overview of theory and techniques of ODMR METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, lnc. All rights of reproduction in any form reserved.

[10]

ODMR OF TRIPLETSTATES

,0 o

----+

t,

.oMo - 4 - +

-

;hw

,so 4 - -

+-+-

-++

--+-+-

singlet

singlet excited state

triplet excited state

ground state

291

's°'- - - 4 +

singlet ground state

FIG. 1. Distribution of electrons over the highest occupied (HOMO) and lowest unoccupied (LUMO) molecular orbital of singlet and triplet states. ISC, Intersystem crossing. [From A. J. Hoff, in "Optical Properties and Structure of Tetrapyrroles" (G. Blauer and H. Sund, eds.), p. 435. de Gruyter, Berlin, 1985.]

in protein research I have benefited much from two recent reviews on the biological applications of ODMR. L2

1.1. Physics of Triplet State Optical transitions of aromatic molecules are usually 7r-Tr* absorptions of the 7r electrons or n-Tr* transitions of the lone 2p pair of heteroatoms (oxygen, nitrogen, etc.). Depending on the spin pairing of the two unpaired electrons, the excited state is either a singlet state (spins antiparallel) or a triplet state (spins parallel), as illustrated in Fig. 1. The unexcited molecule is in the singlet ground state; that is, all electronic orbitals are occupied by a pair of electrons with opposite spin. On excitation by a photon of sufficient energy, one of the electrons of the highest occupied molecular orbital (HOMO) of a molecule may jump to the next higher orbital, the lowest unoccupied molecular orbital (LUMO). During the excitation process the spin state of the excited electron is preserved, because of the law of conservation of angular momentum, so that the excited molecule is still in a S = 0 singlet (SO state and would remain in the singlet manifold if there were no coupling between spin and orbital angular momentum. However, the spin moment and the magnetic moment generated by the orbital motion interact magnetically, and through this spin-orbit interaction orbital angular momentum of the electrons may be converted to spin angular momentum without violating the conservation law. This means that there is a certain probability that the spin vector in the LUMO is inverted (this is allowed by the Pauli principle as the unpaired electrons i A. H. Maki, in "Biological Magnetic Resonance" (L. J. Berliner and J. Reuben, eds.), Vol. 6, p. 187. Plenum, New York, 1984. 2 A. J. Hoff, in "Advanced EPR: Applications in Biology and Biochemistry," (A. J. Hoff, ed.), p. 633. Elsevier, Amsterdam, 1989.

292

PROBES OF METAL ION ENVIRONMENTS

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are in different orbitals). The sum of all individual electron spin vectors now adds up to unity, S = 1, and the multiplicity 2S + 1 is equal to 3, that is, we have a triplet state, the ground state TOof the triplet manifold. (Sometimes the lowest triplet state is denoted TI to indicate that it is an excited state.) The probability of the spin inversion or intersystem crossing (ISC) depends on the strength of the spin-orbit interaction, which for a single atom is proportional to the nuclear charge Z. The molecules that concern us in the section on applications contain only relatively light atoms, so that spin-orbit coupling is weak and ISC in the LUMO is a relatively slow process, comparable with that of deexcitation by fluorescence. In some cases, however, the presence of a heavy metal promotes ISC (Section 3.1). This effect could be of importance for applications of ODMR in metalloprotein research. Often triplet states are formed by radical recombination reactions. The radicals may be generated from a singlet excited state created by illumination or by ionizing radiation. In photosynthesis under conditions in which normal, forward electron transport is blocked, such a recombinational triplet state is formed with high efficiency. Once the molecule is in a triplet state it may remain there for a long time (microseconds to milliseconds for chlorophylls, depending on the temperature), as deexcitation to the singlet ground state again involves a "forbidden" spin flip. The To ---> So transition is much slower than the S~ ~ TOtransition because (1) the electronic orbitals of the initial and the final state are different, and (2) the latter transition presumably occurs through higher energy triplet states, which makes it easier to dispose of the excess energy as heat. The triplet state may decay directly to So under emission of radiation (phosphorescence) or without radiation. When the energy gap between the S~ and To states is comparable to kBT (kB is Boltzmann's constant and T is the temperature), the triplet may decay to So via $1 with emission of delayed fluorescence (Fig. 2). The energy of the TO state is usually appreciably lower than that of the S1 state. This is because the two unpaired electrons have the same spin quantum number and, according to the Pauli principle, cannot move in the same electronic orbital. On average the two electrons are farther apart in the triplet state than in the singlet state; hence, the energy of Coulombic repulsion is less and the state energy is lower. The decrease in repulsion energy is accounted for by introducing the electrostatic exchange energy, - J . For two unpaired spins on one molecule the exchange energy is usually negative ( J > 0), and the triplet state lies lower than the excited singlet state. This means that the wavelength of phosphorescence emission is longer than that of fluorescence emission.

ODMR OF TRIPLETSTATES

[10]

293

s2lc,I--T~r, i 7 "-a"a-~---t -ii'dT"~L---#--l--'l k,

[ P,-~/NR

"~'.~-O-z

12/

i t

"

FiG. 2. Energy level diagram of the singlet and triplet manifold. So, Singlet ground state; Sl and $2, singlet excited states; To and T1, first and second excited triplet states; SA and TA, singlet and triplet absorption, respectively; F and DF, fluorescence and delayed fluorescence, respectively; P, phosphorescence; NR, nonradiative transition; IC, internal conversion. Enlarged To levels: X, Y, and Z, eigen energies of the dipole-dipole interaction; D and E, zero-field splitting parameters. Downward arrows tending to the right, populating probabilities; to the left, decay rates; filled circles, equilibrium populations. Vl, v2, and v3 are the frequencies corresponding to the ([D[ + [Ei)/hand 2[El/h transitions, respectively. (From Ref. 2.)

1.2. Triplet Spin Hamiltonian in Zero Magnetic Field The triplet spin Hamiltonian without external magnetic field comprises interactions involving the magnetic moment of the electrons. These are 2-fold: spin-spin coupling and spin-orbit coupling. The main contribution to the spin-spin coupling o p e r a t o r , / t s s , is the classic magnetic dipoledipole interaction between two electrons: /lss

= ~3\~](g2fl2/z°] It(Si-S2r :3

(sl" r)(s2 r!)r s •

(1)

with g being the electronic g value, fl the electronic Bohr magneton, s I and s2 the magnetic moments of the two electrons, r their distance vector, a n d / x 0 the permeability of vacuum. Equation (1) can be rearranged to /~ss = S" ! ) - S

(2)

where !) is a tensor operator whose elements consist of integrals over the coordinates of the electrons and S is the total spin angular momentum operator S = sl + s2- !) can be diagonalized by a coordinate transformation to its principal axes, a n d / l s s becomes

Hss = _X~2_ r ~ 2 _ Z ~ z 2

(3)

where X, Y, and Z are the principal values of I) and Su (u = x, y, z) the components of S along the principal axes of I). Often, these axes coincide with the molecular symmetry axes.

294

PROBES OF METAL ION ENVIRONMENTS

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In a two-electron approximation the triplet wave functions can be written in symmetry-adapted form ITx) = 2-1/2(fllfl 2 -- OtlOt2)

ITy) = 2-1/2i(fllfl 2 + a l a 2) ITz) = 2-1/2(o(1132 + flla2)

(4)

where ct and fl are the eigenfunctions of the component of the spin operator along the z direction, gz. The functions ITu) (u = x, y, z) belong to different irreducible representations of the point group C2~, which is assumed to be a subgroup of the symmetry point group of the molecule; they are eigenfunctions of g2 with eigenvalue 2 and are to a first approximation degenerate if we neglect the spin-spin interaction. The Hamiltonian [Eq. (2)] lifts the degeneracy, and it turns out that ITu) are eigenfunctions of Hss with eigenvalues X, Y, and Z. The ITs) functions have the property S~IT~) = 0,

SxITy) = -SyIT~) = i]Tz)

(5)

The second relation holds for cyclic permutation of the subscripts x, y, and z. Thus, the triplet component ITs) is an eigenfunction of the operator S~ with eigenvalue zero, that is ITu) corresponds to a situation where the spin angular momentum vector lies in the coordinate plane u = 0. From Eq. (5) it further follows that there is no net magnetic dipole moment associated with any of the triplet substates in zero magnetic field yh
(6a)

but that there is a transition dipole moment present between any two of the triplet substates

= i ,h

(cyclic)

(6b)

where 3' is the gyromagnetic ratio of the electron and h is Planck's constant divided by 2~r. This is an important result as it shows that, in zero magnetic field, population can be transferred from one triplet sublevel to another by applying a resonant electromagnetic field. From Eq. (6b) it follows that the transition probability is proportional to

lyh(TzlB," glr,)l 2

= (

hn,x) z

(cyclic)

(7)

where B 1 is the amplitude of the magnetic component of the driving field, B = B1 cos tot. From Eq. (7) it follows that the microwave transition T, -~ Ts (u, s = x, y, z) is polarized with transition moment along w = u × s. This allows microwave-selection spectroscopy to be performed to which we will turn later in this chapter.

[10]

ODMR OF TRIPLETSTATES

295

The resonance frequencies follow from the eigen energies of Hss, namely, X, Y, and Z. Because X + Y + Z = 0 (the trace of D is zero), it is customary to express the energies in two independent parameters D and E, the zero-field splitting (ZFS) or fine-structure parameters:

D=-~Z,

E=-

(X-r)

(8)

where, by convention, ]D[ -> 3[E[. The physical meaning of the zerofield splitting parameters is that they represent averages over the spatial coordinates x', y', z' of the distance vector r of the two unpaired electrons:

3 (g2flZtZo~ (.r2 - 3z'2\ D = -~ \ ~ ] -r: /

(9)

7

e= - ~ \ ~ 7 , -

Thus, E is a measure of the deviation from axial symmetry about the z axis. The relative order of the energy levels depends on the sign of D and E. For a flat molecule such as chlorophyll, one would expect D to be positive. (The z axis is the axial symmetry axis and is perpendicular to the plane of the molecule, so that the z' component of r is on average much smaller than [rI.) For a rodlike molecule, such as a biradical, D will be negative. 2. Optical Detection of Magnetic Resonance Continuous illumination will generate an equilibrium population of the triplet sublevels, given for light levels that are not too high by

Nu = p,KN/ku,

~uN~ = N T

(I0)

where u = x, y, z; in a commonly adopted notation 1Pu is the probability of transit from the singlet excited state to the uth triplet sublevel with E~ p~ = l, k~ is the decay rate that governs deexcitation from the uth sublevel back to the singlet ground state, K is the overall rate of population of the triplet state, N T is its total population, and N is the number of photoexcitable molecules. Both p, and k~ are determined by molecular symmetry. The N~ values obtained for about equal pu and kx ,ky ~> k z are depicted in Fig. 2. Let us now assume that we have almost totally inhibited spin-lattice relaxation by working at very low temperature. If we then switch on a microwave field of a frequency corresponding to a transition between the y and z level, the field will transfer population from the heavily populated,

296

PROBES OF METAL ION ENVIRONMENTS

[10]

slowly decaying z level to the much less populated, fast decaying y level. Obviously, this transferred population will not remain there but quickly decay to the singlet ground state. A new equilibrium will be established that for a strong enough microwave field is given by Ny = N z = p ' K N / k ' , with p' ½(py + Pz) and k' = ½(ky + kz). If we take py ~- Pz, it is then immediately seen that (Ny + Nz) < (Ny + Nz), because for Icy > k z, 2/(ky + kz) < 1/ky + 1/k z. Although the x level will sense the new equilibrium via the photogeneration cycle of the triplet state, this is a second-order effect. The change in population will lead to enhanced phosphorescence intensity I if the triplet decays radiatively. I will be proportional to E krN,,, where k~, is the radiative decay rate of level u. The microwave field changes the Nu by ANu, so that the change in I is given by A / = kryANy + krAN~ = AN(ky - k~). Thus, for a change in phosphorescence intensity it is necessary that both AN ~ 0 and k~ ~ k z. In our example, E N" = N~ < N T , so the concentration of the singlet ground state is enhanced, as the concentration of the singlet excited state will be negligibly low when conventional light sources are used. For AN ~ 0 and ky ~ k z (k = kr + knr, with knr being the nonradiative decay rate), this will lead to enhanced fluorescence and enhanced singlet ground state absorbance, as both these phenomena are proportional to the ground state population, whereas the absorption of the triplet To ground state will be decreased. In the above we have the essence of ODMR. A sample is continuously illuminated at liquid helium temperatures, preferably below 2.1 K, and simultaneously irradiated by microwaves of a frequency v not far from that corresponding to one of the triplet sublevel spacings: Vl,2 -- (IDI -+ IEI)/h and v3 -- 2E/h (Fig. 2). The frequency v is slowly scanned across one of the frequencies vl,2,3 while either the phosphorescence, (delayed) fluorescence, or absorbance of the sample is monitored. When v is close to or precisely equal to v -- /./1,2,3 the ensemble of triplet states is in resonance with the microwave field and the fluorescence (FDMR), phosphorescence (PDMR), or absorbance (ADMR) will be enhanced, or diminished, depending on the relative values of p~ and k~. An important advantage of ODMR in zero magnetic field compared to conventional EPR in high field, where the absorption of microwaves is monitored, is that the optical probing occurs with quanta of much higher energy than the microwave quantum [e.g., E(orange light, 600 nm or 6 × 10 -5 cm)/E(microwaves, 3 cm) = 5 × 104.] This enhances detector sensitivity enormously. Second, because the modes of detection and excitation are decoupled, the technique is insensitive to noise sources arising from the microwaves (as, e.g., amplitude fluctuation), especially when the transition is saturated. (Note that the optical signal does not disappear on saturation as does the microwave absorption in microwave detection; =

[10]

ODMR OF TRIPLETSTATES

297

this is another advantage of optical detection.) Third, compared to highfield EPR of triplet states one has in zero-field resonance much narrower lines and a concomitant increase in sensitivity, since the anisotropy in resonance conditions associated with an applied magnetic field is absent. Finally, the possibility of probing the resonance at various wavelengths gives much new information, especially for ADMR (Section 2.3). In the next section the theory of ODMR is summarized, following Refs. 1 and 2.

2.1. Quantitative Description of Optically Detected Magnetic Resonance 2.1.1. Time Dependence. For continuous illumination the three triplet sublevels and the singlet ground and excited states form a coupled fivelevel system, which is described by a set of coupled, first-order linear differential equations: l~.o(t) --- -ko[No(t) - Nl(t)] + klNl(t) + ~. kuN.(t) N. l(t) = ko[No(t) - Nl(t)] - (kl + kz)Nl(t) Nu(t) = kzpuNl(t) - [ku + "Zv¢. (W.v + P.v)]Nu(t) + ~o~. (Wo. + Pv.)No(t), u, v = x, y, z No(t) + Nl(t) + "Z~ Nu(t) = N

(lla) (llb) (llc) (12)

Here, Nu, NT = ~ N . , and N are as defined before, No and N1 are the population of the singlet ground state and the singlet excited state, respectively, p. is the populating probabilities, the k values are defined in Fig. 2, Wo, and W,o = W~, exp[(E~ - Ev)/k~T] are the spin-lattice relaxation rates to and from the uth triplet sublevel from and to the vth sublevel, and P,o = Po, is the rate of microwave transition between sublevels u and v. The set of Eqs. (11) and (12) can be analytically solved with only one simplifying assumption: The population of the singlet excited state is under all but the most extreme cases of continuous illumination much smaller than the total number of molecules, N~ ~ N, so that Eq. (12) reduces to No + " Z ~ N , ~ N o + N T . ~ N

(13)

and we may write, with K = k2ko/(ko + kl 4- k2), -ko[No(t) - Nl(t)] + klNl(t)~- - K N o ( t ) k2P,Nl(t) ~ KP,No(t)

(14a) (14b)

Substituting Eqs. (14a) and (14b) in Eqs. (1 la) and (1 lc), respectively, we get with Eq. (13) a set of three independent differential equations. Their steady-state solution gives the ODMR under slow-passage conditions, from which the ODMR frequency and line shape are determined; their

298

PROBES OF M E T A L I O N E N V I R O N M E N T S

[10]

analytic solution yields expressions for transient ODMR, from which the molecular decay rates are extracted. 2 . 1 . 2 . S l o w - P a s s a g e O p t i c a l l y D e t e c t e d M a g n e t i c R e s o n a n c e . The solution of Eqs. (1 la)-(1 lc) and (12) for steady-state conditions is readily obtained for conditions that the W terms are negligible. a. A b s e n c e o f m i c r o w a v e s . N o = koNo/(ko + k I + k2) = K ( N - N T ) / k 2 N o = NK(p./ku)/(1 + KEu pJk.) N o = ~uN~ = NKE~(pJk.)/(1 + KE~ p./k.)

(15a) (15b) (15c)

When K is much smaller than the k. values, we may expand Eqs. (15b) and (15c) into a series, giving N o = NK(pJk.)(1

- K E~ p . / k u + . . . )

N T = NK~u(pJk~)(1

- K~

p~/k. + ...)

(16a) (16b)

F o r small K Eq. (16a) reduces to Eq. (10). Note that the fraction of molecules in the triplet state, N T / N , depends on K but that the relative steady-state sublevel population, N J0N T ,0 does not. b. S a t u r a t i n g m i c r o w a v e s . The steady-state equations for resonant microwaves saturating the u ~ v (u, v = x, y, z) transitions are readily obtained from Eqs. (15) and (16) by substituting for p . and Pv, ½(P~ + Pv), and for k. and kv, ½(k~ + kv), while the third p and k remain unaltered. When two transitions, for example, u ~ v and u ~ w (u, v, w = x, y, z) are saturated, we havek,, = k~ = k w = k = 1 / 3 ~ k u a n d p ~ = 1/3 for all u. The changes in triplet population for microwaves saturating the u ~ v transition are then to first order in K given by AN~,~= N ~ ° - N o -~ - ( k u / k o ) A N ~ °-~ N K ( k ~ p ~ - kop.)/k.(k~ + k~) A N ~ ~ ~- A N ~ ~ + A N ~ ° -~ N K ( p J k .

- p o / k v ) ( k . - ko)/(k. + ko)

(17a) (17b)

whereas the change in population of the third level is zero to first order in K. F r o m Eqs. (16) and (17) we see that the slow-passage ODMR signal for fluorescence or absorbance detection, which is proportional to AN 0 = - A N T , is given by S"°(FDMR, ADMR) oc ( N o - N O ) ( k . - ko)/(k" + kv)

(18)

Thus, as noted above for obtaining an F D M R or ADMR signal, both the equilibrium populations and the molecular decay rates of the two triplet sublevels c o n n e c t e d by microwaves must be unequal. The sign o f the F D M R or A D M R response depends on the relative magnitudes of N~, N~ and k., kv. Note that the microwave-induced change in singlet absorbance

[10]

ODMR OF TRIPLETSTATES

299

(sometimes called SADMR) is opposite in sign to that of the triplet absorbance (TADMR). Note also that generally triplet formation leads to complete bleaching of the S1 +-- So transition. Because the Tn <-- To transitions almost always have a much lower molar extinction coefficient than the S 1 <---So transition, there will be generally little interest in using TADMR instead of SADMR for the determination of the ZFS parameters and the decay rates. The proportionality constant for S'V(FDMR) contains a factor K k 2-1 because the microwave-induced change in fluorescence is proportional to the microwave-induced change in $1 population, AN]'v , which from Eqs. (15) and (17b) is given by AN'{" = K k ~ lANkY ~- - K k ~ 1AN,~V ~ - K2k2 XN funct(p~,o; k,,o) (19)

Thus, for low light conditions such that Eqs. (17a) and (17b) are valid, S(FDMR) is proportional to the square of the light flux, in contrast to S(ADMR), which depends linearly on the light flux (provided K ~ k, for all u holds). This makes it advantageous to use high illumination for FDMR experiments. The phosphorescence P is given by P = c Es U N s , where c is an instrumental constant and k~ the radiative decay rate. For saturating micro1 r waves, k r''o = k~''v = ~(k, + k~). With Eq. (17a) we then obtain for the phosphorescence-detected ODMR signal S,o(PDMR) ocEs krAN~ °

(20)

where s = x, y, z; which on substitution of Eq. (17a) becomes S,o(PDMR) ~ (N O - N,)(k~/k~ 0 r r - ko/ko)k, kv/(k . + ko)

(21)

Thus, a PDMR response requires that the equilibrium populations and the radiative quantum yields k r / k , , Uo/ko, rather than the decay rates, are unequal. 2.1.3. Transient Optically D e t e c t e d M a g n e t i c R e s o n a n c e . The time dependence of the ODMR signal in response to a change in the condition of microwave irradiation (switching them on or off, or applying pulses) is given by the solution of Eqs. ( l l a - l l c ) and (12), simplified by the neglect of the Sl population. The general analytic solution is given in Ref. 3; for the present discussion it suffices to note that the response of the system on switching on or off resonant microwaves connecting x = z or y ~ z or both is given by the sum of three exponentials Ni(t) = £j 3 A. J. H o f f and B. C o r n e l i s s e n ,

~i,j exp(Xjt)

Mol. Phys.

+

45, 413 (1982).

'lt~i

(22)

300

PROBES OF METAL ION ENVIRONMENTS

[10]

where j = 1, 2, 3, i = 0, x, y, z, and ~:j, hi, and "0i are rather cumbrous functions of the rates of decay, spin-lattice relaxation, and microwaveinduced transitions and the rate K (proportional to the light flux) of populating the triplet state. For K ~ 0 and W ~ 0 and in the absence of microwaves, the triplet sublevels are uncoupled, and each sublevel decays after some perturbation according to N.(t)

= Nu(oo) - [Nu(oo) - N.(0)] exp[-ku(t)]

(23)

For pulsed resonant microwaves applied for a time short compared to the fastest sublevel decay time, the return to equilibrium is given by ~kl(g ) = -k. - p.K h z ( K ) = _ k ° _ P° K

K---~O;u ¢ v = x,y, orz

(24a) (24b)

that is, by two exponentials with amplitudes of opposite sign. Provided K is low enough, the third sublevel is not perturbed [Eq. (17)]. The amplitude of the pulse response to first order in K follows from Eq. (23) for the appropriate boundary condition: N.(~) = N K p . / k . and N.(0) - N.(oo) = f ( p o / k ° - p . / k . ) , where f i s a parameter describing the effect of the pulse on the population of sublevels u and v, namely, f = ½ for saturation (high power and/or long pulse) and f = 1 for inversion of N. and No (the maximal effect). Normally, f ~ ½. With N T ( t ) = ~ . N . ( t ) , the change 8 N T ( t ) , and thus S(F,ADMR), is to first order in K given by S(F,ADMR) ~ 8NT(t ) = NT(t ) -- ~u N,(~) - - ~ f ( N ° - X ° ) ( e - k u t - e -kot)

(24c)

whereas for the phosphorescence response the exponentials are weighted by the radiative decay rates: S(PDMR) oc c f ( N o _ N ~o) ( k ~r e - k . t - Uo e - k j )

(24d)

Determination o f a v e r a g e d e c a y r a t e . When saturating microwaves are applied simultaneously to two of the three ODMR transitions, the triplet collapses to one level with average decay rate k = 1/3 ~, k~. The set of the simplified Eqs. ( l l a - l l c ) and (12) reduces to an equilibrium reaction, which immediately yields

NT(t) = N K ( K

+

k)-l{1 - e x p [ - ( K + k)t]}

(25)

for the triplet built up after the onset of illumination at t = 0. Equation (25) permits the evaluation of k by extrapolating to K --~ 0. Obviously, the same equation applies when the triplet levels are coupled not by microwaves but by spin-lattice relaxation at higher temperatures. 4 In fact, 4 M. Nakamizo and T. Matsueda, J. Mol. Spectrosc. 27, 450 (1968).

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ODMR OF TRIPLETSTATES

301

the latter method to determine k is to be preferred, since especially for randomly oriented samples the condition of microwave saturation is difficult to obtain (see below). O n s e t o f s a t u r a t i n g m i c r o w a v e s . When saturating microwaves are applied to the u ~ v transition at t = 0, the characteristic rates of attaining the new equilibrium are, for K ~ 0, ½(k. + ko) and kw (u, v, w = x , y , z). With Eqs. (17) and (23) we obtain the following for the time dependence of the connected sublevels, keeping in mind that N .UO (0) = Not/O (0) = ½NK(pu/k.

+ pJko):

N~°(t) ~ N~V(~) - [NUV(~) - N."°(0)] exp[-½(k. + ko)t] = N K { ( p ~ + p o ) / ( k . + ko) - ½ ( p . / k . - po/ko)(k . - k~)(k. + ko) -1 exp[-½(k~ + kv)t]}

(26a) (26b)

and similarly for N~°(t), whereas as before the population of the third sublevel remains constant. It is now easily shown ''2 that the time dependence of the F D M R and ADMR signals is given by S ~ O ( F , A D M R ) ( t ) o: 8N~O(t) = AN~r~{1 - exp[-½(k~ + k~)t]}

(27)

with AN}v given by Eq. (17b), and that of the PDMR signal by r uu S"°(PDMR)(t) - S"o(PDMR)(~) -= c "Zs ksANs (t)

r 0 _ NO)(kv _ ku ) = r'-ctk 2 ~ . + ko)(N~ (k. + ko)-~ exp[-½(k. + kv)t]

(28)

with S"°(PDMR)(~) given by Eq. (21). It follows that immediately after switching on the microwaves the PDMR signal is not zero, as in FDMR and ADMR, but is equal to S(PDMR)(0) = ~c(N~ ' 0 + N o0) ( k r~ - k~). r This is a consequence of the immediate response of the phosphorescence intensity to the change in the sublevel population brought about by the microwaves, in contrast to the change in So population, which needs to be built up by the photocycle. R e c o v e r y f r o m s a t u r a t i n g m i c r o w a v e s . Substituting in Eq. (26a) N ~ ° ( ~ ) for N~°(0) as the new initial condition at t ~- 0, and N O = N K p J k ~ for N~o(~), one obtains the recovery curve after switching off the saturating u ~ v microwaves: Ns(t) ~ NK{pJk~

+ [(p~ + pv)/(ku + k o) - p~/ks] exp(-k~t)}

(29)

for s = u, v. Again the population of the third sublevel remains unaltered. For the change 8NT(t) in total triplet population we obtain, using Eq. (29), 8NT(t) = N T ( t ) -- NT(~) ~ N K "Z. . . . ~ [(p~ + p o ) / ( k . + k o) - p~/k~] exp(-k~t) =

(N o _

o N~)(k~ + ko)-l[koexp(-k~t)

- k~exp(-kot)]

(30)

302

PROBES OF METAL ION ENVIRONMENTS

[10]

Thus, the decay curve S(F,ADMR) is the sum of two exponentials with opposite amplitudes and rates equal to the molecular decay rates (all assuming K ~ 0). For S(PDMR) we have to weight the exponentials by the radiative decay rates, ck~,and ck r , respectively. Multiplying with k, kv/ k, ko the preexponential factors ko and ku then become proportional to the radiative quantum yields k r / k , and kr/ko, respectively. Compared with Eq. (30), this may be of advantage when one of the decay rates is much smaller than the other, since the quantum yields, being ratios, are bound to be comparable. ~Note that this also applies when comparing with the response to a microwave pulse [Eq. (24d)]. N o optical excitation. As mentioned above, Eq. (23) is strictly true only for very low values of K, since only in the limit of zero optical excitation are the triplet sublevels truly decoupled (provided there is no spin-lattice relaxation). Thus, an ideal situation would be to measure microwave-induced transients for K = 0. This can obviously only be done for phosphorescence and delayed-fluorescence detection; the technique is called microwave-induced delayed phosphorescence (MIDP) 5 or luminescence (MIDL). 6 Essentially, after photogeneration of the triplet state, the light is switched off at time t = 0 and at time t = t' a short pulse of microwaves resonant between sublevels u and v is applied. Population is transferred and the resultant difference in phosphorescence recorded. The pulse-induced change in phosphorescence is then given by APpulse = AP~ + AP o = cf(k r - kv)[N r ~0 exp( - kvt ) - N°exp(-k,,t')]

(31a)

which for k~ >> ko and k , t ' >> 1 reduces to APpuI~ ~ = cf(k r - kr)N°o exp( - kot')

(31b)

Thus, by measuring the amplitude of the pulse-induced change in phosphorescence as a function of t', ko is easily evaluated. For k u >> ko the transient phosphorescence APpu~s~ decays with the fastest rate constant, ku, so that the MIDP experiment yields accurate values of both decay rates. When k, ~ kv a biexponental fit has to be carried out. Note that the microwave pulse need not be saturating, but must be short compared to the fastest decay time for the above simple analysis to apply. The method works best for rather disparate k~.v rates; the light switching has to be done much quicker than either of the decay times. The initial populations N°,o need not be the equilibrium ones, so in principle a laser flash may be used. 5 j. Schmidt, W. S. V e e m a n , a n d J. H. v a n der Waals, Chem. Phys. Lett. 4, 341 (1969). 6 j. G. W e e r s and A. H. Maki, Biochemistry 25, 2897 (1986).

[10]

ODMR OF TRIPLETSTATES

303

O t h e r s c h e m e s . A great many variants of the above schemes to measure the decay rates are possible, in some of which the spin-lattice rates are explicitly introduced (see, e.g., Refs. 1 and 2). Often, the treatment then gets more complex and one has to resort to numerical simulation along the lines discussed in Ref. 3 to evaluate the parameters of interest. 2.1.4. P o p u l a t i n g Probabilities. In the preceding section expressions are given to evaluate radiative and total decay rates from experimental kinetic traces. Here, a few experiments are discussed that allow the populating probabilities to be determined. We limit ourselves first to fluorescence, casu quo absorbance detection. R e s p o n s e to o n s e t o f illumination. The light is switched on at time t = 0 with saturating microwaves connecting two triplet levels in different ways, say, u ~ v and the double resonance x ~ y ~ z combination, while the fluorescence F is monitored. With Eqs. (13) and (15c) we then have 2 F"°(O)/F'°(~)- 1 FXyz(o)/FXyZ(°°) -

1

= (k/NK)NK ~Vps/k s

= k[2(p~ + po)/(k~ + ko) + pw/kw]

(32)

and similarly for F "w or F vw and F (no microwaves). The triplet excitation rate K is eliminated, and from a set of three equations the p values can be solved; as a check E, P,,exp should be close to unity. FXyZ(oo)and F'V(oo) are best measured by continuously irradiating the sample with u ~ v microwaves and switching the u -~ w (or v ~ w) microwaves on and off; the difference signal can be signal-averaged, yielding accurate relative values of F ( ° ° ) . 7 M i c r o w a v e switching u n d e r continuous illumination. The above method works only when the light can be switched on faster than the fastest sublevel decay rate. When this is difficult (e.g., for bacteriochlorophyll triplets), a variant may be used in which the relative fluorescence intensity under various conditions of saturating microwave irradiation is measured. For example, for low K [F~O(oo) - FXyZ(~)]/[ F,W(oo) - FXyZ(oo)] = (1 - kX~°ps/ks)/(1 - kX~Wps/ks)

(33)

with p~O = p~O = ½(Pu + Pv), k~ ~ = k~ ~ -= ½(k, + ko), etc., and similarly for F °w. With E, p , = 1, we again have three independent linear equations from which the p~ values are solved. Note that we now need all three ODMR resonances. 7 W. G. van Dorp, W. H. Schoemaker, M. Soma, and J. H. van der Waals, Mol. Phys. 30, 1701 (1975).

304

PROBES OF METAL ION ENVIRONMENTS

[10]

Simulation o f decay curves. With Eqs. (24c) and (24d) the experimental decay curves can be simulated for assumed values of k, and N O(the latter depend on the p,). Simulating (maximally) three decay curves simultaneously with five parameters (three k, and two p , , as E p, = 1), a best fit yields both the k, and the desired Pu. Note that this procedure works only if great care is taken that the factor f be the same for all three decay experiments. Phosphorescence detection. Dividing the phosphorescence response to a pulse of microwaves under continuous wave (CW) illumination for t = 0 [Eq. (24d)] by the MIDP response to an identical pulse for t = t' and k, >> kv [Eq. (31b)], we obtain AP"v(O)/AP"qt') ~ (N O - N ° ) / N ° exp( - kot')

(34)

and similarly for AP "w (provided k, >> ko). The prefactors are evaluated from a semilog plot versus t', and from them the relative populating probabilities p,/po and P,/Pw can be calculated for known sublevel decay rates. 8 2.1.5. Note o f Caution. A good many of the methods discussed in the previous sections rely on saturation, that is equalization of the populations of one or two transitions. This requires a very low temperature to inhibit spin-lattice relaxation and fairly large microwave powers to make the microwave-induced transition rate much larger than the fastest of the molecular decay rates. The latter requirement is often not met when the output of microwave sweepers (20-40 mW) is used without amplification. As a result large errors of more than a factor of 4 can be made in the evaluation of the molecular decay rates. 3'9 It is absolutely necessary to verify saturation by evaluating the decay rates as a function of applied microwave power. Preferably one should use pulse methods 5'7 to determine the k~ values. When randomly oriented samples are used, such as normally is the case for biological material, 100% saturation can never be achieved. This is a consequence of the polarization of the microwave transitions: the transition probability is proportional to cos 2/3, where/3 is the angle between the microwave field B~, and the transition moment. Hence, molecules whose transition moment is close to perpendicular to B 1 (this is a sizable fraction in view of the sin/3 distribution) have a low probability to be microwave-excited, and their sublevel population is not or is only 8 I. Y. Chan and B. N. Nelson, J. Chem. Phys. 62, 4080 (1975). 9 A. J. Hoff, in "Triplet State ODMR Spectroscopy" (R. H. Clarke, ed.), p. 367. Wiley (Interscience), New York, 1982.

[10]

ODMR OF TRIPLETSTATES

305

very slowly affected by the microwaves. This effect is quite noticeable, even at microwave powers exceeding 1 W at 1.2 K . 3 For such samples the pulse methods seem to be the only reliable way to measure the decay rates. Another pitfall in the determination of the k~ values is the dependence of the apparent decay rates on K, the rate of triplet formation, which is proportional to the light flux. This applies equally to equilibrium and pulse methods, except of course for the MIDP technique. As mentioned, extrapolation to K ~ 0 is not trivial because of the poor signal-to-noise ratio attendant with the requirement that K is much smaller than the slowest molecular decay rate (which can be less than 1 sec-l). Most workers prefer to fit a curve of ku versus K, using relations such as outlined in Ref. 3, but even then the fit in the K = 0 regime is often ambiguous.

2.2. Line Shape, Hole Burning, and Double Resonance The ZFS parameters of a triplet state are sensitive to its environment. The larger the interaction with the environment, the more spread one will find in the values of [DI and IEI. This translates into inhomogeneous broadening of the ODMR lines, which are often close to or even a perfect Gaussian. In molecular crystals ODMR line widths can be as narrow as 1 MHz, but in glassy matrices they often exceed 100 MHz. Such inhomogeneous broadening is demonstrated by so-called hole burning 1° in which a microwave transition, say, the IDI - IEI, is irradiated with constant power at a precisely defined, fixed frequency within the ODMR line, whereas the transition is simultaneously swept with a second modulated source of variable frequency. At the first frequency the ODMR will show a dip, because then the sublevel populations are already more or less equalized and additional power has comparatively little effect. An example is shown in Fig. 3C. The width of the " h o l e " is twice the homogeneous line width or equal to the frequency interval corresponding to the field intensity of the "burning" microwaves IB11, whichever is largest. (The latter situation is undesirable and should be avoided.) When a hole is found in the burned (e.g., IDI - IEI) transition, the IDI + IEI transition does not show a hole but is somewhat decreased in intensity. This is because the particular combination of [DI and IEI values that correspond to the precisely defined frequency of the hole in the IDI [El line sum to values that spread across the whole IDI + [El line. The ODMR line width is not very sensitive to the bandwidth of optical excitation. Usually a broad optical band corresponds to a broad ODMR 10 M. Leung and M. A. EI-Sayed, Chem. Phys, Lett. 16, 54 (1972).

306

~

182.8 193.6 209.1

MH£

[10]

PROBES OF METAL ION ENVIRONMENTS

467 MHz

.....

2

467

MHz

FiG. 3. (A) Double resonance (EEDOR) spectrum of the triplet state of Rhodobacter sphaeroides employing fluorescence detection. The first microwave field was set at 467 MHz, resonant with the IDI - [EI transition, while the second microwave field was scanned from 183 to 210 MHz. (B) IDI - IEI resonance of same, where crosses denote the computed Gaussian normalized to the experimental curve. The slight deviation to lower frequency is due to the ma~gnetic field of the earth. (C) Hole-burning experiment on the 467 MHz resonance of (B), One microwave field was set at 467 MHz while the frequency of a second field was slowly swept through the resonance. [From A. J. Hoff, Biochim. Biophys. Acta 440, 765 (1976).]

line because similar environmental interactions are at work. l 1-13H o w e v e r , selecting a narrow bandwidth of optical excitation (e.g., by using a laser) does not produce significant narrowing of the ODMR line, 14because generally there is little correlation (the effect of a similar perturbation in electronic m o l e c u l e - s o l v e n t interactions on the triplet wave function is different from that on the singlet wave function). In contrast to this lack of correlation, there are slight correlated shifts o f the ZFS values when the wavelength of detection is scanned across the p h o s p h o r e s c e n c e band. 15-17 When this correlation shows a discontinuity, it is indicative of the presence of more than one triplet site (e.g., tryptophans in a protein). In addition to the hole-burning double resonance experiment performed on one O D M R transition, one may carry out a double resonance experiment at two different O D M R frequencies, which is known as elect r o n - e l e c t r o n double resonance (EEDOR). Saturating one, say, the u v, transition equalizes the N ,"v and No"° populations. The population difference N ,"v - N °, is then increased or decreased compared to N O - N ° (or II j. p. Lemaistre and A. H. Zewail, Chem. Phys. Lett. 68, 296 and 302 (1979). n j. van Egmond, B. E. Kohler, and I. Y. Chan, Chem. Phys. Lett. 34, 423 (1975). 13 A. L. Kwiram, in "Triplet State ODMR Spectroscopy" (R. H. Clarke, ed.), p. 427, Wiley (Interscience), New York, 1982. i4 R. L, Williamson and A. L. Kwiram, J. Phys. Chem. 83, 3393 (1979). 15 j. U. von Schlitz, J. Zuclich, and A. H. Maki, J. Am. Chem. Soc. 96, 714 (1974). 16 A. L. Kwiram, J. B. A. Ross, and D. A. Deranleau, Chem. Phys. Lett. 54, 506 (1978). 17 R. L. Williamson and A. L. Kwiram, J. Chem. Phys. 88, 6092 (1988).

[10]

ODMR OF TRIPLETSTATES

307

vice versa for No), with a concomitant change in the intensity of the u -~ w or v ~ w transition. This is often useful to enhance the ODMR line corresponding to two sublevels whose equilibrium populations in the absence of microwaves are nearly equal. An example is shown in Fig. 3A. In addition E E D O R allows one to discriminate between ODMR resonances belonging to the same triplet state (the same site) when in a single resonance experiment more than three ODMR lines are recorded. The latter double resonance experiment is usually carried out by irradiating one transition with amplitude-modulated microwaves at fixed frequency and measuring with CW microwaves the other transitions while applying lockin detection at the modulated frequency. Only those transitions belonging to the same triplet as the first transition will then show up. 2.3. O p t i c a l M i c r o w a v e D o u b l e R e s o n a n c e

Once the ODMR lines of a triplet have been determined, the resonance frequencies are known precisely, and one can investigate the dependence of the intensity of a particular resonance line on the probing wavelength. Thus, one irradiates the sample with (amplitude-modulated) ~8 resonant microwaves of sufficient, preferably saturating intensity, and monitors the (lock-in detected) photodetector output as a function of the probe beam wavelength. The resulting spectra may be called microwave-induced phosphorescence, fluorescence, or absorbance spectra (abbreviated as MIP, MIF, and MIA spectra, respectively). For one particular triplet state, the shape of the spectra does not depend on the selection of the resonance frequency (i.e., v1,2 or v3). Obviously, if more than one triplet state is present, the microwave-induced spectra provide another means to sort out which resonances belong to the same triplet state. Conversely, microwave-induced spectroscopy allows the unraveling of complex optical spectra. MIF spectra are useful for discriminating between various triplet states and for identifying the triplet-carrying molecule, as exemplified by the studies of Beck et al. 19,20on photoinduced triplet states in bacterial photosynthetic membranes. MIA spectra are a case apart, since the~, provide much more information than the MIP or MIF spectra. As discussed in the next section, they represent the difference of the singlet ground state, " n o r m a l , " absorbance spectrum and the spectrum for the system when a triplet state is present. T h e y have therefore been labeled triplet-minuses M. A. E1-Sayed, D. V. Owens, and D. S. Tinti, Chem. Phys. Lett. 6, 395 (1970). 19j. Beck, G. H. Kaiser, J. U. von Schi)tz, and H. C. Wolf, Biochim. Biophys. Acta 634, 165 (1981). 2oj. Beck, J. U. von Schfitz, and H. C. Wolf, Z. Naturforsch. C: Biosci. 38, 220 (1983).

308

PROBES OF METAL ION ENVIRONMENTS

microwaves off

microwaves

[10]

on

FIG. 4. Principle of absorbance-detected magnetic resonance. Filled circles denote relative equilibrium populations of the triplet sublevels, open circles that of the ground state. A saturating microwave field connecting two triplet sublevels (wavy arrow) leads to a new (here, higher) equilibrium value of the singlet ground state population, hence to a change in the absorbance. The same principle holds for fluorescence detection, whereas the phosphorescence is also enhanced by the microwave field. (From Ref. 2.)

singlet absorbance difference (T - S) spectra, rather than by the MIA acronym. 21 2.3.1. Triplet-Minus-Singlet Absorbance Difference Spectra. When a triplet state is present, the absorbance spectrum contains the following contributions: (1) the unperturbed singlet ground state absorbance spectrum (S, ~ So transitions) of all molecules that are not in the triplet state and that do not interact with the molecule that is in the triplet state; (2) the perturbed singlet ground state spectrum of those molecules in a molecular aggregate (comprising proteins) that are not in the triplet state but do interact with the triplet-carrying molecule (generally this interaction will be different when this particular molecule is in the triplet state from that when the molecule is in the singlet ground state); and (3) the absorbance spectrum of the triplet state itself, consisting of Tn ~-- TOtransitions. With square-wave, on-off amplitude-modulated microwaves, the MIA spectrum represents the difference in absorbance of the sample for microwaves on and microwaves off (Fig. 4). It can be shown 21that this difference is proportional to the difference in absorbane with and without the triplet state present. In other words, the MIA spectrum represents the difference of the absorbance of the sample with all molecules in the singlet ground state and that when all molecules of one particular type are excited into the triplet state whose ODMR resonance is being monitored. It is important to note that other triplet states with different values of IDI and IEI and consequently different ODMR resonance frequencies may be present without showing up in the MIA (T - S) spectrum. Their absorbance is not changed by the microwaves, and therefore their contribution to the absorbance cancels in the ADMR-monitored T - S difference spectrum. On the other hand, recording T - S spectra for different ADMR 21 H. J. den Blanken and A. J. Hoff, Biochim. Biophys. Acta 681, 365 (1982).

[10]

ODMR OF TRIPLETSTATES

309

resonance frequencies provides a means to discriminate the resonances belonging to one and the same triplet states, since in general contributions (2) and (3) will be different for different triplet states. The ADMR-monitored T - S spectrum has several features of interest. For noninteracting triplet states it provides a very accurate triplet absorbance spectrum, since that spectrum is given by adding the " n o r m a l " singlet ground state spectrum to the T - S spectrum. For interacting triplet states, for example, those present in a photosynthetic pigment-protein complex, it records these interactions very sensitively and thus provides a unique means to study pigment configuration.

2.3.2. Linear Dichroic Triplet-Minus-Singlet Absorbance Difference Spectroscopy. The microwave transitions between the u and v triplet sublevels are polarized along w -- u x v. This is analogous to an optical transition, whose transition dipole moment usually has a well-defined direction in the molecular frame. Often, the direction of the triplet magnetic resonance transition moments are not as well-known. In chlorophylls, for example, one may be reasonably certain that the z transition moment is perpendicular to the molecule and that the x and y transition moments lie in the plane of the macrocycle, but the precise direction in the plane of the latter was until recently not known. As will be shown below, linear dichroic (LD)-(T - S) spectroscopy provides a means to ascertain the directions of the magnetic transition moments. With this knowledge one may then derive from the LD-(T - S) spectra precise structural information on molecular aggregates. In optical spectroscopy, the transition probability for a transition with transition dipole p is proportional to [E[2lp[2 COS 2 /3, where /3 is the angle between p and the E electric vector of the (polarized) incident light. A similar relation holds for the magnetic microwave transitions between the triplet sublevels. Thus, for a microwave transition moment t~w and an angle /3 between tXr~w and the B1 magnetic vector of the (polarized) microwave field, we have a transition probability IBllZlt~mwl z cos 2/3. It follows that molecules oriented with /Zmwmore or less parallel to B 1 have a much higher transition probability than those oriented about perpendicular to B1. (Of course, for /3 = 90 °, the transition probability is exactly zero.) Hence, for random excitation to the triplet state, molecules oriented in an angular interval d/3 close to/3 = 0 ° will experience a much higher change in their relative triplet concentration on the application of (polarized) resonant microwaves than molecules in an interval d/3 close to /3 = 90 °. Consequently, the distribution of triplet states, which was isotropic before the application of the microwaves, becomes axially anisotropic with the axis parallel to B1 when resonant microwaves are switched on.

310

PROBES OF METAL ION ENVIRONMENTS

fl-----..~ tamp

"~),/

/

Lo/(,.~

]

i]mw :

/x' ,

.I. sample

[I0]

t..'r_M

chromator

I

,

FIG. 5. Schematic drawing of the LD-ADMR experiment. !11, Microwave field vector; prow, microwave transition moment; ~o, optical transition moment; x', y', z', laboratory frame; PEM, photoelastic modulator. Unpolarized light becomes elliptically polarized because of the anisotropic transmittance of the sample induced by the microwave field resonant with an ODMR transition. The ellipticity is analyzed by the PEM and the polarizer. [Adapted from E. J. Lous, Doctoral Dissertation, University of Leiden, The Netherlands (1988).]

The microwave-induced anisotropy in the triplet state distribution can be interrogated with a beam of polarized light. F o r example, let us assume that the optical transition m o m e n t p is parallel to/.tmw, that the microwaves decrease the triplet concentration, and that we interrogate at a wavelength where the singlet ground state has an absorption band and the triplet state does not absorb. Then, for light polarized parallel to BI we will measure a lower transmittance than for light polarized perpendicular to B l . (Along B~ there are fewer triplets, hence more singlet ground states, than perpendicular to B~.) Obviously, the difference in transmittance (which for small changes can be taken equal to the difference in absorbance AA 21 will depend on the angle a between p and Pmw" In the above example, the sign of AA = All - A L would be reversed if ot is not 0 ° as assumed, but 90 °. Going from a = 0 ° to ot = 90 °, at a given angle AA must b e c o m e zero. This is the magic angle ot = 54.7 ° [for which 3 cos 2 a - 1 = 0, see Eq. (35)]. Thus, from the magnitude of AA relative to the magnitude of A we should be able to directly derive ot (Fig. 5). It will be recognized that the above description of the microwaveinduced selection in the triplet state distribution is very similar to that of photoselection. We can therefore partake of the formalism derived for that technique (see, e.g., Ref. 22) to calculate the functional relationship between AA and a. In doing so, we must of course average over all positions of the molecules with respect to B1, assuming a random initial distribution. To simplify this averaging we further assume that the triplets 22 A. Vermeglio, J. Breton, G. Paillotin, and R. Cogdell, Biochim. Biophys. Acta 501, 514 (1978).

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ODMR OF TRIPLETSTATES

311

~I-""'----~R-LD-IT-S)~,-~,--,- " lb"

z'o" '

-0.2

' o<

'

#

:

" #

=

-0.~

FIG. 6. Dependence of the amplitude of the ADMR-monitored T - S and LD-(T - S) spectra and their ratio R as defined by Eq. (35) on the angle ~ between the optical and the microwave transition moment, a = 54.7 ° is the magic angle for which the microwaveinduced linear dichroism is zero (R = 0). [From E. J. Lous, Doctoral Dissertation, University of Leiden, The Netherlands (1988).]

are isotropically excited. This is not strictly true, as the (unpolarized) exciting light beam does not excite molecules oriented such that their optical transition moment for triplet excitation is parallel to the direction of propagation of the light. Nevertheless, because of energy transfer among differently oriented pigments before the excitation is trapped onto the triplet state, and because of scattering at sample cell walls and at impurities and cracks inside the sample, isotropic excitation proves to be a good approximation. Finally, we have employed unpolarized probe light and interrogate the difference All - A± after the light has passed the sample, using a photoelastic modulator and a polarizer. Taking all this into account, it can be shown that the ratio R of the intensity of the LD-(T - S) spectrum [which represents the difference All - A l = (T - S)II - (T - S)±] and the T - S spectrum is given by 23'24 R -

LD-(T-S)_3cos 2or- 1 T- S cos2a + 3

(35)

Equation (35) is plotted in Fig. 6, together with plots of the LD-(T - S) and T - S intensities versus a. It is seen that R is quite sensitive to a, 23 A. J. Hoff, in "Antennas and Reaction Centers of Photosynthetic Bacteria: Structure, Interaction and Dynamics" (M. E. Michel-Beyerle, ed.), p. 150. Springer-Verlag, Berlin, 1985. 24 H. J. den Blanken, R. F. Meiburg, and A. J. Hoff, Chem. Phys. Lett. 105, 336 (1984).

312

PROBES OF METAL ION ENVIRONMENTS

[10]

so that with p r o p e r calibration of the T - S and LD-(T - S) spectra, ot can be determined quite accurately. Of course, all this applies rigorously only for single absorbance bands. If bands with different directions of p vis-a-vis /Xmw overlap, R will have some intermediate value and one will have to simulate the complete LD-(T - S) and T - S spectra to obtain values for the various a angles. In photoselection, one customarily extrapolates to zero intensity of the exciting, selecting light beam. This is because the transition, and therefore photoconversion, probability is proportional to IEI 2 cos 2/3. E v e n for/3 close to 90 °, p h o t o c o n v e r s i o n will be appreciable if the field strength ]E I is high enough. In that case, almost all molecules will be photoconverted, regardless of their orientation, and selection is lost. In microwave selection, the relevant field strength is that of the B 1 field. Thus, to ensure p r o p e r selection one has to measure R as a function of ]B1] and extrapolate for [Bl] ~ 0. This is best done by taking the slope of a graph of the LD(T - S) versus the T - S intensity as a function of IBll. The shape of the L D - ( T - S) spectrum is not dependent on the intensity of B~, so that for the study o f the orientation d e p e n d e n c e of the influence of the triplet state on neighboring pigments, expressed as band shifts, bleachings, and appearing bands in the LD-(T - S) spectrum, it is best to work at comparatively high B~ amplitudes. The angle ~ in Eq. (35) refers to one particular/~mw, say, that corresponding to the x polarized y -~ z transition at frequency (ID] - ]El)/h. Tuning the microwaves to the (]D I + ]EI)/h frequency, that is, the x ~ z or y polarized transition, allows the recording of LD-(T - S) spectra and the determination of R for a different a, namely, the angle % between p and y. When p, x, and y lie in one plane, % = 90 ° - ax, since the triplet spin axes x, y, and z span a Cartesian coordinate frame. If p, x, and y are not coplanar, the two measurements uniquely define the orientation of p in the x,y,z coordinate frame. This is a great advantage o v e r the ordinary photoselection experiment, where just one angle between two transition moments is determined which leaves one with a conical ambiguity. Note that it suffices to record LD-(T - S) spectra for just two of the three possible A D M R transition frequencies. Because the orientations o f all p population probabilities are determined in one and the same coordinate frame, their mutual angular d e p e n d e n c e immediately follows.

2.4. Instrumentation The minimal requirements for an ODMR experiment are a source of light, one o f microwaves, a cryostat, and a detector. An up-to-date discussion of these c o m p o n e n t s can be found in Ref. 1. H e r e we limit ourselves

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ODMR OF TRIPLETSTATES

313

to a few notes and a discussion of special arrangements for ADMR and LD-ADMR. The best excitation light source in the visible or infrared region is an incandescent lamp (e.g., a tungsten-iodine projection lamp) powered by a current-stabilized dc power supply. Mercury or xenon lamps are more intense, certainly in the UV region, but suffer from instabilities in the intensity I, often to a level of AI/I 10 -3 or worse. The same holds for laser excitation. Adequate filtering should be provided. 25 When the excitation beam is broad-banded, it may also serve as probe beam at various wavelengths. Microwaves are supplied preferably by a sweep unit (now available with a range of 0.1-27 GHz) with provisions to scan the frequency over the desired range and to amplitude- or frequency-modulate the output. Usually, the output (20-40 mW) is enough for regular ODMR at liquid helium temperatures without amplification. For measuring the ODMR lines the microwaves are fed into a broadband resonator (e.g., a helix) that can admit the frequency range of interest. When very broad ODMR lines need to be scanned, attention should be paid to frequency-dependent reflections of the microwaves (owing primarily to mismatch between helix and conductor), which may considerably affect the BI field intensity inside the helix and thus the ODMR intensity. Also, without leveling provisions, the sweeper output may considerably depend on frequency, even in a relatively narrow range. When in doubt that certain features are artificial, another helix of different physical characteristics should be used for comparison. If one is interested in the (probe) wavelength dependence in microwave-induced spectroscopy, the microwave frequency is set exactly at resonance and a narrow-band cavity may be used. We have used a loop-gap resonator 26 with slots for optical access (Fig. 7). The cryostat is preferably a double-walled helium bath cryostat equipped with at least two windows. Helium boils at 4.2 K; scattering by the bubbles then prohibits optical detection. By lowering the pressure above the helium bath the temperature of the helium can be lowered under 2.1 K, the so-called h point, below which helium is superfluid and bubbles disappear completely. Thus, adequate pumping facilities should be provided. For FDMR at 4.2 K a simple He vessel may be used in which a light pipe with sample compartment at the end is lowered. 27 Such a light pipe may also be used in a bath cryostat. 28 =

25 M. Kasha, J. Opt. Soc. Am. 38, 929 (1948). 26 W. H. Hardy and L. A. Whitehead, Rev. Sci. lnstrum. 57, 213 (1981). 27 S. J. van der Bent, P. A. de Jager, and T. J. Schaafsma, Rev. Sci. Instrum. 47, 117 (1976). 28 I. Y. Chan, in "Triplet State ODMR Spectroscopy" (R. H. Clarke, ed.), p. 1. Wiley (Interscience), New York, 1982.

314

[10]

PROBES OF METAL ION ENVIRONMENTS

J /

tRt" n H °1 o

o

jJ Coax line

o

o

Light palh

Light path FIG. 7. Loop-gap resonator used for LD-ADMR and pulsed-ADMR experiments. The cavity resonance frequency is tuned by the screw governing the width of the gap. The B~ field is along the cylinder axis of the cavity. Dimensions: inner diameter, 20 mm; height, 60 mm. (From Ref. 24.)

The optical detector depends on the optical mode. For phosphorescence and fluorescence a photomultiplier is used. For ADMR, one may employ a strong probe beam (in fact, the excitation beam may serve as such), providing a sufficiently high number of transmitted quanta for allowing the use o f a photodiode (up to 1100 nm, silicon; 1100 nm to 2/zm, germanium). To observe the resonance by fluorescence or phosphorescence, the wavelength of detection should be separated from the wavelength of excitation by adequate filtering. For ADMR, either a filter or, when the probe wavelength dependence is monitored, a monochromator is used. The sensitivity of the ODMR spectrometer for slow-passage experiments can be considerably enhanced by modulating the amplitude of the microwaves and applying frequency-selective amplification of the photodetector signal combined with lock-in detection. Noise is then reduced to that corresponding with the passband of the amplifier-lock-in detector combination, leading to an increase in the signal-to-noise ratio of several orders of magnitude. For example, in our ADMR spectrometer a AA/A ratio of better than 5 x 10 -7 is routinely achieved. Obviously, the modulating frequency has to be less than the slowest sublevel decay rate. For slowly decaying triplets (k < 10 sec- i), one may be better off by scanning the line with unmodulated microwaves and signal averaging. For kinetic measurements broad-banded amplification of the detector signal and signal averaging are used.

[10]

O D M R OF TRIPLET STATES

obsorbance spectrum ~ (T-S) spectrum

1 2~ ~

- i'7_I L

!IAI

totat tronsmitted

315

light

I 10(k-in l._ amplifier I-

IT-S)

t LD-IT-S)

spectrum

_AA[ x I

I lock-in ~ amplifier

LD-(T-S)

F

~

fm= 31S Hz

=

rences

--

circulator f:t00 kHz I microwo. I I I,icro~ove I I sweep ~ frequency I oscillator J I counter I

tungsten ,0°,he

,,°pbr°g.

IR filter / ÷ shutter HzO I

lamp zso w

recovery

Nz.tiq.kl

}I

J~ ~

-

~,vGcuum

I ~1 -~ I ~ -_

~'

...sample cuvefte~ i ..-split..-split-ring cavity ..i . . . .

~

r -- -

1.1 L1,2,3=tenses

ilion

L3 ~-window bath cr ostQt

"~ t

*preamplifier Jpdarizer tphotoetastic modulQtor (PEM)

/

F[o. 8. Block scheme of the A D M R and L D - A D MR setup. [From E. J. Lous, Doctoral

Dissertation, University of Leiden, The Netherlands (1988).] Finally, as in all modern spectrometers, the instrument is interfaced to a small, dedicated computer that handles monochromator set.ti;ng and data collection and storage. The computer also carries out simple operations such as taking the AI/I ratio (which is important when recording the dependence of the ADMR signal on the probe wavelength). A schematic diagram of our present ADMR setup is shown in Fig. 8. With small modifications it can be used for PDMR and FDMR as well.

2.4.1. Instrumentation for Linear Dichroic Triplet-Minus-Singlet Absorbance Difference Spectroscopy. The instrumentation for LD-(T - S) spectroscopy is quite similar to that for isotropic T - S spectroscopy, and we can refer to Fig. 8 for a description. First of all we need polarized microwaves; therefore, a simple helix is not suitable. We use a split-ring

316

PROBES OF METAL ION ENVIRONMENTS

[10]

or loop-gap cavity as described by Hardy and Whitehead. 26 This design has the advantage that for the rather long wavelength of the microwaves (between 50 and 100 cm) one still has a cavity of manageable dimensions, which fits easily into a four-window liquid helium cryostat. The B~ field is polarized along the vertical and is therefore perpendicular to the horizontal light beam. The probe light is unpolarized. The B 1 field induces an ellipticity (-= Tii - T~) in the transmitted light T, which is detected via a photoelastic modulator (PEM) after the sample followed by a polarizer. The PEM rotates the ellipse spanned by the unequally transmitted light vectors Ell and E± (with respect to BI) by 180° at a frequency of 50 kHz. The analyzer converts this polarization modulation to an amplitude modulation at I00 kHz that is proportional to TII - T± All - A± (T, transmission: A, absorbance) for small differences. The microwaves are modulated at low frequency (say, 315 Hz), as in T - S spectroscopy, so that the light intensity falling on the photodiode is doubly modulated at I00 kHz and 315 Hz. Demodulation at 315 Hz combined with suitable electronic filtering gives the normal T - S signal. Double demodulation at 100 kHz and 315 Hz gives the (T - S)II - (T - S)± LD-(T - S) difference signal. This procedure is illustrated in Fig. 9. The signal-to-noise ratio is enhanced by inserting selective amplifiers in both modulation channels. Scanning the monochromator yields simultaneously the T - S and the LD-(T - S) spectra. As before, the signals are divided by the intensity I to correct for changes in lamp output, monochromator sensitivity, etc., as a function of wavelength. To evaluate correctly the ratio R [Eq. (35)] we must mutually calibrate the T - S and LD-(T - S) signals. This calibration includes amplification factors, lock-in sensitivities, etc. It is best done by simulating the transmitted modulated light by a modulated light-emitting diode (LED). The amplitude of the 100 kHz modulation at specific instrument settings can then be accurately compared with that of the 315 Hz modulation. Care must be taken to avoid as much as possible ellipticities induced by extraneous sources, such as the lamp, cryostat windows, sample cell, etc. In our setup these extraneous ellipticities amounted to less than I% of the LD(T - S) signal. 3. Applications of Optically Detected Magnetic Resonance to Protein Research The principal results of an ODMR experiment are (1) proof of the photoproduction of one or more triplet states, (2) determination of highly accurate values of the ZFS parameters, (3) determination of the individual sublevel molecular decay rates, (4) determination of the sublevel populata

[10]

ODMR OF TRIPLETSTATES I OC sign~, photodiode

AC coupled ond I~nd-po, ss filfered signals

"fro

-fro

O' ,

o fm

2r~x

signo[ pho('odiode

rlTIITTTTn

317

; '~

. ' % , ~

0 fm

-fit

*fl

i

0

A(: coupLed sign(;[ a.¢= (T-S)= -AIT_ $ =-c

2fpEl,I

frequency

0 fm

A{6AI= LD-(T-S)= (/D.,_.)T - (6A,_.)S: = - ((A |l_j.) T -(All_j I $ = - ( a - bl

m1"mTm

;

;

I

If(n-b)

L~.bI~1% •

N

:o '--

~o %"

:o '

t,,,;

~o

'

on

G

z

'

'

'

flare

FIG. 9. Double modulation scheme used in L D - A D M R . (Top) Frequency spectrum of

the signal and its side bands before and after ac coupling and filtering. (Bottom) The photodiode signal is modulated at the frequency of the photoelastic modulator, fPEM = 100 kHz, and that of the microwave field modulation,fro ~ 350 Hz. Q and Q', Q factors of the electronic filters, casu quo, selective amplifiers; AA, microwave-induced change in absorbance; AA ~ A/, the change in transmittance (here negative). Filtering at fm and low-frequency demodulation yield the T - S spectrum. Filtering at 2fPEu --+fro, demodulation at 2frEu, and subsequent demodulation at fro yield the LD-(T - S) spectrum. [From E. J. L o u s , Doctoral Dissertation, University of Leiden, The Netherlands 0988).]

ing probabilities, (5) information on environmental interactions through line shape analysis and hole burning, and (6) spectral information via optical-microwave double resonance experiments. The zero-field splitting parameters and the sublevel populating probabilities and decay rat~s are fingerprints of molecular structure. They are also sensitive to environmental effects, which in addition to line shape analysis and hole burning allows one to use the triplet state as a probe of secondary and tertiary structure of proteins and of pigment environments in photosynthetic structures. Finally MIP, MIF, and especially MIA (T - S) spectra give a wealth of information on the interactions between pigments and between the pigment and its environment. In addition, with LD-(T - S) spectroscopy detailed information on the orientation of the triplet spin axes and on the relative angles of singlet transition moments may be derived. In this section some applications of ODMR for proteins are discussed, with emphasis on the more recent literature. For earlier literature, see Refs. 1, 16, and 29. 29 A. H. Maki, in "Triplet State ODMR Spectroscopy" (R. H. Clarke, ed.), p. 479. Wiley (Interscience), New York, 1982.

318

PROBES OF METAL ION ENVIRONMENTS

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3.1. Optically Detected Magnetic Resonance of Amino Acid and Cofactor Triplet States All proteins contain naturally occurring triplet probes, namely, their aromatic amino acids: phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). Histidine does not show an observable triplet state, whereas Phe is difficult to excite and it loses the excitation easily by energy transfer. Therefore, in practically all applications of ODMR in protein research, mostly from the groups of Kwiram and Maki (University of Washington, Seattle, and University of California at Davis, respectively), attention has focused on the study of the triplet states of tryptophans and tyrosines in vitro and in various proteins, using phosphorescence for the detection of the magnetic resonance. Often, in proteins that contain several Trp and/ or Tyr residues, energy transfer occurs to the aromatic residue with lowest energy, so that only one Trp or Tyr is observed. In some cases, however, the individual aromatic residues could be distinguished, for example, Trp and Tyr in azurin, 3° Trp in bovine serum albumin 3~ and in lysozyme, 32 and Tyr in triply point-mutated lysozyme of the bacteriophage T4, which contains no Trp. 33 Aromatic residues close to active sites of enzymes are expected to be sensitive to substrate or dye binding. Triplet-singlet energy transfer may occur, for example, from Trp to proflavin bound to chymotrypsin, 34which allowed detection of ODMR resonances of Trp via the delayed fluorescence of proflavin, or triplet-triplet transfer may occur between substrate or dye and aromatic residue. Also, signals from coenzymes, such as NAD ÷ and NADH, are readily observed by ODMR and give information on the conformation of the cofactor in the enzyme. 35 In addition to the ku values and the values of D and E, the ODMR line width is an important probe of protein structure. Generally, the lines are broad when the triplet is on a solvent-exposed residue, as various conformers are then frozen in on cooling. 36'37The ODMR line width then reflects the range of conformational fluctuations at physiological temperatures, but now they are not averaged, as in experiments at ambient temper30 K. Ugurbil, A. H. Maki, and R. Bersohn, Biochemistry 16, 901 (1977). 31 S. Y. Mao and A. H. Maki, Biochemistry 26, 3106 (1987). 32 R. L. Williamson and A. L. Kwiram, Biochem. Biophys. Res. Commun. 125, 974 (1984). 33 S. Gosh, L.-H. Zang, and A. H. Maki, Biochemistry J. Chem. Phys. 88, 2769 (1988). 34 A. H. Maki and T.-T. Co, Biochemistry 15, 1229 (1976). 35 j. B. A. Ross, K. W. Rousslang, A. G. Motten, and A. L. Kwiram, Biochemistry 18, 1808 (1979). 36 M. V. Hershberger, A. H. Maki, and W. C. Galley, Biochemistry 19, 2204 (1980). 37 K. L. Bell and H. C. Brenner, Biochemistry 21, 3735 (1982).

[10]

ODMR OF TRIPLETSTATES

319

atures: It is, as it were, a freeze-frame snapshot rather than an uninterpretable blur) 3 Finally, the MIP spectrum can be probed as a function of microwave frequency within the site-broadened ODMR line; this provides some insight in the energies of the conformers. 3.1.1. Heavy Atom Effects. The triplet yield can be appreciably enhanced by complexing a closed shell heavy metal component such as Ag + or methylmercury(II) (CHaHg +) to aromatic residues) ,13,37,38 Because of the large nuclear mass (Z number) of, for example, mercury, spin-orbit interaction and therefore the intersystem crossing efficiency is dramatically enhanced. Orbital overlap between the aromatic residue or base and the metal compound provides the required interaction, which is spinsublevel selective. 39'4° Subtle changes in the binding of the heavy metal component, for example, induced by protein conformational changes, may give rise to large changes in the populating probabilities and the decay rates of the individual triplet sublevels. These changes often interact in parallel, making slow-passage ODMR less sensitive to the effect than rapid-passage ODMR, where the frequency is swept fast across the resonance. 3.1.2. Energy Transfer. Energy transfer processes can be grouped into two classes: dipole-dipole-induced transitions (F6rster mechanism) and exchange-mediated processes. The former are long-range interactions (I/R 3 with R being the donor-acceptor distance) and depend on the spectral overlap of emission and absorbance spectra, whereas the latter are short-range interactions [a exp(-fiR), with fl -~ 1.7 ~.-l] and depend on orbital overlap of the singlet and triplet wave functions. Singlet-triplet [S1 -~ S0(D), To ~- So (A)] and triplet-triplet [To -~ So (D), To "--- So (A)] processes are spin-forbidden. Consequently, the molar extinction coefficient of the acceptor absorbance spectrum is low and the F6rster spectral overlap integral very small. Thus, these processes occur mainly at short distances where the exchange (Dexter) mechanism predominates, which is independent of spectral overlap. Triplet-singlet transfer is equally spin-forbidden, but if the quantum efficiency of phosphorescence is high enough, spectral overlap is sufficient to make the time-integrated probability of transfer comparable to spin-allowed singlet-singlet transfer. Triplet-singlet To --~ So, S~ ~-- So processes may give rise to delayed fluorescence (luminescence) of the acceptor. Because the lifetime of the donor triplet state can be manipulated by resonant microwaves, ODMR transi38 L.-H. Zang, S. Gosh, and A. H. Maki, Biochemistry 28, 2245 (1989). 39 S. Gosh, M. Petrin, A. H. Maki, and L. R. Sousa, J. Chem. Phys. 87, 4315 (1987). 4o S. Gosh, M. Petrin, A. H. Maki, and L. R. Sousa, J. Chem. Phys. 88, 2913 (1988).

320

PROBES OF METAL ION ENVIRONMENTS

[10]

tions within the donor can then be measured by monitoring the acceptor luminescence intensity. An application of this method for horse liver alcohol dehydrogenase has been published. 6 Triplet-triplet transfer has been studied in a benzophenone (D)-naphthalene (A) system in dodecyl sulfate m i c e l l e s . 41-43 It occurs between different Trp residues (126, 138, and 158) in wild-type bacteriophage T4 lysozyme and can be monitored by making use of the slight differences in phosphorescence peak wavelength and ZFS parameters of the three Trp residues that were found in work on mutant T 4 lysozyme in which Trp residues were displaced by Try. 44 The results were compared with the crystal structure of lysozyme.

3.2. Optically Detected Magnetic Resonance in Photosynthesis The use of ODMR in photosynthesis research has proved to be particularly fruitful. By far the most important triplet state in photosynthetic membranes is that generated on the primary electron donor by radical recombination under conditions that forward electron transport is blocked by the prereduction of one of the acceptors (or by its physical deletion): DA1A f

hv

) D*AIA f

<3 psec

) D + A 1 A f --> 3DAlAf

This reaction takes place in the so-called reaction center (RC), a specialized pigment-protein complex. The structure of two bacterial reaction centers has been elucidated in atomic detail by X-ray crystallography. 45-48 D is a bacteriochlorophyll (BChl) dimer, A~ is a bacteriopheophytin (q~A), and A2 is a quinone, QA- There are also another q~ molecule, q~B, and two so-called accessory BChls (BB and BA) whose function is as yet unclear. The pigments subscripted with A and those subscripted with B are arranged in two chains that show C2 symmetry with D on the symmetry axis. Because A 2 is prereduced and cannot normally accept two electrons, 41 S. Gosh, M. Petrin, and A. H. Maki, J. Phys. Chem. 90, 1643 (1986). 42 S. Gosh, M. Petrin, and A. H. Maki, J. Phys. Chem. 90, 5206 (1986). 43 M. Petrin, A. H. Maki, and S. Gosh, Chem. Phys. Lett. 128, 425 (1986). 44 S. Gosh, L.-H. Zang, and A. H. Maki, J. Chem. Phys. 88, 2769 (1988). 45 j. Deisenhofer, O. Epp, K. Miki, R. Huber, and H. Michel, Nature (London) 318, 618 (1985). 46 C.-H. Chang, D. Tiede, J. Tang, U. Smith, J. Norris, and M. Schiffer, FEBS Lett. 205~ 82 (1986). 47 j. p. Allen, G. Feher, T. O. Yeates, H. Komiya, and D. C. Rees, Proc. Natl. Acad. Sci. U.S.A. 114, 5730 and 6162 (1987). 48 T. O. Yeates, H. Komiya, A. Chirino, D. C. Rees, J. P. Allen, and G. Feher, Proc. Natl. Acad. Sci. U.S.A. 85, 7993 (1988).

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the radical pair D+Ai - recombines in about 20 to 50 nsec to either the singlet excited or ground state of D, or with almost 100% yield at cryogenic temperatures to its triplet state, 3D.49 3.2.1. Fluorescence-Detected Magnetic Resonance Spectroscopy. FDMR experiments on a photosynthetic bacterium, first carried out by Clarke et al., 5° have yielded accurate values of IDI and [E[ of the reaction center triplet and of triplets on plant antenna pigments and (bacterio)chlorophyll precursors. Small, but well-observable differences in the ZFS parameters of the reaction center triplet, for example, showed that the primary electron donor was subtly different in closely related species. 51 The earlier work up to 1984 on plant photosynthetic material and on bacterial photosystems has been reviewed 1'2'9'5z and is not discussed here. FDMR measurements on light-harvesting pigments of antenna complexes ofRhodobacter (Rb.) capsulatus Ala+pho - ,53Rb. capsulatus Ala ÷ , and Rb. sphaeroides R26.154 yielded triplet states with distinctly different ZFS parameters, suggested to belong to the two BChl molecules present per subunit antenna protein. FDMR measurements of light-harvesting complexes from barley yielded several ODMR signals of positive and negative sign associated with chlorophylls emitting at different wavelengths. 55'56 The ZFS parameters were compared with model Chl a complexes; it was suggested that the positive FDMR signals were associated with (Chl a" HzO)z dimers and hexacoordinate Chl a of the Chl a-2 pyridine type, whereas the negative FDMR signal resembled that of Chl a . 2H20 complexes. Fluorescence of isolated bacterial RCs is weak, and FDMR experiments are difficult to carry out. Den Blanken et al. 57 w e r e nevertheless 49 p. L. Dutton, J. S. Leigh, and M. Seibert, Biochem. Biophys. Res. Cornmun. 46, 406 (1972). 5o R. H. Clarke, R. E. Connors, J. R. Norris, and M. C. Thurnauer, J. Am. Chem. Soc. 97, 7178 (1975). 5~ A. J. Hoff, Biochim. Biophys. Acta 440, 765 (1976). 52 T. J. Schaafsma, in "Triplet State ODMR Spectroscopy" (R. H. Clarke, ed.), p. 292. Wiley (Interscience), New York, 1982. 53 j. Beck, J. U. von SchOtz, and H. C. Wolf, Chem. Phys. Lett. 94, 147 (1983). 54 A. Angerhofer, J. U. von Schiitz, and H. C. Wolf, Z. Naturforsch. C: Biosci. 411, 379 (1985). 55 G. F. W. Searle, R. B. M. Koehorst, T. J. Schaafsma, B. L. Moiler, and D. yon Wettstein, Carlsberg Res. Commun. 46, 183 (1981). 56 T. J. Schaafsma, G. F. W. Searle, and R. B. M. Koehorst, J. Mol. Struct. 79, 461 (1982). 57 H. J. den Blanken, G. P. van der Zwet, and A. J. Hoff, Biochim. Biophys. Acta 681, 375 (1982).

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able to discern in RCs of Rb. sphaeroides R26 two MIF bands, one at 905 and one at 935 nm. The [D] and IEI values corresponding to the 905 nm band are those of the RC triplet, 3D. The sign of the 905 nm band, however, is negative, which is unexpected if the fluorescence originates from D. Reversal of sign occurs when the fluorescing pigment is not D itself, but an associated pigment from which singlet excitation may travel to D by singlet energy transfer, z'9 The 905 nm fluorescence may stem from a weak concentration of antenna bacteriochlorophylls (less than a few percent of D) that are strongly bound to the RC protein. Formation of 3D closes the reaction center and removes it as a strong quencher, which may strongly enhance the fluorescence of such an antenna pigment and result in a detectable MIF signal when the concentration of 3D is modulated by resonant microwaves. The sign of the 935 nm fluorescence indicates that it originates from D, but the [D[ and [E[ values are not those of 3D (as measured with ADMR or high-field EPR). It was concluded that this fluorescence stems from a minority of reaction centers with a slightly different configuration, perhaps caused by the isolation procedure. Hence, caution should be exerted in analyzing experiments in which the fluorescence of isolated reaction centers is monitored as a probe of the primary photoprocesses. FDMR experiments on isolated reaction centers of Rhodopseudomonas (Rps.) viridis revealed a similar sign reversal of the ODMR transition as a function of monitoring wavelength as observed for Rb. sphaeroides R-26. 58 The sign of the fluorescence-detected ODMR signal of whole cells is positive, 57-59 which poses a problem, as the usual interpretation of such a sign reversal predicts a negative sign of the ADMR signal of reaction centers, contrary to observation. 6° The discrepancy between the signs of the antenna fluorescence- and absorbance-monitored ODMR signals in Rps. viridis was addressed by den Blanken et al. 6° and tentatively attributed to energy transfer from a vibrationally excited SI state of the antenna pigments to the reaction center.

3.2.2. Absorbance-Detected Magnetic Resonance Spectroscopy of Reaction Centers. ADMR is a variant of ODMR that, after a first rather unpromising experiment on a pigment solution, 6~ has been developed in 58 A. Angerhofer, J. U. von Schiatz, and H. C. Wolf, Z. Naturforsch. C: Biosci. 39, 1085 (1984). 59 F. G. van Wijk and T. J. Schaafsma, in "Advances in Photosynthesis Research" (C. Sybesma, ed.), Vol. 2, p. 173. Nijhoff/Dr. W. Junk, The Hague, 1984. 6o H. J. den Blanken, A. P. J. M. Jongenelis, and A. J. Hoff, Biochim. Biophys. Acta 725, 472 (1983). 61 R. H. Clarke and R. E. Connors, Chem. Phys. Lett. 33, 365 (1975).

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Leiden to a high degree of sophistication (reviewed in Refs. 23 and 62). The great advantage of the ADMR variant over fluorescence or phosphorescence detection is that it can always be applied, regardless of quantum yields of emission, provided the lifetime of the triplet state is not too short (this holds for all CW-ODMR techniques) and a sufficient optical density can be attained (OD ~ 0.7 gives maximal signal). Both conditions hold for the photosynthetic triplets, and in the first application of ADMR to isolated bacterial reaction centers 63 it was shown that the sensitivity of ADMR was several orders higher than that of FDMR on the same material. The high sensitivity of the ADMR method opened the way to studies of numerous isolated reaction centers, pigment solutions, etc., both of bacterial and plant origin. Accurate values of IDI, IEI, and k, were determined, 51'63-74 as well as approximate values of the populating probabilities.64'74 The values of IDI for the primary donor triplet in bacterial reaction centers are lower by 20-30% compared to that of the isolated bacteriochlorophyll pigment. 65 From EPR data on the reaction center triplet state in single crystals 75 and from ADMR spectroscopy 76 it was concluded that, at least in Rps. viridis, the triplet state is largely localized on one of the dimer BChls, namely, DA. The difference between the in oivo and in oitro value of [D[ was ascribed to admixture of charge transfer (CT) states of the form 3[BChI÷ • BChl-] to the monomeric 3DA state. 75 The temperature 62 A, J. Hoff, in "Photosynthesis III: Photosynthetic Membranes and Light Harvesting Systems" (L. A. Staehelin and C. J. Arntzen, eds.L Vol. 19, Encyclopedia of Plant Physiology, New Series, p. 400. Springer-Verlag, Berlin, 1986. 63 H. J. den Blanken, G. P. van der Zwet, and A. J. Hoff, Chem. Phys. Lett. 85, 335 (1982). 64 A. J. Hoff and H. Gorter de Vries, Biochim. Biophys. Acta 503, 94 (1978). 65 H. J. den Blanken and A. J. Hoff, Chem. Phys. Left. 96, 343 (1983). 66 H. J. den Blanken, H. Vasmel, A. P. J. M. Jongenelis, A. J. Hoff, and J. Amesz, FEBS Lett. 161, 185 (1983). 67 H. J. den Blanken and A. J. Hoff, Biochim. Biophys. Acta 724, 52 (1983). 68 H. J. den Blanken, A. J. Hoff, A. J. P. M. Jongenelis, and B. A. Diner, FEBS Lett. 157, 21 (1983), 69 H. Vasmel, H. J. den Blanken, J. A. Dijkman, A. J. Hoff, and J. Amesz, Biochim. Biophys. Acta 767, 200 (1984). 7o R. H. Clarke and R. H. Hofeldt, J. Chem. Phys. 61, 4582 (1974). 71 n . J. den Blanken and A. J. Hoff, Chem. Phys. Lett. 98, 255 (1983). 72 A. J. Hoff, Govindjee, and J. C. Romijn, FEBS Lett. 73, 191 (1977). 73 j. Ullrich, A. Angerhofer, J. U. yon Schfitz, and H. C. Wolf, Chem. Phys. Lett. 140, 416 (1987). 74 A. Angerhofer, R. Spee, J. Ullrich, J. U. von Schiitz, and H. C. Wolf, Appl. Magn. Reson. 2, 203 (1991). 75 j. R. Norris, C. P. Lin, and D. E. Budil, J. Chem. Soc., Faraday Trans. 1 83, 13 (1987). 76 E. J. Lous and A. J. Hoff, Proc. Natl. Acad. Sci. U.S.A. 84, 6147 (1987).

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[10]

dependence of the zero-field splitting parameters was investigated with A D M R 73'77 and yielded further proof that it cannot be explained as a thermally activated population of one of the accessory BChls, in accordance with temperature-dependent EPR measurements. 78 ADMR spectroscopy of triplets of carotenoids present in reaction center and antenna complexes of various photosynthetic bacteria yielded accurate IDI and IEI v a l u e s . 79'8° It was even possible to distinguish different naturally occurring carotenoids in the same antenna preparation. 74 In plant reaction centers the values of IDI, fEI, and k, are practically the same as those of monomeric chlorophyll in solution. This can be explained by one of three possibilities: (1) the primary donor of both plant photosystems I and II is a monomeric Chl a molecule, (2) the primary donor is a plane-parallel sandwich (Chl a) 2 dimer with a fully delocalized triplet state (strong exciton coupling), or (3) the primary donor is a dimer on which the triplet state is fully localized on one of the monomeric constituents and does not have CT admixture. In the latter case the term dimer in the sense of two interacting molecules obviously applies only to the singlet and possibly oxidized states of the primary donor. We discuss this further in Section 3.2.4.

3.2.3. Triplet-Minus-Singlet Absorbance Difference Spectroscopy o f Reaction Centers. ADMR was first used to record T - S spectra in 1982 by den Blanken et al. 2~It was immediately clear that this technique permits the recording of low-temperature T - S spectra far more accurately than was possible with conventional flash techniques. Typical examples are the T - S spectra ofRps, viridis (Fig. 10A,B, top) and the T - S spectra of photosystem I and II particles of plants (Fig. 11). The interpretation of these spectra is discussed in the next section. The T - S spectra of carotenoids in various bacterial reaction and antenna complexes were reported in Refs. 79-81, those of plant antenna complexes in Refs. 82 and 83.

3.2.4. Linear Dichroic Triplet-Minus-Singlet Absorbance Difference Spectroscopy. As explained in Section 2.3 LD-(T - S) spectroscopy is 77 V. Aust, A. Angerhofer, P. H. Parot, C. A. Violette, and H. A. Frank, Chem. Phys. Lett. 173, 439 (1990). 78 A. J. Holt and I. I. Proskuryakov, Chem. Phys. Lett. 115, 303 (1985). 79 j. Ullrich, R. Speer, J. Greis, J. U. von Sch~tz, H. C. Wolf, and R. J. Cogdell, Chem. Phys. Left. 155, 363 (1989). 8o V. Aust, A. Angerhofer, J, Ullrich, J. U. von Schiitz, H. C. Wolf, and R. J. Cogdell, Chem. Phys. Lett. 181, 213 (1991). 81 E. J. Lous and A. J. Hoff, Biochim. Biophys. Acta 974, 88 (1989). 82 R. van der Vos, D. Carbonera, and A. J. Hoff, Appl. Magn. Reson. 2, 179 (1991). 83 D. Carbonera, G. Giacometti, C. Agostini, A. Angerhofer, and V. Aust, Chem. Phys. Lett. 194, 275 (1992).

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very similar to that of photoselection, in which an oriented distribution of, for example, photooxidized primary donors is produced by exciting the immobilized unoriented sample with a beam of linearly polarized light of a wavelength corresponding to a particular absorption band, for example, that of D. It has the advantage that, in contrast to photoselection, the dichroic spectrum can be recorded with respect to two axes of reference that are perpendicular to each other. This reduces considerably the ambiguity of unraveling the orientation of the various transition moments (tm) in the T - S spectrum. Complex T - S spectra such as those of photosynthetic reaction centers, where many overlapping features are present such as appearing bands, bleachings, and band shifts to the red and to the blue, can only be interpreted with certainty if the corresponding LD-(T - S) spectra are available. Although a consistent interpretation is sometimes possible without a complete spectral simulation, 84 the full power of T - S and LD-(T - S) spectroscopy is realized when the experimental spectra can be compared with spectra simulated with exciton theory, which describes the interaction between pigment molecules brought about by the electrostatic dipole-dipole coupling between their electronic transition moments. For two identical molecules this interaction causes a shift and a splitting of the excited state, which is now composed of two levels with energies (apart from the shift) given by the original energy plus or minus the interaction energy. The theory is easily extended to n pigments, each pigment interacting differently with the other n - 1 pigments. 85-87For a known pigment configuration the absorption spectrum of the excitonically coupled spectrum is then readily obtained. Generally, the resulting bands are mixtures of the original uncoupled bands, with tm values that are vectorial combinations of the original tm values. Bacterial reaction centers. Exciton calculations based on the crystal structure of the RC of Rps. oiridis 45 yielded satisfactory simulations of its ADMR-monitored T - S and LD-(T - S) spectra 76'88'89 (Fig. 10). The spectral fits led to the conclusion that the triplet is localized on one of the BChl components of D, DA. The bleaching at 990 nm is due to the disappearance of the long-wavelength band of D that is shifted to the red because of excitonic coupling between the two BChls of D. Exciton A. J. Hoff, H. J. den Blanken, H. Vasmel, and R. F. Meiburg, Biochim. Biophys. Acta 806, 389 (1985). 85 A. S. Davidov, "Theory of Molecular Excitons." Plenum, New York, 1981. 86 M. Kasha, H. S. Rawls, and M. A. E1-Bayoumi, Pure Appl. Chem. U , 371 (1965). 87 R. M. Pearlstein, in "Photosynthesis" (Govindjee, ed.), Vol. 1, p. 293, Academic Press, New York, 1982. 88 E. W. Knapp, P. O. J. Scherer, and S. F. Fischer, Biochim. Biophys. Acta 852, 295 (1986). 89 p. O. J. Scherer and S. F. Fischer, Biochim. Biophys. Acta 891, 157 (1987).

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625

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Wavelength (nm) FIG. 11. T - S spectra at 1.2 K of reaction center particles of photosystem I (P700) and photosystem II (P680). The spectra were recorded at the IDI + IE[ transitions at the indicated frequencies and normalized on the major bleaching. (Courtesy Dr. R, van der Vos.)

coupling is much weaker in the triplet state of 3D, so that in a localized 3D state part of the BChl absorption is bleached and part shifts back to the "normal" wavelength of BChl absorption in a protein matrix, giving rise to the large positive band at about 830 nm. The two smaller features at the long- and short-wavelength side of the positive band are attributed to band shifts of the two accessory BChls induced by the change D 3D.21 The small positive band at 872 nm is a triplet-triplet absorption of 3D, whose t m is oriented along the NII-NIv axis in the BChl macrocycle.

FIG. 10. T - S spectra and LD-(T - S) spectra at 1.2 K of Rps. viridis for the two A D M R transitions at ]D[ - IEI (A) and IOl + IEI (B). Dots mark the measured spectra, the solid line are simulations assuming the triplet state of D localized on the D A monomer, and the dashed lines are simulations assuming 3D to be localized on D e . (From Ref. 76.)

328

PROBES OF METAL ION ENVIRONMENTS

[10]

The absence of significant bands in the 790 nm region indicates that the two • pigments, qbA and ~B, are only weakly coupled to D. In addition to the above conclusions, the spectral simulations allowed the investigators to infer that the y spin axis of the localized triplet state is very close to the NI-N m axis at an angle of I0 °, with the x and y spin axes lying approximately in the plane of the BChl macrocycle (sublevel ordering y, x, z for D, E > 0). The calibrated T - S and LD-(T - S) spectra allowed calculations of the position of the Qy tm of all coupled BChls and • molecules in the x,y,z spin axes coordinate frame, and consequently all their mutual angles. With minor differences the qualitative aspects of the above picture hold for all purple bacteria investigated. 9° The T - S spectra of green photosynthetic bacteria and of Heliobacterium chlorum are more complex and have only been tentatively interpreted, 66,9~,92 with the exception of the green gliding bacterium Chloroflexus (Cfl.) aurantiacus, whose RC is very similar to that of purple bacteria. 93'94 The above results show that, once the crystal structure is known, one can draw very detailed conclusions from a simulation of the T - S and LD(T - S) difference spectra. Now that the (fairly simple) exciton treatment appears to be good enough to simulate accurately optical spectra, one might ask whether the reverse is also possible, namely, predicting from a simulation of optical (difference) spectra the crystal structure. For a completely unknown RC this is obviously a tall order, in view of the very large parameter space. For RCs closely related to those of Rps. viridis and Rb. sphaeroides R-26, however, this can indeed be done, as was demonstrated by Scherer and Fischer, 89'95Vasme169'94 and H. Vasmel (unpublished simulations, 1986), who were able to simulate and predict remarkably well the T - S and LD-(T - S) spectra of Cfl. a u r a n t i a c u s 89'94 and of a chemically modified RC ofRb. sphaeroides R-26. 95-97 This opens the prospect of interpreting the T - S spectra of the RC of photosystem 9o j. A. Dijkman, H. J. den Blanken, and A. J. HolT, Isr. J. Chem. 28, 141 (1988). 91 A. J. Hoff, H. Vasmel, E. J. Lous, and J. Amesz, in "Green Photosynthetic Bacteria" (J. M. Olson, J. G. Ormerod, J. Amesz, E. Stackebrandt and H. G. Triiper, eds.), p. 119. Plenum, New York, 1988. 92 j. Vrieze, E. J. van de Meent, and A. J. Hoff, in "The Photosynthetic Bacterial Reaction Center II" (J. Breton and A. Vermrglio, eds.), p. 67. Plenum, New York, 1992. 93 H. Vasmel, R. F. Meiburg, J. Amesz, and A. J. Hoff, in "Progress in Photosynthesis Research" (J. Biggins, eds.), Vol. 1, p. 403. Nijhoff, Dordrecht, The Netherlands, 1987. 94 H. Vasmel, Doctoral Dissertation, University of Leiden, The Netherlands (1986). 95 p. O. J. Scherer and S. F. Fischer, Chem. Phys. Lett. 137, 32 (1987). 96 D. Beese, R, Steiner, H. Scheer, B. Robert, M. Lutz, and A. Angerhofer, Photochem. Photobiol. 47, 293 (1988). 97 A. Angerhofer, D. Beese, A. J. Hoff, E. J. Lous, and H. Scheer, in "Applications of Molecular Biology in Bioenergetics of Photosynthesis" (G. Singhal, ed.), p. 197. Narosa Publ., New Delhi, 1989.

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II, which is believed to be closely related to that of the purple bacteria (see below). P l a n t r e a c t i o n c e n t e r s . The T - S spectra of RC of the plant photosysterns are characterized by a strong bleaching of donor bands at 703 and 682 nm for photosystems I and II, respectively (Fig. 11). In photosystem I a positive band appears at the blue side of this bleaching, close to the wavelength of the absorption of Chl a in vitro. 67 Analogously to the interpretation of the T - S spectra of the purple bacteria, this band was attributed to the appearing absorption of a monomeric Chl a molecule belonging to a primary donor dimer on which the triplet state was localized on the second Chl a , 67 supporting the notion that in photosystem I the primary donor is a dimeric Chl a complex. The above-mentioned positive component is much smaller in the T S spectrum of photosystem II, 68,99,1°° rendering assignment to a dimeric primary donor complex tenuous. More information was sought by LD(T - S) spectroscopy. 98 It turns out that the t m of both the bleaching and the small positive contribution make about the same angles with the triplet x , y axes, which are practically the same as the angles measured for monomeric Chl a in vitro. This makes it very unlikely that the primary donor of photosystem I! is composed of two Chl a molecules with a sizable exciton interaction. It thus appears that even though two Chl a molecules may be bound to the RC protein of photosystem I! in locations similar to those o f the two primary donor BChls in the bacterial RC protein, only one functions as the primary donor. 4. Prospects of Optically Detected Magnetic Resonance Spectroscopy of Metalloproteins Some of the proteins discussed in previous sections contain metals and are technically metalloproteins. This does not mean that they are normally classified as such. This is especially true for the photosynthetic reaction centers that contain a divalent Fe ion. The function o f this ion, however, is not clear; it does not appear to play a significant role in early electron transport. Possibly it functions in setting the redox potential of secondary quinone acceptors, regulating electron transfer between them. It is tempting to envision the application of ODMR to metalloproteins containing a functional metal. It is unlikely that the metal itself can be 98j. Vrieze, P. Gast, and A. J. Hoff, in "Research in Photosynthesis" (N. Murata, ed.), Vol. I, p. 553. Kluwer Academic Publ., Dordrecht, The Netherlands, 1992. 99R. van der Vos, P. J. van Leeuwen, P. Braun, and A. J. Hoff, Biochim. Biophys. Acta in press (1992). i00D. Bernlocher, A. Angerhofer, and B. Robert, Proceedings of the International Symposium on MagneticField and Spin Effects in Chemistry, in press. Konstanz, Germany, 1992.

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studied, as few metals can be excited to a metastable triplet state. E v e n then, fast rates of relaxation and decay may not allow the detection of O D M R signals. More likely, the metal ligands will be amenable for study by O D M R . F o r example, metals are often liganded to histidine residues, and although these are not favorable for O D M R , triplets generated on the histidines m a y be detectable on tyrosines or tryptophans through energy transfer (Section 3.1). It m a y be anticipated that the h e a v y metal effect (Section 3.1. l) will be of importance, providing a clue to the distance between the metal site and a liganded c h r o m o p h o r e . In addition, fastrelaxing paramagnetic metal centers will influence the spin-lattice relaxation time o f the triplet manifold o f nearby aromatic amino acids, which can be verified by temperature-dependent ODMR. This again will provide distances and perhaps in favorable cases the orientation of the chromophore with respect to the metal. Thus, although presently O D M R is not much applied to metalloproteins, the future may see more activity in this field. Acknowledgments This chapter was written during my tenure as a Visiting Fellow at Corpus Christ! College, Oxford. I gratefully acknowledge the hospitality provided both by the College and by the Physical Chemistry Laboratory of the University of Oxford. I am indebted to Profs. J. H. van der Waals and J. Schmidt of the Leiden Centre for the Study of Excited States of Molecules, who over the years assisted with equipment, laboratory space, and stimulating interest. Much of the work carried out in Leiden was performed by Drs. H. J. den Blanken, E. J. Lous, R. van der Vos, and J. Vrieze with skill and unstinting enthusiasm under the auspices of the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Foundation for Scientific Research (NWO). Finally, I am indebted to Elsevier Science Publishers for allowing me to quote several sections of Ref. 2.

[11] Electron Paramagnetic

Resonance

By JOHN R. PILBROW and GRAEME R. HANSON Introduction Electron paramagnetic resonance (EPR) and electron spin resonance (ESR) are s y n o n y m o u s terms I for describing the resonant absorption o f m i c r o w a v e radiation by a paramagnetic substance in a static magnetic i The International EPR Society recommends that EPR be used as the preferred acronym.

METHODS IN ENZYMOLOGY, VOL. 227

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studied, as few metals can be excited to a metastable triplet state. E v e n then, fast rates of relaxation and decay may not allow the detection of O D M R signals. More likely, the metal ligands will be amenable for study by O D M R . F o r example, metals are often liganded to histidine residues, and although these are not favorable for O D M R , triplets generated on the histidines m a y be detectable on tyrosines or tryptophans through energy transfer (Section 3.1). It m a y be anticipated that the h e a v y metal effect (Section 3.1. l) will be of importance, providing a clue to the distance between the metal site and a liganded c h r o m o p h o r e . In addition, fastrelaxing paramagnetic metal centers will influence the spin-lattice relaxation time o f the triplet manifold o f nearby aromatic amino acids, which can be verified by temperature-dependent ODMR. This again will provide distances and perhaps in favorable cases the orientation of the chromophore with respect to the metal. Thus, although presently O D M R is not much applied to metalloproteins, the future may see more activity in this field. Acknowledgments This chapter was written during my tenure as a Visiting Fellow at Corpus Christ! College, Oxford. I gratefully acknowledge the hospitality provided both by the College and by the Physical Chemistry Laboratory of the University of Oxford. I am indebted to Profs. J. H. van der Waals and J. Schmidt of the Leiden Centre for the Study of Excited States of Molecules, who over the years assisted with equipment, laboratory space, and stimulating interest. Much of the work carried out in Leiden was performed by Drs. H. J. den Blanken, E. J. Lous, R. van der Vos, and J. Vrieze with skill and unstinting enthusiasm under the auspices of the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Foundation for Scientific Research (NWO). Finally, I am indebted to Elsevier Science Publishers for allowing me to quote several sections of Ref. 2.

[11] Electron Paramagnetic

Resonance

By JOHN R. PILBROW and GRAEME R. HANSON Introduction Electron paramagnetic resonance (EPR) and electron spin resonance (ESR) are s y n o n y m o u s terms I for describing the resonant absorption o f m i c r o w a v e radiation by a paramagnetic substance in a static magnetic i The International EPR Society recommends that EPR be used as the preferred acronym.

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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field. A paramagnetic substance consists of weakly interacting ions or free radicals that possess permanent magnetic moments originating from electron spin and also, in most cases, including contributions from the electron orbital angular momentum, z-3 In metalloproteins these constituents are usually present as cofactors involving transition metal ions, multiatom clusters, or free radicals, which may have a structural role or more impotantly are intrinsically involved in enzymatic catalysis and/ or electron transfer. Because the tertiary structure of metalloproteins is normally diamagnetic, EPR is a powerful method for characterizing the structure of the paramagnetic cofactor in the resting enzyme, enzyme-substrate intermediates, and product complexes. Apart from scandium and titanium, all of the first row transition metal ions and the second and third row elements molybdenum and tungsten have been found in a wide range of metalloproteins. 4 Sometimes it is possible to replace the catalytically essential diamagnetic, and spectroscopically silent, metal ion such as zinc(II), calcium(II), or magnesium(II) by one that is spectroscopically active, notably cobalt(II) or manganese(II). This provides additional scope for the investigation of a wide range of proteins and enzymes with EPR spectroscopy. 5'6 In many cases magnetic coupling between two or more paramagnetic centers can be observed in the EPR spectra of metalloproteins, which is suggestive of the existence of either metal clusters or an electron transfer chain. Finally many transition metal ions such as copper, manganese, and vanadyl ions are found to bind extrinsically to proteins. This is advantageous because their EPR spectra can often be observed at room temperature. 7 In restricting this chapter to transition metal ions, major areas of EPR in biology concerning spin labeling, spin trapping, and radiation-induced radicals are not considered. This chapter concentrates on basic principles 2 j. R. Pilbrow, "Transition Ion Electron Paramagnetic Resonance." Clarendon Press, Oxford, 1990. za A. Abragam and B. Bleaney, "Electron Paramagnetic Resonance of Transition Ions." Oxford Univ. Press (Clarendon), Oxford, 1970. 3 H. M. Swartz, J. R. Bolton, and D. C. Borg (eds.), "Biological Applications in Electron Spin Resonance." Wiley (Interscience), New York, 1972. 4 j. j. R. Fratlsto da Silva and R. J. P. Williams, "The Biological Chemistry of the Elem e n t s - T h e Inorganic Chemistry of Life." Oxford Univ. Press (Clarendon), Oxford, 1991. 5 B. L. Vallee in "Metal Ions in Biological System" (S. K. Dahr, ed.), p. 1. Plenum, New York, 1955. 6 B. L. Vallee and B. Holmquist, in "Methods for Determining Metal Ion Environments in Proteins, Structure and Function of Metalloproteins" (D. W. Damell and R. G. Wilkins, eds.), Vol. 2, p. 27. Elsevier, New York, 1980. 7 R,C. Sealy, J. S. Hyde, and W. E. Antholine, i n " M o d e m Physical Methodsin Biochemistry" (A. Neuberger and L. L. M. Van Deenen, eds.), p. 69. Elsevier, New York, 1985.

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PROBES OF M E T A L I O N E N V I R O N M E N T S

[11]

of continuous wave EPR (CW-EPR) as applied to metalloproteins. Other chapters in this volume are devoted to a range of specialized topics, such as pulsed electron nuclear double resonance (ENDOR), CW-ENDOR, vanadyl ENDOR spin probes, electron spin echo envelope modulation (ESEEM), iron EPR Spectroscopy, intrinsic and extrinsic paramagnets as probes of metal clusters, and EPR spectrochemical titrations. The information obtained from an EPR spectrum can be divided into two classes: (I) structural information obtained from the spin Hamiltonian parameters and (2) the quantification of the EPR signal intensity. Although most researchers use EPR spectroscopy for the elucidation of structural information, spin quantitation can be used to determine (1) the number of EPR active centers present and the spin state, (2) the redox potentials of a paramagnetic center, and (3) the rate constants for biochemical reactions. Electron Paramagnetic Resonance of Transition Metal Ions The basis for understanding the properties of paramagnetic ions in proteins is the set of closely spaced electronic energy levels that are dependent on the number of d electrons, the symmetry arrangement, and the number and type of neighboring ligand atoms or ions.2-3 Later, we give a brief, largely descriptive, introduction to the most commonly occurring electronic d configurations. Rather more detail than is possible here may be found in a book by one of the authors. 2 In the meantime a general introduction to EPR follows. In the simplest system to introduce EPR, one has an electron spin of ½ and two degenerate energy levels in the absence of a magnetic field. These two levels diverge linearly in an applied magnetic field, and resonance occurs when the microwave quantum of energy exactly equals the spacing between the levels (Fig. 1). Line broadening from neighboring spins, unresolved electron nuclear hyperfine interactions, and relaxation effects means that there is no longer a unique resonance, but an overlapping family of them. This is termed inhomogeneous broadening. There are various possibilities. Sometimes the interactions are isotropic, that is, they do not vary with orientation of the magnetic field relative to the molecule and the resonance is unchanged as the magnetic field is rotated in the case of a crystal. However, anisotropy occurs in most paramagnetic systems and arises from the interactions between the paramagnetic electrons and the surrounding anisotropic electron distributions associated with the neighboring ligand atoms or ions. The energy of interaction (U) between a paramagnetic ion, with magnetic moment ~, and a magnetic field B is U = -g,.a

(1)

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ELECTRON PARAMAGNETICRESONANCE

333

'~C ~0.3cm -I

s=½

Bo~O.3T

Kramers Doublet

Ms = - ~ / ~ ~ 'X, II

dB

ABSORPTION FIRST DERIVATIVE

MAGNETIC FIELD

J~__ ~,~/~

L

/ Bo

~

}.

B

FiG. 1. EPR in a Kramers doublet (spin ½). [Reproduced with permission from T. D. Smith and J. R. Pilbrow, in "Biological Magnetic Resonance" (L. J. Berliner and J. Reuben, eds.), Vol. 2, p. 85. Plenum, New York, 1980.]

In paramagnetism it is assumed that the magnetic moments of the paramagnetic centers are only weakly coupled and that, to a very good approximation, they can be assumed to be isolated from one another. This is true for low concentrations of single transition metal ions or for paramagnetic metal ion clusters where each cluster possesses a net magnetic moment. The relation between the magnetic moment, it, and associated electron spin, $, where both # and S are considered to be quantum mechanical operators, is tz = -g/3S

(2)

where/3 is the Bohr magneton and g is the electronic splitting factor. The latter is characteristic of the environment in which the unpaired electron is located. In contrast to nuclei, the gyromagnetic ratio for free electrons, 3/, that is, the ratio o f angular m o m e n t u m to magnetic moment, is negative. In the case of free radicals, g is close to the free electron value of 2.0023 and nearly isotropic. The potential energy of the dipole in the magnetic field now becomes -- gBS. B

(3)

334

PROBES OF METAL ION ENVIRONMENTS

[11]

Actually it is customary to use the symbol ~ in place of U. ~ is known as the Hamiltonian and in EPR it is usually referred to as the spin Hamiltonian. For a two-level system, in which the electron spin is S = 4, the energies of the two levels obtained by solving the eigenvalue problem ( ~ = E ~ ) with the spin Hamiltonian given in Eq. (3) are E + = +--½gflB

(4)

An EPR transition at a fixed frequency, vc, occurs when the magnetic field is varied until the resonance value of B 0 is reached. Therefore, AE

= E+ -

E_ = hv c = gBB o

(5)

In the two-level example shown in Fig. 1 the levels are described by Ms = ---4. Resonant absorption can be explained as follows. The transition rate for induced transitions (4 ~ -4) and (-4 ~ 4) can be calculated using the rules of quantum mechanics. These are magnetic dipole transitions and occur only when the sample is placed in a magnetic field, B, and where there is a component of the microwave magnetic field, B1, perpendicular to B. The selection rule for the quantum number Ms is [AMs[-- ---1

(6)

In thermal equilibrium, the number of paramagnetic ions in the upper and lower energy states can be calculated from the Boltzmann formula. Because an excess population exists in the lower state, the EPR signal intensity must depend on this Boltzmann population difference. In continuous wave EPR the absorption process disturbs the system from equilibrium only by a very small amount. Spin-lattice relaxation processes, which result from an indirect coupling of the electron spins to the surrounding crystal or molecular lattice, remove the excess energy nonradiatively and maintain the near-equilibrium or steady-state situation. This is characterized by the time T1. Spin-lattice relaxation is very much dependent on temperature (T). The relaxation rate for a number of situations can be written as follows: T1-1 = a T + b T n + c A 3 e a/kr

(7)

where a, b, and c are constants and k is the Boltzmann factor. Equation (7) is one of the very important equations in EPR theory for it underpins considerations as to whether a spectrum can be observed. The first term is very important at very low temperatures. The second term arises from Raman relaxation processes and is important at higher temperatures. For odd-electron ions n = 9, whereas n -- 7 for even-electron ions. The final term (Orbach process) involves relaxation transitions to excited electronic

[11]

ELECTRON PARAMAGNETIC RESONANCE

335

states at an energy A above the ground state. It applies, for example, to high-spin d 5 heme proteins and also to high-spin cobalt(II) (S = ~) centers in cobalt-substituted zinc enzymes. For low-spin d 5 heine proteins the relaxation depends on a fractional dimension of ~, consistent with the dimensionality of the protein backbone. In most cases TI is so short at room temperature that EPR cannot be observed, and experiments often must be performed at the temperature of liquid helium. Insertion of the values of fl and h in appropriate units into Eq. (5)8,9 gives the resonance condition B0 (mT) =

71.448 × vc (GHz) g

(8)

At X-band microwave frequencies, for example, 9.248 GHz, the resonant field position for a free electron (g = 2.0023) will be 330 mT (3300 gauss). The SI unit of magnetic induction is the tesla (T) which is equivalent to 10,000 gauss.

Spin Hamiltonian and Effective Spin The concept of the spin Hamiltonian has been known for a long time ~°-~3 and allows the determination of the energies of the spin states responsible for EPR spectra. It can be written in the conventional way for an ion with effective spin S and nuclear spin ! as = ~FS + flB.g.S + S.A.I + I.Q.I

-

gnflnB.l

(9)

where ~FS represents various second-order and higher terms in electron spin components which give rise to fine structure in the spectrum. Here 13 is the Bohr magneton, as before, fl~ is the nuclear magneton, and S and ! are the electron spin and nuclear spin operators, g is the electron Zeeman interaction matrix, and A is the hyperfine coupling matrix for the interaction between S and the nuclear spin I of the central metal ion. Q (sometimes called P) is the quadrupole tensor, and the last term represents the nuclear Zeeman effect. There can also be an equivalent set of hyperfine, quadrupole, and nuclear Zeeman terms from one or more ligand nuclei. The spin Hamiltonian is another of the important equations in EPR. The condition in Eq. (5) does not always strictly hold, for example, when S > ½or when 8 E. R. Cohen and B. N. Taylor, CODATA Bull. 63, 1 (1986). 9 E. R. Cohen and B. N. Taylor, J. Res. Natl. Bur. Stand. (U.S.) 92, 85 (1987). i0 A. Abragam and M. H. L. Pryce, Proc. Phys. Soc. London, Sect. A 205, 135 (1951). it A. Abragam and M. H. L. Pryce, Proc. Phys. Soc. London, Sect. A 206, 173 (1951). 12 M. H. L. Pryce, Phys. Rev. 80, 1107 (1950). ~3 M. H. L. Pryce, Proc. Phys. Soc. London, Sect. A 63, 25 (1950).

336

PROBES OF M E T A L I O N E N V I R O N M E N T S

[11]

hyperfine coupling, including quadrupole and nuclear Zeeman interactions, are present or at low magnetic fields. The effective spin, S, may be calculated from the number of low-lying paramagnetic energy levels (2S + 1) responsible for the EPR spectrum and which are split up in an applied magnetic field. When S is greater than ½, the ~FS term in the spin Hamiltonian for symmetries lower than cubic is = D [ S z 2 - S ( S + 1)/3] + E(S~ 2 - Sy 2)

(10)

where D and E, which determine the zero-field splittings, arise from axial and rhombic distortions of the ligand field, respectively.

Orientational Dependence

of g Factors and Hyperfine Splittings

In contrast to nuclear magnetic resonance (NMR), the magnetic moments of electrons, and hence g factors, of transition ions in crystalline and molecular environments are frequently very anisotropic; in other words, the EPR properties depend on the orientation the applied magnetic field makes with the molecular framework. For ions in sites of 3- or 4fold symmetry, two g factors, gll and g±, are required to describe EPR spectra. In general, three g factors are needed, and the orientational dependence of the g factor is given by g2 = lx2gx 2 + ly2gy2 + iz2gz 2

(11)

where Ix, ly, and Iz are the direction cosines relating B to the principal g axes. With regard to the electron nuclear hyperfine interaction, in general, three components are needed, and the orientational dependence of the hyperfine interaction is given by a slightly more complicated relation: A2g 2 = Ix2Ax2gx 2 + ly2my2gy 2 + lz2Az2gz 2

(12)

In this chapter only the first-order resonance equation is given, although interpretation of transition metal spectra requires the inclusion of higher order terms, 2 B = B o - A M I / g fl

(13)

where B 0 and g are related by the resonance condition [Eq. (5)]. Hyperfine lines are separated, to a first approximation, by an equal amount A / g f l in field units such as gauss or millitesla. We now consider the commonly occurring d electronic configurations and coupled systems found in biological systems.

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ELECTRON PARAMAGNETIC RESONANCE

337

dr: Vanadium(IV), Molybdenum(V), and Tungsten(V). The d I configuration is more complicated than might first appear since the original electron orbital states, even if not mixed by low-symmetry ligand fields, experience state mixing owing to the spin-orbit coupling of the orbital and spin magnetic moments. In the case of axial symmetry the spin Hamiltonian requires two g factors (gll and g±) and two hyperfine constants (All and A±). In the absence of oxo or sulfido ligands coordinated to the metal ion, the ligands produce a trigonal distortion with the unpaired electron in a metal-based dz2 orbital. However, this situation is uncommon in metalloproteins, and oxo or sulfido ligands usually impose a tetragonal distortion on the metal ion with the unpaired electron in a metal-based dxy orbital. For metaUoproteins containing an oxovanadium(IV) center [stVO(II); S = ½, I = ~], typical anisotropic g and A values are gll "~ 1.94 and g l ~ 1.98-1.99, whereas Aib, assumed negative, is typically around 0.02 cm-1 and A± -~ 0.006 cm -1.14 Naturally occurring molybdenum consists of a mixture of isotopes. The even isotopes, with a total natural abundance of 75%, have I = 0, and the remaining 25%, consisting of 95Mo and 97Mo, have I = ~ and essentially equal hyperfine splittings. Typical values of the hyperfine principal values (All and A±) lie in the ranges 0.004-0.008 and 0.0020-0.0025 cm -~, respectively. It is generally found that gll > g± with gll ~ 1.99 and g± ~ 1.95-1.96, although sulfur coordination can produce gll values equal to or greater than 2. Molybdenum sites in enzymes such as xanthine oxidase are not axially symmetric, and, in many cases, evidence for symmetry lower than orthorhombic is obtained from experiments and associated computer simulations carried out at more than one microwave frequency. ~5 Formate dehydrogenase 16'17 has been shown to contain a tungsten cofactor with a structure similar to that found for the molybdenum cofactor in mononuclear oxomolybdenum enzymes. dS : Low- and High-Spin Iron(Ill) and Manganese(II). The d 5 configuration is an interesting case as it has a half-filled d shell. There are two main possibilities, high spin and low spin. EPR spectra have identified both high-spin (S = ~) and low-spin (S = ½) sites in heme proteins. The review by Palmer TM on the EPR of heine proteins provides a readable introductory account of the theory. 14 N. D. Chasteen, in "Biological Magnetic Resonance" (L. J. Berliner and J. Reuben, eds.), Vol. 3, p. 53. Plenum, New York, 1981. 15 G. L. Wilson, R. J. Greenwood, J. R. Pilbrow, J. T. Spence, and A. G. Wedd, J. Am. Chem. Soc. 113, 6803 (1991). 16 S. Mukand and M. W. Adams, J. BioL Chem. 265, 11508 (1990). i7 S. Mukand and M. W. Adams, J. Biol. Chem. 266, 14208 (1991). is G. Palmer, in "The Porphyrins" (D. Dolphin, ed.), p. 313. Academic Press, New York, 1979.

338

[11]

PROBES O F M E T A L I O N E N V I R O N M E N T S

E/D=0

E/D=113

gY

9x g..L

i8~"'-"O'~ 3,530

0

Zg~. 9967

0.6

J

3.53D~ g,~

o

0.6

0.86 1=,

B(//)

1=_

B(xl

B(z)

11

B(xl B(y7

b FIG. 2. Schematic energy level diagrams for high-spin Fe 3+ in the weak magnetic field limit where resonance is observed only within and not between doublets. (a) E/D = 0; (b) E/D = ]. The effective g factors are indicated. (Reproduced from Ref. 19, with permission.)

To understand the high-spin case, 19 observed, for example, in some heme proteins, g [Eq. (9)] is in all cases nearly isotropic, and the effective spin is the same as the real free ion spin of~. The spin Hamiltonian requires additional terms that are fourth-order polynomials in the components of the spin including ~Fs given by Eq. (10). For some high-spin heme proteins, the three paramagnetic (Kramers) doublets are separated by energies of approximately 20 and 30 cm -1, respectively (see Fig. 2). Consequently resonance is only observed in the lowest of these doublets, yielding glJ -2 and g l = 6, corresponding to axial symmetry (E/D = 0). The condition E/D -- ] leads to g factors ranging from 0.6 to 10.0 (see Fig. 2), though this case is most usually identified by the g ~ 4.3 isotropic resonance from the middle doublet. The high-spin case is also important for mononuclear and magnetically coupled manganese(II) ions. EPR spectra of the 19T. D. Smith and J. R. Pilbrow, in "Biological Magnetic Resonance" (L. J. Berliner and J. Reuben, eds.), Vol. 2, p. 85. Plenum, New York, 1980.

[11]

ELECTRONPARAMAGNETICRESONANCE

339

mononuclear sites consist of a six-line hyperfine splitting of each transition arising from the manganese hyperfine coupling of approximately 9 mT. The low-spin d 5 configuration may be described by the theory for d ~ with some minor sign changes in the equations for g and A principal values. The g factors are anisotropic, lying typically in the range 1.5-3.0. EPR spectra of nitrile hydratasez° consist of two components each having three main features corresponding to the principal g factors. Such spectra are usually observed only at fairly low temperatures. dT : Low- and High-Spin Cobalt(II). Low-spin cobalt(II) has a single unpaired electron in a metal-based dz2 orbital and is found as an intrinsic constituent of the coenzyme vitamin B12 in its reduced form. 21 Identification of ligands axially coordinated to the cobalt ion can be readily identified from ligand hyperfine interactions. High-spin cobalt(II) ions can occur in a range of geometries ranging from octahedral to tetrahedral with five-coordinate geometries in between. The ligand field theory describing the energy levels for cobalt(II) in these geometries has been reviewed by Banci et al. 22 For octahedral high-spin cobalt(II) a doublet is lowest in energy which is very anisotropic. EPR spectra are only observed in the lowest doublet, a situation that can be described by an effective spin of ½. Because of the rather complex interactions involved, the g factors are far removed from 2. In the case of a purely octahedral field, g = 4.3, but in anisotropic cases the values may lie somewhere between 2 and 8. So in this case, the true spin of for the free ion and the effective spin of ½are quite different. Octahedrally coordinated cobalt sites have been found in cobalt(II)-substituted zinc phospholipase C . 23 Distorted tetrahedral cobalt(II) sites have long been identified in cobaltsubstituted enzymes such as carboxypeptidase and carbonate dehydratase. The theory required for the interpretation of EPR spectra from tetrahedral cobalt ions is in principle similar to that for chromium(III) in near octahedral surroundings. For cobalt(II) in a tetrahedral crystal field an orbital singlet is the lowest state with S = ~. In axial symmetry, the spin Hamiltonian consists of the electron Zeeman interaction and ~FS. Zero-field splitting produces two Kramers doublets (separated by 2D), which are further split by a magnetic field B. The g factors are found to 20 y . Sugiura, J. Kuwahara, T. Nagasawa, and H. Yamada, J. Am. Chem. Soc. 109, 5848 , (1987). 21 j. R. Pilbrow, in "BI2" (D. Dolphin, ed.), Vol. 1, p. 431. Wiley (Interscience), New York, 1982. 22 L. Banci, A. Bencini, C. Benelli, D. Gatteschi, and C. Zanchini, Struct. Bond. (Berlin) 52, 37 (1982). 23 R. Bicknell, G. R. Hanson, B. Holmquist, and C. Little, Biochemistry 25, 4219 (1986).

340

PROBES OF METAL ION ENVIRONMENTS

[1 1]

be a little larger than 2, the deviation from 2 arising from the effect of spin-orbit interaction and the low-symmetry ligand field. When the electron Zeeman interaction is very much larger than the zero-field splitting, the system can still be described as spin a. z On the other hand, when the microwave energy is small compared with the zero-field splitting, then each doublet must be considered as having its own spin of ½ and has effective g factors that can be very anisotropic, ranging from about 10 to less than I. Little hyperfine structure is ever observed for distorted tetrahedral cobalt(II), in contrast to octahedral high-spin cobalt(II). The latter situation is what occurs for cobalt(II) replacing zinc in carboxypeptidase and carbonated hydratase. Typical doublet splittings are usually less than 13 cm-l. 24-26The effective g factors from these are not easily distinguishable from those of octahedrally coordinated high-spin cobalt(II) ions referred to previously but are characterized by relatively small hyperfine splittings, when observed, and in this respect are distinguishable from the other case. The resonances are observed only at temperatures near that of liquid helium, and relaxation to the upper doublet causes loss of the signal at about 15 K. Marked differences are found in the EPR spectra on binding of substrates and inhibitors. 27 dS: High-Spin Nickel(II). There are a number of proteins known to contain nickel including urease, nickel hydrogenases, and carbon-monoxide dehydrogenase. The ground state is an orbital singlet with 3-fold spin degeneracy, which is removed by the ligand field and leads to large zerofield splittings. Consequently, nickel(II) is ordinarily EPR silent. However, oxidation or reduction of the nickel(II) ion produces nickel in the oxidation states of +3 and + 1, respectively, producing S = ½ ground states. Use of 61Ni (I = a) has shown that the EPR signals arising from the hydrogenases and carbon-monoxide dehydrogenase structure contain nickel. 33S isotope substitution has shown the presence of sulfur ligands in the coordination sphere of the nickel ion. d9: Copper(lI). Copper(II) is the archetypical example for transition metal ion EPR. In crystals, spectra show a characteristic four-line hyperfine structure since I = ~. There are two isotopes, 63Cu and 65Cu, that have slightly different magnetic moments, and this means that for naturally abundant copper, the spectra from the two isotopes overlap. Characteristic 24 M. B. Yim, L. C. Kuo, and M. W. Makinen, J. Magn. Reson. 46, 247 (1982). 25 L. C. Kuo and M. W. Makinen, J. Am. Chem. Soc. 107, 5255 (1985). 26 M. W. Makinen, L. C. Kuo, M. B. Yim, G. B. Wells, J. M. Fukuyama, and J. E. Kim, J. Am. Chem. Soc. 107~ 5245 (1985). 27 R. A. Martinelli, G. R. Hanson, J. S. Thompson, B. Holmquist, J. R. Pilbrow, D. S. Auld, and B. L. Vallee, Biochemistry 28, 2251 (1989).

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ELECTRONPARAMAGNETICRESONANCE

341

but generally poorly resolved spectra are observed for frozen solutions of copper enzymes. There are a great many copper-containing proteins and enzymes, which are generally grouped into three main classes according to EPR characteristics rather than function. Type I copper(II) is recognized by an intense blue color correlated by the absorption spectrum in the 600 nm region. It has an extinction coefficient about 100 times that commonly encountered in simple copper(II) chelates. The type 1 sites are now understood to be distorted four-coordinate, "tetrahedral-like." Although the g factors appear normal, with gll > g± > 2, the resolved parallel hyperfine splitting, All, has an unusually small value, namely, less than 0.01 cm-1. 28 Type 2 copper(II) possesses visible absorption and EPR spectral properties compatible with those of simple copper(II) amino acid and peptide chelates and is an essential constituent of the multicentered copper(II) proteins. It has been argued that the designation, type 2, should be reserved for cases where type 1 copper(II) is also present. Typical g factors are gLI ~" 2.2 and g . ~ 2 . 0 2 . 29 Type 3 copper centers are thought to consist of pairs of copper(II) ions in close proximity that are strongly antiferromagnetically coupled. Examples include so-called type 3 copper found in the multicopper oxidases and the nondetectable coppers in hemocyanin. Solomon e t al. 3° have reviewed this field extensively with particular emphasis on the hemocyanins. Both ceruloplasmin sl and hemocyanin show evidence for dipolar coupled pairs of copper(II) ions after treatment with nitric oxide. The dipolar interaction produces a zero-field splitting of the triplet state and separates the singlet state from the "center of gravity" of the triplet state, For copper(II) ion pairs, the dipolar splittings are approximately 0.1 cmwhen the internuclear distance r is about 0.3 nm and approximately 0.01 cm -~ when r is around 0.7 nm. This may be compared with the magnitudes of the typical electron Zeeman interaction at 10 GHz and g = 2 of approximately 0.3 cm -~ and the typical copper nuclear hyperfine interaction of approximately 0.015 cm -~. Poorly resolved lines for the allowed and forbidden transitions (sometimes called half-field lines) usually occur when exchange coupling is weak. Studies of cases in which two metal ions share a common ligand include copper(II)-copper(II) pairs following migration of copper(II) ions to the vacant zinc binding site of bovine erythrocyte 28 K. W. Penfield, R. R. Gay, R. S. Himmelwright, N. C. Eickman, V. A. Norris, H. C. Freeman, and E. I. Solomon, J. Am. Chem. Soc. 103, 4382 (1981). 29 j. A. Fee, Struct. Bond. (Berlin) 23, 1 (1975). 30 E. I. Solomon, K. W. Penfield, and D. E. Wilcox, Struct. Bond. (Berlin) 53, 1 (1983). 31 F. R. Van Leeuwen, R. Wever, and B. F. van Gelder, Biochim. Biophys. Acta 315, 200 (1973).

342

PROBES OF METAL ION ENVIRONMENTS

[11]

superoxide dismutase 32 and the binuclear copper active site of mollusc and arthropod hemocyanin or Neurospora tyrosinase. 33,34

Magnetically Coupled Systems In general there are two types of magnetic interactions between metal centers, namely, dipole-dipole coupling and superexchange coupling. The former interaction is a through-space interaction and varies as the inverse cube of the distance between the magnetic centers. The observation of dipole-dipole coupling in xanthine oxidase has allowed the relative disposition of the redox centers to be determined and provides direct evidence of an intramolecular electron transfer chain. 35 A model of the subunit of xanthine oxidase showing the relative disposition of the redox centers determined from magnetic interaction measurements is given in Scheme I.

Mo lI~3_A. [2Fe-(2S] (I) . .14. . --4 . . . . [2Fe7-2S] (II)

o".,'~,,

16-+4A

/

%% %

•

/

16-+ 4"~

•

"FAD • SCHEME I

Superexchange can be further classified into antiferromagnetic and ferromagnetic coupling and involves the overlap of molecular orbitals on each metal center with a bridging ligand. The isotropic exchange coupling constant J (negative for antiferromagnetic coupling) is a measure of the extent to which the electrons are coupled and can be determined in favorable circumstances with variable-temperature EPR spectroscopy. When multiple centers become paramagnetic the coupling is via spin-spin coupling and is inferred from splittings, half-field transitions, and loss of microwave power saturation. A detailed analysis of magnetic interactions and the determination of J couplings is provided in the book entitled EPR of Exchange Coupled Systems by Bencini and Gatteschi. 36 32 j. S. Valentine, M. W. Pantoliano, P. J. McDonnell, A. R. Burger, and S. J. Lippard, Proc. Natl. Acad. Sci. U.S.A. 76, 4245 (1979). 33 R. S. Himmelwright, N. C. Eichman, C. D. LuBien, and E. I. Solomon, J. Am. Chem. Soc. 102, 5378 (1980). 34 R. S. Himmelwright, N. C. Eichman, C. D. LuBien, K. Lurch, and E. I. Solomon, J. Am. Chem. Soc. 102, 7339 (1980). 35 M. J. Barber, J. C. Salerno, and L. M. Siegel, Biochemistry 21, 1648 (1982). 36 A. Bencini and D. Gatteschi, " E P R of Exchange Coupled Systems." Springer-Verlag, Berlin, 1990.

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ELECTRONPARAMAGNETICRESONANCE

343

The most common examples in which these interactions occur are the iron-sulfur-containing metalloproteins, and, consequently, we concentrate on these. However, there are many other examples of exchange coupling of metal centers in metalloproteins, for example, the binuclear iron oxo-proteins of which hemerythrin and uteroferrin are good examples, the type 3 copper centers, the molybdenum-iron cofactor in nitrogenase, and the nickel-iron-sulfur clusters in hydrogenase. A large number of ferredoxins have been purified to homogeneity and have been characterized with a wide range of spectroscopic techniques, 37-39 including EPR spectroscopy. To date the following types of iron-sulfur clusters have been identified: [Fe-Cys4] ~-,2- (S = ~, 2); [2Fe-2S] 2+'1+ (S = 0, ½); [3Fe-4S] 1+'° (S = ½, 2); and [4Fe-4S] 3+'2+;z÷'1+ (S = ½, 0, ½). In clusters with more than a single iron atom, there are two or more tz-sulfido bridges with cysteine completing the tetrahedral coordination geometry about the iron atoms. Histidine, aspartic acid, or serine may also provide the terminal ligating atoms rather than cysteine. An example of this is where one histidine coordinates to each iron in the [2Fe-2S] cluster in the Rieske center. 4° The facile interconversion of [3Fe-4S] I+'° and [4Fe-4S] 3+,2+;2+'1+ has also been demonstrated by EPR spectroscopy. Proteins containing these clusters have been shown to be involved not only in electron transfer, but also in catalyzing biochemical reactions of, for example, aconitase41 and lactoyl-CoA dehydratase. 42 Representative spectra from the paramagnetic spin states are shown in Fig. 3. Spectra of the [Fe-Cys4] ~- sites in rubredoxins consist of resonances around g = 4.3, typical of highspin iron(Ill) in a tetrahedral environment where the resonance occurs within the middle paramagnetic doublet state (see Fig. 2). Antiferromagnetic coupling between the iron(II) (S] = 2) and iron(Ill) ($2 = ~) ions in the [2Fe-2S] ~+ cluster yields the following spin states, ST = 1S1 + s21,1sl+s2 II, . . . , Is -s l,=½, 2, ~, -z, ' and ~, the lowest state being S = ½. For the [2Fe-2S] 1+ clusters, the energy of first excited state (~J > 200 cm -1, for the plant proteins) is sufficiently large that thermal population of the excited spin states does not complicate the EPR spectrum. For these systems, g factors ranging from approximately -

37 R. Cammack, D. S. Patil, and V. M. Fernadez, Biochem. Soc. Trans. 13, 572 (1985). 38 H. Beinert, Biochem. Soc. Trans. 13, 542 (1985). 39 R. Cammack, Adv. Inorg. Chem. 38, 281 (1992). 4o R. J. Gurbiel, C. J. Batie, M. Sivaraja, A. J. True, J. A. Fee, B. M. Hoffman, and D. P. Ballou, Biochemistry 28, 4861 (1989). 4l H. Beinert and M. C. Kennedy, Eur. J. Biochem. 11t6, 5 (1989). 42 R. D. Kuchta, G. R. Hanson, B. Holmquist, and R. H. Abeles, Biochemistry 25, 7301 (1986).

344

PROBES OF METAL ION ENVIRONMENTS 2.2 1

g factor 2.0 1.9

2.1 I

1

1

r

l

I

[1 1]

1.8 1

I

I

(a) 2 [4Fe-4S] I+

\ (b) [3Fe-4S] ~+

-

~

-

[4Fe-4S]l+

(d) [4Fe-4S] 3÷

2Fe-2S]l +

I

0.30

a

J

0.32

I

J

0.34

I

i

0.36

I

a

0.38

B(T) FIG. 3. Representative EPR spectra for iron-sulfur clusters in ferredoxins. (a) CIostridium pasteurianum; (b) Desulfooibrio gigas; (c) Bacillus stearothermophilus; (d) Chromatium oinosum high potential iron-sulfur protein (HIPIP); and (e) Mastigocladus laminosus. All spectra were recorded at X-band frequencies at temperatures between 10 and 20 K. (Adapted from Ref. 37, with permission.)

1.8 to 2.2 are found. The reader is referred to the article by Bertini e t ai. 43 for a description of the antiferromagnetic coupling in the more

complex [3Fe-4S] 1÷'° and [4Fe-4S] 3+'2+;2+'1+ clusters. There have been reports of a new [6Fe-6S] 6+'5+;5+'4+'4+'3+ cluster in 43 I. Bertini, F. Briganti, and C. Luchinat, lnorg. Chim. Acta 175, 9 (1990), and references therein.

[11]

ELECTRONPARAMAGNETICRESONANCE

345

ferredoxins purified from Desulfovibrio vulgaris 44 strain Hildenborough and Desulfovibrio desulfuricans 45 (ATCC, Rockville MD, strain 27774). The fully oxidized protein (+6 state) appears to be diamagnetic. In the +5 state the cluster exists in two magnetic forms: 10% is low-spin S = ½, and the remainder is in a high-spin S = g spin state. A one-electron reduction yields a g ~ 16 resonance, presumably from an S = 4 spin state. Further reduction to the +3 state yields a mixture of two S = 1 ground states. EPR spectra of the iron-sulfur clusters are observed only at temperatures below about 50 K because of rapid spin-lattice relaxation at higher temperatures. In addition, higher spin multiplets will be populated at higher temperatures. At low temperatures the signal strength is proportional to 1/T, as may be inferred from Eq. (7) for isolated paramagnetic doublets. In proteins with more than one cluster, resolution of the signals from each cluster often requires variation of the temperature, microwave power, and redox potential. Experimental

Instrumental Factors To observe EPR the sample is placed in a resonant microwave cavity in an electromagnet and irradiated with microwaves. EPR has traditionally been carried out by fixing the microwave frequency, usually X-band or 9-10 GHz, and by varying the magnetic field. Historically, the second major frequency used in EPR was 35 GHz (Q-band) as it was traditionally believed it would always provide greater g factor resolution. However, at Q-band microwave frequencies the spectral line widths are frequently larger than those observed at X-band frequencies. The line width variation as a function of microwave frequency has been interpreted in terms of a statistical distribution of spin Hamiltonian parameters. 46'47 A correlated g and A strain 48-5° model provides a simplified picture of the line width variation as a function of the nuclear spin quantum number and the micro44 A. J. Pierik, W. R. Hagen, W. R. Dunham, and R. H. Sands, Eur. J. Biochem. 206, 705 (1992). 45 I. Moura, P. Tavares, J. J. G. Moura, N. Ravi, B. H. Huynh, M.-Y. Liu, and J. LeGall, J. Biol. Chem. 267, 4489 (1992). 4~ W. R. Hagen, D. O. Hearshen, R. H. Sands, and W. R. Dunham, J. Magn. Reson. 61, 220 (1985). 47 W. R. Hagen, D. O. Hearshen, R. H. Sands, and W. R. Dunham, J. Magn. Reson. 61, 233 (1985). 48 W. Froncisz and J. S. Hyde, J. Chem. Phys. 73, 3123 (1980). 49 j. R. Pilbrow, J. Magn. Reson. 58, 186 (1984). 50 j. S. Hyde and W. Froncisz, Annu. Rev. Biophys. Bioeng. 11, 391 (1982).

346

PROBES OF METAL ION ENVIRONMENTS

[11]

wave frequency for S = ½ and I > 0 systems. In a simple form of the theory, the important equation describing the anisotropic line width o-i, ( i = x , y , z ) is z ori2 = O'Ri2 + [Agi/g i ~,o(n) + AAiMI]2

(14)

The O-Riterm represents residual line widths arising from dipolar broadening and/or unresolved metal and ligand hyperfine interactions, whereas A g / g and AA terms represent the half-widths of the strain-induced distributions of the g and A values. An important consequence of Eq. (14) is that increased resolution can be obtained at lower microwave frequencies. In particular, it is found that the best resolution for many type 2 copper proteins and inorganic compounds lies somewhere in the range 2-6 GHz. There are a number of experimental and instrumental aspects that can affect the line shape of an EPR spectrum. These include the choice of the modulation frequency and modulation amplitude, microwave power, quality factor of the cavity, sample temperature, and sample preparation. Although adequate descriptions of these effects have been provided in the books by Swartz et al. 3 and Pilbrow, 2 it is pertinent to provide a brief description of the optimum conditions for measuring EPR spectra. Normally a modulation frequency of 100 kHz is employed in EPR. However, when the line width of the paramagnetic resonance is very small, side bands can occur that can be eliminated by reducing the modulation frequency. Optimum resolution is obtained when the modulation amplitude is no larger than one-tenth of the line width. Unfortunately, for very weak spectra with poor signal-to-noise ratios higher modulation amplitudes must be employed to improve the signal-to-noise ratio. Clearly there is a trade-off between spectral resolution and the ability to detect the spectrum in the first instance. For most detectors the signal intensity is proportional to the square root of the microwave power (P). On increasing the microwave power the population difference between the two levels in a Kramers doublet decreases, causing a decrease in the signal amplitude. PI/2 is defined as being half the microwave power required to saturate an EPR resonance and is characteristic of the paramagnetic species. Under certain conditions P1/2 c a n be related to the spin-lattice relaxation time T1.24 The quality factor Q0, for a cavity, or other microwave resonator, is defined as the ratio of microwave energy stored over the amount energy dissipated and is directly proportional to the signal intensity. Typically for X-band rectangular cavities Q is of the order of 2000-5000. The predominant factor affecting the magnitude of Q is the introduction of dielectric materials into the cavity that can absorb the electric component ol" the microwave energy. Clearly, this presents a problem for measuring aqueous solutions, which have a significant microwave dielectric constant at room

[11]

ELECTRON PARAMAGNETIC RESONANCE

347

temperature. The use of "flat cells" or 1 mm sample tubes at X-band microwave frequencies reduces absorption of microwaves to an acceptable level, though Q will be reduced causing a decrease in the ultimate sensitivity. Dielectric loss is not a problem at low temperatures or at lower microwave frequencies. The factors governing the choice of temperature have already been mentioned.

Sample Preparation Sample preparation is not generally a problem for biological materials as aqueous solutions yield good glasses when frozen. Moreover, it is almost impossible to have protein concentrations greater than 2 mM where intermolecular dipole-dipole broadening can occur. Even at these high concentrations the diamagnetic protein backbone will prevent these magnetic interactions from occurring. It is important that samples be frozen outside the magnet because, in highly anisotropic systems, the electron magnetic moments can be partially aligned, leading to artifacts in the spectrum owing to unknown spatial distributions of the molecules. When dealing with frozen samples researchers should be aware that EPR tubes can explode as a result of ice expansion or trapped liquid oxygen. In our laboratories, samples are frozen gradually so that on thawing the sample the ice expands up the tube and not outward. Even with these precautions it is possible for tubes to explode, and in these cases it is necessary to plunge the tubes into boiling water for an instant to thaw the sample against the walls of the tube.

Rapid Freeze Quenching Rapid freeze quenching is a technique by which a reaction mixture can be frozen very quickly ( - 3 msec) and subsequently characterized by a range of spectroscopic techniques, including EPR spectroscopy. This technique has proved to be a very useful method for trapping enzyme-substrate intermediates. The basic instrumentation includes a stop-flow instrument equipped with an exit jet where the solution is sprayed onto the surface of an isopentane-liquid nitrogen slush bath. The frozen droplets are collected and packed into an EPR tube for subsequent study. This method has allowed various intermediates [very rapid, rapid type I, and rapid type II molybdenum(V) signals] to be characterized in the oxidation of xanthine to uric acid catalyzed by xanthine oxidase.

Isotope Substitution The observation of both metal and ligand hyperfine coupling can greatly aid the characterization of metal binding sites in metalloproteins. For

348

[1 1]

PROBES OF METAL ION ENVIRONMENTS

9.1125 GHz

I 3080

I

I

~

i 3180

I

I

r

r

I 3280

I

I

I

I 3380

I

I

I

I 3480

Gauss

FIG.4. Rapidtype 1 signalgeneratedfrom l-methylxanthinerecordedat 150 K, showing the signalfrom native xanthineoxidase. [Reproducedwith permissionof Dr. G. L. Wilson from Ph.D. Thesis, Latrobe University(1988).]

instance, the relative abundance of molybdenum isotopes with a nonzero nuclear spin (I = ~) is 25%, and consequently the intensity of the hyperfine lines is less than 10% of that for the I -- 0 isotopes (Fig. 4). 95Mo isotope enrichment (to -80%) provides greater sensitivity and resolution of the hyperfine resonances (Fig. 5a). Multifrequency EPR in conjunction with computer simulation studies (Fig. 5a-c) has identified a monoclinic molybdenum site for the rapid type I intermediate with the following g and A principal values: gl, 1.9890; g2, 1.9699; g3, 1.9647; Aj, 61,7; A2, 24.8; A 3 , 24.8 × 10 -4 cm-]; with the angle between the g] and A 3 directions, a]3, being 20°. The spectra in Fig. 5 also show coupling to two distinct deuterium (I = 1) exchangeable protons. The use of ]70, ]3C, and 338 isotopes in the characterization of molybdenum(V) intermediates and model complexes {[MoO2L]-, cis-[MoO(OH)L], [MoOSL]-, and cis-[MoO(SH)L], where L H 2 = N,N'-dimethyl-N,N'-bis(2-mercaptophenyl)ethylenediamine} for these sites has provided the structures and reactions (Scheme II) for the intermediates observed during the oxidation of xanthine to uric acid. Analysis of Electron Paramagnetic Resonance Spectra and Determination of Spin Hamiltonian Parameters An important step in the characterization of metal binding sites is the accurate determination of the anisotropic spin Hamiltonian parameters.

[11]

ELECTRON PARAMAGNETICRESONANCE

349

[Mo(IV)O(SH)(OR)] +

RH [Mo(VI)OS(RH)I

[Mo(Vl)OS]

-

e-

- e-,

[Mo(V)OS(OR)I Very Rapid

- RH

[MoO(SH)(OR)] Rapid

- ROH

/HH+

SCHEME II

Computer simulation of the anisotropic EPR spectra is a method of quantifying complex overlapping spectra. In addition, second-order hyperfine contributions, ligand hyperfine, quadrupole, nuclear Zeeman, and lowsymmetry effects add to the complexity of the spectrum. Simulation of the anisotropic EPR spectrum (S) measured as a function of magnetic field (B) and at a constant frequency (vc) is performed using Eq. (15)2: 2 ¢r/2 n'/2

I

S( c, ):cZZ Z Z Z i=1 O=O•=O M I = - I M N aib(M N) (gl2)f[vc - vo(B), o"u] A cos 0 A•

(15)

where the constants C and ai involve experimental parameters and the natural abundance of the metal ion isotopes which have a nonzero nuclear spin; for example, the two copper isotopes 63Cu and 64Cu have I = ~. The b(MN) terms are the binomial coefficients for magnetically equivalent nitrogens, namely, 1 : 3 : 6 : 7 : 6 : 3 : 1 for three equivalent nitrogens and 1 : 4 : 10 : 16 : 19 : 16 : 10 : 4 : 1 for four equivalent nitrogens. (gl 2) is the spatially averaged expression for the transition probability s~ needed in powder simulations, and f is the Gaussian line shape function. In the line shape function f, vo(B), multiplied by Planck's constant, is the actual energy difference between the energy levels and is evaluated by solving the eigenvalue problem with an appropriate spin Hamiltonian [Eq. (9)]. The fundamental reason for using Eq. (15) is given elsewhere. 2'49 The line width, o-~, as a function of the polar angles 0 and ~, required for randomly orientated (frozen solution) spectral simulations, is assumed to behave in an analogous manner to hyperfine structure. Correlated g and A strain in frequency space is used to calculate the actual line width (o i, i = x, y, z) as a function of vo(B) and the nuclear spin quantum number M I [Eq. (14)]. 51 j. R. Pilbrow, Mol. Phys. 16, 307 (1969).

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,, !,

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[11]

ELECTRON PARAMAGNETICRESONANCE

351

Further details concerning the use of computer simulation in the analysis of anisotropic and isotropic EPR spectra may be found in a book by one of the authors. 2 The International EPR Society has established a database of existing computer simulation programs, a copy of which may be obtained from Professor R. Cammack. 52 Spin Quantitation Spin concentrations may be determined by either the "absolute" or "comparison" methods. The accuracy to which the spin concentration measurement can be made is governed by a number of experimental parameters and a knowledge of the transition probability. A comprehensive discussion of the factors effecting the spin concentration measurements has been published. 53'54 By far the most commonly used technique is the comparison method, which is described below. The comparison method relies on a comparison of the EPR signal intensity of an unknown compound I(Exp), with that of a reference compound I(Exp)r. Providing the resonant cavity is critically coupled and identical sample geometries, via the filling factor ('0) and the loaded quality factor (QoL), are employed, the number of spins in an unknown sample (Nou) can be related to the number of spins in a reference sample (Nor) through . Uoo =

c;lvijl 2

I(Exp)u ×

(16)

where C,=

T S(S + 1) RG MA p1/Z

52 Department of Biomolecular Sciences, King's College, Hill Road, Camden, London W8 7AH, UK. E-mail address: [email protected]. 53 S. S. Eaton and G. R. Eaton, Bull. Magn. Reson. 1, 130 (1979). 54 M. L. Randolph in "Biological Applications in Electron Spin Resonance" (H. M. Swartz, J. R. Bolton, D. C. Borg, eds.), p. 119. Wiley (Interscience), New York, 1972.

FIG. 5. Experimental ( ) EPR spectra and simulations ( - - - ) of the l-methylxanthine rapid type I signal from 9SMo-enriched xanthine oxidase (95Mo, 70-80 atom %) in IH20 buffer, 150 K, recorded at (a) 9.113 GHz, (b) 3.591 GHz, and (c) 2.3139 GHz. Arrows indicate the resonance position of the F A D H ' radical. [Reproduced with permission of Dr. G. L. Wilson from Ph.D. Thesis, Latrobe University (1988).]

352

PROBES OF M E T A L ION E N V I R O N M E N T S

[11]

The subscripts u and r refer to the unknown and reference samples, respectively, and I V,TI2 is the transition matrix element squared and is proportional to the product of the anisotropic transition probability (g2) and I(ils+_lJ>l 2. c ' is a normalization constant involving the instrument settings: receiver gain (RG), modulation amplitude (MA), microwave power (P), temperature (T), and the electron spin (S), assuming the sample obeys the Curie law. Because the double integral of a computer-simulated spectrum is directly proportional to the anisotropic transition probability 2 [Eq. (15)], we can replace I v,jI 2 in Eq. (16) with the double integral of the computersimulated spectrum. Thus, the number of spins in an unknown sample (N0u) can be related to the number of spins in a reference sample (Nor):

I(Exp)u N0u = Nor C,ui(Sim)u

×

C~I(Sim)r l(ExP)r

(17)

where I(Exp) and I(Sim) are the doubly integrated experimental and simulated spectra. It is very important to have identical sample geometries (7/) and Q0L factors for both the standard and unknown samples when determining spin concentrations using the comparison method. One way of ensuring this is to combine both the standard and the unknown samples in a single EPR tube and measure their signals. However, more often than not, the EPR signals overlap, and this creates difficulties in the determination of the intensity of each component. This may be overcome by either successive sample replacement in a single cavity or by the use of a double cavity. Obtaining identical sample geometries ('0) and Q0L factors with the successive sample replacement method is quite difficult and can lead to large errors in the determination of spin concentrations. By far, the preferred method is that of the double cavity (normally a dual TEio4 rectangular cavity is employed). One important point which should be noted when using a dual cavity is that the individual cavities may have different modulation amplitude calibrations and microwave magnetic field strengths (B~). The former is simply solved by calibrating each cavity, whereas the latter can be overcome by first measuring the standard and unknown samples in the front and back cavities and then swapping the samples and repeating the measurement. Thus, the equation for determining the spin concentrations becomes

r

_i(Exp)ul _

Nou = Nor /LC'~lI(Sim)~l ×

Crli(Slm)r 1 ,• i(Exp)u2 C~21(Sim)r2]1/2 × × i(ExP)rl C,u2i(Sim)u2 I(EXP)r2 J, (18)

[12]

EPR SPECTROSCOPYOF IRON

353

where the subscripts 1 and 2 refer to the front and back cavities, respectively. An important consequence of this method is that the EPR spectrum of the standard sample should be well understood. The use of solid copper(II) sulfate is not recommended as there are dipole-dipole interactions between the copper(II) ions in addition to the interactions described in Eq. (9), and the quality factor of the cavity will be different from that of aqueous frozen solution samples. A good choice is the copper(II) complex with ethylenediaminetetraacetic acid (EDTA). Conclusions EPR spectroscopy as a technique for characterizing metal binding sites in metalloproteins has proved invaluable. The theory presented here for the analysis of the spin Hamiltonian parameters and the method of spin quantitation should provide the reader with an introduction to the use of EPR spectroscopy in metalloprotein research. However, the reader is strongly advised to read the relevant literature in order to gain a full understanding of the technique. In short, the future looks bright for EPR in the characterization of metal binding sites in metalloproteins.

[12] E l e c t r o n P a r a m a g n e t i c R e s o n a n c e S p e c t r o s c o p y of Iron Complexes and Iron-Containing Proteins

By RICHARD CAMMACK and CrtRISTOPHER E. COOPER Introduction Among the transition metal ions in biochemistry, iron has the richest variety of electron paramagnetic resonance (EPR) spectra. These include spectra of low-spin and high-spin ferric iron and of metal clusters. This chapter concentrates on practical aspects of the detection, identification, and quantitation of iron-containing proteins in biochemical systems, including enzymes, other proteins, and whole tissues. The reader is referred to previous articles in this series on the general principles of EPR by Palmer ~ and on transition metal EPR by Fee 2 and Pilbrow and Hanson. 3 A number of preparative methods have been de1 G. P a l m e r , this s e r i e s , Vol. 10, p. 594. 2 j . A. F e e , t h i s s e r i e s , Vol. 49, p. 512. 3 j . R. P i l b r o w a n d G. R. H a n s o n , this v o l u m e [11].

METHODS 1N ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All fights of reproduction in any form reserved.

[12]

EPR SPECTROSCOPYOF IRON

353

where the subscripts 1 and 2 refer to the front and back cavities, respectively. An important consequence of this method is that the EPR spectrum of the standard sample should be well understood. The use of solid copper(II) sulfate is not recommended as there are dipole-dipole interactions between the copper(II) ions in addition to the interactions described in Eq. (9), and the quality factor of the cavity will be different from that of aqueous frozen solution samples. A good choice is the copper(II) complex with ethylenediaminetetraacetic acid (EDTA). Conclusions EPR spectroscopy as a technique for characterizing metal binding sites in metalloproteins has proved invaluable. The theory presented here for the analysis of the spin Hamiltonian parameters and the method of spin quantitation should provide the reader with an introduction to the use of EPR spectroscopy in metalloprotein research. However, the reader is strongly advised to read the relevant literature in order to gain a full understanding of the technique. In short, the future looks bright for EPR in the characterization of metal binding sites in metalloproteins.

[12] E l e c t r o n P a r a m a g n e t i c R e s o n a n c e S p e c t r o s c o p y of Iron Complexes and Iron-Containing Proteins

By RICHARD CAMMACK and CrtRISTOPHER E. COOPER Introduction Among the transition metal ions in biochemistry, iron has the richest variety of electron paramagnetic resonance (EPR) spectra. These include spectra of low-spin and high-spin ferric iron and of metal clusters. This chapter concentrates on practical aspects of the detection, identification, and quantitation of iron-containing proteins in biochemical systems, including enzymes, other proteins, and whole tissues. The reader is referred to previous articles in this series on the general principles of EPR by Palmer ~ and on transition metal EPR by Fee 2 and Pilbrow and Hanson. 3 A number of preparative methods have been de1 G. P a l m e r , this s e r i e s , Vol. 10, p. 594. 2 j . A. F e e , t h i s s e r i e s , Vol. 49, p. 512. 3 j . R. P i l b r o w a n d G. R. H a n s o n , this v o l u m e [11].

METHODS 1N ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All fights of reproduction in any form reserved.

354

PROBES OF METAL ION ENVIRONMENTS

[12]

scribed by Ballou 4 (rapid-freezing), Beinert et al. 5 (low-temperature spectroscopy), Beinert 6 (mitoch0ndrial electron transfer proteins), Dutton 7 (redox potentiometry), and Cammack 8 (cyanobacterial iron-sulfur proteins). Other helpful reviews of the EPR technique include those on general instrumental methods by Eaton and Eaton, 9 on EPR of biological systems by Sealy et al. 1° and Kosman and Bereman, 11 and on iron proteins by Smith and Pilbrow.12 The theory of the EPR of transition ions has been reviewed in a book by Pilbrow. t3 The principle of EPR spectroscopy is similar to that of nuclear magnetic resonance (NMR), but using electrons instead of nuclear spins. The unpaired electrons lead to paramagnetism in the sample. The differences between the two techniques are mainly due to the much larger magnetic moment of the electron, compared with the proton. The conventional EPR spectrometer employs a microwave cavity, in which the sample sits, that resonates at a fixed microwave frequency, v. This allows a considerable enhancement of the microwave field, and hence sensitivity, but prevents the acquisition of a spectrum in the conventional way by measuring absorption as a function of frequency. Instead, the applied magnetic field, B o, is swept, by means of an electromagnet. The energy (AE) at which resonant absorption by an unpaired electron occurs is AE=

hv = g~aBo

where the proportionality constants h and/-~B are Planck's constant and the Bohr magneton for the electron, respectively. Thus the resonance magnetic field (Bo) is inversely related to the g factor, a spectroscopic 4 D. P. Ballou, this series, Vol. 54, p. 85. 5 H. Beinert, W. H. Orme-Johnson, and G. Palmer, this series, Vol. 54, p. l 11. 6 H. Beinert, this series, Vol. 54, p. 133. 7 p. L. Dutton, this series, Vol. 54, p. 411. s R. Cammack, this series, Vol. 167, p. 427. 9 G. R. Eaton and S. S. Eaton, in "Analytical Instrumentation Handbook" (G. W. Ewing, ed.), p. 467. Dekker, New York, 1990. l0 R. C. Scaly, J. S. Hyde, and W. E. Antholine, in "Modern Physical Methods in Biochemistry, Part A " (A. Neuberger and L. L. M. V. Deneen, eds.), p. 69. Elsevier, Amsterdam, 1985. 11 D. Kosman and R. Bereman, in "Spectroscopy in Biochemistry" (J. E. Bell, ed.), p. 57. CRC Press, Boca Raton, Florida, 1981. t2 T. D. Smith and J. R. Pilbrow, in "Biological Magnetic Resonance" (L. J. Berliner and J. Reuben, eds.), p. 85. Plenum, New York, 1980. z3 j. R. Pilbrow, "Transition Ion Electron Paramagnetic Resonance." Oxford Univ. Press, Oxford, 1990.

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E P R SPECTROSCOPY OF IRON

355

parameter that is characteristic of the paramagnetic iron complex studied. The g factor is easily calculated by rearranging the above equation: g - 71.4484v _ _ B0

where B 0 is in millitesla (mT) and v is in gigahertz (GHz). The characteristic appearance of the EPR spectrum derives partly from the technical necessity for sweeping the magnetic field rather than the microwave frequency; this has consequences for the simulation and integration of EPR spectra. It has the effect of making the high-field (low g factor) features appear broad compared with those at low field. Moreover, in order to improve sensitivity, the EPR spectrum is acquired by modulation of the applied magnetic field B 0 and phase-sensitive detection. As a result the spectrum is obtained as the first derivative or first harmonic. For iron proteins, low temperatures (liquid nitrogen or liquid helium) are normally required to observe an EPR spectrum. Characteristics of Electron Paramagnetic Resonance Spectra To observe resonant absorption in EPR, there must be two energy levels differing in energy by by, and the transitions between the levels must be allowed. A combination of different electrostatic and magnetic interactions between electrons and nuclear spins gives the EPR spectra of iron their characteristic shapes. The major contributions to the splittings between the energy levels available to the unpaired electron are as follows (Fig. 1).

Spin-Orbit Coupling. Spin-orbit coupling interactions with the electrons of other orbitals of the iron atom and its ligands (fine structure) cause the effective g factor to differ from the free-electron value of 2.0032. Zeeman Interaction. Magnetic interactions with the applied field (Zeeman interaction) normally gives rise to the EPR phenomenon. Electrons aligned with the applied magnetic field have a lower energy than those aligned against it; microwave quanta are of the right energy to stimulate transitions from one state to the other. Zero-Field Splitting. If there is more than one unpaired electron in the ion, as in high-spin Fe m, electrostatic interactions between the unpaired electrons give rise to zero-field splittings. Thus, even in the absence of an applied magnetic field there are differences in energy between the unpaired electrons. The magnitude of the zero-field splittings is often so great that the energy levels are split by more than the energy of a microwave quantum, so as to prevent resonance being observed. Fortunately,

356

PROBES OF METAL ION ENVIRONMENTS

[12]

MS

+s/2

/

L

iI iI / iI iI

4D

iI iI

~ "- ±3/2

Increasing Bo field

Zero-field splitting

Zeeman interaction

Hyperfme splitting

FIG. 1. Illustrations of the effects of splittings of the energy levels of unpaired electrons in high-spin Fem. The diagram is not to scale; the hyperfine interactions are much smaller (of the order of 10 MHz) compared with the Zeeman splitting (9 GHz at X-band) and the zero-field splittings (D = 300 GHz for metmyoglobin).

for any paramagnetic state with an odd number of unpaired electrons an EPR spectrum is always in principle detectable. 13 This is not necessarily the case if there is an even number of unpaired electrons. Hyperfine Interaction. Magnetic interactions with nuclear spins give rise to the hyperfine interaction. In general, interaction with a nuclear spin I will cause a splitting into (21 + 1) lines. Thus, two lines should be observed for the iron isotope 57Fe, which has a nuclear spin I = ½. Superhyperfine Interaction. Magnetic interactions with ligands to the iron that have nuclear spins also cause splittings (sometimes called super-

[12]

EPR SPECTROSCOPYOF IRON

357

hyperfine interactions); the most important is 14N (I = 1) which splits the EPR spectra into three lines. In this case the ligand can be identified by substituting with one containing ~SN (I -- ½), when only two lines will be observed. The hyperfine interactions are often so weak that they are less than the line width of the spectrum, which prevents their detection by conventional continuous wave EPR. They may, however, be detected by electron nuclear double resonance (ENDOR) spectroscopy or by electron spin echo envelope modulation (ESEEM) in pulsed EPR. Dipole-Dipole and Exchange Interactions. Interactions with other unpaired electrons in the protein over distances of the order of 5-15 ,~ result in changes in the line shape of the spectrum owing to dipole-dipole and exchange interactions. At closer distances (<3 ,~) the exchange interaction dominates, resulting in a coupled spin system (see below). The first five types of interactions above are anisotropic, which means that the magnitude of the splittings depends on the orientation of the molecular axes to the applied B0 field. In most biological samples we have no control over this orientation as the samples consist of frozen proteins in a random orientation. The spectrum is thus distributed over a range of magnetic field. Zero-field splittings in particular give rise to extremely anisotropic splittings, so that for ions with S > 1 the spectrum may be spread over a wide range of B 0 field (see below).

Chemistry of Iron in Relation to Magnetic Properties Iron occurs in at least three oxidation states in biology, FeII (ferrous) with the d 6 electron configuration, d 5 Fem (ferric), and d 4 Fe TM (ferryl). Of these, only Fe xIIhas an odd number of unpaired electrons, which means that it has degenerate electron levels which can be split by a magnetic field and are readily detectable by EPR. The other valence states may be magnetic, but it often happens that the energy level splittings are greater in magnitude than the energy of the microwave quantum, hu, and the spectra are undetectable. The spin state of the iron ions has a great influence on the EPR spectra (Fig. 2). The d electrons, each have a spin S -- ½, are distributed among five orbitals. Because of electrostatic repulsion there will be a tendency for them to occupy different orbitals. If they do, the ion is said to be in the high-spin state. Thus, for Fe m, the five electrons would each occupy a different orbital, giving a net spin S = ~. For FeII, two of the six electrons pair up, giving a net spin S -- 2. However, interactions with the ligand field causes the d energy levels to be separated. If the ligand field is strong enough, the electrons will prefer to pair up in the levels of lowest energy.

358

[12]

PROBES OF METAL ION ENVIRONMENTS

Octahedralcoordination

Tetrahedralcoordination A

,dl& k"

X

i

y

x

High spin []

Fe

II

Fe

es

Total spin, S = T5 Weak ligand field

Hi.gh spin

Low spin

eg

HI

H

Fe

Fe

1

0

F~ u Fff

~g

"TA

2

2

±

2

2

Strong ligand field

FI~. 2. d electron energy levels and electron distributions for iron in octahedral and tetrahedral coordination.

This is the low-spin case. Low-spin Fe Itl and FeII have total spins S = ½ and S = 0, respectively. An intermediate spin state, S -- 3, is possible but rare. The magnitude of the ligand-field splitting A, which determines the spin state, depends on the types of ligands and their geometry. Iron in a tetrahedral coordination is always high spin. In octahedral coordination, which approximates the situation in heme proteins, iron may be either high spin or low spin, depending on the nature of the axial ligands. Spin state interconversions depending on substitution of the axial ligand are common, as in cytochrome P-450, which changes from low spin in the substrate-free form to high spin in the substrate-bound form. 14 It is even possible for the iron to change its spin state (from high to low) on cooling from room temperature to liquid helium temperature; this change, for example, in hemoglobin, may be observed by optical spectroscopy as well as by magnetic susceptibility measurements. 15 14 S. Sligar, Biochemistry15, 5399 (1976). 15 T. Iizuka and M. Kotani, Ado. Biophys. 1, 157 (1970).

[12]

E P R SPECTROSCOPY OF IRON

359

For high-spin Fe nI, the five unpaired d electrons couple together to give three doublets, with S = -½, ___a, ___~. The extent to which these electron energy levels are populated depends on the zero-field splittings between the levels and on the temperature (see below).

Information Obtained from Electron Paramagnetic Resonance Spectroscopy

Identification of Iron as Responsible for Spectra The identification of an EPR spectrum of an unknown paramagnet as arising from an iron compound is assisted by enrichment with the isotope 57Fe (I = ½; natural abundance 2.2%). Unlike radioactive labeling, a high proportion of the isotope must be introduced. The techniques to do this are the same as used in M6ssbauer spectroscopy. In a protein it may be done by removal of iron and reconstitution with the iron isotope, or, if that is not possible, by growing the organism on a growth medium containing the isotope. If the spectrum is narrow, substitution with 57Fe may cause a significant hyperfine broadening, as observed in the ferredoxins 16 and in the nickel-iron cluster of carbon-monoxide oxidoreductase.17 The magnitude of the splitting depends on the extent of the spin polarization onto the iron nucleus, that is, the extent to which the nucleus " s e e s " the unpaired electron. If the spectrum is broad, the hyperfine interactions may instead be observed by ENDOR. 18The number of iron nuclei associated with the paramagnetic center may be determined by simulation of the spectrum. In ENDOR, each distinct 57Fe interaction will give rise to a separate pair of lines, which can be distinguished.

Quantitation of Iron in Sample Unlike many forms of spectroscopy, such as optical spectroscopy, EPR spectra are usually readily quantifiable from first principles, that is, there is no extinction coefficient for an EPR spectrum. This makes it a very valuable tool in determining the stoichiometry of metal centers in 16 j. C. M. Tibris, R. L. Tsai, I. C. Gunsalus, W. H. Orme-Johnson, R. E. Hansen, and H. Beinert, Proc. Natl. Acad. Sci. U.S.A. 59, 959 (1968). 17 S. W. Ragsdale, H. G. Wood, and W. E. Antholine, Proc. Natl. Acad. Sci. U.S.A. 82, 6811 (1985). Is B. M. Hoffman, Accts. Chem. Res. 24, 164 (1991).

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PROBES OF METAL ION ENVIRONMENTS

[12]

an enzyme, especially as, in contrast to atomic absorption, the method is selective for only those iron molecules contributing to the characteristic EPR signal of the protein.

Redox Potential of Iron Center A method of estimating the midpoint potentials of membrane proteins, by poising in the presence of mediators and measuring the EPR spectra, has been described by Dutton. 7 This method is also effective for soluble iron proteins in complex systems or where the optical spectra are difficult to detect. Usually if the iron protein is detectable in one oxidation state, for example, Fe nl, the other state is undetectable under the same EPR conditions. Thus, the EPR signal disappears or appears on reduction of the sample. This method can also be used to resolve overlapping EPR spectra of centers that have different redox potentials (see, e.g., Refs. 19 and 20).

Coordination State~Nature of Iron Ligands The EPR spectrum is a function of the electron distribution and geometry of the paramagnetic molecule. Some of the terms used to describe the shape of anisotropic EPR spectra (cubic, axial, or rhombic) and the coordination geometry (octahedral, tetrahedral, square-planar) derive from studies of transition ions in inorganic crystals. 21In principle, information about coordination geometry and types of ligands (e.g., histidine) can be derived from the shape of the EPR spectra. 22 Unfortunately, because of the distortions from ideal symmetry that occur in proteins, it is not possible to give a rigorous description of the g factors in terms of ligand field theory. However, theory can give useful insights when comparing, for example, the spectra of various types of low-spin ferric hemes.23 Electron Paramagnetic Resonance Properties of Different Types of Iron

Low-Spin Iron(Ill) For low-spin hemoproteins such as most respiratory cytochromes and substrate-free cytochrome P-450, the ligand field is strong enough to make i9 R. A. Rothery and W. J. Ingledew, Biochem. J. 261, 437 (1989). 2o S. W. Meinhardt, R. B. Gennis, and T. Ohnishi, Biochim. Biophys. Acta 975, 175 (1987). 21 A. Abragam and B. Bleaney, "Electron Paramagnetic Resonance of Transition Ions." Oxford Univ. Press, Oxford, 1970. 22 R. Calvo, J. Magn. Reson. 26, 445 (1977). 23 W. D. Biumberg and J. Peisach, in "BioInorganic Chemistry" (R. Dessy, J. Willard, and L. Taylor, eds.), p. 271. American Chemical Society, Washington, D.C., 1971.

[12]

EPR SPECTROSCOPYOF IRON

361

the iron low spin. In this case the g factors lie between 4 and 0. The reasons for these rather large excursions from the " t r u e " g factor of 2.0 for an S = ½ system are outside the scope of this chapter but have been explained in an accessible way by Palmer. 24 The anisotropy of the spectrum, given by the spread of g factors, varies considerably between proteins. The cytochromes P-450 have relatively small anisotropy (rhombicity), with g factors typically between 2.5 and 1.7. By contrast some btype cytochromes have highly anisotropic spectra (maximum rhombicity), with highest g factors approaching 4.0. The shape of the low-field line is sharply cut off near g = 4 and has been aptly described as a "folded line shape". 25 In that case the other g factors are usually so low as to be undetectable. Blumberg and Peisach 23 pioneered a method of plotting the EPR spectroscopic parameters of low-spin ferric hemoproteins, representing the rhombicity versus tetragonal (or axial) field, derived from the g factors. The position of the protein in the diagram was related to the nature of the axial ligands to the iron. Such diagrams are a useful indication of coordination of the iron site. However, such diagnoses require further confirmation since variations in g factors may also be the result of steric hindrance of the ligands. 26

High-Spin Iron(III) As stated previously the zero-field splitting in high-spin Fe uI (S = ~) significantly affects the g factor observed. The zero-field splitting may be described in terms of two parameters: D, the axial splitting, and E, the rhombic splitting. The coordinate axes for these terms may be chosen so that the ratio ~ = E/D can take values between 0 and ~. The case where h = 0 corresponds to an axially symmetric ligand field; the case where h = ~ corresponds to the state of maximum rhombicity (i.e., the least symmetrical). For a given spin state the expected g factors may be calculated as a function of h (Fig. 3). From the diagrams in Fig. 3 a number of properties are apparent, for example, that the maximum expected g factor is equal to 4S (10 in this case). For the case of axial symmetry there are prominent EPR features at gx = gy = 6 and gz = 2, arising from the S = ---½ doublets. An example is metmyoglobin (Fig. 4a). Where there is a small degree of rhombicity, the g factors gx and gy become split around 6, as in catalase (see Fig. 10) and the siroheme-containing nitrite reductase (Fig. 4b). As h increases, the spectrum broadens out, as in desulforedoxin (Fig. 24 G. Palmer, Biochem. Soc. £rans. 13, 548 (1985). 25 j. Salerno, Biochem. Soc. Trans. 13, 611 (1985). 26 F, A. Walker, D. Reis, and V. L. Balke, J. Am. Chem. Soc. 106, 6888 (1984).

362

PROBES OF METAL ION ENVIRONMENTS 10

[12] 9.68

8 6

1+5/2> 4 2 I

0

I

0.86 0.61

10

g factor

8

]+3/2>

6 4.29 4.29 4.29

0

~

I

I

10

9.68

8 6

I+_I12> 4

2

o86

0 / 0

a

=

0.11

0.22 X =

I 0.61 0.33

E/D

FIG. 3. Rhombogram for S = ~ (high-spin ferric iron). All possible g factors are represented by the three curves in each box. Each iron complex will have a specific value of ~; reading vertically up from this gives the value of the three principal g factors (gx, gy, and gz). The EPR spectrum of a randomly oriented frozen sample will therefore be distributed between the three g factors. Signals are possible from transitions not only in the lowest energy doublet (---½), but also in the higher doublets (_+~ and -+{). As the temperature increases the higher

[12]

EPR SPECTROSCOPYOF IRON

363

4c, h = 0.08), and may become progressively more difficult to detect. As the extreme rhombic case, h = ~, is approached, narrower signals appear at g = 4.29, arising from the S = ___adoublets, as in rubredoxin (Fig. 4d) and transferrin (Fig. 10). This is associated with a much weaker feature near g = 9.7, arising from the S = +--½doublet. ~The g = 4.3 lines are frequently sharp and intense, as all possible orientations of the iron protein (gx, gy, and gz) contribute to the signal. The majority of nonheme Fe tlI proteins are high spin. Their spectra commonly show high rhombicity, with g ~ 4.3. Some, such as lipoxygena s e , 27 a r e axial, and there are rare examples such as nitrile hydratase 28 that have low-spin Fem.

Ferrous-Nitrosyl Complexes As already stated, it is not usually possible to observe EPR signals from Fe n owing to large zero-field splittings in the high-spin state, and the absence of unpaired electrons in the low-spin state (although see Hendrich and Debrunner z9 for exceptions). However, it is possible to render such iron sites (both in heme and nonheme iron proteins) EPRdetectable by the addition of nitrogen monoxide (nitric oxide, NO) as a ligand. Because NO contains an unpaired electron, the resulting nitrosyl complex is usually an odd-electron system. EPR spectra of the complexes often show splittings into three lines owing to the 14N (I = 1) nucleus of the NO; the use of 15NO (I = ½) results in two lines and confirms that the splitting observed is due to nitrogen from the NO. To generate nitrosyl complexes the addition of nitrite and a reducing agent such as ascorbate (or dithionite) is usually sufficient. 3° The direct addition of NO to proteins under anaerobic conditions, while sometimes 27 S. Slappendel, G. A. Veldink, J. F. G. Vliegenthart, R. Aasa, and B. G. MalmstrOm, Biochim. Biophys. Acta 667, 77 (1981). 28 y . Sogiura, J. Kuwahara, T. Nagasawa, and H. Yamada, J. Am. Chem. Soc. 1119, 5848 (1987). 29 M. P. Hendrich and P. G. Debrunner, Biophys. J. 56, 489 (1989). 3o T. Yonetani, H. Yamamoto, J. E. Erman, J. S. Leigh, Jr., and G. H. Reed, J. Biol. Chem. 247, 2247 (1972).

doublets will become more populated and their signal sizes will increase. Any doublet that has any of its three g factors equal to 0 will not contribute to the EPR spectrum, as the line width will be infinite and therefore the intensity zero (e.g., no lines will be seen from -+3 and +_~)when h = 0). [Reproduced with permission from W. R. Hagen, Adv. Inorg. Chem. 38, 165 (1992).]

364

PROBES OF METAL ION ENVIRONMENTS

[12]

g factor 10 8 6 if i i i

5

4 [

3

2 i

"-v

i

i

i

i

i

i

i

50

100

150

200

250

300

350

i

400

Magnetic field, mT

FIG. 4. EPR spectra of high-spin ferric iron proteins. (a) Metmyoglobin; (b) Cucurbita pepo nitrite reductase; (c) Desulfooibrio gigas desulforedoxin; (d) D. gigas rubredoxin. Conditions of measurement: temperature 12 K, microwave power 20 mW, frequency 9.2 GHz, modulation amplitude 1 mT, modulation frequency 100 kHz.

[12]

EPR SPECTROSCOPYOF IRON

365

necessary, must be done carefully as NO is a highly reactive ligand and can displace other intrinsic ligands to the iron. 31 A signal that often appears in cell extracts or in enzyme preparations treated with nitrite (Fig. 5a) is the axial signal around g = 2.03, which is due to nitrosyl iron-sulfur complexes of the type Fe(RS)2(NO) 2. This signal is prominent because of its narrow width, and it can be detected even at room temperature. In some cases the NO is of biogenic origin. Another signal that may cause confusion is the spectrum of NO itself, which appears with a g factor just less than 2, detectable at helium temperat u r e s . 32

NO complexes o f h e m e iron (S -- ½) are usually centered around g -- 2, and their line shapes are often complicated by the presence of more than one species, as in the R-state of hemoglobin (Fig. 5b). If a three-line spectrum occurs around g = 2.00, this is often an indication that the ligand opposite the NO is either weakly bound or absent, as in T-state hemoglobin (Fig. 5c). This triplet spectrum is sometimes an indication of denatured protein. On the other hand, further triplet splitting of each of the three lines, giving nine lines in all, is a strong indication that the proximal ligand from the protein is a histidine (Fig. 5d). These nitrosyl iron complexes just described are derived from lowspin Fe H and thus have S = ½. However, there are some complexes that are derived from high-spin Fe n and have S = 3. These have broader spectra with gx ~ gy ~ 4, gz = 2. An example is nitrosyl lipoxygenase. 33 Another commonly encountered complex of this type is nitrosyl ferrous EDTA. This has a sharp axial spectrum with g = 4.0 and 2.0.

Coupled Spin Systems Iron-Sulfur Clusters. The iron-sulfur proteins that have been known for many years comprise clusters of iron and sulfide, coordinated directly to the protein by cysteine sulfur ligands. 6'8'34 These cysteine-ligated proteins generally yield one of two different types of EPR spectra. The first is a signal in the reduced state with average g factors around 1.96, as in [2Fe-2S) + or [4Fe-4S] + clusters of ferredoxins (Fig. 6a). The second occurs in the oxidized state around g = 2.01-2.04, as in [3Fe-4S] ÷ clusters or in the [4Fe-4S] 3÷ clusters of high-potential iron-sulfur proteins. The 31 R. H. Morse and S. I. Chan, J. Biol. Chem. 255, 7876 (1980). 32 T. H. Stevens, G. W. Brudvig, D. F. Bocian, and S. I. Chan, Proc. Natl. Acad. Sci. U.S.A. 76, 3320 (1979). 33 M. J. Nelson, J. Biol. Chem. 262, 12137 0987). 34 W. R. Hagen, Adv. lnorg. Chem. 38, 165 (1992).

366

[12]

PROBES OF METAL ION ENVIRONMENTS g factor 2.05

2

1.95

% b

I

I

I

I

I

305

315

325

335

345

Magnetic field, mT FIG. 5. EPR spectra of nitrosyl iron complexes. (a) Nitrosyi iron-sulfur complex; (b) R-state hemoglobin; (c) T-state hemoglobin (in the presence of inositol hexaphosphate); (d) nitrosyl HMP (E. coli hemoglobin-like protein). Conditions of measurement: (a-c) temperature 74 K, microwave power, 10 mW, frequency 9.2 GHz, modulation amplitude 0.3 mT, modulation frequency 100 kHz; (d) as for (a-c) except temperature 34 K, microwave power 2 roW, modulation amplitude 0.2 mT.

[12]

E P R SPECTROSCOPY OF IRON

367

Mixed-valence 2Fe clusters g factor 2.2

1.6

1.8

2

I

a Parsley ferredoxin

b Benzene dioxygenase

ygenase

I

t

I

I

I

I

[

290

310

330

350

370

390

410

Magnetic field, mT FIG. 6. EPR spectra of mixed-valence binuclear iron complexes. (a) Reduced [2Fe-2S] ferredoxin from parsley; (b) reduced"Rieske-type" [2Fe-2S] cluster in benzene dioxygenase of Pseudomonas putida; (c) methane monooxygenase of Methylococcus capsulatus (Bath); note that the latter spectrum has an additional signal from a free radical at g = 2.00. Temperature of measurement was 30 K for (a) and (b), 10 K for (c); other conditions of measurement were as follows: microwave power 20 mW, frequency 9.2 GHz, modulation amplitude 10 mT, modulation frequency 100 kHz.

368

PROBES OF METAL ION ENVIRONMENTS

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Rieske-type [2Fe-2S] clusters, which have S and N coordination from cysteine and histidine ligands, 35 are recognizable by their characteristic g factors, around g = 2.01, 1.90, and 1.80, in the reduced state (Fig. 6b). It is now known that there is greater variation in the structures of iron-sulfur clusters and that different types of iron-sulfur clusters can give a range of other EPR signals. For example, some reduced [4Fe-4S] clusters have odd-electron ground states with higher spins such as 6, z, and ~-, or are mixtures of such states. These have g factors up to 10, 14, or 18, respectively. 34 To detect the EPR signal from an unknown iron-sulfur protein, it is necessary to search over a wide range of magnetic field, and in different redox states. Iron-sulfur clusters usually have very efficient electron-spin relaxation mechanisms 36 and are typically detected at temperatures below that of liquid nitrogen. Exceptions are the rubredoxins and some of the reduced [2Fe-2S] ferredoxins (e.g., adrenal ferredoxin), which can be detected at temperatures near ambient, if sufficiently concentrated samples (>0.1 mM) are used. The reduced [3Fe-4S] clusters have even-spin states, probably S = 2, which in some cases are detectable as a broad line around g = 12. 37

Binuclear Oxygen-Bridged Iron Centers. In addition to the/x-sulfidobridged iron-sulfur clusters, there are proteins which contain /z-oxobridged iron dimers. These include the invertebrate oxygen carrier hemerythrin, the iron-containing methane monooxygenase, and purple acid phosphatase. Like the [2Fe-2S] ferredoxins these clusters are antiferromagnetically coupled, giving even-spin or zero-spin ground states for the fully reduced and fully oxidized proteins. The mixedvalence F e I I - F e m states are paramagnetic, with S = ½, like the [2Fe-2S] clusters. They may be distinguished from the iron-sulfur clusters in that they have all their g factors less than 2.038 (Fig. 6c). Moreover, they can readily be reduced to the FelI-Fe n state, and so the characteristic of their redox properties is that their signals appear as they are reduced, then disappear again on further reduction. In some cases, such as ribonucleotide reductase, the F e n - F e In state is relatively unstable and difficult to detect. 35 j. A. Fee, K. L. Findling, T. Yoshida, R. Hille, G. E. Tarr, D. O. Hearshen, W. R. Dunham, E. P. Day, T. A. Kent, and E. Miinck, J. Biol. Chem. 259, 124 (1984). 36 j..p. Gayda, P. Bertrand, A. Deville, G. More, J. F. Gibson, and R. Cammack, Biochim. Biophys. Acta 581, 15 (1979). 37 W. R. Hagen, W. R. Dunham, M. K. Johnson, and J. A. Fee, Biochim. Biophys. Acta 828, 369 (1985). 3s j. Sanders-Loehr, in "Iron Carriers and Iron Proteins" (T. M. Loehr, ed.), p. 373. VCH, Weinheim, 1989.

[12]

EPR SPECTROSCOPYOF IRON

369

An unexpected recent finding has been that the diferrous states of reduced binuclear iron complexes in deoxyhemerythrin azide, 39 methane monooxygenase,4° and ribonucleotide reductase 4~ all give intense EPR spectra at g ~ 16, detected at 4.2 K. The spectra are particularly pronounced when recorded in parallel mode (see later). These spectra have been interpreted 39as being due to a weakly ferromagnetically coupled pair of Fe H ions. It is also possible to see spectra with high g factors from coupled binuclear Fe-Cu centers (S = 2) in cytochrome o x i d a s e . 29'42,43 Polynuclear Iron. The presence of large numbers of ferric iron atoms can give rise to very broad (> 1,800 mT) EPR spectra that are both difficult to detect and difficult to quantify. Examples include the iron storage proteins ferritin and hemosiderin, 44 which can contain as many as 4000 iron atoms per molecule. These spectra contain both ferromagnetic (anisotropic) and superparamagnetic (isotropic) contributions arising from uncompensated spins in the iron core; it is necessary to understand the magnetic properties of the core as a whole to explain them. A somewhat simpler signal, isotropic around g -- 2, is observed from the superparamagnetic core iron in bacterioferritin molecules, although the line width is still very large. 45

Practical Considerations

Spectrometers Standard EPR spectrometers operate at X-band microwave frequency ( - 9 GHz). Microwave bridges are also commercially available to operate at microwave frequencies of S-band ( - 4 GHz) and Q-band (-35 GHz). The effect of these different frequencies is to alter the relative strengths of the effects of g factors and zero-field splittings (which scale with frequency) and hyperfine and electron spin-spin splittings (which are relatively insensitive to frequency). In general, lower frequencies are useful in resolving hyperfine interactions and higher frequencies in resolving g 39 M. P. Hcndrich, L. L. Pearce, L. Que,Jr., N. D. Chasteen, and E. P.Day~ J. Am. Chem. Soc. 113, 3039 (1991). 4o M. P. Hendrich, E. P. Munck, B. G. Fox, and J. D. Lipscomb, J. Am. Chem. Soc. 112, 5861 (1990). 41 j. B. Lynch, C. Juarez-Garcia, E. Munck, and L. Que, J. Biol. Chem. 264, 8091 (1989). 42 W. R. Hagen, Biochim. Biophys. Acta 708, 82 (1982). 43 C. E. Cooper and J. C. Salerno, J. Biol. Chem. 267, 280 (1992). 44 M. Weir, T. J. Peters, and J. F. Gibson, Biochim. Biophys. Acta 828, 298 0985). 45 M. R. Cheesman, F. H. A. Kadir, J. A1-Basseet, F. A1-Massad, J. Farrar, C. Greenwood, A. J. Thomson, and G. R. Moore, Biochem. J. 286, 361 (1992).

370

PROBES OF METAL ION ENVIRONMENTS

[12]

factors. At higher frequencies, the sensitivity should in principle increase with the Boltzmann factor, which increases the proportion of electrons that undergo resonant absorption. However, at the same time the size of the cavity and hence the sample size decrease. Tuning of Q-band resonators is also more difficult. Hence, unlike NMR, increased magnetic field does not generally lead to enhanced sensitivity. For lower frequencies the requirement for larger sample diameters has been overcome by the use of loop-gap resonators and other cavity designs with high filling factors. Conventional EPR spectrometers operate in such a way that the microwave field B~ applied to the sample in the cavity lies perpendicular to the static magnetic field B 0. For integer-spin paramagnets such as high-spin Fe n (S = 2), the EPR transition probabilities are often greatest when measured in parallel mode, BoI]B ~. Parallel-mode measurements require a special cavity, such as a bimodal cavity (see Hagen 4z) or alternatively a small cylindrical cavity which can be mounted on its side in the cryostat. Sample Preparation

The dielectric microwave loss of ice is much less than that of liquid water, so that relatively wide tubes can be used for low-temperature work. The tubes commonly used for X-band EPR spectroscopy have an internal diameter of approximately 3 mm and are about 150 mm long. The wider the bore, the greater the signal intensity, as the quantity of sample in the sensitive region of the cavity is roughly proportional to the square of the tube diameter. Tubes for Q-band spectroscopy have internal diameters about 1 ram. Tubes may be purchased ready-made or may be fabricated from high-purity quartz tubing of suitable size. Each batch of the tubing should be thoroughly cleaned and checked for spurious EPR signals. The bottom end of each tube is sealed off evenly without any bulges or thickening, and the top end is lightly flame-polished. For quantitative work the bore (internal diameter) of the tubes must be known since the volume measured is proportional to the square of the diameter. Precision bore quartz tubes are available from the Wilmad Glass Co. Alternatively, tubes can be calibrated in the laboratory and the EPR signals of different samples corrected for their internal diameter. Calibration can be done to an accuracy of about ---2% by filling the tubes with water to a known length and weighing the water. The diameter can then be marked on the tube with a diamond scriber. Samples can be injected into the sample tubes with microliter syringes fitted with long needles, which can also be used to stir the sample. Alternatively, small magnetic stirrers are commercially available. Freezing can be performed either slowly (in liquid nitrogen) or more rapidly in liquid

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EPR SPECTROSCOPYOF IRON

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methanol or isopentane, precooled in liquid nitrogen. In the absence of rapid freezing techniques, 4 the fastest time to add a reagent, stir, and freeze an EPR sample is of the order of 5 sec. One must be aware of possible artifacts arising from freezing; the most common of these is the pH change associated with the freezing of buffers, especially Tris, phosphate, and pyrophosphate. 46

Concentrations of Sample Required As mentioned previously different types of iron enzymes exhibit widely different EPR spectra. The ability to detect EPR spectra from an iron protein is therefore critically dependent on the nature of the spectrum. Figure 7 illustrates this by showing four spectra of different iron EPR signals, all at the same concentration (40/xM) from the same enzyme (the Escherichia coli flavohemoprotein iron reductase, HMp47'48). The EPR signal height is inversely proportional to the square of the line width. Therefore, the same concentration of iron will be more readily detected if the line width is narrow (e.g., the nitrosyl complex). Low-spin heme complexes are easy to detect if they have narrow line widths (the DTT complex), but hard if they are very broad (e.g., the cyanide complex). A broad signal can still be easily detectable if it contains a sharp component (e.g., the met-HMP complex). Both the sharpness of the signal and the increase in the EPR intensity with increasing g factor tend to make highspin heme easier to detect than an equivalent concentration of low-spin heme. In general high-spin and sharp low-spin signals are detectable down to levels of 1 /xM. At these concentrations, however, it is necessary to run the spectra of the EPR tubes prior to adding the sample, as contamination in the quartz can result in similar size signals, notably in the g = 4.3 region. Iron nitrosyl complexes and sharp F e - S clusters with narrow line spectra may be detectable at even lower concentrations. Of course, higher concentrations are also necessary to improve signal-to-noise ratios, to get accurate values for g factors, and to resolve detailed features of the spectrum. Broader low-spin centers and even-spin systems will require higher concentrations. Polynuclear iron centers with signals as broad as 2000 mT (e.g., ferritin, hemosiderin) will require the highest attainable concentrations. 46 D. L. Williams-Smith, R. C. Bray, M. J. Barber, A. D. Tsopanakis, and S. P. Vincent, Biochern. J. 167, 593 (1977). 47 S. G. Vasudevan, W. L. Armarego, D. C. Shaw, P. E. Lilley, N. E. Dixon, and R. K. Poole, Mol. Gen. Genet. 226, 49 (1991). 48 N. Ioannidis, C. E. Cooper, and R. K. Poole, Biochem. J. 288, 649 (1992).

372

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PROBES OF METAL ION ENVIRONMENTS

7.0 6.0

Met

5.0

4.0

~

3.5

3.0

I

I

g factor 2.5

2.00

I

1.75

I

I

~

Cyanide

Nitrosyl

I

I

I

I

i

I

I

I

50

100

150

200

250

300

350

400

M a g n e t i c Field (roT)

FIG. 7. Sensitivity of different iron protein complexes to detection by EPR. Bacterial hemoglobin (HMP) protein samples were prepared as follows: Met, protein used as purified; cyanide, protein + 5 mM sodium cyanide; DTT, protein + 5 mM dithiothreitol; nitrosyl, protein + 5 mM sodium nitrite followed by 5 mM sodium dithionite. All samples were 40 p.M heme b. EPR conditions: temperature 30 K, microwave power 20 mW, microwave frequency 9.36 GHz, modulation frequency 100 KHz, modulation amplitude 1 roT, receiver gain 1 × 105, time constant 0.33 sec, sweep time 2.4 mT/sec. Spectra displayed are average of two scans.

Oxidation and Reduction For oxidoreductases, substrates may be used as oxidants or reductants. Alternatively, potassium ferricyanide, K 3 F e ( C N ) 6 , is often useful as a general-purpose nonspecific oxidant, and sodium dithionite, Na2S204, as a reductant. Ferricyanide has a broad EPR signal around g = 3 that interferes with the spectra of some iron complexes. An alternative oxidant is ammonium persulfate. With such strong oxidants it is important to check that oxidative damage to the protein has not occurred. Commercial sodium dithionite is about 80% pure (the contaminants are mostly sodium sulfite and sodium carbonate). If greater purity is required it can be recrys49 C. E. McKenna, W. G. Gutheil, and W. Song, Biochim. Biophys. Acta 1075, 109 (1991).

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EPR SPECTROSCOPYOF IRON

373

Argon

\

\ p

Quart2

Millivolts

Stirrer Electrodes

Calomel,,," Platinum

FIG. 8. Apparatus for preparation of EPR samples under an argon atmosphere. The apparatus is constructed of glass. Connections to the glass manifold are sealed with epoxy or very short lengths of butyl rubber tubing. To minimize drying of samples, the gases are bubbled through water. The miniature magnetic stirrer is of the type used for oxygen electrodes. Long syringe needles are used to purge with gas the vessels in the side arms and the EPR tube prior to filling. The needles are inserted into the apparatus through short, close-fitting lengths of wider bore tubing which pierce the septum stoppers. The outflow of argon gas through the tubes minimizes the entry of air into the apparatus.

tallized. 49 R e d u c t a n t s s u c h as d i t h i o n i t e r e q u i r e o x y g e n - f r e e c o n d i t i o n s (see B e i n e r t et al.5); a r g o n gas c a n b e b l o w n o v e r the s u r f a c e of the s o l u t i o n in the t u b e , t h r o u g h a n o t h e r s y r i n g e n e e d l e . F i g u r e 8 s h o w s a s i m p l e a p p a r a t u s for p e r f o r m i n g E P R r e d o x t i t r a t i o n s o n a n i r o n c e n t e r . T h i s m e t h o d o l o g y has b e e n f o u n d to be suitable for m a n y

374

PROBES O F M E T A L I O N E N V I R O N M E N T S

[12]

types of manipulations of oxygen-sensitive samples and for adjustments of redox potentials. It does not require vacuum conditions and relies on a steady flow of oxygen-free, water-saturated gas to exclude oxygen. Argon gas from the cylinder is usually of sufficient purity. The gas regulator is of the low-leakage type with a metal diaphragm, and the gas is passed through glass and metal tubing. We use narrow-bore stainless steel tubing with compression fittings. Solutions in the apparatus are freed of oxygen by either bubbling with the gas or blowing gas over the surface. Equilibration between the gas and liquid phases is slow but is assisted by vigorous stirring with small magnetic followers. The vessel for adjusting redox potentials shown has a maximum volume of 2-10 ml and is designed for a small minimum sample volume, so all the solution is usable. The potential is measured with a small platinum disk electrode (3 mm diameter), fused into the base of the cell, and a calomel reference electrode glued into the cell with epoxy. A mixture of mediators is used in the solution, as described by Dutton. 7 Solutions of oxidants such as ferricyanide or reductants such as dithionite are made up in the side vessels. Solutions are transferred into the vessel and between the various side arms, using Hamilton gas-tight syringes with fixed needles, 25 cm long. Because the tip of the syringe is always in the gas space, leakage of oxygen is avoided. The sample is transferred in the same way to the EPR tube and frozen with a freezing bath of i sopentane or methanol, cooled with liquid nitrogen. For addition of reductant or oxidant, trial and error is needed to find the concentration required and time of reduction. Typical conditions for reduction of an electron transfer protein would be reduction with 5 mM dithionite solution for 2 min befor freezing. If reduction is very slow it may be assisted by the addition of a mediator such as methyl viologen. When using dithionite and methyl viologen, one must also be aware of the possibility of observing spurious free radical (g = 2.0) signals from sulfur radicals or methyl viologen radical cation.

Conditions for Running Spectra The effects of varying the different instrumental parameters, such as the microwave power, magnetic field modulation amplitude, scan speed, filter time constants, and instrumental gain, on the EPR spectrum have been described by Palmer 1 and by Fee. 2 There is compromise to be made between maximum sensitivity (signal-to-noise ratio) and resolution of the spectrum. Because the samples are usually frozen it is always possible to enhance the signal-to-noise ratio by repetitive scanning and signal averaging. This is straightforward with the computer systems on modern spectrometers. By ~making n scans the signal-to-noise ratio is enhanced by

[12]

EPR SPECTROSCOPYOF IRON

375

n 1/2. In practice a limitation to sensitivity is commonly set by the presence

of contaminant metal ions which contribute spurious signals. The contamination may be in the cavity (in which case it will not vary with sample temperature), in the cryostat, or in the sample itself (in which case its signals may increase or decrease at lower temperature, depending on the type of paramagnetic impurities). Signals arising from the cavity or glassware may be subtracted out of the sample spectrum with the aid of the computer, if another spectrum is taken, under identical conditions, of a sample containing only water.

Temperature The electrons excited by the microwave field lose energy to their surroundings by a process of spin-lattice relaxation. This returns them to the ground state and allows a continuous steady-state absorption of microwaves to be observed. The relaxation process is characterized by a relaxation time, T1 which is analogous to the same effect in NMR spectroscopy. In many iron complexes, T1 is usually extremely rapid at room temperature, causing extreme broadening of the EPR lines. This is why in order to see EPR from iron proteins it is usually necessary to cool the sample to cryogenic temperatures, sometimes down to the boiling point of liquid helium (4.2 K). Moreover, lower temperatures give increased signal size owing to the increased Boltzmann factor. However, if the temperature is too low, the spin-lattice relaxation becomes inefficient and the signal size decreases at high microwave power as the number of electrons in the ground and excited states becomes equal; this phenomenon is termed microwave power saturation. Studies of the microwave power saturation characteristics of an EPR signal can yield information about the environment of the unpaired electron; for example, if another unpaired electron is nearby (<2 nm) the relaxation rate will be faster and the signal more difficult to saturate. The necessity for cryogenic temperatures prevents experiments from being made on living tissues and cells; kinetic measurements can, however, be made by rapid-freezing methods .4 The characteristic temperature dependencies of EPR signals may be exploited to distinguish components in the spectrum of a complex mixture. Commercial temperature controllers generally use a flow of cryogenic gas, either nitrogen (for temperatures down to 77 K) or helium (down to 4.2 K). For lower temperatures it is possible to reduce the pressure over liquid helium with a large vacuum pump, though such temperatures are rarely needed for iron proteins. The temperature of the sample is measured with a thermocouple positioned in the gas stream, as close to the sample as possible. There are inevitably temperature gradients, particularly in

376

PROBES OF METAL ION ENVIRONMENTS

[12]

the flow cryostats which fit inside the cavity, as these cannot be silvered. The sample temperature may be checked periodically with a thermal sensor inside an EPR tube. It is preferable to use an enclosed system, to prevent the condensation of air inside or around the sample. Oxygen itself is paramagnetic, and condensed 02 gives broad but intense background signals.

Simulation of Spectra It is possible to simulate an EPR spectrum for an iron complex, by computation from assumed values of g factors, hyperfine splittings, zerofield splittings, etc. The parameters are adjusted to give the best fit between the observed and computed spectrum. For simple cases the computer simulates the spectrum iteratively. Computer simulation has a number of uses. It may be a more accurate way to determine the spectroscopic parameters. It may be employed to quantify the signal of one component of a complex of noisy EPR spectra from one component. Simulations can also be used to resolve and quantify spectra made up of several overlapping components. Care must be taken, however, and there are special difficulties in accurately simulating anisotropic spectra with unresolved hyperfine interactions. A full discussion is beyond the scope of this chapter. However, there is no need to "reinvent the wheel" when it comes to writing programs for the simulation of EPR spectra. Some simulation programs are available commercially, and a software exchange database is available from the authors' laboratory. Quantitation of Electron Paramagnetic Resonance Signal Intensities To determine if one is studying a paramagnetic signal from the protein complex desired (and not a minor impurity), it is essential to be able to quantify the number of spins in an EPR signal. The general principles of integration have been described previously by Fee 2 and by Randolph. 5° The process of subtracting background signals and integrating is greatly assisted by means of a computer, and the software provided with the spectrometer is usually capable of doing this. The area under the absorbance peak of an EPR signal is directly proportional to the number of spins. Because EPR spectra are generally recorded as first derivatives, all that is required to quantify an EPR signal is to integrate the spectra to obtain the "absorbance-type" spectrum, then integrate again to obtain the area under this peak (correcting for baseline drift after each integration). This 50 M. L. Randolph, in "Biological Applications of Electron Spin Resonance" (H. M. Swaa'tz, J. R. Bolton, and D. C. Borg, eds.), p. 119. Wiley (Interscience), New York, 1972.

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EPR SPECTROSCOPYOF IRON

377

area is then compared to a standard solution of spins (e.g., 1 mM CuSO4 + 10 mM HC1 + 2 M NaCIO4) run under identical conditions in the spectrometer. Both sample and standard must be run under conditions where they are not saturated with microwave power, that is, where the signal size is proportional to the square root of the microwave power. However, in its simple form this method is strictly true only for samples and standards of narrow line width at g = 2. Modifications have to be made for the wide variety of different EPR signals detected in iron enzymes.

Low-Spin Iron Complexes The low-spin heroes and reduced iron-sulfur proteins that have S = ½ are the easiest signals to quantify by EPR. Simply comparing the double integral to that of a g = 2 standard gives a good approximation. However, increased accuracy is obtained by allowing for the fact that the transition probability at any field is proportional to the g factor of the spectrum. In practice this can be done by dividing the complete double integral by the average g factor (gay) calculated by the approximation used in Aasa and V~inng~trd.s~ Aasa and V~inng~rd also show that it is possible to quantify an EPR spectrum from the area under an isolated peak, which is useful if part of a spectrum overlaps other EPR signals. This is frequently the case in proteins as the g = 2 region may contain radical or copper signals; for example, in cytochrome-c oxidase the Cu a signal interferes with the double integration of the hemp a signal. 52 This method is also necessary if the complete spectrum is too broad to integrate, as the gx peak is at too low a g factor to be detectable (as is the case with many low-spin hemp complexes such as cytochrome c and the bacterial HMP cyanide complex; see Fig. 7). In this case the method of calculating gay may be modified by using an approximate determination of the missing g factors, as described by de Vries and Albracht: 3 It is desirable to use standards that are as similar in spectral line shape as possible to the sample being studied, and the two samples should be run under the same measurement conditions. Thus, although Cu(II) is fine for Fe-S clusters, it is usually preferable to use a low-spin hemp standard for S = ½hemp proteins. Horse heart metmyoglobin readily forms an azide complex with an optical extinction coefficient of 123 mM -1 cm -~ at 420 nm and can thus be used to construct a standard curve to calculate the concentration of the unknown sample. 51 R. Aasa and T. V~inng~rd, J. Magn. Reson. 19, 308 (1975). 5z R. Aasa, S. P. J. Albracht, K.-E. Falk, B. Lanne, and T. V~inng~rd, Biochem. Biophys. Acta 422, 260 (1976). 53 S. de Vries and S. P. J. Albracht, Biochim. Biophys. Acta 546, 334 (1979).

378

PROBES OF METAL ION ENVIRONMENTS

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High-Spin Iron Complexes

For accurate quantification of high-spin iron complexes, it is necessary to know the zero-field splitting parameters D and E. In most biological cases the value of these parameters is such that it is not possible to assume that only the ground state is populated) 4 Therefore, one needs to be able to calculate the Boltzmann distribution (e -ae/kr) of spins in the different energy levels to determine the population contributing to the EPR spectrum (in high-spin heme, when E / D ~- O, these are usually those in the lowest energy level, but in high-spin nonheme iron complexes, E / D -~ and signals are usually seen from the middle g -- 4.3 doublets). In the case of rubredoxin the signal at g = 4.3 increases as the temperature is raised from 2 to 10 K. 55 As the temperature rises further the populations of the different energy levels become similar, and the double integrals become independent of the exact value of the zero-field splitting. Thus, at 77 K the double integrals of the EPR signal at g = 4.3 give similar values (for the same iron concentration) for a wide variety of protein and nonprotein iron complexes. It is especially important to quantify the EPR signal at g = 4.3 accurately before concluding that it is an intrinsic component of a protein. Although this signal can be due to iron in the active site of a protein (Fig. 4), signals in this region are observed in most samples owing to nonspecifically bound iron. Indeed, the majority of iron complexes likely to be present as contaminants in biological samples give rise to such a signal (e.g., Fe-EDTA, Fe-ascorbate, Fe-citrate). A similar signal is seen if iron is adventitiously bound to proteins such as albumin. Differences seen in the signal on substrate binding56can help to assign the signal to an active role in enzyme structure and function. In whole cells the g = 4.3 signal is much smaller and may in part represent the cellular low molecular weight iron pool. There are problems when attempting to quantify signals from lowmolecular-weight iron complexes such as siderophores 57 and low molecular weight sub strates (e.g., Fe-ascorbate, Fe-citrate, Fe-nitrilotriacetate) for enzymes that catalyze iron transport and/or reduction. When aqueous solutions of these compounds are frozen, the iron complex freezes out 54 j. Peisach, W. E. Blumberg, S. Ogawa, E. A. Rachmilewitz, and R. Oltzik, J. Biol. Chem. 246, 3342 (1971). 55 j. Peisach, W. E. Blumberg, E. T. Lode, and M. J. Coon, J. Biol. Chem. 246, 5877 (1971). 56 L. Que, J. D. Lipscomb, R. Zimmerman, E. Miinck, N. R. Orme-Johnson, and W. H. Orme-Johnson, Biochim. Biophys. Acta 452, 320 (1976). 57 y . Mino, T. Ishida, T. N. Ota, M. Inoue, K. Nomoto, T. Takemoto, H. Tanaka, and Y. Suguira, J. Am. Chem. Soc. 105, 4671 (1983).

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EPR SPECXROSCOPYOF IRON

379

from the ice crystals, resulting in locally high concentrations of iron molecules that can magnetically interact. This phase separation can result in a significant underestimation of the iron concentration. With proteins this problem does not usually occur because the protein molecules physically separate the iron ions. In complexes the chelator/iron ratio may be increased until it approximates the magnetic dilution seen in proteins; the consequent increase in the EPR signal may be monitored. Alternatively, the samples can be frozen as a glass (in high glycerol or sucrose concentrations), although this then means one is inevitably studying a nonphysiological system. A suitable standard for high-spin heme iron signals is horse heart metmyoglobin (optical extinction coefficient 188 mM -1 c m -1 at 408 nm; D = 10.0 cm -1, E = 0), provided the pH is 6.5 or below to avoid formation of low-spin complexes. Excess ammonium persulfate or ferricyanide may be used to oxidize the myoglobin if it is not already fully oxidized from the bottle. If ferricyanide is used the metmyoglobin must be passed down a column, as ferricyanide has a broad EPR signal at low temperatures. Serum transferrin58 can be used as a standard for high-spin (g = 4.3) nonheme iron (D = 0.32, E = 0.1). Apotransferrin quantitatively binds added ferrous sulfate with an extinction coefficient of 2.5 mM -1 cm -l at 465 nm relative to the iron-free protein.

Even-Spin Systems (S = 2, 3, etc.) More and more proteins are now being shown to have even-spin systems. Unlike signals from odd-spin systems, EPR signals from even-spin systems usually represent only a fraction of the total spins; therefore, to quantitate the total spins it is necessary to determine exactly which spin transition is responsible for the detectable signal and then simulate the spectrum. 29'34It is usually necessary to use a parallel mode EPR spectrometer to improve signal intensity; the comparison of the intensities between parallel and perpendicular mode spectra also assists in the simulation. High-spin ferrous EDTA and ferrous hexahydrate can be used as S = 2 standards, z9,42although there are methods to relate the signal size to oddspin standards. 29Owing to the difficulty in quantifying these systems from first principles, it is sometimes necessary to infer the concentration by other methods, for example, converting the signal to an odd-spin form that can be measured accurately. 59 58 R. A. Pinkowitz and P. Aisen, J. Biol. Chem. 247, 7830 (1972). 59 B. C. Hill, T.-C. Woon, P. Nicholls, J. Peterson, C. Greenwood, and A. J. Thomson, Biochem. J. 224, 591 (1984).

380

PROBES OF METAL ION ENVIRONMENTS

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Quantitation of Signals from Whole Cells If an iron protein is a major constituent of a cell it may sometimes be studied by EPR without purification. This makes it possible to examine and quantify proteins in their natural environment. Difficulties may be encountered owing to weak signal strengths and the presence of overlapping signals. Judicious use of signal averaging will alleviate the former problem, and computer-assisted spectral subtraction the latter. Figure 9 illustrates how this can be used to determine the amount of a ferredoxin from Pseudomonas putida 6° that has been expressed in Escherichia coli. In this case the spectrum of the purified ferredoxin was used as a reference, though in principle simulated spectra could also be used.

Errors in Quantitation It is obvious that there are errors involved with measuring the concentration of spins in an EPR sample. Under optimum conditions (e.g., a pure protein at high concentration with the same protein used as a standard) the errors would be not much larger than pipetting errors. Realistically a 5% error is probably the best obtainable. Having to integrate broad spectra at low concentrations can produce errors of 20% or more, primarily owing to problems in baseline subtraction. Electron Paramagnefic Resonance Spectroscopy of Iron Proteins in Vivo EPR has advantages in both selectivity and sensitivity over NMR when it comes to detecting signals in vivo. As there are relatively few paramagnets in a cell or tissue, it is frequently possible to study individual iron enzymes in whole tissues and cells without resort to extensive signal averaging. 6'61-64 However, as opposed to NMR, iron EPR has the fundamental problem of the requirement for the measurement to be made at low temperature (liquid nitrogen or liquid helium). Therefore, one can only obtain snapshots of a dead system, rather than monitor a live system. Still this situation is no worse than is obtained with freeze-clamping. 6o p. j. Geary, F. Saboowalla, D. S. Patil, and R. Cammack, Biochem. J. 217, 667 (1984). 6~ H. Beinert, in "Iron-Sulfur Proteins" (W. Lovenberg, ed.), p. 61. Academic Press, New York, 1977. 62 N. R. Orme-Johnson, R. E. Hansen, and H. Beinert, J. Biol. Chem. 249, 1928 (1974). 63 R. Bache, P. M. Kroneck, H. Merkle, and H. Beinert, Biochim. Biophys. Acta 722, 417 (1983). e4 M. J. Payne, L. F. J. Woods, P. Gibbs, and R. Cammack, J. Gen. Microbiol. 136, 2067 (1990).

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E P R SPECTROSCOPY OF IRON

381

g factor 2.25

2.20

I

I

2.15 i

2,10

2.05

2.00

I

I

I

d. Purified ferredoxln

290

1.95 I

1.90 |

1.85 I

1.80

1.75

I

I

/~

I

]

I

I

I

300

310

320

330

340

Magnetic

I 350

I 360

1 370

l 380

Field (mT)

FIG. 9. In vivo quantitation of iron-sulfur clusters. Escherichia coil cells into which DNA from a cloned ferredoxin sequence ([2Fe-2S] from Pseudomonas putida ML2) has been genetically engineered were subjected to EPR study (a). The cells were frozen and thawed to make them more permeable, and 5 mM sodium dithionite was added to ensure full reduction of the iron-sulfur clusters. Also, 5 mM EDTA was added to broaden the manganese signals, although there is still some interference from a six-line manganese spectrum detectable. The spectrum is different from the spectrum of the same number of wild-type cells (b), suggesting that the F e - S cluster has been incorporated into the expressed cloned protein. This is confirmed by subtracting the two spectra. The difference observed (c) is primarily due to the ferredoxin spectrum (although there are also differences in the amount of free radicals present at g = 2.0). By comparison with the spectrum of the purified ferredoxin from Pseudomonas putida ML2 (d), it is possible to quantify the amount of F e - S centers successfully expressed in Escherichia coli, either by comparing one of the peak minus troughs or by integrating the spectrum (or parts of it). The safest way, however, is to subtract the purified protein spectrum from that of the cloned ferredoxin until there is no visible ferredoxin spectrum left, and then quantify the amount of the subtracted signal. EPR conditions: temperature 30 K, microwave power 2 mW, microwave frequency 9.335 GHz, modulation frequency 100 KHz, modulation amplitude 0.5 naT, receiver gain 2.5 × l05, time constant 0.33 sec, sweep time 1.2 mT/sec. Spectra displayed are average of five scans.

382

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PROBES OF METAL ION ENVIRONMENTS

g factor 7.06.0 I

I

5.0

4.0

3.5

3.0

2.5

2.00

1.75

I

I

I

I

l

I

I

Mitochondrial iron-sulfur proteins

Methemoglobin

Brain

Ceruloplasmin

I,

60

I

+ I,

1 O0

I,

I

140

t

I

~

l

180

~

I,

I,

220

I

,

I,

260

I,

I,

I,

300

I,

340

Blood

t,

l,

350

l,

I

420

Magnetic Field (roT) Flo. 10. EPR spectra of rat tissues. Different tissue samples were removed and frozen in EPR tubes within 5 min. EPR conditions: temperature 12 K, microwave power 20 mW, microwave frequency 9.35 GHz, modulation frequency 100 KHz, modulation amplitude 1 mT, receiver gain 1.25 × 105, time constant 0.33 sec, sweep time 2.4 roT/see. Spectra displayed are average of 10 scans.

Figure 10 illustrates the variety of iron proteins that can be detected from whole rat tissues at liquid helium temperatures; similar spectra are obtained with mouse or human tissue. Delays of the order of hours in obtaining the tissues postmortem before freezing result in changes such as oxidation of the iron-sulfur clusters and reaction of formate with heine proteins such as catalase. In the heart, high-spin heme signals (g = 6) are present and assigned to metmyoglobin and methemoglobin. Variations in the metmyoglobin concentration are likely to result from and/or initiate free radical damage in the heart. 65'66 Therefore, a direct monitor of these levels in the whole 65 j. M. C. Gutteridge, FEBS Lett. 201, 291 (1986). 66 j. Kanner and S. Harrel, Arch. Biochem. Biophys. 237, 314 (1975).

,

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EPR SPECTROSCOPYOF IRON

383

tissue is of great interest. At g = 2 the spectra are dominated by the mitochondrial iron-sulfur centers. These are all present in the reduced state; the only center that is therefore not detectable is center 3 of succinate dehydrogenase, which is EPR-detectable in the oxidized state. 62 By varying the temperature it is possible to detect all the five clusters present in complex I (NADH dehydrogenase) and center 1 in succinate dehydrogenase. It is harder, although still possible, to detect the signals from the Rieske Fe-S cluster in the mitochondrial bcl complex and the [4Fe-4S] signal from the electron-transferring flavoprotein ubiquinone oxidoreductase (ETF).5 EPR therefore provides a direct probe for these redox centers in whole tissue. This is likely to prove valuable in the study of disorders in iron-sulfur enzymes, for example, mitochondrial myopathies of muscle tissue. 67 In the liver sample the high-spin hemP signals are dominated by that of catalase. A low-spin signal is also seen from cytochrome P-450 (at g = 2.415, 2.24, and 1.91), and there are iron-sulfur clusters around g = 2. Clearly the possibility exists of monitoring spin state changes associated with substrate binding to cytochrome P-450 in whole tissues. The brain signals are similar to those seen in the heart. The ability to detect the different iron-sulfur centers in NADH dehydrogenase is likely to be useful in the study of neurological disorders associated with damage to this enzyme (e.g., Parkinson's disease68). The blood spectrum shows signals at g = 6 from methemoglobin. As most of the cells are erythrocytes, no mitochondrial signals are detected, although a small signal is present at g = 2 from the copper protein ceruloplasmin. At g = 4.3 there is a characteristic signal from the iron-transport protein transferrin. Interestingly, there are signals at g = 4.3 in all the tissues studied. These are not all due to transferrin, as the line shape is different; we have also shown that these signals are still present in mice that are genetically completely deficient in transferrin (J. Shergill, R. Simpson, and C. E. Cooper, unpublished observations, 1992 ). These signals may therefore include contributions from other nonheme iron proteins or low molecular weight iron species. EPR studies of serum can be used to probe the disease state; it is possible to detect differences in EPR signals from healthy and iron-overloaded patients (C. E. Cooper and A. Bomford, unpublished observations, 1992 ). It is possible to place pieces of tissue directly in EPR tubes and freeze them or to core out frozen postmortem material. Pieces of tissue can be 67 j. A. Morgan-Hughes, A. H. V. Schapira, J. M. Cooper, I. J. Holt, A. E. Harding, and J. B. Clark, Biochim. Biophys. Acta 1018, 217 (1990). 68 A. H. V. Schapira, J. M. Cooper, D. Dexter, J. B. Clark, P. Jenner, and C. D. Marsden, J. Neurochem. 54, 823 (1990).

384

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loaded into the tubes with the assistance of low-speed centrifugation. H o w e v e r , quicker results can be obtained by using freeze-clamped materials ground into a p o w d e r and then packed into EPR tubes while in the frozen state. In all cases, however, it is not clear how much of the sample is sitting in the sensitive part o f the cavity, and for an accurate comparison the signals must be normalized against an internal standard. Any of the signals mentioned above can be used as standards, provided they are known not to be a target for the disease in question. It is possible that while freezing whole tissue cellular damage may arise, and a consequent change in the E P R spectra is expected. This is especially possible for rapidly turning o v e r pools, for example, the postulated 1-2/xM low molecular weight iron pool in cells. 69 H o w e v e r , small g = 4.3 signals are still observed in a wide variety o f bacteria and mammalian cell lines frozen rapidly (1-2 sec) in liquid methanol; the cells were all viable prior to freezing, and little damage is likely to occur on freezing. Therefore, it remains possible that these signals are due to the low molecular weight iron pools in the cell. Certainly, many of the candidates for these pools would be expected to have g = 4.3 signals. 7° Acknowledgments We would like to thank Andy White for technical assistance, and Ruth Williams and John Wrigglesworth (King's College London) for helpful comments on the manuscript. C.E.C. is grateful for a King's College Research Fellowship. We thank the following(all at KCL) for preparing the samples used for the figures: Jasvinder Shergill (Fig. 10), Nikolaos Ioannidis and Robert Poole (Figs. 5 and 7), and Hal-Meng Tan and Jeremy Mason (Fig. 9) and SERC for financial support. 69R. R. Crichton and M. Charloteaux-Waters, Eur. J. Biochem. 164, 485 (1987). 7oS. Pollack, T. Campana, and J. Weaver, Am. J. Hematol. 19, 75 (1985).

[13] I n t r i n s i c a n d E x t r i n s i c P a r a m a g n e t s of Metal Clusters

By

BRIAN

as Probes

J. HALES

Introduction Protein-bound metal ions or clusters can be studied by a wide variety o f spectroscopic techniques. The majority o f these techniques probe local effects, monitoring the electronic or magnetic properties of either the METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

384

[13]

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loaded into the tubes with the assistance of low-speed centrifugation. H o w e v e r , quicker results can be obtained by using freeze-clamped materials ground into a p o w d e r and then packed into EPR tubes while in the frozen state. In all cases, however, it is not clear how much of the sample is sitting in the sensitive part o f the cavity, and for an accurate comparison the signals must be normalized against an internal standard. Any of the signals mentioned above can be used as standards, provided they are known not to be a target for the disease in question. It is possible that while freezing whole tissue cellular damage may arise, and a consequent change in the E P R spectra is expected. This is especially possible for rapidly turning o v e r pools, for example, the postulated 1-2/xM low molecular weight iron pool in cells. 69 H o w e v e r , small g = 4.3 signals are still observed in a wide variety o f bacteria and mammalian cell lines frozen rapidly (1-2 sec) in liquid methanol; the cells were all viable prior to freezing, and little damage is likely to occur on freezing. Therefore, it remains possible that these signals are due to the low molecular weight iron pools in the cell. Certainly, many of the candidates for these pools would be expected to have g = 4.3 signals. 7° Acknowledgments We would like to thank Andy White for technical assistance, and Ruth Williams and John Wrigglesworth (King's College London) for helpful comments on the manuscript. C.E.C. is grateful for a King's College Research Fellowship. We thank the following(all at KCL) for preparing the samples used for the figures: Jasvinder Shergill (Fig. 10), Nikolaos Ioannidis and Robert Poole (Figs. 5 and 7), and Hal-Meng Tan and Jeremy Mason (Fig. 9) and SERC for financial support. 69R. R. Crichton and M. Charloteaux-Waters, Eur. J. Biochem. 164, 485 (1987). 7oS. Pollack, T. Campana, and J. Weaver, Am. J. Hematol. 19, 75 (1985).

[13] I n t r i n s i c a n d E x t r i n s i c P a r a m a g n e t s of Metal Clusters

By

BRIAN

as Probes

J. HALES

Introduction Protein-bound metal ions or clusters can be studied by a wide variety o f spectroscopic techniques. The majority o f these techniques probe local effects, monitoring the electronic or magnetic properties of either the METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

[13]

I N T R I N S I C A N D EXTRINSIC P A R A M A G N E T I C PROBES

385

cluster or its immediate surroundings. However, distant interactions can also be investigated. For example, because paramagnetic metals have a relatively large magnetic moment, the magnetic dipolar effects of these metals extend over tens of angstroms and, as such, influence the magnetic properties of distant paramagnets. Therefore, a secondary paramagnet can be used as a probe to investigate the structure of a paramagnet innate to a protein. In this chapter, the theory and methodology behind employing secondary paramagnetic probes to investigate biological metal clusters are discussed. The discussion is restricted to the study of biological metal clusters only and will not focus on the study of free radicals such as intrinsic semiquinones and flavins or extrinsic nitroxide labels, even though investigation of the latter systems follow directly from the theory presented. On the other hand, the secondary paramagnet used to probe the biological metal clusters can be either intrinsic or extrinsic to the system and does not have to abide by the above restriction, that is, it can be either a paramagnetic metal ion or a free radical. Finally, although both exchange and dipolar interactions can occur between the biological metal cluster and the paramagnet probe, this chapter predominantly focuses on the latter since it is the more common interaction observed in biological systems, and it is also the form of interaction that directly yields distance and structural information.

Theory of Dipolar Interactions In biological samples, a major source of interaction between two different paramagnetic species (i and j) arises from dipolar coupling. The electronic magnetic dipole momentum vector (/~i) of species i can be written as l.t i =

TihJi

=

- gi/3Ji

(1)

where Yi is the magnetogyric ratio, $i is the total angular momentum vector, gi is the g factor, and/3 is the Bohr magneton. The interaction energy (~0) between a pair of dipoles i and j separated by a distance vector r can be written as

'~'ij =

r-3[(/xi"/-¢j) -- 3r-2 (/xi" r)(/~j-r)] = Y i T j h 2 r - 3 ( A + B + C + D + E + F)

(2)

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PROaES OF METAL ION ENVIRONMENTS

[13]

where A B C D E F

= = = = = =

JziJz/(1 - 3 cos 20ij) -¼[(Jxi - iJyi)(Jxj + iJyj) + (J~i + iJri)(Jxj - iJya.)](l - 3 cos 20ii) - a [ ( J x i + iJyi)Jai + (J~j + iJy:)Jzi] sin 0ij cos Oi:e-ie, ij -][(JxiiJyi)Jzj + (Jxj - iJyj)Jzi] sin 0ucos Oij ei~'U -a4[(Jxi + iJyi)(Jx j + iJyj)] sin2Oije-2ieoiJ - ?~[(Jxi - iJyi)(Jxj - iJyj)] sin 20ii e 2i¢0~:

and 0~j and ~bij are the spherical coordinate angles defining the vector r. In the presence of a large external magnetic field, B0, the magnetic dipoles of species i and j will precess about B 0 with angular frequencies ¢.oi and toj defined' by ¢.0i "~"

")/iBo

and

toj = y j B o

(3)

and with spin-lattice relaxation times T~; and T~j., respectively. It will be assumed that species i is the biological metal cluster under investigation and species j is the extrinsic or intrinsic perturbing paramagnet. In this situation, species i experiences a magnetic field that is the sum of the static external magnetic field B 0 plus a time-dependent local field. The time variation of the local field results from the relaxation, or spin flipping, o f j at a rate 1/T~j. Under the condition where this rate of relaxation is in resonance with the precession rate of i alone (i.e., to~T~j ~ 1) or with the superposition rate representing either the sum or the absolute difference of the precession rates of the two species [i.e., (toi + %)T1j -~ 1 o r ](to i - toj)]Tlj. ~ 1], s p e c i e s j will be coupled to species i and induce relaxation in it. Bloembergen 1 and Abragam 2 derived the expression in Eq. (4) showing the influence that paramagnetic s p e c i e s j has on the spin-lattice relaxation rate, T~, of species i when the two species are coupled by magnetic dipolar interactions: ( l/Tli)dipolar ~- Jj( Jj + 1){~bij2[2Tjj/(1 + (to i - toj)2Tlj2)] + ~acij2[2Tlj/(1 + toiZTlj2)] + ~eij2[2Tlj/(1 + (coi + %.)2T1~2)]}

where bu 2 = ~i2~/j 2 h2r-6(1 -- 3 COS20ij) 2 Cij2 _~_ 4~i2,~j2 h2r -6 sin 20ij cos 20ij eij2 = s~/i2y2 h2r-6 sin 40ij I N. Bloembergen, Physica 15, 386 (1949). 2 A. Abragam, Phys. Rev. 911, 1729 (1955).

(4)

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INTRINSIC AND EXTRINSIC PARAMAGNETIC PROBES

387

The first term in Eq. (4) arises from the B term in the dipolar interaction energy expression [Eq. (2)], the second from the C and D terms, and the third from the E and F terms. Although the original derivation assumed species i to be a nucleus with I ~ 0, Eq. (4) is equally valid in describing relaxation induced by dipolar interaction between two paramagnetic species.

Spectroscopic Detection From Eq. (4) it is obvious that one parameter which can be used to monitor the presence of dipolar interactions between paramagnetic species is the change in Tli of species i induced by the presence of speciesj. Three major techniques we discuss for the determination of Tli are progressive power saturation (PPS), saturation recovery (SR), and spin echo-detected recovery (SEDR). Of the three, PPS is the most commonly used since it requires no special instrumentation other than a conventional electron paramagnetic resonance (EPR) spectrometer (with as wide a power range as possible) and, for most applications, a variable-temperature cryostat (capable of temperatures down to -3.8 K, if possible). P r o g r e s s i v e P o w e r Saturation

Portis 3 and Castner 4 first evaluated the relationship between power saturation and relaxation times of paramagnetic resonances. In general, the amplitude, A, of the derivative EPR spectrum is related to the incident microwave power, P, by the expression A = KPI/2/(1 + P / P m ) hI2 PI/2 = 1/gZT1T2

(5)

K is a proportionality factor, P1/2 is the microwave power needed for halfsaturation of the signal, and Tz is the spin-spin relaxation time. Exponent b in Eq. (5) is referred to as the inhomogeneity factor. For inhomogeneously broadened (Gaussian) lines, such as those observed for most metalloproteins, b -- 1, whereas homogeneously broadened (Lorentzian) lines yield b = 3. Equation (5) can be rearranged 5 to A/p1/2 = K(Pl/2)b/2/(P1/2 + p)b/2

3A. M. Portis, Phys. Rev. 91, 1071 (1953). 4T. G. Castner, Jr., Phys. Rev. 115, 1506(1959). 5 H. Beinert and W. H. Orme-Johnson, in "MagneticResonance in BiologicalSystems" (A. Ehrenbery,B. G. Malmstr6m,and T. Vanngfird,eds.), p. 221.Pergamon,Oxford,1967.

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PROBES OF METAL ION ENVIRONMENTS

[13]

and, by taking the logarithm of each side, yields log(A/P z/2) = _ b/2 log(P1/2 + P ) + b/2 log(P]/2) + log(K) = - b / 2 log(P]/2 + P) + constant

(6)

The value of Pz/2 can be determined from progressive saturation experiments by plotting Eq. (6) as log(A/P 1/2) versus log(P). In such a plot, two linear regions are observed, one at low power [i.e., no observed saturation or P ~ Pl/2 and log(A/P ]/2) = log(K)] and the second at high power [i.e., pronounced saturation or P >> P1/2 and log(A/P 1/2) = - b / 2 log(P) + constant]. The value of the power at which the straight lines representing these two linear regions intersect is P]/2, and the slope of the second linear region yields - b / 2 . Figure 1 shows examples of plots of Eq. (6) for both homogeneously and inhomogeneously broadened lines. The disadvantage in using the PPS technique for the determination of T1 is that PI/2 also depends on g and T2 [see Eq. (5)] as well as the quality factor of the microwave cavity and passage effects. Passage effects can

10

. . . . . . .

I

. . . . . . .

"1

. . . . . . .

"1

. . . . .

"1'1

. . . . . . .

I

. . . . . . .

"1

. . . . . . .

t_

4) O

a. Q.

E

.11

.

• O--~L

.......

00

I

101"

......

J

.......

10 2

I

.....

10 3

Relative

d

10 4

.......

I

10 5

.......

I

......

10 6

07

Power

FIG. 1. Saturation plots of A / P i/2 versus P (on log scale) for both Gaussian (dashed curve) and Lorentzian (dotted curve) line shapes using Eq. (6) in the text with b = 1 and 3, respectively. Both curves were plotted assuming the same value of PI/2 of l0 s. Note that straight lines fitted to both curves in the low-power (P ~ PI/2) and high-power (P >> P1/2) regions intersect at P~/2-

[13]

INTRINSIC AND EXTRINSIC PARAMAGNETIC PROBES

389

be eliminated as a contributing factor by determining P1/2 a s a function of modulation amplitude. Ways of minimizing the effects of the other factors are discussed below. Saturation and Spin Echo-Detected Recovery

Unlike progressive power saturation, pulsed techniques such as inversion recovery from electron spin echo (ESE) or saturation recovery provide direct means for the measurement of T1. In SR, the growth of an EPR absorption is monitored with low observation microwave power levels following a saturating microwave pulse. On the other hand, the determination of T1 with ESE requires selected pulse sequences. One example is the series based on the use of a two-pulse 90°-180 ° sequence. In a simplified description of this sequence, an initial 90 ° pulse rotates the magnetization vector (M~) from its equilibrium position along the (+)z axis into the xy plane, whereas the second 180° pulse inverts the direction of the magnetization but keeps it in the xy plane, thus allowing rephasing to occur. In the second series of pulses, the magnetization vector is rotated back into the ( + ) z direction (90° pulse) then finally rotated into the ( - ) z direction (180 ° pulse). In this example, T~ is the time needed for the magnetization, M z, to again reach its equilibrium value following the last pulse. These recovery methods are complicated by the necessity for elimination of the effect of spectral diffusion. Spectral diffusion, 6 which occurs in inhomogeneously broadened lines, is the spreading of saturation from spins that are in resonance with the microwave irradiation to whose that are not. Because the detected relaxation is not associated with a single spin packet, the true 1/T~ must be determined by using a series of saturating pulses of variable width. In these measurements, 1/T1 decreases in a nearly exponential manner with increasing durations of the saturating microwave pulse and levels off as the spectral diffusion channels become saturated. Furthermore, when using the SR technique, the relaxation rate is also perturbed by the observation power level, P, such that (1/T)observed = (1/T)actual +

CP

(7)

where C is a proportionality constant. Therefore, whenever these techniques are employed, I/T~(observed) must be determined as a function of both the observation power level and the duration of the saturating microwave pulse. 6 y . C. Zhong and J. R. Pilbrow, J. Magn. Reson. 97, 111 (1992).

390

PROBES OF METAL ION ENVIRONMENTS

[13]

Experimental Procedures As stated earlier, it will be assumed that species i is the intrinsic metal cluster under investigation while speciesj is the paramagnet whose presence perturbs the relaxation time of i. It is assumed that species j is either an intrinsic or extrinsic free radical, metal ion, or cluster. As discussed below, in order to measure the effect that species j has on 1/Tli , there must be a way of switching on and off the magnetic interaction between them. Intrinsic P r o b e s

Equations (2) and (4) demonstrate that two paramagnetic species, that are dipolar coupled, perturb the relaxation rates of one another. Therefore, when investigating magnetic interactions between two species, either can serve as the monitor of the interaction while the other is considered the perturber. All that is required of the monitoring species is that it possess a detectable EPR signal that exhibits measurable saturation. Obviously, metals in diamagnetic oxidation states cannot be used, whereas those with integer spin may be unsuitable as monitors since they are often EPRsilent. In the situation where both species i andj are intrinsic paramagnetic metal ions or clusters with half-integer spin states, either can be used as the monitor. Typically, changes in I / T l of the monitoring species are determined while the paramagnetism of the other species is changed, usually through oxidation or reduction, switching it between diamagnetic and paramagnetic states. When the second species is a free radical, the situation is slightly different. Because free radicals have relaxation rates significantly longer than those of typical metal ions (in our case, species i), it is often more convenient to reverse the situation, using the free radical as the monitor of the magnetic interaction. In this case, the intrinsic metal ion under investigation (species i) is regarded as the perturber, and only its oxidation state needs to change. Extrinsic P r o b e s

As with intrinsic probes, extrinsic probes can be either free radicals or metal ions. However, because free radicals usually induce magnetic interactions only over a short range, they are much less often used as extrinsic probes. The most notable exception to this has been the use of nitroxide spin labels specifically bound to sites on membranes or metalloproteins.

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391

Metal ions are often much more effective as extrinsic probes than free radicals. There are several reasons for this. First, as can be seen in Eq. (4), the magnitude of the effect that a perturbing species j has on the relaxation rate of species i depends on Jj(Jj + 1), where Jj is the total angular momentum of species j. For a free radical, the orbital angular momentum is quenched and the value of J is approximately equal to the spin of a free electron (J ~ S = ½). However, for metal ions this need not be the case. For example, lanthanides have been widely used as shift reagents in nuclear magnetic resonance (NMR) spectroscopy. These metal ions can similarly function as extrinsic probes in EPR spectroscopy. Because the crystal field environment about the metals produces only a small magnetic perturbation, their orbital angular momentum is not quenched, and, correspondingly, J can be very large. The second important reason why some rare earth metals serve as good perturbers is that they have spin-lattice relaxation times, Tlj, in a range that allows them to be effective relaxing agents. Using an ion with the proper TIj is very important. Too long a T~j (such as observed for Gd 3÷) will tend to cause a dominance of the b term in Eq. (4), as well as the A term of Eq. (3), resulting in an apparent loss of signal intensity of species i. On the other hand, too short a T1j (characteristic of an ion such as Pr 3÷) causes a dominance of the e term in Eq. (4) but with low magnitude of its overall perturbation effects on T~i. In light of these considerations, experience has shown that one of the best ions to use as an extrinsic perturber is Dy 3÷ (J = ~ ; tl ~ o3i where toi is the X-band precession frequency of a free electron).

Interpretation of Data

Distance Determination Equation (4) demonstrates that dipolar interaction between two paramagnets induces an increase in the observed rate of relaxation of each. Equation (4) further shows that for a species i, this increase depends on the quantities ~/i, Yj, Jj, toi, toj, T1j, r, and Oii. The quantities "Yi, Yj, Jj, ¢'°i, toj, and TI~ are all set by the paramagnetic species being investigated, as well as by the perturber, whereas r and 00 are functions of the geometry of the system being probed. The dependency of the latter terms is discussed in more detail below. In general, all three terms in Eq. (4) are important. However, some situations allow a simplification of the expression. For example, H y d e

392

PROBES OF METAL ION ENVIRONMENTS

[13]

and Rao 7 showed that when Dy 3+ (species j ) interacts with a free radical (species i) such as a nitroxide spin label, the middle c term dominates, or (1/Tli) = ~ J j ( J j + 1)yi2yj2h2r-6[2Tlj/(l

+ toi2Tlj)] sin 20ij c o s 20ij

(8)

To gain information about the magnetic interaction between species i and

j, 1/Tli must be determined. Because relaxation rates are additive, (1/Tli)observe d = (1/Tli)actua I + (1/Tli)dipolar

(9)

where (1/Tli)actua I is the rate in the absence of perturbing species j. Obviously, one way to obtain (1/Tli)dipola r is to determine the difference in relaxation rates of species i in the presence and absence of species j, or (1/Zli)dipolar = A(1/TIi ) = (1/Tli)observe d -- (1/Tli)actua I

(10)

In other words, (1/Tli)dipolar c a n be obtained by determining the change in relaxation rates of species i when the paramagnetism of speciesj is turned on and off. The paramagnetism is most easily changed through oxidation or reduction, when species j is an intrinsic metal cluster, or by simple addition and deletion o f j from the medium, when it is extrinsic. Once the value of (1/Tli)dipolar is determined, it can be used along with Yi, Yj, Ji, t°i, %, TIJ, and Oij in Eq. (4) or Eq. (8) to deduce the value of r, the distance between species i andj. It should be remembered that Eqs. (4) and (8) express the influence that one species j has on the relaxation rate of species i. Whereas species i andj are often in a 1 : 1 ratio in intrinsic interactions, extrinsic perturbers are normally added to the medium in large excess of the concentration of species i. This means that the resultant (1/Tli)dipolar term arises from the summation of Eq. (4) over r and Oij for all possible speciesj in the system. Assuming that speciesj is Dy 3+, Eq. (8) can be written 8 as (1/Tli)dipolar = C[Dy 3+] ~ r-6

(11)

where C is a proportionality constant, [Dy 3+] is the Dy 3+ concentration, and the summation, Y~ r -6, is taken over all of the individual Dy 3÷ ions. Knowing the structure of the protein containing species i, the value of the summation can be determined by integration over the distribution (r and 0ij) of Dy 3+ ions. Four different situations have been considered by Innes and Brudvig s in deriving equations for this summation, namely, both surface-bound and random distributions of Dy 3÷ when species i is either a soluble or a membrane-bound spheroidal protein. Simplifications 7 j. S. Hyde and K. V. S. Rao, J. Magn. Reson. 29, 509 (1978). s j. B. Innes and G. W. Brudvig, Biochemistry 28, 1116 (1989).

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INTRINSIC AND EXTRINSIC PARAMAGNETIC PROBES

393

of these interactions have been discussed 9 using the assumption that the protein containing species i has a spherical structure rather than the more complex spheroidal structure. Similar derivations for systems where species j is an intrinsic probe have been performed.I° However, these later calculations are complicated by the presence of phase memory in the determination of relaxation rates. To obtain absolute distances from Eq. (11), the value of C must be determined. This can be accomplished by using paramagnetic protein standards where both the structure and the type of interaction between species i and j are known. Cytochrome c and myoglobin nitroxide have been used TM as standards for soluble proteins, and the reaction center from Rhodobacter sphaeroides provides a good model for membranebound proteins: When choosing standards, it is important to remember that Eq. (4) contains the quantities Yi and cog, which depend on the spin state of species i. Therefore, the value of C in Eq. (11) assumes 12that the standard has the same J as the unknown species i.

Leigh Effect In addition to increases in the rate of relaxation, dipolar magnetic interaction may also induce spectral broadening. Of particular mention is the situation where i and j are two well-defined, fixed paramagnets and species i, the monitoring species, has a T 1 much longer than that of the perturbing species. As already mentioned, this situation will tend to cause a dominance of the b term in Eq. (4), as well as the A term of Eq. (2), resulting in an apparent loss of signal intensity of species i. For example, if species i is a free radical, its spectrum may be significantly broadened as a result of its magnetic interaction with a metal ion. According to Eq. (4), the magnitude of this broadening depends on 0ij, the angle subtended by r, the vector connecting species i to each species j, and the direction of the external magnetic field, B0. Therefore, each species j will have a different value of 0;j and, correspondingly, induce a different amount of broadening. Under these conditions, the observed resultant effect is not an overall broadening of the spectrum of the free radical but, instead, an apparent decrease in its amplitude, implying a decrease in spin concentration. As explained by Leigh,~3 the true spin concentration does not actually decrease. What happens is that, because the observed spectrum is actually 9 M. E. Oliver and B. J. Hales, Biochemistry 32, 6058 (1993). l0 D. J. Hirsh, W. F. Beck, J. B. Innes, and G. W. Brudvig, Biochemistry 31, 532 (1992). 11 M. E. Oliver and B. J. Hales, J. Amer. Chem. Soc. 114, 10618 (1992). 12 H. Blum, J. S. Leigh, and T. Ohnishi, Biochim. Biophys. Acta 626, 31 (1980). t3 j. S. Leigh, Jr., J. Chem. Phys. 52, 2608 (1970).

394

PROBES OF METAL ION ENVIRONMENTS

[13]

a composite of all possible spectra of the radical with different values of Oij, the individual component spectra are significantly broadened from spin-spin interactions, except for those spectra of free radicals that are oriented with 0ii close to the "magic angle" (i.e., cos 2 0;j ~ k), where the dipolar A term and relaxation b term go to zero. The intensity of the resultant spectrum is dominated by the unbroadened radicals within the magic angle. Therefore, in this situation, the extent of the decrease in the spectrum, rather than the extent of increase in 1/Tj, is a better gauge of the degree of magnetic interaction between the two paramagnetic species.

Spectral Diffusion and Contribution of Te to Pm It has already been mentioned that spectral diffusion can contribute to (1/T~)observeawhen recovery techniques are employed to measure relaxation rates. ~4 However, when using the PPS technique, the situation is slightly different owing to the more complex relationship between 1/TI and the measured quantity, PI/2. Absolute values of T~ from PPS experiments are difficult to obtain since it requires both the use of relaxation standards for the determination of TI and/'2 as well as the calibration of the quality factor of the resonance cavity. Because relaxation rates are additive [Eqs. (9) and (10)], (PI/2)dipolar =

APv2

= (Pl/2)observed -- (Pl/2)intrinsic

(12)

with the assumption that T2 is constant for all the measurements. For an extrinsic probe, combining Eqs. (I I) and (12) yields

APv2 = CA[Dy3+] ~] r -6

(13)

where A[Dy 3+ ] represents the change in the concentration of the paramagnetic probe. Plots of AP~/2 versus A[Dy 3+] should yield a straight line whose slope is C ~] r -6. In conjunction with the equations derived by Innes and Brudvig 8 or by Oliver and Hales 9 along with the standardized value of C, the value of this slope can be used to obtain the distance of the intrinsic paramagnet to the surface of its protein. For intrinsic interactions, the effects of scalar exchange coupling can become important, and the assumption that T2 is constant may no longer be valid.l° The dipolar contribution to T2 is similar to that found for TI in Eq. (4), and the resultant relaxation rates can similarly be expressed as (l/Tl)observed = (1/Tl)scalar + (1/Tl)dipola r + (1/T1)actual (I/T2)observed = (l/T2)scalar + (l/T2)dipola r + (1/T2)actual

(14) (15)

14 W. F. Beck, J. B. Innes, J. B. Lynch, and G. W. Brudvig, J. Magn. Reson. 91, 12 (1991).

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Equations (14) and (15) show that both observed rates are the results of contributions from the scalar, dipolar, and unperturbed (actual) rates. For intrinsic paramagnets where the orientations of species i and j are fixed by the system, the magnitude of the magnetic interaction specifically depends on the angle 0ij in Eq. (4). In this situation, Eqs. (14) and (15) should be represented as a summation of the rates corresponding to all possible orientations yielding an observed rate that will be nonexponential. Controls and Precautions

To obtain accurate values of (1/T l/)dipolar, several precautions should be taken. Because relaxation times are often highly temperature dependent, accurate temperature regulation is important. This is especially true at liquid He temperatures and/or when the PPS technique is used, where multiple data sets must be recorded at the same temperature over an extended period of time. Regulation of the solvent composition is important when using extrinsic probes. Variables such as pH or salt concentrations, which are known to influence protein structure, must be controlled. Similarly, because extrinsic paramagnets may bind to the intrinsic protein, changes in pH or ionic strength can have dramatic effects on the magnitude of this binding and, therefore, on the extent of the dipolar interaction. Different complex forms of the extrinsic probe may have different binding constants with the intrinsic protein. For example, dissolving DyC13 in buffered water produced solvated Dy 3+ , which can often exhibit strong ionic strength effects. Using chelators such as EDTA (ethylenediaminetetraacetic acid) or HEDTA [N-(2-hydroxyethyl)ethylenediaminetriacetic acid], which produce negatively charged or neutral complexes, respectively, often minimizes ionic strength effects and reduces the extent of external binding of the probe to proteins or membranes. Finally, precaution should also be taken to control for possible concentration-dependent effects of an extrinsic probe. When D y 3 + is used as the probe, the control experiments would employ the use of the diagmagnetic analog, La 3÷, to determine whether the presence of any rare earth ion induces relaxation effects.

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ION ENVIRONMENTS

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[14] E l e c t r o n P a r a m a g n e t i c R e s o n a n c e Spectroelectrochemical Titration

By KIM E. PAULSEN, MARIAN T. STANKOVICH, and ALLEN M. ORVILLE

Introduction The use of spectroelectrochemical methods to elucidate the energetics of biological electron transfer reactions has become common practice among researchers interested in characterizing the redox properties of biological systems. Simply stated, spectroelectrochemistry is the combined use of redox potentiometry and spectroscopy to determine the important thermodynamic parameters of an electron transfer reaction. These parameters include the formal potential value for the redox reaction (E°'), the number of electrons transferred in the reaction (n), and the free energy change for the reaction (AG°'). Aside from the obvious importance in understanding the energetics of single or multiple electron transfer reactions in a biological system, there are other interesting aspects to this kind of investigation. For example, the technique can be used to determine the effect that substrate or product binding has on the formal potential values of enzyme systems and to determine the binding constants for these interactions. The technique can also be used to make comparisons between different proteins containing a similar redox active center, in order to determine the magnitude to which different structural components around the redox active site affect the energetics of electron transfer for that particular type of center. Similarly, comparisons between the redox potential values of proteins subjected to site-directed mutations around the redox active si~e enables one to ascertain the importance of various amino acid residues for the energetics of that electron transfer reaction. Some innovative spectroelectrochemical methods have included the use of visible and electron paramagnetic resonance (EPR) spectroscopies for the quantitation of redox species present during potentiometric titration. The visible spectroelectrochemical technique has proved useful for the formal potential determinations of proteins that contain one chromophoric redox center since the amount of each redox species can be quantitated from its visible absorption spectrum. However, many protein systems contain multiple redox active centers with visible absorption spectra that overlap, and thus it may not be possible to resolve the absorbance change arising from each species unless their formal potential values are METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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well separated. In addition, certain redox active centers do not exhibit visible absorption spectra, or exhibit spectra but have very small molar absorptivity values, so that resolving the amount of each redox species present is not possible using visible spectroelectrochemical methods. EPR spectroelectrochemistry is an attractive alternative for such systems, since many biological redox centers are EPR active in one or more redox states and can therefore be quantitated from their EPR spectrum.

Electron Paramagnetic Resonance Spectroelectrochemical Cells In this section two novel EPR spectroelectrochemical cells designed in our laboratory are described. However, other EPR spectroelectrochemical cells of different construction have been designed and are described in previous literature. ~-3 Initially, the main body of each cell is made anaerobic by subjecting it to cycles of argon and vacuum over a period of 2 hr. The main body of the cell is maintained under positive argon pressure throughout the titration to minimize oxygen leakage. After the cell is made anaerobic, the experimental solution is partially reduced, by using either mediated electrochemical reduction or a chemical reductant such as dithionite. The protein and redox indicators are allowed to equilibrate, and the potential is measured. The potentials are measured at a temperature in the range from 4 ° to 25 °. Both cells described here have been designed so that after potential equilibration has taken place a quantitative aliquot of experimental solution is withdrawn from the electrochemical pool by a syringe. This syringe is located on a greased ball-and-socket joint which rotates from the experimental cell to a section of the cell containing an EPR tube in which the aliquot is then deposited and frozen in liquid nitrogen. The sample is further reduced, and the process is repeated. Thus, each point in the EPR spectroelectrochemical titration is represented by its own EPR sample. The cell design allows quantitative samples of experimental solutions to be taken under completely anaerobic conditions. In addition, repeated freezing and thawing of the experimental solution, which quickly denature many proteins, are avoided. This cell type was designed with these parameters in mind, so that quantitative aliquots of solution can be transferred anaerobically into EPR tubes and frozen, while the solution in the electrochemical pool remains unperturbed. The first cell to be described (Fig. 1 p. L. Dutton, this series, Vol. 54, p. 411. 2 S. R. Harder, B. A. Feinberg, and S. W. Ragsdale, Anal. Biochem. 181, 283 (1989). 3 C. J. Kay and M. J. Barber, Anal. Biochem. 184, 11 (1990).

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A

I1

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6

9

11 FIG. 1. (A) EPR spectroelectrochemical cell. 1, Syringe containing sodium dithionite; 2, syringe to transfer aliquot from experimental cuvette to EPR tube; 3, gas line to top section; 4, stopcock to top section; 5, ball-and-socket joint; 6, gas line to main cell; 7, reference electrode; 8, working electrode; 9, main body of cell; 10, electrochemical pool; 11, EPR tube; 12, gas line to bottom section; 13, auxiliary electrode. (B) EPR spectroelectrochemical cell with visible capability. The numbering for the cell parts is identical to that in (A), except that 10 is a visible cuvette and 13 is a holder for a Cary spectrophotometer.

1A) has been designed for species in which it is not advantageous to obtain visible spectra during titration. By avoiding the need for a visible cuvette with a relatively short path length, the electrochemical pool was made larger, which allowed for the use of a three-electrode system and more efficient stirring. The second cell to be described (Fig. 1B) has been designed for species in which it is advantageous to obtain visible spectra during titration. It contains only a two-electrode system for potentiometric measurement and has less efficient stirring owing to the limited area inside the visible cuvette.

Standard Spectroelectrochemical Cell The first EPR spectroelectrochemical cell we describe is depicted in Fig. 1A as it looks when a transfer of experimental solution is made from

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Fig. 1. (continued)

the main body of the cell (9) to an EPR tube (11) and then frozen. During titration and equilibration, the transfer syringe (2), located in the top section of the cell, and the bottom section of the cell containing the EPR tube are disconnected from the main cell. Once potential equilibration has taken place, the top and bottom sections are made anaerobic according to the procedures described below. The main body of the cell (Fig. 1A) contains a three-electrode system, such that coulometric reduction (oxidation) may be used during potentiometric titration. The three-electrode system consists of silver/silver chloride reference (7) and auxiliary (13) electrodes, and a gold working electrode (8). The ends of the electrodes are submerged in the experimental solution located in the electrochemical pool (10). This portion of the cell is cylindrical, with a 1 cm diameter and a height of 3 cm. The electrodes are made to extend deep into the electrochemical pool, without interfering with a small glass-encased stir bar. In this way, a minimal amount of experimental solution is needed to cover the electrodes, so that the number of aliquots of solution which can be taken, after each potential equilibration has taken place, is maximized. A gas-tight Hamilton syringe (1) containing a chemical reductant (or oxidant) can be inserted into the side arm of the main cell, if this method of reduction is preferred over coulometric

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reduction. The needle of the syringe must touch the side of the cell, above the experimental solution, so that the cell can be tipped and the aliquot of solution rinsed off the end of the needle by the experimental solution. After the main body of the cell is made anaerobic (6) the silver/silver chloride electrodes and the dithionite syringe are introduced into the cell under positive argon pressure. Once the solution has been partially reduced and has come to equilibrium, an aliquot of solution is transferred from the electrochemical pool into an anaerobic EPR tube and frozen. The transfer procedure will be described for a single point in the potentiometric titration. Above the main body of the cell is a top section, connected to the main cell by a greased ball joint that is clamped into its socket (5). This section of the cell has a stopcock (4), which keeps the main cell anaerobic during the electrochemical measurement, and a port through which a gastight Hamilton syringe, containing a 42-50 cm needle, can be inserted via a septum (2). The Hamilton syringe is equipped with a threaded plunger such that quantitation and the maintenance of an anaerobic environment are assured. When the transfer of solution to an EPR tube is necessary, the top portion of the cell and the transfer syringe are subjected to cycles of argon and vacuum, until the top section is anaerobic (3). At this time, the stopcock to the main cell is opened, the top section is rotated via the greased ball-and-socket joint, and the transfer syringe is used to withdraw an aliquot from the equilibrium solution in the electrochemical pool. Below the main body of the cell is a bottom section, connected to the main cell by a stopcock that keeps the main cell anaerobic during the electrochemical measurement. A hollow glass T section equipped with a degassing stopcock is connected to the main cell via a section of Norprene tubing. On the other end of the T is an EPR tube (ll) connected via a section of Norprene tubing. The bottom section is subjected to cycles of argon and vacuum (12) and is opened to the main cell. The greased ball joint is rotated, the transfer needle is inserted down into the EPR tube, the solution is deposited, and the needle is withdrawn. The stopcock between the main cell and bottom section is shut, and the EPR tube, while still connected, is frozen in liquid nitrogen and removed. The transfer syringe is fully removed from the top section of the cell, after the stopcock between the main cell and top section is shut. The transfer syringe is cleaned, and a new EPR tube is attached in preparation for the next sample transfer. The cell is then ready for an additional electrochemical measurement. It was of interest to determine the extent to which anaerobiosis could be maintained in this cell. The potential of the experir0ental solution in the electrochemical pool was measured after each solution transfer to an EPR tube and was no more than 0.005 V more positive after each transfer had taken place.

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Spectroelectrochemical Cell with Visible Capability An EPR spectroelectrochemical cell that has the capability for coordinated visible measurement during redox titration, as well as for potential and EPR measurements, is shown in Fig. lB. This cell is useful for correlating visible and EPR reduction spectra during potentiometric titration. The main body of the cell (9) consists of a visible cuvette which serves as the electrochemical pool (10), with a path length of 0.58 cm and the capacity to hold 2.9 ml of experimental solution. A silver/silver chloride reference electrode (7) and a gold working electrode (8) are used for potential measurement. Because of size limitations in the visible cuvette, an auxiliary electrode could not be fit into the electrochemical pool, so that only chemical reduction (or oxidation) could be used during potentiometric titration. A gas-tight Hamilton syringe (1) containing a chemical reductant (or oxidant) is located in a side arm of the cell. The protein solution is tipped into the side arm during titration so that it can come into contact with an aliquot of reductant. The experimental method and transfer procedures are similar to those described for the first EPR spectroelectrochemical cell and are not repeated here. This cell exhibited virtually no oxygen leakage when kept under positive argon pressure. This was determined with a solution of partially reduced pyocyanine (Era = 0.000 V, at pH 7.5 and 4°). The pyocyanine solution was monitored using visible spectroscopy and potentiometry, for a period of 2 hr, after which time no appreciable reoxidation of pyocyanine was detected.

Summary Both cell designs minimize denaturation and loss of a protein sample by avoiding the repeated transport of experimental solution between the electrochemical pool and an EPR tube. In addition, repeated freeze/thaw cycles of the sample are avoided since a new aliquot of the experimental solution is withdrawn and frozen to define each titration point. This is important since many proteins can be quite unstable owing to repeated freeze/thaw cycles that occur in other EPR potentiometric titration cells. Glycerol and ethylene glycol may temporarily stabilize the protein during freeze/thaw cycles, but such reagents should be left out of the experimental solution, if possible, since they may slow the rate of potential equilibration, especially at cooler temperatures (4°). These cells also allow for anaerobic sample transfer from the electrochemical pool directly to the EPR tube. It is very important for quantitative work that all EPR tubes used during a titration fill to the same height in the tube when filled with a specific volume of solution, in order to minimize error arising from sample

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height inconsistencies in the sample cavity. Although a maximum of only five to seven EPR tubes containing 250-300/zl can be acquired from these cells during a single titration, the tubes contain protein which is of high integrity, since protein manipulation and the total period of titration are minimized. Therefore, depending on the volume of aliquots taken during potentiometric titration, it may be necessary to perform more than one titration to gain sufficient data for formal potential value determinations, but the samples that result are more likely to represent the true state of the protein. Electrochemical Aspects of Electron Paramagnetic Resonance Spectroelectrochemistry Many aspects of redox potentiometry have been covered in other articles from previous volumes in this series, ~.4 so an extended discussion of this technique will not be repeated. Only those topics of specific relevance to the EPR spectroelectrochemical technique are discussed here. Electrochemical Equations

In redox potentiometry, the solution of interest is titrated either coulometrically or chemically in order to achieve reduction (or oxidation) of the redox center. At each point in the titration, the potential of the working electrode with respect to the reference electrode is measured, and the concentrations of oxidized ([Ox]) and reduced ([Red]) species are determined. In the case of EPR spectroelectrochemistry, the quantitation is achieved by double integration of the EPR spectrum for the paramagnetic species of interest. Consider the redox reaction: oxidized + ne- .~ reduced The number of electrons transferred during the redox reaction (n) and the formal potential value for the electron transfer (E °') are determined from a Nernst plot of the measured potential values (Emeas) versus the log([Ox]/ [Red]) for each point in the titration by using the Nernst equation (25°): Em~as = E °' + (0.059/n) log([Ox]/[Red])

(1)

A formal potential value indicates a potential value versus the standard hydrogen electrode (SHE), but under specified conditions o f p H , temperature, and buffer type and concentration (ionic strength). Reporting redox potentials this way is necessary in biological work since at no time does 4 G. S. Wilson, this series, Vol. 54, p. 396.

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one work under conditions specified for the standard hydrogen electrode. Rather, the conditions employed are those that are optimal for the biological system of interest. A Nernst plot is the most straightforward procedure for displaying spectroelectrochemical data of species for which only one electron is transferred during the redox reaction. Either the oxidized or reduced species is paramagnetic, and this species can be quantitated from its EPR spectrum, whereas the concentration of the nonparamagnetic species can be calculated by subtracting the concentration of the paramagnet present at each point in the titation from the initial or total concentration of protein present at the beginning of titration. Of course, the only other requirement is that the initial concentration of protein can be determined in some way, for example, by using a molar absorptivity value in the UV/visible absorption spectra or by using an assay to determine the total concentration of protein. Therefore, for a one-electron transfer there is at most only one unknown concentration present, and the data can be fit by a straightforward Nernstian analysis. Several redox active centers carry out two separate electron transfers during redox titation. In certain cases the intermediate or one-electron reduced species is formed to quite a large extent, indicating that the formal potential values for each electron transfer are widely separated. 5 In addition, the intermediate species may also be the only species that exhibits a quantifiable EPR signal. In this case there are two unknown concentrations, those of the oxidized and fully reduced redox species. The analysis used to determine the amount of each redox species present during the titration is less straightforward than that of a simple Nernstian analysis. A commonly used approach for presenting data from a redox titration that involves the formation of an intermediate species is to plot the fraction of the intermediate species present versus the measured potential value for each point in the titration. The fraction of intermediate species (Fq) present at any point in the titration can be represented by Fr I = [I]/([O] + [I] + [R])

(2)

where I, O, and R represent the intermediate, oxidized, and fully reduced redox species, respectively. If Eq. (2) is divided through by [I], then Fq = 1/([O]/[I] + 1 + [R]/[I])

(3)

5 W. M. Clark, "Oxidation-Reduction Potentials of Organic Systems." Robert E. Krieger Publ., New York, 1972.

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The ratios of [O]/[I] and [R]/[I] can be represented by rearranging the Nernst equations for the first and second electron transfers such that [O]/[I] =

exp[(nF/RT)(E

[R]/[I] =

e x p [ ( n F / R T ) ( E 2 °' -

-

El°')]

(4)

E)]

(5)

and

so that the final form of Eq. (2) can then be written as Fri

= 1/{I +

exp[(nF/RT)(E

-

El°')] +

e x p [ ( n F / R T ) ( E 2 °' -

E)]}

(6)

where n is the number of electrons transferred, F is the Faraday constant, R is the gas constant, Tis the temperature in kelvins at which the potentials are measured, E represents the measured potential values during titration, and E1°' and E2°' are the formal potential values for the first and second electron transfers, respectively. The typical approach for plotting the data is to use computer-generated lines in order to fit the experimental data, by using different values of El °' and E2°', until a best fit is observed. The formal potential values are defined by the equation which represents this line. Equations representing the fraction of oxidized and fully reduced species present during titration can be derived and plotted in the same fashion. 6,7 Redox Mediator

Titrants and Indicators

Because the redox centers of most proteins are surrounded to some extent by protein, direct communication between the redox center and the working electrode may not be possible without mediation. A redox mediator titrant is a small redox molecule which has a formal potential value quite different than that of the protein. The mediator titrant is reduced at the working electrode, at a formal potential value much more negative than that of the protein redox center, and it, in turn, quickly transfers its electron(s) to the redox centers of the protein. During the potentiometric portion of the redox titration, redox indicators are present in the solution in order to equilibrate with the protein redox centers and communicate the potential of the protein redox center to the working electrode. Redox indicators are chosen so that their formal potential values are close to the formal potential values of the protein redox center. The maximal redox buffering capacity of an indicator is reached when the ratio of [Ox]/[Red] is between approximately 10 and 6 M. Teixeira, I. Moura, A. V. Xavier, J. J. G. Moura, J. LeGall, D. V. DerVartanian, H. D. Peck, Jr., and B. Huynh, J. Biol. Chem. 264, 16435 (1989). 7 S. Mukund and M. W. W. Adams, J. Biol. Chem. 265, 11508 (1990).

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0.1 or when the indicator is within the limits of being 90% oxidized and 90% reduced. Thus, the maximal redox buffering capacities for one-electron and two-electron indicators are +-0.060 and -+0.030 V of the formal potential values for their electron transfers, respectively. The criteria for choosing mediator titrants and indicators that can be successfully used for redox titrations have been addressed previously, t Some imporant properties that mediator titrants/indicators must have in order to be used for redox titration are also discussed here. A mediator titrant/indicator molecule must not bind to the redox center of interest. An important indication that one of these molecules may be binding to the redox center and changing its immediate environment is that the EPR signal is perturbed so that the position or shape of the signal on the EPR spectrum has changed. This type of perturbation indicates that the electronic environment of the redox center has significantly changed, so that a shift in redox potential has probably also occurred. Therefore, a redox mediator titrant or indicator must react quickly and reversibly with the redox center without perturbing the redox potential of that center. In addition, these molecules must be chemically stable in all redox states, and it is most useful if they do not exhibit an EPR spectrum that interferes with the EPR signal of interest. Many mediator titrant/indicator redox potentials have been reportedS'8; however, it is worthwhile to determine these values independently under the conditions at which they are used in the experiment of interest. The concentrations of biological redox species present during EPR spectroelectrochemical titration may range from 20 to 600 ~M. The concentration of biological species used in the experiment is dependent on the characteristics of the redox center and the intensity of its EPR signal. It is useful to use an indicator concentration that is 10-20% of the total concentration of biological redox species present for potentiometric titration. Each indicator that is used to cover a portion of the anticipated potential range of reduction should be at this concentration. This indicator concentration allows for a reasonable equilibration time in which to make the potential measurement, while minimizing possible binding problems of the indicator to the redox center which may occur at higher indicator concentrations. There may be anywhere from 1 to 13 or more indicators present in the experimental solution, depending on how wide a potential region is titrated. The mediator titrant (e.g., methyl viologen) may be present at higher concentrations than the concentration of indicators in the experimental solution, for example, from 50 to 100% of the total 8 p. A. Loach, in "Handbook of Biochemistry and Molecular Biology" (G. D. Fasman, ed.), 3rd Ed., Vol. 1, p. 122. CRC Press, Boca Raton, Florida, 1985.

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concentration of biological redox species present, in order to ensure quick and complete reduction of the redox center during the reductive portion of the titration. M e t h o d s o f R e d u c t i o n (Oxidation)

Some consideration must be made as to what method of reduction (or oxidation) will be used during a potentiometric titration. If an EPR spectroelectrochemical cell contains a three-electrode system, with an auxiliary electrode as well as working and reference electrodes, then a coulometric titration is an attractive alternative to chemical methods of reduction, since chemical methods of reduction may produce by-products that can affect the stability of the protein, the stability of the protein redox center, or the pH of the solution. During coulometric reduction, the mediator titrant transfers electrons from the working electrode to the protein redox center at a potential more negative than that of the redox center being reduced. It is usually informative to titrate a redox center without any indicators present initially, in order to determine the number of electrons that the redox center takes up during reductive titration. It is still necessary to use a mediator titrant in order to facilitate quick and reversible reduction of the redox center; however, the mediator titrant itself does not remain reduced, whereas indicators are reduced and equilibrate with the protein system, thus taking up reducing equivalents themselves. The number of reducing equivalents taken up by the redox center during titration can be calculated from O = nFN

(7)

where Q is the number of coulombs added during titration, F is the Faraday constant, and N is the number of moles of redox center present for titration. The total number of equivalents needed for reduction can be determined by plotting the intensity of the EPR spectra for the species of interest versus the number of equivalents added (n) during titration. Chemical reductants (oxidants) retain their popularity as potentiometric titrants. Sodium dithionite (E °' = -0.660 V versus SHE, at pH 7.0, 25°, and 10 nM) 9 has been universally used as a reductant of protein redox centers. It is convenient to choose a concentration of stock dithionite solution such that only a very small volume of dithionite must be added to achieve reduction. A dithionite solution must be made and stored under anaerobic conditions, in order to retain its reducing strength for the length of time required by one titration. In addition, it is important to maintain 9 S. G. Mayhew, Eur. J. Biochem. 85, 535 (1978).

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a constant concentration of dithionite in solution (no oxidation occurring) during titrations in which it is desirable to know the number of equivalents needed to fully reduce the protein (without indicators present). In this case, it is necessary to standardize the dithionite solution, since some portion of the solution is typically oxidized when the solution is made. It is necessary to standardize dithionite solutions not only because of possible oxidation which may occur when making the solution, but also because solid sodium dithionite is not a primary standard. A dithionite solution may be standardized by titrating an indicator that exhibits a visible absorption spectrum. The concentration of dithionite used to reduce the indicator can be calculated from a known molar absorptivity value for the indicator in the visible absorption region. The reducing strength of dithionite solutions may vary for many reasons. For instance, the sulfinate radical anion, which is the actual reducing agent present in a dithionite solution, dimerizes and causes the redox potential of the dithionite solution to become more positive at concentrations above 10 nM. In addition, the equilibria governing the redox potential of the dithionite solution are pH dependent, so that at lower pH values the redox potential value becomes increasingly more positive. 9 Finally, a well-buffered solution is also necessary when working with dithionite, since a protein solution titrated by dithionite will otherwise become increasingly more acidic. Potassium ferricyanide (E °' = +0.425 V, at pH 7.0, 25°, and 0.1 M phosphate buffer) ~° has been a popular oxidant of protein redox systems for many years. One important drawback to its use has been noted in the redox titration of certain iron-sulfur clusters. It has been found that some 4Fe-4S clusters are converted to 3Fe-xS clusters owing to oxidative damage caused by ferricyanide titration. H Therefore, in this case it is wise to use another chemical oxidant, perhaps 2,6-dichlorophenolindophenol (Em = +0.237 V, at pH 7.0, 25°, and 0.1 M phosphate buffer), or mediated coulometric oxidation.

Criteria for Equilibrium An often underestimated but very important aspect of potentiometric titration is the ability to determine whether a redox system is at true equilibrium at each point in the titration. The time needed for potential equilibration, after the addition of reductant, varies according to the protein and indicators present in the system, since each indicator equilibrates at different rates with different protein redox centers. The equilibration time is dependent on the structure and charge of the indicators and redox 10 j. E. O'Reilly, Biochim. Biophys. Acta 292, 509 (1973). 11 M. Bruschi, Biochem. Biophys. Res. Comrnun. 91, 623 (1979).

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centers and how compatible they are structurally and electronically. For instance, if a redox center has acquired a negative charge or has a highly electronegative surrounding environment, then a negatively charged indicator may not ever achieve a true equilibrium with the redox center owing to electrostatic repulsion. This type of nonequilibrium problem can be seen experimentally as a potential that will not stabilize within any reasonable period of time. An analysis of the Nernst plot may also indicate that such a problem exists, since the slope may not reflect the number of electrons transferred in the redox reaction. Essentially, a good criterion for establishing that equilibrium has been reached is that the potential remain constant, with stirring, for at least 15-20 rain. This may take minutes or hours depending on the kinetics of the redox center and indicator equilibration. There is an important balance between the time it takes for equilibration of the indicator with the redox center, in order to establish a stable potential, and minimizing the total period of titration such that protein denaturation and oxygen leakage do not become significant problems. Spectroscopic Aspects of Electron Paramagnetic Resonance Spectroelectrochemistry The primary focus of this discussion is to describe how the concentration of a paramagnetic redox species is determined from its EPR spectrum and to also address specific problems that may arise during the determination. Thus, EPR is used as a quantitative tool in EPR spectroelectrochemistry, in order ultimately to determine the concentrations of all redox species present during redox titration. This discussion also focuses primarily on low-temperature (liquid helium) work, since most EPR-active redox systems are observable at these temperatures. A discussion about other aspects of EPR is avoided here, since other articles in this volume and other volumes have addressed these topics 12'~3 and several books have been dedicated to this form of spectroscopy, t4,z5

Quantitation in Electron Paramagnetic Resonance At each point in an EPR spectroelectrochemical titration, after potential equilibration of the experimental solution has taken place, an EPR 12 H. Beinert, W. H. Orme-Johnson, and G. Palmer, this series, Vol. 54, p. 111. 13 j. A. Fee, this series, Vol. 49, p. 512. 14 C. P. Poole, Jr., "Electron Spin Resonance." Wiley (Interscience), New York, 1983. 15 p. F. Knowles, D. Marsh, and H. W. E Rattle, "Magnetic Resonance of Biomolecules." Wiley (Interscience), New York, 1976.

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tube is filled with experimental solution and an EPR spectrum taken of the redox species of interest. Because an EPR spectrum is the first derivative of the absorption signal, the EPR signal is then integrated twice, usually by computer, in order to determine the area under the absorption signal, which is then described as the number of spins present in that particular sample. The actual concentration of the redox species is calculated by comparing the integrated signal of the unknown species to the integrated signal of a species in a standard solution of known concentration. Thus, the molar concentration of the unknown species can be calculated from

Mu = M~(HmJHmu)(P~l/2/Pul/2)(Gs/Gu)(Iu/geavu)/(Is/geovs)

(8)

where the subscripts u and s represent the unknown and standard, respectively, M is the molar concentration, Hm is the modulation amplitude of the magnetic field, P is the microwave power, G is the spectrometer gain setting, I is the integral of the absorption signal, and geav is the average integrated intensity factor. 13,16 To minimize the error introduced by each of these factors during the quantitation of the unknown species, it is useful to run both the standard and unknown species, if possible, under the same conditions of modulation amplitude, power, etc. If it is necessary to take EPR spectra of the standard and unknown species at different temperatures, another factor can be added to Eq. (8) that reflects the fact that the signal intensity or the magnitude of the second integral is inversely proportional to the temperature as long as both signals are following the Curie law in the temperature region of measurement. 17 According to Aasa and V~inng~rd,16 when the standard used for quantitation and the redox species under study have different g values, an integrated intensity factor may be calculated which takes into account the transition probability connecting two energy states and the g value related to that transition. An average of the integrated intensity factor over all directions of the oscillating magnetic field perpendicular to the static magnetic field should give the total integrated intensity of an EPR spectrum; however, a good approximation for the average integrated intensity factor, ge, v, is given by ge~v ~- -zs[(gx2 + gy2 +

gzZ)/3]l/z + [½(gx + gy + gz)/3

(9)

The reader is directed to the discussion by Aasa and V~inng~rd, who 16 R. Aasa and T. Vfinng~rd, J. Magn. Reson. 19, 308 (1975). 17 S. Fujita, "Statistical and Thermal Physics, Part II." Robert E. Krieger Publ., Malabar, Florida, 1986.

410

PROBES O F M E T A L I O N E N V I R O N M E N T S

[14]

developed Eq. (9), which accounts for the experimental error involved in using a standard with different g values than those of the unknown species under study. Two standards (S = ½) widely used for low-temperature quantitation in EPR are the Cu(I1) complexes Cu(CIO4)2 and Cu(EDTA) 2-, both of which are usually made to a Cu 2+ concentration of 1.0 mM. The Cu 2+ EPR spectrum exhibits an axial signal around g = 2 and hyperfine splitting owing to interaction of the electron spin with the spin of its nucleus. A procedure generously supplied by Helmut Beinert (Medical College of Wisconsin, Milwaukee, WI) for the preparation of a Cu(C104) 2 standard solution entails the dissolution of very pure copper metal in a minimum amount of HCI and then making the final solution to a concentration of 1.0 mM in Cu 2+' ion, 10 mM in HCI, and 2 M in sodium perchlorate.

Additional Experimental Considerations for Electron Paramagnetic Resonance Quantitation One important experimental consideration for quantitation in EPR is that the EPR spectrum must be scanned under conditions in which the signal is not saturated. At constant temperature, the intensity or double integral of the signal will remain proportional to the square root of the power, while the power is increased, as long as the signal does not saturate. Thus, the ratio of signal intensity (I) to the square root of power (pV2) will remain constant in the nonsaturating microwave power region, such that

I1/(pl~)j = I2/(p1~)2

(10)

If the EPR signal becomes smaller in intensity than expected as the power is increased, then the signal is becoming saturated and no longer reflects the actual concentration of species present in the sample. It is therefore important to characterize the saturation behavior of each redox center, before choosing the parameters of temperature and power at which quantitation will be performed. Another consideration involves that of interfering EPR signals from multiple redox centers in a protein. Often there are several EPR-active species present in a sample, and this may result in difficulty in quantitation of redox species if the signals arising from these species overlap. To a first approximation, the EPR spectrum of the component parts can be simulated based on g values and line width considerations. The simulated features can then be subtracted from the experimental data and the resultant integrated. In this fashion, a quite complicated EPR spectrum can

[14]

EPR SPECTROELECTROCHEMISTRY

411

be deconvoluted and quantitated. An impressive application of this procedure is demonstrated by Jollie and Lipscomb. 18,~9 A final consideration which may affect the formal potential value determinations of redox centers by EPR spectroelectrochemistry is that the measured potential values are taken at a temperature in the range of 4 ° to 25 °, but the quantitation of redox species is done at liquid helium temperature, after the EPR tube is frozen in liquid nitrogen. Therefore, any temperature-dependent equilibria may change during the freezing process. For example, the formal potential values of many redox centers exhibit pH dependence, and certain buffers exhibit a relatively large change in pH on freezing to liquid nitrogen temperature. The equilibrium constant for the redox reaction and, thus, the formal potential value will be affected by any pH change that occurs when the EPR sample is frozen after potential equilibration. Therefore, the concentrations of redox species will be different from when the potential of the experimental solution was measured to when the samples are frozen and the concentations are determined by EPR spectroscopy. The magnitude of the experimental error associated with the formal potential value determination in this case is dependent on how large the change in pH is for the buffer in which the experiment is performed. Williams-Smith et al. completed a study in which the apparent change in pH of various buffer solutions was measured on freezing to liquid nitrogen temperatures. They found that buffers such as phosphate, pyrophosphate, and Tris exhibited relatively large changes in pH on freezing, whereas buffers such as HEPES, bicine, and tricine were much less affected. 2° The amount of error present in any EPR spectroelectrochemical formal potential determination will depend on the number of problems encountered during the quantitation process. There is a certain amount of error inherent in any EPR quantitation owing to instrumental limitations. 2~ A wide range of redox potential errors have been reported by researchers, and they tend to fall within the range of -+0.010 to +-0.035 V of the reported formal potential values. Acknowledgments This work was supported by National Institutes of Health Grants GM29344(to M.T.S.) and GM24689(to John D. Lipscomb). 18 D. R. Jollie and J. D. Lipscomb, J. Biol. Chem. 266, 21853 (1991). 19 H. Beinert and S. P. J. Albracht, Biochim. Biophys. Acta 683, 245 (1982). 2o D. L. Williams-Smith, R. C. Bray, M. J. Barber, A. D. Tsopanakis, and S. P. Vincent, Biochem. J. 167, 593 (1977). 21 D. C. Warren and J. M. Fitzgerald, Anal. Chem. 49, 250 (1977).

412

PROBES O F M E T A L I O N E N V I R O N M E N T S

[15]

[15] M a g n e t i c S u s c e p t i b i l i t y By CH. BUTZLAFF, A. X. TRAUTWEIN, and H. WINKLER Introduction The measurement of the magnetic susceptibility is a major tool for determining the electronic properties of paramagnetic, possibly spin-coupled transition metal centers in biomolecules and related analogs. It is a method complementary to M6ssbauer, electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), magnetic circular dichroism (MCD), nuclear magnetic resonance (NMR), and X-ray absorption spectroscopies. 1 From temperature- and field-dependent measurements it is possible to derive information about the spin state o f a paramagnetic center as well as the exchange coupling between metal ions or between a metal ion and a radical. For this purpose analysis of the measured data with an appropriate model Hamiltonian is necessary. In exchange coupling, the magnetic state of metal ions in biological systems is a key chemical parameter, because the overlap of electron orbitals which governs exchange coupling also controls electron transfer processes. Systematic studies of molecular magnetism have something in common with many other areas of spectroscopy insofar as they serve two objectives: (I) to learn how to deduce the spectroscopic properties of a system theoretically from its structure by going back as far as possible to the first principles of quantum mechanics and (2) to draw conclusions about the geometric conformation and electronic configuration from the spectroscopic findings. The above-mentioned systematics cannot be determined by studying native and mutated proteins alone, because the possibilities of manipulating these systems are limited. Therefore, the efforts of bioinorganic chemistry to provide suitable model compounds are indispensable for any progress to be made in this area of research.

Magnetic Susceptibility The magnetic susceptibility of a system describes its response to a magnetic field. In general the magnetic susceptibility is given by a real I A . X . T r a u t w e i n , E . Bill, E . L . B o m i n a a r , a n d H . W i n k l e r , 1 (1991).

METHODS IN ENZYMOLOGY, VOL. 227

Struct. Bonding, (Berlin) 78,

Copyright © 1993 by Academic Press. Inc. All fights of reproduction in any form reserved.

[151

MAGNETICSUSCEPTIBILITY

413

symmetric tensor X, which relates the magnetization ~t to the magnetizing field/4: = X/t

(1)

The tensor X can be decomposed into a diagonal part and a traceless part X = ½Tr(x)E + F

(2)

with F = X - ~ T r ( x ) E and Tr(F) = O. E denotes the unit tensor. In a powder or liquid, averaging over the angles between the principal tensor axes and the magnetizing field leads to a disappearance of F. Then only the component of the magnetization in field direction remains, which is given by MH -=- ½ T r ( x ) H = x H

(3)

with X = l[Xxx + Xyy + Xzz]. Usually all values are given in terms of the molar susceptibility, defined by rn

where m denotes the molar mass and P the mass density of the substance. The dimensions of Xmol are given as cm3/mol in the cgs or as m3/mol in the SI system, respectively. The magnetization, being a macroscopic quantity, can also be obtained from the molecular magnetic moment tz through MR(T) =

NAP m

(~H)T

(5)

and, thus, Xmol(T) =

NA(I~H)T/H

(6)

where NA is Avogadro's number and (/ZH)x the ensemble average of the molecular magnetic moments parallel t o / 4 at temperature T. To continue, either one can calculate the expectation values of the operator 12 = -(/~ + gES)/~a with gsdenoting the spin-only g factor, L the total angular momentum, and S the total spin, which requires

414

PROBES OF METAL ION ENVIRONMENTS

[15]

knowledge of the molecular wave functions, or one can make use of the relation 2

\ix o OH/

(7)

where only the derivative of the appropriate Hamiltonian li~ with respect to H is needed. By denoting its eigenvalues by e5 one obtains for the magnetic moment

(l~H)j = -tz--~oOH

(8)

and correspondingly (~)T

=

1 Ej (de~OH) exp( - ej/k B T) /x0 Ej exp( - e / k B T)

(9)

or

Xii,~ol(T) =

1 NA "Zj [Oej(Hi)/OH i] e x p ( - ej/k B T) /~0 H Ej exp( - ej/kB T)

(10)

with i equals x, y, z a n d / 4 , denoting the condition t h a t / t points in the direction of the corresponding axis. Equation (10) is the basis of all further calculations of the magnetic susceptibility, presented in the following sections. Assuming that e1 can be expanded as power series in B = tz0H,

= e)o)+ C ) B +

+

. . .

one gets

Oej= ( 1 ) + OB ej

2Be}Z) + . . .

and e x p ( - eJkBT) = e x p ( - e(°)/ksT)(1 - Be}I)/kBT + " ") and finally, from Eq. (9), for the term linear in B: ($Xn)T =

B Ej [(e}l))Z/kBT - 2e) z)] exp( - e}°)/kaT) ~j exp( -- e}°)/ksT)

01)

2 j. H. van Vleck, "The Theory of Electric and Magnetic Susceptibilities." Oxford Univ. Press, London, 1932.

[15]

MAGNETICSUSCEPTIBILITY

415

It is assumed here that the material has no residual moment in the absence of the external field B. This condition may be expressed by Ej eJl) e x p ( - eJ°)/kBT) = 0. 3 Equation (11) is the well-known van Vleck formula. 2 It is relevant in the absence of saturation. If the magnetization is purely of orientational origin the Langevin formula for the molar susceptibility, Xmo~(T) -

"~ NAn 2~tt/~/x o

3ka T

(12)

is obtained in the high temperature limit, with/x 0 being the permeability and neff the so-called effective Bohr magneton number. 2.3 It is therefore customary to quote results of magnetic susceptibility measurements generally in terms of the effective magnetic moment t~efr = neff~B, which is related to the molar susceptibility ×tool by /.6ef t =

3kB/tZoNA~BZ)I/2(XmolT)I/2tZB

(13)

Inserting the numerical values in cgs units yields /xeff = 2.828(Xmotc~sT)l/21~a

(14)

and correspondingly, inserting of the values in SI units yields

tXeff = 797.5(XmoLsIT)l/E~B

(15)

For a state with the net spin S the spin-only value of/%rr is expected to be /x~ff = [4S(S + 1)]l/2/xB

(16)

Susceptometer

Principles of Superconducting Quantum Interference Magnetization Measurements The determination of the magnetic susceptibility is based, in gefieral, on effects produced by the macroscopic magnetic moment of the sample. This can be the force exerted by a magnetic field gradient and measured, for example, in a Faraday balance or the electric voltage induced in a coil when the sample is moved. Typical devices that use the second principle are the Foner susceptometer and the superconducting quantum interferE. A. Boudreaux and L. N. Mulay, "Theory and Applications of Molecular Paramagnetism." Wiley, New York, 1976.

416

PROBES OF METAL ION ENVIRONMENTS

Superconducting solenoid

b Superconducting

uSarnple

t

[15]

V I _

A

I le

Pick up coil array

[-

Apptied fietd B

FIt~. 1. Magnet configuration (a) and sensor (second-derivative coil) configuration (b) of the M P M S - S Q U I D susceptometer.

ence device (SQUID) susceptometer. Figure 1 shows how the pickup coils and the superconducting solenoid are arranged in the magnetic property measurement system (MPMS)-SQUID susceptometer by quantum design. The superconducting coils are wound in second-derivative configuration in which upper and lower single turn are counterwound with respect to the two-turn center coil. This configuration strongly rejects interference from nearby magnetic sources and allows the system to function without any benefit from a superconducting shield. 4 This enables not only temperature-dependent but also field-dependent measurements to be made. The normal measurement technique used in the MPMS is to position the sample below the sensing coils and then to raise the sample through the coils. The signals induced in the second-order derivative coils are coupled to a SQUID sensor through an isolation transformer. The sample is then measured by repeatedly moving it upward a particular distance and reading the voltage from the SQUID detector. The function of a SQUID detector is based on the concept of fluxional quantization in a superconducting ring and on tunneling through a "weak link" (Josephson contact) between two superconductors. An exact explanation lies beyond 4 A. C. Bruno, C. Ribeiro, J. P. v o n d e r Weid, and O. G. Symko, J. Appl. Phys. 59, 2584 (1986).

[15]

MAGNETICSUSCEPTIBILITY

417

amplltude/V

O O O O O O O O ¢D ¢D O O O ®

O ¢5 O O ¢D O O O ¢D O O ~9 O O ¢D

- - L [ I I I I I I I I l i I I I I I I I I I [ I I I I L I I I I [ I I I IuL I I L I [ I

-2

-1

0

1

2

posltlon/cm FIG. 2. Response of a cylindrical palladium r e ~ n c e field of 0.1 T.

sample at 295 K with an external

the scope of this chapter. Readers interested in this field are referred to Ref. 5. The SQUID voltage is read at numerous points (e.g., 64), and the values are stored as a function of sample position. At each position the voltage reading is typically repeated several times, and many vertical scans are averaged to improve the measurement precision. The response of the detecting system to a point sample is given by Res ~ M

.[R 2 + (A + Z)2] 3/2

(R 2 + Z2) 3/2 + [R 2 + ( - A

+ Z)2] 3/2

(17) with R being the radius of the detection coils, A the coil separation, and Z the sample position. M is the magnetization of the sample in the direction of the magnetizing field. Equation (17) is the sum of the responses of four single-wound coils, located at the positions - A , O , O , A . The sign of the response of the inner coils is opposite to the sign of the response of the upper and lower coil. This is due to the different winding directions. Figure 2 shows the response of a cylindrical palladium sample, 2.5 mm in diameter and 2.5 mm in length, which can be regarded as a point 5 0 . V. Lounasmaa, "Experimental Principles and Methods Below 1 K , " Chap. 7. Academic Press, New York, 1974.

418

PROBES OF METAL ION ENVIRONMENTS

[15]

la,f~/la 2.2

2.0

1.6

1.6 J

J

I

~

!

50

I

I

I

I

I

I00

I

I

I

I

F

r

150

I

r

i

I

200

r

i

i

i

!

250

i

i

~

i

300 T/K

Fit3.3. Effective moment/zeff of [(CH3)2NHCH2CHzNH(CH3)2]CuCI4 .

sample. Comparison of this signal with the values given by the N1ST (National Institute o f Standards and Technology) for a platinum sample provides the right calibration factor. In our case we work mainly with magnetically highly diluted samples. Therefore, it is useful to check the calibration with a similar standard. F o r this purpose one can use a sample of [(CH3)2NHCHzCH2NH(CH3)2]CuCI4 . The temperature-dependent measurement from 2 to 300 K gives a temperature-independent magnetic moment o f 1.88 ~B (Fig. 3). A fit with the spin Hamiltonian yields an average g value of 2.17(I). A diamagnetic contribution o f - 2 0 0 x 10 -6 cm 3 mol-1 and a temperature-independent paramagnetism of 60 × 10 -6 cm 3 mo1-1 was assumed. 6 A simulation of the measured E P R spectrum (Fig. 4) gives the g values (g = 2.26, 2.195, 2.073), with an average of 2.176. This is in excellent agreement with the g value determined by the susceptibility measurement. In the literature 7 g values of 2.168 measured by E P R and of 2.149 determined by a susceptibility m e a s u r e m e n t are reported. The first value is in good agreement with our values, whereas the second value is too small. In this analysis 7 a diamagnetic contribution o f - 2 . 1 7 x 10 -6 cm 3 mol -~ was assumed. 6 A. Weiss and H. Witte, "Magnetochemie." Verlag Chemie, Weinheim, 1973. 7 D. B. Brown, V. H. Crawford, J. W. Hall, and W. E. Hatfield, J. Chem. Phys. 81, 1303 (1977).

[15l

MAGNETIC SUSCEPTIBILITY

419

g ¢octor 2.5

2.3

i

2.2

2.1

2.0

i

i

,

I

1.6 t

1.9

Z

2/

dX7 dB

!

260

[

I

280

[

I

300

'

'

320

J

*

3z,O

I

!

360

380

B[mT]

FIG. 4. EPR measurement of [(CH3)2NHCH2CH2NH(CHa)2]CuC14 (bottom trace). The top trace shows the corresponding simulation with g equal to 2.26, 2.195, and 2.073 and line widths of 5, 5, and 3.5 mT, respectively.

Sample Holder

The sample holder is made of a quartz tube, 4 mm outer diameter, 3 mm inner diameter, and 20 cm length. The tube is closed in the middle for holding the sample. The sample volume itself is enlarged to form a cylinder of 6 mm length and 6 mm diameter. At the lower and upper end, two rings, made of Delrin, are fixed for getting the right horizontal position. The material of the sample holder is quite important with respect to the background signal. Day et al. s have shown that quartz is a good material for sample holders because it does not contain detectable levels of fluorine or proton nuclear spins, which would otherwise be the case with most plastic materials. The sample holder is fixed to the lower part of the sample support tube of 1.2 m in length and 3 mm in diameter, as shown in Fig. 5. The upper part of the tube is made of stainless steel, whereas the lower part is made of brass to minimize the contribution in the SQUID detector caused by the sample support tube magnetization. Owing to its geometry s E. P. Day, T. A. Kent, P. A. Lindahl, E. Mi)nck, W. H. Orme-Johnson, H. Roder, and A. Roy, Biophys. J. 52, 837 (1987).

420

PROBES OF METAL ION ENVIRONMENTS

Sample

[15]

S u p p o r t Tube

Sample

Holder

~ t . a n c e

Ring

FIG. 5. Sample holder and sample support tube.

the sample holder gives a symmetric response in the SQUID detection system. This is important if one is interested in absolute magnetization measurements, where the amplitude of the background signal must be determined (see section on data acquisition). Sample Handling. The sample is introduced into the sample holder with a long spatula. The sample holder is weighed before and after filling. After filling, the sample holder is flushed carefully with N2 to remove molecular oxygen. The sample holder is then inserted into the upper part of the susceptometer tube. This part works as an air lock and is separated from the rest of the susceptometer tube by an "air lock vent." The air lock is then purged of air and backfilled three times with helium gas. Then the air lock vent can be opened, and the sample is lowered into its measurement position, which ought to be symmetric with respect to the detection coils. Frozen samples are loaded as follows. The sample is kept under liquid nitrogen in a Dewar flask. Care has to be taken that no liquid nitrogen remains in the sample holder when inserting the sample into the susceptometer tube. The sample holder is mounted quickly into the air lock, which is purged of air and backfilled with helium gas three times. Finally the sample holder containing the frozen sample is lowered into the cold instrument. The complete transfer of the sample holder from the liquid nitrogen Dewar system into the susceptometer takes less than 1 min. The reverse transfer of the frozen sample from the cold instrument to the liquid nitrogen

[15]

MAGNETIC SUSCEPTIBILITY

421

Dewar system is accomplished accordingly. It is important to have the temperature inside the instrument adjusted well below liquid nitrogen temperature during loading and unloading, to avoid thawing of the sample during the relatively slow insertion and removal process. Experimental Conditions. A typical powder sample contains 30 mol of material with S = { substance in a volume of 0.05 c m 3. Table I summarizes significant data for the MPMS susceptometer.

Data Acquisition The complete measurement with a temperature or field scan is fully controlled using a computer. A computer program with information about the required temperature and field values, holding times, and number of measurement points runs automatically. For determining the response of the sample holder, which is part of the sample signal, the whole procedure has to be repeated with an empty sample holder under identical conditions. For each temperature or field scan the sample travels several times step by step through the detection coils, as described above. After each step, the voltage at the SQUID detector is read several times (typically three times) and averaged. There are typically 64 steps for a travel length of 4 cm. This procedure can be repeated several times and averaged. The instrument adjusts the sensitivity automatically so that the output voltage at the SQUID sensor is in the range of ---10 V. This means that for later data evaluation the information about the sensitivity region must be stored. Within a temperature scan different sensitivity regions are used. All data concerning travel length, number of data points, number of repetitions, temperature, field, etc., have to be stored. TABLE I DATA FOR MAGNETIC PROPERTY MEASUREMENT SYSTEM SUSCEPTOMETER Parameter

Data

Temperature region Applied field Range of detectable magnetic moments Measuring time Amount of liquid He Differential sensitivity~

2-400 K -+5.5 T +2 × 10 -3 A m 2 10 hr for temperature scan of 2-300 K 5 liters/day 10 T M A m E in 2 T field

o The given sensitivity should be understood as a detection limit. For absolute measurements of the magnetic moment of a sample, a lower limit of the sample concentration is 0.25/zmol of S = ½ particles? This limit is due to the problem of separating the diamagnetic contribution of the sample holder and the uncertainties in weight or cleanness of the sample from the true signal.

422

PROBES OF METAL ION ENVIRONMENTS

[15]

amplltude/V

10

-2

-I

0

1

2 pos 1 t t on/cm

FIG. 6. Measurement of C30H52N6S2OlsCI2Fe~n at 90 K. The applied field was 1 T. Squares represent the uncorrected and circles the corrected data. Both curves are scaled independently to given an amplitude smaller than -+10 V. The solid line is given by the fit using Eq. (17) and assuming an additional linear drift and offset in the voltage.

The M P M S standard software fits each response curve (see, e.g., Fig. 2) with the r e s p o n s e function given in Eq. (17). The amplitude, corrected for the sensitivity region and multiplied by the appropriate calibration factor, gives the magnetization of the sample. This p r o c e d u r e has to be repeated with the e m p t y sample holder. The two magnetizations are subtracted f r o m one another, thus yielding the magnetization of the sample alone. F o r magnetically dilute samples, which is usually the case with biological materials, the r e s p o n s e c u r v e of the u n c o r r e c t e d data often does not h a v e the ideal pattern s h o w n in Fig. 2. Therefore the data cannot be fitted with the r e s p o n s e function given in Eq. (17). Figure 6 represents m e a s u r e m e n t s o f C30HszN682015CI2Fe2 TM at 90 K; the field was 1 T. The raw data (squares) cannot be described by Eq. (17). We h a v e d e v e l o p e d a c o m p u t e r p r o g r a m that solves this p r o b l e m by making a p p r o p r i a t e data corrections. First the sample and the e m p t y sample holder m u s t be m e a s u r e d under identical conditions. The sample holder m u s t be m o u n t e d at exactly the same position in both cases. 9 Then 9 The program is able to correct for small deviations in the two positions.

[15]

MAGNETICSUSCEPTIBILITY

423

1.10 1.06 1.06 4--

. . . . .

1.02

= o

----

•:-.1.......~L..-'--- ---~

....

1.00 : .....

m~

"-~_

0.96

,,,__

..U9 . m

" " -'" - "" "'" .~" 1.27mm

0.92 0.90

0

1

2

3

t~ Sompte

S

6

7

8

9

Length/1~m

FIG. 7. R e s p o n s e of the M P M S - S Q U I D s u s c e p t o m e t e r as a function of sample size relative to a point source. T h e sample diameter was varied from 1.27 to 8.89 m m in steps of 1.27 m m .

the program converts the data points for both cases to the same sensitivity region. In general, the sensitivity regions of the sample and of the empty sample holder measurement are not the same. Afterward the program subtracts the data point per point for each sample position yielding the corrected data (circles in Fig. 6). This procedure applies for each temperature or applied field. It is obvious that the corrected data have a pattern corresponding to Fig. 2. Now it is possible to fit the data with the response curve of Eq. (17).1° The solid line shows the corresponding fit, the amplitude of which, calibrated with the sensitivity factor and the calibration factor, represents the magnetization of the sample. The response function of Eq. (17) is valid only for a point source. Expanded samples yield the same response pattern, but with a lower amplitude. The arrangement of the coils would give no response for an infinitely long rod. Therefore, a correction factor has to be taken into account. Figure 7 shows the response, relative to the point source, as a function of sample size.lJ ~0 A n offset and a linear drift of the voltage has to be a s s u m e d and fitted additionally. i1 M. B. S i m m o n d s , " E f f e c t of sample size on axial r e s p o n s e . " Q u a n t u m Design, San Diego, CA, 1990.

424

[15]

PROBES OF METAL ION ENVIRONMENTS

Theoretical Concepts Spin Hamiltonian

The magnetic (Zeeman) and spin-orbit interaction of an electronic system with spin S and angular momentum £ is described by the Hamiltonian 12

= x/?,g + aBg(f. + g~3)

(18)

For an orbitally nondegenerate and energetically well-separated ground state, ~ can be regarded as perturbation of the ligand field interaction and may then be written as (19) with go = geSij - 2 h A i j

(20)

Dig = _ h2Aij

(~0[t/[~,)(~nltj 1~0)

A°= .,,oE

en-Eo

where ~0, ~ , are the eigenfunctions and E 0, E. the eigenvalues of the ligand field operator. Because g and D are symmetric matrices, ~ can be given in its principal axes system by = D

Sz z -

S(S

+ 1) + ~ (Sx

-

Sy 2)

~- l~B E

giBiSi

i=x,y,z

(21)

D denotes the zero-field splitting parameter [D = D z - I ( D x - Dr)] and E the rhombicity [ E = ½(Dx - D r ) . Equation (21) is called the spin Hamiltonian and is derived for a nondegenerate ground state from second-order perturbation theory. It was originally introduced to EPR by Abragam and Pryce.13 With this method the quantitative behavior of paramagnetic complexes under the influence of an external field can be described, with only a few exceptions. The method is applicable as long as ligand field and spin-orbit interactions produce a group of ground state atomic levels whose internal splitting is small compared to the energy separation of all other levels produced by the ligand 12 M. Weissbluth, " A t o m s and Molecules." Academic Press, New York, 1978. 13 A. Abragam and M. H. L. Pryce, Proc. R. Soc. London A 205, 135 (1951).

[15]

MAGNETICSUSCEPTIBILITY

425

field. This is often the case in coordination complexes of transition metals. At sufficiently low temperatures, when only this low-lying group of levels is appreciably populated, it alone determines the magnetic properties of the system. Even in the absence of an external field, the (2S + 1)-fold degeneracy of the ground state is partly removed by the zero-field splitting, which has the form of an electrostatic quadrupole interaction and is derived from the admixing of excited orbital states into the ground state in second order by spin-orbit interaction. When rhombic distortion is present any degeneracy in integer spin systems is totally lifted, but at least a 2-fold degeneracy is left in half-integer spin systems as a consequence of Kramers' theorem. Kramers degeneracy will, however, be removed by the action of the Zeeman interaction/ZB/lg~. The eigenvalues ej and eigenstates I~j), j = 1. . . . . (2S + 1) can be derived by diagonalization of the spin Hamiltonian matrix. The eigenstates can be expressed in terms of ISms): 1%) = ~

CJmslSms)

(22)

ms

where C~s represents the expansion coefficients of I~j) in the basis of ISms). From the eigenvalues the magnetic susceptibility can be calculated according to Eq. (10).

Exchange Interaction A molecule containing two or more paramagnetic centers must be described by a Hamiltonian that takes into account the interaction between these centers. For a dimer with two paramagnetic centers of spin S l and 2, the appropriate Hamiltonian is given by ~l = $11S2

(23)

where J denotes the interaction tensor. J can be decomposed into a scalar part J E , with E being the unit tensor, and a traceless tensor J'. J' includes the spin-dipole interaction ~4and anisotropic exchange interaction terms. J describes an isotropic exchange interaction which is given by

t4 R. E. Coffman and G. R. Buettner, J. Chem. Phys. 83, 2393 (1979).

426

PROBES OF METAL ION ENVIRONMENTS

[15]

the H e i s e n b e r g - D i r a c - v a n Vleck (HDVV) spin Hamiltonian2,15a6: ~ i = J S 1$2

(24)

Different interaction mechanisms have been proposed, and a detailed discussion can be found in a n u m b e r of excellent review papers 17-~9 or in the original work. 15,16,20Since the 1970s synthetic chemistry has produced a wealth o f spin-coupled binuclear and polynuclear transition metal complexes, which have been employed for systematic studies of the structural d e p e n d e n c e of exchange coupling. 21,22 In the major part of these complexes, the paramagnetic centers are in definite valence states and possess good local spin quantum numbers. In these systems, the coupling is, to good approximation, described by the H D V V Hamiltonian. In most o f the exchange-coupled systems, the paramagnetic sites are separated by one or more bridging ligands. The intervening ligands play an essential role in the coupling mechanism, which is termed superexchange. The exchange coupling constants IJ] are, owing to the indirect character of the coupling, generally small at the chemical energy scale (i.e., <500 cm-1). Experimental21.22 as well as theoretical investigations 23 show that the superexchange coupling is strongly structure dependent, that is, relatively small changes in bond angles or distances may even reverse the sign of j.24 S p i n H a m i l t o n i a n f o r S l = $2 = 1 D i m e r

As an example we present the spin Hamiltonian for a S 1 = $2 = I dimer. The tensors g and ,I are assumed to be diagonal. Therefore ~ can be written as = ~ I + ~](~II + ~III 15 W. Heisenberg, Z. Phys. 38, 411 (1926); W. Heisenberg, Z. Phys. 39, 499 (1926); W. Heisenberg, Z. Phys. 41, 239 (1927); W. Heisenberg, Z. Phys. 49, 619 (1928). 16 p. A. M. Dirac, Proc. R. Soc. London Ser. A 112, 661 (1926); P. A. M. Dirac. Proc. R. Soc. London Ser. A 123, 714 (1929). 17 j. B. Goodenough, "Magnetism and the Chemical Bond." Wiley, New York, 1963. 18 p. W. Anderson, "Magnetism," (G. T. Ratio and H. Suhl, eds.), Vol. 1. Academic Press, New York, 1963. 19 A. P. Ginsberg, Inorg. Chim. A. Rev. 5, 45 (1971). P. W. Anderson, Phys. Reu. 79, 350 (1950). z~ R. D. Willet, D. Gatteschi, and O. Kahn (eds.), "Magneto-Structural Correlations in

Exchange Coupled Systems." Reidel, Dordrecht, The Netherlands, 1985. 22R. Holtzelmann, K. Wieghardt, U. Fl6rke, H. J. Haupt, D. Wetherburn, J. Bonvoisin, G. Blondin, and J. J. Girerd, J. Am. Chem. Soc. 114, 1681 (1992). 23E. L. Bominaar and R. Block, Physica 121(B + C), 109 (1983). 24 p. Knopp and K. Wieghardt, lnorg. Chem. 30, 4061 (1991).

[15]

MAGNETIC SUSCEPTIBILITY

427

with

(S/x2 -

iz -- 3 "}-

i=1,2 ~iI = tzB ~

i=1,2

S iy2)

g~SoBj

(25)

j=x,y,z

~III

=

E

JjSIjS2j

j=x,y,z

F o r the calculation o f the matrix elements o f ~ i and ~ n w e use 12 1 "8 ' (lm's[Sx[lms) = ~72( ms,ms+l "~ 8m's,ms-I)

i "8 ' (lmglSyllms) = ~7"~( ms,ms-I -- 8m~s,ms+l)

(26)

(lm~lSz[lms) = msSm's,ms and t

(lm'sllms2lSl;x,y,zllmsllms2) and for

t

p

= (lmsllSl;x,y,zllms,)Smsz,msz

~lII

(lm'sllm 'sz[Sl;xS2;xllmsllmsz) =

l(Srn'sl,msl+l -~

F

msi,msl_l)(Sms2,ms2+ 1 -~-

(lm~l lm~zlSl;ySa;yllmsl lrns2) = 1

--5(--Sm'sI,mSI+I q-

(lrnsllms21Sl;zS2;z]lmsllms2)

8m'sI,msI_I)(--Sm~s2,ms2+I

"q-

= mslms28m's|,msl~m's2,ms2

8rn's2,rns2_l)

~m's2,ms2_I) (27)

~ l , ~ I I , and ~iii are s u m m a r i z e d in Tables I I - I V . ~Dimer in Table V r e p r e s e n t s the simple case with Jx = Jr = Jz = J, Bx = By = O, B z = B, E l = E 2 = 0, D 1 = D 2 = D, and gl = g2 = g. Diagonalization o f

+

+

"-A +

r

+

+

"'A +

I

0 u~

+

,,,w-, I "'A +

<

I "-A +

+

I

+

I

+

+

I

-

~ 428

'7'

I

r

~'II

~

~

~1

I

,,+

~+

t~ O

=~.

~z F-,

r',~ .4<

oZ II II

~+

~.+

I

I

o

429

-?

,@ I

f

+

+

I

+

=

I

0

+

+

f

,J

X

+

r

,<

+

I

+

r

I

I

-

I

°

430

I

7'

4-

I

~q ~L ~o I

I

% I

LL

~

z

~q

4-

X

I

÷

q~

"4-

% 4-

$ I

I

I

432

PROBES OF METAL ION ENVIRONMENTS

~Dimer

[15]

leads to the following eigenvalues 2

e9=-~D+2gI~BB+

J

1 e8 = - - ~ D +gtZBB + J e7 =

-~D - ~J +

~

8J 2 +

~-D~-~D

-

J

+

D+

J

1

136 = -- ~ D - gtZBB + J 2 ~5 = -~ D - 2gtzBB + J

(28)

1

e4 = --~D +gtZBB-- J 2 e3 =~D

- J

1 e2 = -~D-gtZBBet = --~D--~J-

J +

D-

J

+

D+

J

The magnetic susceptibility can be calculated by using Eq. (I0) with Z = ~i e-flei, ~

X=

=

1/kB T, NAgtZB {2e -#e9 -- 2e -~5 + e -#~8 + e -/3~4 - -

Z

e -/3e6

--

e-#~2} (29)

Figure 8 shows the D and J dependence of the eigenvalues ej with the applied field of 1 T being parallel the z axis. Examples Susceptibility o f a S 1 = 52 = 1 D i m e r

An example for a $1 = $2 = 1 system is the/z-hydroxobis (/z-carboxylato)divanadium(III) complex described by Knopp and Wieghardt. 24 The two V III ions are bridged by an hydroxo and two carboxylato groups, leading to antiparallel spin coupling of the two paramagnetic V III ions. The measured magnetic susceptibility is shown in Fig. 9, with the solid curve representing the spin Hamiltonian analysis. The data reflect the typical behavior of an antiparallel spin-coupled dimer with ground state S = 0. The increase at temperatures below 10 K is due to paramagnetic impurity. The solid line has been recalculated with J = 75 cm-l, D = 4

[15]

MAGNETIC SUSCEPTIBILITY

ej/crn -1

433

e j / c m -1

20 --r-

J=lO~

20 "ID=5cm- 1 £

£5

i0

~8

~6

~3

-tO ~

£4

-i0

e

-20 t

.

.

0

.

.

.

i

.

.

4

2

.

.

6

.

lO

8

o

2

6

4

a

10 D i c t a -1

J l c m -1

FXG. 8. Energy eigenvalues for a Sj = S 2 = 1 dimer; g~ = g2 = 2, Dj = D 2 = D, and Jx = J~. = Jz = J. The applied field of 1 T lies parallel the z axis.

X =Jcm 3 mole4 0.008

0.006

0.004

0.002

0.0

i

0

I

i

I

50

I

n

I

i

I

I00

I

I

I

I

I

150

I

I

I

I

I

200

I

L

I

I

i

250

I

L

I

1

300 T/K

FUG. 9. Magnetic susceptibility of L2V~n(/z-OH)(ox-carboxylato)2 (L is 1,4,7-trimethyl1,4,7-triazacyclononane).

434

PROBES OF METAL ION ENVIRONMENTS

[15]

cm -I, and g = 1.9, a diamagnetic contribution of -53.1 × 10 -5 cm 3 mo1-1, and a paramagnetic impurity of 3.6%. The D and g values are typical for V m ionsY

Susceptibility of Porphyrin Radical Complex In a series of oxoferryl porphyrin radical complexes the spin coupling between the ferryl iron (S = 1) ion and the porphyrin radical (S' = ½) could be proved conclusively 26 by applying EPR and M6ssbauer spectroscopy. However, this procedure failed in the case o f [TPpivPFelVO] + V(TPpivP is tetra phenyl "picket-fence" porphyrin) for reasons given below. Therefore, as an alternative we have measured the susceptibility of 10/xmol [TPpivPFetVO] ÷ (Fig. 10). 27 The measurement yields an effective magnetic moment of 3.4/x B at 100 K which corresponds to a spin S = a. The oxoferryl porphyrin with spin S = I would give a magnetic moment of 2.9/.t B . A fit of the whole temperature scan with the spin Hamiltonian formalism described in above provides a coupling constant of J = (4 --- 2) cm -~ and a zero-field splitting parameter of D = (18 --+ 4) cm -1. For the ratio of J/D ~- ¼significant anisotropy for the spin expectation value is expected.28 Therefore, the EPR absorption extends from approximately 0.3 T to ~, thus making the EPR signal undetectably weak. The M6ssbauer measurements, detected so far in the temperature and field range T -> 4.2 K and B -> 1 T, are not sensitive to the relatively small value of J = 4 cm -~.

Spin-Frustrated Copper(I1) Trimer The trinuclear complex [L3Cu3(Im)3](CIO4) 3 (L is 1,4,7-trimethyl-l,4,7triazacyclononane and Im is imidazolate) has been characterized by X-ray crystallography as an equally spaced imidazolate-bridged copper trimer. 29 The three copper ions are arranged at the corners of an equilateral triangle with a Cu . - . Cu separation of 0.529 nm. The structure is shown in Fig. 11. The temperature dependence of the magnetic moment/xeff of the complex is shown in Fig. 12. The data reveal antiparallel spin coupling A. Bencini and G. Gatteschi, in "Transition Metal Chemistry" (G. A. Melson and B. N. Figgis, eds.), Vol. 8, Springer-Verlag, Berlin, 1982. 26 D. Mandon, E. Weiss, E. Bill, Ch. Butzlaff, A. X. Trautwein, K. Jayaraj, A. Gold, and J. Terner, J. lnorg. Biochem. 43, 292 (1991). 27 E. Bill, X. Q. Ding, E. L. Bominaar, Ch. Butzlaff, A. X. Trautwein, D. Mandon, R. Weiss, and A. Gold, J. Inorg. Biochem. 43, 332 (1991). R. Rutter, L. P. Hager, M. Hendrich, M. Valentine, and P. Debrunner, Biochemistry 23, 6809 (1984). 29 Ph. Chaudhuri, I. Karpenstein, M. Winter, Ch. Butzlaff, E. Bill, A. X. Trautwein, U. Fl6rke, and H. J, Haupt, J. Chem. Soc., Chem. Commun. 4, 321 (1992).

[15]

MAGNETIC SUSCEPTIBILITY

435

.,./1% 4 r __3

~

G

C

C

G

~

G

F/

C

C

~

C

O

S=I

!

1

I

, i

0

I

i

I

,

t

I

,

20

l

1

,

40

1

1

i

,

i

,

I

i

I

i

I

i

I

,

60

80

i

I

l

i

100

I

l

I

I ,

120 T/K

FIG. 10. Effective magnetic m o m e n t #eft of the [TPpivPFelVO] + radical complex with J = 4 c m - k Both c u r v e s are calculated with D = 18 cm -1 and g = 2.1.

C9 C14 N6 CI

)N3 )C5 N3o "°ClS

C17

~

[ulct

FiG. 11. Crystal structure of [L3Cu3(Im)3](CIO4) 3 .

436

[15]

PROBES OF METAL ION ENVIRONMENTS

m

3,0

2.5

2.0

-I

0

i

~

i

!

50

I

t

!

I

I

I

i00

I

i

I

!

I

150

I

I

I

t

200

I

I

I

I

I

250

I

I

I

f

300 T/K

FIG. 12. Effective magnetic moment/z~fr of [L3Cu3(Im)3](CIO4)3.

behavior. The low-temperature value of/Zeff = 1 . 8 / z B corresponds to a S = ½ground state with g = 2.1. The temperature-dependent data were fitted with the spin Hamiltonian using D = 0 cm-1 and ~ m = JtStS2 + J2SIS3 + J3S2S3, leading to a single, isotropic coupling constant J~ = J2 = J3 = J 0f75 cm-1. Because of the three equal and positive 3° coupling constants, spin frustration occurs. This is due to the fact that not all of three required antiparallel spin coupling schemes with equal strength can be satisfied simultaneously.29

Conclusion

In this chapter we have delineated that the field and temperature dependence of the magnetization even of rather dilute samples can be readily measured by means of a SQUID system. From these data the spin and the zero-field splitting of its paramagnetic centers can be deduced as well as the strength of the exchange coupling between centers. The main difficulties in extracting this information lie in the proper removal of contributions from paramagnetic impurities and the exact determination of the metal concentration. The safest way to solve these problems is to combine the results with those obtained from complementary methods, such as EPR and Mfssbauer spectroscopy. 30 Our notation gives antiparallel coupling for a positive coupling constant. This notation differs from other notations.

[16]

MULTIFIELD

SATURATION

MAGNETIZATION

437

Acknowledgments This work was supported by the Deutsche Forschungsgemeinshaft. We thank K. Wieghardt, Ph. Chaudhuri, R. Weiss, D. Mandon, and co-workers for providing us with the relevant complexes and for many fruitful discussions.

[16] M u l t i f i e l d S a t u r a t i o n M a g n e t i z a t i o n o f M e t a l l o p r o t e i n s By EDMUND P. DAY Introduction The multifield saturation magnetization technique ~measures all of the paramagnetism present in a metalloprotein sample with greater resolution than previous susceptibility measurements. The required sample volume is on the order of 200/.d with a protein concentration of more than 0.5 mM. [This is the amount of material required for spin S = ½. Higher spin states can be studied at lower concentrations. The magnetization signal is proportional to S(S + 1) at high temperatures when the Curie law is obeyed.] This thermodynamic measurement extends magnetic susceptibility measurements to lower temperatures (down to 2 K) and higher magnetic fields (up to 5.5 T). The new methodology has better resolution than a susceptibility measurement, but it lacks the high resolution of resonance techniques such as electron paramagnetic resonance (EPR), low-temperature magnetic circular dichroism (MCD), and M6ssbauer spectroscopy. Proper interpretation of magnetization data requires careful sample preparation aimed at eliminating magnetic contamination. Careful sample characterization is required as well to detect residual magnetic impurities. When the magnetic properties of a metalloprotein are complex, it is best to lay the groundwork for the multifield saturation magnetization measurement by first using the high-resolution resonance techniques. This new methodology, which detects all of the paramagnetism in a sample, complements the resonance techniques since the resonance techniques do not detect all forms of paramagnetism. Although the magnetic properties of a metalloprotein are not themselves fundamental to its biological function, these properties are a signature of unpaired electrons which are fundamental to metabolism. The 1 E. P. Day, T. A. Kent, P. A. Lindahl, E. Mtinck, W. H. Orme-Johnson, H. Roder, and A. Roy, Biophys. J. 52, 837 (1987).

METHODS IN ENZYMOLOGY, VOL, 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

[16]

MULTIFIELD

SATURATION

MAGNETIZATION

437

Acknowledgments This work was supported by the Deutsche Forschungsgemeinshaft. We thank K. Wieghardt, Ph. Chaudhuri, R. Weiss, D. Mandon, and co-workers for providing us with the relevant complexes and for many fruitful discussions.

[16] M u l t i f i e l d S a t u r a t i o n M a g n e t i z a t i o n o f M e t a l l o p r o t e i n s By EDMUND P. DAY Introduction The multifield saturation magnetization technique ~measures all of the paramagnetism present in a metalloprotein sample with greater resolution than previous susceptibility measurements. The required sample volume is on the order of 200/.d with a protein concentration of more than 0.5 mM. [This is the amount of material required for spin S = ½. Higher spin states can be studied at lower concentrations. The magnetization signal is proportional to S(S + 1) at high temperatures when the Curie law is obeyed.] This thermodynamic measurement extends magnetic susceptibility measurements to lower temperatures (down to 2 K) and higher magnetic fields (up to 5.5 T). The new methodology has better resolution than a susceptibility measurement, but it lacks the high resolution of resonance techniques such as electron paramagnetic resonance (EPR), low-temperature magnetic circular dichroism (MCD), and M6ssbauer spectroscopy. Proper interpretation of magnetization data requires careful sample preparation aimed at eliminating magnetic contamination. Careful sample characterization is required as well to detect residual magnetic impurities. When the magnetic properties of a metalloprotein are complex, it is best to lay the groundwork for the multifield saturation magnetization measurement by first using the high-resolution resonance techniques. This new methodology, which detects all of the paramagnetism in a sample, complements the resonance techniques since the resonance techniques do not detect all forms of paramagnetism. Although the magnetic properties of a metalloprotein are not themselves fundamental to its biological function, these properties are a signature of unpaired electrons which are fundamental to metabolism. The 1 E. P. Day, T. A. Kent, P. A. Lindahl, E. Mtinck, W. H. Orme-Johnson, H. Roder, and A. Roy, Biophys. J. 52, 837 (1987).

METHODS IN ENZYMOLOGY, VOL, 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

438

PROBES OF METAL ION ENVIRONMENTS

[16]

magnetic properties of the active site of a metalloprotein yield information about its oxidation state, ligands, and, for coupled systems, bridging structures. Interpretation relies on comparison with model compounds of known structure.

Sensitivity The paramagnetic signal of a metaUoprotein sample is typically more than 100 times weaker than that o f a paramagnetic salt owing to the dilution of the paramagnetic active site by the protein. For this reason, saturation magnetization measurements of metalloproteins must deal with several sources of magnetic noise which can be ignored in the study of low molecular weight paramagnetic inorganic compounds. These background noise sources include (1) the nuclear susceptibility of spin I = ½nuclei such as the protons of water, (2) paramagnetic molecular oxygen dissolved in airsaturated water, and (3) low levels of ferromagnetic impurities found in all sample holder materials. Techniques for detecting the presence of each of these contaminating signals are described in this chapter along with procedures for minimizing their contribution to the saturation magnetization measurement.

Resolution As important as the need for improved sensitivity in the measurement of the magnetic properties of a metalloprotein is the requirement for improved resolution. Resolution is required to distinguish the contributions of the active site paramagnetism of interest from the contributions o f paramagnetic impurities and/or contaminating redox states of the protein. In addition, there are cases when the paramagnetic properties of a given redox state of the metalloprotein are unexpectedly complex. These can be properly interpreted only if the magnetic measurement has the resolution required to distinguish contributions from different paramagnetic species when they are simultaneously present.

Theory The improved resolution of multifield saturation magnetization measurements over earlier susceptibility measurements arises because the shape of a saturation magnetization curve changes with spin. The curves of Fig. 1 represent the equation of state of each spin for spins from S = ½ to S = ~ when there is a high degree of symmetry at the paramagnetic site. For a particular spin, the plot of magnetization (M) against/3H/kT yields a universal saturation (Brillouin) curve (13 is the Bohr magneton

[16]

MULTIFIELD SATURATION MAGNETIZATION

.

.

.

.

I

. . . .

I

. . . .

5.0

439

I . . . .

~----~5/2-

4.0

~ j / - - 2

3.0

~ ~ - - ~ 3 / 2 .

2.0

1-

1.0

--------1/2-

0.0

~-'

0.00

I 0.50

'

[

I

1.00

1.50

~ 2.00

I H/kT FIG. 1. Saturation magnetization (Brillouin) curves for S = ~, 2, ~, 1, and ½. The magnetization in units of Bohr magnetons (/3) is plotted against/3H/kT. The curves were calculated from Eqs. (1)-(3) with D = E/D = 0 in Eq. (1).

and k is the Boltzmann constant). The magnetization shown in Fig. 1 is seen to depend only on the spin and on the ratio of the field alignment energy (/3H) to the thermal energy tending to disrupt the alignment (kT). The greater the spin the easier it is fully to align (saturate) the paramagnetic spin with increasing field or decreasing temperature. Therefore, the curves have a sharper " k n e e " as the spin is increased. Clearly, the magnitude of the saturation signal increases with increasing spin as well. For the present discussion, which is focused on resolution, we want to concentrate of the form of the curves, imagining that we have no information as to the amount of each spin. One way to focus on the differences in the shapes of the Brillouin curves is to normalize the curves to give identical Curie law slopes at high temperatures. This is shown in Fig. 2. The curves were drawn assuming 1 mol of spin S = ½. The amount of each higher spin [ns = (a4)/S(S + 1)] was lowered to give the same high-temperature (Curie law) slope as 1 mol of spin S = ½. The increased resolution of saturation magnetization measurements over magnetic susceptibility measurements arises from the dif-

440

[16]

PROBES OF METAL ION ENVIRONMENTS

1.0

~1/2-

0.8 ~0.5 0.3

~

0.0 . 0.00

. . .

I . . . .

0.50

5/2

/

. . . . .

1.00

I

. . . .

1.50

2.00

[~H/kT Fro. 2. Same saturation magnetization curves as in Fig. 1 scaled to give identical hightemperature (Curie law) slopes. One mole o f spin S = ½ is shown. The amount o f each higher spin has been decreased by the factor n s [ns = (])/S(S + 1)].

ferences at low temperatures in the form of the saturation curves with spin shown in Fig. 2. Magnetic susceptibility measurements focus on the high-temperature, field-independent (Curie law) slope common to all of these curves. Spin H a m i l t o n i a n a n d Z e r o - F i e l d Splitting

The spin Hamiltonian used to calculate the saturation magnetization curves shown in this chapter is = D[Sz 2 - S ( S + 1)/3 + (E/D)(Sx 2 - Sy2)] + fl ~i=x,y,z giSiHi

(1)

where gi, Si, Hi are the x, y, and z components of the g tensor, the electron spin operator, and the applied magnetic field, respectively; D and E / D are the zero-field splitting parameters. The x y z coordinate frame is fixed relative to the molecule. This spin Hamiltonian is appropriate for paramagnets with well-isolated spin multiplets. Even when this is not true, for example, when Iol is not small compared with the spin-orbit coupling

[16]

MULTIF1ELD SATURATION MAGNETIZATION

441

constant, the spin Hamiltonian formalism serves as a unifying description of saturation magnetization, EPR, MCD, and M6ssbauer measurements. The Brillouin curves shown in Figs. 1 and 2 result when D = E/D = O.

Theoretical Saturation Magnetization Curves The superconducting susceptometer measures the component of the macroscopic magnetic moment (M) of the sample parallel to the applied field (H). The calculation of this signal begins with the contribution of a single molecule [m(O, ~b)] to the macroscopic moment. The applied field direction relative to the molecular frame is specified by the polar angles (0, ~b). The magnetization of the molecule is then calculated using the thermodynamic expression re(O, 6) = -E~[OE~(O, +)/OH] exp[-E~(O, cb)/kTl/Y~, exp[-E~(O, 49)/kr] (2) where E~(O, 4~) (with a = 1. . . . ,2S + 1) are the energy levels of the spin multiplet for the specified molecular orientation. The partial derivatives with respect to field of the energy levels [OE~(O, +)/OH] are found by diagonalizing the Hamiltonian [Eq. (1)1 twice for slightly different field values. The average of Eq. (2) over 0 and 4) is multiplied by the total number of molecules in the sample (N) to yield the signal per sample:

M = (N/4 7r) f re(O, ~b) dO d~b

(3)

We normally assume orthorhombic symmetry for the quadratic spin Hamiltonian. Hence, the angular average need only be over an octant of the unit sphere. Magnetization curves calculated using a 15-point grid within this octant are usually well within 1% of those calculated using higher density grids.

Multifield Saturation Magnetization Data For a given spin, the universal saturation (Brillouin) curve shown in Fig. 1 breaks into a family of curves when zero-field splitting is introduced. This is shown for a spin S = 2 system in Fig. 3. In this case data were collected on a sample of oxidized Mn-superoxide dismutase from Thermus thermophilus at four fixed fields (4.0, 2.0, 1.0, and 0.5 T) over the temperature range from 2 to 200 K. z The appearance of the multifield saturation magnetization data as a nested family of curves when data are collected at four fixed fields arises from zero-field splitting and the fact the x axis depends on H/T. The lowest temperature at each field is 2 K. With a field 2 j. Peterson, J. A. Fee, and E. P. Day, Biochim. Biophys. Acta 1079, 161 (1991).

442

PROBES OF METAL ION ENVIRONMENTS

4.0

I

I /

3.0-

v

[16]

I

I

I

f

//

2.0-

1,0

0.0 0.00

t

I

I

I

I

0.25

0.50

0.75

1.00

1.25

1.50

13H/kT FIG. 3. Saturation magnetization of native manganese-superoxide reductase from

Thermus thermophilusplotted in units of Bohr magnetons against flH/kT. Data were collected at four fields (~ 4.0, [] 2.0, © 1.0, and A 0.5 T) over the temperature range from 2 to 200 K. The parameters used to calculate the solid lines from the spin Hamiltonian [Eq. (1)] are S = 2, D = 2.4 cm -I, E/D = 0.1, and g = 2. The amount o f S = 2 is 49 nmol, and XR2 equals 1.3. The dashed curve is the S = 2 saturation magnetization (Brillouin) curve calculated with D = E/D = 0 and g = 2. [Data adapted with permission from J. Peterson, J. A. Fee, and E. P. Day, Biochim. Biophys Acta 1079, 161 (1991).] o f 4T, flH/kT is a p p r o x i m a t e l y 1.4 at this t e m p e r a t u r e . W h e n t h e field is h a l v e d to 2T, flH/kT is h a l v e d at this l o w e s t t e m p e r a t u r e (2K) to a p p r o x i m a t e l y 0.7. S i m i l a r l y , this is r e p e a t e d f o r t h e n e x t field, w h i c h is h a l v e d to 1 T. T h e r e s u l t is a set o f c u r v e s w h i c h r e a c h h a l f as f a r o n t h e flH/kT a x i s a s t h e field is h a l v e d e a c h t i m e . T h e f a c t t h e c u r v e s c o l l e c t e d at d i f f e r e n t f i x e d fields d o n o t lie o n t o p o f o n e a n o t h e r is d u e to t h e e f f e c t s o f t h e z e r o - f i e l d s p l i t t i n g (D a n d E/D). T h e s a t u r a t i o n m a g n e t i z a t i o n c u r v e s f o r s p i n S = ½ a r e a l w a y s s u p e r i m p o s e d ( s e e F i g s . 6B a n d 10B) b e c a u s e z e r o - f i e l d s p l i t t i n g d o e s n o t e x i s t f o r this spin. W h e n z e r o - f i e l d s p l i t t i n g is i n t r o d u c e d t h e B r i l l o u i n c u r v e ( s h o w n a s a d a s h e d line in F i g . 3) is n o t o b s e r v e d d i r e c t l y . H o w e v e r , , fitting to t h e o b s e r v e d s e t o f s a t u r a t i o n m a g n e t i z a t i o n c u r v e s will d e t e r m i n e t h e s p i n (S), t h e a m o u n t o f s p i n (IS]), t h e a v e r a g e g v a l u e , a n d the z e r o - f i e l d s p l i t t i n g (D a n d E/D). It is n o t p o s s i b l e to o b t a i n a fit to this multifield

[16]

MULTIFIELD SATURATION MAGNETIZATION

443

family of curves using a different spin, even after varying the fit parameters ([S], D, E/D) over their full range. (The parameter g must be kept in the vicinity of g = 2. If g ranges freely it is possible to achieve a fit with the wrong spin but with physically meaningless values of g.) In this sense, the Brillouin curve can be uniquely constructed from the information obtained in the fitting process. Resolution Requires Multifield Fit

The fit to a single field of saturation magnetization data is not unique. For example, if we fit just the highest field of Fig. 3 (4 T) assuming, incorrectly, that the spin is S = 2, we obtain the fit shown in Fig. 4A. That this fit is incorrect can be seen only by examining the magnetization curves at all four fields fit with parameters locked at these values with the intercepts and amount of spin free to vary (shown in Fig. 4B). A repeat of this process of fitting the highest field only and then calculating the fit to all four fields using parameters derived from the single field fit yields the results shown in Fig. 4C,D when the correct spin S = 2 is assumed. In judging these fits it is important to examine the quality of the fit over the entire temperature range. To do this graphically requires two different plots. In Fig. 4 the magnetization versus flH/kT plots exhibit the quality of the fits at low temperatures. The xT versus T (X = M/H) plots of the insets in Fig. 4 exhibit the quality of the fits at high temperatures. There is substantial scatter in the data in the insets because the paramagnetic signal is decreasing with increasing temperature (being proportional to inverse temperature), making the fixed, temperature-independent noise of the instrument increasingly apparent at high temperatures. Even though there is substantial scatter in all of the data shown in the insets, the difference is readily apparent between the excellent fit (for S = 2) shown in the inset of Fig. 4D relative to the poor fit (for S = 2) shown in the inset of Fig. 4B. We want to understand why the xT versus T plot is so sensitive an indicator of whether the proper spin has been assumed in fitting multifieid magnetization data. The susceptibility (X = M/H) of a sample containing both paramagnetic and diamagnetic contributions is given by X = C/T + B

(4)

where C measures the strength of the paramagnetism and B that of the temperature-independent diamagnetism. C is the Curie law slope and B the intercept in a plot of X versus inverse temperature. Multiplying by temperature we have XT = C + BT

(5)

444

PROBES OF METAL ION ENVIRONMENTS

[16]

If the diamagnetic contribution is set to zero (B = 0), a plot of xT versus T will be a horizontal line with magnitude C. If the correct spin is assumed when fitting the multifield data set, it is possible to minimize the quality of fit parameter (XR2) while at the same time choosing the intercept at each field to be zero. This is shown in the inset of Fig. 4D, where the scatter in the data is centered on the horizontal theoretical line as expected. When the incorrect spin is assumed, the minimum in XR2 can be found only by assigning nonzero values to the diamagnetic intercepts. This results in a steep slope coming from the BT term of Eq. (5) in a xT versus T plot. Moreover, this incorrect diamagnetic term differs from field to field, as none of the calculated magnetization curves matches the form of the data. The result shown in the inset of Fig. 4B is typical of attempts to fit multifield data with the incorrect spin with the intercepts free. In general, we find the ×T versus T plot to be one of the best ways to judge the quality of the overall fit graphically and to be a sensitive indicator of the incorrect spin. We shall see later that it can also be a sensitive indicator of the presence of a second spin (compare Fig. 10A,B). Clearly it is necessary to collect data at several fields (e.g., four) and fit the entire multifield family of curves with a single parameter set in order to determine the spin uniquely. The minimum number of fields required by this strategy is two: the highest field the instrument can attain (5.5 T with the present instrument) and a low field. When the field is low enough (usually less than 0.5 T) the susceptibility versus inverse

FIG. 4. D e m o n s t r a t i o n exercise to illustrate the importance o f multifield data for determining the spin from the form o f the data. A single field o f data can be fit with two different spins (A, C), w h e r e a s four fields of data can only be fit with a unique spin (B, D). Data are from Fig. 3 for native m a n g a n e s e - s u p e r o x i d e d i s m u t a s e from Thermus thermophilus. (A) High-field (4.0 T) data of Fig. 3 fit a s s u m i n g a spin o f ~. The main figure is a plot o f magnetization in SI microunits per sample against [3H/kT. The parameters used to calculate the solid c u r v e are S = ~, D = - 0 . 7 cm -t, E/D = 0.2, and g = 2. T h e inset s h o w s the s a m e data and theory plotted as susceptibility (X = M/H) multiplied by the temperature v e r s u s t e m p e r a t u r e ( x T v e r s u s T) in SI microunits per sample. The a m o u n t o f S = ~ for this singlefield fit is 35 nmol, a n d XR2 equals 1.3. (B) All four fields fit with the parameters found from the single-field fit o f (A) T h e poor fit (XR2 = 31) s h o w s that S = ~ is not the spin for this sample. T h e a m o u n t o f S = ~ for this poor fit is 31 nmol. (C) Identical analysis to (A) with spin S = 2. T h e p a r a m e t e r s used to calculate the solid curve are S = 2, D = 2 cm -1, E/D = 0.25 and g = 2. T h e a m o u n t o f S = 2 for this single-field fit is 48 nmol, a n d XR2 equals 1.0. (D) All four fields fit with the p a r a m e t e r s found from the single-field fit o f (C). T h e excellent fit (XRz = 2.4) s h o w s that spin S = 2 is the correct spin. The appropriate w a y to determine the p a r a m e t e r s for this data set would be to fit all four fields at once. This was not done in this illustrative exercise. The appropriate parameters for these data are given in Fig. 3.

[16]

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[16]

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[16]

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temperature curve is independent of field down to the lowest temperature attainable by the instrument (2 K with the present instrument). Any field in this low-field region will serve as the second field. Fields between these two limits increase the resolution when more than a single spin is present. We routinely collect as many fields as we can in a single day (six with the present instrument and current software). In the chemical literature susceptibility data are presented against temperature. Typically this is done as both 1/X against temperature and either [xT ]~/2(p-eff)or xT(/xef2) againstltemperature on the same plot. With the multifield saturation magnetization technique it is necessary in addition to present a plot of either M against flH/kT or × against 1/T to exhibit the quality of the data and the fit at low temperatures. If this is not done, even a poor fit at low temperatures can appear to be an adequate fit if shown only as either 1/X or ×T(~f 2) against temperature. It is also common practice to measure the metal content of a sample and to convert the raw magnetization data (SI microunits per sample) to molar magnetization data (SI units per mole) before proceeding. We begin our analysis, instead, with the raw, holder-corrected difference data (sample minus control) plotted as signal per sample rather than per mole. The fitting process is then used to determine the number of moles of each spin that is present from the form of the data. This emphasizes that the sample is, more often than not, a composite of several different spins. In such cases it is not possible to present the signal per mole at the start since different spins will have different numbers of moles. Once the analysis is completed and contaminating spins have been subtracted, it is possible to present the signal from a single spin in SI units per mole. When this is done the amount of spin is that found from fitting the magnetization data, not from metal analysis or from protein concentration measurements. In this way the saturation magnetization measurement stands on its own in measuring the spin (S), spin concentration (IS]), gyromagnetic ratio (g), and zero-field splitting (D and E/D). Comparison can then be made with independent measurements of the amount of metal or protein in the sample to determine the spin per metal or per protein. Equipment Multifield saturation magnetization studies of metaUoproteins are made possible by the commercial availability of a fully automated superconducting susceptometer. ~ This type of instrument was originally developed for 3 Quantum Design, 11578 Sorrento Valley Road, Suite 30, San Diego, CA 92121. Telephone: (619) 481-4400.

448

PROBES OF METAL ION ENVIRONMENTS

[16]

SAMPLE ~ SUSPENSION SAMPLE SQOID

DETECTOR~I~

fi

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PICKUP COIL

SQUIDRESPONSE Schematicdiagramof the pickupcoil geometryand insertionsignalof the superconducting susceptometer.SQUID, Superconductingquantuminterferencedevice. FIG. 5.

biophysical measurements by Edmund P. Day and John S. Philo at Stanford University (Stanford, CA) in the low-temperature physics group of William M. Fairbank in the late 1960s and early 1970s. 4-s The instrument measures changes in magnetic flux directly. A signal proportional to the total magnetization of the sample is found by recording the detector output as the sample is moved through the detection coils as shown schematically in Fig. 5. The area under the curve is a measure of the magnetization of the sample. The instrument is calibrated by performing the same measurement on a susceptibility standard traceable to the National Institute of Standards and Technology. Because this calibration depends only on the fixed detection coil geometry and on the flux quantum, there is no need 4 E. P. Day, in " L o w TemperaturePhysics--LT 13" (K. D. Timmerhaus, W. J. O'Sullivan, and E. F. Hammei, eds.), Vol. 4, p. 550. Plenum, New York, 1972. 5 E. P. Day, Ph.D. Dissertation, StanfordUniversity, Stanford,California (1972). 6 j. S. Philo and W. M. Fairbank, Rev. Sci. lnstrum. 48, 1529 (1977). 7 j. S. Philo, Ph.D. Dissertation, StanfordUniversity, Stanford,California(1977). s E. P. Day and J. S. Philo, in "Near Zero: New Frontiers of Physics" (J. D. Fairbank, B. S. Deaver, Jr., C. W. F. Everitt, and P. F. Michelson, eds.), p. 442. Freeman, New York, 1987.

[16]

MULTIFIELD SATURATION MAGNETIZATION

449

to check the calibration continually. This is in contrast to a Faraday balance, where sample positioning can change the calibration and repeated calibration is required. Saturation magnetization can be measured directly by the superconducting susceptometer, which responds to changes in magnetic flux. A Faraday balance, on the other hand, responds to the force exerted by a field gradient on a paramagnetic sample. The signal of a Faraday balance goes to zero at saturation when OM/OH goes to zero. The paramagnetic signal measured by a superconducting susceptometer is a maximum at saturation. Thus, although both instruments are comparable when it comes to making susceptibility measurements, the superconducting susceptometer is uniquely suited to saturation magnetization studies. The commercial superconducting susceptometer can be programmed to collect data at several fixed fields up to 5.5 T, each over the temperature range from 2 to 200 K. (The temperature range of the susceptometer extends to 400 K, but the frozen metaUoprotein sample in an uncapped quartz bucket sublimes at 210 K.) For instance, the present commercial instrument3 can record six fixed fields of data consisting of fifty temperature points at each field, all in less than 1 day of unattended operation. The reliability, speed, and convenience of this unattended data collection represent major gains over the first commercial instrument, 9 which was three times slower and required manual operations to change from data collection above 5 K to data collection over the range from 2 to 5 K. It is now easier and faster to collect six complete, fixed fields of data than it was to collect two complete, fixed-field data sets on the original commercial superconducting susceptometer. Commercial software ~° is now available to aid in the interpretation of multifield saturation magnetization data. The software can model data with a single paramagnetic species (one monomer), a single coupled site (one dimer), or any combination of these. In particular cases, judicious use of this software makes it possible to resolve data when more than one paramagnetic species is present. The software will also perform Monte Carlo simulations to determine the uniqueness of the parameters, their uncertainties, and their correlations. Background Signals The techniques required to exploit the sensitivity of a superconducting susceptometer are at least as important as the commercial availability of 9 Biomagnetic Technologies incorporated (BTi), 9727 Pacific Heights Boulevard, San Diego, CA 92121. Telephone: (619) 453-6300. This company was formerly S.H.E. Corporation. l0 WEB Research Co., 6716 Samuel Rd., Edina, MN 55439-1053. Telephone: (612) 942-6276.

450

PROBES OF METAL ION ENVIRONMENTS

[16]

a sensitive and automated instrument. Indeed the new instrument is not substantially more sensitive than the Faraday balances developed by Tweedle and Wilson u and by Petersson and Ehrenberg ~2for metalloprotein measurements. What is new today is both the multifield saturation magnetization measurement and the full implementation of a range of procedures aimed at controlling background signals and at fully characterizing the metalloprotein sample itself. I = ½Nuclei

An important source of noise in metalloprotein measurements that had not been recognized earlier is the nuclear susceptibility of the slowly relaxing protons in water. ~The ratio of the square of the magnetic moment of the proton to that of the electron is 2.3 x 10-6. This represents the relative contribution of the proton to the Curie law (high-temperature) signal of a paramagnet. Because this number is so small, the susceptibility of the proton had been ignored in earlier metalloprotein measurements. However, the 110 M concentration of protons in water means that these contribute to the Curie law slope an equivalent signal to that of a concentration of 0.25 mM spin S = ½electrons. This is a substantial signal compared to that of the active site in a metalloprotein experiment where the metalloprotein concentration, typically, is on the order of 0.5 mM. The contribution of the nuclear susceptibility of the protons in water cannot be eliminated by subtracting a matched control because the proton relaxation is very slow (hours) and changes in the presence of the metalloprotein magnetization under study.~ Deuteration of the buffer eliminates the problem. Because the deuteron has a quadrupole moment, it relaxes rapidly independently of the magnetization under study, and its susceptibility can be subtracted by using a matched control. Hysteresis introduced by protons remaining in a metalloprotein sample or residing in a plastic sample holder can be readily measured. Half of the data points can be collected upon cooling with alternate points collected on warming. Hysteresis introduced by slowly relaxing protons will result in these alternate points being offset (see Figs. 7B, 8B, and 9). The holdercorrected difference data (sample minus control) can be examined for this signature of hysteresis. If the metalloprotein signal is large relative to this hysteresis, the hysteresis can be ignored. The procedure of collecting half the data points on cooling and alternate points on warming works well below 35 K, where the heat capacity of the helium gas or liquid (below 4.3 K) used to control the temperature of the instrument dominates that II M. F. Tweedle and L. J. Wilson, Rev. Sci. lnstrum. 49, 1001 (1978). 12 L. Petersson and A. Ehrenberg, Rev. Sci. lnstrum. 56, 575 (1985).

[16]

MULTIFIELD SATURATION MAGNETIZATION

451

of the metal of the walls of the sample chamber. Above 35 K the susceptometer works far better on warming than on cooling. Consequently, we routinely collect alternate points on cooling (warming) below 35 K and all of the points above 35 K on warming.

Molecular Oxygen Paramagnetic (S = 1) molecular oxygen dissolved in air-saturated buffers contributes a signal comparable to that of the metalloprotein and cannot be ignored (see Fig. 7B).1 If the oxygen content of the sample and its matched control can be made identical, 13 then the oxygen signal will be subtracted on taking the difference between the sample and its control (see Figs. 6 and 7). The difficulty with this procedure is in determining by measurement that the sample and its control contain identical amounts of oxygen. The alternative procedure of eliminating oxygen from both sample and control has the advantage that the absence of oxygen in the control can be verified by measurement of the saturation magnetization of the control. Consequently we routinely remove oxygen from both sample and control in most of our studies of metalloproteins. The freeze-pump-thaw procedure has the advantage of removing molecular oxygen dissolved in the buffer without changing the concentration of the protein solution in the process. The alternative procedure of bubbling oxygen-free argon or nitrogen through or over the sample may dry the sample, altering its concentration. For the thaw step of the freeze-pump-thaw procedure it is important to backfill the atmosphere above the frozen sample with oxygen-free argon or nitrogen and thaw slowly to avoid sample loss as oxygen bubbles out of the sample. This bubbling is avoided by pumping only on the frozen sample followed by slow thawing under an atmosphere of oxygen-free gas. The presence of any remaining paramagnetic oxygen is detected by examining the saturation magnetization data of the matched control. It is crucial that the control be prepared at the same time as the sample and that it be handled in the same manner. For example, we routinely subject both the sample and control to the freeze-pump-thaw procedure at the same time in the same gas manifold to ensure that both have identical oxygen contents. This is especially important when the expected signal of the active site of the metalloprotein is S = 1, which would be indistinguishable from contaminating oxygen dissolved in the buffer. This is the case, for example, when nickel(II) is being studied. 13 D. M. Dooley, J. A. Landin, A. C. Rosenzweig, W. G. Zumft, and E. P. Day, J. Am. Chem. Soc. 113, 8978 (1991).

452

PROBES O F M E T A L ION E N V I R O N M E N T S

[16]

Ferromagnetic Impurities Sample holders are etched overnight in 10% hydrofluoric acid to eliminate detectable ferromagnetic impurities commonly found on all sample holder materials. Remaining ferromagnetic impurities can be detected by looking for a field dependence in the Curie law susceptibility intercept. 1 The high-temperature data (>50 K, for example) are plotted as X (M/H) against inverse temperature (l/T) and fitted to a straight line for each of the magnetic fields (H). The intercepts found from the straight-line fit will be independent of H if there are no ferromagnetic impurities present. When ferromagnetic impurities are present, it is important to work at high magnetic fields (>0.15 T) to saturate the impurities and to avoid the hysteresis they contribute at low fields. It is also difficult to collect isotherm data (data collected at a fixed temperature over a wide field range) when ferromagnetic impurities are present. Data collected at low temperatures (<70 K) and at fixed high fields (>0.15 T) can be used to study multifield saturation magnetism of metalloproteins in the presence of low levels of ferromagnetic impurities.

Multiinstrument Sample Holder It is important to characterize the metalloprotein sample directly using resonance techniques before studying its saturation magnetization. EPR, MCD, and M6ssbauer measurements are used to verify by measurement that the sample is in the appropriate redox state. These measurements are also used to quantitate both the levels of unwanted redox states and the levels of unwanted paramagnetic impurities. For Fe-containing proteins we collect MOssbauer spectra of the 57Fe-enriched sample after it has been prepared in the magnetization sample holder. For these experiments we use a plastic sample holder since the quartz holder is opaque to the y rays of the Mrssbauer spectrometer. Iron(II) impurities are routinely detected by M6ssbauer spectroscopy. EPR spectra are collected on parallel samples because we have not yet developed an EPR spectrometer which can accept the relatively large diameter (8 mm) sample holders used in the saturation magnetization measurement. EPR spectroscopy routinely detects Fe(III) and Mn(II) impurities. EPR spectra should be collected on all samples, including those which are not expected to show an EPR signal. In this way the absence of magnetic contamination can be shown by measurement. Optical measurements on parallel samples are used similarly to characterize the sample. The plastic holder used in experiments combining saturation magnetization and Mrssbauer spectroscopy is machined from a Delrin rod to form a bucket (8 mm outer diameter, 8 mm height, and mass of approximately

[16]

453

MULTIFIELD SATURATION MAGNETIZATION

130 mg) with a pair of holes near the top for the suspension thread. The plastic buckets are acid-etched overnight in 10% hydrofluoric acid to remove ferromagnetic impurities. The thread used to suspend the holder is lightly greased at each end before tying the knot through the holder hole in order to prevent sample moving up the threads by capillary action during loading. Because the holders exhibit a S = 1 signal (see Fig. 9), we routinely measure each empty holder and subtract this background magnetization from the filled sample or control to give holder-corrected data. The holder-corrected data is then subtracted to yield the holdercorrected difference data [(sample minus its holder) minus (control minus its holder)].

Fitting Software Input to the computer program used to fit the saturation magnetization data consists of a file containing the difference data to be fit and a file containing the input parameters.I° The input parameter file contains the initial values for the spin Hamiltonian parameters, upper and lower bounds on these parameters, and flags controlling the fitting process. The data can be modeled with a single paramagnetic species (one monomer), a single coupled site (one dimer), or any combination of these. The output data file contains the calculated saturation magnetization at each field and temperature of the input data in addition to the input data. The output parameter file contains the amount of each paramagnetic spin and its spin Hamiltonian parameters. This file also reports the intercepts at each field and the reduced X2 (XR2) of the fit. The parameters indicating the quality of fit (X2 and XR2) are defined as X 2 ~- ~ i = l , n

riE/ori 2

and

XR2 =

X2/(n -

nfree)

(6)

where n is the number of data points, ri is the ith residual between the data and the theory, o-i is the uncertainty of r;, and nfree is the number of free parameters used to fit the data. To calculate XR2, the fitting software must estimate the value of tr;. A least-squares fit of a quadratic curve to the first nine data points at a specified field is performed. The root mean square (RMS) value of the nine residuals is defined as the tr of the fifth data point. This process is repeated for data points two through ten to determine the uncertainty of the sixth data point. The process is continued until the uncertainty of each data point has been determined. The first four data points are assigned the uncertainty of the fifth, and a similar process is followed for the final four data points at each field.

454

PROBES OF METAL ION ENVIRONMENTS

[16]

This method of estimating the uncertainty in the data is based on the observation that scatter introduced by changing the temperature within the susceptometer dominates the noise. Repeated measurement once the temperature is set underestimates the uncertainty. The observed scatter measured on returning to a specified temperature after collecting data at other temperatures is essentially the same as found by the analysis described in the previous paragraph. Data Analysis Next we present the full details of the data analysis underlying the published magnetic properties of Pseudomonas stutzeri nitrous oxide reductase. Here we present the data analysis as a case study of the techniques involved in a multifield saturation magnetization study. The original paper should be consulted for the interpretation of these results in light of what is known about nitrous oxide reductase.~3 In the experiment that we discuss as a case study, the same amount of dissolved molecular oxygen was present in both the protein sample and its matched control. Saturation magnetization data were collected at four fields (5.5, 2.75, 1.375, and 0.2 T) over the temperature range from 2 to 200 K on both the sample (shown in the main plot of Fig. 6A) and its matched control (shown in Fig. 7A). These data sets were subtracted to give the raw difference data (shown in the inset of Fig. 6A). Next, the raw difference data were fit assuming the only spin present was that of the S -- ½ Cu(II) site of the protein seen by EPR. The fit assumed the g values measured by EPR. The result is shown in Fig. 6B. For these plots the data are presented as magnetization in SI microunits per sample plotted against/3H/kZ. With the fitting complete, the amount of S = ½paramagnetism is known from the fit. This amount of spin can then be used to calculate the magnetization per mole and the result presented in units of Bohr magnetons (as was done in Ref. 13). The quality of the fit shown in Fig. 6B (Xg2 = 2.8) could be improved somewhat by doing a two-spin fit assuming the second spin was S -- 1 molecular oxygen. The improved fit (XR2 = 1.7) uses three additional parameters (the amount of S = 1 and the zero-field splitting parameters D --- 4.5 cm -1 and E/D = 0 with g locked at g = 2). With the S = 1 component present, the amount of S = ½determined by the fit decreased from 415 to 394 nmol. The resulting 4% uncertainty in the amount o f S = ½ [405(15) nmol] is substantially less than the 14% uncertainty arising from ambiguities in the molecular weight of the protein discussed in Ref. 13. In earlier susceptibility studies of metalloproteins, data were collected on a diamagnetic reference state such as the apoprotein in order to subtract

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0

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~.,.,.,

•~ ~~

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~

::, .-~

[16]

MULTIFIELD SATURATION MAGNETIZATION

457

out the diamagnetic background. This was an essential part of the measurement when the susceptibility was measured at a single temperature or over a limited temperature range. With little or no temperature variation the only way to sort out the paramagnetic and diamagnetic contributions was to measure the diamagnetic contribution carefully in a separate experi~ ment and subtract. Here we are collecting data over a broad temperature range at several magnetic fields. The paramagnetic signal of interest varies with temperature. The signal arising from a mismatch in the diamagnetism of the sample and its control will be constant in temperature and linear in field. The diamagnetic mismatch signal is eliminated by setting the Curie law intercept at each field to zero. In this way it is possible to remove the diamagnetic contribution to the signal without measuring it in a separate experiment. In the current methodology separate examination of the matched control is used to test for (1) the presence of dissolved molecular oxygen in the buffer, (2) the presence of other unexpected paramagnetic contaminants in the buffer, and (3) hysteresis owing to remaining protons. Subtraction of the matched control removes these unwanted background paramagnetic signals as well as the small S = 2 signal present in the quartz holders (see Figs. 8B and 9). The matched control shown in Fig. 7A contains dissolved molecular oxygen. This was by design in order to match the oxygen content of the protein sample. To present the oxygen signal, we have subtracted a typical control (shown in Fig. 8A). The result is shown in the inset of Fig. 7A and in Fig. 7B. In this case there are large offsets from field to field owing to the large diamagnetic mismatch between the control for the nitrous oxide reductase experiment (Fig. 7A) and the typical control (Fig. 8A). When these difference data are fit and the appropriate Curie law intercepts subtracted, however, there is no difficulty in isolating the temperaturedependent signal arising from the paramagnetic oxygen (shown in Fig. 7B). The small S -- 2 paramagnetic signal of the typical control is shown in Fig. 8B. This small amount of S -- 2 paramagnetism is always seen in empty quartz holders (see Fig. 9) and is the minimum signal seen in our controls. This small background quartz signal is removed on taking the difference between the sample and its matched control. The relatively large hysteresis seen in Fig. 8B arises from protons remaining in the buffer. It is often the case that controls will show some hysteresis (usually less than that shown here) but not enough to affect the larger protein signal found by taking the difference between the sample and its matched control. When this hysteresis interferes with the interpretation of the protein signal, more thorough deuteration of the sample and its control is required.

0 0 0,1

,,,I .... I .... I .... I .... I .... r .... I .... I .... f .... t .... I,,,

o o

o []

-r"

o

o

~

m-

= .o II

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,,,~i,,,J,,,i

,,,,J,,,

0

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~

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d

d

d

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°

(eldu.J~S..L/Pd)l~ ,,,I,,

o

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~ ~ ,~ ~-ae

.,

I,,,I,,,I,,,I,

g

o o o o <)

o f~

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= ~

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q

i

(elduJEs

J./p

d)lAI

c~ 0

0

[16]

MULTIFIELD SATURATIONMAGNETIZATION

I

0.35

~

L

I

I

L <>

0.30

I

459

t

<>

<>

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<)

0.25

0..

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<>

0.15 v

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0.05 -

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t + I 9v+4'~-¢~+1 I * I+ v4,l *'~'1

0.00 0.00

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13H/kT FIG. 9. High-field (5.5 T) data over the temperature range from 2 to 200 K for both an empty Delrin (©) and an empty quartz (O) sample holder. The Delrin data are hysteretic owing to the slowly relaxing, nuclear spin I = ½protons in the plastic. The quartz data are not hysteretic because quartz does not have any I = ½nuclei. The solid lines were found by fitting four fields of data for each empty holder (only one field is shown). The Delrin signal is S = 1 and probably arises from molecular oxygen present in the plastic. We do not know the source of the small S = 2 signal of quartz. F o r c o m p l e t e n e s s and c o m p a r i s o n w e h a v e s h o w n the high-field (5.5 T) data o f an e m p t y q u a r t z h o l d e r and that o f an e m p t y Delrin holder o n the s a m e g r a p h in Fig. 9. T h e S --- 1 signal o f Delrin is an o r d e r o f m a g n i t u d e larger than the spin S = 2 signal o f quartz. F o r this r e a s o n w e m e a s u r e e a c h e m p t y Delrin holder and s u b t r a c t as explained a b o v e in the s u b s e c t i o n o n m u l t i i n s t r u m e n t sample holders. E a c h o f the data sets o f Figs. 6 t h r o u g h 9 w e r e e x a m i n e d for f e r r o m a g netic impurities. T h e Curie law intercepts [B o f Eq. (4)] o f the raw differe n c e d a t a w e r e f o u n d at e a c h field b y fitting a straight line to e a c h g r a p h o f the h i g h - t e m p e r a t u r e susceptibility d a t a plotted against inverse t e m p e r a ture. T h e s e intercepts w e r e e x a m i n e d for their field d e p e n d e n c e to determine the a m o u n t o f f e r r o m a g n e t i c impurities p r e s e n t in the sample holders. T y p i c a l l y , the field d e p e n d e n c e w a s less than 3% o f the Curie law intercepts for these sample holders, w h i c h had b e e n t h o r o u g h l y e t c h e d o v e r -

460

PROBES OF M E T A L ION E N V I R O N M E N T S

[16]

night in 10% hydrofluoric acid. At this low level the ferromagnetic impurities make a negligible contribution and can be ignored. The raw data presented here in Figs. 6A, 7A, and 8A are simpler than the raw data presented in Ref. 1. The earlier data were collected using an instrument 9 that had an atmosphere of helium in the sample region. This affected the data since the helium displaced by the sample gave a detectable signal (see Ref. I). This signal changed substantially when the sample chamber was being pumped to reach low temperatures. In the new instrument 3 the sample chamber is separated from the helium used to control the temperature of the sample-chamber walls. Only low-pressure helium (rather than atmospheric pressure helium) is present in the sample chamber to establish thermal contact between the sample and the chamber walls. The magnetization of the low-pressure helium displaced by the sample during data collection is below the sensitivity of the instrument. The resulting raw data shows no change as the new instrument shifts automatically under computer control between temperatures below 4.2 K (where liquid helium is used to manipulate sample-chamber wall temperature) and above 5 K (where helium gas at atmospheric pressure is used for this purpose).

Single Spin Fits The spin of the sample is determined by trial and error by fitting the raw difference data using a single spin theoretical model. The fitting program is run using a several hundred iteration limit with generous bounds on each : of the three, free, nonlinear parameters ((g) = gx = g y = gz, D, E/D) for an assumed spin (S) and with the Curie law intercepts free to vary. (The data are fit using the average value of g. This does not necessarily mean that g is isotropic. Multifield saturation magnetization data generally do not have the resolution to decide the issue of the isotropy of the components of g~) The appropriate spin is indicated when the g value found from the fit is on the order of 2. If g is substantially larger than 2 a higher spin is indicated. I f g is substantially lower than 2 a lower spin is indicated. Once a spin has been chosen which results in an output g value of approximately 2, two other important characteristics of the fit can be examined to determine whether the sample contains only one spin. A single spin sample will yield an appropriate xT versus T plot (see the insets of Figs. 4D and 6B) and will yield a low value for the quality of fit parameter (X2).

Two Spin Fits Our experience shows that a multifield saturation magnetization data set can be fit with four to five free parameters. In this accounting we are

[16]

MULTIFIELD SATURATION MAGNETIZATION

461

ignoring the Curie law intercepts, one per field, which are also found from the fit and which deal with the diamagnetic mismatch in the data set. When the sample contains a single spin species, the fitting process determines the spin (S), the amount of spin ([S]), the g value, and the zero-field-splitting parameters (D and E/D). In cases where the data contain more than a single spin species, the fitting must be carried out without increasing the number of free parameters. This limits the measurement severely, since the two spins (Sl and 82) and the amounts of each ([S~] and [82] ) and one g value (gl, say) take up the free parameters, leaving no room to determine the interesting magnetic properties of the two different spin species. We are left in the position of having to determine all but one of the spin species from other measurements. Once this has been done, and the saturation magnetizations of each of the species have been subtracted, the singlespin fitting process should yield a clean fit. As a general rule, then, twospin fits are not appropriate for fitting saturation magnetization data. The most common case where a two-spin fit is appropriate involves the study of a metalloprotein in a S = ½state. In this case the spin S~ = ½ EPR signal can be used to determine the three components of its g~ tensor. It is then possible to determine the amount ([S~]) of this spin as well as the second spin ($2) and its amount ([$2]), g2 value, and zero-field splitting (D2) with the components of g~ locked at their EPR values. As an example we consider the published magnetization study of the oxidized [3Fe-4S] cluster of ferredoxin II from Desulfovibrio gigas.~4The results of a single spin S = ½ fit to the raw difference (sample minus control) data are shown in Fig. 10A. The xT versus T plot shown in the inset of Fig. 10A is not a very good fit because there is a second spin present in addition to the S = ½paramagnetism assumed by the fit. The second spin is a spin S = ~ ferric impurity common to preparations of iron-containing proteins. The presence of a high-spin ferric impurity was indicated by a signal at g = 4.3 in the EPR spectrum for this sample. The results of a two-spin fit to the same data are shown in Fig. 10B. This fit assumed both a spin $1 = ½species and a spin $2 = ~ impurity. It is clear, and dramatically so from the improved ×T versus T plot shown in the inset of Fig. 10B, that the two-spin fit works. The amount of ferric impurity determined by the fit is 2,6%. (This two-spin fit is not unique. Fits of comparable quality were obtained assuming the second spin to be either S = ~ or S = 2. It is the EPR signal at g = 4.3 which establishes that the impurity is spin S = ~. The two-spin fit to the magnetization then gives an excellent measure of the amount of spin S = ~ which is present.) 14 E. P. Day, J. Peterson, J. J. Bonvoisin, I. Moura, and J. J. G. Moura, J. Biol. Chem. 2,63, 3684 (1988).

,

t

/

,

/

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,

l

,

l

,

l

,

l

,

l

,

Id

l

,

l

,

l

,

I

,

o

1~

E ~ ~.~.~

= o ~ - .~ . - ~

~

~ ~'~"

II ~ ¢~ ~ _ . ~ . ~ , i ~

~

o

~

~.

~

~.,~';

'~"~ 00

~0,~ ~~

~ ~ t~ ~

I'~

I

I

I

I

I

I

I

~'f ~

~

~

n'l

~ ~

~ =,~

o,

-

o ~

°

~

=~

~

.

~,~,.~O~ II

,~ ~_ ~

.,., o

..

,,.,

=

"

_~ ~

.

/~.AI

tu~uwEs ~ P d ) l ~ l

,lo, l,l,l,l,l,l,l,r,l,l,l,

o

.1., ~

,,

,..,

~.=.-~°-=~;;=.~-,.4 E

1%

i

: ~

I°°

{

I

o

°

.

"~=~

c~

<

c~

d

(, IeH ,_wes

c~

o

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o

cS

d

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[17]

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

463

Conclusions Multifield saturation magnetization studies of metalloproteins have been rare until now. This can be expected to change. A fully automated susceptometer with the requisite sensitivity is commercially available. Software required for fitting the data is commercially available, as are inexpensive high-speed computers capable of thoroughly analyzing a given data set. The fundamental techniques for identifying and minimizing background signals have been discussed in this chapter. With these tools and procedures in place, we can expect increasing application of this new methodology. This methodology is unique in detecting all of the magnetic sites in a given sample. Proper interpretation of multifield saturation magnetization data, however, can still be tricky. Good judgment is required to deal properly with impurities. The technique does not lend itself to exploratory work on magnetically complicated systems because the methodology lacks the resolution of resonance techniques. Even so, several "discoveries" have been made using this methodology after thorough measurements with EPR and M6ssbauer spectroscopies had laid the groundwork. Saturation magnetization measurements were then able to answer a single, well-defined question that remained. Acknowledgments I thank Jim Peterson who contributedto the methodology,ThomasA. Kent who made helpful critical commentson the manuscript, and Mariana S. Sendova who drew Fig. 5. This research was supported by National Institutes of Health Grant GM32394.

[1 7] C o m b i n i n g M 6 s s b a u e r S p e c t r o s c o p y w i t h I n t e g e r S p i n Electron Paramagnetic Resonance

By

ECKARD MONCK, KRISTENE K. SURERUS,

and

MICHAEL P. HENDRICH

Introduction In 1978 we wrote an article in this series about 57Fe M6ssbauer spectroscopy of electron carrier proteins ~that focused on the relationship between M6ssbauer spectroscopy and electron paramagnetic resonance (EPR). Since then, these correlations have been frequently utilized to untangle 1 E. M0nck, this series, Vol. 54 [20].

METHODS IN ENZYMOLOGY,VOL. 227

Copyright © 1993by Academic Press, Inc. All fights of reproduction in any form reserved.

[17]

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

463

Conclusions Multifield saturation magnetization studies of metalloproteins have been rare until now. This can be expected to change. A fully automated susceptometer with the requisite sensitivity is commercially available. Software required for fitting the data is commercially available, as are inexpensive high-speed computers capable of thoroughly analyzing a given data set. The fundamental techniques for identifying and minimizing background signals have been discussed in this chapter. With these tools and procedures in place, we can expect increasing application of this new methodology. This methodology is unique in detecting all of the magnetic sites in a given sample. Proper interpretation of multifield saturation magnetization data, however, can still be tricky. Good judgment is required to deal properly with impurities. The technique does not lend itself to exploratory work on magnetically complicated systems because the methodology lacks the resolution of resonance techniques. Even so, several "discoveries" have been made using this methodology after thorough measurements with EPR and M6ssbauer spectroscopies had laid the groundwork. Saturation magnetization measurements were then able to answer a single, well-defined question that remained. Acknowledgments I thank Jim Peterson who contributedto the methodology,ThomasA. Kent who made helpful critical commentson the manuscript, and Mariana S. Sendova who drew Fig. 5. This research was supported by National Institutes of Health Grant GM32394.

[1 7] C o m b i n i n g M 6 s s b a u e r S p e c t r o s c o p y w i t h I n t e g e r S p i n Electron Paramagnetic Resonance

By

ECKARD MONCK, KRISTENE K. SURERUS,

and

MICHAEL P. HENDRICH

Introduction In 1978 we wrote an article in this series about 57Fe M6ssbauer spectroscopy of electron carrier proteins ~that focused on the relationship between M6ssbauer spectroscopy and electron paramagnetic resonance (EPR). Since then, these correlations have been frequently utilized to untangle 1 E. M0nck, this series, Vol. 54 [20].

METHODS IN ENZYMOLOGY,VOL. 227

Copyright © 1993by Academic Press, Inc. All fights of reproduction in any form reserved.

464

PROBES OF METAL ION ENVIRONMENTS

[17]

the complex MOssbauer spectra of proteins with multiple Fe sites and have played a crucial role in the discovery of Fe3S 4 clusters. 2 Exploitation of the connection between these two techniques has also provided fundamental insights into the nature of protein active sites. Thus, MOssbauer and EPR studies lead to the conclusion that the active site of Escherichia coli sulfite reductase contains a siroheme prosthetic group covalently linked to a Fe4S 4 cluster. 3 This conclusion has subsequently been confirmed by X-ray crystallographic studies. Owing to the utility of combining MOssbauer and EPR studies, we are prompted to further develop this theme here. The previous article ~also described the benefits of studying the MOssbauer spectra of compounds with integer electronic spin (non-Kramers systems) in strong applied magnetic fields. Such studies, described for the S = 2 state of reduced cytochrome P-450, were just emerging as a powerful probe of the electronic structure of non-Kramers systems. As metals in states with integer spin were not suspected to yield EPR signals, a useful connection between EPR and MOssbauer spectroscopy was apparently nonexistent. However, more recent observations have changed this picture considerably. 4-s Thus, integer spin EPR signals have been detected and analyzed for many metalloproteins and synthetic complexes s that mimic active site structures. Not surprisingly, there are close correlations between the MOssbauer and EPR spectra of integer spin systems, just as previously shown for Kramers systems. Moreover, newly developed methods for the analysis of the integer spin EPR line shape 4-8 have made it feasible to determine spin concentrations with a precision approaching that achieved for Kramers systems. In this chapter we further describe the correlations between MOssbauer and EPR spectroscopies and indicate the potential for these methods for the quantitative analysis of non-Kramers systems. Throughout this chapter we assume that the reader is familiar with the concepts and nomenclature outlined in the previous account on MOssbauer spectroscopy. 2 M. H. Emptage, T. A. Kent, B. H. Huynh, J. Rawlings, W. H. Orme-Johnson, and E. Miinck, J. Biol. Chem. 255, 1793 (1980). J. A. Christner, E. M0nck, P. A. Janick, and L. M. Siegel, J. Biol. Chem. 256, 2098 (1981). 4 M. P. Hendrich and P. G. Debrunner, Biophys. J. 56, 489 (1989). 5 M. P. Hendrich, E. Miinck, B. G. Fox, and J. D. Lipscomb, J. Am. Chem. Soc. 112, 5861 (1990). 6 M. P. Hendrich, L. L. Pearce, L. Que, Jr., N. D. Chasteen, and E. P. Day, J. Am. Chem. Soc. 113, 3039 (1991). K. K. Surerus, M. P. Hendrich, P. D. Christie, D. Rottgardt, W. H. Orme-Johnson, and E. Miinck, J. Am. Chem. Soc. 114, 8579 (1992). s C. Juarez-Garcia, M. P. Hendrich, T. R. Holman, L. Que, and E. MOnck, J. Am. Chem. Soc. 11.3, 518 (1991).

[17]

COMBINING MOSSBAUERAND EPR SPECTROSCOPIES

465

M6ssbauer and Electron Paramagnetic Resonance Spectroscopy of Non-Kramers Systems In this section we describe the connections between M6ssbauer and EPR spectroscopy for systems with integer electronic spin. For mononuclear systems the state of most interest is the high-spin (S = 2) ferrous ion. However, the considerations developed below apply also to highspin (S = 2) Fe 4+ and to metal clusters with other spins such as observed in iron-oxo proteins (hemerythrin, ribonucleotide reductase) and nitrogenase (the P cluster in the state pox). The discussion is best framed in the spin Hamiltonian formalism, and to be as concrete as possible we discuss the specific case of an S = 2 system. The electronic part of the pertinent spin Hamiltonian is commonly written as 'g = D

Sz 2 -

S ( S + 1) + ~ (Sx 2 - Sy 2)

-b/3S.g. H

(1)

Here D and E are the axial and rhombic zero-field splitting (ZFS) parameters, respectively, and g is the electronic g tensor. For a sample with randomly oriented molecules, such as protein in frozen solution, the orientation of the electronic x, y, z coordinate system of Eq. (1) relative to a crystallographic coordinate system is usually not known. Such information can only come from spectroscopic studies of single crystals. However, one can always choose an electronic coordinate system x, y, z, such that the parameter E / D is confined to 0 -< E / D <- ~. For simplicity, we assume that the orientations of the ZFS and g tensors are the same. For highspin Fe z+ the principal values o f g are generally confined to 2.0 < gi < 2.3. Figure 1 shows, for D < 0, the zero-field energies resulting from Eq. (I) plotted against E / D . In the following we focus on the two lowest levels. Using the standard representation IS,M) = I2,M) -= [M), the zero-field eigenstates and the associated energies of these two levels can be written a s 4 [2+) = a+([+2) + I-2))/21/2 + a-10) [2-) = ( 1 + 2 ) - I-2))/2 '/2

E+ = - 2 ( 0 2 + 3E2) 1/2 E_ = - 2 D (2)

where a -+ ~ {1 --- [1 - ~(E/D)2]}1/2/21/2. The energy difference between the two levels, A = E_ - E + , can be conveniently approximated as A -~ 3 D ( E / D ) z. Now, if a is smaller than the microwave quantum (hv 0.3 cm-1 at X-band) one may observe an EPR transition between the two states. The probabilities for EPR transitions are obtained by standard procedures, namely, by computing the matrix elements of [(2+[13S • g. H~]2-) 2, where H1 is the magnetic component of the microwave field.

466

[17]

PROBES OF METAL ION ENVIRONMENTS

M=..~0

"-'-

2D

D

S=2

~ ,

0

M=+2

-2D

0

0.05

~

0.1

0.15 E/D

0.2

i

12->

i

,

I

i

0.25 H ('13

i

i

T

I

0.5

12->

0.25

0.3

FiG. 1. Energy levels of an S = 2 system for H = 0 and D < 0 according to Eq. (1). F o r D = - 1.8 cm -] and E/D = 0.23 an EPR transition between the two lowest levels (A = 0.28 cm -l) can be observed at X-band frequencies (hv ~- 0.3 cm-l). For E = 0, and with an applied field along z, the levels can be labeled with the magnetic quantum n u m b e r M. The inset s h o w s the EPR transition between the [2+>and [2-) levels.

For systems with half-integral electronic spin only the matrix elements involving Sx and Sy are nonzero, and thus the selection rules for EPR transitions are AM = ___1. For the levels considered here, on the other hand, only the matrix element involving S z is finite; (2÷rSz[2 -) = 2a ÷ because Sz([+2) - I - 2 ) ) = 2([+2) + I - 2 ) ) a n d <0[Szl2-> = 0. Consequently, only the component of 1-I1 fluctuating along z is effective. These expressions show that the EPR transition between the [2 +> and 12-) levels has AM = 0 (not AM = ---2, --+4 as frequently described in the literature).

[17]

COMBINING Mt)SSBAUER AND E P R SPECTROSCOPIES

467

However, transitions between levels of an integer spin system do not necessarily have AM = 0. For instance, if we choose values of D and E such that the two highest levels of Fig. 1 are split in energy by less than 0.3 cm -1, a AM = +-1 transition can occur. We have analyzed such a transition for an S = 3 system. 8 In the presence of a static magnetic field the 12+-) levels split quadratically as shown in the inset of Fig. 1. This nonlinear field dependence is due to a competition between the zero-field and Zeeman interactions to define the direction of the magnetic moment. In contrast, levels of halfintegral spin systems split linearly with the magnetic field (for flH ~ D) because the direction of the magnetic moment is defined by the magnetic field alone. As a result, the resonance condition for integer spin doublets with the field along the z axis is (hv) 2 = A2 + (geff]3H) 2

(3)

where geff = 4a+gz is an effective g value and where gz is the component of the g tensor of Eq. (1); for E/D < 0.15 the effective g value is near 8. The above-mentioned fundamental differences between integer and halfintegral spin systems give rise to dramatically different EPR spectra. To give the reader a flavor for the variety of integer spin EPR resonances, we have displayed in Fig. 2 traces of spectra obtained for nitrogenase, cytochrome-c oxidase, and the hydroxylase of methane monooxygenase. Two characteristic features readily distinguish the spectra of integer from half-integral spin metal species. First, as a consequence of the selection rule AM = 0, integer spin signals grow in intensity when H1 is oriented parallel to H. Second, because doublets of integer spin systems are split in zero field by A which may be near A = 0.3 cm-1, EPR resonances may be observed near H = 0. The interpretation of integer spin EPR spectra is also quite different from that of half-integral spin spectra. The assignment of a signal to a specific metal species requires consideration of all aspects of the spectrum and not just the position of the resonance alone. The nonlinear field dependence causes highly skewed line shapes. Moreover, a nonzero value of A will result in a shift of the resonance to lower field, away from geff. Thus, marking the g value of some feature of the spectrum is of marginal value, since that g value is not equal to g~tr of Eq. (3). Hendrich and Debrunner 4 have discussed the orientation dependence of the EPR resonances, and they have described procedures for fitting the line shapes of the spectra. Data obtained for a variety of metalloproteins and synthetic complexes have shown that heterogeneity in the local environment of the metal often leads to substantial broadening of the resonances. This heterogeneity should not be confused with "sample

468

[17]

PROBES OF METAL ION ENVIRONMENTS g = 15.5

I

.._._

~

A 9.3

f

a

"r

? 16.0

"¢3

I

xO.5

-2/ I

0

I

!

1 O0

C

I

I

200

300

H (mT) FIG. 2. X-band (9.1 GHz) EPR spectra of the P clusters of nitrogenase 7 (A), beef heart cytochrome-c oxidase (B), 4 and the diferrous hydroxylase of methane monooxygenase (C). 5 The spectra were recorded in parallel mode, that is, the magnetic component of the microwave field was fluctuating parallel to the static field. The spectra are plotted for equal instrumental conditions (T = 4 K) and sample concentrations (0.2 mM).

heterogeneities" that biochemists laboriously try to remove by using, for example, column techniques. The latter type of heterogeneity may arise from denatured protein or protein isozymes. The heterogeneity discussed here is an intrinsic property of the metal site; it may arise from a continuum of conformational subsites of the protein lattice or from random solvent-protein interactions. These heterogeneities affect the orbital states and, via spin-orbit coupling, cause the ZFS parameters D and E to be distributed. This, in turn, yields a distribution of A in Eq. (3). Without adequate treatment of the line shapes (by computer simulations), quantitation of the spin concentration, a major goal in biochemical EPR spectroscopy, is not possible. For the discussion of the Mrssbauer spectra we amend Eq. (1) with terms describing the hyperfine interaction of the 57Fe nucleus with the electronic environment:

~hf

=

S" A" I + I" P" I - g~8,H" I

(4)

where S . A . I describes the magnetic hyperfine interactions, and -gn~n H" I is the nuclear Zeeman term. The I. P . I term describes the interaction of the quadrupole moment, Q, of the nuclear excited state ( / = ]) with the electric field gradient tensor [principal components Vxx,

[17]

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

Vyy, Vz~ ; r~ = (Vxx usually written as ~Q =

469

Vyy)/Vz~]. In its principal axis form this term is

eQVzz 1---~[31z2 - I(I + 1) + rt(Ix2 - Iy2)]

(5)

In the absence of magnetic effects, the M6ssbauer spectrum of an iron site generally consists of a two-line pattern, a quadrupole doublet, the splitting of which is given by AEQ =

2

1 +-

(6)

As discussed previously, 1 the magnetic splitting observed in a M6ssbauer spectrum is determined by the effective magnetic field He~r = Hin t hH, where Hin t =

- (S}. A/gnfl n

(7)

is the internal magnetic field and where (S) is an appropriately taken expectation value of the electronic spin S. Because we previously ~ discussed the meaning and applications of Eq. (7) in considerable detail, we do not dwell on it here. Let us focus again on the two lowest levels of the S = 2 system and assume that we conduct M6ssbauer experiments at temperatures where the other three states are not populated. (This restriction is not essential for the following arguments.) We assume that the electronic spin relaxation rate is slow on the M6ssbauer time scale (-10 MHz); for metalloproteins this condition is usually fulfilled at T < 4.2 K. In the slow relaxation limit each spin level of Eq. (1) contributes its own M6ssbauer spectrum. Therefore, we need to compute (S) for each electronic level. Using the eigenstates given in Eq. (2) the reader may verify that (Si) = 0 for the two levels, in accord with the general result that singlet states do not have a permanent moment, - fig. (S), in the absence of an external magnetic field. An applied field mixes the two electronic states through the Zeeman term of Eq. (1). The mixing is proportional to the matrix elements of the Zeeman interaction taken between the two electronic levels, that is, proportional to (2+[S/[2-). These are the same elements which control the EPR transition probability! Because (2+1Sxl2 -) ~ (2+lsrl2 -) ~ 0 and (2+1Szl2 - } = 2a + ~ 2, mixing is most effective for those molecules of the sample that have their z axis, defined by Eq. (1), parallel to the applied field. In Fig. 3 we have plotted (solid lines) the expectation values (Sx), (Sy), and <Sz) for the lowest level, 12+), as the function of the applied field for fields of moderate strength (fill <~ ID[); the finite values for the x and

470

PgOBES OF METAL ION ENVIRONMENTS

[17]

1.9

1.4

i

0.9

0.4

Y -0.1

,

0

,

,

,

I

0.1

J

,

,

.

I

.

.

.

.

0.2

I

0.3

.

.

.

.

I

0.4

.

.

.

.

0.5

H (T) FIG. 3. Solid curves give expectation values (Sx), <Sy), and (Sz) for the [2+) level of an S = 2 system with D = -2.5 cm -1, E/D = 0.23, and g = (2.0, 2.0, 2.0) with the magnetic field applied along x, y, and z, respectively. Circles correspond to fields for which the MOssbaner spectra of Fig. 4 were computed. Dashed curves give (Sz) computed for E/D = 0.23 --- O'E/D,with O'E/o = 0.055.

y c o m p o n e n t s result f r o m mixing with the M = ---1 levels o f the S = 2 manifold. T h e 12-) state will p r o d u c e a similar graph, e x c e p t that its (Sz) value is positive. A t l o w fields, (S z) o f Fig. 3 has the slope -8flgza+2/A. F o r small values o f A the e x p e c t a t i o n value (Sz) saturates easily, and the M O s s b a u e r s p e c t r u m exhibits its m a x i m a l H i n t = -(Sz)Az/(gnl~n) at low applied field. T h u s , if o n e o b s e r v e s the full internal field in applied fields b e l o w 0.5 T, o n e c a n infer that the electronic splitting A is such that E P R transitions are o b s e r v e d at X - b a n d ; w e a d d r e s s the limit A ~ 0 below. B e c a u s e an E P R - a c t i v e state p r o v i d e s additional s p e c t r o s c o p i c a c c e s s to the s y s t e m u n d e r study, this is i m p o r t a n t i n f o r m a t i o n for the biochemist. E v e n w i t h o u t a detailed u n d e r s t a n d i n g o f the line shape, the signal amplitude c o n v e y s i m p o r t a n t i n f o r m a t i o n in r e d o x titrations and substrate binding studies.

[17]

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

471

Figure 4 shows theoretical M6ssbauer spectra computed along the "magnetization" curve of Fig. 3 for those values of H indicated by the circles on the (Sz) curve. Note that the magnetic splitting increases rapidly with increasing applied field and that the final six-line pattern has developed essentially for H = 0.5 T. From such patterns the M6ssbauer spectroscopist can construct the (Sz) curve of Fig. 3 and also deduce that (Sx) and (Sy) are small. This knowledge, in turn, allows one to determine the ZFS parameters D and E/D and to predict whether an EPR signal can be observed at S-, X-, or Q-band. Conversely, the observation of an integer [

l

l

l

I

I

l

l

l

l

i

l

l

l

l

1.0T

0.5 T

0.2T

0.1T

0.05 T

O.OT

I

-6

l

-4

I

I

-2 0 2 VELOCITY (mm/sec)

I

I

I

I

I

4

I

I

6

FIG. 4. Theoretical M6ssbauer spectra o f the 12+) level of Fig. 1 computed along the "magnetization" curve of Fig. 3. In zero field the spectrum consists of a quadrupole doublet. The spectra originating from the 12-) level are very similar to those of the 12+) state.

472

PROBES OF METAL ION ENVIRONMENTS

[17]

spin EPR signal gives considerable insight regarding the general features of the M6ssbauer spectrum. This knowledge may allow one to correlate a M6ssbauer component with the EPR-active species for metalloproteins containing more than one metal center with integer electronic spin.

Example: Desulfovibrio gigas Ferredoxin II Ferredoxin II (Fd II) from Desulfovibrio gigas is an electron transfer protein that has been studied with a variety of spectroscopic techniques. The protein contains an Fe3S 4 cluster that can be stabilized in two oxidation states. 9 In the oxidized state, the cluster contains three high-spin ferric sites that are antiferromagnetically coupled to yield an S = ½ground state. The X-ray structure of oxidized Fd II has revealed that, in accord with the spectroscopic data, the Fe3S 4 cluster has a structure like the cubane Fe4S4 clusters except that one Fe site is unoccupied.l° The reduced Fe3S4 cluster has a ground state with electronic spin S = 2. The M6ssbauer spectra of this state have been analyzed in detail by Papaefthymiou et al.,9 and in the following we describe some of their results in light of the discussion of the preceding section. These authors have also reported an EPR spectrum of reduced Fd II; here we provide spectral simulations for these data. Figure 5 shows M6ssbauer spectra of Fd II recorded in zero field (curve C) and in applied fields of 0.25 (curve B) and 1.0 T (curve A). The zero-field spectrum consists of two quadrupole doublets, labeled I and II, with a 2:1 intensity ratio. Doublet I has AEQ = 1.47 mm/sec and an isomer shift 8 of 0.46 mm/sec. The two identical sites associated with this doublet constitute a valence delocalized Fe2÷-Fe 3÷ pair. Site II, on the other hand, has parameters typical of high-spin Fe 3+ with tetrahedral sulfur ligation (AEQ = 0.52 mm/sec and 8 = 0.32 mm/sec). Analysis9 of a large set of spectra recorded in strong applied fields yielded D = - 2.5 cm -~ and E/D = 0.23. The graph shown in Fig. 3 was constructed with these parameters. The arguments presented in the preceding section all apply to the analysis of the Fd I1 spectra, except that we have to compute one M6ssbauer spectrum for each distinct Fe site. (The spectra of the delocalized pair were found to be indistinguishable under all experimental conditions.)

9 V. Papaefthymiou, J.-J. Girerd, I. Moura, J. J. G. Moura, and E. MOnck, J. A m . Chem. Soc. 109, 4703 (1987). J0 C. R. Kissinger, E. T. Adman, L. C. Sieker, and L. H. Jensen, J. A m . Chem. Soc. 1109 8721 (1988).

[17]

C O M B I N I N MOSSBAUER G AND EPR SPECTROSCOPIES i

l

l

l

l

l

l

l

l

l

l

l

II

473

l

V f--

0.0 0.5

r" A= 1

.

0

T

~

Z 0 0.0 0 r~

< 0.5

H =0.25 T

0.0 2.0

C

4.0

I

-6

I

I

-4

I

I

I

-2 0 2 VELOCITY (mm/sec)

I

I

I

I

t

4

I

I

6

FIG. 5. Low-temperature M6ssbauer spectra of the S = 2 state of the Fe3S4 cluster of reduced D. gigas Fd II. The zero-field spectrum (4.2 K, C) consists of two doublets with 2 : 1 intensity ratio. Spectra shown in (A) and (B) were recorded in parallel applied fields at 1.5 K. The theoretical spectra displayed above the 1.0 T spectrum show the contributions of the delocalized Fe2+-Fe3÷ pair (site I) and the localized Fe 3+ (site II),

Figure 6 shows two EPR spectra of Fd II recorded at 4.2 K with a bimodal cavity. For recording trace A in Fig. 6 the cavity was tuned such that the microwave field H1 was fluctuating parallel to the static field H (parallel mode), whereas trace B was obtained in the standard transverse

474

PROBES OF METAL ION ENVIRONMENTS

[17]

g=18

I

A

S

" ~ . . . . ~.~

-1-

"10 =

I

0

I

1oo

I

I

200

I

I

300

I

400

H (mT) FIG. 6. X-band EPR spectra of the S = 2 state of the Fe3S4 cluster of reduced D. gigas Fd II recorded at 4 K in parallel (A) and perpendicular (B) mode (M. P. Hendrich, I. Moura, J. J. G. Moura, K. K. Surerus, E. M~nck, unpublished results). The dashed curves are theoretical spectra computed with the parameters quoted in the text.

mode. Note that the minima of the resonances do not occur at the same field in the two spectra, showing that one cannot obtain the effective g value by simply marking the minima of the curves. The dashed lines are the result of computer simulations using the S -- 2 Hamiltonian of Eq. (1) with D = - 2.5 c m - ~, E/D = 0.23, and g = (2.0, 2.0, 2.0). To produce the correct shapes of the spectra we have assumed that the parameter E/D has a Gaussian distribution centered around the mean E/D = 0.23 with trE/D = 0.055. The quoted parameters yield a mean A of 0.38 cm-1 and a A distribution as shown in Fig. 7. The amplitude of the theoretical curve in Fig. 6A was adjusted to fit the experimental curve. Absolute spin quantitation of the sample relative to a standard, Fe(II)-doped zinc fluorosilicate, gave a spin concentration of 6.6 mM S = 2 spin, which compares well with the cluster concentration of 7.7 mM determined by metal analysis. The relative intensities of the theoretical spectra of Fig. 6 are predicted by the theory without use of any additional parameters. We have marked in Fig. 7 the energies of the microwave quantum at X- and Q-band frequencies. It can be seen that only a small fraction, 15%, of the molecules of the sample have a A sufficiently small to permit transitions at X-band ! Figure 7 demonstrates forcefully that determination of the spin concentration of the sample is not possible without knowledge of the distribution of A, because most of the molecules do not contribute

[17]

475

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

X

Q

N,--

0

._Z, ,0 Q.

/

\ I

0

0.3

0.6

I

I

0.9 A (cm "1)

1.2

1.5

FIG. 7. Distribution of A values for the Fe3S 4 cluster of Fd II as obtained from the analysis of the spectra of Fig. 6. The vertical lines indicate the energies of the microwave quantum at X- and Q-band frequencies. Only those molecules having A values smaller than the microwave quantum at X-band contribute to the EPR spectra of Fig. 6. This graph demonstrates that one cannot obtain the spin concentration from an integer spin EPR signal without analysis of the line shapes.

to the spectrum at X-band. Note that for Fd II virtually all molecules in the sample would contribute to the spectrum if the spectrum were recorded at Q-band (-36 GHz). Finally, it is exceedingly difficult to estimate, even within a factor of 10, the spin concentration of an integer spin signal by mere inspection of the signal strength. However, from simulations of EPR spectra, a quantitative analysis of the signal can be accomplished even when the fraction of the molecules observed is only 15%, as is the case for Fd II. In many instances, the spin concentration will be known when M/Sssbauer spectra of the same sample are recorded under suitable conditions. We have stressed that the line shape of integer spin EPR signals is quite sensitive to heterogeneities, as expressed by distributions of the ZFS parameters D and E/D. The Mrssbauer spectra reflect these heterogeneities as well. The dashed lines in Fig. 3 were computed for E/D values of Fd II which correspond to the standard deviation of the Gaussian that fits the EPR spectra of Fig. 6. It can be seen that (Sz), and therefore Hint , is quite sensitive to variations in E/D during the rising portion of the curves. Because of the variations in Hi,t the MOssbauer spectra broaden.

476

PROBES I

I

-6

I

I

I

I

I

-4

OF I

METAL I

I

I

I

l

l

I

ION

ENVIRONMENTS

l

l

1

l

I

[17]

l

I

I

-2 0 2 VELOCITY (mm/sec)

I

4

I

I

6

FIG. 8. Theoretical spectra of site I of reduced D. gigas Fd II at 0.15 T and 1.5 K assuming E/D = 0.23 --+ O'E/D. The solid line was computed assuming trE/D = 0 while the simulation represented by the crosses is a convolution of spectra with a Gaussian distribution assuming

OrE/D ----- 0 . 0 5 5 .

Figure 8 shows two 1.5 K spectra c o m p u t e d with the p a r a m e t e r s of Fd II; for clarity we h a v e s h o w n only the s p e c t r u m of site I. The s p e c t r u m r e p r e s e n t e d b y the solid line was c o m p u t e d under the a s s u m p t i o n that O'E/D = 0, w h e r e a s for the c o m p u t a t i o n r e p r e s e n t e d by the crosses we h a v e c o n v o l u t e d spectra along a Gaussian distribution with O-E/D = 0.055 as d e t e r m i n e d b y EPR. (To test w h e t h e r O-E/D can be extracted f r o m the M f s s b a u e r spectra we h a v e added s o m e r a n d o m noise to the s p e c t r u m a n d a s k e d one of our first-year graduate students to extract O'E/o. H e estimated O'E/D as 0.05.) Spectra in Limit A = 0 W e h a v e r e p o r t e d a c o m b i n e d M 6 s s b a u e r and E P R study for the state pox of the P clusters o f nitrogenase that describes an integer spin s y s t e m for which the lowest two levels are separated in energy b y A < 0.01 c m - 1.7 W e found that A -< 0.001 c m - l for the P clusters of the proteins f r o m Azotobacter vinelandii, Kiebsiella pneumoniae, and Clostridium pasteurianum, and that A ~ 0.01 c m - 1for the Xanthobacter autotrophicus protein. Figure 2A shows a parallel m o d e s p e c t r u m o f the X. autotrophicus protein. N o t e the sharpness o f the r e s o n a n c e line. This sharpness is a characteristic feature of spectra for which flH ,> A; under these conditions the spectra are quite insensitive to distributions in A, and the transition probability

[17]

COMBINING MOSSBAUER AND E P R SPECTROSCOPIES

477

peaks sharply at g~ff = hv/flH with an intensity proportional to (A/fill) z. Thus, the signal vanishes in the limit A -- 0. (For the S = 2 system discussed above, A = 0 occurs for E -- 0. For E = 0 the two levels have magnetic quantum numbers M = + 2 and M = - 2 when the magnetic field is applied along z, and EPR transitions are forbidden because AM = --_4.) For many years we thought that A values as small as 0.0l cm-~ would be unlikely for metal complexes of biological interest. This is still true for the high-spin ferrous system. H o w e v e r , more recent studies in various laboratories have dealt with metal clusters having S > 2. We have pointed o u t 7 that a multiplet with spin S describable by Eq. (l) has at least one pair of levels for which A ~ IDI(E/D) s. Because E/D is confined to 0 -< E/D <- ½, small A values are likely to be observed for multiplets with large S. Consider, for instance, an exchange-coupled pair of high-spin (S = ~) Fe 3÷ sites. The exchange coupling produces a series of multiplets with S -- 0, l, 2, 3, 4, and 5, where S is the dimer spin. For the iron sites of Fe2S 2 clusters and the clusters of iron-oxo proteins typical D values are about 1 cm-~. It is thus very likely that some of the states with S -> 2 have a pair of levels with a small A. For example, we have observed a sharp resonance at geff = 8.0 for the excited S = 2 multiplet of the Fe 3+ - F e 3÷ cluster of the hydroxylase component of methane monooxygenaseH; for the transition observed we found that A ~ 0.03 cm-~. (The multiplets with S > 3 are too high in energy to be populated at temperatures when the spin-lattice relaxation is slow enough to permit observation of an E P R signal.) The P clusters of nitrogenase have been quite a challenge for the spectroscopist. For nearly 15 years it was thought that a P cluster contains four Fe sites, and that the P cluster state pox had half-integral electronic spin, S >- ~. For A _< 0.001 cm -~ the two nearly degenerate levels of pox behave essentially like a Kramers doublet, and it is therefore understandable that it took many years before the nature of the electronic state was recognized. The distinction between a Kramers doublet of a system with half-integral electronic spin and a pair of nearly degenerate levels of a non-Kramers system may appear esoteric to a biochemist. H o w e v e r , the structural implications for the two cases differ substantially. If pox were a Kramers system, spectroscopic and redox data would suggest that nitrogenase contains four Fe4S4-type P clusters. The recognition of pox as a non-Kramers system implies that each P cluster contains eight Fe sites IIB. G. Fox, M. P. Hendrich, K. K. Surerus, K. K. Andersson, W. A. Froland, J. D. Lipscomb, and E. Mfinck, J. Am. Chem. Soc. 115, 3688 (1993).

478

PROBES OF METAL ION ENVIRONMENTS

[17]

and that the protein contains two such superclusters. The latter view is supported by X-ray crystallographic studies.~2 Concluding Remarks Integer spin EPR will become an important tool for the studies of the active sites of iron-containing metalloproteins. In our laboratories we have focused on combined EPR and M6ssbauer studies of systems yielding M6ssbauer spectra that could be analyzed with high accuracy and confidence in the framework of the spin Hamiltonian formalism. Using zerofield splitting parameters from the M6ssbauer analysis, we have been able to assign EPR transitions with confidence and have refined the methods of EPR data analysis (see, e.g., Ref. 8). Integer spin EPR, in turn, has aided us in recognizing the fundamental features of some systems; in particular, it has helped us to solve the problem of the P cluster state pox. We now have sufficient experience in analyzing integer spin EPR signals that we can venture into studying noniron systems. It may be useful here to list some of the iron-containing proteins and model complexes for which integer spin EPR has been observed. The M6ssbauer spectra of the S -- 2 states of reduced Fe3S4 clusters are quite similar, and, not surprisingly, most clusters exhibit an EPR signal in this state. For those Fe3S4 proteins for which an EPR signal has not been observed at X-band, Q-band EPR is most likely to yield positive results. Integer spin EPR has been reported for a diverse class of proteins, including deoxymyoglobin as prepared or after flash-off of CO 4 or O2,13 beef heart 4,14 and yeast 4 cytochrome-c oxidase, nitrogenase, 7 iron-oxo proreins, 5'6'1~ desulfoferredoxin, ~5 and Fe(II)-substituted alcohol dehydrogenase.~6 Integer spin EPR has also been reported for model complexes of rubredoxin ~7 and iron-oxo proteins. TM One distinction should be made regarding the EPR properties of the listed proteins. The signals observed for myoglobin, 4 the oxidized hydroxylase of methane monooxygenase, ~ and K. pneumoniae and A. vinelandii nitrogenase 7 originate from transit2 j.

Kim and D. C. Rees, Science 257, 1677 (1992). 13 M. P. Hendrich and P. G. Debrunner, J. Magn. Resort. 78, 133 (1988). 14 W. R. Hagen, Biochim. Biophys. Acta 708, 82 (1982). 15 I. Moura, P. Tavares, J. J. G. Moura, N. Ravi, B. H. Huynh, M.-Y. Liu, and J. LeGall, J. Biol. Chem. 265, 21596 (1990). 16 E. Bill, C. Haas, X.-Q. Ding, W. Maret, H. Winkler, A. X. Trautwein, and M. Zeppezauer, Eur. J. Biochem. 180, 111 (1989). 17 M. T. Werth, D. M. Kurtz, Jr., B. D. Howes, and B. H. Huynh, lnorg. Chem. 28, 1357 (1989). ~8A. S. Borovik, M. P. Hendrich, T. R. Holman, E. Miinck, V. Papaefthymiou, and L. Que, Jr., J. Am. Chem. Soc. 112, 6031 (1990).

[18]

VOLTAMMETRY OF REDOX-ACTIVE CENTERS

479

tions between excited spin levels. These signals are not easily correlated with the corresponding M6ssbauer spectrum because the electronic spin has generally intermediate relaxation rates at temperatures (10-20 K) where the excited spin levels are appreciably populated; for intermediate relaxation rates the M6ssbauer spectra are broad and ill-resolved. M6ssbauer spectra are observed in the slow fluctuation limit if the electronic spin relaxes with a rate slower than 106/sec. Thus, if one observes a M6ssbauer spectrum in the slow fluctuation limit, one can be assured that EPR spectra will be observed in the slow fluctuation limit as well. A survey of the literature on M6ssbauer spectroscopy suggests that a variety of other iron-containing proteins are likely to exhibit integer spin EPR at either X- or Q-band frequencies. Based on the progress that has been made in the last few years we anticipate that this technique will be a valuable tool for the biochemist and biophysicist. By applying M6ssbauer and EPR spectroscopy to proteins with multiple iron-containing sites and using the methodology discussed above, one should be able to untangle quite complex situations. Acknowledgments The work described here was supportedby grants fromthe NationalInstitutesof Health (GM-22701) and the National Science Foundation(MCB-9096231).

[18] V o l t a m m e t r i c S t u d i e s o f R e d o x - A c t i v e C e n t e r s in M e t a l l o p r o t e i n s A d s o r b e d o n E l e c t r o d e s

By FRASER A. ARMSTRONG, JULEA N. BUTT, and ARTUR SUCHETA Introduction In recent years it has been shown that redox proteins can be induced to interact directly with an electrode surface and display reversible electrochemistry in the same way as many smaller molecules. ~,2 This has suggested the possibility of using dynamic electrochemical methods such as cyclic voltammetry to examine the many intricate functional properties of redox-active centers in proteins. In this chapter we describe a particular strategy, thus far demonstrated to be applicable for investigating labile I F. A. Armstrong, Struct, Bonding (Berlin) 72, 137 (1990). 2 A. M. Bond and H. A. O. Hill, in "Metal Ions in Biological S y s t e m s " (H. Sigel and A. Sigel, eds.), Dekker, N e w York, 1991.

METHODS IN ENZYMOLOGY,VOL. 227

Copyright © 1993by Academic Press, Inc. All rightsof reproduction in any form reserved.

[18]

VOLTAMMETRY OF REDOX-ACTIVE CENTERS

479

tions between excited spin levels. These signals are not easily correlated with the corresponding M6ssbauer spectrum because the electronic spin has generally intermediate relaxation rates at temperatures (10-20 K) where the excited spin levels are appreciably populated; for intermediate relaxation rates the M6ssbauer spectra are broad and ill-resolved. M6ssbauer spectra are observed in the slow fluctuation limit if the electronic spin relaxes with a rate slower than 106/sec. Thus, if one observes a M6ssbauer spectrum in the slow fluctuation limit, one can be assured that EPR spectra will be observed in the slow fluctuation limit as well. A survey of the literature on M6ssbauer spectroscopy suggests that a variety of other iron-containing proteins are likely to exhibit integer spin EPR at either X- or Q-band frequencies. Based on the progress that has been made in the last few years we anticipate that this technique will be a valuable tool for the biochemist and biophysicist. By applying M6ssbauer and EPR spectroscopy to proteins with multiple iron-containing sites and using the methodology discussed above, one should be able to untangle quite complex situations. Acknowledgments The work described here was supportedby grants fromthe NationalInstitutesof Health (GM-22701) and the National Science Foundation(MCB-9096231).

[18] V o l t a m m e t r i c S t u d i e s o f R e d o x - A c t i v e C e n t e r s in M e t a l l o p r o t e i n s A d s o r b e d o n E l e c t r o d e s

By FRASER A. ARMSTRONG, JULEA N. BUTT, and ARTUR SUCHETA Introduction In recent years it has been shown that redox proteins can be induced to interact directly with an electrode surface and display reversible electrochemistry in the same way as many smaller molecules. ~,2 This has suggested the possibility of using dynamic electrochemical methods such as cyclic voltammetry to examine the many intricate functional properties of redox-active centers in proteins. In this chapter we describe a particular strategy, thus far demonstrated to be applicable for investigating labile I F. A. Armstrong, Struct, Bonding (Berlin) 72, 137 (1990). 2 A. M. Bond and H. A. O. Hill, in "Metal Ions in Biological S y s t e m s " (H. Sigel and A. Sigel, eds.), Dekker, N e w York, 1991.

METHODS IN ENZYMOLOGY,VOL. 227

Copyright © 1993by Academic Press, Inc. All rightsof reproduction in any form reserved.

480

PROBES OF METAL ION ENVIRONMENTS

[18]

F e - S clusters, which could be more widely exploited for deciphering complicated reactivity that is linked to electron transfer. Our discussion focuses on application; the fundamental principles and methods of dynamic electrochemistry are described in several excellent textbooks. 3'4 We first outline some general features of voltammetric methods that are potentially useful for studies of redox sites in proteins.

General Information from Voltammetric Experiments Cyclic voltammetry is a widely used technique that provides a broad "snapshot" of the redox chemistry of molecules in solution. By analogy with a spectrum, in which the presence of certain species is evident from energy absorption or emission at a characteristic energy, a voltammetric response arises from exchange of electrons that is most readily observed at some characteristic potential. The current-voltage profile thus constitutes a signal which can be assigned to a particular species (redox couple) that is identifiable by complementary studies. Voltammetry is dynamically interactive, that is, reactions can be both induced by application of a potential and analyzed through the current response. Complicated activity may be visualized in both the time and potential domains. Information is obtained on reduction potentials, electrode kinetics, and the relevant parameters of coupled reactions including catalysis) '4 A variety of other dynamic electrochemical methods can be employed to reveal more detailed mechanistic information. For example, in pulse methods, the current is monitored (or measured at some time interval) after stepping the applied potential to a new value; in hydrodynamic methods the current is measured under varying conditions of enforced convection. It is important to note the contrast with potentiometry, which is a static technique concerned with determining the populations of species at redox equilibrium (i.e., measuring the potential at which no net current flows). The useful potential range is wide and continuous, being restricted only by the practical limits for electrical breakdown of electrode and solvent, not by properties of mediators and titrants. To date, the chemistry of biological species generated at potentials below approximately -550 mV [versus standard hydrogen electrode (SHE)], which is the practical limit for dithionite at pH 7, has remained almost unexplored. This is despite the likelihood that such strong reductants may be important inter3 A. J. Bard and L. R. Faulkner, "Electrochemical Methods, Fundamentals and Applications." Wiley, New York, 1980. 4 Southampton Electrochemistry Group, "Instrumental Methods in Electrochemistry." Ellis Horwood, Chichester, 1985.

[18]

V O L T A M M E T ROF Y REDOX-ACTIVE CENTERS

481

mediates in biological systems (which are rarely at equilibrium). By contrast, voltammetry with neutral aqueous solutions (at carbon electrodes) is feasible at potentials below - 1 V and up to at least +800 mV. Although voltammetry can yield an instructive picture of complex reactivity, it provides no information on structure. Even so, the task of characterizing a labile active site by spectroscopic methods is made much easier since it is possible to define the precise conditions (reagent concentration, potential) under which a particular state can exist. Advantages of Immobilizing Protein Molecules on Electrodes Important experimental and interpretative refinements become possible if we confine the molecules under study to the electrode surface, as depicted in Fig. 1. The idealized concept is as follows. (1) Redox-active protein molecules are adsorbed strongly at the surface of an electrode. Electron transfer between the electrode and the protein active site(s) is reversible in the electrochemical sense, that is, Nernstian equilibrium is maintained at each value of the applied potential. (2) Adsorption occurs with minimal disruption of the native conformation so that native functional properties are conserved. (3) The coverage is monolayer or lower, and the active site in each protein molecule acts independently, but identically, to its counterparts in adjacent molecules. (4) Formation of such an array may depend critically on coadsorption of other complex molecules. These are represented by the symbol A. Because the protein molecules are absent from the electrolyte, and none effectively leave the electrode surface, all the charge that crosses

electrons

ELECTRODE

i

f

~

l

r •

metal ions ~') ~ligands substrates

ELECTROLYTE

FIG. 1. Concept of the molecular arrangement formed by electron transfer proteins and coadsorbates at the surface of an electrode. Electrons can be freely exchanged between the electrode and redox centers within the protein. Binding sites in the protein, which can be influenced or triggered by the status of the redox centers, are available to reagents that diffuse freely in the solution.

482

PROBES O F M E T A L I O N E N V I R O N M E N T S

[18]

the interface reversibly is accounted for in what can be regarded as a closed system. Analysis of the voltammetry yields quantitative information on reactivities of specific redox sites, information which for labile systems may be difficult to obtain by conventional methods. We first consider a simple electrontransfer system that is not complicated by coupling to further reactions such as reversible binding or catalysis. In this case, the specific advantages of having all the protein molecules confined to the electrode surface are as follows.

High Sample Economy. The amount of material required to form a monolayer on a typical small electrode surface corresponds to approximately I pmol. Thus, a large number of experiments can be conducted on valuable samples in limited supply. Precise Control of Redox Status of Centers. The status of all active sites can be addressed and fine-tuned on a rapid time scale. This stems from the fact that waves observed for well-behaved surface-confined systems are compact (even at high scan rates), reflecting the exhaustive conversion of redox species. This is to be contrasted with the voltammetric response typically observed for a planar electrode contacting a thick layer of freely diffusing redox-active species, for which the current is finite and diffusion-limited at a sufficiently large overpotential. Figure 2 describes the theoretical waveform (discussed by Laviron 5) for the ideal case, namely, a reversible diffusionless electron-transfer reaction that is uncomplicated by species heterogeneity, intersite interaction, or coupled chemistry. 5 The separation AEp between oxidation and reduction peaks (Epa - Ep¢) is 0 mV, and the theoretical half-height width 8 is 91/n mV at 25 ° (where n is the number of electrons transferred in the elementary process). However, even in the limit of irreversible (sluggish) electrode kinetics, the wave envelope remains compact; 8 for reduction becomes 62.5/Oma mV [or 62.5/ (1 - a ) n a mV for oxidation], where a is the transfer coefficient and n a is the number of electrons transferred in the rate-determining step. Waveform Analysis. For a protein molecule in solution, the voltammetric waveform can be complicated by variation in the geometry of diffusion to the electrode surface. 2 Such effects cause waves to broaden and flatten as the number and size of suitable interaction sites on the electrode surface decrease. This is not a problem for an immobilized protein. In the case of single, isolated redox couples, the following easily measured parameters are analytically important: average peak potentials [(Epa + Ep¢)/2] correspond to the reduction potential E °', areas of waves give the finite charge that is exchanged between electrode and protein 5 E . L a v i r o n , J. Electroanal. Chem. 101, 19 (1979).

[18]

483

VOLTAMMETRY OF REDOX-ACTIVE CENTERS

E

-0.30

b o

-0.20

(/)

m c o ~z o

E

a

-0.10 0.00 0.10 0.20 0.30 -250

- 150

-50

n(E-Epeak)

50

/

150

250

mV

FIG. 2. Ideal cyclic voltammogram for the case of a reversible, surface-bound redox couple with rapid electron transfer kinetics, in the absence of coupled chemical reactions and interactions between redox centers. The resulting waveform is described by the expression I = -[nEF2oAF/(RT)]O(1 + 0) -2, w h e r e n is the number of electrons exchanged, F is the Faraday constant (96,484.5 coulombs per gram-equivalent), ~ the scan rate (taken as velocity, i.e., positive or negative, depending on the scan direction), F is the surface concentration of redox center, R is the gas constant, T is the absolute temperature, and 0 = e x p [ n F ( E E°')/(RT)]. T h e waveforms resulting from actual experiments should approach this ideal shape at scan rates slow enough that the electron exchange rate between the electrode and the redox couple is not rate-limiting. The width at half-height 8 is 2 ln[3 + 2(2)I/2]RT/(nF), or 82.8/n mV at 0 °.

active sites, peak separations AEp (if above 0) can be used to derive rate constants for electron exchange, and the parameter ,3/n is an indicator of the degree of species heterogeneity and intersite interactions. 5 Complex waveforms arise if two or more redox couples have similar reduction potentials. However, these may be deconvoluted by computer calculations such as nonlinear regression or by simulations based on the fundamental parameters. Thus, for multicentered proteins, one may readily determine the stoichiometries of specific centers, their electron capacity, E °' values, and electrontransfer facilities. Next we extend our discussion to coupled reactions. If electron transfer induces a rearrangement of the structure of an active site, the voltammogram may be perturbed. The immobilized protein method becomes particularly useful for studying oxidation state-dependent interactions with extraneous molecules, since the protein-coated electrode can be transferred between solutions containing the reagent of interest. Such reactions include metal ion uptake and release, ligand binding, and substrate transformation. This brings us to outline further features of the surface-confined voltammetric method as follows.

484

PROBES OF METAL ION ENVIRONMENTS

[18]

Sensitivity for Study of Interactions between Active Sites and Exogenous Reagents. The extremely small number of protein molecules under observation makes it possible to observe and quantify reactions which occur between the protein active site and reagents contained in the electrolyte at very low concentration. Kinetic Analysis of Coupled Processes. Because the electrode and the redox centers are in direct electronic communication, all the related chemical reactivity can be induced and monitored in the time-domain mode through control of the electrode potential. By analyzing the waveform over a range of scan rates, it is possible to plot the course of coupled reactions. Digital simulation can be employed to obtain rate constants for these processes. For a surface-confined redox enzyme, the catalytic turnover of substrate greatly amplifies the current-potential response. Because the enzyme is coupled directly to a continuously variable and tunable applied potential, analysis of the form of this response under stationary and rotating electrode conditions leads to detection and definition of subtle mechanistic phenomena. These include the effects of electron distribution or ordered binding that may be important in regulation. Basic Considerations The central problem is to identify the conditions of electrode surface, electrolyte, and coadsorbate (if required) that are necessary to achieve and optimize adsorption of the protein and observation of voltammetric signals. This is a complicated, largely uncharted area, and a "trial and error" search, guided by available information on the physical properties of the protein, is executed. Many demands must be met to achieve a stable film that is useful for studies: the protein must remain conformationally intact while retaining internal mobilities that are essential for function; electron exchange between the active sites and the electrode must be fast; the microenvironments of active sites should be uniform throughout the adsorbed protein population; and access for extraneous reagents must be unimpeded. In favorable cases it is possible to obtain an independent assessment of the degree of conformational change induced by adsorption. Bowden and co-workers have shown that an electroactive monolayer of cytochrome c adsorbed on tin oxide (in the presence of phosphate at pH 7) displays optical absorption bands that are characteristic of the native Fe(III) and Fe(II) forms. 6 It should also be emphasized that the capability for facile electron exchange with an electrode is not so strongly correlated with protein size as is commonly assumed. Two examples illustrating this 6 M. Collinson a n d E. F. B o w d e n , Anal. Chem. 64, 1470 (1992).

[18]

VOLTAMMETRY OF REDOX-ACTIVE CENTERS

485

point are the structurally related Fe-S flavoproteins fumarate reductase and succinate dehydrogenase, the membrane-extrinsic forms of which have molecular weights of around 100,000. Each of the enzymes exhibits a remarkably high level of electrocatalytic activity when adsorbed at carbon electrodes. 7'8 Visualizing and Quantifying Complicated Redox Activities To illustrate how information is obtained from these experiments, we refer to our own endeavors to study the complicated redox-linked activities of iron-sulfur (Fe-S) clusters in proteins.9 It is now recognized that these centers may perform vital roles aside from electron transfer and storage, roles that include acid-base catalysis and regulation of iron metabolism at the level of mRNA. 1°'11 We need to learn more about the factors that determine reactivities such as metal ion and ligand exchange as may occur at a labile cluster, that is, a cluster difficult to study by conventional means. As an example, we consider some of the reactions that are possible for a protein-bound [3Fe-4S] cluster. These are indicated in Scheme I. The [3Fe-4S] cluster is to be considered as a tridentate ligand that may exist in up to four oxidation levels) 2-~4 Binding of a single metal ion M may occur to the [3Fe-4S] core in one or more of the oxidation levels to produce cubane-type products exemplified by the [4Fe-4S] cluster [M -- Fe(II)].10 The incoming metal ion will require further coordination by a ligand (X) that is possibly added to or exchanged readily for others (L). It is obvious that electron transfer, metal ion binding, and ligand binding activities must be interlinked, and as such could provide the basis for an intricate catalytic/regulatory system. The experimental challenge is to identify pathways and quantify the chemistry within this maze of possibilities. Voltammetry with an adsorbed protein is well suited to de7 A. Sucheta, B. A. C. Ackrell, B. Cochran, and F. A. Armstrong, Nature (London) 356, 361 (1992). 8 A. Sucheta, R. Cammack, J. H. Weiner, and F. A. Armstrong, Biochemistry 32, 5455 (1993). 9 F. A. Armstrong, in "Advances in Inorganic Chemistry" (A. G. Sykes and R. Cammack, eds.), Vol. 38. Academic Press, San Diego, 1992. 10 H. Beinert and M. C. Kennedy, Eur. J. Biochem. 186, 5 (1989). N R. Cammack, in "Advances in Inorganic Chemistry" (A. G. Sykes and R. Cammack, eds.), Vol. 38. Academic Press, San Diego, 1992. 12 S. Ciurli and R. H. Holm, lnorg. Chem. 30, 743 (1991). 13 j. N. Butt, F. A. Armstrong, J. Breton, S. J. George, A. J. Thomson, and E. C. Hatchikian, J. Am. Chem. Soc. 113, 6663 (1991). 14 j. N° Butt, A. Sucheta, F. A. Armstrong, J. Breton, A. J, Thomson, and E. C. Hatchikian, J. Am. Chem. Soc. 113, 8948 (1991).

486

1 S

S

[18]

PROBES OF M E T A L I O N E N V I R O N M E N T S

S

2 S

S

S

3 S

e-

S

S

e-

M

e-

5

M-~ 6

M

i X

v

8

s:..q'~.s

X

v

~:';',,..s Fe"

• \sf/-

9

Pe ~Fe

, \,/-

L-~ 12

e-

l

L~,~ 11

e-

i

X

v

10

s,.,";',,.s -~%7s /"~*-

L.,~ 13

7

e-

i

X

v

~..";',,..s - \~/-

L.~ 14 i

X? L

,~r

X? L

15

X? L

~v

e-

16

X? L

,~r

e-

17

~v

e-

oxidation level of [3Fe-4S] core 1+

SCHEME

0

1-

2-

I. Pathways (1-17) of redox, metal binding, and ligand binding reactions based on the [3Fe-4S] cluster. Solid arrows indicate established reactions or ones detected by voltammetry.

tecting and deciphering coupled chemistry that appears chaotic when viewed by more conventional methods. Outline of Experimental Methods We have found that a number of proteins adsorb spontaneously at "edge"-oriented pyrolytic graphite (PGE) electrodes in the presence of suitable coadsorbates such as neomycin or polymyxin. The PGE electrode is a small block of pyrolytic graphite mounted on a brass rod and embedded with epoxy in a Teflon sheath. The block is oriented in such a way that the working surface is an " e d g e " plane perpendicular to the aromatic

[18]

V O L T A M M E T ROF Y REDOX-ACTIVE CENTERS

487

"basal" plane. Abrasion of the edge plane by cutting or polishing in air generates hydrophilic oxides that are weakly acidic. 15 Neomycin and others in the aminocyclitol family of antibiotics possess a specific arrangement of various groups, particularly - N H 3 ÷ groups, located on a complex, often rigid, structure in such a way perhaps as to fulfill requirements of preorganization in binding to complex, complementary surfaces) In the presence of these reagents, ferredoxins and many other protein molecules coadsorb on the PGE electrode surface to give an electroactive film with a coverage of one monolayer or lower.16 The electrochemical cell that we have used frequently is shown in Fig. 3. The central water-jacketed compartment housing the reference electrode (typically saturated calomel) is linked via Luggin capillary tips to four pots each containing a platinum (Pt) wire or gauze counterelectrode. Such an arrangement allows the response of the protein film to a variety of reagents to be observed; the coated PGE electrode is simply transferred from one pot to another. The pots are thermostatted by immersing the lower half of the cell in a circulating bath. For cyclic voltammetry, the electrodes are connected to a standard potentiostat, and data are typically recorded on an X - Y recorder. For digitally based techniques such as square-wave voltammetry, a commercial instrument such as the BAS 100B electrochemical analyzer (Bioanalytical Systems, West Lafayette, IN) is useful. We have favored an analog-based system wherever the exploratory nature of an experiment has required flexibility (which is the situation in most cases). The capability of performing rotating-disk voltammetry (requiring an electrode rotator) is also to be recommended since reactions of adsorbed molecules with reagents in solution may depend critically on mass transport to the electrode. The following method for preparing a protein film has been consistently successful for a number of ferredoxins, and we outline it here with the understanding that we have not endeavored to optimize the technique. The PGE electrode is first polished with an aqueous slurry of 1/xm A1203 on cotton wool and then sonicated in cold water. Immediately following this, 1/~1 of ice-cold protein solution (containing coadsorbate as necessary) is spread across the electrode surface using a glass rod drawn to a fine tip. A typical coating solution contains 100/xM protein and the required concentration of coadsorbate in 0.1 M NaCI buffered at pH 7. The coated electrode is then introduced to a pot containing buffer-electrolyte at the 15 F. A. Armstrong, P. A. Cox, H. A. O. Hill, V. J. Lowe, and B. N. Oliver, J. Electroanal. Chem. 217, 331 (1987). 16 F. A. Armstrong, J. N. Butt, S. J. George, E. C. Hatchikian, and A. J. Thomson, FEBS Lett. 259, 15 (1989).

488

PROBES O F M E T A L I O N E N V I R O N M E N T S

[18]

! i

t. ~

I

a:zz

g_f" FIG. 3. Diagram of the electrochemical cell typically used in metalloprotein experiments. The centrally located reservoir houses a reference electrode. It is water-jacketed to maintain a standard reference temperature. Liquid junctions, terminating in Luggin capillary tips (see inset), connect the reference reservoir with each component cell. Platinum gauze attached to the wall of each sample pot serves as the counterelectrode. Each sample pot can be independently purged of atmospheric oxygen by bubbling an inert gas (usually argon) through a flexible tubing capillary gas line. This enters the pot through a side arm, which also serves as the passageway for the counter electrode lead. A single working electrode can be rapidly transferred between pots, facilitating multisample studies. By immersing the lower part of the cell in a circulating bath, temperature control can be accomplished.

d e s i r e d a p p l i e d p o t e n t i a l , a n d t h e e x p e r i m e n t is c o m m e n c e d . A n a l t e r n a t i v e m e t h o d o f film f o r m a t i o n t h a t h a s a l s o p r o v e d s u c c e s s f u l is to i n t r o duce a freshly polished electrode into a pot containing a dilute (-1/xM) s o l u t i o n o f t h e p r o t e i n a n d c o a d s o r b a t e in t h e b u f f e r - e l e c t r o l y t e , to c y c l e t h e p o t e n t i a l o r p o i s e it f o r a s h o r t t i m e in a r e g i o n t h a t is f a v o r a b l e f o r a d s o r p t i o n , a n d t h e n to t r a n s f e r t h e c o a t e d e l e c t r o d e to a s e c o n d p o t ( n o t containing the protein) for study.

[18]

VOLTAMMETRY OF REDOX-ACTIVE CENTERS

489

We have made the following observations with ferredoxins from Desulfovibrio africanus, Azotobacter vinelandii, and others which typically require neomycin as coadsorbate: no signals are observed if neomycin is absent from the coating solution; if neomycin is present during coating but absent from the pot solution, the signals diminish rapidly; and if neomycin is present in both the coating and the pot solution, the signals persist. The results show that coadsorption of neomycin is necessary for adsorption of the protein. As neomycin undergoes net desorption, as occurs if it is absent from the solution, the protein molecules also desorb. On the other hand, loss of signals is suppressed if neomycin is present in the solution. From a number of studies with various proteins, we have noted that film formation depends on a number of factors. Success is more likely if the ionic strength is 0.2 M or lower, if the temperature is low (typically 0°), and if the sample of interest is of very high purity (because there would be competition for occupancy of sites on the electrode). We have also found that the extent of adsorption and the redox properties of the centers in the adsorbed protein molecules may be sensitive to the applied potential. Any such dependence must always be identified prior to more detailed investigations.

Analysis and Interpretation

Imaging Redox Centers in Proteins. Figure 4 shows voltammograms obtained for four different ferredoxins absorbed on PGE electrodes. These are typically measured on the fourth or fifth cycle. Although dominated by the contribution from capacitance, the Faradaic components are readily revealed and can be studied in greater detail by expanding the scale. Three of the ferredoxins display several distinctive couples, thus showing that there is multiple electron-transfer activity. The low potential at which a number of these couples occur (below -550 mV) shows that they are inaccessible to dithionite and hence may not be addressed by straightforward chemical titrations. Furthermore, in contrast to more conventional investigations of such strong reductants, a sufficiently anaerobic environment is easy to maintain. The first step in obtaining quantitative information from such results is to superimpose a baseline on the voltammogram. For a simple electrontransfer reaction that is not complicated by coupled chemistry, the baseline consists only of the capacitive components of the sweep. To a reasonable degree, the form of the baseline can be obtained by carrying out an identical voltammetric experiment on a freshly polished electrode placed directly into electrolyte solution. After taking the baseline into consideration,

490

PROBES OF METAL ION ENVIRONMENTS

T

b

T4.A

C

I

i

-1000

I

[

I

I

[

0

[18]

I

E /mY vs. S.H.E.

FIG. 4. Adsorbed film voltammetry of four ferredoxins: (a) Desulfovibrio africanus Fd III, 200 mV/sec (expanded view of oxidative sweep is also shown); (b) Azotobacter vinelandii Fd I, 20 mV/sec; (c) Thermodesulfobacterium commune Fd, 200 mV/sec; (d) Clostridium pasteurianurn Fd, 200 mV/sec. Films prepared from a coating solution containing 100 v.M ferredoxin, 2 mM neomycin, and 100 p.M EGTA (0.1 M NaCI, buffered at pH 7.4) were placed into an electrolyte solution at 0 ° also containing 2 mM neomycin and 100/zM EGTA (0.1 M NaC1, buffered at pH 7.4). Redox couples are labeled according to the discussion in the text.

values of E °', 8, AEp and the ratio ipc/ipa c a n each be determined, and the areas of the waves estimated. An illustration of the procedure is given in Fig. 4a, for the case of ferredoxin III (Fd III) from Desulfovibrio africanus. As isolated, this protein contains one [3Fe-4S] and one [4Fe-4S] cluster, and we refer to it as 7Fe-Fd III.17 As recorded in buffer-electrolyte containing EGTA, 17 F, A. Armstrong, S. J. George, R. Cammack, E. C. Hatchikian, and A. J. Thomson, Biochem. J. 264, 265 (1989).

[18]

VOLTAMMETRY OF REDOX-ACTIVE CENTERS

491

the ratio of areas under oxidative waves A', B', and C' is 1 : 1 : 2 [the prime notation refers to the fact that these are adsorbed redox couples; they may differ (usually marginally) in potential from the same couple measured in free solution]. 13'16Couple A' with ipc/ipa 1 and 8 = 100 mV conforms well to the prediction for a reversible one-electron process within a homogeneous noninteracting population of redox centers. The fact that AEp is less than 30 mV even at a scan rate of 500 mV/sec demonstrates that electron transfer is fast. The areas under the waves are in accord with values expected for about one electroactive monolayer of protein molecules. The component waves of couple B' are broader, indicative of interaction between redox centers or heterogeneity that might be due to varying microenvironments. Couple C' is clearly more complex since ipc/ipa is less than l, and 8 for the oxidative wave is approximately 50 mV, close to the predicted value for a concerted two-electron process. For Fd III, and for the 7Fe-ferredoxin from Azotobacter vinelandii (Fig. 4b), couples A' and B' have been established to coincide with couples A and B observed for the protein in bulk solution. These couples have been assigned as [3Fe-4S] l+/° and [4Fe-4S] 2+/1+ respectively, on the basis of coulommetric titration and electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies. 17-19 The ferredoxin from Thermodesulfobacterium commune is not well characterized at the time of writing, but its voltammetry is shown in Fig. 4c because it illustrates how the technique permits a complex situation to be visualized. The protein has been reported to contain a [3Fe-4S] cluster that takes up Fe(lI) to form [4Fe-4S]. 2° Following the information given by the voltammetry, EPR spectroscopic examinations showed that couples A~' and A2' are each associated with the normal 1+/0 reaction of [3Fe-4S] clusters (reaction I of Scheme I), A2' being of unusually high potential (180 mV), whereas only AI' is transformable by M(II) (see below). It seems, therefore, that the protein can accommodate two chemically distinct [3Fe-4S] clusters. The simplest voltammogram is displayed by the ferredoxin from Clostridium pasteurianum (Fig. 4d). The single pair of slightly distorted waves =

18 F. A. Armstrong, S. J. George, A. J. Thomson, and M. G. Yates, FEBS Lett. 234, 197 (1988). 19 S. E. Iismaa, A. E. V~izquez, G. M. Jensen, P. J. Stephens, J. N. Butt, F. A. Armstrong, and B. K. Burgess, J. Biol. Chem. 266, 21563 (1991). 10 B. Guigliarelli, P. Bertrand, C. More, P. Papavassiliou, E. C. Hatchikian, and J. P. Gayda, Biochim. Biophys. Acta 810, 319 (1985).

492

PROBES OF METALION ENVIRONMENTS

[18]

arises because the two [4Fe-4S] 2+/1+ clusters in this protein have reduction potentials that are very similar in value. 2~ Couple C', as observed clearly for Fd III and for A z o t o b a c t e r ferredoxin (Fig. 4a,b), is particularly intriguing since the E °' value is pH dependent and very low (in the region o f - 7 0 0 to - 8 0 0 mV at pH 7). By comparing the area o f C ' waves with the area generated by the one-electron couple A', it can be determined that two electrons are transferred.16 F o r Fd III, this is most evident from the oxidation wave; the broadness of the reduction wave indicates some degree of additional complexity in this process. (On a cautionary note, current arising from an electron-transfer reaction that is dependent on a preceding or intermediary chemical process may not appear as a distinctive wave. This situation can usually be identified by varying the scan rate, as a slow scan will enable a chemical proess to remain kinetically effective and lead to observation o f a more regular waveform.) E v e n in the case o f A z o t o b a c t e r ferredoxin, for which couple C' lies very close in potential to couple B' (the [4Fe-4S] 2+/1+ cluster in this protein has an unusually low E °' value), the narrow shapes of the C' waves make its quantification quite easy. Furthermore, in the case of Fd III, couple C ' disappears in concert with couple A' when the [3Fe-4S] cluster is transformed into [M3Fe-4S] by uptake of M(II). 13 We have therefore suggested that it corresponds to the couple [3Fe-4S] °/2- with uptake o f H +, that is, generation of what is formally an all-Fe(II) cluster. Couples A' and C ' thus correspond to reactions 1 and (2 + 3) of Scheme I, whereas couple B' corresponds to reaction 9 with M = Fe and X = cysteine R S - . It is likely that the couple observed at very low potential for the ferredoxin from T. c o m m u n e is also associated with a [3Fe-4S] cluster since reaction with various M(I1) ions causes it to disappear in concert with couple AI'. Investigating Coupled Chemical Reactions That Are Slow on Voltammetric Time Scale. A good example of a coupled reaction that is slow on

the voltammetric time scale is given in Fig. 5, which shows successive voltammograms (positive scan direction only) of a film of Fd III following transfer to a pot containing Fe(II), Zn(II), or Cd(II)/3 Over several scans we observe a decrease in the amplitudes of couples A' and C ' and appearance of a new couple that we term D'. Focusing first on the reaction with Fe(II), it was shown by coulometric studies and product analysis by EPR and MCD studies that the [3Fe-4S] ° cluster takes up Fe(II) to form a second [4Fe-4S] 2+ cluster. 22 This is reaction 5 of Scheme I. This new 21E. T. Smith, D. W. Bennett, and B. A. Feinberg, Anal. Chim. Acta 251, 27 (1991). 22S. J. George, F. A. Armstrong, E. C. Hatchikian, and A. J. Thomson, Biochem. J. 264, 265 (1989).

[18]

V O L T A M M E T ROF Y REDOX-ACTIVE CENTERS

493

cluster has unusual magnetic properties, being a S = a system in the reduced state, and necessarily having noncysteinyl ligation, presumably in the position of the new Fe subsite. The reduction potential of the new cluster (couple D', reaction 9) is very similar to that of the indigenous, stable [4Fe-4S] z+/l+ cluster; hence, it appears as an increase in the amplitude of couple B'. Similar products (with different reduction potentials, however) were proposed for the reactions with Zn(II) and Cd(II). 13 This experiment provides a simple test for detecting redox-linked transformations of clusters. Similar experiments with A z o t o b a c t e r ferredoxin have shown that no such facile transformations occur. On the other hand, experiments with T. c o m m u n e ferredoxin reveal easily that part of the [3Fe-4S] cluster population (AI' along with the broad couple at very low potential, but not A2') transforms readily. 23 In such a case the voltammetric data are analyzed as follows. 13Inspection of the final voltammogram shows that the area of D' is equal to the area of the initial wave A' before the reaction. This demonstrates 1 : 1 interchange of one-electron couples. By vertically displacing successive voltammetric sweeps, the course of the transformation can be monitored; in this case a plot of log[A/] (the difference in current as measured for two potential values) versus time is linear and has an identical slope for the attenuation of couples A' and C' and the increase in amplitude of couple D'. This establishes that the reaction conforms to first-order kinetics and that the three processes are interrelated. However, one cannot determine real rate constants in this way because the rate of interconversion among centers may depend critically on oxidation level. In this case, for example, formation of [4Fe-4S] clusters occurs only when the [3Fe-4S] cluster is in the " 0 " oxidation level. This can be demonstrated readily by introducing a film of Fd III into M(II) at 0 mV, holding at this potential for around 5 min, then rapidly cycling once. The voltammogram then observed is unchanged from that of the 7Fe form. In contrast, if the coated electrode is introduced at a potential of -250 mV (i.e., switching the [3Fe-4S] cluster to the " 0 " oxidation level) and held for several seconds, a single cycle reveals that reaction has occurred. Given the importance of maintaining potential control over the various redox centers, we can observe the ease with which this requirement can be achieved with adsorbed protein voltammetry. For example, if we wish to study reaction 5 and determine the equilibrium constants for various M(II) ions, it is essential to apply a potential that maintains [3Fe-4S] at the " 0 " oxidation level while maintaining [M3Fe-4S] as " 2 + . " This effectively isolates the chemical equilibrium that is of interest. The proce23 j. N. Butt, F. A. Armstrong, J. Breton, and A. J. Thomson, unpublished observations (1992).

494

PROBES OF METAL ION ENVIRONMENTS

[18]

t Z 1 pA Fe

I 1 I i

Cycle number

zn

~ 2 ',~

II

1I

-4C

•

i

1

2

i

.

i

$1' Cd

1

4 I I ! I

o -2 I

.','.; ..'

I I

-1000

I

]

I

1

-500

I

i

l

I

I

0 E/mV vs. SHE

•

3 4 5 Cycle number

Cycle number

•

•

i

6

j

7

[18]

VOLTAMMETRY OF REDOX-ACTIVE CENTERS

495

dure is straightforward for reactions that are slow by comparison with voltammetric scan rates. Using our described reactions as examples, the 7Fe ferredoxin-coated electrode is introduced to a solution containing a known concentration of M(II) at a " b a s e " potential that is midway between the E °' values of reactions 1 and 9. Poising the electrode at this potential precisely defines the correct oxidation states of the clusters involved. After stirring the solution for sufficient time to allow equilibrium to be established, the status of the clusters is determined from a rapid voltammetric cycle. The ratio of surface populations of reactant versus product at each concentration of M(II) can be determined by measuring the relative amplitudes of the respective voltammetric signals. (How this is done will depend on the wave shapes observed.) The dissociation constant K d is calculated by fitting data to Eqs. (1) or (2). Although {[M3Fe-4S] 2+} _ {i(II)} {[3Fe-4S] °} Kd ({[M3Fe-4S]2*}-~ = log{M(II)} - log K d l o g \ {[3Fe_4S]0} ]

(1) (2)

the scatter of data may be large, measurements are rapid, and the sample economy means that experiments can be repeated a large number of times where necessary. The system can be tested by varying the " b a s e " potential; for example, it is possible to check that the equilibria being measured are valid properties of the protein and are not influenced by changes in the electrical properties of the electrode-solution interface. With the above procedure we were able to measure the affinity of the [3Fe-4S] ° cluster for various M(II) ions without interference from coupled redox reactions. Results showed that for Fd III, the binding of M(II) to [3Fe-4S] ° runs according to the Irving-Williams series, that is, Cd(II) > Zn(II) >> Fe(II). Although the identity of the noncluster ligand(s) to M is

FIG. 5. (Left) Oxidative s c a n s (successive cycles) of a film of 7Fe-ferredoxin III on transfer to solutions containing M(II) ions. (Right) Corresponding semilog plots showing time c o r r e s p o n d e n c e of appearance of w a v e s D' and disappearance of w a v e s A ' . T h e potential was held briefly at approximately +50 m V prior to c o m m e n c e m e n t of scanning at 470 m V / s e c (temperature 0°). E a c h determination involved m e a s u r e m e n t of the difference in current at two potentials as indicated. T h e s e are as follows. Fe 2+ (300/xM): + , i-129 rnV -- i-229 mY, couple A ' ; &, i_393mV - i-250 my, couple D ' ; O, i_651 m v - i-554 mv, couple C ' . Zn 2+ (10 /.LM): + , L_129mv - i-229mV, couple A ' ; 0 , i_48,1mv - i_570mv, couple D ' ; O, i_655 mv - i_579mV, couple C ' . Cd 2+ (10 p.M): + , i_129mv - i_229mv, couple A ' ; II, i_64]rnv i-56s mv, combination of couples C ' and D ' . [Reprinted with permission from J. Am. Chem. Soc. 113, 6663 (1991). Copyright 1991 A m e r i c a n Chemical Society.]

496

PROBES OF METAL ION ENVIRONMENTS

[18]

yet to be established, this study has indicated the biological feasibility of F e - S clusters containing Zn. Investigating Coupled Chemical Reactions That Are R a p i d on Voltammetric Time Scale. If a redox-linked chemical reaction occurs on a time scale that is rapid compared to the voltammetric scan rate, it is not possible to separate the coupled process from the electron transfer. A simple case to consider is the rapid and reversible binding of a reagent to a center in either of two oxidation states. If the affinity for the reagent differs between oxidation states, then a single redox couple is observed with an E °' value that is dependent on the concentration of reagent. Because equilibrium is always established within the time scale of measurement, the result is similar to that which would be obtained by potentiometry. An example of such a system is given by the reaction of the 3Fe cluster of Fd III with a single thallium(l) ion.14 This reaction can be described by a thermodynamic cycle, in this case a " b o x " composed of reactions 1, 5, 8, and 4 of Scheme I. With TI(I) concentrations over the range 10-5 to 10-1 M, and employing scan rates up to 500 mV/sec, the waves arising from couple A' remain unchanged in shape or size as compared to the initially formed film, but the observed reduction potential shifts to a more positive value E°'obs. Equilibrium constants for reactions 4 and 5 are obtained by fitting data to Eq. (3): E°'obs =

E °' + (2.3 R T / F ) log{1 + [Q]/Kdred)/(1 + [Q]/Kd°X)}

(3)

in which Q represents the reagent that is binding to the cluster. A graph of E°'obs against log[Q] is sigmoidal with asymptotic limits at the E °' values for the two isolated couples, in this case reactions 1 and 8. The reduction potential for reaction 8 is now obtained from Eq. (4): E°'~s) = E°'~1) + (2.3 R T / F )

log(Kd°X/Kdred)

(4)

For TI(I) binding to the [3Fe-4S] cluster of Fd III, at an ionic strength of 0.5 M, such an analysis showed Kdred = 1.5 /xM, Kd°x = 34 mM, E °' = -177 mV, and E°'xl = +81 mV. The results demonstrate the point (to be illustrated further below) that even weak binding of reagents to an active site is detectable by the voltammetric method. On the basis of these data, a solution sample of oxidized Fd III (i. e., with [3Fe-4S] 1+) containing an appropriately high concentration of TI(I) was prepared for EPR spectroscopy. The resulting spectrum showed that the g = 2.01 signal characteristic of the species [3Fe-4S] 1+ had been replaced by a rhombic siknal, thus supporting the proposal that TI(I) interacts directly with the [3Fe-4S] core. The observation of near-ideal waveform even at quite high scan rates shows that entry and release of TI(I) are very rapid.

[18]

VOLTAMMETRY OF REDOX-ACTIVECENTERS

-l-o.1.A_ ~ ~ ~D' '-

a

lo., A

b

-

497

-

I

I

I

I

-800 -600 -400 -20o

E /mV vs. S.H.E.

FIG. 6. Adsorbed film voltammetry of (a) D. africanus 8Fe-FdlII, 10 mV/sec, 100/~M Fe(II). The film was transferred into a solution containing Fe(II) and 347 mM mercaptoethanol and scanned at (b) 10, (c) 200, and (d) 500 mV/sec. The coating solution was as described in Fig. 4. The electrolyte solution contained 2 mM neomycin, 0.2 M NaC1, buffered at pH 8, 0°. Redox couples are labeled according to the discussion in the text.

Extracting Kinetic Information. A f u r t h e r e x a m p l e illustrates h o w kinetic d a t a can be o b t a i n e d if rates o f the c o u p l e d p r o c e s s e s are c o m p a r a b l e to the v o l t a m m e t r i c scan rate. T h e case we h a v e c h o s e n again involves F d I I I , and w e c o n s i d e r the reversible binding o f a ligand, ethanol 2thiolate, to the t r a n s f o r m e d [ 4 F e - 4 S ] cluster in b o t h the 2 + and 1 + oxidation levels. 24 As a t h e r m o d y n a m i c cycle, the situation is that o f a b o x c o m p r i s i n g r e a c t i o n s 9, 13, 16, and 12 o f S c h e m e I. T o study this s y s t e m , a film o f the 7 F e - f e r r e d o x i n is first t r a n s f o r m e d into the 8Fe f o r m as d e s c r i b e d a b o v e , and the e l e c t r o d e is t h e n transferred to solutions containing v a r i o u s c o n c e n t r a t i o n s o f m e r c a p t o e t h a n o l o v e r the p H range 8 - 8 . 5 . Results are s h o w n in Fig. 6. U n d e r slow scan conditions (Fig. 6b), the 24j. N. Butt, A. Sucheta, F. A. Armstrong, J. Breton, A. J. Thomson, and E. C. Hatchikian, J. Am. Chem. Soc. 115, 1413 (1993).

498

PROBES OF METAL ION ENVIRONMENTS

[18]

single pair of waves composed of overlaying couples B' and D' splits into two, with one remaining at around -390 mV and the other shifting to a lower potential E°'obs, the value of which is dependent on the calculated concentration of thiolate anion. No changes are observed if a film of the original 7Fe-Fd III is scanned in the mercaptoethanol solution. It may therefore be deduced that it is the transformed [4Fe-4S] cluster (not the indigenous [4Fe-4S] cluster) which reacts with thiolate. The variation in observed E °' value with thiolate concentration is of the same form as Eq. (3). If the voltammetry is performed at a fast scan rate (in this case typically 500 mV/sec or higher), the position of the new couple becomes insensitive to thiolate concentration, whereas the oxidation wave is observed to be smaller than the reduction component. This occurs because the scan rate is now sufficiently fast to isolate the reversible redox couple (F') arising from the thiolate-ligated cluster, in this case reaction 16 of Scheme I. The distortion now observed on the low-potential side of the oxidation wave of couple B' arises because reoxidation of the remaining labile cluster population is made more favorable by rapid recombination of the product [4Fe-4S] 2+ with thiolate. Combining Eqs. (3) and (4), the equilibrium constants for reactions 12 and 13 can be determined. From these studies we were able to determine that binding of thiolate to the oxidized cluster [ K d ( L ) °x = 28/zM] is much stronger than binding to the reduced cluster [KarL)red = 97 mM]. This feature manifests itself clearly in the form of the significant negative shift in reduction potential from -396 mV (D') to -585 mV (F'). Interestingly, we found that we could not prepare a sample of the thiolate-ligated reduced cluster in solution for spectroscopic studies. This reflects the need for an intolerably high concentration of mercaptoethanol coupled with the requirement for a very low potential. As a voltammetric transient, however, the "virtual" existence of the thiolate-ligated reduced cluster is clearly demonstrated. With an intermediate scan rate, the kinetics of reactions 12 and 13 are revealed more closely. A third oxidation wave ( * , Fig 6c) is now clearly observed, the position and size of which vary with scan rate and thiolate concentration. As the thiolate concentration increases, this wave shifts to more negative potential but becomes smaller by comparison with oxidation wave F'. As the scan rate is increased, the wave becomes smaller and shifts to higher potential, merging with oxidative wave B'. Further analysis of the voltammetry is now approached by computer simulation. In this case an iterative program has been used, based on an effectively constant concentration of thiolate maintained at the electrode surface by the large buffering capacity of the rapidly established thiol/thiolate equilibrium.

118]

499

V O L T A M M E T ROF Y REDOX-ACTIVE CENTERS 0.35 (0.25 k..

L)

0.15

O0 (/)

0.05

CO CO C"

-0.05

E

-0.25

'O

-~.~" -~

...'" ./~N/

-0.15

-0.35 -450

I

-350

I

-250

I

I

- 150

/X,E

-50

I

I

50

150

250

/ mV

FIG. 7. Simulated voltammogram showing behavior typical for a box composed of reactions 9, 13, 16, and 12 in Scheme I in which L reacts reversibly with [4Fe-4S] 2÷ and [4Fe-4S] 1÷ at an intermediate scan rate. Simulation is for experiments shown in Fig. 6 but with a different ligand concentration and scan rate v = 100 mV/sec. " O n " rate constants are 3.2 x 104 M -I sec -l for [4Fe-4S] 2÷ and 3.09 M -1 sec -1 for [4Fe-4S] l÷. " O f f " rate constants are 0.9 sec -1 for [4Fe-4S] 2÷ and 0.3 sec -l for [4Fe-4S] 1÷. The large on rate for the oxidized cluster gives rise to the wave marked with an * in Fig. 6c. Electrochemical rate constants for couples B', D', and F' are also varied to optimize the fit. The potential (AE) axis is referenced against the reduction potential of the transformed [4Fe-4S] couple (D') as measured in the absence of ligand. Values of the current are presented in dimensionless form {I/[n2F2oAF/(RT)]} (see Fig. 2 for meaning of terms). Component signals arising from couples B' (heavy dashed line), F ' (dotted line), and the highly distorted D' (light dashed line) reveal the nature of broadening observed for the total current (solid line) near the potential characteristic for the reduction peak F'.

The current at each value of applied potential is determined by applying the Butler-Volmer equation to each redox couple B', D', and F' and by considering the change in populations of clusters caused by ligand exchange. By generating simulated voltammograms based on varied rate constants for the processes involved, a best fit of kinetic constants can be found. Such rate constants (electrochemical as well as homogeneous) lead to the optimum reproduction of experimental traits (e.g., position and size of the waves) over the range of experimentally varied parameters such as reagent concentrations and scan rate. The result of one such simulation, displaying each component of the voltammogram, is shown in Fig. 7. It can be noted that coupled electron transfer reactions may

500

PROBES OF METAL ION ENVIRONMENTS

[18]

produce current contributions of sign contrary to simple expectation. For example, close inspection shows that one reaction yields a weak but noticeable reduction peak in the direction of increasing potential. From this particular set of experiments it was determined that the kinetic origin of the difference in affinities of [4Fe-4S] 2+ and [4Fe-4S] l+ clusters for thiolate (and hence also of the decrease in reduction potential in the presence of the ligand) lies in an approximately 104-fold increase in the rate of binding to the oxidized cluster. Concluding Remarks Although considerable effort may be required to establish conditions for obtaining a stable, electroactive film, the voltammetric approach can lead to the detection and clarification of chemistry that is not revealed by other methods. A wide spectrum of information on dynamic systems can be derived, ranging from a rapid "image" of the redox chemistry of centers in a protein to the determination of equilibrium and kinetic constants for coupled reactions. It permits an extensive exploration of reactivities with small amounts of material and is useful in the characterization of labile systems for which critical conditions must be met for preparation of spectroscopic samples. Acknowledgments We thank Dr. Edmond Bowden for communicatingresults prior to publication. This work has been supported by grants fromthe Exxon EducationFoundation,The Petroleum Research Fund administeredby the AmericanChemicalSociety,and the NationalScience Foundation (MCB-9118772).

[19]

ELECTROCHEMICAL STUDIES OF METALLOENZYMES

501

[19] D i r e c t a n d I n d i r e c t E l e c t r o c h e m i c a l I n v e s t i g a t i o n s of MetaUoenzymes

By H. ALLEN O. HILL and NICHOLAS I. HUNT Introduction It is now possible to investigate ~-4 virtually all redox metalloproteins by electrochemical methods. There is no difficulty in achieving the electrochemistry of small redox proteins such as cytochromes, ferredoxins, blue copper proteins, and flavodoxins. Direct electrochemistry proceeds without the need for an electron transfer shuttle, or mediator, between the redox center of the protein and the electrode. However, at most metal electrodes the presence of a promoter ~ is required. Such a compound binds to the electrode surface and, while not itself taking part in the electron transfer process, encourages electron transfer with the protein to proceed. Over the years, many promoters have been reported. 6'7 They are all hi- (or multi-) functional molecules of the type X - Y (Fig. 1): X is a substituent which allows binding to the metal electrode surface (e.g., a pyridyl, phosphine, sulfhydryl, or thioether); Y (e.g., a carboxyl or pyridyl group) interacts transiently with some part of the protein surface. The extension ( - ) should be such that it does not permit Y to bind to the electrode surface; it does not appear to matter6 whether it is aliphatic or aromatic in nature. There is some evidence 8'9 that really clean surfaces do not require promoters for the electrochemistry of some proteins to proceed. However, in most laboratory environments, traces of impurities are adsorbed at the electrode surface, and hence electron transfer of the protein or its ability to bind to the surface, or indeed both, is inhibited. i F. A. A r m s t r o n g , H. A. O. Hill, and N. J. Walton, Q. Rev. Biophys. 18, 261 (1986). 2 F. A. A r m s t r o n g , H. A. O. Hill, and N. J. Walton, Acc. Chem. Res. 21, 407 (1988). 3 F. A. A r m s t r o n g , Struet. Bonding (Berlin) 72, 137 (1990). 4 A. M. B o n d and H. A. O. Hill, Met. Ions Biol. Syst. 27, 431 (1991). 5 M. J. E d d o w e s and H. A. O. Hill, J. Chem. Soc., Chem. Commun., 771 (1977). 6 p. M. Allen, H. A. O. Hill, and N. J. Walton, J. Electroanal. Chem. 178, 69 (1984). 7 F. A. A r m s t r o n g , P. A. Cox, H. A. O. Hill, V. J. Lowe, and B. N. Oliver, J. Electroanal. Chem. 217, 331 (1987). 8 E. F. B o w d e n , F. M. Hawkridge, and H. N. Blount, J. Electroanal. Chem. 161, 355 (1984). 9 S..-C. Sun, D. E. Reed, J. K. Cullison, L. H. Rickard, and F. M. Hawkridge, Mikrochim. Acta 3, 97 (1988).

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993by AcademicPress, Inc. All rights of reproduction in any form reserved.

502

[19]

PROBES OF METAL ION ENVIRONMENTS

.s-~. /

"°°c~~s

HS__~~NH 2

/

Na2S .

/

/-ooo.

/ / /

/

/

/

/

/

. ~ - ~ ~)../-~

,

/ / /

$~COOH

,

sv

COOH

c~-a,0-c~ Lys-Gly-Cys

/ / /

FIG. 1. Structures and proposed surface conformations of surface modifiers for the promotion of protein electrochemistry.

[19]

ELECTROCHEMICAL STUDIES OF METALLOENZYMES

503

Techniques have been developed for elaborate modification of metal electrodes by multifunctional X - Y adsorbates to act as promoters: (1) If a di- or tripeptide is chosen to act as the promoter, normally with the sulfur of a cysteine as X, Y can be chosen so that a negatively charged group (as in a glutamate-containing peptide) or a positively charged group (as in a lysine-containing peptide) is present, the former "attracting" positively charged proteins, such as cytochrome c, and the latter negatively charged proteins, such as plastocyanin. (2) By choosing Y such that it can bind metal ions, for example, Mg 2+, Cr(NH3)63+, or even Pt(NH3)64+, negatively charged proteins such as plastocyanin, ferredoxin, or flavodoxin are encouraged to bind to the modified electrode surface long enough for electron transfer to occur. (3) Provided X is such that the molecule binds tightly to the electrode surface (e.g., a sulfhydryl or a phosphine), a monolayer of the promoter can be created, either by dipping the metal electrode in a solution of the promoter and then washing the electrode to remove unbound molecules or, for example, by electrochemically reducing a disulfide bond to induce binding to the metal electrode. With such a large selection of promoters, it is possible to achieve the electrochemistry of any small redox protein. None fail to give good electrochemistry, and results are often such that the half-wave potential corresponds to that assumed to be the thermodynamic potential of the protein. However, one must bear in mind that the experimental value reported is for the protein-promoter-electrode interaction; if this is significant, for example, when a metal ion is used as an ancillary promoter, then differences may be observed. Such redox potentials are not without interest: it may be that the redox potential of, for example, the complex l° of magnesium ions with plastocyanin, may provide a more meaningful description of the complex of the protein in the photosynthetic unit than the redox potential of the 'free' protein. It should be noted that the behavior of proteins at absolutely clean metal surfaces (a situation rarely encountered) is complex: transient short-lived electrochemistry may be observed in such systems. Irreversible adsorption on the metal surface with or without a marked change in potential frequently occurs. The purpose of promoters is to avoid such problems: the electrochemistry is well-behaved and reversible without the need to follow complex experimental procedures. Nonmetal electrodes can also be used in electrochemical studies of biological molecules. Perhaps the most extensive investigations have been made with graphite electrodes. It was found 1° that the relevant electro-

l0 F. A. Armstrong, H. A. O. Hill, B. N. Oliver, and D. Whitford, J. Am. Chem. Soc. 107, 1473 (1985).

504

PROBES OF METAL ION ENVIRONMENTS

[19]

chemistry at edge-plane pyrolytic graphite electrodes, with and without added metal ions (depending on the charge of the protein being studied), was well behaved. Edge-plane pyrolytic graphite has, at its surface, a variety of oxygenated functional groups including carboxylates. Depending on the state of oxidation of the freshly prepared surface, the electrochemistry of cytochrome c and, with added metal ions, plastocyanin, etc., is quasi-reversible. In an attempt to make use of better defined electrode surfaces, materials such as metallic ruthenium dioxide have also been employed with some success. 11 The results obtained with cytochrome c were similar to those reported at the same time as the first use of promoters, namely, tin-doped indium oxide, ~2 and followed up some time later by o t h e r s . 8,13

It would be misleading to give the impression that these results were straightforward. Of course there were, and still are, problems concerning the exact nature of the electrode surface. Attempts have been made to examine the detailed nature of the surfaces, for example, by ellipsometry TM and, more recently, by scanning tunneling microscopy 15; an exact description in atomic terms still eludes us. It appears the problems were, at one level, more mundane. Under a host of conditions, peak-shaped electrochemical responses could not be obtained. Often, when a promoter was used, rather " p o o r " electrochemistry resulted; moreover, when the electrode surface was modified in a variety of ways, the electrochemical results indicated poor electrochemistry when essentially no electrochemistry would have been expected. When the results were reassessed 4'i6-18 in terms of the behavior of the electroactive species at a microelectrode, that is, one having dimensions of the order of 1/.tm, they appeared consistent with rapid electron transfer rates. The difference was due to the effect II M. A. Harmer and H. A. O. Hill, J. Electroanal. Chem. 170, 369 (1984); M. A. Harmer, and H. A. O. Hill, J. Electroanal. Chem. 189, 229 (1985). 12 p. Yeh and T. Kuwana, Chem. Lett., 1145 (1977). 13 E. F. Bowden, F. M. Hawkridge, J. F. Chlebowski, E. E. Bancroft, C. Thorpe, and H. N. Blount, J. Am. Chem. Soc. 104, 7641 (1982). 14 D. Elliott, A. Hamnett, O. C. Lettington, H. A. O. Hill, and N. J. Walton, J. Electroanal. Chem. 202, 303 (1986). 15 A. J. Mayne, A. R. Avery, J. Knall, T. S. Jones, I. G. Blackham, L. Pinheiro, T. R. I. Cataldi, H. A. O. Hill, G. A. D. Briggs, J. B. Pethica, and W. H. Weinberg, J. Chem. Soc., Faraday Trans. in press (1993). 16 F. A. Armstrong, A. M. Bond, H, A. O. Hill, 1. S. M. Psalti, and C. G. Zoski, J. Phys. Chem. 93, 6485 (1989). 17 F. A. Armstrong, A. M. Bond, H. A. O. Hill, B. N. Oliver, and I. S. M, Psalti, J. Am. Chem. Soc. 111, 9185 (1989). i8 A. M. Bond, H. A. O. Hill, D. J. Page, N. J. Walton, and I. S. M. Psalti, Eur. J. Biochem. 191, 737 (1990).

[19]

ELECTROCHEMICAL STUDIES OF METALLOENZYMES

505

Radial Limit

/

--

f

,1 /

"\

"~"\ \

\

\

site destructionl [ sitegeneration

z

\

/

E~

\

sitedestruction1J sitegeneration

E Linear Limit

FIG. 2. Schematic representation of an electrode surface depicting the conversion of radial to linear diffusion as the density of surface electroactive sites increases.

of radial diffusion (Fig. 2) of the electroactive species to the microelectrode as compared to the normally observed linear diffusion at macroelectrodes. The cyclic voltammogram of proteins whose diffusion to a microelectrode is radial in manner i'esembles, albeit superficially, poor electrochemistry at a macroelectrode. Thus, it was suggested that, owing to the nonuniformity of an electrode surface, only an array of microscopically sized sites are actually electroactive to protein molecules, such that diffusion to these sites occurs in a radial manner. Detailed analysis of the experimental

506

PROBES OF METAL ION ENVIRONMENTS

[19]

results showed that the electrochemistry of the proteins corresponded, under all conditions, to situations in which the heterogeneous electron transfer rate was very fast. Another phenomenon that was elucidated by consideration of radial diffusion was the time dependence of the electrochemistry, whether in situations corresponding to small amounts of promoter on the electrode surface, or with mixtures of promoters, or indeed with the mixture of a promoter and an inactive adsorbate. Even the gradual addition of increasing amounts of an adjunct promoter, for example, Cr(NH3)63+, to a ferredoxin solution produced a time-dependent electrochemistry. Obviously this could be associated with the rearrangement of, for example, the Cr(NH3)63+-protein complex, but many of the changes are now thought to be due either to the rearrangement of the promoters on the electrode surface or to dissolution of the promoter followed by readsorption. In most cases, it is relatively easy to envisage how a mixture of promoter and inhibitor could, with time, rearrange to a set o f " i s l a n d s , " corresponding to an array of microelectrodes on the electrode surface. There are a few examples of situations where the reverse happens, ~9 that is, where two different promoters rearrange such that the electrochemistry of a protein alters from a response corresponding to an electrode consisting of an array of microelectrodes to that characteristic of linear diffusion to a macroelectrode. Many, if not all, redox proteins have no biological function on their own: they are associated with other redox proteins, assembled in electron transport systems in membranes or allied with redox enzymes in pursuit of some metabolic process. It therefore appeared sensible to attempt to investigate electrochemically protein-protein complexes. The systems studied 2° were the complexes formed between cytochrome c and plastocyanin and between cytochrome c and cytochrome bs. The availability of electrodes that were "selective" toward the electrochemistry of the given proteins, for example, edge-plane pyrolytic graphite for cytochrome c and gold coated with the tripeptide Cys-Lys-Cys for plastocyanin, enabled the behavior of the complexes to be understood. It appears that the protein which binds to the electrode surface, for example, cytochrome c at edgeplane pyrolytic graphite, acts as an adjunct promoter, holding plastocyanin or cytochrome b5 in such an orientation that electron transfer to the latter can occur. This is consistent with zinc-cytochrome c or indeed many proteins which have an overall charge opposite to that of plastocyanin, acting in the same manner. 19 H. A. O. Hill and G. A. Lawrance, J. Electroanal. Chem. 270, 309 (1989). 20 S. Bagby, P. D. Barker, L.-H. Guo, and H. A, O. Hill, Biochemistry 29, 3213 (1990).

[19]

ELECTROCHEMICAL STUDIES OF METALLOENZYMES

507

There is one important point that emerged from these and related studies. The structures of the complexes must be considered as dynamic; they move with respect not only to each other, but to the electrode surface. The initial description of electron transfer at the electrode envisaged a static arrangement of the protein, or proteins, at the surface but now one must consider a more mobile array of the protein(s) essentially moving in a lateral manner over the electrode surface until a configuration is reached where electron transfer to, or from, the surface is rapid. Such a dynamic view of the electrode-protein structure must involve motions within the protein. Indeed, recent studies on the electrochemistry of genetically engineered 2~ variants of cytochrome c and of its complexes with cytochrome b5 indicate that subtle structural changes within cytochrome c affect the electrochemistry. Such procedures, however, have not generally proved successful in the probing of metalloenzymes. This is essentially due to the same problems encountered in studying protein electrochemistry, namely, low diffusion coefficients of the biological molecule in aqueous solution as a result of the size of the molecules, which leads to low Faradaic currents due to the redox species; rapid and often irreversible adsorption of the molecule at the electrode surface; and a redox center "buried" within the protein and thus shielded for all but a few electrode-protein orientations. The electrochemical study of enzymes has proved more difficult since they are generally substantially larger and more flexible than proteins, and therefore each of the above problems is more apparent. In addition many enzymes are closely associated with membranes and possess highly lipophilic surfaces, which further hinders aqueous studies. Redox enzymes may be classified as being intrinsic or extrinsic in nature.: An extrinsic redox enzyme requires an associated redox protein (cofactor) as part of the electron transfer process, and therefore there must exist one or more sites for its binding at the enzyme surface (and correspondingly one or more electron transfer pathways through the enzyme between redox center and binding site). Such a redox site should enable interaction with an electrode surface to occur and thus facilitate the heterogeneous electron transfer. Therefore, in order to enable communication between an enzyme and the electrode, the surface of the latter must bind the enzyme and prevent denaturation. An intrinsic redox enzyme is one in which electron transfer associated with the catalyzed event is contained within the confines of the active site, that is, the enzyme lacks a "natural" long-range electron transfer pathway. Thus, achieving 21 A. Burrows, L.-H. Guo, H. A. O. Hill, G. McClendon, and F. Sherman, Eur. J. Biochem. 202, 543 (1991).

508

PROBES OF METAL ION ENVIRONMENTS

[19]

la r---

,re

J

|

FIG. 3. Conventional low-volume electrochemical cell. (a) Saturated calomel electrode as reference electrode. (b) Working electrode. (c) Platinum gauze counterelectrode. (d) Luggin capillary. (e) Potassium chloride solution (0.1 M). (f) Working solution (300/zl). heterogeneous electron transfer with an electrode may require that (1) the • site o f the catalytic reaction be close to the e n z y m e surface, (2) the e n z y m e be able to deform without losing its activity, (3) the electrode surface (with or without a promoter) " p r o j e c t " into the enzyme, or (4) electron transfer pathways be introduced by modification. Experimental Procedures The standard electrochemical technique, dc cyclic voltammetry, uses a three-electrode system and potentiostatic control. 22 The conventional electrochemical cell is shown in Fig. 3 and has a working volume o f 300/zl. The reference electrode generally consists of a saturated calomel electrode (SCE), and is housed in a separate compartment from the working solution, connected via a 0.1 mm Luggin capillary, which prevents mass transfer between the chambers. All solutions in the electrochemical cell must contain millimolar concentrations of an electrolyte, commonly potassium chloride, to act 22 as the main charge carriers in the system. In addition, the working solution generally contains millimolar concentrations o f a buffer to ensure p H stability. All electrolytes and substrates must be pure, and all solutions must be prepared with the use o f highpurity water (possessing a resistivity o f 18 MI~ cm). Solutions are degassed prior to electrochemical experimentation with argon or nitrogen to purge 22 Southampton Electrochemistry Group, "Instrumental Methods In Electrochemistry." Ellis Horwood, Chichester, 1985.

[19]

ELECTROCHEMICAL STUDIESOF METALLOENZYMES

509

oxygen from the system, which would otherwise result in a large reduction peak around -400 mV (versus SCE). In addition, a flow of the inert gas over the surface of the working solution during experimentation reduces the risk of airborne contamination to the system. A potential window is scanned between the working and reference electrodes, and the current passed between the working and counterelectrodes is monitored. The counterelectrode consists of a 1 cm 2 piece of platinum gauze; the working electrode (2 to 3 mm in diameter) is constructed by sealing the electrode material (e.g., a gold rod) within a Teflon or glass shroud with the use of an epoxy resin, such as Araldite. All working electrodes are polished with high-purity alumina or diamond paste (graded down to 0.03/xm particulate size) in order to achieve high surface homogeneity. In addition, when a metal electrode is to be used, electrochemical cycling in a 0.1 M inorganic acid, typically sulfuric acid, is usually undertaken prior to each experiment in order to remove 23 surface impurities. Slightly different procedures are employed when a microelectrode is used as the working electrode. A wire or fiber (typically 1 to 25 /zm in diameter) can be sealed directly into a glass shroud, without the requirement of an adhesive. Because a far smaller current is obtained at a microelectrode, there are severely reduced ohmic losses as a result, 24 and thus only a two-electrode configuration is required; current passed at a reference electrode would be negligible. The potential reference for the system may be provided by addition of a redox species possessing a precisely known redox potential at the termination of an experiment. A background cyclic voltammogram consists of a non-Faradaic current due to the capacitive nature of the system (Fig. 4a). On addition of a redox-active species, a Faradaic current is observed if electrochemical communication is achieved with this species (Fig. 4b). From the cyclic voltammograms of a redox-active species, thermodynamic information such as the reversible potential for the heterogeneous electron transfer process is obtained, and kinetic information such as the heterogeneous rate constants of electron transfer may be 22 calculated.

Indirect Enzyme Electrochemistry Indirect electrochemical communication between an electrode and a metalloenzyme may be achieved using an associated redox protein as a mediator, in other words, as an electron transfer shuttle between the 23j. p. Hoare, J. Electrochem. Soc. 131, 1808 (1984). 24S. Pons and M. Fleischmann,Anal. Chem. 59, 1391 (1987).

510

[19]

PROBES OF METAL ION ENVIRONMENTS

(-

...)

I

I

-0.2

I

0.2 ~A

I

0.0 Potential/V (SCE)

I

I

0.2

FIG. 4. Typical cyclic voltammograms (a) for non-Faradaic current of the electrolyte solution and (b) on addition of the redox-active species.

enzyme and the working electrode. Thus, the reduction of dioxygen by cytochrome oxidase may be observed owing to the catalytic regeneration of cytochrome c at a modified gold electrode. 25 A solution of I00/zM horse heart cytochrome c in a phosphate buffer, in the presence of dioxygen, exhibits a quasi-reversible electrochemical response at a bis(4-pyridyl) disulfide-modified gold electrode over scan rates of 1-200 mV sec -j (Fig. 5a). A plot of the peak current against v ~/2, where v is the scan rate (V sec-l), is linear, as defined by the Randles-Sev~ik equation for quasireversible systems. 22 On addition of 0.21 /zM cytochrome-c oxidase to the solution, a large current appears at reducing potentials (Fig. 5b) owing to the catalytic regeneration of the ferricytochrome by the enzyme: Cyt-c oxidase tea + 0 2 + 4H + ~ cyt-c oxidase °x + 2H20 kca, Cyt-c oxidase °x + cyt c(II) ~ cyt-c oxidase tea + cyt c(III) Cyt c(III) + n e - ---> cyt c(II)

(at the electrode)

(I) (2) (3)

Using the approach of Nicholson and Shain, 26the second-order homogeneous rate constant for the reaction between cytochrome c and cytochrome-c oxidase was calculated. From the theoretical working curve 26 relating i¢/i a to the dimensionless parameter (kcat/a) 1/2, a range of kcat/a 25 p. D. Barker, J. O. D. Coleman, H. A. O. Hill, N. J. Walton, and D. Whitford, Soc. Trans. 14, 130 (1986). 26 R. S, Nicholson and I. Shaln, Anal. Chem. 36, 706 (1964).

Biochem.

[19]

ELECTROCHEMICAL STUDIES OF METALLOENZYMES

511

0.5 NA I -0.1 0.0 0.1 0.2 Potential/V(SCE)

C

J FIG. 5. Cyclic voltammograms showing (a) direct electrochemistry of horse heart cytochrome c at a bis(4-bipyridyl) disulfide-modifiedgold electrode and (b) catalytic response on addition of cytochrome-c oxidase.

values for several potential scan rates can be determined, where ic is the catalytic current observed on addition of enzyme, io is the diffusion current, calculated from the reversible c y t o c h r o m e c system [Eq. (3)], kcat is the pseudo-first-order rate constant, and a equals nFv/RT, where F is the Faraday constant, R the universal gas constant, and T the temperature (K). The calculated values of kcat/a are then plotted against v -1, for a range of cytochrome-c oxidase concentrations, each gradient,providing an estimate for kcat for that e n z y m e concentration. Finally, the gradient from a plot of k~at against e n z y m e concentration provides the secondorder homogeneous rate constant for the reaction between c y t o c h r o m e c and c y t o c h r o m e - c oxidase. The value obtained, 3 × 106 M - I sec-i, agreed well with values previously obtained from stopped-flow experiments. An alternative, and theoretically more rigorous, approach to calculating homogeneous second-order rate constants has been developed 27 by 27p. N. Bartlett, P. Tebbutt, and R. G. Whitaker, Prog. React. Kinet. 16, 55 (1991).

512

PROBES OF METAL ION ENVIRONMENTS

[19]

TABLE I SURVEY OF METALLOENZYME ELECTROCHEMISTRY

Metal/active center

Enzyme Alcohol dehydrogenase

Zn

Alcohol dehydrogenase CO acceptor oxidoreductase CO dehydrogenase Cytochrome-c oxidase Cytochrome-c peroxidase Enoate reductase Flavocytochrome b 2 (lactate dehydrogenase)

Heme/PQQ b Fe-S/Mo Fe-S/Ni Heme/Cu Heme Fe-S/FAD b Heme/FMN b

Flavocytochrome c552

Heme/FAD

Galactose oxidase D-Gluconate dehydrogenase

Cu Fe-S/heme/FAD

Hydrogenase

Fe-S

Laccase

Cu

Lysyl oxidase Nitrate reductase Nitrite reductase p-Cresol methylhydroxylase

Cu Heme/Mo/FAD Cu Heme/FAD

Peroxidase

Heme

Succinate dehydrogenase Sulfite oxidase Xanthine oxidase

Fe-S/FAD Mo/heme Fe/Mo/FAD

Nature of electrochemical response a Adsorbed, mediated, s Adsorbed, direct, s Adsorbed, direct, s Ferrocene mediated, s Adsorbed, direct, ns Cytochrome c mediated, s Direct, s Mediated, s Ferrocene or cytochrome c mediated Adsorbed, mediated, s Immobilized, direct, s Direct, ns, catalytic with substrate Mediated, s Adsorbed, mediated, Adsorbed, direct, s Adsorbed, mediated, s Adsorbed, direct, ns Mediated, s Adsorbed, direct, ns, catalytic with substrate Adsorbed, direct, s Direct, ns Mediated, s Direct, s Azurin mediated, s Direct, ns, catalytic with substrate Ferrocene mediated, s Adsorbed, mediated, s Mediated, ns Adsorbed, direct, s Adsorbed, direct, s Mediated, s Ferrocene mediated, s Immobilized, mediated, s Immobilized, direct, s

Refs. 30, 31 32 33 34 35 25, 36 37-39 40 41 30, 42, 43 44 45 46, 47 48 33, 49 50, 51 35 43, 52 53, 54 55 56 57 58 59 60 61 30, 42, 62-64 65 66-73 74 75, 76 77 30, 78-81 82, 83

a Substrate required (s)/not required (ns) for electrochemical response. b pQQ, Pyrroloquinoline quinone; FAD, flavin adenine dinucleotide; FMN, llavin mononucleotide.

[19]

ELECTROCHEMICAL STUDIES OF METALLOENZYMES

513

Bartlett et al. Their method takes into consideration the enzyme-substrate reaction, in addition to the mediator-enzyme reaction, by the introduction of Michaelis-Menten kinetics to provide the first-order rate constant for the regeneration of the enzyme by substrate. With the assumption that sufficient substrate is present so that its concentration gradient across the diffusion layer at the electrode is negligible, a set of second-order differential equations can be produced, describing the transport and kinetics of the various species in the system. A number of approximate analytical solutions are then derived, each corresponding to different concentrations of enzyme, mediator, and substrate and to different relative rates of the enzyme-mediator and enzyme-substrate reactions. Thus, mediator, enzyme, and substrate titrations are performed under electrochemical conditions in order to determine what range of these concentrations result in the case that is required for measurement of the desired variable. The kinetics of the mediated electrochemical enzyme system can then be derived with ease, negating the need to produce a large number of plots as in the application of the Nicholson and Shain approach. A cofactor-mediated enzyme electrochemical system is substantially simplified if a low molecular weight species is used to replace the redox protein as the mediator, with the constraint that rapid heterogeneous electron transfer kinetics with the enzyme and the electrode are retained. In addition, an ideal mediator should require a low overpotential to regeneration and should be stable with respect to pH, temperature, redox state, and oxygen. The organic dyes frequently used in spectrophotometric studies are generally unsuitable for electrochemical work owing to their readiness to autoxidize, their instability on reduction, and a frequent pHdependent redox potential. In enzyme studies the use of a ferrocene as an electrochemical mediator has received much attention2s'29 (and see Table I), 30-83 since its first use in mediating the electrochemically controlled 28 A. E. G. Cass, G. Davis, M. J. Green, and H. A. O. Hill, J, Electroanal. Chem. 190, 117 (1985). 29 E. Liaudet, F. Battaglini, and E. J. Calvo, J. Electroanal. Chem. 293, 55 (1990). 30 j. j. Kulys, Biosensors 2, 3 (1986). 31 H. Yamanaka and M. Mascini, Anal. Lett. 25, 983 (1992). 32 S. Miyamoto, T. Murakami, A. Saito, and J. Kimura, Biosens. Bioelectron. 6, 563 (1991). 33 T. Ikeda, S. Miyaoka, F. Matsushita, D. Kobayashi, and M. Senda, Chem. Lett. 5, 847 (1992). 34 A. P. F. Turner, W. J. Aston, I. J. Higgins, J. M. Bell, J. Colby, G. Davis, and H. A. O. Hill, Anal. Chim. Acta 163, 161 (1984). 35 E. T. Smith, S. A. Ensign, P. W. Ludden, and B. A. Feinberg, Biochem. J. 285, 181 (1992). 36 W. J. Albery, A. E. G. Cass, and Z. X. Shu, Biosens. Bioelectron. 5, 379 (1990). 37 F. A. Armstrong and A. M. Lannon, J. Am. Chem. Soc. 109, 7211 (1987). 38 R. M. Paddock and E. F. Bowden, J. Electroanal. Chem. 260, 487 (1989).

514

PROBES OF METAL ION ENVIRONMENTS

[19]

catalytic oxidation of glucose by the enzyme glucose oxidase. 84 Ferrocene is stable in both redox states in aqueous solution, possesses a pH-independent redox potential (Ev2 = + 165 mV versus SCE), is only slowly reactive with oxygen, and shows rapid reversible electrochemistry at an electrode. Most significantly, a large number of ferrocene derivatives can be readily

39 H. Assefa and E. F. Bowden, Biochem. Biophys. Res. Commun. 139, 1003 (1986). 4o H. Simon, J. Bader, H. GOnther, S. Neumann, and J. Thanos, Angew. Chem., Int. Ed. Engl. 24, 539 (1985). 41 A. E. G. Cass, G. Davis, H. A. O. Hill, and D. J. Nancarrow, Biochim. Biophys. Acta 828, 51 (1985). 42 j. j. Kulys and A. S. Samalius, Bioelectrochem. Bioenerg. 10, 385 (1983); J. J. Kulys, A. S. Samalius, and G. J. S. Svirmickas, FEBS Lett. 114, 7 (1980). 43 N. K. (~6nas, A. K. Pocius, and J. J. Kulys, Bioelectrochem. Bioenerg. 12, 583 (1984). 44 S. L. Staskeviciene, N. K. (~6nas, and J. J. Kulys, Anal. Chim. Acta 243, 167 (1991). 45 L.-H. Guo, H. A. O. Hill, D. J. Hopper, G. A. Lawrance, and G. S. Sanghera, J. Biol. Chem. 265, 1958 (1990). 46 j. M. Dicks, W. J. Aston, G. Davis, and A. P. F. Turner, Anal. Chim. Acta 182, 103 (1986). 47 p. D. Hale and T. A. Skotheim, Synth. Met. 28, C853 (1989). 48 T. lkeda, K. Miki, F. Fushimi, and M. Senda, Agric. Biol. Chem. 51,747 (1987); T. Ikeda, M. Miki, F. Fushimi, and M. Senda, Agric. Biol. Chem. 52, 1557 (1988). 49 T. lkeda, F. Fushimi, K. Miki, and M. Senda, Agric. Biol. Chem. 52, 2655 (1988). 50 M. R. Tarasevich, Bioelectrochem. Bioenerg. 6, 587 (1979). 51 H. A. O. Hill and I. J. Higgins, Philos. Trans. R. Soc. London 302A, 267 (1981). 52 V. T. Taniguchi, B. G. Malmstr6m, F. C. Anson, and H. B. Gray, Proc. Natl. Acad. Sci. U.S.A. 79, 3387 (1982). 53 M. R. Tarasevich, A. I. Yaropolov, V. A. Bogdanovskaya, and S. D. Varfolomev, Bioelectrochem. Bioenerg. 6, 393 (1979). 54 C.-W. Lee, H. B. Gray, F. C. Anson, and B. G. MalmstrOm, J. Electroanal. Chem. 172, 289 (1984). 55 I. V. Berezin, V. A. Bogdanovskaya, S. D. Varfolomeev, M. R. Tarasevich, and A. I. Yaropolov, Dokl. Akad. Nauk S S S R 240, 615 (1978); A. I. Yaropolov, B. Malovik, S. D. Varvolomeev, and I. V. Berezin, Dokl. Akad. Nauk S S S R 249, 1399 (1979). 56 K. Govindaraju, B. U. Nair, T. Ramasami, and D. Ramaswamy, J. lnorg. Biochem. 29, 111 (1987). 57 C. J. Kay, L. P. Solomonson, and M. J. Barber, Biochemistry 30, 11445 (1991). 5a Z. H. L. Abraham, R. Early, H. A. O. Hill, D. J. Lowe, P. de Oliveira, and B. E. Smith, in preparation (1993). 59 H. A. O. Hill, 13. N. Oliver, D. J. Page, and D. J. Hopper, J. Chem. Soc,, Chem. Commun., 1469 (1985). 60 L.-H. Guo, H. A. O. Hill, G. A. Lawrance, and G. S. Sanghera, J. Electroanal. Chem. 266, 379 (1989). 61 j. E. Frew, M. A. Harmer, H. A. O. Hill, and S. I. Libor, J. Electroanal. Chem. 201, 1 (1986). 62 G. J. Moody, G. S. Sanghera, and J. D. R. Thomas, Analyst 112, 65 (1987). 63 j. j. Kulys and R. A. Vidziunaite, Anal. Lett. 16, 197 (1983). T. Tatsuma, Y. Okawa, and T. Watanabe, Anal. Chem. 61, 2352 (1989). 65 V. J. Razumas, A. V. Gudavi~ius, and J. J. Kulys, J. Electroanal. Chem. 151, 311 (1983); V. J. Razumas, A. V. Gudavi~ius, and J. J. Kulys, J. Electroanal. Chem. 198, 81 (1986).

[19]

ELECTROCHEMICAL STUDIES OF METALLOENZYMES

515

synthesized, with substituent groups being easily altered such that interactions with a particular enzyme may be encouraged. Direct Electrochemistry The first reports of the direct electrochemistry of enzymes involved studies with flavoenzymes. However, because in most cases the prosthetic group is not covalently bound to the protein, and is often observed to dissociate, especially at an electrode surface, it is likely that it acts as a mediator in the electron transfer process. Similarly, some reports 7s suggested that direct electron transfer of a number of enzymes had been observed at conducting organic salt electrodes, such as tetrathiafulvalinium-tetracyanoquinodimethanide (TTF-TCNQ). It has now been generally accepted that the organic groups of the electrode possess sufficient solubility for slight dissolution to occur, and the resulting enzyme electro-

66 j. j. Kulys, V.-S. A. Laurinavi~ius, M. V. Pesliakiene, and V. V. Gurevi~iene, Anal. Chim. Acta 148, 13 (1983). 67 T. Tatsuma and T. Watanabe, Anal. Chim. Acta 242, 85 (1991). 68 T. Tatsuma and T. Watanabe, J. Electroanal. Chem. 310, 149 (1991). 69 H. Iwai and S. Akihama, Chem. Pharm. Bull. 34, 3471 (1986). 7o H. Durliat, A. Courteix, and M. Comtat, Bioelectrochem. Bioenerg. 22, 197 (1989). 71 j. j. Kulys and R. D. Schmid, Bioelectrochem. Bioenerg. 24, 305 (1990). 72 U. Wollenberger, V. Bogdanovskaya, S. Bobrin, F. Scheller, and M. R. Tarasevich, Anal. Left. 23, 1795 (1990). 73 j. Zhao, R. W. Henkens, J. G. Stonehuerner, J. P. O'Daly, and A. L. Crumbliss, J. Electroanal. Chem. 327, 109 (1992); J. G. Stonehuerner, J. Zhao, J. P. O'Daly, A. L. Crumbliss, and R. W. Henkens, Biosens. Bioelectron. 7, 421 (1992). 74 A. Sucheta, B. A. C. Ackrell, B. Cochran, and F. A. Armstrong, Nature (London) 356, 361 (1992). 75 p. A. Nader, S. S. Vives, and H. A. Mottola, J. Electroanal. Chem. 284, 323 (1990). 76 L. A. Coury, Jr., B. N. Oliver, J. O. Egekeze, C. S. Sosnoff, J. C. Brumfield, R. P. Buck, and R. W. Murray, Anal. Chem. 62, 452 (1990); L. A. Coury, Jr., R. W. Murray, J. L. Johnson, and K. V. Rajagopalon, J. Phys. Chem. 95, 6034 (1991). 77 A. E. G. Cass, G. Davis, M. J. Green, and H. A. O. Hill, J. Electroanal. Chem. 190, 117 (1985). 78 W. J. Albery, P. N. Bartlett, M. Bycroft, D. H. Craston, and B. J. Driscoll, J. Electroanal. Chem. 218, 119 (1987). 79 R. M. Ianniello, T. J. Lindsay, and A. M. Yacynych, Anal. Chem. 54, 1980 (1982). so K. McKenna and A, Brajter-Toth, Anal. Chem. 59, 954 (1987). el H. Okuma, H. Takahashi, S. Sekimukai, K. Kawahara, and R. Akahoshi, Anal. Chim. Acta 244, 161 (1991). 82 E. Watanabe, H. Endo, T. Hayashi, and K. Toyama, Biosensors 2, 235 (1986). 83 O. Doblhoff-Dier and G. A. Rechnitz, Anal. Lett. 22, 1047 (1989). 84 A. E. G. Cass, G. Davis, G. D. Francis, H. A. O. Hill, W. J. Aston, I. J. Higgins, E. V. Plotkin, L. D. L. Scott, and A. P. F. Turner, Anal. Chem. 56, 667 (1984).

516

PROBES OF METAL ION ENVIRONMENTS

[19]

chemical reaction takes place via a mechanism involving heterogeneous catalysis by the organic salt, as originally proposed by Kulys. 3°'85Probably the first genuine studies of the direct electrical communication with an enzyme involved the copper-containing laccase. 53,54 Lee et al. adsorbed fungal laccase A from Polyporous versicolor directly onto pyrolytic edgeplane graphite electrodes and observed the direct electroreduction ofdioxygen, catalyzed by the enzyme. A reversible electrochemical response for the adsorbed enzyme, in the absence of dioxygen, was produced in a solution saturated with either 2,9-dimethylphenanthroline or 4,4'-bipyridine acting as promoters. As discussed in the introduction, direct electrochemistry of a metalloenzyme may be encouraged with the use of promoters, via the formation of favorable electrostatic interactions between the enzyme and the electrode surface. For example, Armstrong described the electrochemical reduction of hydrogen peroxide, catalyzed by cytochrome-c peroxidase (CCP). 37 The electrode reaction was promoted by the addition of an aminoglycoside. The aminoglycosides are a family of bactericidal antibiotics that are both water soluble and stable in solution (Fig. 6). They consist of a hexose nucleus with amino sugars attached by glycosidic linkages, resulting in molecules possessing spatially arranged NH3 + functionalities on a quasirigid skeleton. Cytochrome-c peroxidase is a b-type heme-containing metalloenzyme that utilizes cytochrome c2 as an electron donor. A number of acidic (CO2-) residues are present on the surface of CCP, and it was shown that the amine functionalities of the aminoglycosides neomycin and gentamycin readily interact with the acidic groups. In solution a film formed at the surface of an edge-plane graphite electrode, approaching monolayer coverage of this enzyme-promoter complex. Thus, the aminoglycosides promoted the nondenaturative adsorption of the negatively charged CCP at a similarly charged electrode. On addition of hydrogen peroxide to the solution a large catalytic current was passed at the working electrode due to the reduction of the peroxide by the enzyme. Paddock and Bowden have observed the catalytic reduction of the peroxide by CCP adsorbed directly onto edge-plane graphite electrodes, without the presence of promoters. 38 Many reports have been published concerning electrochemical communication with peroxidases (see Table I). Much of the interest lies in the field of biosensors and the use of peroxidase as a peroxide sensor. Taken a stage further, Kulys et al. adsorbed horseradish peroxidase (HRP) directly onto SnO electrodes 66 before addition of a further enzyme which in the presence of substrate produced a peroxide as one of the products. In this s5 j. j. K u l y s , Enzyme Microb. Technol. 3, 344 (1981).

[19]

ELECTROCHEMICAL STUDIES OF METALLOENZYMES

517

Ho O,

a

NH2

HO - - ~ 7 ~ ' ~

HO

.o O

HO

0

H~O~NH2~__O_~

OH

NH2 b

II OH H2NCH~._~~

OH

CH3

e

NH

OH

0

NH~

O ~ O ~NHCH~)H H OH NH2 C

NH2

H O ~

NH21 ~ 2

NH

0'~'~/l~n2

2

0-~/NH

HO

....

0 --~OH ~''v

2

HO _._.~..7 ~--.v

HO HO

OH

FIG. 6. Members of the aminoglycoside family: (a) glucosamine, (b) dihydroxystreptomycin, (c) neamine, (d) neomycin, (e) gentamycin, (f) ribostamycin.

518

PROBES OF METAL ION ENVIRONMENTS

[19]

manner xanthine oxidase,62.66 uricase,67 and alcohol dehydrogenase6s have been coimmobilized with HRP to act as sensors for their respective substrates. The great interest in the application of enzymes in the production of sensor devices 86 has provided much of the impetus for the advances that have been made 87,ss in the immobilization of enzymes at an electrode. The entrapment of an enzyme in a carbon paste electrode, with the use of a paraffin to provide the paste, has been reported on numerous occasions (see Table I). Similarly, much research is being undertaken in the field of polymer electrodes,SS such that an enzyme may be entrapped in a polymer matrix, for example, a polypyrrole, at an electrode surface. Finally, enzymes may be chemically bound to a surface via, for example, a carbodiimide- or cyanuric chloride-modified graphite electrode. 87 The direct reversible electrochemistry of the dehydrogenase, p-cresol methylhydroxylase (PCMH), from P s e u d o m o n a s putida, has been observed 6° between the heme of the flavocytochrome and an edge-plane graphite electrode. Here the reversible electrochemical response was obtained due to oxidation and reduction of the enzyme itself, the presence of substrate not being required for communication between electrode and enzyme to be observed. Diffusion-controlled heterogeneous electron transfer was modulated by the concentration and type of promoter used, ranging from simple cations through to polyamines and aminoglycosides. PCMH is found in the periplasm of certain pseudomonads, where it catalyzes the dehydrogenation and hydration of p-cresol and homologs to the corresponding alcohol, which is then further dehydrogenated to the aldehyde or ketone. A buffered solution containing l0 mM spermine, a linear polyamine, and 35/zM PCMH produced a reversible cyclic voltammetric response (Fig. 7b) at an edge-plane graphite electrode. The midpoint potential ( - + 10 mV versus SCE) was in good agreement with the potential determined by potentiometric titration. On addition of 3 mM p-cresol to the solution, a large catalytic current was observed at oxidizing potentials (Fig. 7d) owing to the repetitive regeneration of the reduced enzyme (Fig. 8) by the substrate as a result of the dehydrogenation of p-cresol by the enzyme. A linear current against substrate ¢oncentration response was obtained up to 0.5 mM p-cresol. Similarly, if p-cresol was replaced by the intermediate alcohol p-hydroxybenzyl alcohol, a catalytic current response was still obtained, though with reduced peak currents. The necess6A. P. F. Turner, I. Karnbe, and G. S. Wilson, "Biosensors, Fundamentalsand Applications." Oxford Univ. Press, Oxford, 1986;A. E. G. Cass (ed.), "Biosensors,A Practical Approach." Oxford Univ. Press, Oxford, 1990. s7V. J. Razumas,J. J. Jasaitis, and J. J. Kulys,Bioelectrochem. Bioenerg. 12, 297 (1984). ss H. D. Abrufia, Coord. Chem. Rev. 86, 135 (1988).

[19]

ELECTROCHEMICAL STUDIES OF METALLOENZYMES

519

C

-&

,

olo

i

,~

-o.2

I

o

i

012

Potential/V(SCE)

FIG. 7. Direct electrochemistry of p-cresol methylhydroxylase. (a) Response in buffered solution at an edge-plane graphite electrode in the presence of a promoter. (b) Response on addition of enzyme. (c) As (b), but at reduced sensitivity. (d) Catalytic response on addition of p-cresol to solution. Reproduced with permission from J. Electroanal. Chem. 2 ~ , 379 (1989).

sity of the presence of a cationic promoter was shown by the lack of a Faradaic response at an edge-plane graphite electrode in a buffered solution containing both PCMH and p-cresol (Fig. 9a), but, on addition of Cr(NH3)63+, neomycin, or gentamycin to the solution, a large catalytic

CHO

PCMHox

S

~0H

FIG. 8. Schematic representation of the electrochemically controlled catalytic conversion of p-cresol to p-hydroxybenzaldehyde by the enzyme p-cresol methylhydroxylase (PCMH).

520

PROBES OF METAL ION ENVIRONMENTS

[19]

b

j I_

-0.3

I

|

|

t

0.0

!

I

0.3

Potential/V (SCE) Fro. 9. Cyclic voltammograms illustrating (a) lack of response observed for a solution of p-cresol methylhydroxylase and p-cresol and (b) catalytic response obtained on addition of Cr(NH3)63÷ to the solution. Reproduced with permission from J. Electroanal. Chem. 266, 379 (1989).

current was immediately passed at the working electrode (Fig. 9b). The high specificity o f the e n z y m e was revealed when no catalytic current was observed if the substrate was replaced with o- or m-cresol. Although the direct electrochemistry of many redox enzymes can be observed at an electrode, this is one of only a very few reported instances o f the direct electrochemical communication between an electrode and an enzyme, in the absence o f substrate. Similarly, the direct electrochemis-

[19]

E L E C T R O C H E M I C A L S T U D I E S OF M E T A L L O E N Z Y M E S

521

tries of two iron-sulfur enzymes have been reported in the absence of substrate. 35 The reduction potentials of the hydrogenase from Clostridium pasteurianum and the carbon monoxide dehydrogenase from Rhodospirillum rubrum were obtained by square-wave voltammetry at edge-plane graphite electrodes in the presence of cationic promoters. For both enzymes, the reduction potential was about -640 mV versus SCE, and one electron was transferred per redox center, as determined from the peak width at half-height, which is characteristic of low-potential [4Fe-4S] clusters. The enzyme sulfide : cytochrome-c oxidoreductase, or flavocytochrome c552, from the purple sulfur bacterium Chromatium vinosum is involved in the photosynthetic oxidation of sulfur. It contains a flavin adenine dinucleotide (FAD) center and two hemes. Direct electrochemistry of the heme of the enzyme is observed at an edge-plane graphite electrode in the presence of polyvalent cation promoters, including aminoglycosides.45 On addition of substrate, sulfide, a catalytic current is observed. The substrate is first oxidized at the FAD center of the enzyme, then, following an intramolecular electron transfer to one of the hemes, the enzyme is reoxidized at the electrode. In a few instances, direct unpromoted electrical communication to an enzyme may be achieved at an unmodified electrode. A catalytic response in the presence of substrate is obtained with D-gluconate dehydrogenase (GADH) adsorbed on carbon paste electrodes. 49 GADH from Pseudomonas fluorescens is a membrane-bound enzyme that contains an FAD center, an Fe-S cluster, and a c-type heme. Similarly, this enzyme and alcohol dehydrogenase, a quinohemoprotein from Gluconobacter suboxydans, have been adsorbed at carbon or metal electrodes and catalytic responses in the presence of the respective substrates produced) 3 Succinate dehydrogenase (SDH) is a mitochondrial electron transport enzyme containing one FAD and three Fe-S centers. When SDH is adsorbed TM directly onto an edge-plane graphite electrode, a catalytic current is observed in the presence of succinate. However, on addition of fumarate to the solution, "troughs" appear in the cyclic voltammograms, such that an increase in the driving force produces a decrease in the rate of reduction. It is postulated that substrate binding to or product release from SDH is allowed only during the period when the active site is oxidized, which means the enzyme possesses a degree of control over the electron flow in the system. In conclusion, the techniques that have been developed since the late 1970s in the study of protein electrochemistry are now being applied in the study of enzyme systems. Although electrochemical communication with over 20 metalloenzymes has been achieved, most studies have been directed toward the development of biosensors, in which the enzyme is immobilized at the electrode surface and provides a substrate-dependent

522

PROBES OF M E T A L ION E N V I R O N M E N T S

[20]

current response. The application of electrochemical techniques for providing kinetic information on enzyme systems is only slowly developing, but if progress is similar to that made with metalloproteins, the results should be promising. Acknowledgments We thank the A.F.R.C. for a studentshipto N.I.H., and the S.E.R.C., M.R.C., Leverhulme Trust, E. P. Abraham Trust, and MediSenseInc. for financialsupport.

[20] P u l s e R a d i o l y s i s By G. ARTHUR SALMON and A. GEOFFREY SYKES Introduction The technique of pulse radiolysis, which was originally developed to study fast primary processes in radiation chemistry, ~-4 is the radiation chemical analog of flash photolysis) ,6 In essence, the technique employs an intense pulse of ionizing radiation, usually of submicrosecond duration, to generate a high concentration of reactive intermediates, the chemical reactions of which may be followed by UV-VIS spectrophotometry. Time resolution down to 10 nsec is easily achieved, and in several instances this has been reduced to the picosecond domain. 7-9 For metalloprotein studies it is, however, also important that processes extending over seconds can be followed. Other modes of detection have been used including conductivity, 1° electron paramagnetic resonance

I M. S. Matheson and L. M. Dorfman, J. Chem. Phys. 32, 1870 (1960). 2 R. L. McCarthy and A. McLachlan, Trans. Faraday Soc. 56, 1187 (1960). 3 j. p. Keene, Nature (London) 188, 843 (1960). 4 j. W. Boag and R. W. Steel, Br. Empire Campaign Rep. 38 (Part II), 251 (1960). 5 R. G. W. Norrish and G. Porter, Nature (London) 164, 658 (1949). 6 G. Porter, Proc. R. Soc. London A 200, 284 (1950). 7 M. J. Bronskill, R. K. Wolff, and J. W. Hunt, J. Chem. Phys. 53, 4201 (1970). 8 C. D. Jonah, Rev. Sci. lnstrum. 46, 62 (1975). 9 y . Tabata, J. Tanaka, S. Tagawa, Y. Katsumura, T. Ueda, and K. Hasegawa, J. Fac. Eng. Univ. Tokyo Ser. B 34, 619 (1978). l0 K.-D. Asmus and E. Janata, in "The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis" (J. H. Baxendale and F. Busi, eds.), NATO Advanced Study Institute Series, D, p. 91. Reidel Publ., Dordrecht, The Netherlands, 1982.

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

522

PROBES OF M E T A L ION E N V I R O N M E N T S

[20]

current response. The application of electrochemical techniques for providing kinetic information on enzyme systems is only slowly developing, but if progress is similar to that made with metalloproteins, the results should be promising. Acknowledgments We thank the A.F.R.C. for a studentshipto N.I.H., and the S.E.R.C., M.R.C., Leverhulme Trust, E. P. Abraham Trust, and MediSenseInc. for financialsupport.

[20] P u l s e R a d i o l y s i s By G. ARTHUR SALMON and A. GEOFFREY SYKES Introduction The technique of pulse radiolysis, which was originally developed to study fast primary processes in radiation chemistry, ~-4 is the radiation chemical analog of flash photolysis) ,6 In essence, the technique employs an intense pulse of ionizing radiation, usually of submicrosecond duration, to generate a high concentration of reactive intermediates, the chemical reactions of which may be followed by UV-VIS spectrophotometry. Time resolution down to 10 nsec is easily achieved, and in several instances this has been reduced to the picosecond domain. 7-9 For metalloprotein studies it is, however, also important that processes extending over seconds can be followed. Other modes of detection have been used including conductivity, 1° electron paramagnetic resonance

I M. S. Matheson and L. M. Dorfman, J. Chem. Phys. 32, 1870 (1960). 2 R. L. McCarthy and A. McLachlan, Trans. Faraday Soc. 56, 1187 (1960). 3 j. p. Keene, Nature (London) 188, 843 (1960). 4 j. W. Boag and R. W. Steel, Br. Empire Campaign Rep. 38 (Part II), 251 (1960). 5 R. G. W. Norrish and G. Porter, Nature (London) 164, 658 (1949). 6 G. Porter, Proc. R. Soc. London A 200, 284 (1950). 7 M. J. Bronskill, R. K. Wolff, and J. W. Hunt, J. Chem. Phys. 53, 4201 (1970). 8 C. D. Jonah, Rev. Sci. lnstrum. 46, 62 (1975). 9 y . Tabata, J. Tanaka, S. Tagawa, Y. Katsumura, T. Ueda, and K. Hasegawa, J. Fac. Eng. Univ. Tokyo Ser. B 34, 619 (1978). l0 K.-D. Asmus and E. Janata, in "The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis" (J. H. Baxendale and F. Busi, eds.), NATO Advanced Study Institute Series, D, p. 91. Reidel Publ., Dordrecht, The Netherlands, 1982.

METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

[20]

PULSE RADIOLYSIS

523

(EPR), 11 and polarography, 12 but kinetic spectrophotometry is by far the most versatile and the most used for studies on proteins, and we confine our consideration to this method. The value of the pulse radiolysis technique to studies on proteins and enzyme systems stems from the fact that the radiolysis of water and aqueous solutions provides a means of generating, in a controlled way, a wide range of one-electron oxidizing and reducing agents which may be used to characterize and study protein function. Essentially three kinds of studies have been performed. In the selective probe technique, inorganic oxidizing radicals such a s ( S C N ) 2 - : , Br2 ~-, I2 ~ , C12 ~-, C O 3 v , and S e O 3 ~have been used to identify the essential amino acids which, if damaged, lead to inactivation of a protein. The method has been reviewed by Adams and Wardman 13 and Bisby et a l ) 4 Perhaps the most extensive application of the technique to protein chemistry involves the exploration of the properties of intermediate oxidation states of metal ions in metalloproteins, which either are involved in electron transport chains or are vital to enzyme activity. In this kind of study the intermediate, usually unstable, oxidation state of the protein is generated either by selective one-electron reduction or by oxidation of the reduced protein using one of the selective one-electron oxidants referred to above. A review of this type of study has been made by Buxton) 5 Finally, considerable use of the technique has been made to study electron transfer processes involving redox proteins. The method is closely related to the study of intermediate redox states mentioned above, but in this case particular emphasis is placed on observing electron transfer between sites of different reduction potential in a protein, such as occurs in the multisite copper-containing protein ascorbate oxidase, or on observing electron transport to or from the natural redox site in a protein and an artificially introduced redox moiety in a well-characterized site on the protein. The pulse radiolysis method is closely related to the pioneering work by Gray and colleagues, 16who used flash photolysis to study ruthenium-copper (Run-Cu u) electron transfer in azurin modified by complexing Ru(III)(NH3)5 to histidine-83 of the protein. H A. D. Trifunac, in "The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis" (J. H. Baxendale and F. Busi, eds.), NATO Advanced Study Institute Series, D, p. 163. Reidel Publ., Dordrecht, The Netherlands, 1982. 12 K.-D. Asmus and E. Janata, in "The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis" (J. H. Baxendale and F. Busi, eds.), NATO Advanced Study Institute Series, D, p. 115. Reidel Publ., Dordrecht, The Netherlands, 1982. 13 G. E. Adams and P. Wardman, Free Radicals Biol. 3, 53 (1977). 14 R. H. Bisby, R. B. Cundall, and A. K. Davies, Photochem. Photobiol. 28, 827 (1978). 15 G. V. Buxton, Adv. lnorg. Bioinorg. Mech. 3, 131 (1984). 16 N. M. Kostic, R. Margalit, Chi-Ming Che, and H. B. Gray, J. Am. Chem. Soc. 105, 7765 (1983).

524

PROBES OF METAL ION ENVIRONMENTS

[20]

Radiation Chemical Basis When a beam of high-energy electrons (-> 1 MeV) traverses water or an aqueous solution, it results in the ionization of water molecules along the tracks of the electrons. Although a major proportion of the ionization events involve single ion pairs, in a proportion of the events the secondary electron has sufficient energy to bring about further ionizations, and this results in a number of ion pairs being formed in close proximity, that is, in a so-called spur. Thus, along the track of the primary electron we have randomly distributed regions of ionization that may contain one to five ion pairs, the distribution being in favor of single ion pairs. Thermalization of the secondary electrons occurs within 10-~2 sec, and trapping and solvation of the electrons takes place very rapidly, leading to the formation of hydrated electrons eaq-. Within about 10-14 sec the H2O+ radical cation generated in the ionization events undergoes the very fast ion-molecule reaction (1), which results in the formation of the H2 O + + H 2 0 ~

"OH + H30 +

(1)

hydroxyl radical and the hydroxonium, or hydrogen, ion. Thus, within 10-12 sec of the passage of the high-energy electron through the aqueous system the situation may be summarized as in Eq. (2). The numbers in (4.8 × 10-7)H2O---> (4.8 x 10-7)eaq- + (4.8 × 10-7)'OH + (4.8 × 10-7)H +

(2) parentheses are G values, that is, the number of moles of product formed or reactant consumed per joule of energy absorbed by the system, in this case the aqueous solution. Although the SI unit version of the G value used here is rapidly gaining acceptance, it should be noted that the alternative unit of molecules/(100 eV) is still in frequent use. The relationship between the two units is G/mol j - i = 1.037

×

10 -7

G'/molecules (100 eV) -1

Within 10-7 sec radical-radical reactions within the spurs lead to the formation of molecular products, and the surviving free radicals are essentially homogeneously distributed. At this stage the radiolysis of water is summarized by Eq. (3). (4.23 × 10-7)H20 ---->(2.8 × 10-7)eaq- + (6.2 x 10-8)H • + (2.8 × 10-7)'OH + (2.8 × 10-7)H + + (7.3 × 10-8)H202

(3)

For historical reasons, the radicals surviving after 10 -7 s e c are known as primary radials. The G values shown in parentheses represent the yields of these products (mol j-l), which would be scavenged by low

[20]

PULSE RADIOLYSIS

525

concentrations (<1 mmol dm -3) of a suitable reactant. Hydrated electrons (E ° = - 2 . 9 V) and H. (E ° = - 2 . 3 V) are powerful reducing agents and . O H (E ° = 2.8 V) a powerful oxidant. Thus, initially following the passage of a short duration pulse of ionizing radiation through an aqueous solution we have approximately equal concentrations of eaq-, "OH, and H + and an approximately 5 times lower concentration of H-. To use the radiolysis of water as an effective tool for the study of protein systems, it is desirable to be able to convert these primary species so that we have either a single reducing or a single oxidizing radical. Several systems where this may be largely achieved are considered next.

Systems Yielding Selected Reactants Oxidizing Radicals Hydroxyl radical. The most convenient way of obtaining an almost totally oxidizing system is to saturate the solution with nitrous oxide, which converts eaq- to "OH via reaction (4). The concentration of NzO eaq- + N 2 0

> N 2 + O-

H20

) "OH + O H -

(4)

in a saturated solution at 20 ° is about 2.5 × 10 -2 mol dm -3, and under these conditions G(-OH) = 5.7 × 10 -7 mol J-~; however, the G value is somewhat dependent on the concentration of the free radical scavenger.17 Nitrous oxide does not react rapidly with hydrogen atoms, so in NzO-saturated solutions in the p H range 3-11 about 10% of the total yield of radicals remains as H.. Below pH 3, the reaction of eaqwith H + competes effectively with reaction (4), and saturation with N20 is no longer a satisfactory means of achieving oxidizing conditions. Selective oxidizing radicals. Although •O H is a convenient oxidant for many studies, it is a very powerful oxidant (E ° = +2.8 V) and acts both by hydrogen abstraction and electron transfer. It is often desirable to employ a more selective oxidant. This is usually achieved by incorporating a second solute in the solution, in addition to N20, to react with the •O H and convert it to the more selective agent. Suitable solutes are azide ion, thiocyanate ion, bromide ion, and iodide ion, which generate, respectively, N 3. (E ° = 1.33 V), ( S C N ) 2 ~- (E ° = 1.33 V), Br2: (E ° = 1.66 V), and Iz 7 (E ° -~ 1.0 V) radicals, reactions (5)-(8). Generation of SO47 (E ° = 2.4 V)

x7R. H. Schuler, A. L. HartzeU, and B. Behar, J. Phys. Chem. 85, 191 (1981).

526

PROBES OF METAL ION ENVIRONMENTS • O H + N 3- ~

O H - + N3"

•O H + S C N - --~ O H - + S C N . . •O H + B r - ~ O H - + B r . . •O H + I - ---~ O H - + I . .

(5)

SCNBr-

I-

[20]

(SCN)2 ~

" Br 2~-

" 12~

(6) (7) (8)

m a y be achieved by radiolysis o f nitrogen- or argon-saturated solutions o f p e r o x o d i s u l f a t e ion ( - 10 -3 mol dm-3), which reacts with eaq- by reaction (9) (k = 1.2 x 101° d m 3 mol -l s e c - l ) . In this case, it is n e c e s s a r y to (9)

eaq- + 82082- "~ 5 0 4 7 + 5042-

add a b o u t 10 -2 mol d m -3 tert-butanol to convert - O H to the relatively unreactive tert-butanol radical, reaction (10). C o m p a r e d with . O H , SO4 ~ H3 H3C--C--OH

I

CH 3

~ H3 + .OH--~ HzC--C--OH

I

+ H20

(10)

CH 3

has a m u c h lower t e n d e n c y to undergo h y d r o g e n abstraction or addition reactions and reacts mainly b y electron transfer.

Reducing Radicals Hydrated electron. To use eaq- as a reductant it is usual to add tertbutanol to the d e o x y g e n a t e d solution so as to c o n v e r t •O H , as described above. U n d e r normal conditions, [tert-butanol] ~ 10 -2 M , H a t o m s will remain with a b o u t 25% of the yield o f eaq-, but they can be eliminated b y increasing the tert-butanol concentration to a p p r o x i m a t e l y 1 mol dm -3. Carbon dioxide radical anion• The CO2 ~ radical (E ° = - 1 . 9 V) is a versatile reductant that can be generated as the only radical species b y radiolysis o f N 2 0 - s a t u r a t e d solutions containing 10 -2 mol dm -3 sodium formate• In this system, . O H and H . are c o n v e r t e d to CO2 -~ by reactions (11) and (12)• O t h e r m o r e selective reducing agents m a y be generated by •O H + H C O 2- --~ H 2 0 + CO2 ~H" + H C O 2- --) H 2 + CO2 ~

(11) (12)

adding to the f o r m a t e / N 2 0 solution a third reactant X in lower concentration than the f o r m a t e but at a concentration such that electron transfer f r o m CO2 ~ to X is c o m p l e t e on a fast time scale, reaction (13). A p~/xticuCO2 ~- + X - - * CO 2 + X ~-

(13)

[20]

PULSE RADIOLYSlS

527

larly valuable solute in this respect is methyl viologen (1,1 '-dimethyl-4,4'bipyridinium dichloride, M V '÷) which is converted to the radical cation (MV "+, E ° = -448 mV) in reaction (13). As well as being a more selective agent because of its much more positive potential compared to eaq- or COz 7 , MV "+has the additional advantage that in the absence of an oxidant it is very long-lived. Thus, it can be used to reduce quantitatively a protein even when the reactivity with the protein is low or the concentration of the protein very low. Other solutes which can be used to generate selective reducing radicals by reaction (13) are the quinones, flavin derivatives, and nitroaryl compounds. The one-electron reduction potentials of many of the radicals derived from these solutes have been tabulated by Wardman. TM Alcohol radicals. A number of the radicals derived from alcohols by the attack of .OH radicals, reaction (14), have been used as selective reductants, the most used being the radical derived from 2-propanol (E ° = - 1.5V). •OH + RIR2CHOH---> H20 + R1RzCOH

(14)

Technique All pulse radiolysis facilities currently operating employ spectrophotometric detection rather than the spectrographic method that was used in some of the early experiments. The methods used are derivatives of those described by Keene. 19The sample is irradiated with a short pulse, usually 10-100 nsec duration, of high-energy electrons, and an optical analyzing system measures the change in light absorption due to the species generated by the radiation. Detection systems are set up so that the recorded signal corresponds to the change in signal from the photomultiplier as a function of time, rather than the absolute signal versus time. The absorbance change at time t, usually measured from the start of the radiation pulse, is given by

A(t) = - l o g I1

Vs(t)]

J

where V0 is the signal from the photodetector before the radiation pulse and Vs(t) is the recorded signal at time t. Thus, traces of absorbance change as a function of time are recorded. Absorption spectra of the transient species existing at time t are obtained by measuring the absorb18 p. Wardman, J. Phys. Chem. Ref. Data 18, 1637 (1989). 19 j. p. Keene, J. Sci. lnstrum. 41, 493 (1964).

528

PROBES OF METAL ION ENVIRONMENTS 10

i

T

i

[20]

i

o') d

E

50 psec/div.

o

0

350

I

I

I

I

400

450

500

550

600

Wavelength/rim FIG. 1. Spectrum produced by the pulse radiolysis of an N20-saturated 15.2/zM solution of deactivated ribonucleotide reductase containing 10 mM sodium azide. (Inset) Pulse radiolysis trace taken at 410 nm.

ance at the given time from a series of traces recorded at a set of wavelengths. Traces of absorption versus time are usually transferred to a computer for kinetic analysis. The inset to Fig. 1 shows a trace obtained at 410 nm in a study 2° of the oxidation by azide radical of ribonucleotide reductase in which the tyrosyl radical has first been inactivated by treatment with hydroxyurea. The trace shows the formation, which is complete in about 150/zsec of a species absorbing at 410 nm, and the spectrum shown in Fig. 1 is obtained from traces such as this by recording the absorbance at 250/zsec. In this case the absorption with hma x equal 410 nm is due to the formation of the tyrosyl radical, whereas the absorption around 5 I0 nm is due to the radical derived from tryptophan. The extent of the radiation-induced absorption is, of course, dependent on the radiation dose absorbed by the system. Spectra such as that shown in Fig. 1 are, therefore, presented either as absorbance normalized to a standard dose, usually 1 krad [in SI units 10 Gy (gray)] or, preferably, as the product of the G value for the species produced and its molar absorptivity, e. Figure 2 shows a first-order kinetic treatment of the trace shown as the inset to Fig. 1. For convenience, it is customary to use conditions such that the reaction of a radical with the protein can conform to pseudo20 K.-Y. Lam, K. Govindaraju, J.-Y. Han., G. A. Salmon, and A. G. Sykes, J.C.S. Dalton Trans., in press (1993).

[20]

PULSE RADIOLYSIS -8.5

. . . .

,

. . . .

,

. . . .

,

529 . . . .

,

.... O0

-8.0

0

-7.5

CO ~:(e~O0-

- / I II - 7 . 0 .<

--=

o

CO

o

-6.5

-6.0 --5.5

.

0

.

.

.

i

20

.

.

.

.

i

40

.

.

.

.

i

60

.

.

.

.

I

80

.

.

.

.

100

Time/t~se¢ FIG. 2. First-order kinetic plot of the trace shown as inset to Fig. 1.

first-order kinetics. This is done by adjusting the radiation dose absorbed by the sample so that the concentration of the radical generated is less than 10% of that of the protein. Thus, it is necessary to know the radiation dose absorbed by the sample. This is achieved by the use of a chemical dosimeter, that is, a system that produces a known absorbance for a given absorbed dose (see below). It is outside the scope of the present discussion to consider the details of detection and data handling systems. For more detail, see Refs. 21-23.

Dosimetry Most pulse radiolysis systems have a means of measuring the dose delivered in individual pulses. These are based on measuring the total charge of electrons passing through the sample. Two systems for achieving this are the secondary emission chamber (SEC) 24 and the inductive beam 21 M. C. Saner, in "The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis" (J. H. Baxendale and F. Busi, eds.), NATO Advanced Study Institute Series, D, p. 35. Reidel Publ., Dordrecht, The Netherlands, 1982. 22 G. Roffi, in "The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis" (J. H. Baxendale and F. Busi, eds.), NATO Advanced Study Institute Series, D, p. 63. Reidel Publ., Dordrecht, The Netherlands, 1982. 23 D. C. Foyt, in "The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis" (J. H. Baxendale and F. Busi, eds.), NATO Advanced Study Institute Series, D, p. 213. Reidel Publ., Dordrecht, The Netherlands, 1982. 24 S. I. Taimuty and B. S. Deaver, Rev. Sci. lnstrum. 32, 1098 (1961).

530

PROBES OF METAL ION ENVIRONMENTS

[20]

Gas //

ID I ,-C

\

FIG. 3. Apparatus for deaerating and saturating protein solutions with argon or N20 in readiness for pulse radiolysis. A, All-glass syringe; B, protein sample; C, sintered frit; D, water; and E, gas outlet via bubbler.

monitor. 25 With both monitors it is necessary to relate the reading to the absorbed dose by carrying out chemical dosimetry (for a general description, see Ref. 26). For most purposes the thiocyanate dosimeter is employed 26 in which an oxygen-saturated solution of 10 mM potassium thiocyanate is subject to pulse radiolysis and the absorption of the (SCN)2 ~ radical, which is formed in reaction (6), is measured at 475 nm. The factor relating absorbance to absorbed dose for this system is established, z6 and hence the beam monitor can be calibrated. z5 B. Vojnovic, Radiat. Phys. Chem. 24, 517 (1985). E. M. Fielden, in "The Study of Fast Processes and Transient Species by Electron Pulse Radiolysis" (J. H. Baxendale and F. Busi, eds.), NATO Advanced Study Institute Series, p. 49. Reidel PubI., Dordrecht, The Netherlands, 1982.

[20]

PULSE RADIOLYSIS

(

531

C II I

i J

I- I ica-I i--

J

FIG. 4. Pulse radiolysis cell and flow system. A, S13 socket connection to gas supply; B, pressure regulation system; C, irradiation cell; D, two-way tap; E, delivery tube; F, drain; G, all-glass syringe; and H, protein solution.

Solution Preparation and Handling For pulse radiolysis studies it is necessary to prepare solutions in either phosphate or sodium tetraborate buffers, depending on the pH desired, as many other buffer reagents react rapidly with the primary radiolytic species. Generally the solutions must be deaerated by bubbling with either nitrous oxide or argon, and with most protein solutions this requires extreme care. A concentrated solution of protein is prepared by FPLC (fast protein liquid chromatography) followed by ultradialysis against airfree buffer Solutions using an Amicon (Danvers, MA) cell with a YH5 membrane and a pressure of 55 psi provided by nitrogen gas. A volume of buffer is bubbled with the appropriate gas in the apparatus shown in Fig. 3 for about 30 rain to ensure deaeration and saturation with the bubbling gas. During the bubbling period, the solution is driven into the syringe from time to time to ensure complete removal of air and to ensure that the barrel is well wetted with solution. Then an aliquot of the concentrated solution of protein is added to the buffer using a micropipette, which has been well purged with the bubbling gas, and bubbling is continued very gently for a further 5-10 min. The solution is then forced by gas pressure into the syringe, which is capped before being introduced to the apparatus for filling the pulse radiolysis cell (Fig. 4). This procedure is satisfactory

532 .,.i

....

PROBES OF METAL ION ENVIRONMENTS i ....

a

i ....

), -

a ....

i ....

I ....

i ....

i ....

ji -

i...

,.,i

605nm

e~

m

ID

o

....

i ....

i ....

i ....

i ....

....

i . . . .

b

w

..i

i ....

i ....

i ....

..i

! ....

i ....

i ....

c

i ....

X -

i ....

i ....

i ....

i ....

i ....

X -

....

i ....

20 psec per dlv.

....

[20]

l ....

i ....

i ....

i ....

~ ....

i ....

i ....

i ....

~ ....

i.~j

i ....

i ....

i ....

i ....

i ....

i,.,

557nm

l ....

i ....

20 gsec per dlv.

i ....

i ....

i ....

• ,.i

557nm

....

i ....

i ....

d

.m 'u

r ....

X -

i ....

i ....

557nm

~8 12.

to .Q ig

O v-

J ,,i

....

I ....

i ....

i ....

i ....

i ....

100 msec per dlv.

i ....

i ....

i ....

i ~

d CD ~D

O

....

i ....

i ....

i ....

i ....

i ....

i ....

20 msec per dlv.

FIG. 5. Pulse radiolysis of cytochrome bJmethyl viologen solutions: (a) at 605 rim, showing the fast formation and decay of the MV .+ absorption; (b) at 557 nm, showing the formation of reduced Fe(II) protein; (c) of the K56H mutant, showing the stability of the Fe(II) absorption at 557 nm; and (d) of the Ru-modified K56H mutant in t h e Fe(III)Ru(III) form, showing the decay of the Fe(II) absorption at 557 nm in the second stage of the reaction. Experiments were performed at 19°, pH 7.0, and I = 0.100 M.

for most proteins, but in some cases, where the protein is very sensitive to denaturation by bubbling, more complex arrangements are needed.

Pulse Radiolysis Applied to Study o f Long-Range Electron Transfer in Proteins In this section we describe the use of pulse radiolysis to study longrange electron transfer in proteins. We use recent studies 27 of rutheniummodified cytochrome b2 core mutants as an example of this procedure. As mentioned earlier, complexing ruthenium to a histidine residue of 27 E. Lloyd, Ph.D. Thesis, The University of Newcastle upon Tyne, U.K. (1991).

[20]

PULSE RADIOLYS1S

533

a metalloprotein provides a convenient means of introducing a second redox site onto the surface of a protein. Theory predicts that rate constants for intramolecular electron transfer between metal sites in rutheniummodified metaUoproteins decrease exponentially with distance. However, the detailed nature of the pathway for electron transfer, for example, through-bond, through-hydrogen-bond, or through-space, can also affect the rate. A combination of ruthenium modification and site-directed mutagenesis can be applied to a number of metalloproteins, enabling a strategic examination of the effects of distance and polypeptide structure on the electron transfer rate constant to be carried out. We have applied this technique to three mutants of cytochrome b 2 (lysine-56--* histidine, lysine51 ~ histidine, and asparagine-42 ~ histidine). Pulse radiolysis of wild-type cytochrome b2 in N20-saturated solutions containing 12 mM sodium formate and 5 x 10 -4 M methyl viologen (MV z+) allows the reduction of the protein by MV "+ to be monitored, reaction (15). Figure 5a shows a trace of the decay of MV +" followed at 605 nm, MV + + C y t . F e ( I I I ) ~ MV 2+ + Cyt.Fe(II)

(15)

whereas Fig. 5b shows a trace of the corresponding growth of the reduced form of cytochrome b2 observed at 557 nm. Repetition of such measurements at several concentrations of protein allows the rate constant of reaction (15) to be evaluated. The rate constants observed at the two wavelengths are in good agreement, (3.00 - 0.01) x 10 9 M -1 sec -1 at 605 nm and (2.9 --- 0.2) x 10 9 M - ! s e c -1 at 557 nm. Similar measurements were carried out with the mutants. Figure 5c shows that, in the absence of ruthenium substitution, the reduced form of the protein is stable over a period in excess of 1 sec. When similar experiments are conducted on ruthenium-modified cytochrome bz in which the ruthenium is in the oxidized Ru(III) state, then the fast reduction of the protein by MV "+occurs at both the Fe(III) and Ru(III) centers, reactions (16) and (17). Thus, there are two semireduced forms of the protein, MV "+ + Cyt.Fe(III)Ru(III) ~ MV 2+ + Cyt-Fe(II)Ru(III) MV '+ + Cyt-Fe(III)Ru(III)~ MV 2+ + Cyt.Fe(III)Ru(II)

(16) (17)

Cyt. Fe(II)Ru(III) and Cyt. Fe(III)Ru(II), where the former is a metastable product and the latter a stable form. Subsequently a slow intramolecular electron transfer is observed as the Fe(II), which absorbs at 557 nm, is oxidized back to Fe(III), reaction (18). Figure 5d is a pulse radiolysis Cyt-Fe(II)Ru(III) ~ Cyt. Fe(III)Ru(II)

(18)

trace demonstrating the occurrence of this process for the rutheniummodified K56H cytochrome bz.

534

PROBES OF METAL ION ENVIRONMENTS

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[eyt.Fe(llllRu(llll]//JM FIG. 6. Second stage of reaction in studies on ruthenium-modified K56H cytochrome b 2 in the F e ( I I I ) R u ( I I I ) f o r m , illustrating the d e p e n d e n c e o f kobs for the reoxidation of F e ( I I ) o n the c o n c e n t r a t i o n o f oxidized protein, at 19°, p H 7.0, and 1 = 0.100 M.

In addition to the intramolecular process, an intermolecular reaction of Cyt. Fe(II)Ru(III) with the fully oxidized protein results in the reoxidation of the Fe(II), reaction (19). The dependence of the observed firstCyt.Fe(II)Ru(III) + Cyt. Fe(III)Ru(III) --~ Cyt.Fe(III)Ru(III) + Cyt.Fe(III)Ru(II)

(19)

order rate constant on oxidized protein follows Eq. (20). Thus a plot of kobs = k18 + klg[Cyt.Fe(III)Ru(III)]

(20)

kobs versus the concentration of oxidized protein should yield a straight line with intercept k~8 and slope k19. Such a plot is shown in Fig. 6 for the dependence of kobs on protein concentration for the rmhenium-modified K56H cytochrome b2 mutant and yields k18 = (3.5 - 0.7) sec -I and k19 = (1.4 _ 0.1) x 106 M -1 sec -1. Values of kl8 observed for the other mutants studied are as follows: His-51Ru(NH3)5, 2.4 sec -l, and His-42Ru(NH3)5, 78 sec -1. Discussion of the implication of these results is outside the scope of the present review. Nevertheless, this study clearly illustrates the value of the pulse radiolysis technique for studying electron transfer reactions.

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PHYSICAL METHODS TO LOCATE METAL ATOMS

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[21] Physical Methods to Locate Metal Atoms in Biological Systems By MICHEL THELLIER, CAMILLE RIPOLL, CARMEN QUINTANA, FRAN~OISE SOMMER, PIERRE CHEVALLIER,and JACK DAINTY 1. Introduction Metal elements are involved in a number of cellular processes such as enzyme activation, transmembrane transport, water exchange, signal transduction, and intercellular communication. For instance, the alkaline and alkaline earth cations Na ÷, K +, Mg 2÷, and Ca 2÷ play a key role in the functioning of living systems; Cu, Fe, Zn, or Mn may be associated with proteins; heavy metals and radioactive Sr 2÷ or Cs ÷ can be dangerous pollutants; and lithium or cobalt may be used as specific inhibitors of biological processes. Quantitatively mapping, or depth-profiling, metal atoms in biological systems can thus help toward an understanding of physiological mechanisms. A variety of physical methods are progressively being adapted to the study of biological specimens. In the following, we examine X-ray fluorescence, analytical electron microscopy (X-ray microanalysis, electron energy loss spectrometry, and Auger spectrometry), analysis of tracks originating from nuclear events, nuclear probes, and secondary ion imaging methods. Among these different methods, some can detect most chemical elements, whereas others apply to only a few elements. Some will discriminate between the isotopes of a chemical element, and others will not. Some will be useful mainly on a histological scale, whereas, in other cases, the spatial resolution may be good enough for considering subcellular localizations. In vivo measurements may be possible in a few cases, but, most often, the methods are meant for fixed preparations. It is then clearly requisite that the techniques used for sample preparation (fixation, dehydration, staining) do not disturb the natural distribution of the metal ions under observation. 2. Preparation of Fixed Specimens In fixation of specimens, there is the double requirement (1) to preserve the cellular structures and (2) not to displace the substances under analysis. This causes no particular difficulty when studying nondiffusible substances; one may simply prepare the specimens (most often tissue sections) according to conventional methods for histology and cytology. However, the addition of heavy elements [such as osmium METHODS IN ENZYMOLOGY, VOL. 227

Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

536

PROBES OF METAL ION ENVIRONMENTS

[21]

(Os), lead (Pb), or uranium (U)] may be unsuitable in some cases; for instance, they can increase the risk of interference or decrease the sensitivity by increasing the background when observations are performed using analytical electron microscopy. With diffusible substances, which is the case with most metal ions, one has to prevent them from being mobilized during sample preparation. Two main approaches have been followed with this aim in view, namely, cryomethods and precipitation methods.

2.1. Cryomethods of Sample Preparation The various cryomethods employed are summarized in Fig. 1. Minute pieces of tissue ( - 1 mm 3) are sampled. These samples are cryofixed by quick freezing via various possible methods (e.g., immersion in a cryogenic fluid, projection of a refrigerated fluid onto the sample, projection of the sample onto a cooled metal plate). Freezing has to be extremely rapid (freezing rate of the order of 105 K sec -~) in order that the ice crystals thus formed remain smaller than the spatial resolution of the analytical method used, that is, approximately 0.5/.tm with the ionic microscope and the nuclear probe, 0.1 /xm with X-ray analysis, and 0.01 p.m with electron energy loss. The cryofixed material may be cryosectioned (sections a few microns thick) or ultracryosectioned (very thin sections). Obtaining 2 to 3 /~m thick cryosections is not straightforward, especially with plant samples. Performing ultracryosectioning is clearly even more difficult. Cryofractures of cell cultures grown on silicon substrates have also been carried out. 1When using analytical instruments equipped with cold sample stages and transfer systems for the frozen samples, one may perform the elemental analyses directly on the frozen hydrated specimens. This has been done in some cases with analytical electron microscopy; however, the irradiation damages produced by the electron beam on the frozen hydrated samples limit the sensitivity of the X-ray analyses. Although there is no theoretical obstacle to the analysis of frozen hydrated specimens by ion microscopy, this still has not often been attempted2; the first experiments 3 have demonstrated the feasibility of using a cold stage in an ionic microscope, but one must still study the consequences of the interaction of the primary ions with the more or less amorphous ice in the sample on the quality of the measurements. 1 S. Chandra and G. H. Morrison, Biol. Cell 74, 31 (1992). 2 M. T. Bernius, S. Chandra, and G. H. Morrison, Rev. Sci. lnstrum. 56, 1347 (1985). 3 S. Chandra, M. T. Bernius, and G. H. Morrison, Anal. Chem. 58, 493 (1986).

[21]

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Usually analyses are carried out using dehydrated specimens. Freezedrying is the most frequent means of dehydration. 4'5 With animal tissues, freeze-drying has been shown to be appropriate for the subsequent utilization of most methods of analyses (e.g., analytical electron microscopy, autoradiography and its alternatives, neutron capture radiography, nuclear probes). When the cells have big vacuoles, such as in most plant tissues, it is questionable whether the vacuolar contents remain immobilized, where they were, during ice evaporation or whether they collapse against the cytoplasmic layer. The concentration values obtained from freeze-dried specimens are clearly expressed relative to the dry weight of tissue; to express the concentration values relative to the total (i.e., fresh) mass of tissue, one has to evaluate the water content of all the cell compartments (usually 6 in the range of 60 to 85% with animal tissues, and up to more than 95% in the plant cell vacuoles). Freeze-dried cryosections or cryofractures have also been tried 1'7 for SIMS (secondary ion mass spectrometry) microscopy. However, reliable SIMS microscopy measurements require the analyzed surfaces to be as perfectly flat as possible, whereas freeze-drying causes the sample surface to become fairly uneven. This is especially true for plant tissues with highly vacuolated cells. Moreover, freeze-dried sections tend to curl, thus spoiling their adhesion to the conductive substrate (e.g., metallic disk, silicon wafer), which renders SIMS imaging difficult or impossible. To overcome the latter difficulty, it has recently been proposed 8 that freeze-dried cryosections be mounted on an indium substrate and firmly pressed against it; because indium is very malleable, even at low temperatures, this results in a very good adhesion of the sections. To our knowledge, however, this method has still not been tested with plant samples. As alternatives to the freeze-drying of frozen sections, one may embed the freeze-dried or freeze-substituted tissue fragments at low temperature9-14; then dry-cut sections (carried out at low temperature) can be used for microanalysis. It has recently been shown ~3 that 70% of the 4 p. Echlin, J. Microsc. Biol. Cell. 22, 215 (1975). 5 N. Roos, in "Electron Probe Microanalysis: Applications in Biology and Medicine" (K. Zierold and H. K. Hagler, eds.), p. 17. Springer-Verlag, Berlin, 1989. 6 T. Von Zglininicki and M. Bimmler, J. Microsc. 146, 77 0987). 7 S. Chandra and G. H. Morrison, this series, Vol. 158, p. 157. 8 E. W. Sod, A. R. Cooker, and G. H. Morrison, J. Microsc. 160, 55 (1990). 9 R. Wroblewski and J. Wroblewski, Histochemistry 81, 469 (1984). 10j. Wroblewski, R. Wroblewski, and G. M. Roomans, J. Electron. Microsc. 9, 83 (1988). IIE. Edelman, in "Electron Probe Microanalysis: Application in Biology and Medicine" (K. Zierold and H. K. Hagler, eds.), p. 33. Springer-Verlag, Berlin, 1989. 12 R. Wroblewski, J. Wroblewski, S. O. Wikstr6, and M. Anniko, Scanning Microsc. 4, 781 (1990). 13 E. Edelman, J. Microsc. 161, 217 (1991). 14 C. Quintana, J. Electron Microsc. Techn. 18, 411 (1991).

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

539

cellular potassium remained in muscle tissues after they were freezesubstituted and cryoembedded with Lowicryl resins (Bio-Rad, Hemel Hempstead, UK). Improving such cryotechniques (e.g., by performing cryoembedding at lower temperatures) might result in an almost total conservation of the diffusible substances within the tissues under study. It is noteworthy that the density of the Lowicryl resins is close to that of the water (I g cm -3) which they have replaced in the tissues. The concentration values which are measured with Lowicryl-embedded tissues thus practically correspond to concentrations expressed with reference to the fresh mass of tissues. 2.2. Precipitation Methods

Immobilization of a diffusible metal cation may be achieved by precipitating it in situ. The technique consists of adding a precipitating reagent to the fixative mixture, then dehydrating, embedding, and sectioning the sample as usual. The method is simple in its operational procedure, but it suffers from several difficulties and possible causes of artifacts (for a recent critical review, see Ref. 15). Nonnegligible losses of cations may occur during the process of chemical fixation and precipitation, depending on the relative rates of penetration of both reagents (fixative and precipitating) into the cell structures. When the penetration rates of the two reagents are high and not too different from one another, this favors the cations being precipitated close to where they were.16 The higher the number of precipitates and the smaller their size, the better is the spatial resolution of the method; this is obtained by using a high concentration of the precipitating reagent. There is no universal precipitating reagent, and the choice of reagent depends on the nature of the metal under study. For instance, calcium may be precipitated by oxalate, fluoride, or pyrophosphate. 17For sodium, calcium, and magnesium, one may use the well-known pyroantimonate method, 16 a method having in fact many variants. TM Potassium may be precipitated using sodium salts of perchlorate or of cobaltinitrite. Clearly, the use of a given precipitating agent may cause almost total precipitation of some cations and only partial precipitation of others. More generally, the precipitation of the free cations is governed by the following rules: (1) those ions are precipitated whose actual concentration reaches the value of the saturation concentration, and (2) the lower the solubility product of the insoluble salt and the higher the concentration of the precipitating reagent, the lower is the saturation concentration. In 15 p. 16 j. 17 j. 18 p.

Mentr6, Th~se de Doctorat d'Etat, Universit~ Pierre et Marie Curie, Pads (1991). A. V. Simson and S. S. Spicer, J. Histochern. Cytochem. 23, 575 (1975). C. Meyran, F. Graf, and G. Nicaise, Tissues Cell 16, 269 (1984). Mentr6 and F. Escaig, J. Histochem. Cytochem. 36, 49 (1988).

540

PROBES OF METAL ION ENVIRONMENTS

[2 1]

biological systems, there are metals (e.g., Fe, Zn, Cu) that are not present in the form of free ions but chiefly complexed or chelated by organic molecules. Even such metals may be displaced from their associations with cellular components if the stability constant of these associations is low enough. Therefore, depending on its nature and concentration, the precipitating reagent may result in precipitating mainly the free and loosely bound cations.~5 Only precipitated cations can be detected by electron microscopy, precipitation thus playing the part of a contrasting procedure. The precipitated cations will also be those most easily detected by the methods of analytical electron imaging (which have a low sensitivity), because the cation concentration is much higher in the precipitates. The methods with a higher sensitivity will usually detect and image both the precipitated and the chelated cations. This is typically the case with SIMS microscopy; however, owing to the existence of important matrix effects with this analytical method (cf. Section 7.3), it might be possible to discriminate (at least in the most favorable cases) between the free and the chelated in vivo forms of a given cation. To our knowledge, no experiment with that aim has yet been performed, but it might be worthwhile to try. Owing to their simplicity of use, precipitation methods have sometimes been preferred to more tedious cryomethods for the preparation of biological specimens for the mapping of diffusible substances. In doing so, one must be well aware of the complex mechanisms involved in the precipitation processes, in order to avoid misinterpreting the images obtained. It has also been suggested that precipitation methods be used together with cryosubstitution. For instance, cobaltinitrite has been added to ethanol for the precipitation of potassium, ~9and oxalic acid to acetone for that of calcium, 2° with ethanol and acetone being used as cryosubstitution solvents.

3. Analytical Electron Microscopy

3.1. Bases of Analytical Electron Microscopy 3.1.1. Introductory Statements. The term analytical electron microscopy (AEM) implies the ability of the electron microscope to generate quantitative information from a well-characterized area of the specimen. From the information thus provided, one may obtain information about the sample elemental composition (H excepted). In electron microscopy, t9 M. E. V. Van Steveninck and R. F. M. Van Steveninck, J. Microsc. 122, 259 (1981). 20 G. Nicaise, I. Gillot, A. K. Julliard, E. Keicher, B. Blaineau, J. Amsellen, J. C. Meyran, M. I. Hernandez-Nicaise, B. Ciapa, and C. Glezal, Scanning Microsc. 3, 199 (1989).

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

541

the analytical methods that can be used to detect metal elements in biological systems are X-ray microanalysis (XRMA), electron energy loss spectrometry (EELS), and Auger electron spectrometry (AES). The sensitivity of these different methods is not good enough for them to be considered as trace-analysis techniques. Thus, the terms microanalysis and nanoanalysis refer to the micro and nano dimensions of the minimal volume analyzed. The sensitivity and the spatial resolution of XRMA, EELS, and AES are not the same. These different methods of analysis are thus complementary to one another, and they are also complementary to the other analytical methods described in this chapter. None of the methods of analytical electron microscopy is suitable for discriminating between the isotopes of a chemical element. 3.1.2. Physical Bases of Analytical Electron Microscopy and Analytical Instruments. The interaction of a high-energy electron with a sample atom can be elastic (i.e., the incident electron practically loses no energy but is scattered at a large angle from its initial trajectory) or inelastic (in which case the incident electron gives part of its energy to the target atom, and the deflection angle is usually much less). Incident electrons with energies in the range of 10 to 100 keV, as usually employed in electron microscopy, have a high probability of ionizing the inner shells of the impinged atoms, with emission of "secondary electrons" (Fig. 2A). Deexcitation of such ionized states may then occur by X-ray emission (Fig. 2B) or by a process of internal energy conversion (Fig. 2C), which results in the emission of an electron from a more outer shell (Auger electron). The probabilities of the radiative and of the Auger deexcitation processes

hv A /r

l:l

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FIo. 2. Atomic excitation and deexcitation processes. (A) E x a m p l e of atom ionization by a fast incident electron ( 0 ) at the level of an inner shell (1) hole, ([]) with emission of a s e c o n d a r y electron. (B) Deexcitation by emission of an X-ray photon with h u = E~ - E r . (C) Nonradiative deexcitation by emission of an A u g e r electron (energy E = E I - Ev - Er).

542

PROBES OF METAL ION ENVIRONMENTS

[21]

are complementary. The high-Z elements (Z being the atomic number) have a higher probability for the radiative process, and the Auger process is favored for low-Z elements. There are several types of analytical instruments, namely, the electronprobe microanalyzer (EPMA), the conventional transmission electron microscope (CTEM), the scanning electron microscope (SEM), and the scanning transmission electron microscope (STEM). The EPMAs are meant for X-ray analysis of bulk objects. They are equipped either with a wavelength dispersive spectrometer (WDS) or with an energy dispersive spectrometer (EDS). They function in the mode "fixed probe." Location of the analyzed area is carried out either by optical microscopy or using a TV camera. The first EPMA was built in France 2~'22in 1949, and the instruments were commercialized by the CAMECA Company (Courbevoie, France) in and after 1956. SEMs have been equipped with WDS X-ray detectors z3 since 1956 and with EDSs since the 1970s. Fixed probe or scanning analyses, as well as mappings, can be recorded, whereas the corresponding structural images are obtained from the secondary or the backscattered electrons. The STEM was created in 1970 at the Enrico Fermi Institute of the University of Chicago. 24 Using a new high-brightness field-emission electron gun, a 0.5 nm electron probe intense enough for obtaining scanning transmission images of thin objects was manufactured. One advantage of this type of instrument compared to the CTEMs is their ability to acquire simultaneously, in a single scan, several different signals issuing from the specimen; for instance, they can be equipped both with electron energy loss spectrometers and with EDS X-ray detectors. In the late 1970s, combined CTEM/STEM microscopes began being commercialized. These instruments were equipped with a condenser-objective lens whose sphericity and chromatic aberration coefficients were of the same order of magnitude in both modes, conventional and scanning. When using conventional electron guns the resolution of the scanning transmission images was 1.5 nm, whereas it was 1 nm with LaB 6 electron guns. Some of these instruments are now equipped with field-emission electron guns, which further improves their performance. Today medium voltage (200, 300, or 400 kV) CTEM/STEMs are available. 3.1.3. Measurements Performed in Analytical Electron Microscopy. The term analytical electron microscope (AEM) is often used with the restricted meaning of STEM or even of CTEM/STEM. In an AEM, the 21 R. 22 R. 23 V. 24 A.

Castaing and A. Guinier, Proc. 1st !ntern. Conf. Electron Microsc. Delft, 60 (1949). Castaing, Th~se de l'Universit6 de Paris, publications ONERA 55, 1 (1951). E. Coslett and P. Duncumb, Nature (London) 177, 1172 (1956). V. Crewe and J. Wall, J. Mol. Biol. 48, 375 (1970).

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PHYSICAL METHODS TO LOCATE METAL ATOMS

543

signals (unscattered, elastically and inelastically scattered electrons, and X-ray photons) are recorded using appropriate detectors in the form of a current signal, I. This current is usually a function of three independent variables, r (position in the space of the object), 0 (detection angle), and E (energy) or h (wavelength). 25 By integrating in the solid angle of signal collection, 0, two different types of intensity measurements, Ir(AE) and IaE(r), may be performed. With Ir(AE) one obtains the electron or X-ray spectrum characteristic of the sample area under analysis. This spectrum is a histogram of the distribution of the intensities as a function of the electron energy loss values (in EELS) or of the wavelength (in X-ray) or energy values (in X-ray or Auger electron emission). With digitized data, the spectrum is the histogram of the numbers of counts measured in a series of detection channels. With IaE(r), the result is an energy-filtered image, or "elemental mapping." It shows the two-dimensional distribution of a given energy loss of an electron or energy of a characteristic X-ray photon or Auger electron, representing the image of the distribution of the corresponding chemical element in the imaged area. In SEM and STEM instruments, this image is acquired by scanning the electron beam on the object and recording the characteristic X-ray or electron signals. In EPMA, the electron beam is immobile while the object is mechanically moved in x and y directions. In CTEM, stigmatic (instead of scanning) EELS images can be recorded (filtered electron transmission electron microscope, FETEM). In all these cases, signal recording can be analog or digital.

3.2. Auger Electron Spectrometry When deexcitation of an ionized atom occurs via emission of Auger electrons, the energy, E, of the electrons depends on three energy levels (El, El,, and El,,) of the atom (see Fig. 2C), with E=

E , - Ev - Er

The Auger energy spectrum is thus characteristic of the sample elemental composition, but this spectrum can also provide information concerning molecular bonds when a valence energy level is involved in the transition. Auger analysis is well adapted to the study of light elements such as carbon, nitrogen, and oxygen. Moreover, since the energy of the Auger electrons is weak, those produced far below the sample surface are absorbed and do not get out. Therefore, Auger spectrometry is a method for surface analysis, representative of only a few atomic layers below the sample surface. Up to now, this type of analysis has been performed only 25 C. Colliex, in "Advances in Optical and Electron Microscopy" (R. Barer and V. E. Coslett, eds.), Vol. 9, p. 65. Academic Press, London, 1984.

544

PROBES OF METAL ION ENVIRONMENTS

ho

[21]

h

FIG. 3. Total X-ray emission spectrum: intensity I as a function of wavelength h, representing a set of characteristic peaks on the bremsstrahlung continuum (beginning at h0).

with massive objects, using a SEM. Because the applications to biological specimens are extremely few, 26 we do not describe this method in more detail.

3.3. X-Ray Spectrometry 3.3.1. X-Ray Emission Characteristics. When deexcitation of an ionized atom occurs via X-ray emission, the energy, E, of the X-ray photon, and also its frequency, v, or wavelength, h, depends on two energy levels, El and Ev, of the atom (see Fig. 2B), with c

E= hv= h-~= E i - Ev (h is Planck's constant). It is noteworthy that the probabilities for the different possible transitions Ev/Et to occur are not equal, and some transitions are not allowed. In addition, interferences may occur between L or M rays of heavy elements and K rays of lighter elements. This is the case, for instance, with sulfur K~ (E = 2.307 keV) and molybdenum Ms rays (E = 2.293 keV). Moreover the total X-ray spectrum observed (Fig. 3) corresponds to the superimposition of the characteristic spectrum of the atoms present in the sample onto a continuous bremsstraMung specz6 A. Lignell, R. Kaln, G. M. Roomans, A. von Hofsten, and O. Vingsbo, Scanning Microsc. 3, 57 (1989).

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

545

trum. The bremsstrahlung is due to the deceleration of the incident electrons in the coulomb field of the target atom nuclei. The bremsstrahlung energies range from the value of the initial energy of the incident electrons to zero, but the bremsstrahlung is much more intense at low compared to high energies. Using appropriate detectors, analysis of the X-ray spectra makes it possible to detect any chemical element whose atomic number is above 4. 3.3.2. X-Ray Spectrometers. WDS spectrometers (Fig. 4) consist of an analyzer made of a curved crystal which diffracts the incident X-ray photons, a proportional counter, and a single-channel analyzer (SCA). The X-ray diffraction occurs according to Bragg's law 2d sin a = nk where d is the interreticular distance, a the photon incident angle, and h the X-ray wavelength. In a fully focusing spectrometer, the sample, the analyzing crystal, and the proportional counter are arranged in a circle called the focusing or Rowland's circle. The emission X-ray spectrum, Ir(Ah), is obtained by progressively varying the incidence angle, a, of the X-rays on the crystal. The crystals are given a linear movement in order to keep the "take-off" angle constant. The information is acquired in serial mode. With such an instrument, one may detect all the elements between boron and uranium. The energy resolution can be as good as 5 eV, depending on the analyzer crystal. Ell

\o IV

V

D

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Fxc. 4. Schematic representation of an X-ray WDS spectrometer. (1) Sample; (2) crystal analyzer; (3) proportional counter; (4) Rowland's circle; (I) preamplifier; (II) amplifier; (III) single-channel analyzer (SCA); (IV) internal clock; (V) counter; (VI) integrater; (VII) recorder; ~, takeoff angle; HT, high tension; EB, electron beam.

546

[21]

PROBES OF M E T A L I O N E N V I R O N M E N T S

The EDS X-ray spectrometers (Fig. 5) possess a reverse-bias p-i-n semiconductor lithium-drifted silicon crystal detector which collects the emitted spectrum. When an X-ray photon is absorbed in the semiconductor, it creates electron hole pairs; the number of pairs thus created is proportional to the energy of the X-ray photon. The resulting charge pulse signal is converted to a voltage pulse signal in a charge-sensitive preamplifier. This voltage pulse signal is then amplified and sent to a multichannel analyzer (MCA). The resulting voltage distribution is displayed on a cathode ray tube (CRT) or an x/y recorder. Note that the information is thus acquired in a simultaneous mode, and the EDS spectrometer is not a fully focusing detector. The content of the MCA memories can be transmitted to a computer and stored in a file memory for further processing. Software packages appropriate to peak identification and quantification are commercially available. The Si(Li) detector, which is cooled at 77 K, is usually separated from the microscope column by a thin beryllium window. Because of X-ray absorption in this window, the detection of chemical elements lighter than Na is not possible. However, windowless EDS spectrometers (or spectrometers with very thin windows) also exist; they can detect chemical elements with Z greater than 4, as it is the case with the WDS spectrometers.

EB

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FIG. 5. Schematic representation of an X-ray EDS spectrometer. (1) Sample; (2) Si(Li) crystal detector; (3) cryostat; (I) preamplifier; (II) amplifier; (III) baseline control; (IV) analog/digital converter; (V) multichannel analyzer; (VI) pileup rejector; EB, electron beam.

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

547

The energy resolution of EDS spectrometers depends on the X-ray energy; it is approximately 150 eV at 5.9 keV (Mn Ks). This poor energy resolution is responsible for peak interference. In qualitative analysis, this can be overcome by using the higher energy peaks. For instance, the identification of Mo in the presence of S may be resolved by considering Mo K s . In quantitative analysis, the overlapping of peaks is a more difficult problem, and one has to remove the interferences. For instance, for measuring low calcium concentrations with the Ca K~ peak in the presence of potassium, the interfering K K~ peak must be subtracted. Compared to WDS, the EDS instruments have the advantages of a smaller data collection time, a smaller size, and a better quantum and geometrical collection efficiency. On the other hand, WDS instruments have a better energy resolution and a larger signal-to-background ratio. The SEM/EPMA instruments possess both a WDS and an EDS. Only EDS detectors are used in the CTEM/STEM microscopes. 3.3.3. Quantitative Analysis. X-Ray spectrometry can be used with both thick and thin objects. For quantitative analysis, the continuous bremsstrahlung spectrum must be subtracted. Different background subtraction methods exist (e.g., the linear interpolation method in WDS and the nonlinear Kramer method and digital filter methods in EDS). With massive objects, one has to correct the measured intensities for the atomic number, fluorescence effects, and absorption effects. With thin objects, the fluorescence and absorption effects may be assumed to be negligible. If C~ and Cy are the mass fractions of two chemical elements, x and y, and Sc~ and S,.y the corresponding characteristic X-ray signals, then a relative quantitative analysis can be obtained by use of the Cliff-Lorimer relationship 27: C~

S=

In this equation, Kxy is the Cliff-Lorimer constant, which may be calculated or measured by use of appropriate standard samples containing known concentrations of x and y. This relative quantitative method becomes an absolute quantitative method when all the chemical elements, j, present in the samples, can be analyzed. In this case, one may state

Ec .= 1 J

27 G. Cliff and G. W. Lorimer, J. Microsc. 103, 203 (1975),

548

PROBES OF METAL ION ENVIRONMENTS

[21]

and thus obtain a number of independent mass-fraction equations just equal to the number of chemical elements in the samples. With biological samples, one may also use the absolute method of Hall, 28 whose basic equation is written

W - Wo where W is the continuum intensity of a region free from characteristic peaks, W0 is the contribution of the surroundings, A and Z are the mass and charge numbers (with parentheses indicating the mean value of the expression), K is a parameter whose value has to be determined using standards with a known composition, and the other symbols have already been defined. The detection limit depends on the number of X-ray photons detected per unit of mass and unit of time, the background at the peak under consideration, the counting time, and the statistical treatment used. With massive objects, routine X-ray spectrometry allows the detection of concentrations in the range of 50 to 500 ppm with a WDS detector, and 0.02 to 0.2% with an EDS, in volumes of the order of 1/.,m3. With thin objects, the lateral resolution is close to the diameter of the electron beam; the detection limit, in routine measurements, is of the order of 1 mmol kg -~ (i.e., -600 atoms in a volume of 10-15 cm 3) with a counting time of 100 sec. 3.3.4. Filtered X-Ray Images. In the scanning mode, filtered X-ray images may be recorded with both types of spectrometers, WDS and EDS. One begins by selecting the wavelength or energy value of the more intense characteristic photon of the chemical element under analysis. In a WDS, the analyzing crystal is positioned at an angle corresponding to the maximal intensity. In an EDS, an energy window enclosing the maximum of the peak is chosen. In analog images, the signals delivered by the single- or multichannel detector at each point of the sample, during one or several scans, are used to modulate the electron beam of the CRT. The image is visualized on the instrument screen and recorded on photographic film. Because the background is not subtracted, the information yielded is qualitative: the brightest areas correspond to the highest concentrations of the studied element, and the black areas mark regions where this element is not present. For the major elements, the time for acquiring an image is of the order of a few minutes, whereas it can be up to 30 min for the minor elements. The emitted signals may be digitized by means of an analog-to-digital converter (ADC) and stored in the mem28 T. A. Hall, Scanning Microsc. 3, 46 (1989).

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

549

C o u n t s xlooo Counts xlO00 CARBON-K

•"IRON-M23

10C

11

:

~A

5

LCIUM-L23

LEL

i

..

\

X"""°°E"'K

OXYGEN-K

50 300 .

.

400

.

500 .

. 600

700 ~ E

\

(eV)

ELi ;TI~;

::,, O

100

200

300

400

500 --'

600 '

7 O0':~E

FI~. 6. Typical high energy loss spectrum from an unstained section of the oocyte of

Planorbarius embedded in a mixture of Epon and Araldite. The selected area, typically 100 x 100 nm2, contains a yolk platelet, with a dense organized aggregation of ferritin molecules (specimen courtesy of A. Y. Jeantet and C. Quintana). The spectrum shows the main features: elastic peak, low energy loss (LEL) region at about 25 eV, followed by a regularly decreasing backgroundover which successiveedges for the marked elements can be distinguished. Changes in the intensity scale occur between each of the major contributions. [Reproduced from C. CoUiex, C. Jeanguillaume, and C. Mory, J. Ultrastruct. Res. 88, 177 (1984), with permission].

ory of a computer. With an EDS the simultaneous recording of several images is possible. For obtaining quantitative images, the background has to be recorded and subtracted.

3.4. Electron Energy Loss Spectrometry 3.4.1. Characteristics o f lnelastically Scattered Electron Spectrum. Several kinds of characteristic peaks are observed in an E E L S spectrum (Fig. 6). There is a high maximum at zero energy loss which corresponds to the elastically scattered electrons. Several peaks in the low energy losses (in the range of 1 to 50 eV) correspond to the excitation of valence and conduction electrons. A series of characteristic peaks in the high energy loss region (from 50 eV to approximately 2 keV) contain the information relative to atom core levels and may thus be used for elemental analysis. A n y chemical element with Z greater than 2 can be detected.

550

PROBES OF METAL ION ENVIRONMENTS

[21]

The characteristic peaks are superimposed on a continuous background owing to multiple inelastic events. 3.4.2. E E L S Spectrometers. The first EELS spectrometer was developed for a CTEM instrument by Castaing and Henry 29 in 1962. It consists of a triangular magnetic prism associated with an electrostatic mirror. The spectrometer, placed at the exit of the objective lens, allows the production of achromatic stigmatic images of the specimen and energy loss (i.e., filtered) images. This spectrometer was first commercialized by the French company Sopelem (in 1967), then by the German company Zeiss (Oberkochen, Germany) (with the EM902 CTEM). A three-magnet l) filter was developed by Zanchi et al. 3° in 1975 for high-voltage (1 MeV) microscopes. A new 120 kV CTEM equipped with an f~ filter is now available (Zeiss EMg12 l-l). In STEM and CTEM/STEM instruments, the spectrometer most often is a magnetic prism located at the extremity of the column. It consists of a curved electromagnetic lens giving the energy loss spectrum of the electrons. This spectrometer is well adapted for treating analytical problems with a spatial resolution at the nanometer level. The spectrometer is optically coupled with the microscope (in a manner which is not the same for STEM and CTEM/STEM). In most EELS spectrometers, the recording system is a serial electron detector located several centimeters in front of the exit slits. The electron detector is made of a plastic scintillator and a photomultiplier coupled to a threshold discrimination system. With this system single electron counting is possible, and there is a large dynamic range. EELS spectra can also be recorded using a parallel detection system (PEELS). In most cases, the PEELS detectors consist of semiconductor cellular devices, namely, charge-coupled devices (CCDs) and photodiode arrays (PDAs). According to Chapman et al., 31 the two-dimensional (2D) CCDs and the PDA onedimensional elongated cells would be more appropriate for image and spectrum recording, respectively. 3.4.3. Quantitative Analysis. Quantification without standards can be achieved by application of a theoretical model relating the number of atoms of a given chemical element per unit area to the net signal corresponding to this element. For obtaining this net elemental signal, the background has to be subtracted. The exact shape of the background in the various parts of the spectrum is still not very well known, and different models are used for fitting the different regions of the spectrum. The detection limit depends on the background, which in turn depends on the specimen thick29 R. Castaing and L. Henry, C.R. Acad. Sci. Paris Ser. B 255, 76 (1962). 30 G. Zanchi, J. P, Perez, and J. Sevely, Optik 43, 495 (1975). 37 j. N. Chapman, A. J. Craven, and C. P. Scott, Ultramicroscopy 28, 108 (1989).

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

551

ness. The specimen thickness is thus the main factor controlling the detection sensitivity in the measurement of low concentrations of chemical elements in biological samples. In 1987, Shuman and Sombyo 32announced that it was possible to detect as few as 3 Ca atoms in standard organic samples, by use of a TEM/STEM/PEELS instrument with a spot 10 nm in diameter. 3.4.4. Comparison of XRMA with EELS. Calcium measurements have been performed in plastic-embedded insulin crystals using different types of spectrometers (X-ray, EELS, and PEELS). 33The total acquisition time, for similar values of the signal-to-noise ratio, was 5 sec for PEELS, 60 sec for EDS, and 1500 sec for EELS. From the X-ray spectrum, the Ca concentration in insulin was estimated close to 150 mmol kg-1 (dry weight), and with an acquisition time of 1500 sec the detection limit for PEELS should be about 1.5 mmol kg -1. In Ca measurements performed on embedded sections of pancreatic islets of Langerhans, and in Fe measurements on cryosections of erythrocytes, PEELS was advantageous only when using sections of the embedded material 0.5 h in thickness (where h is the mean free path for inelastic collision). This excludes the possibility of using freeze-dried cryosections, 1.2 h in thickness, which are normal in XRMA analyses. The relative sensitivities of PEELS and XRMA as a function of the atomic number of the chemical element under study 34 are plotted in Fig. 7.

3.5. Biological Applications of Analytical Electron Microscopy The biological applications of AEM are already many, 35-4° especially in relation to medical and physiological problems. XRMA has been used in pathology, 36-38 physiology, 39 toxicology, 36 and cytochemistry. 4° It has been used, for instance, (1) for the detection of exogeneous particles retained in the lungs (in relation to silicosis or inhalation of asbestos fibers), 36 (2) for the detection of abnormal deposits of Ca in cells and 32 H. Shuman and A. P. Sombyo, Ultramicroscopy 21, 23 (1987), 33 R. D. Leapman and R. L. Ornberg, UItramicroscopy 24, 251 (1988). R. D. Leapman and J. A. Hunt, Microsc. Microanal. Microstruct, 2, 231 (1991). 35 C. Quintana and C. Nicaise, J. Electron Microsc. Tech. 18, 424 (1991). 36 G. M. Roomans, Scanning Microsc. 4, 1055 (1990). 37 p. Galle, in "Advances in Nephrology" (J. Helige, J. Crosnier, J. P. Grunfeld, and M. H. Maxwell, eds.), Vol. 12, p. 404. Yearbook Medical Publ., Chicago, 1983. 38 j. D. Shelburne, J. A. Tucker, V. L, Rogli, and P. Ingram, in "Microprobe Analysis in Medicine" (P. Ingrain, J. D. Shelburne, and V. L. Rogli, eds.), p. 55. Hemisphere, New York, 1989. 39 A. Le Furgey, M. Bond, and P. Ingram, Ultramicroscopy 24, 185 (1988). 4o A. T. Summer, J. Electron Microsc. Tech. 9, 99 (1988).

552

PROBES OF METAL ION ENVIRONMENTS

[21]

(/) X 12a W o

10-

l L23

8-

65

_J W W

6

_

K

4

2 .

0

I

I

5

10

"

I 15

I 20

AtomicNumber(Z)

I 25

.

.

.

30

FIG. 7. Relative sensitivities, S/o's, of EELS and XRMA (with an energy dispersive spectrometer, EDXS) as a function of the atomic number for K and L23 shell excitation. S, signal; o-~, standard deviation of the estimated signal.

tissues or of pathological intracellular accumulation of metals (e.g., Fe in hemosiderosis, 36 A1 in various tissues of hemodialysis patients, 37 and Cu in Wilson's disease41), and (3) for studying diseases related to anomalous distribution of diffusible ions 36 (e.g., distribution of CI and Ca in cystic fibrosis, unusually high value of the Na/K ratio in neoplasms but not in other types of proliferating cells, general increase of electrolyte concentration in aging cells, and abnormally low Zn concentration in sperm nuclei of infertile humans). The applications of X-ray microanalysis in physiology have recently been reviewed. 39 Quantitative X-ray images have now been obtained in a few cases 42 (Fig. 8). EELS has been applied mainly to the analysis of highly concentrated elements in small volumes of material. For applications to the detection of mobile ions (Na +, K ÷, Mg 2÷, Ca2+), freeze-dried cryosections of cryofixed tissues are too thick for EELS measurements; ultrathin sections of freezesubstituted and cryoembedded tissues are more suitable. Analyzing potassium in a carbon matrix is not feasible, because there is an interference between the L edge of potassium and the K edge of carbon. The biological data acquired with EELS spectrometry have already been reviewed. 43,44 41 K. Tousimis and J. Adler, J. Histochem. Cytochem. 11, 40 (1963). 42 A. Le Furgey, S. D. Devilla, D. A. Kopf, J. R. Sommer, and P. Ingram, J. Microsc. 165, 191 (1992). 43 C. Jeanguillaume, Scanning Microsc. 1, 437 (1987). 44 G. T. Simon and Y. M. Heng, Scanning Microsc. 2, 257 (1988).

[2 1]

PHYSICAL METHODSTO LOCATEMETALATOMS STEM

SULPHUR

SODIUM

MAGNESIUM

CHLORINE

POTASSIUM

553

PHOSPHORUS

CALCIUM

FIG. 8. STEM image and quantitative XRMA images from kidney glomerular mesangial cells (magnification, × 3880). (Reproduced from Le Furgey et al.,42with permission from the Royal Microscopical Society.)

It has been claimed that, using a C T E M ( F E T E M 902 Zeiss) instrument, it was possible to produce P and Ca images in various cell organeUesY H o w e v e r , almost all of the images were obtained from only one preedge image for calculation and subsequent subtraction of the background below the characteristic peaks, and it has been demonstrated 46'47 that only one preedge subtracted image provides the mass thickness image rather than the chemical image of phosphorus. In STEM, the background subtraction with multiple preedge images is easier than in F E T E M . Using P E E L S detection considerably shortens the time necessary to acquire images, which also decreases irradiation damage. 4. Synchrotron Radiation-Induced X - R a y Fluorescence Analysis 4.1. P h o t o n - l n d u c e d X - R a y Emission 4.1.1. X - R a y Fluorescence and Synchrotron Radiation-Induced X - R a y Fluorescence Analysis. It has been pointed out (see Section 3.1.2) that, after excitation of an inner electronic shell, atoms can deexcite with

45F. P. Ottensmeyer, D. W. Andrews, A. L. Arsenault, G. T. Simon, and G. C. Weatherly, Scanning 10, 227 (1988). 46C. Colliex, C. Jeanguillaume, and C. Mory, J. UItrastruct. Res. 88, 177 (1984). 47R. D. Leapman, Ann. N.Y. Acad. Sci. 483, 326 (1986).

554

PROBES OF M E T A L ION E N V I R O N M E N T S

[21]

simultaneous emission of X-rays. Instead of using electrons for atom excitation, one may also use energetic photons (i.e., X-rays). In fact, atom excitation with photons has some interesting properties. Photons mostly interact with inner shell electrons, and the photoelectric cross section varies approximately as Z 4 (where Z is the atomic number of the target atom). On the other hand, ionization cross sections induced by charged particles rougly vary as Z -4 and are more important for the outer than the inner shell electrons by orders of magnitude. X-Ray induced X-ray fluorescence (XRF) has long been used for elemental analysis. However, with conventional X-ray sources, the sensitivity was usually too low for useful applications to biological samples. The advent of synchrotron radiation (SR) has largely pushed down the detection limits achievable (far under the parts per million limit for most elements) and stimulated very important changes in the classic XRF apparatus. Synchrotron radiationinduced X-ray fluorescence analysis (SRXRF) is still under development, and there is still much to be done to reach the degree of refinement commonly found with other techniques. The most promising development is the realization of a microprobe with a lateral resolution in the micron range. SR offers many interesting features which make it an ideal excitation mode of X-ray fluorescence. On deviation in bending magnets or other insertion devices, relativistic electrons (or positrons) radiate energy (SR). The energy spectrum is continuous from the far-infrared and extends more or less into the X-ray regime, depending on the machines. Large fluxes are still available at 100 keV photon energy in the new dedicated facilities [e.g., about 1013 photons sec -1 mrad -~ in a 0.1% bandwidth are expected in the wiggler line of the European synchrotron radiation facilities (ESRF) at this energy]. The spectral brilliance of the source (photons sec -~ mm -2 mrad -2 in a 0.1% bandwidth) is several orders of magnitude higher than that of the best X-ray tubes. The SR is naturally collimated in the vertical plane, the angle of divergence being typically less than 0.1 mrad. In addition, this radiation is highly polarized in the orbital plane of the electrons, which can be used to reduce considerably scattering processes from the sample. Finally, this very low vertical divergence makes an efficient monochromatization of the photon beam possible. The excitation energy can thus be continuously adjusted and chosen so as to enhance the observation of a given element. Consequently, the background under the fluorescence peaks is considerably reduced, improving the minimum detectable limits (MDL). 4.1.2. Application of Synchrotron Radiation to Elemental Analysis. The use of sagitally curved crystals as monochromators enables one to

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

555

easily concentrate SR on a small spot of submillimeter size. Thus, SR can readily be used for elemental analysis of trace elements in small samples. As an example, Fig. 9 presents the spectrum of Friend leukemia cell nuclei. The cells were exposed for 15 min to an anticancer drug containing a gold atom, tTAuP, at a concentration of 1 /xM. The excitation energy was set at 19 keV, and the spectrum was recorded with a Si(Li) detector. For the sample preparation, 1.5 x 10 6 cells were collected and lysed with nonionic detergent to separate the nuclei. The gold L X-ray spectrum shows up nicely, and a MDL of 60 pg of gold was calculated for a 1000sec exposure. Characteristic lines of other constituents of the cell also appear (Fe, Cu, Zn, Br) as well as Sr that was added as an internal standard. During this work it appeared that Zn might be a good indication of the number of cells in the beam, and this shows the interest of the multielement aspect of this analytical method. The very important peak near 18-19 keV is due to Rayleigh and Compton scattering by the sample. If a white beam excitation had been used, the background under the fluorescence peaks would have been everywhere as high as this peak, and, in this sample, gold would have been very difficult to observe. In fact, although the X-ray spectrum was recorded at 90° to the excitation beam and in the plane of polarization where Rayleigh and Compton scattering are at their minima for horizontally polarized light, this undesirable peak is still quite important in the spectrum. This severely contributes to the MDL because of the associated background and saturation of the detection. Note that with the new generation dedicated machines this scattering can be reduced by

103

~ 102 0

101 0

5

10 Energy (keV)

15

20

FIG. 9. SRXRF spectrum of a pellet of Friend leukemia cell (FLC) nuclei. The cells growing in the exponential stage were exposed for 15 min to tTAuP [(8-thiotheophyllinate)(triphenylphosphine)gold(I)] at a concentration of 1 /,M, and, after separation, the cell nuclei were excited with a 19 keV synchrotron radiation beam.

556

PROBES OF METAL ION ENVIRONMENTS

[21]

at least a factor of 10. The argon peak which appears in the left part of the spectrum is due to the air path traveled by the X-ray beam. In fact SRXRF can be performed under air, which is of considerable interest for biological samples. Such a study would not have been possible using conventional XRF, nor perhaps even with particle-induced X-ray emission (PIXE) (see Section 6.2.1) where the MDL has been found to be 50 times higher with 1.9 MeV protons (e.g., 2.3 ng for Pt). 48

4.2. Synchrotron Radiation-Induced X-Ray Fluorescence Microprobes SRXRF microprobes are now under development in every SR center. Some projects are already under operation, but all will profit from the progress made in recent years in the field of X-ray optics. In giving examples of existing microprobes, we do not intend to be exhaustive but only to present various approaches in the design of such facilities. It must also be kept in mind that the performances depend very much on the source brilliance, so that direct comparisons are difficult. The National Science Laboratory (NSL; Brookhaven, NY) project is one of the most advanced, and many experiments in various fields (geology, biology, astrophysics, etc.) have been performed since 1985. 49,50 The equipment consists of a monochromator (Si channel-cut) and an ellipsoidal 8:1 focusing mirror working at grazing incidence. This leads to a 60 × 20/xm 2 image of the light source, which can be reduced to 5 to 30 ~m with a pinhole collimator a few millimeters upstream of the target. About 6 x 109 10-keV photons sec -1/zm -2 are expected for 500 mA beam current. The Synchrotron Radiation Source (Daresbury, UK) project was started in 1984,51 and there have since been very important improvem e n t s ) 2 The apparatus now uses a focusing monochromator bent in an ellipsoidal mold. At 15 keV the beam size (full width at half-maximum, FWHM) is around 15 x 20/.tm 2 with a flux of a few million photons sec -~ /zm -2 under usual running conditions. With this apparatus trace elemental

4s F. Sommer, P. Chevallier, H. Tapiero, P. Massiot, P. Galle, I. Silvestro, P. Arizti, and D. Picot, Vacuum 42, 801 (1991). 49 B. M. Gordon and K. W. Jones, Nucl. Instrum. Methods Phys. Res., Sect. B B10-11, 293 (1985). 50 j. R. Chen, E. C. T. Chao, J. A. Minkin, J. M. Back, K. W. Jones, M. L. Rivers, and S. R. Sutton, Nucl, lnstrum. Methods Phys. Res., Sect. B B49, 533 (1990). 51 M. Prins, S. M. Kuipper, and M. P. A. Viegers, Nucl. Instrum. Methods Phys. Res., Sect. B B3, 246 (1984). s2 F. Van Langevelde, O. K. Bowen, G. H. J. Tras, R. D. Vis, A. Huizing, and D. K. G. De Boer, Nucl. Instrum. Methods Phys. Res., Sect. A A292, 719 (1990).

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PHYSICAL METHODS TO LOCATE METAL ATOMS

557

distributions have been carried out in line scans of biological samples, 53 but with 2 min acquisition time per pixel. The Photon Factory (Tsukuba, Japan) project 54 was proposed as a Walter Type I optical system to focus a monochromatic beam. The spot size is of a few tens of square microns at 10 keV with enough flux to perform trace element analyses. The Lawrence Berkeley Livermore (Berkeley, CA) project 5s is based on using multilayer spherical reflectors to monochromatize and to focus the SR. Two such reflectors are used in a Kirkpatrick-Baez geometry to avoid most of the spherical aberrations. A 10 × 10/xm 2 spot size was obtained at 10 keV with a flux of about 3 × 10 7 photons sec -~ /xm-2. MDLs as low as a few femtograms for a 60-sec exposure per spot for elements like Ni have been reported. 56 The Laboratoire pour l'Utilisation des Rayonnements Electromagngtiques (LURE, Orsay, France) SRXRF microprobe, still under construction, uses Bragg-Fresnel lenses (BFL) as a monochromator and focusing optics. Two lenses are mounted in a Kirkpatrick-Baez geometry. This system seems to be the most promising way to achieve a real SR microprobe. Preliminary results have shown a beam spot of 1.7/zm in diameter for a 100/xm entrance beam aperture and a total number of photons of around 104, which is remarkable for a machine of such low brilliance. 57 Up to now SR microprobes have not been able to achieve a lateral resolution better than 10 p~m, which is quite insufficient for most biological applications. Available fluxes are in the range of around 107 photons sec -I ~m -2, which is not enough for trace element study at the parts per million level with short exposure times (12 to 2 sec per pixel). Nevertheless, great improvements can be expected soon. The use of wigglers or undulators, instead of bending magnets, would result in a gain of typically 2 (to 3) or 4 (to 6) orders of magnitude, respectively, in the photon flux on the target; moreover, it has been proved that a micron size beam spot can be reached with BFL. These real microprobes will use the advantages of SR, namely, the very little energy released in the sample compared to electrons and 53 G. H. J. Tras, F. Van Langevelde, and D. R. Vis, Nucl. lnstrum. Methods Phys. Res., Sect. B B$II, 343 (1990). 54 y . Goshi, S. Aoki, A. Ida, S. Hayahowa, H. Yamasi, and K. Sahurai, Adv. X-Ray Anal. 31, 495 (1988). 55 j. H. Underwood, A. C. Thompson, Y. Wu, and R. D. Giauque, Nucl. lnstrum, and Methods Phys. Res., Sect. A A266, 296 (1988). 56 A. C. Thompson, J. H. Underwood, Y. Wu, R. D. Giauque, K. W. Jones, and M. L. Rivers, Nucl. Instrum. Methods Phys. Res., Sect. A A266~ 318 (1988). 57 A. Erko, E. Khzmalian, L. Panchenko, S. Redkin, V. Zimenko, P. Dez, P. Chevallier, C. Kahn Maler, A. Freund, and B. Vidal, "X-Ray Microscopy 1990," London, 3-7 September, 1990.

558

PROBES OF METAL ION ENVIRONMENTS

[21]

protons, and will allow the mapping of elements at the sub-parts per million level, in air, and nondestructively, in a reasonable time.

4.3. X-Ray Microtomography With computerized tomography one can obtain a cross section of a three-dimensional object. With the microbeam already available it has been possible to reconstruct such images with a resolution in the range of 10 /zm. For example, Spanne and Rivers 58 obtained the image of a freeze-dried caterpillar head slice. The tomograph dimensions consisted of 177 x 177 pixels of 30/xm spatial resolution. Data acquisition time (211 translations x 276 rotations) was 82 min. Faster systems have also been reported using CCDs as position-sensitive detectors. Only rotation of the sample is needed in this case, and the resolution is that of the spacing of the detector cells. 59 The unique possibility offered by monochromatic SR to take such images just under and over the absorption edge allows enhancement of the contrast for the mapping of a given element.

5. Detection of Tracks of Nuclear Reactions, Neutron Capture Radiography

5.1. Nuclear Reactions When an incident radiation, x, interacts with a target atom nucleus, X, to produce another nuclide, Y, and one or several radiations, y, this is called a nuclear reaction, and it is written symbolically X(x,y)Y. The probability for such a process to occur is usually characterized by its cross-section, or, which corresponds to the equivalent surface area offered by each individual atom nucleus X to the interaction with radiation x. The usual unit of cross section is the barn, with 1 barn = 10-28 m 2. The cross section of a given nuclear reaction often strongly depends on the energy of the incident radiation, x. The energies carried off by the products, y and Y, of the nuclear reaction are characteristic of this nuclear reaction and may thus serve in its identification. For different nuclear reactions, the incident radiation, x, as well as the produced radiation, y, may be photons (3' rays), neutrons, or light atom nuclei [protons, deuterium or tritium nuclei, helium nuclei (also termed a-particles), etc.]. 5s p. Spanne and M. L. Rivers, Nuclear lnstrum, and Meth. B24-25, 1063 (1987). 59 B. P. Flannery, H. W. Deckman, W. G. Roberge, and K. L. D'Amico, Science 237, 1439 (1987).

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PHYSICAL METHODS TO LOCATE METAL ATOMS

559

5.2. Neutron Capture Radiography 5,2.1. Principle of Neutron Capture Radiography. The neutron capture radiography (NCR) method is based on using nuclear reactions where (1) the incident particles are neutrons (usually " s l o w " neutrons, i.e., neutrons whose kinetic energies are comparable to those of atmospheric molecules at normal temperature, or even below) and (2) there is at least one heavy ionizing particle (with a nonnegligible range in matter) produced in the reaction. Relevant nuclear reactions are thus (n,p) or (n,a) nuclear reactions, or else neutron-induced fissions usually termed (nf). Examples 6° of such nuclear reactions with metals are as follows: 6Li(n,a)3H 39K(n,a)36C1 4°K(n,p)4°Ar 4°K(n,a)37Cl 4°Ca(n,ot)37Ar 235U(n,f)

or or or or or or

= = = = = =

941.4 barn 4.3 mbarn 4.4 barn 0.39 barn 2 mbarn 583 barn

% % % % % %

6Li in natural Li = 7.5 39K in natural K = 93.26 4°K in natural K = 0.012 4°K in natural K = 0.012 4°Ca in natural Ca = 96.94 235U in natural U = 0.72

but similar reactions also exist with nonmetal elements, especially the following: l°B(n,a)VLi 14N(n,p)14C 1 7 0 ( n , ot)14C 32S(n,ot)29Si 338(n,p)33p 33S(n,ot)3°Si 35Cl(n,p)35S

or = 3836 barn or = 1.83 barn or = 0.24 barn or = 4 mbarn or = 2 mbarn or = 0.14 barn or = 0.48 barn

% t°B in natural B = 19.9 % 14N in natural N = 99.63 % 170 in natural O = 0.038 % 32S in natural S = 95.02 % 33S in natural S = 0.75 % 33S in natural S = 0.75 % 35C1 in natural CI = 75.77

More details about the principle of NCR and its application to biology may be found in previous review articles. 61,62 In principle, the imaging o f a nuclide by NCR consists o f tightly pressing the specimen containing the nuclide against an appropriate detecting film, then irradiating the whole arrangement with neutrons. The particles induced in the reactions leave characteristic tracks in the detector; the distribution o f the tracks is representative of the distribution o f the nuclide under study in the specimen. In its final result, NCR thus somewhat resembles autoradiography, with the important difference that, with NCR, the detected nuclei are usually stable. When considering the nuclear reac60 F. W. Walker, D. G. Miller, and D. F. Reiner, " C h a r t of the Nuclides," 13th Ed., General Electric, San Jose, California, 1984. 61 M. Thellier, T. Stelz, and J. C. Wissocq, J. Microsc. Biol. Cell. 27, 157 (1976). 62 M. Thellier, M. Laurent-Pettersson, F. Martini, and C. Ripoll, Neutron News 2, 23 (1991).

560

PROBES OF METAL ION ENVIRONMENTS

[21]

tions with the metals listed above, it clearly appears that 39K and 4°Ca are not very good candidates for labeling experiments since (1) their cross sections for (n,a) reaction are extremely low and (2) they are the nuclides with by far the largest isotopic abundance in natural K and Ca, respectively. Moreover, even in bone, it may be calculated that the natural calcium would contribute only a small density of tracks, not much above background. On the other hand, 6Li, 4°K, and 235U, whose cross sections for neutron capture are much higher and whose concentrations in living tissues are extremely low under natural conditions, are thus appropriate to be used in labeling experiments and for imaging by NCR. In fact, with metals, so far most biological applications of NCR have been with lithium (Fig. 10), but other biological applications of NCR exist with nonmetallic elements, especially B and N. 5.2.2. Practical Procedures for Neutron Capture Radiography. The NCR method usually does not require the use of very thin and perfectly flat specimens. In most cases tissue cryosections (a few microns thick) are prepared and laid on rigid slides. Adhesion to the slide may be obtained by application of a transient pressure or by touching the slide with the finger immediately below the section (thus slightly rewarming the section without thawing it). Then the sections are lyophilized, stuck on their slide, covered with an appropriate detector tightly pressed against the slide, and irradiated with neutrons. To limit the occurrence of background tracks, slides are used in which the nuclear reactions listed in Section 5.2.1 do not take place in significant amounts. Normal glass slides (containing

FIG. 10. NCR imaging of the distribution of lithium in the brain of a mouse treated with Li (daily injections of 15 mmol kg -l for 3 consecutive days). The darker each part of the image, the richer in lithium is the corresponding area of brain tissue. (Reproduced, with permission, from Thellier et al. 61)

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

561

much boron) are thus not appropriate. Quartz slides are better, although they are much more expensive. In the past photographic films have been used as detectors, but they were fogged by the/3 and y rays accompanying neutron irradiation. Nowadays, use o f " solid-state" track detectors, made of insulating materials is preferred. For biological applications, two main types of detectors are employed: cellulose nitrate films (e.g., the LR 115 and CN 85 detectors from KodakPathr, Paris, France) and polycarbonate foils (e.g., the CR 39 detectors from Pershore Mouldings, Pershore, Great Britain). After neutron irradiation, the detector and the slide carrying the specimens are separated from one another. The specimens are stained using the conventional methods of histology, then photographed. Usually, the development of the latent tracks in the detectors is performed by etching these detectors in a strongly alkaline solution. This enlarges the latent tracks into small holes visible by optical microscopy. For instance, the CR 39 detectors are usually etched in 6.25 M sodium hydroxide at 70° for 1 hr. Similar conditions may also be used for the cellulose nitrate detectors. However, milder conditions are sometimes preferred, such as 2.5 M NaOH at room temperature. The etching time is then much longer (20 to 30 hr), but it is easier to adjust it to the precise value such that the tracks corresponding to the nuclide under study have exactly the desired size. The etched detectors are photographed, and this photograph of the distribution of the tracks may be compared with a photograph of the histological section. From the track density at each point of the detector, one can calculate the concentration of the nuclide under study at the corresponding site of the histological preparation. 63 Note that it is often preferred to begin counting the tracks after a mild etching and then to further etch the detectors before photographing their surface. There are many alternatives to the general procedure just described. For instance, using a very thin detector (0.8 ~m thickness), it was possible 64 to avoid separating the detector from the specimen for staining and etching, thus obtaining a better correlation between the distribution of tracks and the structure of the specimen than by comparing photographs. With usual detectors, one may use specimens ashed into spodograms (i.e., preserving the general structure of the tissue). The NCR method may also be applied to cell cultures or to microdrops of liquid samples (down to 63 M. Thellier, E. Hennequin, C. Heurteaux, F. Martini, M. Pettersson, T. Fernandez, and J. C. Wissocq, Nucl. Instrum. Methods Phys. Res., Sect. B B30, 567 (1988). 64 R. G. Zamenhof, S. C16ment, K. Lin, C. Lui, D. Ziegelmiller, and O. K. Harling, Strahlenther. Onkol. 165, 188 (1989).

562

PROBES OF METAL ION ENVIRONMENTS

[21]

0.5/xl in volume) enriched with gelatin or other types of gel. The CR 39 foils are fairly rigid; they can thus play a double role of slide carrying the specimens and of detector. 5.2.3. Performance of Neutron Capture Radiography. NCR is not a general method of analytical imaging, since it can detect only a restricted number of nuclides (listed in Section 5.2.1); however, it is practically nondestructive and has the advantage of detecting and mapping stable nuclides. The method discriminates between isotopes. For instance, with lithium, it detects 6Li very well but not 7Li. This has made it possible 65 to perform a kinetic analysis of lithium transport in the mouse brain (by studying 6Li/TLi isotopic exchange) despite the lack of any radioisotope of lithium with an appropriate half-life. Calculating the local concentrations of the nuclide of interest in the specimen, from the corresponding track densities in the detector, is not straightforward. The reason is that, when a given nuclear reaction emits particles in the specimen, the probability for these particles to reach the detector depends on a number of parameters (e.g., particle energy, sample thickness, and the local values of water content, density, and mean atomic mass number in the specimen). However, by combining appropriate modeling of the problem with the use of adequate calibration samples, one can obtain fairly accurate evaluations of the local nuclide concentrations. 63 The sensitivity of the quantitative evaluations depends on the cross section o-, of the nuclear reaction under consideration. With lithium, using the purified isotope 6Li, 10 /zm thick tissue sections, and a fluence of I017 neutrons m -2, the sensitivity is of the order of I ppm (dry weight) for 0.1 mm z of tissue examined. From the nuclear reactions listed in Section 5.2.1, it is clear that interference problems may arise. Detectors exist that are sensitive only to heavy particles, 66 and thus will detect fissions but not (n,a) or (n,p) reactions. With the usual detectors, one may determine etching conditions such that the proton tracks are much smaller than the a tracks, which makes it easy to discriminate between (n,p) and (n,a) reactions. For a given type of nuclear reaction, for instance, (n,a), the energy is not the same for the different reactions, and the latent tracks which they leave in detector are thus not exactly alike; the general method for accurate discrimination is then based on etching kinetics, 67 but it is fairly cumbersome. The problem is usually much simpler with biological specimens, 65 C. Heurteaux, C. Ripoll, S. Ouznadji, H. Ouznadji, J. C. Wissocq, and M. Theilier, Brain Res. 547, 122 (1991). 66 B. S. Carpenter and C. H. Cheek, Anal. Chem. 42, 121 (1970). 67 G. Baroni, S. Di Liberto, S. Petrera, G. Romans, and C. Sgarbi, Nucl. lnstrum. Methods 113, 545 (1974),

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

563

since, under natural conditions, practically only the reactions l°B(n, a)TLi and 14N(n,p)lac contribute nonnegligible densities of big and small tracks, respectively. Then, if one wishes, for instance, to map lithium in a lithiumenriched specimen, it is always possible to use non-lithium-enriched controis for evaluation of the background of big (mainly boron) tracks, then to subtract this background from the measurements. An alternative consists of intercalating a sheet of absorbing material between sample and detector, such that all the short-range a particles are absorbed and only the long-range 3H particles of the 6Li(n,a)3H reaction may reach the detector. The presence of the proton tracks of IaN may be an advantage, since it makes it possible to calculate the local nitrogen concentration at each point of the preparation and to use it as a reference for expressing the local concentration of other nuclides (g of lithium per g of nitrogen, for instance). Another possible advantage of the proton tracks of nitrogen is that they outline the histological structure of the specimen 68 on the detector, thus facilitating the correlation of the distribution of big tracks (of 6Li, for instance) with the specimen structure. When one is interested in the nitrogen tracks of the specimen, it is clearly preferable to use nitrogenless detectors (such as the CR 39 detectors), as cellulose nitrate detectors would give a heavy background of small tracks. There is, however, the problem with the CR 39 detectors that the size of the proton tracks increases rapidly in the course of etching, and, thus, with slightly overetched detectors, the proton and a tracks may begin to become confused with one another. Therefore, when one is not interested in the nitrogen signal, we recommend the use of cellulose nitrate detectors; it is then much easier to define etching conditions such that the protons contribute an almost continuous background of tiny tracks on which the big a tracks appear with a very good contrast. In most cases, the neutrons used for NCR come from nuclear reactors. With conventional reactors, there is a small proportion of neutrons with relatively high energies; they are responsible for various nuclear events (fast-neutron reactions, recoils of atom nuclei, especially protons) that may leave tracks likely to be confused with those of the reactions listed in Section 5.2.1. In our group, we have therefore used either "thermal" neutrons at the nuclear centers of Studsvik (Sweden) or StraSbourg (France) or, even better, "cold" neutrons at the nuclear center of Saclay (France) or at the European Institute Laue-Langevin in Grenoble (France). For reliability of the quantitative evaluations, it is important that the neutron flux be sufficiently homogeneous. This may be checked by disposing calibration samples close to the experimental ones, by examining the 68 M. L a u r e n t - P e t t e r s s o n , B. Delpech, and M. Thellier, Histochem. J. 24, 939 (1992).

564

PROBESOF METALION ENVIRONMENTS

[2 1]

background of the detecting films, or by using gold foil activation. One may also improve the homogeneity of the neutron irradiation over a specimencarrying slide by rotation of the slide during the irradiation. The lateral resolution of the method is limited by the diameter of the tracks. The theoretical limit of resolution is equal to the diameter of the latent tracks, that is, of the damaged area of the detector following the impact of radiation issued from a nuclear reaction; it is of the order of a few tens of nanometers. 69 It has been claimed that these latent tracks should be observable directly, after thinning the detector sufficiently for viewing in the electron microscope. However, when the tracks have been enlarged by appropriate chemical treatment (see Section 5.2.2) in order to make them visible in optical microscopy, their diameter (hence, the practical limit of lateral resolution) is usually a fraction of a micron for the small proton tracks and 1 to several microns for the large a tracks. The range of the particles issued from the nuclear reactions listed in Section 5.2.1 is of the order of a few to a few tens of microns in organic material. This gives the limit of resolution in depth of the NCR method. To improve the resolution in depth, the only possibility is the use of thin samples; however, this would be at the expense of sensitivity. The processing of the NCR detectors is carried out in daylight, which is much easier than processing photographic emulsions for autoradiography. When considering the delays involved in sending the samples to a nuclear reactor, neutron irradiating, getting back the samples from the reactor, and etching the detectors, the time required for obtaining an image with the N C R method is usually up to a few days. For measuring the densities of tracks on the detectors, the use of an automatic image analyzer makes this operation more rapid, and probably also more reliable, than direct observation at the microscope by eye.

5.3. Possible Detection o f Metals by Tracks o f Nuclear Reactions with Charged Particles Nuclides may also be involved in (p,a) or (d,o0 nuclear reactions, when irradiated with accelerated protons or deuterons. Often the cross section of these reactions reaches maximal values for well-defined values of the energy of the incident particles; also, the energies of the emitted particles are characteristic of the reactions. With the light elements (Z < 15), the proton or deuteron energies required for these reactions are 69A. G. Malmon, J. Theor. Biol. 9, 77 (1965).

[2 i I

PHYSICAL METHODS TO LOCATE METAL ATOMS

565

not too high (from 1 to 4 MeV) and can thus be attained using a Van de Graaf particle accelerator. Examples of such nuclear reactions with metals are as follows: 6Li(d,2a)

Energy d -- 2 MeV

Energy o~ = 14 MeV

7Li(p,2o0

Energy p = 2.3 MeV

Energy a = 8.9 MeV

Energy p = 1.01 MeV Energy p 0.59 MeV

Energy ~/ = 1.63 MeV Energy a = 2.26 MeV

Energy p = 0.59 MeV

Energy a = 2.26 MeV

23Na(p,~y)2°Ne

ZDNa(p,a)Z°Ne

[

In principle, this should make it possible to image these metals in a manner similar to that with NCR (except that the irradiation would be with protons or deuterons instead of neutrons). It is only necessary to find a detecting film sensitive to the emitted a particles, but insensitive to the incident protons or deuterons, at the energies of these particles. The advantage would be that it would be possible to image separately the different stable isotopes of a given metal (for instance, 6Li and 7Li). In fact, to our knowledge, nobody has tried to make use of this possibility. The reason is probably that the same nuclear reactions can now be detected, and used for imaging, with the nuclear microprobe (see Section 6).

6. Nuclear Microprobe Analysis

6.1. Interactions of Protons or Other Light Nuclear Particles with Matter In a nuclear microprobe, a beam of monoenergetic light nuclear particles is focused on the specimen under study. The incident particles may be protons (in which case the nuclear microprobe is often termed a "proton microprobe") or other light nuclear particles such as deuterons, helium3, or c~ particles. When the beam of incident particles interacts with the matter of the specimen, various atomic and nuclear processes take place. The atomic events may be compared with those described after irradiation with electrons (Section 3) or with X-rays (Section 4): inner atom shells are excited with emission of secondary electrons, then the holes thus created are filled with less strongly bound electrons from higher shells, with simultaneous emission of X-rays or of Auger electrons. The incident particles may also be scattered by interaction with the Coulomb field of the specimen atom nuclei, or they may produce nuclear reactions with these atom nuclei (see Sections 5.1 and 5.3). The nuclear microprobe takes advantage of all these possibilities of interaction, giving rise to

566

[21]

PROBES OF METAL ION ENVIRONMENTS

ATOMIC INTERACTIONS

PIXE SEM

Incident particle ~ BSA

~

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FIG. 11. Main microanalytical methods based on atomic and nuclear interactions between a beam of charged particles and matter. See text for abbreviations.

specific analytical methods (Fig. 11), namely, mainly, particle-induced X-ray emission (PIXE), scattering analysis (SA), and nuclear reaction analysis (NRA). By scanning the sample surface with a thin beam of incident particles, one may image the distribution of the chemical element (possibly of the isotope) under analysis. The secondary electrons produced in the atomic interactions, or the bombarding particles themselves after emerging from the specimen, may be collected and used to obtain structural images. 6.2. Microanalytical Methods Used in a Nuclear Microprobe 6.2.1. Particle Induced X-ray Emission. A typical PIXE spectrum is given in Fig. 12. In a manner similar to that described above for X-ray spectrometry (Section 3.3.1), a continuous background, whose main component is the bremsstrahlung of the secondary electrons, is superimposed on the X-ray peaks characteristic of the elemental composition of the specimen under study. The background is particularly intense at the lowest X-ray energies; it decreases rapidly with increasing X-ray energies and becomes negligible for energies above a threshold value, T, which depends on the energy, E, of the incident particles and which corresponds to the maximal energy transferred from an incident particle to a free electron. When the biological specimen is thin enough (thickness below 0.5 mg cm-2), it may be assumed (I) that the energy of the incident particles is not significantly decreased in passing through the specimen and (2) that the self-absorption (i.e., the absorption of the emitted X-rays within the

[21]

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Energy (keY) FIG. 12. PIXE spectrum of a human brain sample. A droplet of brain homogenate was dried on a 2 /~m thick polycarbonate film; it was then irradiated with a 2.4 MeV proton beam (I = 10 nA, t = 1000 sec). Sr was used as an internal standard, and a 300/~m thick Mylar film was put in front of the Si(Li) detector. (Fran~oise Sommer, unpublished data.)

sample) is negligible. Under such conditions, after subtraction of the background, the intensity of each X-ray peak, I A, is directly proportional to the superficial density o f the corresponding element under analysis. With noninfinitely thin samples, the energy of the incident particles progressively decreases in the depth of the sample. Therefore, the X-ray emission cross section has to be integrated over the energy range of the incident particles, and X-ray absorption corrections have to be calculated. In standard practice, the incident particles have an initial energy in the range of 1 to 3 MeV. Owing to their high X-ray emission cross sections and the low background produced, protons are usually used for PIXE analysis. The X-rays produced under particle bombardment are detected using Si(Li) detectors; the energy resolution of these detectors is not as good as that of crystal spectrometers, but they have the advantages of a high counting efficiency and relatively small size. Using such Si(Li) detectors with a Be window, the lightest element which can be analyzed is sodium. The chemical elements with Z below or above 40 are measured by their K and L X-rays, respectively.

568

PROnES OF METAL ION ENVIRONMENTS

[21]

To minimize interferences by low-Z elements, an absorber of the Xrays which they produce may be put in front of the detector. To improve the sensitivity of the detection of trace elements, selective absorbers of the X-rays emitted by more abundant elements may be used. The detection limit is defined as being equal to three times the standard deviation of the background. The best detection limits are obtained for thin samples put on a thin backing (usually a foil of a polymer, such as Mylar or polycarbonate). With a backing below 2 /xm in thickness, free of impurities or interfering elements, the detection limit is close to 1 ppm with a particle macrobeam (>100 /zm in diameter) and a time of analysis of 600 sec. When using a proton microbeam (<100 /zm in diameter), the time of analysis has to be increased by a factor of 10 to 100 to obtain the same detection limit. For data processing, the various corrections (self-absorption in the matrix, attenuation in absorbers, detector efficiency, shape of the background) can be performed using software packages such as PIXAN 7° or GUPIXE. 71 Since the pioneer work of Johansson e t a l . , 72 PIXE microanalysis has been used extensively for biological applications. The simultaneous detection of several trace elements (heavier than sodium) has been achieved with a sensitivity of approximately 1 ppm in microdrops of biological fluids, tissue sections, and c e l l s . 73 For instance, the composition of sweat has been compared in normal children and in children suffering from cystic fibrosis74: among eight elements analyzed (Na, C1, K, Ca, Mn, Cu, Zn, and Br), five (Na, CI, Ca, Zn, and Br) were observed to have significantly different concentrations in the two groups of subjects under study. 6.2.2. Scattering Analysis. An incident particle (mass m, charge z, energy E0) can be elastically scattered by interacting with the coulomb field of an atom nucleus (mass M, charge Z) of the target. The cross section, or(0), of this elastic interaction and the energy, E, of the scattered particle, are well-defined functions of the scattering angle, 0, and of the characteristic parameters M, Z, m, z, and E0. Moreover, especially when the incident particles are protons and with low-Z targets (Z < 20), high cross sections and resonances may be observed even with particle ener70 E. Clayton and C. C. Ryan, Nucl. lnstrum. Methods Phys. Res., Sect. B B49, 161 (1990). 71 j. L. Campbell, W. J. Teesdale, J. A. Maxwell, J. X. Wang, and L. J. Cabri, Nucl. Instrum. Methods Phys. Res., Sect. B B44, 347 (1990). 72 T. B. Johansson, R. Akselsson, and G. J. F. Legge, Nuel. lnstrum. Methods 84, 141 (1970). 73 G. L. Allan, J. Camakalis, and G. J. F. Legge, Nucl. Instrum. Methods Phys. Res., Sect. B B54, 175 (1991). 74 F. Sommer and B. Massonnet, Nucl. Instrum. Methods Phys. Res., Sect. B B22, 201 (1987).

[21]

PHYSICAL METHODS TO LOCATE METAL ATOMS

569

gies, E0, below the coulomb barrier of the target. Scattering analysis (SA), consisting of detection of the scattered particles, allows one to obtain the elemental composition of the target. There is also a possibility of discriminating between isotopes, because they differ in M value. SA is termed backscattering analysis (BSA) for deflection angles larger than 90 °, or forward scattering analysis (FSA) for deflection angles smaller than 90°. The scattered particles are detected using solid-state surface barrier silicon detectors, and possibly also annular detectors (for detection at an angle of 180°). With thin specimens, the energy spectrum is composed of well-individualized peaks, each peak corresponding to the presence of a given chemical element (or an isotope) in the sample. With thicker specimens, the peaks broaden (as a consequence of both the incident and the scattered particles progressively losing energy while passing through the specimen). With very thick homogeneous specimens, the energy spectrum tends toward a continuum presenting a series of steps, each of which corresponds to the presence of a given chemical element in the sample. Using the so-called Rutherford approximation for evaluation of the cross section, o-(0), and taking into consideration the stopping power of the sample matter for the incident particles, appropriate modeling allows one to determine absolute values of concentrations. 75 However, because the Rutherford approximation is not very accurate with incident protons of low energy (and with heavier incident particles of low or medium energies), the quantitative analyses are usually carried out by comparing the experimental specimens with standards of known chemical composition and thickness. The detection limits are of the order of a few parts per million at best. This is especially the case when carrying out BSA with heavy incident particles of low energy for the detection of a heavy chemical element in a light matrix. FSA is usually used for the detection of light chemical elements in thin targets. The selectivity of the method, defined as the minimum energy separation of units of atom mass, is better for light elements and for high values of the parameters m, 0, and E0. A typical spectrum of backscattered protons with a 2 /~m thick plant sample is shown in Fig. 13; for the light elements the neighboring peaks are well separated, and the isotopes (for instance, 12C and 13C) are discriminated from one another. 76 Nondestructive depth-profiling measurements may also be performed using SA, with a depth resolution of the order of a few nanometers in the most favorable cases. 75 L. R. Doolittle, Nucl. Instrum, Methods Phys. Res., Sect. B 119, 344 (1985). 76 p. Massiot, F. Sommer, M. Thellier, and C. Ripoll, Nucl. Instrum. Methods Phys. Res., Sect. B 1166, 250 (1992).

570

PROBES OF METAL ION ENVIRONMENTS

121]

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FIG. 13. Backscattering spectrum of a plant sample. The plants (Lemna gibba L.) were embedded in Epon; 2/zm thick sections were laid on a copper grid, covered with a thin gold layer, and irradiated with a 3 MeV proton beam (1 = 1.5 nA; t = 900 sec; diameter = 100 p.m). The scattered charged particles were detected using a silicon barrier detector set at a scattering angle of 165°. (Reproduced from Massiot et al., 76 with permission.)

For biological specimens, SA is usually less sensitive than PIXE. It has therefore been mainly used to get information about the major chemical elements of the matrix (H, C, N, O). In a study of the fixation of metal ions by the mycorrhizal fungi of a bracken fern growing in polluted areas, Watt and G r i m e 77 have used simultaneously BSA and PIXE to study the distribution of heavy metals (Ba, Cd, Pb) in longitudinal root sections. 6.2.3. Nuclear Reaction Analysis. When a sample is irradiated with a beam of protons or other light ions, nuclear reactions (see Section 5.1) occur. The radiations produced in these nuclear reactions may be light atom nuclei (see Section 5.3), y rays [(P,3') reactions], or both [such as (p,p'y) (p,t~y), or (d,py) reactions]. Detecting the particles or the y rays (termed "prompt y rays") produced in the irradiation thus provides information about the chemical (and isotopic) composition of the specimen at the point hit by the incident particles. The detection of the prompt 3' rays is termed PIGE (particle-induced y-ray emission). Large NaI scintillators may be used for this or, better, solid-state Ge(Li) detectors when a high 77 F. Watt and G. W. Grime, Nucl. lnstrum. Methods Phys. Res., Sect. B B54, 123 (1991).

[21]

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Fla. 14. PIGE spectrum of the same human brain sample as described in Fig. 12. The 3' rays were detected using a Ge detector. (Fran~oise Sommer, unpublished data.)

energy resolution is required. The cross sections of a number of nuclear reactions producing y rays have been experimentally determined, and it was decided at the Third International Conference on Chemical Analysis (Namur, Belgium, 8-12 July 1991) to compile all these data. With the light elements, high cross sections (resonances) have been found to exist for particular energy values of the incident particles (in the range of a few hundreds of kiloelectron volts to a few megaelectron volts). PIGE is thus especially well suited to the detection of light elements and isotopes. An example of a prompt y-ray spectrum, obtained by irradiating a drop of a brain homogenate with 2.4 MeV protons, is shown in Fig. 14. The concentration Cx of a chemical element, at the point x of the specimen hit by the incident beam, may be calculated from the intensity, N x , of the corresponding y ray, by use of the equation SxNx Cx = C~t S~t Ns, where C~t is the concentration of the same element in an appropriate standard, Nst the y-ray intensity of the standard, and Sx and S~t the stopping power of the specimen studied and of the standard for the incident particles. For the light elements, by using the strongest and narrowest reso-

572

PROBES OF METAL ION ENVIRONMENTS

[21]

nance peaks, one may carry out analYSeS in depth; when the energy of the incident particles is progressively increased above the resonance value, the nuclear reaction under consideration will occur deeper and deeper in the specimen. For instance, the nuclear reaction 7Li(p,y)SBe exhibits a resonance line (12 keV in width) at the proton energy of 441 keV, and the emitted y rays have energies of 14.7 or 17.6 MeV; this reaction has been used TM to determine Li concentration profiles over 10 /~m in depth of a specimen with a resolution in depth better than 150 nm. The charged particles which are possibly emitted in the nuclear reactions may be detected using solid-state surface barrier silicon detectors. The detectors are protected from scattered incident particles by use of thin metal foils. Because of the energy loss of the emitted particles within the specimen, the handling of such data is more difficult than for y rays. Interference problems may occur. NRA has been used mainly to detect light elements in thin targets. Once again, the energy loss of the incident particles within the specimen may be used to perform in-depth analyses. On the whole, NRA is a technique complementary to PIXE; with the incident energies used (below 4 MeV) only the elements with an atomic number below 15 can be detected with a good sensitivity. In this range of atomic numbers, the isotopes of a given element are also discriminated from one another. The detection limits usually lie between 10 and 100 ppm. In a biological application, using the resonances of the nuclear reaction 19F(p,ocy)160, it has been possible to obtain the concentration profile of fluorine in dental enamel 79 with an in-depth resolution of 0.7/~m.

6.3. Nuclear Microprobe Instrumentation and Operative Procedures The first nuclear microprobe 8° was built by Cookson in 1972. Now at least 40 nuclear microprobes are functional worldwide. Small electrostatic accelerators are used to produce the charged particle beams; most often they are Van de Graaff particle accelerators, in the range of 0.5 to 4 MV, single or double stage. These instruments are manufactured by National Electric Corporation (NEC, Middleton, WI), General Ionex Corporation and High Voltage Engineering (Amersfoort, The Netherlands). The investment cost is comparable to that for other methods such as SIMS (see Section 7) or STEM (see Section 3). For instance, NEC manufactures a 1.7 MV tandem accelerator, occupying an area of only 5 × 2 m z, for less than $400,000. A focusing system consisting of magnetic quadrupole doublet lenses is manufactured by Oxford Microbeam (Oxford, UK). With 78 C. E n g e l m a n n , I. Golicheff, and M. Loevillet, Silic. Ind. 3, 69 (1974). 79 U. L i n d h and A. B. Tvet, J. Radioanal. Chem. 59, 167 (1980). 8o j. A. C o o k s o n and F. D. Pilling, U.K. At. Energy Auth., Harwell Lab. [Rep.] AERE-R R6300 (1972).

[2 1]

PHYSICAL METHODS TO LOCATE METAL ATOMS

573

these various instruments, the charged particles used for irradiating the specimen are usually protons for PIXE, PIGE, and SA, deuterons for NRA, and 3He or 4He for SA and sometimes for NRA. The particle beams are collimated, in order to eliminate the particles scattered on the walls of the accelerator tube; they are then focused, most often using two to four quadrupole magnetic lenses. With the Oxford 81 and Melbourne 82 nuclear probes, microbeams have been obtained with an optimal lateral resolution of 0.3/xm. For elemental imaging, either the microbeam is scanned using electrostatic or magnetic steerers, or the sample is moved with stepping motors. In any case, the scan is continuous, and the data are recorded pixel by pixel. For each pixel, sophisticated electronics make it possible to record simultaneously the data produced by the different microanalytical methods (PIXE, NRA, SA, etc.). The quantitative data thus collected over the scanned area may be converted to concentration values or color intensities. The time required to acquire an image is relatively long (in the range of 1 hr to several hours), especially for trace element analysis. For biological applications, one advantage of the nuclear microprobe is that it can provide information on trace element concentrations in each individual cell, for instance, red cells 83 (Fig. 15), instead of averaging values from large numbers of cells (as is the case with many other analytical methods). Using energetic incident particles makes it possible to work with the sample in air. For instance, Deconninck has used an external macrobeam for PIXE 84 and for NRA 85 measurements in vivo, and Doyle has carried out BSA depth profiling and STIM (see Section 6.4) on samples in air. 86 These possibilities have still not so far been used very often because of the difficulty of maintaining good spatial resolution when the beam passes through the exit foil and through the air path between exit foil and sample. Solving these problems will make it possible to work routinely with frozen hydrated specimens or even with living cells or tissues. With specimens consisting of histological sections, the problems for sample preparation (especially for the detection of mobile substances) are

81 G. W. Grime, M. Dawson, M. Marsh, I. C. Mac Arthur, and F. Watt, Nucl. lnstrum. Methods Phys. Res., Sect. B B54, 52 (1991). 8: G. J. F. Legge, P. M. O'Brien, R. M. Sealock, G. L. Allan, G. Bench, M. Monoley, D. N. Jamienson, and A. P. Mazzolini, Nucl. lnstrum. Methods Phys. Res., Sect. B B30, 252 (1988). 83 U. Lindh, Nucl. Instrum. Methods Phys. Res., Sect. B B54, 160 (1991). 84 G. Deconninck, J. Radioanal. Chem. 12, 162 (1972). 85 G. Deconninck, IEEE Publ. 76CH 1175-9 NPS, 533 (1976). 86 B. L. Doyle, Nucl. lnstrum. Methods Phys. Res., 218, 20 (1983).

574

PROBES OF METAL ION ENVIRONMENTS

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% t~

J-

"L,

Mercury FIG. 15. Surface plot of Hg concentrations in one red cell from a patient suffering from the metal syndrome. The scan size was 10.5 x 10.5/zm 2 and the beam spot 1.5 x 1.5/xm 2 (I = 30 pA). (Reproduced from Lindh, 83 with permission.)

the same as for the other methods of analytical imaging (see Section 2). For quantitative evaluation, preparation of standards for PIXE is similar to that for analytical electron microscopy (see Section 3). With SA, although quantification may be absolute, it is often preferable to use standards whose composition and thickness are as close as possible to those of the experimental samples. As there are very few matrix effects with NRA, the standards may be prepared using any kind of known matrix. The histological preparations being electrically insulating, it is necessary to cover the sample with a thin conductive (carbon or metal) layer to avoid accumulating charges when they are irradiated under vacuum. The process of irradiation may damage the specimen. 87 The temperature increase of a biological sample under a proton macrobeam has been studied both experimentally, using a radiation thermometer, 88 and theoretically89; both measures agree, giving a temperature increase in a conductive sample close to 30° under irradiation with a proton beam 1/xm in diameter and I00 pA in intensity, and close to 40 ° for the same short integrated charge with a proton beam 0.1 ~m in diameter. A significant loss of hydrogen, carbon, sulfur, and chlorine was observed during ion beam analysis of freeze-dried kidney tissue with a current density of 1.8 10 -13 A / z m -2, s7 j. A. Cookson, Nucl. lnstrum. Methods Phys. Res., Sect. B B30, 324 (1988). F. Gioystein and F. W. Richter, Nucl. lnstrum. Methods Phys. Res., Sect. B B22, 45 (1987). 89 F. Van Langevelde, Natl. Lab: Rep. Vrise Univ. V-U84-2 (1984).

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whereas no loss of sulfur and chlorine occurred with lower current densities. 9° Continuous scanning minimizes the loss of light elements (H, C, O) which occurs when analyzing nylon foil specimens. 91 Helium gas cooling may also help to minimize sample damage. 74

6.4. Structural Imaging: Scanning Electron Microscopy and Scanning Transmission Ion Microscopy In nuclear microanalysis, the conventional methods for imaging the specimen structure consist of using a high-magnification optical microscope or collecting the secondary electrons to build an electron microscopy image (SEM) of the sample. Structural images obtained from the incident ions after their interaction with a thin specimen are now being obtained. This method has been termed scanning transmission ion microscopy (STIM). In the scanning of the specimen with the beam of incident particles, STIM consists of measuring the energy loss of these particles after they have passed through the specimen. When the particle detector is exactly aligned with the beam axis, the energy loss in the sample is a very sensitive measurement of the sample density (even with a beam intensity as low as 1 fA, one obtains statistically good measurements in a short time), and the technical efficiency is 100%, the lateral resolution being limited only by the mechanical and electrical fluctuations of the beam line. So far the optimal spatial resolution is about 50 nm, that is, approximately one order of magnitude better than that for the analysis. STIM images of biological specimens have now been obtained, especially with the Melbourne microprobe. 92 By recording STIM images at various rotation angles, tomography also may be achieved. 93 7. Secondary Ion Mass Spectrometry and Microscopy of Metallic Elements

7.1. Introductory Remarks The biological applications of SIMS (secondary ion mass spectrometry) microscopy7'94-96have increased in number in parallel with the progressive 9o B, J. Kirby and G. J. F. Legge, Nucl. lnstrurn. Methods Phys. Res., Sect. B BS4, 98 (1991). 91 M. Cholewa, G. Bench, B. J. Kirby, and G. J. F. Legge, Nucl. lnstrum. Methods Phys. Res., Sect. B B54, 101 (1991). 92 H. W. Lefevre, R. M. S. Schofield, G. S. Bench, and G. J. F. Legge, Nucl. lnstrum. Methods Phys. Res., Sect. B B54, 363 (1991). 9~ A. E. Pontau, A. J. Antolak, D. H. Morse, A. A. Ver Berkmoes, J. M. Brase, D. W. Heikkinen, H. E. Martz, and I. D. Proctor, Nucl. lnstrum. Methods Phys. Res., Sect. B B40, 646 (1989). 94 p. GaUe, Ann. Phys. Ft. 10, 287 (1985). 95 M. S. Burns, Ultramicroscopy 24, 269 (1988).

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improvement of the instrumentation. SIMS microscopy is among the most sensitive methods of analytical imaging existing at present, 97 and it thus appears to be a particularly attractive technique for metal imaging in biological systems. Because metal ions are usually fairly mobile, the preparation of the samples raises difficult problems. However, these problems have already been approached in some detail by Chandra and Morrison, 7 and they are not substantially different from the problems of sample preparation encountered with other methods of analytical imaging (see Section 2).

7.2. Principle of Secondary Ion Mass Spectrometry and Microscopy Bombarding a sample with a beam of medium weight or heavy ions a few kiloelectron volts in energy (termed the primary ions) results in the sputtering of the most superficial atomic layers of the sample. In part, the sputtered matter is ionized ("secondary ions") and may serve for analytical and imaging purposes. Two closely related techniques based on secondary ion emission have been developed. In secondary ion mass spectrometry (SIMS) the secondary ions (which are characteristic of the elemental and isotopic composition of the sample) are sorted out by mass spectrometry. In SIMS microscopy, not only are the secondary ions collected and mass filtered in a mass spectrometer, but they are also used to build images of their distribution over the sample surface. There are in fact two different modes for forming the images: the ion microscope (also termed stigmatic) mode and the scanning ion microscope (also termed microprobe) mode. In the microscope mode, a relatively wide surface area of the sample is illuminated with a defocused primary ion beam (beam diameter of the order of 100 /zm), and a real image is formed with the mass-selected secondary ions, using a stigmatic optical device. The lateral resolution then depends on the settings of the optical systems, and is usually of the order of 1 /zm. In the scanning mode, the primary ion beam is finely focused (microprobe) and rastered over the sample surface. The intensity of the mass-filtered secondary ion current is used to modulate the brightness of the spot of a CRT rastered in synchronism with the microprobe. The lateral resolution then depends only on the size of the microprobe, which is usually in the range of 50 to 300 nm; any isotope of any chemical element (including the lightest ones such as Li and Be) may, in principle, be detected and imaged with good sensitivity. 96 M. Theilier, C. Ripoll, and J. P. Berry, Eur. Microsc. Anal. 11, 9 (1991). 97 R. W. Linton and J. G. Goldsmith, Biol. Cell 74, 147 (1992).

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7.3. Sputtering Process Among the emitted secondary ions, some are positive and others negative. Chemical elements with a small value of their ionization potential, which is the case for most metal elements, are sputtered mainly in the form of monovalent cations. On the other hand, elements that have a high electron affinity escape from the sample surface preferentially in the form of monovalent anions. The few metal elements which have a nonnegligible electron affinity may thus also be sputtered as anions (e.g., Cu- or Au-). To a much lesser extent, some elements are ejected in the form of multicharged ions or (especially from biological matrices) polyatomic clusters. The ionization yield, ~/A, of a given atom, A, is defined as being the ratio of the number of ejected ions, A+, to the total number of ejected A particles (ionic, neutral, and clusters) originating from a given surface area of sample. The ionization yield depends on the nature of the primary ions. The emission of positive secondary ions is known TM to be favored by the use of oxygen (02 +) primary ions, whereas the use of Cs + as primary ions favors the emission of negative secondary ions. Primary ions of noble gases (Ar + , Kr +) or from liquid metal sources (Ga + , In +) induce ionization yields much lower than those with oxygen or cesium. 98 The sputtering yield, S, defined as being the mean number of ejected particles per incident primary ion, depends very much on the chemical composition of the specimen. The physics of the production of secondary ions under the impact of the primary ions is fairly complicated and still not completely understood. 99'~°° The dependence of the sputtering yield, and also the ionization yield, on the chemical composition of the specimen is rather unprecisely termed the "matrix effect." It has been claimed that the ionization yield is not very different for different metals (such as Na, K, Ca, or Mg) in various biological matrices) m However, several authors have reported appreciable matrix effects in plant and animal specimens, m2 In our opinion, no comprehensive study of the matrix effects in biological tissues has so far been performed. 98 M. Schuhmacher, H. N. Migeon, and B. Rasser, in "SIMS VIII" (A. Benninghoven, K. T. F. Janssen, J. Tiirnpner, and H. W. Werner, eds.), p. 49. Wiley, New York, 1992. 99 M. L. Yu and N. Lang, Nuel. lnstrum. Methods Phys. Res., Sect. B B14, 403 (1985). 100 Z. Sroubek, in "SIMS V I " (A. Benninghoven, A. M. Huber, and H. W. Werner, eds.), p. 17. Wiley, Chichester, 1988. lm M. S. Burns, in "Analysis of Organic and Biological Surfaces" (P. Echlin, ed.), p. 259. Wiley, Chichester (1984). t02 G. O. Ramseyer and G. H. Morrison, Anal. Chem. 55, 1963 (1983).

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7.4. Secondary Ion Mass Spectrometry and Microscopy Instrumentation A number of papers have already been devoted to SIMS microscopy instrumentation.~°3'~°4 Most of the applications to biology have been carried out using the IMS 3f or IMS 4f CAMECA (Courbevoie, France) instruments. 7'~°~'1°4 An important parameter for all these instruments is their mass resolution, M/AM, namely, the ratio of the analyzed mass, M, to the minimal mass difference, AM, that can be discriminated. The primary ions are produced in the primary ion source, also termed primary ion gun, of the ion microscope. A "duoplasmatron" produces fairly intense ion currents from reactive or nonreactive gases (e.g., 02 or Ar) by creating an electric discharge between a cold cathode and an extractive anode and confining the plasma thus obtained in both a magnetic and an electric field. Cesium ions are produced either from liquid cesium or from cesium chromate. In the latter case (in the CAMECA instruments), a cesium vapor is generated by heating a tablet of C s 2 C r O 4 at 400°; this vapor is directed toward a small tungsten slab heated at 1100° (termed the ionizer), where the Cs atoms are ionized. Cesium ion currents up to several hundred nanoamperes are easily obtained with such a device. Finally, the liquid metal ion sources (LMIS) are based on subjecting a small volume of a melted metal (Ga, In, Cs) to a strong electrostatic field; the metal is positively polarized with respect to the surroundings, this results in forming a Taylor cone from which an intense emission of ions occurs. The advantages of LMIS are that it is very bright and can be finely focused (down to 10 nm). ~°5 Any ion microscope possesses one or several of the ion sources just described. The primary ions are directed (well focused or partly defocused) onto the sample surface in the "primary column," where they are also accelerated up to the required energy (in the range of 5 to 20 keV, or even more in the case of a Ga LIMS). In the SIMS microscopy instruments which can be used in a scanning mode, the primary column also contains an ion beam deflection device. After interaction of the primary ions with the sample surface, the secondary ions thus produced enter the secondary optics. In the scanning ion microscopes, the secondary optics consist of a collection lens and a 103A. Benninghoven, F. G. Rudenauer, and H. W. Werner, "Secondary Ion Mass Spectrometry: Basic Concepts, Instrumental Aspects, Applications and Trends." Wiley, New York, 1987. j04 M. T. Beruius and G. H. Morrison, Reo. Sci. Instrurn. 58, 1789 (1987). 105 F. G. Rudenauer, in "SIMS I V " (A. Benninghoven, J. Okano, R. Shimizu, and H. W. Werner, eds.), p. 133. Springer-Verlag, Berlin, 1984.

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mass spectrometer. In the stigmatic ion microscopes, apart from electrostatic lenses and a mass spectrometer, the secondary optics contain preset combinations of stop apertures allowing the selection of the size of the image field and the value of the lateral resolution (see Section 7.5). In all cases, the mass spectrometer is a main component of the secondary optics. Three different types of mass spectrometers are in use: quadrupole mass filters, double focusing (electrostatic and magnetic sectors) spectrometers, and time-of-flight (TOF) spectrometers. The quadrupole mass filters are used mainly in SIMS instruments; e.g., VG (Burgess Hill, UK), Riber (Courbevoie, France), Atomika (Oberschleissheim, Germany), Kratos (Urmston, UK) and in scanning ion microscopes; e.g., VG, UC-HRL (Chicago, IL)1°4; they are well suited to high vacuum and fast mass switching capabilities, but they are fairly electrically noisy and their mass resolution is not very good (M/hdPl< 1000)) 06 The double focusing mass spectrometers have been (and still are) mounted on ion microscopes (CAMECA) or on scanning ion microscopes Hitachi (Tokyo, Japan), VG, CAMECA; they have very good mass resolution (M/AM > I0,000), and they have a good efficiency for secondary ion collection, because of their strong extraction fields. 1°4A°6 The TOF spectrometers are characterized by excellent signal transmission. In addition to the detection of low-mass ions, they can achieve a rather inexpensive determination of high-mass radicals (M > 500). TOF spectrometers also make possible the determination of several mass values simultaneously, 1°7 but this has been shown to be possible with a magnetic sector spectrometer as well. 1°8 The final main components of a secondary ion microscope are the detection device and the image forming system. The secondary ion currents are measured using either an electron multiplier or a Faraday cup. It has already been stated that, in scanning ion microscopy, the image is formed via the modulation of the spot brightness in a CRT. In stigmatic ion microscopy, the image is usually formed using a microchannel plate-fluorescent screen assembly. The systems for acquisition and digitization of the secondary ion image are main components of the image-forming devices in SIMS microscopy. 109 Only digital images are generated in scanning ion microscopy. With the ~06y . Homma, in "SIMS VI" (A. Benninghoven, A. M. Huber, and H. W. Werner, eds.), p. 717. Wiley, Chichester, 1988. 107 K. G. Standing, in "SIMS V I " (A. Benninghoven, A. M. Huber, and H. W. Werner, eds.), p. 225. Wiley, Chichester, 1988. 108G. Slodzian, B. Daigne, F. Girard, F. Boust, and F. Hillion, C.R. Acad. Sci. Ser. 2 311, 57 (1990). 109R. W. Odom, B. K. Furman, C. A. Evans, Jr., C. E. Bryson, W. A. Peterson, M. A. Kelly, and D. H. Wayne, Anal. Chem. 55, 574 (1983).

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ion microscope, either camera-based ~10or resistive anode encoders ~°9have been used to produce digital images. Once acquired, the digital images are treated by standard image processing.

7.5. Quality of Analytical Images The detection sensitivity is determined mainly by the "useful yield", 7A, which is the ratio of the number of ions, A±, collected on the detector in a given time to the number of atoms, A, sputtered from the sample during the same time. The useful yield is the product of the ionization yield, YA, and the instrumental transmission, "O, which combines the efficiencies of collection, transmission, and detection of the microscope. For instance, consider a homogeneous sample containing C atoms per unit of volume, and let XA be the atomic fraction of atom A. To have a 95% chance of detecting one ion A, we must sputter 3/rA atoms A. These ions are contained in a volume V = 3/(TACXA). With ~'A = 0.01 (which is already a high useful yield), C = 6 × 1022 atoms cm -3, and x A 10-6, one can easily calculate that the sputtered volume, V, is equal to 5 × 10-3/zm 3. This corresponds to sputtering a depth of only one atomic layer (-0.3 nm) over a square surface slightly less than 5 × 5/zm 2. To have a 95% chance of measuring the same atomic concentration with a statistical precision of 10%, we would have to sputter a depth of one atomic layer over a square surface of 25 × 25/xm 2. This approximate calculation shows that SIMS is well suited to microanalysis, that is, to measuring metal concentrations of the order of 1 ppm. This is illustrated by the data in Table I, calculated from values tabulated by Burns, 1~° assuming that dry organic matter contains approximately 6 × 10z2 atoms cm -3. For analytical imaging, the surface of the sputtered area has to correspond to one image pixel, that is, to approximately 0.5 × 0.5/xm 2 (the order of magnitude of the lateral resolution of an ion microscope). The same calculation as above then shows that one has to sputter a depth of 30 nm to detect one atom A, and a depth of 1 p.m to measure the concentration of species A at the parts per million level. This is clearly unacceptable, but it shows that concentrations of the order of 100 ppm (corresponding to sputtering a depth of 10 nm) can be easily measured. The precision in the localization of the analyzed area is thus limited by the concentration of the element being analyzed and by the statistical precision, m Maximizing the ionization yield and improving the =

110j. C. Olivo, E. Kahn, S. Halpern, C. Brian~on, P. Fragu, and R. Di Paola, J. Microsc. 156~ 105 (1989). m G. Slodzian, in "SIMS V I " (A. Benninghoven, A. M. Huber, and H. W. Werner, eds.), p. 3. Wiley, Chichester, 1988.

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TABLE I MINIMAL CONCENTRATIONS OF METALS DETECTABLE BY SECONDARY ION MASS SPECTROMETRY MICROSCOPY COMPARED TO MEAN CONCENTRATIONS IN WHOLE DRY BLOODa Concentrations (atomic ppm)

Metal

Minimal detectable by SIMS microscopy

Mean value in whole dry blood

Li Ba Mg V Fe Co Cu Ni Zn Pb Cd Hg

0.004 0.004 0.018 0.018 0.08 0.28 0.8 1.7 2.4 2.8 24 24

0.3 0.13 175 0.03 714 0.02 1.4 0.005 9.4 0.07 0.004 0.0025

a Calculated from values tabulated by Burns.I°~

instrument transmission are thus necessary for achieving high-resolution imaging microanalysis.l~2 The time required for acquiring an image is also an important parameter. Let O be the time for sputtering a depth z in a sample, tr the surface area being sputtered, ip the intensity of the primary ion current, e the elementary electronic charge, C the total atomic concentration (C close to 6 × 1022 atoms cm-3), and S the sputtering yield. One may then write @ = zo-eC/(ipS)

In the ion microscope mode, all the image pixels are acquired simultaneously. Hence, the time required to detect one atom A per pixel (z = 0.3 nm), for a mean A concentration of 100 ppm, is O = 0.1 sec, if the current density (/p/O-) is 1 mA cm -2 and the sputtering yield S = 3. In the scanning mode, the pixels are recorded sequentially; therefore, for a concentration of element analyzed per unit area identical to that in the preceding calculation, the time required to sputter the same depth of material will be increased by a factor equal to the number of pixels per image (in the range of 103 to 105), that is, in the range of 1 min to 3 hr. nz R. Levi-Setti, J. M. Chabala, and Y. L. Wang,

Ultramicroscopy 24, 97 (1988).

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PROBES OF METAL ION ENVIRONMENTS

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This means that, in the scanning mode, the time for image acquisition is acceptable only with small image fields, but it does not mean that the stigmatic mode always allows the fastest image acquisition. The reason is that, in the microscope mode, the lateral resolution is improved at the expense of the instrumental transmission, whereas, with a scanning microscope, the lateral resolution depends only on the size of the probe (the instrumental transmission remaining at its maximal value). For a high lateral resolution and a moderate image field, the scanning mode thus becomes more efficient (and consumes less matter) than the microscope mode. m The best lateral resolution that can at present be achieved with a commercial IMS 4f CAMECA microscope is of the order of 500 nm. Images in the range of 200 to 300 nm, 113or even better than 100 nm, 108,114,115 have, however, been obtained. It is noteworthy that with an ion microscope, one may obtain a good lateral resolution and a good mass resolution at the same time. To be really useful, the analytical images have to be specific, that is, free from interferences. With biological specimens, interferences arise mainly from the production of organic clusters during the sputtering proc e s s . 116'117 The major interferences (e.g., 24Mg+ and r2C2+, 55Mn+ and C4H7 ÷, Fe ÷ and CaO + or C4H8+) are now well known and have been tabulated. A mass resolving power of the order of 2000 is sufficient to discriminate most interferences) ~7 As has been indicated above (Section 7.4), such a resolution in mass is easily obtained with a double focusing spectrometer, but not with a quadrupole mass filter. If, after a first recording performed with a high-resolution mass spectrum, it can be proved that the interfering ions represent only a minor part of the signal under consideration, then it is clearly possible to obtain valuable images even with a low mass resolution (hence, also with high transmission) of the secondary optics.

7.6. Quantification of Images The analytical images may be quantified with adequate statistical precision depending on the concentration of the studied element and on the useful yield of the measurements (see Section 7.5). Given the matrix 1~3M. Schuhmacher, B. Rasser, H. N. Migeon, and G. Slodzian, in "SIMS VI" (A. Benninghoven, A. M. Huber, and H. W. Werner, eds.), p. 177. Wiley, Chichester, 1988. i~4 R. Levi-Setti, G. Crow, and Y. L. Wang, in "Scanning Electron Microscopy" (O. Johari, ed.), Vol. 2, p. 535. SEM, AMF O'Hare, IL, 1985. ll5 M. T. Bernius, Y. C. Ling, and G. H. Morrisson, J. Appl. Phys. 60, 1094 (1986). z~6M. Truchet, Th~se de Doctorat d'Etat, Universit6 Paris VI, Paris (1982). 117M. S. Burns, Anal. Chem. 53, 2149 (1981).

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effects, absolute quantification is usually not straightforward. Calibration standards, with a chemical composition as close as possible to that of the specimens under study, have sometimes given good results. 1°2'H8 The dependence of the sputtering yield on the chemical composition of the sample is a common problem in the interpretation and quantification of analytical images, z19For instance, it has been reported that the sputtering rates of different cell organelles are not equal to one another, both in plant ~19 and animaP 2° tissues. The sputtering rate values have been estimated for each image pixel by carrying out "burn-through" maps 119, that is, by measuring, at each point of the sample, the time necessary to pierce it through down to the backing. The implanting of BF3 + ions has been used to correct for matrix effects in the quantification of calcium in sections of bean root tips. J2~ Using plastic-embedded animal tissues implanted with Be + ions, it has been demonstrated that only negligible differences in the useful yield occurred in different parts of the tissue. 122 Using carbon as a reference element, and assuming that the useful yield was the same for carbon and for the other chemical elements under analysis, a method of relative quantification of the ion images has been worked out with plasticembedded sections of human thyroid tissue. 123 It has also been proposed that potassium and phosphorus be used as reference elements for a large variety of biological specimens.l°2

7.7. Depth Profiling and Three-Dimensional Imaging With SIMS, it is possible to record the concentration-depth profile of an analyte immediately below the sample surface (one-dimensional microanalysis). One may think of achieving three-dimensional imaging by storing time sequentially recorded two-dimensional images. The method suffers from many drawbacks (e.g., matrix effects, development of surface topography, contrast errors, redeposition) that are likely to alter the reliability and the resolution of the images. 106,124However, it has been claimed that all these effects could be assumed to remain practically negligible as long as the depth of totally sputtered material and the superficial irregularities were less than the lateral resolution of the microscope. TM In this lib M. S. Burns-Bellhorn, Anal. Biochem. 92, 213 (1979). H9 A. J. Patkin, S. Chandra, and G. H. Morrison, Anal. Chem. 54, 2507 (1982). ~20R. W. Linton, M. E. Farmer, P. Ingrain, S. R. Walker, and J. D. Shelburn, Scanning Electron Microsc. 111, 1191 (1982). 12t W. C. Harris, S. Chandra, and G. H. Morrison, Anal. Chem. 55, 1959 (1983). 122j. T. Brenna and G. H. Morrison, Anal. Chem. 58, 1675 (1986). n3 p. Fragu, C. Brian~on, S. Halpern, and E. Larras-Regard, Biol. Cell 62, 145 (1988). t24 F. G. Rtidenauer and W. Steiger, in "SIMS V I " (A. Benninghoven, A. M. Huber, and H. W. Werner, eds.), p. 361. Wiley, Chichester, 1988.

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respect, it is good strategy to work with a scanning microscope on small surface areas.

7.8. Biological Applications of Secondary Ion Mass Spectrometry and Microscopy Metal imaging was among the earliest applications of SIMS microscopy in biological systems. 125Recent literature on biological SIMS studies has been extensively reviewed by Linton and Goldsmith97: in the period 1980-1991, 84 papers have dealt with Na, K, Ca, and Mg, whereas 94 have dealt with trace or toxic elements (i.e., the metals AI, Ag, Au, Ba, Bi, Cd, Ce, Co, Cr, Cs, Cu, Fe, Ga, Hg, In, La, Li, Mn, Ni, Pb, Pu, Sn, Sr, Th, Ti, Tm, U, and Zn). Metal imaging by SIMS microscopy has been involved in many different areas of research, for example, toxicology and pharmacology, 126ophthalmology, 127biology and pathology of hard tissues (bones, teeth), 128environmental sciences and pollution studies, ~z9and animal 13° and plant "1 physiology. We give as examples one or two possible applications of SIMS microscopy to metal studies in plants. The use of stable isotopes as tracers might provide new information on the symplastic and apoplastic routes of nutrient exchange in plant roots. 132The possibility of finely imaging the distributions of the main mineral cations in plant tissues, and their possible changes under different experimental conditions, would help very much in the study of signal perception and transduction in higher plants. J33Preventing the mobilization of the metal ions during preparation of plant samples is particularly difficult. Figure 16 shows the distributions of Ca and Mg in transverse sections of the cotyledons of young flax plants, as prepared using the pyroantimonate precipitation 125p. Galle, Ann. Phys. Biol. Med. 4, 84 (1970), 126 S. Duckett and P. Galle, in "Metal Ions in Neurobiology and Psychiatry" (S. Gabay, J. Harris, and B. T. Ho, eds.), p. 345. Alan R. Liss, New York, 1985. 127 M. S. Burns, D. M. File, K. T. Brawn, and D. G. Flaming, Brain Res. 220, 173 (1981). lZ8 A. Lodding, J. G. Nor6n, and L. G. Petersson, in "SIMS V I " (A. Benninghoven, A. M. Huber, and H. W. Werner, eds,), p. 865. Wiley, Chichester, 1988. ~29C. Chassard-Bouchaud, F. Escaig, P. Boumati, and P. Galle, Biol. Cell 74, 59 (1992). 130 S. Chandra, C. S. Fullmer, C. A. Smith, R. H. Wasserman, and G. H. Morrison, Proc. Natl. Acad. Sci. U.S.A. 87, 5715 (1990). 131 A. Jauneau, C. Morvan, F. Lefebvre, M. Demarty, C. Ripoll, and M. Thellier, J. Histochem. Cytochem. 40, 1183 (1992). 132D. Lazof, R. W. Linton, R. J. Volk, and T. W. Rufty, Biol. Cell 74, 127 (1992). 133M. Thellier, M. O. Desbiez, C. Ripoll, M. Demarty, and A. Monnier, in "Signal Perception and Transduction in Higher Plants" (R. Ranjeva and A. M. Boudet, eds.), p. 133. SpringerVerlag, Berlin, 1990.

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FIG. 16. Ionic images of Ca and Mg in transverse sections of cotyledons of 4-day-old flax seedlings, as prepared using the pyroantimonate precipitation method. (a, b) Ca and Mg images of the same area of tissue in the palisade parenchyma. (c, d) Ca and Mg images of the same area of tissue at the level of a leaf vein. (Reproduced from Jauneau el al., 134 with permission.) Bar: 50/~m.

method. 134 In Fig. 16a,c Ca underlines the cell contours; Mg is almost totally absent from the walls of the epidermal and vessel cells (Fig. 16b,d, respectively), but it appears as bright intracellular spots, which might correspond to aleurone bodies in the course of being degraded. 134 134 A. Jauneau, C. Ripoll, C. Rihouey, M. Demarty, and M. Thellier, C.R. A c a d . Sci. I!I 315, 179 (1992).

586

PROBES OF METAL ION ENVIRONMENTS

[21]

8. Concluding Remarks about Metal Atom Location in Biological Systems The methods described in this chapter may be used to localize metals in biological tissues, but they are usually unable to detect whether the metal under study is ionized or not under natural conditions. In most cases, these methods are not restricted to the study of metals only, but rather they can be extended to the detection of other (sometimes of any) chemical elements. Because the different methods have fairly different performances, it is usually advisable to use a combination of several different, complementary methods, instead of a single one, for tackling a given problem. At present, we are in a phase of rapid evolution of these methods and of their application to biology. There are methods, such as analytical electron microscopy, which have already played a major role in the past and which may be expected to remain extremely important in the future, with still improving performances. Other methods, which were little or not at all employed in biology up to a few years ago, are now rapidly developing. This is especially the case with SRXRF and the use of specific nuclear reactions, nuclear probes, and analytical ion microscopes. It is noteworthy that (with the exception of SRXRF) the latter methods are well adapted to the detection of stable isotopes. In fact, it is no longer a scientific problem to replace the use of radioactive isotopes by stable isotopes for labeling and imaging purposes (including multilabeling); however, there remains an economical problem because highly purified, stable isotopes are still very costly. Biologists would be most interested in the possibility of carrying out in vivo measurements. Methods such as the nuclear probe may become amenable to in vivo measurements in the future. However, it is unlikely that the in vivo approaches will ever give performances (e.g., spatial resolution, accuracy) equal to those attainable with fixed preparations. Therefore, it is likely that both approaches (in vivo and fixed samples) will remain complementary for a long time in the future. With all the physical methods we have been considering, it is becoming more and more possible to study the behavior of small mobile substances including metal ions, and not only that of bigger organic components. However, it has to be stressed that the study of mobile substances is not only a problem of physical methods of detection; there is also the crucial" (and very difficult) problem of the preparation of the samples to be examined, since it is absolutely necessary that the natural distribution of these mobile substances not be disturbed.

Contributors to Volume 227 Article numbers are in parentheses following the names of contributors. Affiliations listed are current.

of Pediatrics, University College London Medical School, London WC1E 6JJ

PHILIP AISEN (7), Department of Physiology

and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 FRASER A. ARMSTRONG (18), Department of Chemistry, University of California, lrvine, lrvine, California 92717 CHRISTOPHER J. BENDER (7), Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461 MARCELINO BERNARDO (6), Corporate Research Laboratory, Exxon Research and Engineering Company, Annandale, New Jersey 08801, and Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794 JULEA N. BUTT (18), Department of Chemistry, University of California, Irvine, Irvine, California 92717 CH. BUTZLAFE (15), lnstitut fiir Physik, Medizinische Universitgitzu Liibeck, D-23538 Liibeck, Germany RICHARD CAMMACK (12), Metals in Biology and Medicine Centre, Division of Life Sciences, King's College, London W8 7AH, United Kingdom GERARD W. CANTERS (9), Department of Chemistry, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands" N. DENNIS CHASTEEN (8), Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824 PIERRE CHEVALLIER (21), LURE, Universitd de Paris-Sud, F-91405 Orsay, France JOSEPH E. COLEMAN (2), Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510 CHRISTOPHER E. COOPER (12), Department

JACK DAINTY (21), Laboratoire des Proces-

sus loniques Cellulaires, Physiologie Cellulaire, Signaux et Rdgulations, CNRS URA 203, Universitd de Rouen, F-76134 Mont-Saint-Aignan, France EDMUND P. DAY (16), Department of Phys-

ics, Emory University, Atlanta, Georgia 30322 S. FORSI~N (5), Physical Chemistry 2, Chemical Centre, University of Lund, S-221 00 Lund, Sweden CARLOS F. G. C. GERALDES (3), Depart-

ment of Biochemistry, University of Coimbra, P-3049 Coimbra, Portugal BRIAN J. HALES (13), Department of Chem-

istry, Louisiana State University, Baton Rouge, Louisiana 70803 GRAEME R. HANSON (11), Centre for Mag-

netic Resonance, The University of Queensland, Brisbane, Queensland, Australia 4072 MICHAEL P. HENDRICH (17), Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 CORNELIS W. HILBERS (9), Laboratory of Biophysical Chemistry, Nijmegen SON Research Center, University of Nijmegen, 6525 ED Nijmegen, The Netherlands H. ALLEN O. HILL (19), Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, United Kingdom ARNOLD J. HOEF (10), Department of Biophysics, Huygens Laboratory, Leiden University, 2300 RA Leiden, The Netherlands NICHOLAS 1. HUNT (19), Inorganic Chemistry Laboratory, University of Oxford, Oxford OXI 3QR, United Kingdom vii

viii

CONTRIBUTORS TO VOLUME 227

of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 ARTUR SUCHETA (18), Department of Chemistry, University of California, IrS. LINSE (5), Physical Chemistry 2, Chemivine, lrvine, California 92717 cal Centre, University of Lund, S-221 O0 KRISTENE K. SURERUS (17), Department of Lund, Sweden Chemistry, Carnegie Mellon University, DUARTE MOTA DE FREITAS (4), Department Pittsburgh, Pennsylvania 15213 of Chemistry, Loyola University, ChiA. GEOFFREY SYKES (20), Department of cago, Illinois 60626 Chemistry, University of Newcastle, ECKARD M~2NCK(17), Department of ChemNewcastle upon Tyne NE1 7RU, United istry, Carnegie Mellon University, PittsKingdom burgh, Pennsylvania 15213 MICHEL THELLIER (21), Laboratoire des ALLEN M. ORVILLE (14), Department of Processus loniques Cellulaires, PhysioloBiochemistry, University of Minnesota gie Cellulaire, Signaux et Rdgulations, Medical School, Minneapolis, Minnesota CNRS URA 203, Universitd de Rouen, F55455 76134 Mont-Saint-Aignan, France KIM E. PAULSEN (14), Department of HANS THOMANN (6), Corporate Research Chemistry, University of Minnesota, MinLaboratory, Exxon Research and Engineapolis, Minnesota 55455 neering Company, Annandale, New Jersey 08801, and Department of Chemistry, JOHN R. PILBROW (11), Department of State University of New York at Stony Physics, Monash University, Victoria, Brook, Stony Brook, New York 11794 Australia 3168 A. X. TRAUTWEIN (15), Institutfiir Physik, CARMEN QUINTANA (21), Centro Nacional Medizinische Universitiit zu Liibeck, Dde Microelectr6nica, C.S.I.C., 28006 Ma23538 Liibeck, Germany drid, Spain DAVID) L. TURNER (1), Department of CAMILLE RIPOLL (21), Laboratoire des ProChemistry, Southampton University, cessus loniques Cellulaires, Physiologie Southampton S09 5NH, United Kingdom Cellulaire, Signaux et Rdgulations, CNRS URA 203, Universitd de Rouen, F- MARX VAN DE KAMP (9), Laboratory of Biophysical Chemistry, Nijmegen SON Re76134 Mont-Saint-Aignan, France search Center, University of N(jmegen, G. ARTHUR SALMON (20), Cookridge Radia6525 ED N~imegen, The Netherlands tion Research Centre, The University of SYBREN S. WIJMENGA (9), Laboratory of Leeds, Cookridge Hospital, Leeds LS16 Biophysical Chemistry, Nijmegen SON 6QB, United Kingdom Research Center, University of NUmeHELENA SANTOS (1), Instituto de Tecnologen, 6525 ED Nijmegen, The Netherlands gia Q~imica e Biol6gica, Universidade H. WINKLER (15), Institut fiir Physik, MediNova de Lisboa, 2780 Oeiras, Portugal zinische Universitiit zu Liibeck, D-23538 FRAN~OISESOMMER (21), CENATS, Institut Liibeck, Germany de Physique Nucldaire, Universitd Claude ANT6NIO V. XAVIER (1), Instituto de TecnoBernard, F-69622 Villeurbanne, France logia Qt~imica e Biol6gica, Universidade Nova de Lisboa, 2780 Oeiras, Portugal MARIAN T. STANKOVICH (14), Department C. JOHANSSON (5), Physical Chemistry 2,

Chemical Centre, University of Lund, S221 O0 Lund, Sweden

METHODS IN ENZYMOLOGY

VOLUME I. Preparation and Assay of Enzymes

Edited by SIDNEY P. COLOWICKAND NATHAN O. KAPLAN VOLUME II. Preparation and Assay of Enzymes

Edited by SIDNEY P. COLOWICKAND NATHAN O. KAPLAN VOLUME III. Preparation and Assay of Substrates

Edited by SIDNEY P. COLOWICKAND NATHAN O. KAPLAN VOLUME IV. Special Techniques for the Enzymologist

Edited by SIDNEY P. COLOWICKAND NATHAN O. KAPLAN VOLUME V. Preparation and Assay of Enzymes

Edited by SIDNEY P. COLOWICKAND NATHAN O. KAPLAN VOLUME VI. Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques Edited by SIDNEY P. COLOWICKAND NATHAN O. KAPLAN VOLUME VII. Cumulative Subject Index

Edited by SIDNEY P. COLOWICKAND NATHAN O. KAPLAN VOLUME VIII. Complex Carbohydrates

Edited by ELIZABETH F. NEUFELD AND VICTOR GINSaURG VOLUME IX. Carbohydrate Metabolism

Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation

Edited by RONALD W. ESTABROOKAND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure

Edited by C. H. W. HIRS VOLUME XII. Nucleic Acids (Parts A and B)

Edited by LAWRENCE GROSSMANAND KIVIE MOLDAVE VOLUME XIII. Citric Acid Cycle

Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids

Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids

Edited by RAYMOND B. CLAYTON VOLUME XV|. Fast Reactions

Edited by KENNETH KUSTIN

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VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)

Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)

Edited by DONALD B. McCORMICK AND LEMUEL D. WRIGHT VOLUME XIX. Proteolytic Enzymes

Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D)

Edited by LAWRENCE GROSSMANAND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques

Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A)

Edited by ANTHONY SAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B)

Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEEF VOLUME XXVI. Enzyme Structure (Part C)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEEE VOLUME XXVII. Enzyme Structure (Part D)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEEE VOLUME XXVIII. Complex Carbohydrates (Part B)

Edited by VICTOR G1NSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXII. Biomembranes (Part B)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX

Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)

Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XXXV. Lipids (Part B)

Edited by JOHN M. LOWENSTEIN

. . °

METHODS IN ENZYMOLOGY

Xlll

VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XXXlX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B)

Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C)

Edited by W. A. WOOD VOLUME XLIII. Antibiotics

Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes

Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B)

Edited by LASZLOLORAND VOLUME XLVI. Affinity Labeling

Edited by WILLIAM B. JAKOBY AND MUIR WILCHEK VOLUME XLVII. Enzyme Structure (Part E)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLVIII. Enzyme Structure (Part F)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFE VOLUME XLIX. Enzyme Structure (Part G)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C)

Edited by VICTORGINSBURG VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism

Edited by PATRICIA A. HOEEEE AND MARY ELLEN JONES VOLUME LII. Biomembranes (Part C: Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LUSTER PACKER VOLUME LIII. Biomembranes (Part D: Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIV. Biomembranes (Part E: Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER

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METHODS IN ENZYMOLOGY

VOLUME LV. Biomembranes (Part F: Bioenergetics)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVI. Biomembranes (Part G: Bioenergetics)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVII. Bioluminescence and Chemiluminescence

Edited by MARLENE A. DELUCA VOLUME LVIII. Cell Culture

Edited by WILLIAM B. JAKOBY AND IRA PASTAN VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME LX. Nucleic Acids and Protein Synthesis (Part H)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME 61. Enzyme Structure (Part H)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 62. Vitamins and Coenzymes (Part D)

Edited by DONALD B. McCORMICK AND LEMUEL D. WRIGHT VOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods) Edited by DANIEL L. PURICH VOLUME 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by DANIEL L. PURICH VOLUME 65. Nucleic Acids (Part I)

Edited by LAWRENCE GROSSMANAND KIVIE MOLDAVE VOLUME 66. Vitamins and Coenzymes (Part E)

Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME 67. Vitamins and Coenzymes (Part F)

Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME 68. Recombinant DNA

Edited by RAY Wu VOLUME 69. Photosynthesis and Nitrogen Fixation (Part C)

Edited by ANTHONY SAN PIETRO VOLUME 70. Immunochemical Techniques (Part A)

Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE VOLUME 71. Lipids (Part C)

Edited by JOHN M. LOWENSTEIN VOLUME 72. Lipids (Part D)

Edited by JOHN M. LOWENSTEIN VOLUME 73. Immunochemical Techniques (Part B)

Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS

METHODS IN ENZYMOLOGY

XV

VOLUME 74. Immunochemical Techniques (Part C) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV-LX Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 76. Hemoglobins Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONE

VOLUME 77. Detoxication and Drug Metabolism Edited by WILLIAM B. JAKOBY VOLUME 78. Interferons (Part A) Edited by SIDNEY PESTKA VOLUME 79. Interferons (Part B) Edited by SIDNEY PESTKA VOLUME 80. Proteolytic Enzymes (Part C) Edited by LASZLO LORAND VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by LESTER PACKER VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by LEON W. CUNNINGHAM AND DIXIE W. FREDERIKSEN VOLUME 83. Complex Carbohydrates (Part D) Edited by VICTOR GINSBURG VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 85. Structural and Contractile Proteins (Part B" The Contractile Apparatus and the Cytoskeleton) Edited by DIXIE W. FREDERIKSEN AND LEON W. CUNNINGHAM VOLUME 86. Prostaglandins and Arachidonate Metabolites Edited by WILLIAM E. M. LANDS AND WILLIAM L. SMITH VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereochemistry, and Rate Studies)

Edited by DANIEL L. PURICH VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by LESTER PACKER VOLUME 89. Carbohydrate Metabolism (Part D) Edited by WILLIS A. WOOD VOLUME 90. Carbohydrate Metabolism (Part E) Edited by WILLIS A. WOOD VOLUME 91. Enzyme Structure (Part I) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF

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VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME 94. Polyamines

Edited by HERBERTTABORAND CELIA WHITE TABOR VOLUME 95. Cumulative Subject Index Volumes 61-74, 76-80

Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)] Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)] Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 99. Hormone Action (Part F: Protein Kinases)

Edited by JACKIE D. CORBIN AND JOEL G. HARDMAN VOLUME 100. Recombinant DNA (Part B)

Edited by RAY Wu, LAWRENCEGROSSMAN, AND KIVIE MOLDAVE VOLUME 101. Recombinant DNA (Part C)

Edited by RAY Wu, LAWRENCEGROSSMAN,AND KIVIE MOLDAVE VOLUME 102. Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins) Edited by ANTHONYR. MEANS AND BERT W. O'MALLEY VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides)

Edited by P. MICHAELCONN VOLUME 104. Enzyme Purification and Related Techniques (Part C)

Edited by WILLIAMB. JAKOBY VOLUME 105. Oxygen Radicals in Biological Systems

Edited by LESTER PACKER VOLUME 106. Posttranslational Modifications (Part A)

Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 107. Posttranslational Modifications (Part B)

Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by GIOVANNIDI SABATO,JOHN J. LANGONE,AND HELEN VAN VUNAKIS

M E T H O D S IN E N Z Y M O L O G Y

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VOLUME 109. Hormone Action (Part I: Peptide Hormones)

Edited by LUTZ BIRNBAUMERAND BERT W. O'MALLEY VOLUME 110. Steroids and Isoprenoids (Part A)

Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 111. Steroids and Isoprenoids (Part B)

Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 112. Drug and Enzyme Targeting (Part A)

Edited by KENNETH J. WIDDERAND RALPH GREEN VOLUME ll3. Glutamate, Glutamine, Glutathione, and Related Compounds

Edited by ALTON MEISTER VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A)

Edited by HAROLDW. WYCKOEE, C. H. W. HIRS, AND SERGE N. TIMASHEEF VOLUME 115. Diffraction Methods for Biological Macromolecules (Part B)

Edited by HAROLDW. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by GIOVANNIDI SABATO,JOHN J. LANGONE, AND HELEN VAN VUNAKIS VOLUME ll7. Enzyme Structure (Part J)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFE VOLUME 118. Plant Molecular Biology Edited by ARTHURWEISSBACHAND HERBERT WEISSBACH VOLUME 119. Interferons (Part C)

Edited by SIDNEY PESTKA VOLUME 120. Cumulative Subject Index Volumes 81-94, 96-101 VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME 122. Vitamins and Coenzymes (Part G) Edited by FRANKCHYTIL AND DONALDB. McCoRMICK VOLUME 123. Vitamins and Coenzymes (Part H)

Edited by FRANK CHYTIL AND DONALDB. McCoRMICK VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides)

Edited by P. MICHAELCONN VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER

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METHODS IN ENZYMOLOGY

VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by LESTER PACKER VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology) Edited by JERE e. SEGRESTAND JOHN J. ALBERS VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by JOHN J. ALBERSAND JERE P. SEGREST VOLUME 130. Enzyme Structure (Part K)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 131. Enzyme Structure (Part L)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and CellMediated Cytotoxicity) Edited by GIOVANNIDl SABATOAND JOHANNESEVERSE VOLUME 133. Bioluminescence and Chemiluminescence (Part B)

Edited by MARLENEDELUCA AND WILLIAMD. MCELROY VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton) Edited by RICHARDB. VALLEE VOLUME 135. Immobilized Enzymes and Cells (Part B)

Edited by KLAUS MOSBACH VOLUME 136. Immobilized Enzymes and Ceils (Part C)

Edited by KLAUS MOSBACH VOLUME 137. Immobilized Enzymes and Cells (Part D)

Edited by KLAUS MOSBACH VOLUME 138. Complex Carbohydrates (Part E)

Edited by VICTORGINSBURG VOLUME 139. Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins) Edited by ANTHONY R. MEANS AND P. MICHAELCONN VOLUME 140. Cumulative Subject Index Volumes 102-119, 121-134 VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids)

Edited by P. MICHAELCONN AND ANTHONYR. MEANS VOLUME 142. Metabolism of Aromatic Amino Acids and Amines

Edited by SEYMOURKAUFMAN VOLUME 143. Sulfur and Sulfur Amino Acids

Edited by WILLIAMB. JAKOBYAND OWEN GRIFFITH VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular Matrix)

Edited by LEON W. CUNNINGHAM

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VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular Matrix)

Edited by LEON W. CUNNINGHAM VOLUME 146. Peptide Growth Factors (Part A) Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 147. Peptide Growth Factors (Part B)

Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 148. Plant Cell Membranes

Edited by LESTER PACKER AND ROLAND DOUCE VOLUME 149. Drug and Enzyme Targeting (Part B)

Edited by RALPH GREEN AND KENNETH J. WIDDER VOLUME 150. Immunochemical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) Edited by GIOVANNI DI SABATO VOLUME 151. Molecular Genetics of Mammalian Cells

Edited by MICHAEL M. GOTTESMAN VOLUME 152. Guide to Molecular Cloning Techniques

Edited by SHELBY L. BERGER AND ALAN R. KIMMEL VOLUME 153. Recombinant DNA (Part D)

Edited by RAY WU AND LAWRENCE GROSSMAN VOLUME 154. Recombinant DNA (Part E)

Edited by RAY Wu AND LAWRENCE GROSSMAN VOLUME 155. Recombinant DNA (Part F)

Edited by RAY WU VOLUME 156. Biomembranes (Part P: ATP-Driven Pumps and Related Transport: The Na,K-Pump) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 157. Biomembranes (Part Q" ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 158. Metalloproteins (Part A)

Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 159. Initiation and Termination of Cyclic Nucleotide Action

Edited by JACKIE D. CORBIN AND ROGER A. JOHNSON VOLUME 160. Biomass (Part A: Cellulose and Hemicellulose)

Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 161. Biomass (Part B: Lignin, Pectin, and Chitin)

Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 162. Immunochemical Techniques (Part L: Chemotaxis and Inflammation) Edited by GIOVANNI DI SABATO

XX

METHODS IN ENZYMOLOGY

VOLUME 163. Immunochemical Techniques (Part M: Chemotaxis and Inflammation) Edited by GIOVANNIDI SABATO VOLUME 164. Ribosomes

Edited by HARRY F. NOLLER, JR., AND KIVIE MOLDAVE VOLUME 165. Microbial Toxins: Tools for Enzymology

Edited by SIDNEY HARSHMAN VOLUME 166. Branched-Chain Amino Acids

Edited by ROBERT HARRIS AND JOHN R. SOKATCH VOLUME 167. Cyanobacteria

Edited by LESTER PACKERAND ALEXANDERN. GLAZER VOLUME 168. Hormone Action (Part K: Neuroendocrine Peptides)

Edited by P. MICHAELCONN VOLUME 169. Platelets: Receptors, Adhesion, Secretion (Part A)

Edited by JACEK HAWIGER VOLUME 170. Nucleosomes

Edited by PAUL M. WASSARMANAND ROGER D. KORNBERG VOLUME 171. Biomembranes (Part R: Transport Theory: Cells and Model Membranes) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 172. Biomembranes (Part S: Transport: Membrane Isolation and Characterization) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 173. Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 174. Biomembranes [Part U: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 175. Cumulative Subject Index Volumes 135-139, 141-167 VOLUME 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dynamics) Edited by NORMANJ. OPPENHEIMERAND THOMAS L. JAMES VOLUME 177. Nuclear Magnetic Resonance (Part B: Structure and Mechanism)

Edited by NORMANJ. OPPENHEIMERAND THOMAS L. JAMES VOLUME 178. Antibodies, Antigens, and Molecular Mimicry

Edited by JOHN J. LANGONE VOLUME 179. Complex Carbohydrates (Part F)

Edited by VICTOR GINSBURG VOLUME 180. RNA Processing (Part A: General Methods)

Edited by JAMES E. DAHLBERGAND JOHN N. ABELSON

METHODS IN ENZYMOLOGY

xxi

VOLUME 181. RNA Processing (Part B: Specific Methods)

Edited by JAMES E. DAHLBERGAND JOHN N. ABELSON VOLUME 182. Guide to Protein Purification

Edited by MURRAYP. DEUTSCHER VOLUME 183. Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences Edited by RUSSELL F. DOOLITTLE VOLUME 184. Avidin-Biotin Technology

Edited by ME1RWILCHEKAND EDWARD A. BAYER VOLUME 185. Gene Expression Technology

Edited by DAVID V. GOEDDEL VOLUME 186. Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants) Edited by LESTER PACKERAND ALEXANDERN. GLAZER VOLUME 187. Arachidonate Related Lipid Mediators

Edited by ROBERTC. MURPHY AND FRANK A. FITZPATRICK VOLUME 188. Hydrocarbons and Methylotrophy

Edited by MARY E. LIDSTROM VOLUME 189. Retinoids (Part A: Molecular and Metabolic Aspects)

Edited by LESTER PACKER VOLUME 190. Retinoids (Part B: Cell Differentiation and Clinical Applications)

Edited by LESTER PACKER VOLUME 191. Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 192. Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 193. Mass Spectrometry

Edited by JAMES A. McCLOSKEY VOLUME 194. Guide to Yeast Genetics and Molecular Biology

Edited by CHRISTINEGUTHRIE AND GERALD R. FINK VOLUME 195. Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase

Edited by ROGER A. JOHNSON AND JACKIE D. CORBIN VOLUME 196. Molecular Motors and the Cytoskeleton

Edited by RICHARDB. VALLEE VOLUME 197. Phospholipases

Edited by EDWARDA. DENNIS VOLUME 198. Peptide Growth Factors (Part C)

Edited by DAVID BARNES, J. P. MATHER, AND GORDON H. SATO

xxii

METHODS IN ENZYMOLOGY

Volume 199. Cumulative Subject Index Volumes 168-174, 176-194 VOLUME 200. Protein Phosphorylation (Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression) Edited by TONY HUNTER AND BARTHOLOMEWM. SEFTON VOLUME 201. Protein Phosphorylation (Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases) Edited by TONY HUNTER AND BARTHOLOMEWM. SEFTON VOLUME 202. Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes) Edited by JOHN J. LANGONE VOLUME 203. Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs) Edited by JOHN J. LANGONE VOLUME 204. Bacterial Genetic Systems

Edited by JEFFREY H. MILLER VOLUME 205. Metallobiochemistry (Part B: Metallothionein and Related Molecules) Edited by JAMES F. RIORDANAND BERT L. VALLEE VOLUME 206. Cytochrome P450

Edited by MICHAELR. WATERMANAND ERIC F. JOHNSON VOLUME 207. Ion Channels

Edited by BERNARDORUDY AND LINDA E. IVERSON VOLUME 208. Protein-DNA Interactions

Edited by ROBERT T. SAUER VOLUME 209. Phospholipid Biosynthesis

Edited by EDWARDA. DENNIS AND DENNIS E. VANCE VOLUME 210. Numerical Computer Methods

Edited by LUDWIG BRAND AND MICHAELL. JOHNSON VOLUME 211. DNA Structures (Part A: Synthesis and Physical Analysis of DNA) Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 212. DNA Structures (Part B: Chemical and Electrophoretic Analysis of DNA) Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 213. Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation) Edited by LESTER PACKER VOLUME 214. Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis)

Edited by LESTER PACKER VOLUME 215. Platelets: Receptors, Adhesion, Secretion (Part B)

Edited by JACEK J. HAWIGER

METHODS IN ENZYMOLOGY

xxiii

VOLUME 216. Recombinant DNA (Part G)

Edited by RAY Wu VOLUME 217. Recombinant DNA (Part H) Edited by RAY Wu VOLUME 218. Recombinant DNA (Part I)

Edited by RAY Wu VOLUME 219. Reconstitution of Intracellular Transport

Edited by JAMES E. ROTHMAN VOLUME 220. Membrane Fusion Techniques (Part A)

Edited by NEJAT DOZGONE~ VOLUME 221. Membrane Fusion Techniques (Part B)

Edited by NEJAT DOZGONE~ VOLUME 222. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors) Edited by LASZLOLORAND AND KENNETH G. MANN VOLUME 223. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors) Edited by LASZLOLORANDANn KENNETH G. MANN VOLUME 224. Molecular Evolution: Producing the Biochemical Data

Edited by ELIZABETH ANNE ZIMMER, THOMAS J. WHITE, REBECCA L. CANN, AND ALLAN C. WILSON

VOLUME 225. Guide to Techniques in Mouse Development

Edited by PAUL M. WASSARMANAND MELVIN L. DEPAMPHILIS VOLUME 226. Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins) Edited by JAMES F. RIORDANAND BERT L. VALLEE VOLUME 227. Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins) Edited by JAMES F. RIORDANANn BERT L. VALEE VOLUME 228. Aqueous Two-Phase Systems (in preparation)

Edited by HARRY WALTERAND GOTE JOHANSSON VOLUME 229. Cumulative Subject Index Volumes 195-198,200-227 (in preparation) VOLUME 230. Guide to Techniques in Glycobiology (in preparation)

Edited by WILLIAMJ. LENNARZAND GERALD W. HART

xxiv

METHODS IN ENZYMOLOGY

VOLUME 231. Hemoglobins (Part A: Biochemical and Analytical Methods) (in preparation) Edited by JOHANNES EVERSE, ROBERT M. WINSLOW, AND KIM D. VANDEGRIFF

VOLUME 232. Hemoglobins (Part B: Biophysical Methods) (in preparation) Edited by JOHANNES EVERSE, ROBERT M. WlNSLOW, AND KIM D. VANDEGRIFF

AUTHOR INDEX

587

Author Index Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A Aasa, R., 220, 221(74), 226, 229(95), 363, 377,409 Abeles, R. H., 343 Abilgaard, F., 274, 275(117) Abragam, A., !10, 123, 124(26), 125(26), 132(26), 138(26), 209, 211(45), 212(45), 221(45), 229(45), 279, 331, 331(2), 335, 336(2), 345(2), 360, 386, 424 Abraha, A., 82, 85, 90(14), 96(14), 99, 99(14), 100(48), 101-102, 102(50), 106(52) Abruha, H. D., 518 Ackerman, J.J.H., 18 Ackrell, B.A.C., 485, 512(74), 513(74), 515, 521(74) Adams, G. E., 523 Adams, M. W., 337 Adams, M.W.W., 186, 404 Adler, J., 552 Adler, M., 252, 280(28) Adman, E. T., 153,263,283(75), 472 Adorn, J. D., 18 Agostini, C., 324 Agresti, D. G., 60 Ahmed, R. H., 235 Aisen, P., 78, 226-227, 229(95), 235, 239, 240(13), 243(13), 379 Akahoshi, R., 512(81), 513(81), 515 Akihama, S., 512(69), 513(69), 515 Akke, M., 274, 276(115), 278(115) Akselsson, R., 568 Al-Basseet, J., 369 Albery, W. J., 512,512(78), 513(36, 78), 515 Albracht, S.P.J., 377,411 Allan, G. L., 568, 573 Allen, J. P., 320 Allen, P. M., 501 Allendorfer, R. D., 54 Allerhand, A., 62

Allis, J. L., 90, 102(39) Allured, V. S., 286 A1-Massad, F., 369 Alsaadi, B. M., 55 Amann, B. T., 273,283(108) Amarouche, R., 104 Ambler, R. P., 254, 255(47) Ambruso, D. R., 235,243 Amesz, J., 323, 328, 328(66, 69) Ammann, H., 104 Amsellen, J., 540 Anderson, B. F., 228 Anderson, C. F., 84 Anderson, K. W., 287(145), 288 Anderson, P. W., 426 Anderson, R., 191,223(7) Anderson, R. E., 224 Andersson, K. K., 477,478(11) Andersson, T., 47, 107, 114(1), 115-116, 118, 118(1), 271 Andrews, D. W., 553 Andrews, S. K., 101, 102(51) Anger, G., 224 Angerhofer, A., 321-324, 324(73, 74), 328329 ,~ngstr6m, J., 259 Anniko, M., 538 Anson, F. C., 512(52, 54), 513(52, 54), 514, 516(54) Antholine, W. E., 331,354, 359 Antolak, A. J., 575 Aoki, S., 557 Aramini, J. M., 118 Arean, C. O., 74 Arizti, P., 556 Armarego, W. L., 371 Armitage, I. M., 17, 21,21(2), 22, 23~17), 24, 24(2), 25(17), 26, 27(2), 29(1, 2, 20), 3031, 31(20), 32, 37(51), 39, 40(51) Armstrong, F. A., 283,479, 485,487,487(1), 490-491, 491(13, 16, 17), 492, 492(13,

588

AUTHOR INDEX

16), 493, 493(13), 496(14), 497, 501, 503-504, 507(2), 512, 512(74), 513(37, 74), 515,516(37), 521(74) Arntzen, C. J., 323 Arsenault, A. L., 553 Arumugam, S., 96, 97(43) Asmus, K.-D., 522-523 Assefa, H., 512(39), 513(39), 514 Astlind, J. S., 195, 196(18) Aston, W. J., 512,512(46), 513(34, 46), 514, 514(84), 515 Atherton, N. M., 124, 209 Attanasio, D., 234, 237(7), 239(7) Atwater, F. M., 217 Aue, W. P., 2 Auld, D. S., 340 Aust, V., 324 Avery, A. R., 504 Avison, M. J., 104

B Babcock, G. T., 200, 203(24), 205(24), 211, 215, 215(52), 216(63) Bachmann, P., 2, 10(13) Back, J. M., 556 Bader, J., 512(40), 513(40), 514 Bagby, S., 271,279(104), 506 Bailey, D. B., 24 Baker, E. N., 228 Baker, G. J., 220, 222(78) Baker, H. M., 228 Balch, A. L., 7 Baleja, J. D., 269 Balke, V. L., 361 Ballou, D. P., 343,354, 371(4), 375(4) Balshi, J. A., 89, 103(32), 105, 106(63) Banei, L., 1, 10(46), 11(49), 14(59), 15(46, 47), 256, 258(51), 259(51), 261(51), 270(51), 339 Bancroft, E. E., 504 Bank, J. F., 227 Banks, R. D., 62, 63(76), 66(76) Baram, A., 109 Barber, M. J., 342, 371, 397, 411, 512(57), 513(57), 514 Bard, A. J., 480 Barer, R., 543

Barker, P. D., 506, 510 Baroni, G., 562 Barry, B. A., 215, 216(63) Barry, C. D., 1, 15(8), 52 Bartholdi, E., 2 Bartlett, P. N., 511,512(78), 513(78), 515 Bartucka, V. J., 18 Batie, C. J., 343 Battaglini, F., 512 Bauer, R. E., 207 Baumgarten, M., 226 Bax, A., 3, 8(39), 246, 250, 252-253, 266, 267(87), 270(40), 276(32, 33), 278(33), 279(24), 280(24, 26) Baxendale, J. H., 522-523, 529-530 Bayley, P. M., 118 Beck, J., 307, 321 Beck, W. F., 393-394, 394(10) Becker, E. D., 26 Beese, D., 328 Behar, B., 525 Beinert, H., 191, 219, 223-225, 343, 354, 359, 365(6), 373(5), 380, 380(6), 383(5, 62), 387, 408, 411,485 Belford, R. I., 196 Bell, J. E., 354 Bell, J. M., 512, 513(34) Bell, S. J., 37 Bench, G., 573,575 Bencini, A., 339, 342, 434 Bender, C. J., 200, 203(24), 205(24), 215, 216(63) Benecky, M. J., 170 Benelli, C., 339 Bennett, D. W., 492 Benninghoven, A., 577-580, 582-584 Bereman, R., 354 Berendsen, H.J.C., 289 Berezin, I. V., 512(55), 513(55), 514 Berg, J. M., 273, 283(108) Bergeron, M., 104 Berliner, L., 232, 234(1), 241 Berliner, L. J., 45, 46(15), 291,297(1), 337338, 354 Bernado, M., 122-123, 137, 138(44), 140, 142, 153(44), 155(14), 156(48), 160-161, 162(67), 163(67), 168(44), 171, 171(44), 173(65, 82), 176(22), 178(22), 179, 180(82), 184(82), 186, 186(82) Bernius, M. T., 536, 578, 579(104), 582

AUTHOR INDEX Bernlocher, D., 329 Berry, J. P., 575 Bersohn, R., 254, 255(48), 318 Bertini, I., 1, 2(4), 7(34), 9(43), 10(46), 11(49), 13(43, 51), 14(51, 59, 64), 15(46, 47, 68), 49, 50(43), 55(43), 120, 256, 258(51), 259, 259(51), 261(51), 270(51), 344 Bertrand, P., 368,491 Bicknell, R., 339 Biehl, R., 182, 199, 218 Bieth, J., 73 Biggins, J., 328 Bill, E., 412, 434, 436(29), 478 Bimmler, M., 538 Birch, N. J., 104 Birnbaum, E. R., 73 Bisby, R. H., 523 Blackham, I. G., 504 Blaineau, B., 540 Blake, C.C.F., 57, 62, 63(76), 66(76) Blauer, G., 291 Bleaney, B., 49, 50(41), 51(41), 123, 124(26), 125(26), 132(26), 138(26), 209, 211(45), 212(45), 221(45), 229(45), 331, 331(2), 336(2), 345(2), 360 Bloch, F., 191 Block, R., 426 Bloembergen, N., 386 Blondin, G., 426 Blount, H. N., 501,504 Blum, H., 393 Blumberg, W. D., 360 Blumberg, W. E., 230, 378 Boag, J. W., 522 Bobrin, S., 512(72), 513(72), 515 Bobsein, B. R., 21 Bocian, D. F., 39, 40, 41(54), 365 Bodenhausen, G., 8, 123, 129(20), 246, 264(4), 265(4), 266(4), 279(4) Boelens, R., 247 Bogdanovskaya, V., 512(72), 513(72), 515 Bogdanovskaya, V. A., 512(53, 55), 513(53, 55), 514, 516(53) Bogumil, R., 235, 237, 238(12), 239(12), 240(12) Bolton, J. R., 124, 212, 214(53), 217(53), 331,331(3), 345(3), 351,376 Bomford, A., 383 Bominaar, E. L., 412, 426, 434

589

Bonci, A., 77 Bond, A. M., 479, 501,504 Bond, M., 551,552(39) Bonvoisin, J., 426, 461-462 Boon, K., 235 Borg, D. C., 331,331(3), 345(3), 376 Boroske, E., 219 Borovik, A. S., 478 Borso, M., 18 Bothner-By, A. A., 11, 18(7), 31 Boudet, A. M., 584 Boudreaux, E. A., 415 Bouhoutous-Brown, E., 116 Boulanger, Y., 32, 104 Boumati, P., 584 Boust, F., 579, 582(108) Bowden, E. F., 484, 501,504, 512, 512(39), 513(38, 39), 514, 516(38) Bowen, D., 63 Bowen, O. K., 556 Box, H. C., 181 Boyd, J., 2, 34, 37(47), 258,259(57, 58), 261, 261(57, 58), 262(72), 266, 267(86) Boyer, P. D., 57 Brajter-Toth, A., 512(80), 513(80), 515 Brase, J. M., 575 Braunlin, W, H., 47, 115 Braunschweiler, L., 3, 8(22) Brawn, K. T., 584 Bray, R. C., 371,411 Brayer, G. D., 75 Brenna, J. T., 583 Breton, J., 310, 311(23), 323(23), 485, 491(13), 492(13), 493, 493(13), 496(14), 497 Briancon, C., 580, 583 Bridsall, W. J., 73 Briganti, F., 11, 13(51), 14(51, 65), 344 Briggs, G.A.D., 504 Briggs, J. M., 52 Brodin, P., 116-118 Bronskill, M. J., 522 Brophy, P. J., 89, 90(36) Brown, D. B., 418 Brown, I. M., 122, 123(10), 128(10) Brown, R. D. III, 47, 270 Brown, T. G., 163, 226-227 Browne, D. T., 40 Brudvig, G. W., 227, 365, 392-393, 393(8), 394, 394(8, 10)

590

AUTHOR INDEX

Brumbaugh, J. F., 204 Brnmfleld, J. C., 512(76), 513(76), 515 Bruno, A. C., 416 Bruschi, M., 407 Bryant, R. G., 18, 25(13), 41(13), 43(13), 116 Bryson, C. E., 579 Brzeska, H., 86 Buck, R. P., 512(76), 513(76), 515 Budil, D. E., 323 Buettner, G. R., 425 Buhlmann, C., 123, 176(21), 178(21) Bfinzil, J.C.G., 45, 47(16), 52(16), 78(16) Burger, A. R., 342 Burns, M. S., 575, 577, 578(101), 581(101), 582, 584 Burns, P. D., 55 Burns-Bellborn, M. S., 583 Burris, R. H., 167 Burrows, A., 507 Burstein, D., 93 Burton, D. R., 47 Busi, F., 522-523, 529-530 Busse, S. C., 4, 6(26), 14(26, 63), 261, 266(74) Buster, D. C., 89, 103(35) Butt, J. N., 485, 487, 491, 491(13, 16), 492(13, 16), 493,493(13), 496(14), 497 Butzlaff, Ch., 434, 436(29) Buxton, G. V., 523 Bycroft, M., 512(78), 513(78), 515

C Cabri, L. J., 568 Cacheris, W. P., 76, 89 Calvo, E. J., 512 Calvo, R., 360 Camakalis, J., 568 Cammack, R., 224,343,344(37), 354,365(8), 368, 380, 485,490, 491(17) Campana, T., 384 Campbell, I. D., 2, 34, 37(47), 52, 57(48, 49), 58, 58(48, 49), 59(48), 60(49), 61(48), 266, 267(86) Campbell, J. L., 568 Cannon, J. C., 235 Canters, G. W., 247, 248(13, 15, 16), 254, 255(49), 263,263(13), 264(13), 272(111),

273, 275(112, 113), 277(13), 283, 283(75), 284(137, 138), 285(138), 289 Capozzi, F., 7, 9(43), 13(43), 15(68), 259 Carafoli, E., 73 Carbonera, D., 324 Cardin, A. D., 18 Carey, M., 25 Carmichael, A., 235 Carpenter, B. S., 562 Case, D. A., 247, 250(18), 288(18) Casey, D., 235 Cass, A.E.G., 512, 512(41, 77), 513(36, 41, 77), 514, 514(84), 515 Castaing, R., 542, 550 Castner, T. G., Jr., 387 Castro, M.M.C.A., 85, 89, 103(35) Cataldi, T.R.I., 504 Cavanagh, J., 266, 267(88) Cav6, A., 47 (~6nas, N. K., 512(43, 44), 513(43, 44), 514 Chabala, J. M., 581 Chakrabarti, P., 254, 269(44, 45), 270(4346) Chan, I. Y., 304, 306, 313 Chan, S. I., 226-227, 365 Chan, T. M., 4 Chandra, S., 536,538,538(1), 576(7), 577(7), 578(7), 583-584 Chandreshekar, T. K., 215,216(63) Chang, C.-H., 320 Chao, E.C.T., 556 Chapman, J. N., 550 Charloteaux-Waters, M., 384 Chassard-Bouchard, C., 584 Chasteen, N. D., 232-234, 234(1), 235,239, 240(13), 241, 242(15), 243(13, 15), 337, 464, 478(6) Chaston, S.H.H., 221 Chaudhuri, Ph., 434, 436(29) Chazin, W. J., 247, 250(18), 252, 274, 276(! 15), 278(115), 288(18) Che, Che-Ming, 523 Cheek, C. H., 562 Cheesman, M. R., 369 Chen, D. M., 60 Chen, J. R., 556 Chert, J.-S., 168 Cheng, H., 10, 14(44) Chestnut, D. B., 214 Cheung, T.T.P., 18

AUTHOR INDEX Chevallier, P., 556-557 Chirino, A., 320 Chiu, M. L., 14, 16(62) Chlebowski, J. F., 17, 29(1), 30, 504 Cholewa, M., 575 Choppin, G. R., 45-46, 47(16), 52(16), 78(16) Christie, P. D., 464, 468(7), 477(7), 478(7) Christner, J. A., 464 Chu, S. C., 89, 103(32) Chu, S. C.-K., 105, 106(63) Chuj6, R., 3, 9(23), 11, 14(23), 14(60), 15(48) Ciapa, B., 540 Cieslar, C., 247 Ciurli, S., 9, 13(43), 485 Clark, J. B., 383 Clark, W. M., 403,405(5) Clarke, R. H., 304, 306, 313, 317, 319(13), 321,321(9), 322-323 Clarkson, R. B., 196 Clayton, E., 568 Cl6ment, S., 561 Cliff, G., 547 Cline, J. F., 155, 158(63), 226, 227 Clore, G. M., 246-247, 252-253, 256, 270(40, 41), 273,275(107), 278(35) Clubb, R. T., 252, 280(28) Co, T.-T., 318 Cochran, B., 485, 512(74), 513(74), 515, 521(74) Coffman, R. E., 425 Cogdell, R., 310, 311(23), 323(23), 324 Cohen, E. R., 335 Cohn, M., 63 Colby, J., 512,513(34) Cole, T., 215 Coleman, J. E., 17, 19, 21-22, 22(14), 23(17, 23), 25(17), 26, 29(1, 14), 30, 30(14), 31, 31(14), 33, 33(23), 35(46), 37(23), 39(14), 40(38), 42(14, 39), 263,269 Coleman, J.O.D., 510 Coleman, P. M., 153 Collet, S. A., 25 Colliex, C., 543,549, 553 Collinson, M., 484 Collison, D., 235 Colman, P. W., 46 Comtat, M., 512(70), 513(70), 515 Concar, D. W., 282 Connick, R. E., 48

591

Connors, R. E., 321-322 Cook, R. J., 183, 186 Cooker, A. R., 538 Cookson, J. A., 572, 574 Coon, J., 378 Cooper, C. E., 369, 371, 383 Cooper, J. M., 383 Cornelissen, B., 299, 304(3), 305(3) Cornelius, J. B., 168, 234-235, 241(5), 242(5), 244(5) Corson, D. C., 67, 69(87) Coslett, V. E., 542-543 Costa, H. S., 14, 16(61) Cote, C. E., 270 Courteix, A., 512(70), 513(70), 515 Coury, L. A., Jr., 512(76), 513(76), 515 Cox, B. G., 96, 97(43) Cox, P. A., 487, 501 Craston, D. H., 512(78), 513(78), 515 Craven, A. J., 550 Crawford, J. L., 25 Crawford, V. H., 418 Crewe, A. V., 542 Crichton, R. R., 384 Crosnier, J., 551,552(37) Crow, G., 582 Crumbliss, A. L., 512(73), 513(73), 515 CuUison, J. K., 501 Cundall, R. B., 523 Curli, S., 259

D Dahl, C. E., 273 Dahlin, S., 259 Dahlquist, F. W., 250 Dahr, S. K., 331 Daigne, B., 579, 582(108) Dalai, N. S., 207 Dalgano, D. C., 73, 74 Dallas, J. L., 61 Dalton, L. R., 166, 211 D'Amico, K. L., 558 Darnell, D. W., 331 Davidov, A. S., 325 Davies, A. K., 523 Davies, E. R., 128, 133(37) Davies, R. E., 45

592

AUTHOR INDEX

Davis, D. G., 8, 82, 90(15), 102(15) Davis, G., 512, 512(41, 46, 77), 513(34, 41, 46, 77), 514, 514(84), 515 Davoust, C. E., 181,226, 240 Dawson, J. H., 47 Dawson, M., 573 Day, E. P., 368, 419, 421(8), 437, 441-442, 448, 450(1), 451,451(1), 452(1), 454(13), 455, 460(1), 461-462, 464, 478(6) Deaver, B. S., 529 Deaver, B. S., Jr., 448 de Bernard, B., 118 De Boer, D.K.G., 556 de Boer, E., 235 de Boer, G., 53 de Boer, J.W.M., 53 Debrunner, P., 434 Debrunner, P. G., 363,369(29), 379(29), 464, 467(4), 478,478(4) Dechter, J. J., 80 Deckman, H. W., 558 Deconninck, G., 573 de Haas, G. H., 47, 78 Deisenhofer, J., 320, 325(45) de Jager, P. A., 313 DeKoch, R. J., 234-235 Delaglio, F., 252, 280(26) Delpech, B., 563 Delville, A., 86 Demarco, A., 252 Demarty, M., 584-585 den Blanken, H. J., 308, 310(21), 311(22), 314(22), 321-322, 322(57), 323,324(21), 325, 327(21), 328, 328(66, 69), 329(67, 68) Den Hollander, J. A., 104 Deranleau, D. A., 306, 317(16) Derbyshire, W., 214 de Ropp, J. S., 7, 10(45), 13(53), 15(66), 259, 261(61) DerVatanian, D. V., 404 Desbiez, M. O., 584 Dessy, R., 360 Detellier, C., 80, 81(5), 82(5), 83(5), 86(5) Detlefsen, D. J., 247, 250(17), 252, 280(28) Devilla, S. D., 552 Deville, A., 368 de Vries, S., 377 Dexter, D., 383 Dez, P., 556

Dick, R., 74, 76(99) Dicks, J. M., 512(46), 513(46), 514 Dickson, R. C., 21 Dijkman, J. A., 323,328, 328(69) Dikanov, S., 142 Dikanov, S. A., 244 Di Liberto, S., 562 Dill, K., 62 Dimicoli, J. L., 73 Diner, B. A., 323, 329(68) Ding, X. Q., 434, 478 Dinse, K. P., 218 Di Paola, R., 580 Dirac, P.A.M., 426 Di Stefano, D. L., 13,258, 259(55), 261(55) Dixon, D. W., 283 Dixon, N. E., 371 Dixon, R. M., 90, 102(39) Doan, P. E., 136, 156(42) Doblhoff-Dier, O., 512(83), 513(83), 515 Dobson, C. L., 2 Dobson, C. M., 45, 52-53, 57, 57(48, 49), 58, 58(48, 49), 59(48), 60(49), 61(48), 62 Doi, K., 226, 229(95) Dolphin, D., 337, 339 Dooley, D. M., 47, 270, 451,454(13), 455 Doolittle, L. R., 569, 570(76) Dorfman, L. M., 522 Dorio, M. M., 121, 122(4), 218 Dorr, R. G., 227 Dorus, E., 79, 82, 90(14), 96(14), 99(14), 102, 106(52) Dorus, W., I02, 106(52) Doyle, B. L., 573 Doyle, T., 217 Doyle, W. T., 195 Drabikowski, W., 86 Drakenberg, T., 47, 107, 113, 114(I), 115, 115(13), I16-I18, 118(I, 17), 263, 266 (78, 80), 267(78, 80), 274, 276(I14), 278(I 14) Driscoll, B. J., 512(78), 513(78), 515 Driscoll, P. C., 271,279(103, 104), 283 Dubach, J., 235,243 DuBois Murphy, P., 18 Duckett, S., 584 Dulcic, A., 204 Dumont, M. F., 220, 222(76), 223(76) Duncumb, P., 542 Dunham, W. R., 345, 368

AUTHOR INDEX Durliat, H., 512(70), 513(70), 515 Dutton, P. L., 321,354, 374(7), 397,402(1), 405(1) Dwek, R. A., 1-2, 15(1, 10), 45, 47, 47(10), 54(10), 62

E Eaton, G. R., 232,235,243,351,354 Eaton, S. S., 232,235,243,351,354 Echegoyen, L., 235 Echlin, P., 538,577, 578(101), 581(101) Eddowes, M. J., 501 Edelman, E., 538, 539(13) Edelstein, N., 207 Edwards, M. W., 3 Egekeze, J. O., 512(76), 513(76), 515 Ehrenberg, A., 190, 195, 196(18), 215, 216(63), 230, 450 Eich, G., 8 Eichman, N. C., 342 Eickman, N. C., 341 Eisenegger, J., 202 EI-Bayoumi, M. A., 325 Elenz, E., 102, 106(52) Eley, C.G.S., 74 Elgavish, G., 45, 49, 55(42) Elliott, D., 504 Ellis, K. J., 46 Ellis, P. D., 17-18, 24, 25(13), 41(13), 43(13) Elmd6n, K., 116, 118 El-Sayed, M. A., 305,307 Emerson, S. D., 3, 10(25), 11(25), 12(25), 14(25) Emptage, M. H., 464 Endo, H., 512(82), 513(82), 515 Engelmann, C., 572 Engeseth, H. R., 17, 21(5), 263,265(85) Englander, S. W., 2-3, 9(40), 13(24, 40, 54), 14(24), 258, 259(55, 56), 261, 261(55, 56), 275, 276(120) Ensign, S. A., 512, 513(35), 521(35) Epp, O., 320, 325(45) Erbes, D. L., 167 Eriksson, I. R., 211,214(49), 215(49) Eriksson, L.E.G., 190 Erman, J. E., 7, 10(35), 15(66), 259, 363 Ernst, R. R., 2-3, 7(33), 8(22, 38), 10(13),

593

32, 34(42), 123, 129(20), 133, 246-247, 264(4), 265(4), 266(4), 268, 279(4) Escaig, F., 539, 584 Espanol, M. T., 79, 90, 93, 94(40), 95(40), 96(40), 99(40), 102, 103(38, 40), 105(40), 106(52) Esposito, G., 77,271,279(106) Ettinger, R., 283 Evans, C. A., Jr., 579 Evans, C. H., 46 Evans, P. R., 62, 63(76), 66(76) Evelhoch, J. L., 37, 39, 40, 41(54) Evelo, R. G., 244 Everitt, C.W.F., 448 Ewing, G. W., 354 Eyring, L., 45, 49, 55(42) F Fairbank, W. M., 448 Fairbrother, W. J., 63, 65(81), 66(81), 74(81) Falk, K.-E., 377 Fan, C., 136, 156(42), 219, 227 Farach, H. A., 207,209(43) Farmer, B. T. II, 253, 270(38) Farmer, M. E., 583 Farrar, J., 369 Farrar, T. C., 26 Farver, O., 191 Faulkner, L. R., 480 Fee, J., 191,223(7) Fee, J, A., 24, 153,225,341,343, 353,368, 374(2), 376(2), 408, 409(13), 441 Feher, G., 122, 190-191, 192(9, 13), 209(6), 223(6), 320 Feinberg, B. A., 14, 397, 492, 512, 513(35), 521(35) Feng, Y., 3, 9(40), 13(24, 40, 54), 14(24), 258, 259(55, 56), 261,261(55, 56) Fernandez, T., 561,562(63) Fernandez, V. M., 343, 344(37) Fernlund, P., 116 Ferrari, R. P., 235 Ferrer, J. C., 10, 15(47) Fesik, S. W., 32 Fessenden, R. W., 214-215 Fiat, D., 1, 48, 54 Fielden, E. M., 530 Fields, B. A., 259, 288(71), 289(71)

594

AUTHORINDEX

Figgis, B. N., 433 File, D. M., 584 Finazi Agro, A., 254, 255(49) Findling, K. L., 368 Fisher, S. F., 321,325, 328, 328(89) Fitzgerald, D. W., 21 Fitzgerald, J. J., 235 Fitzgerald, J. M., 411 Flaming, D. G., 384 Flanagan, H. L., 167 Flannery, B. P., 558 Flavell, K. J., 104 Fleischmann, M., 509 Fleser, A., 104 Fl6rke, U., 426, 434, 436(29) Folkers, P.J.M., 77 Forman-Kay, J. D., 252, 256, 278(35) Forrer, J., 150, 166, 202-203 Fors6n, S., 17, 47, 80, 107, 110, 113, 114(!, I1), 115, 115(13), 116-118, 118(1, 17), 263, 266(78), 267(78), 271 Forte, C. P., 32 Fossel, E. T., 32, 93, 103(32) Fox, B. G., 369, 464, 468(5), 477, 478(11) Foyt, D. C., 529 Fragu, P., 580, 583 Francavilla, J. F., 235 Francis, G. D., 514(84), 515 Frank, H. A., 324 Frausto da Silva, J.J.R., 281,331 Freed, J. H., 121, 122(4), 209, 218 Freedman, L. P., 21 Freeman, A. J., 222 Freeman, H., 226 Freeman, H. C., 153, 259, 272(111), 273, 288(71), 289(71), 341 Freeman, R., 9, 97, 101(45) Frenkiel, T., 9 Freund, A., 557 Frew, J. E., 512(61), 513(61), 514 Frey, M. H., 32, 34(42), 268 Freysoldt, F., 150 Fritz, J., 191,223(7) Froland, W. A., 477, 478(11) Froncisz, W., 150, 202, 345 Fujita, S., 409 Fukushima, E., 205 Fukuyama, J. M., 340 Fullmer, C. S., 73,584 Furey, W. F., 25

Furman, B. K., 579 Fushimi, F., 512(49), 513(49), 514, 521(49) Fyfe, C. A., 109

G

Gabay, S., 584 Gabel, S. A., 103 Gadhavi, P. L., 269 Gaffney, B. J., 220 Galle, P., 551,552(37), 556,575,577(94), 584 Galley, W. C., 318 Gallicchio, V. S., 79 Gao, Y., 258, 259(57, 58), 261(57, 58) Gardner, K. H., 21, 23(23), 33(23), 37(23), 269 Gari6py, J., 73 Garner, C. D., 235 Gasparovich, C., 103 Gatteschi, D., 339, 342, 426, 434 Gay, R. R., 341 Gayda, J.-P., 368, 491 Geary, P. J., 380 Gemperle, C., 139 Gennis, R. B., 360 George, S. J., 485, 487, 490-491,491(13, 16, 17), 492, 492(13, 16), 493(13) Geraldes, C.F.G.C., 74, 76, 76(99), 82, 85, 88-90, 90(13, 16), 92(16), 102(16), 103, 103(35, 38), 104(54), 105(16), 106(16), 257 Gerday, C., 86 Gere, E. A., 191, 192(9, 13) Gerfen, G. J., 235, 241,242(15), 243(15) Gerothanassis, I. P., 114 Gerstein, B. C., 18 Geschwind, S., 209, 222 Gesmar, H., 274, 275(117) Gettings, P., 19, 22(14), 29(14), 30, 30(14), 31(14), 39(14), 42(14, 36) Geuze, H. J., 212 Giacometti, G., 324 Gibbs, P., 380 Gibson, J. F., 368-369 Giedroc, D. P., 21-22, 23(17), 25(17) Gillot, I., 540 GiUum, W. O., 18, 19(9) Ginsberg, A. P., 426

AUTHOR INDEX Gippert, G. P., 247,250(18), 288(18) Girard, F., 579, 582(108) Girerd, J.-J., 426, 472 Glasel, J. A., 1, 15(8), 52 Glezal, C., 540 Glickman, J., 31 Glickson, J. D., 60-61 Gloystein, F., 574 Glusker, J. P., 254 Gold, A., 434 Gold, M. H., 15 Goldberg, M. S., 252,280(28) Goldblum, N., 57 Golding, R. M., 49, 51(40), 53(40) Goldsmith, J. G., 576 Golicheff, I., 572 Good, B. W., 54 Goodall, K. G., 271,279(104) Goodenough, J. B., 426 Goodman, C. M., 32 Gooley, P. R., 252,275(34), 278(34) Goran Eriksson, L. E., 195, 196(18) Gordon, B. M., 556 Gordy, W., 124,215,216(61), 217 Goiter de Vries, H., 323 Gosh, S., 318-320 Goshi, Y., 557 Govindaraju, K., 512(56), 513(56), 514, 528 Govindjee, 323,325 Grady, J. K., 235 Graf, F., 125, 539 Graham, H. C., 63, 65(81), 66(81), 74(81) Grampf, G., 197 Grande, H. J., 168 Grandjean, J., 86 Grant, D. M., 283, 284(139) Gray, C. W., 74, 76(99) Gray, D. M., 74, 76(99) Gray, H. B., 153, 226, 512(52, 54), 513(52, 54), 514, 516(54), 523 Green, M. J., 512, 512(77), 513(77), 515 Greenwood, C,, 369, 379 Greenwood, R. J., 337 Greiner, S. P., 226 Greis, J., 324 Gribnau, M.C.M., 220,222(76), 223(76) Griesinger, C., 247 Griffith, J. M., 212 Grime, G. W., 570, 573 Grisham, C. M., 78, 84, 86, 87(22, 25)

595

Groeneveld, C. M., 283, 284(137, 138), 285(138) Grohman, K., 10, 14(44) Gronenborn, A. M., 246-247, 252-253,256, 270(40, 41), 273, 275(107), 278(35) Gruff, E. S., 19, 43(15) Grundstr6m, T., 116-118 Grunfeld, J. P., 551,552(37) Grupp, A., 122-123 Gschneider, K. A., Jr., 49, 55(42) Gudavi~ius, A. V., 512(65), 513(65), 514 Gueron, M., 1, 55 Guigliarelli, B., 491 Guinier, A., 542 Gullans, S. R., 104 Gullapalli, R. P., 105 Gunsalus, I. C., 191,223(7), 359 Gtinthard, H. H., 203 Gunther, H., 512(40), 513(40), 514 Guo, L.-H., 507, 512(45, 60), 513(45, 60), 514, 518(60), 521(45) Guo, L-H., 506 Gupta, R. K., 80, 90(10), 99(10), 106(10) Gurbiel, R. J., 343 Gurevi~iene, V. V., 512(66), 513(66), 515, 516(66), 518(66) Guss, J. M., 259, 272(111), 273, 288(71), 289(71) Gutheil, W. G., 372, 373(49) Gutteridge, J.M.C., 382

H Haas, C., 478 Haberkon, R. A., 7, 18(9), 19(9) Hagen, W. R., 168, 345, 363, 365, 368, 368(34), 369, 379(34, 42), 478 Hager, L. P., 434 Hagler, H. K., 538 Hale, P. D., 512(47), 513(47), 514 Hales, B. J., 393,394(9) Hali, F. C., 254, 255(49) Hall, D. O., 224 Hall, J. W., 418 Hall, L., 63 Hall, T. A., 548 Halle, B., 110, 271 Halpern, S., 580, 583 Halton, M. P., 49, 51(40), 53(40)

596

AUTHOR INDEX

Halvorsen, Y. D., 21 Hammel, E. F., 448 Hamnett, A., 500 Hampton, D. A., 207 Handschumaker, M., 40 Handy, G. W., 62, 63(76), 66(76) Hanna, M. W., 234-235 Hanna, P. M., 235, 239, 240(13), 241, 242(15), 243(13, 15) Hansen, R. E., 223,359, 380, 383(62) Hanson, G. R., 339-340, 343,353 Harbison, G. S., 19, 43(15) Harder, S. R., 14, 397 Harding, A. E., 383 Hardy, W. H., 313 Harling, O. K., 561 Harmer, M. A., 504, 512(61), 513(61), 514 Harmsen, B.J.M., 77 Harrel, S., 382 Harris, D. C., 227 Harris, E. A., 209 Harris, J., 584 Harris, W. C., 583 Harrison, S. C., 25, 269 Harrowell, P. R., 272(111), 273 Hartmann, S. R., 122 Hartzell, A. L., 525 Hasegawa, K., 522 Haser, R., 62, 63(76), 66(76) Hatchikian, E. C., 485, 487, 490-491, 491(13, 16, 17), 492, 492(13, 16), 493(13), 496(14), 497 Hatfield, W. E., 418 Haupt, H. J., 426, 434, 436(29) Havel, T. F., 273 Hawk, R. M., 105 Hawkridge, F. M., 501,504 Hayahowa, S., 557 Hayashi, T., 512(82), 513(82), 515 Hayer, M. K., 89, 90(36), 97, 101(46) Hearshen, D. O., 345, 368 Heikkinen, D. W., 575 Heisenberg, W., 426 Helige, J., 551,552(37) Heller, C., 215 Henderson, T. A., 125, 143(33), 168(33), 222 Hendrich, M., 434 Hendrich, M. P., 363,369, 369(29), 379(29), 464, 467(4, 8), 468(5, 7), 474, 477-478, 477(7), 478(4, 6, 7, 11)

Heng, Y. M., 552 Henkens, R. W., 512(73), 513(73), 515 Hennequin, E., 561,562(63) Henry, L., 550 Heritage, J. E., 34, 37(47), 266, 267(86) Hernandez-Nicaise, M. I., 540 Hershberg, R. D., 47, 78 Hershberger, M. V., 318 Heurteaux, C., 561-562, 562(63) Higgins, I. J., 512, 512(51), 513(34, 51), 514, 514(84), 515 Hilbers, C. W., 53, 77, 247, 248(13), 263, 263(13), 264(13), 277(13) Hilion, F., 579 Hill, B. C., 379 Hill, H. A. O., 254, 255(47), 263, 271, 279(103, 100), 283, 283(75), 479, 487, 501,503-500,506-507,507(2), 510, 512, 512(41, 45, 51, 59-61, 77), 513(34, 41, 45, 51, 59-61, 77), 514, 514(84), 515, 518(60), 521(45) Hille, R., 368 Hillion, F., 579, 582(108) Himmelwright, R. S., 341-342 Hinckley, C. C., 43 Hiraoki, T., 118 Hirsh, D. J., 393, 394(10) Ho, B. T., 584 Hoare, J. P., 509 Hodges, R. S., 73 Hoentzsch, C., 196, 197(20) Hofeldt, R. H., 323 Hofer, P., 122-123, 128(13), 139, 139(13), 150(47), 183(13) Hoff, A. J., 121,202, 241,244, 291,293(2), 297(2), 299, 303(2), 300, 304(3), 305(3), 306, 308, 308(2), 310(21), 311, 314(22), 321, 321(2, 9), 322, 322(57), 323-324, 324(21), 325, 325(76), 327(21, 76), 328, 328(66, 69), 329, 329(67, 68) Hoffman, B. M., 125, 168(32), 219-220, 221(79), 224, 226-229, 229(95), 240, 343, 359 Hoitink, C.W.G., 283 Holden, H. M., 11,259, 261(68) Holloway, C. E., 235 Holm, R. H., 18, 19(9), 485 Holman, T. R., 464, 467(8), 478 Holmquist, B., 331,339-340, 343 Holt, I. J., 383

AUTHOR INDEX Holtzelmann, R., 426 Holz, R. C., 15 Homma, Y., 579 Hong, X., 283 Hoops, S. C., 287(145), 288 Hopper, D. J., 512(45, 59), 513(45, 59), 514, 521(45) Hore, P. J., 276 Horrocks, W. de W., Jr., 44, 47, 52 Horrocks, W.D.J., 120 Horvath, S. J., 273 Houseman, A.L.P., 219 Howard, J. B., 14, 261,266(74) Howard, J. W., Jr., 18 Howes, B. D., 478 Hubbard, P., 107, 110(5) Huber, A. M., 577, 579-580, 582-584 Huber, R., 247, 248(13, 15, 16), 263(13), 264(13), 277(13), 283,320, 325(45) Hughes, M. S., 104 Huhta, D. W., 286 Huizing, A., 556 Hunt, J. A., 551 Hunt, J. B., 22 Hunt, J. W., 522 Hurst, G., 200, 201(23), 222 Hurst, G. C., 125, 143(33), 168(33) Hfittermann, J., 220, 235, 237, 238(12), 239(12), 240(12) Hutton, W. C., 84, 87(22) Huynh, B., 404 Huynh, B. H., 345,464, 478 Hyberts, S. G., 252, 280(28) Hyde, J. S., 120, 126, 150, 168(35), 185, 186(89), 190, 195,196(18), 197,202,211, 214(49), 215(49), 217, 219(30), 220(69), 226(4), 331,345, 354, 392

lanniello, R. M., 512(79), 513(79), 515 Ida, A., 557 Ikeda, T., 512, 512(49), 513(33, 49), 514, 521(33, 49) Ikura, M., 252, 266, 267(87), 276(32, 33), 278(33) Imoto, T., 57 Inagaki, F., 45, 56(14) Inglewood, W. J., 360

597

Ingram, P., 551-552, 552(39), 583 Inners, R. R., 18 Innes, J. B., 392-393,393(8), 394, 394(8, 10) Inoue, M., 378 Inoue, Y., 3, 9(23), 14(23, 60) Ioannidis, N., 371 Isaacson, R. A., 191,209(6), 223(6) Ishida, T., 378 Iwafune, K., 11, 14(60), 15(48) Iwai, H., 512(69), 513(69), 515

J Jackson, J. A., 53 Jacobson, B. L., 11,259, 261(68) Jakobsen, H. J., 18 James, T. L., 246 Jamienson, D. N., 573 Janata, E., 522-523 Janeau, A., 584-585 Janick, P. A., 464 Janssen, K.T.F., 577 Jardetzky, O., 282, 283(129) Jasaitis, J. J., 518 Jayaraj, K., 434 Jeanguillaume, C., 549, 552-553 Jeanloz, R. J., 11 Jeener, J., 2, 10(13) Jeffrey, F.M.H., 89 Jensen, C. F., 18 Jensen, G. M., 41 Jensen, L. H., 57,472 Jensen, P., 227 Jin, H.-Y., 168 Johansson, C., 115, 117-118, 263, 266(80), 267(80) Johansson, T. B., 568 Johari, O., 582 Johnson, B. A., 21, 23(17), 25(17) Johnson, J. L., 512(76), 513(76), 515 Johnson, L. N., 57 Johnson, M. K., 368 Johnston, E. R., 283,284(139) Jollie, D. R., 411 Jonah, C. D., 522 Jones, C. R., 56 Jones, K. W., 556 Jones, T. S., 504 Jones, W., 103, 104(54)

598

AUTHORINDEX

Jongenelis, A.P.J.M., 322-323, 328(66), 329(68) Jonsson, N.B.-H., 37 Juarez-Garcia, C., 369, 464, 467(8) Juilliard, A. K., 540 Junk, W., 322

K

Kadir, F.H.A., 369 K/igi, J.H.R., 32-33, 34(42, 44), 268 Kahn, E., 580 Kahn, O., 426 Kahn Maler, C., 557 Kaiser, G. H., 307 Kallenbach, N. R., 275, 276(120) Kaln, R., 544 Kanner, J., 382 Kappl, R., 235, 237, 238(12), 239(12), 240(12) Kaptein, R., 247 Karlstr6m, G., 47 Karpenstein, I., 434, 436(29) Karube, I., 518 Kasha, M., 313,325 Katsumura, Y., 522 Kawahara, K., 512(81), 513(81), 515 Kay, C. J., 397, 512(57), 513(57), 514 Kay, L. E., 73, 250, 252, 266, 267(87), 279(24), 280(24-26) Ke, Y., 118 Kearns, D. R., 56 Keating, K. A., 7 Keating, K. M., 22 Keeler, J., 9 Keene, J. P., 522, 527 Keicher, E., 540 Keijzers, C. P., 167, 168(74), 220, 222(76), 223(76), 235 Kelly, C. M., 286 Kelly, M. A., 579 Kennedy, M. C., 219, 224-225, 343,485 Kent, T. A., 368, 419, 421(8), 437, 450(1), 451(1), 452(1), 460(1), 464 Kessler, H., 252 Keutmann, H. T., 273 Kevan, L., 121, 122(5), 165(5), 209, 213 Kilfoil, V. J., 273,283(108) Kim, E. E., 31, 40(38), 42(38)

Kim, J., 229, 478 Kim, J. E., 340 Kim, L., 235 Kimura, J., 512, 513(32) King, T. E., 226 Kirby, B. J., 575 Kirk, K., 80, 96(11), 97(1 I), 99(11), 103 Kirk, T. K., 14 Kirste, B., 121, 207, 236, 239(8), 240(9), 243(8) Kispert, L. D., 121, 122(5), 165(5), 209, 213 Kissinger, C. R., 472 Kitchen, N. A., 263, 283(75) Klaassen, A.A.K., 235 Klevit, R. E., 273 Kleywegt, G. J., 247 Knall, J., 504 Knapp, E. W., 321,325 Knetibuhl, F. K., 220 Knopp, P., 426, 431(24) Knowles, P. F., 270, 408 Kobayashi, D., 512,513(33), 521(33) Koch, S. A., 19, 43(15) Kochoyan, M., 273 Koehler, K. A., 115 Koehorst, R.B.M., 321 Koenig, S. H., 47, 78,270 Kohler, B. E., 306 Kohzuma, T., 512(58), 513(58), 514 Kolbe, W., 207 Komiya, H., 320 Komoroski, R. A., 105 Koning, R.N.H., 77 Koningsberger, D. C., 273, 275(113) Kopf, D. A., 552 KOrdel, J., 274, 276(115), 278(115) Kosen, P. A., 270, 271(99) Kosman, D., 354 Kostelnick, R. J., 18 Kostic, N. M., 523 Kotani, M., 358 Kraft, K., 200, 201(23) Kraulis, P. J., 269 Kreilick, R., 200, 201(23), 222 Kreiter, A., 220 Kretsinger, R. H., 67, 73 Krezel, A. M., 11,259, 261(68) Krieger, Robert E., 403, 405(5), 409 Krizek, B. A., 273, 283(108) Kuchel, P. W., 103

AUTHOR INDEX Kuchta, R. D., 343 Kuipper, S. m., 556 Kultat, M., 512(70), 513(70), 515 Kulys, J. J., 512,512(42-44, 63, 65, 66, 71), 513(30, 42-44, 63, 65, 66, 71), 514-516, 516(30, 66), 518, 518(66) Kuntz, I. D., 73 Kuo, L. C., 340 Kurachi, K., 57 Kurreck, H., 121,207 Kurtz, D. M., 478 Kuwahara, J., 339, 363 Kuwana, T., 504 Kwiram, A. L., 166, 211,306, 317(16), 318, 319(13)

L Labotka, R. J., 93, 94(40), 95(40), 96(40), 99(40), 103, 103(40), 104(54), 105(40) Lam, K.-Y., 528 La Mar, G. N., 2, 3(25), 7(27, 36), 10(25, 45), 11(25), 12(25), 13(53), 14(15, 62, 63), 14(25, 27), 15(66), 16(62, 71), 55, 120, 259,261,261(61), 266(74) Landin, J. A., 451,454(13), 455 Landis, C. R., 286 Lang, N., 577 Lanne, B., 377 Lannon, A. M., 512, 513(37), 516(37) Larras-Regard, E., 583 Larson-Raznikiewicz, M., 64 Laszlo, P., 80, 81(5), 82(5), 83(5), 86, 86(5) Lauble, H., 225 Laue, E. D., 269 Laukein, G., 202 Laurent-Pettersson, M., 559, 563 Laurinavi~ius, V.-S.A., 512(66), 513(66), 515, 516(66), 518(66) Laviron, E., 482 Lawrance, G. A., 506, 512(45, 60), 513(45, 60), 514, 518(60), 521(45) Lazof, D , 584 Leapman, R. D., 551,553 Lecomte, J.T.J., 16 Led, J. J., 274,275(117) Lee, C.-W., 512(54), 513(54), 514, 516(54) Lee, J., 11

599

Lee, K., 14, 16(62) Lee, K.-B., 2, 14(15) Lee, L., 45, 67, 70-71, 72(86), 73 Leer, J. C., 254, 255(47) Lefebvre, F., 584 Lefevre, H. W., 575 Le Furgey, A., 551-552, 552(39) LeGall, J., 2, 13(12), 14(12), 345,404, 478 Legge, G.J.F., 568, 573,575 Leigh, J. S., 97, 321,393 Leigh, J. S., Jr., 48, 283,284(135, 136), 363, 393 Lemaistre, J. P., 306 Lemons, J. F., 53 Leniart, D. S., 195 Lenkinsky, R. E., 45, 46(15), 60-61, 89 Leonard, R. T., 46 Lepre, C. A., 247, 250(18), 288(18) Lesk, A. M., 271,279(106) Lettington, O. C., 504 Leung, M., 305 Levine, B. A., 45, 73-74 Levine, W. G., 153 Levi-Setti, R., 581-582 Levitt, M. H., 9 Levy, H. R., 62 Levy, M. A., 229 Lewis, W. B., 53 Leyh, T. S., 235 Li, P. M., 226 Liao, P. F., 122 Liaudet, E., 512 Libor, S. I., 512(61), 513(61), 514 Liepinsh, E., 253,270(37-39) Lignell, A., 544 Lilja, H., 17, 113, 115(13) Lilley, P. E., 371 Lin, C. P., 323 Lin, K., 561 Lindahl, P. A., 419, 421(8), 437, 450(1), 451(1), 452(1), 460(1) Lindh, U., 572-573,574(83) Lindman, B., 80, 110, 114(11), 271 Lindsay, T. J., 512(79), 513(79), 515 Lindstr6m, 215,216(63) Lindstr6m, M., 115 Ling, Y. C., 582 Linse, S., 116, 118, 263, 274, 276(114), 278(114) Linton, R. W., 576, 583-584

600

AUTHOR INDEX

Lipari, G., 279-280 Lippard, S. J., 80, 342 Lipscomb, J. D., 369, 378, 411,464, 468(5), 477, 478(11) Lipscomb, W. N., 25 Lismaa, S. E., 491 Liss, Alan R., 584 Little, C., 339 Littlechild, J. A., 64 Liu, C. S., 18, 19(9) Liu, M.-Y., 345, 478 Lizuka, T., 358 Llin~is, M., 252 Lloyd, E., 532 Loach, P. A., 405 Lodding, A., 584 Lode, E. T., 378 Loehr, T. M., 227, 368 Loevillet, M., 572 Lommen, A., 247, 248(13), 263, 263(13), 264(13), 272(111), 273, 275(112, 113), 277(13), 283 London, R. E., 82, 90(15), 102(15), 103,274, 280(119) Long, K. M., 90, 103(38) Long, M. M., 101, 102(51) Lord, K. A., 235 Lord, R. C., 18, 19(9) Lorimer, G. W., 547 Lounasmaa, O. V., 417 Lous, E. J., 310-311,317, 323-324, 325(76), 327(76), 328 Lovenberg, W., 380 Lowe, V. J., 487, 501 Lu, P., 47, 78 LuBien, C. D., 342 Lubitz, W., 199, 207, 207(37), 218(37) Luchinat, C., 1, 2(4), 7(34), 9(43), 11(49), 13(43, 51), 14(51, 65), 15(68), 49, 50(43), 55(43), 256, 258(51), 259, 259(51), 261(51), 270(51), 344 Lucken, E.A.C., 126, 168(36) Ludden, P. W., 512, 513(35), 521(35) Lui, C., 561 Lure, V., 226 Lurch, K., 342 Luthjen, L. H., 528 Lutz, M., 328 Luz, Z., 48, 109 Lynch, J. B., 369, 394

M Mac Arthur, I. C., 573 Maciel, G. E., 18 Mackenzie, N. E., 252, 275(34), 278(34) MacLennam, D. H., 73 Mair, G. A., 57 Maki, A. H., 216, 291, 297(1), 302, 302(1), 303(1), 306, 312(1), 317, 317(1), 318319, 319(1), 320, 320(6), 321(1) Makinen, M. W., 234, 237(7), 239(6, 7), 340 Malloy, C. R., 89, 103(35) Malmon, A. G., 564 MalmstrOm, B. G., 153, 363, 387, 512(52, 54), 513(52, 54), 514, 516(54) Mandon, D., 434 Mao, S. Y., 318 Marchetti, P. S., 18, 25(13), 41(13), 43(13) Marcus, R. A., 281 Maret, W., 478 Margalit, R., 523 Margoliash, E., 227 Marinetti, T. D., 53, 62 Marion, D., 7, 252, 276(32) Markham, G. D., 235 Markley, J., 4 Markley, J. L., 11, 13(55), 86, 219, 259, 261(68) Marmorstein, R., 25,269 Marsden, C. D., 383 Marsh, D., 408 Marsh, M., 573 Marshall, A. G., 109 Martin, C. T., 158,227 Martin, R. B., 46, 47(21), 56(21) Martin, S, R., 118 Martinelli, R. A., 340 Martini, F., 559, 561,562(63) Martinsen, J., 125, 168(32), 220, 221(79) Martz, H. E., 575 Mascagni, P., 77 Mascini, M., 512, 513(31) Mason, J., 80, 81(4), 82(4), 83(4), 263, 267(77) Massiot, P., 556, 569 Massonnet, B., 568, 575(74) Matheson, M. S., 522 Mathews, B. W., 46 Matsueda, T., 300 Matsushita, F., 512, 513(33), 521(33)

AUTHOR INDEX

Matwiyoff, N. A., 103 Mauk, A. G., 10, 15(47), 283 Maxwell, J. A., 568 Maxwell, M. H., 551,552(37) Mayhew, S. G., 406, 407(9) Mayne, A. J., 504 Mazzolini, A.P., 573 McCarthy, R. L., 522 McClendon, G., 507 McConnell, H. M., 214-215 McCracken, J., 234-235, 241(5), 242(5), 244(5) McDonagh, J. M., 115 McDonnell, P. J., 342 McDowell, C. A., 207 McGarvey, B. R., 50 McGuiri, M. A., 270 McIntosh, L. P., 250 McKenna, C. E., 372, 373(49) McKenna, K., 512(80), 513(80), 515 McLachlan, A., 522 McLachlan, S. J., 2, 14(15) McLaughlin, A. C., 283, 284(136) McLean, P. A., 229 McMichens, R. B., 83, 84(17), 86(17), 97(17), 98(17), 101(17), 102(17) MeMillin, D. R., 17, 21(5), 263,265(85) McPherson, A., 75 McRee, D. E., 25 Meares, C. F., 46 Mehring, M., 122-123, 128(13), 139(13), 150, 183(13) Meiboom, S., 48 Meiburg, R. F., 308, 311(22), 314(22), 325, 328 Meier, B. H., 2, 10(13) Meinhhardt, S. W., 360 Melson, G. A., 433 Mentr6, P., 539, 540(15) Merkle, D. L., 273,283(108) Merret, M., 62, 63(76), 66(76) Merz, K. M., 188, 287(145) Messerle, B. A., 268, 276 Messerschmidt, A., 247, 248(13, 15, 16), 263(13), 264(13), 277(13) Messori, L., 9, 11(51), 13(43, 51), 14(51,65), 78,259 Meyran, J. C., 539 Michel, H., 320, 325(45) Michelson, P. F., 448

601

Migeon, H. N., 577, 582 Miki, K., 320, 325(45), 512(49), 513(49), 514, 521(49) Miller, D. G., 559 Mims, W. B., 121, 122(8), 126, 128(8), 131(8), 132(8), 133(8), 134, 135(8), 141, 143, 149(8, 34), 167, 167(49), 176(34), 225,241 Ming, L.-J., 15 Minkin, J. A., 556 Mino, Y., 378 Miyamoto, S., 512, 513(32) Miyaoka, S , 512, 513(33), 521(33) Miyazawa, T., 45, 56(14) Mizukami, H., 259 Mo, S., 101, 102(50), 105 MObius, K., 181-182, 199,206,207(37), 218, 218(37), 219 Moench, S. J., 4, 6(26), 14(26) Molinari, H., 77,271,279(106) Moiler, B. L., 321 Monaco, H. L., 25 Monnanni, R., 11, 13(51), 14(51, 65) Monoley, M., 573 Montalione, G. T., 252 Mooberry, E. S., 259 Moody, G. J., 512(62), 513(62), 514, 518(62) Moore, G. R., 2, 62, 74,261,262(72), 369 Moore, J. M., 247,250(18), 288(18) Morallee, K. G., 44, 57(2) More, C., 491 More, G., 368 More, K. M., 235,243 Morgan-Hughes, J. A., 383 Morrill, T. C., 45 Morris, G. A., 97, 101(45) Morrison, G. H., 536, 538, 538(1), 576(7), 577, 577(7), 578, 578(7), 579(104), 582583,583(102), 584 Morse, D. H:, 575 Morse, R. H., 365 Mortensen, L. E., 168 Morvan, C., 584 Mory, C., 549, 553 Moss, G. P., 52 Mota de Freitas, D., 79, 82, 85, 90, 90(13, 14, 16), 92(16), 93, 94(40), 95(40), 96(14, 40), 99, 99(14, 40), 100(48), 101-102, 102(16, 50), 103, 103(38, 40), 104(54), 105, 105(16, 40), 106(16, 52), 263

602

AUTHOR INDEX

Motta, A., 77,271,279(106) Motten, A. G., 318 Mottola, H. A., 512(75), 513(75), 515 Moulton, G. C., 207 Moura, I., 345,404, 461-462, 472, 474, 478 Moura, J.J.G., 345,404, 461-462,472,474, 478 Mukand, S., 337 Mukund, S., 404 Mulay, L. N., 415 Mulks, C. F., 236, 239(8), 243(8) Muller, L., 7 Muller, N., 3 Mullinger, R., 224 Miinck, E., 368-369, 378, 419, 421(8), 437, 450(1), 451(1), 452(1), 460(1), 463-464, 467(8), 468(5, 7), 469(1), 472, 474, 477(7), 477-478, 478(7, 11) Murakami, M., 93, 96(42) Murakami, T., 512, 513(32) Murata, M., 272(111), 273 Murphy, E., 82, 90(15), 102(15) Murray, R. W., 512(76), 513(76), 515 Murthy, H.M.K., 40 Mustafi, D., 234, 237(7), 239(6, 7) Muus, L. T., 209 Myers, R. J., 21

Neff, P., 77 Nettesheim, D. G., 14 Neuberger, A., 331,354 Neuhaus, D., 32-33, 34(42, 44), 246, 258(3), 268,279(3) Neumann, S., 512(40), 513(40), 514 Newton, M. E., 197, 202, 219(30) Nguyen, D. T., 273 Nicaise, G., 539-540, 551 Niccolai, N., 77, 271,279(106) Nicholls, P., 379 Nicholson, L. K., 252, 280(26) Nicholson, R. S., 510 Nieboer, E., 44-45, 46(11), 47(11), 57(2) Nieves, J., 235 Niklas, J. R., 196, 197(20), 207 Nirmala, N. R., 252, 280(28) Nitta, K., 118 Nomoto, K., 378 Nooren, I.M.A., 77 Noren, J. G., 584 Norris, J., 320 Norris, J. R., 321,323 Norris, V. A., 272(111), 273, 341 Norrish, R.G.W., 522 North, A.C.T., 1, 15(8), 52, 57 Norwood, T. J., 34, 37(47), 266, 267(86) Nuckolds, C. E., 67

N

O

Nader, P. A., 512(75), 513(75), 515 Nagahashi, G., 46 Nagasawa, T., 339 Nageswara Rao, B. D., 63 Nair, B. U., 512(56), 513(56), 514 Nakamizo, M., 300 Nanai, N., 11, 14(60), 15(48) Nancarrow, D. J., 512(41), 513(41), 514 Nar, H., 247, 248(13, 15, 16), 263(13), 264(13), 277(13), 283 Narula, S., 21, 23(23), 33(23), 37(23), 269 Nassau, K., 134 Nelsestuen, G. L., 116 Nelson, B. N., 304 Nelson, D. J., 73 Nelson, M. J., 228-229, 229(95), 365 Nelson, W. H., 217 Neff, D., 246, 250(2), 252

O'Brien, P. M., 573 O'Daly, J. P., 512(73), 513(73), 515 Odom, R. W., 579 Ogawa, S., 378 Ogino, T., 104 Oh, B.-H., I1, 13(55), 219, 259, 261(68) O'Hara, P. B., 78 Ohnishi, T., 360, 393 Okano, J., 578 Okawa, Y., 512(64), 513(64), 514 Okuma, H., 512(81), 513(81), 515 Oliver, B. N., 487, 501, 503-504, 512(59, 76), 513(59, 76), 514-515 Oliver, M. E., 393,394(9) Oliver, M. V., 10, 11(49), 15(46) Olivo, J. C., 580 Olson, J. M., 328 Oltzik, R., 378

AUTHOR INDEX O'Malley, P. J., 211,215(52) Omichinski, J. G., 273,275(107) Oparil, S., 101, 102(51) Opella, S. J., 47, 78 Oppenheimer, N. J., 246 O'Reilly, J. E., 407 Orme-Johnson, N. R., 378, 380, 383(62), 387,408,419,421(8) Orme-Johnson, W. H., 191, 228-229, 229(95), 354, 359, 373(5), 437, 450(1), 451(1), 452(1), 460(1), 464, 468(7), 477(7), 478(7) Ormerod, J. G., 328 Orr, R. V., 18 Orton, J. W., 212 Osawa, A., 3, 9(23), 11(48), 14(23), 15(48) Oschkinat, H., 247 O'Sullivan, W. J., 448 Ota, T. N., 378 Ottensmeyer, F. P., 553 Otting, G., 34, 37(49), 246, 250(2), 252-253, 268,270(36-39, 90), 276 Otvos, J. D., 14, 17, 21, 21(2, 5), 24, 24(2), 27(2), 29(2, 20), 30-31, 31(20), 37(51), 39-40, 40(51), 263,265(85) Ouznadji, H., 562 Ouznadji, S., 562 Overbeck, H. W., 101, 102(51) Overhauser, A., 191 Owen, J., 209 Owens, D. V., 307

P Paddock, R. M., 512, 513(38), 516(38) Padilla, A., 247 Padmanabhan, S., 84 Page, D. J., 504, 512(59), 513(59), 514 Paillotin, G., 310, 311(23), 323(23) Palmer, A. G. III, 266, 267(88) Palmer, G., 191,223(7), 337, 353-354, 361, 373(5), 374(1), 408 Pan, T., 21, 23(23), 33, 33(23), 35(46), 37(23), 269 Panchalingham, K., 101 Panchenko, L., 557 Pandya, K. I., 273,275(113)

603

Pantoliano, M. W., 342 Papaefthymiou, V., 472, 478 Papavassiliou, P., 491 Parello, J., 47 Parot, P. H., 324 Pasternak, C. A., 78, 79(1), 84 Pastore, A., 77, 271,279(106) Patil, D. S., 343,344(37), 380 Patkin, A. J., 583 Patterson, A., Jr., 283 Paulsen, M., 84 Payne, M. J., 380 Pearce, L. L., 464,478(6) Pearlstein, R. M., 321,325 Pecht, I., 191 Peck, H. D., Jr., 404 Pecoraro, V. L., 247, 250(17) Peisach, J., 143, 153, 167, 225-226, 234235,241,241(5), 242(5), 244(5), 360, 378 Pekar, J., 97 Penfield, G. E., 341 Penfield, K. W., 341 Peng, J., 252, 280(28) Peng, J. W., 252, 279(27), 280(27, 29) Perez, J. P., 550 Peric, M., 204 Perkins, S. J., 62 Pesliakiene, M. V., 512(66), 513(66), 515, 516(66), 518(66) Peters, J. A., 82, 90(13) Peters, T. J., 369 Peterson, J., 379, 441-442,461-462 Peterson, W. A., 579 Petersson, L., 215,216(63), 224, 450, 584 Pethica, J. B., 504 Petrera, S., 562 Petrin, M., 319-320 Pettegrew, J. W., 101 Pettersson, M., 561,562(63) Pfenninger, S., 150, 166, 202 Phillips, A. W., 62, 63(76), 66(76) Phillips, D. C., 57 Philo, J. S., 448 Piccioli, M., 9, 13(43), 15(68), 78, 259 Picot, D., 556 Pielak, G. J., 258, 259(57, 58), 261(57, 58), 282 Pierik, A. J., 345 Pike, M. M., 89, 103(32) Pilbrow, J. R., 124, 212, 221, 331, 332(la),

604

AUTHOR INDEX

336(1), 337-340, 345, 345(1a), 349, 349(I, 49), 351(la), 353-354, 389 Pilling, F. D., 572 Pinheiro, L., 504 Pinkowitz, R. A., 379 Plato, M., 199, 206, 207(37), 218(37) Pletcher, C. H., 116 Piotkin, E. V., 514(84), 515 Pocius, A. K., 512(43), 513(43), 514 Poel, W.A.J.A,v.d., 122 Pollack, S., 384 Pons, S., 509 Pontau, A. E., 575 Poole, C. P., 192, 194(14), 197(14), 207, 209(43) Poole, C. P., Jr., 408 Poole, R. K., 371 Porteous, R., 2 Porter, G., 522 Portis, A. M., 387 Post, J.F.M., 101 Poupko, R., 109 Prince, H. P., 74 Prins, M., 556 Proctor, I. D., 575 Proskuryakov, I. I., 324 Pryce, M.H.L., 335,424 Psalti, I.S.M., 504 Ptashne, M., 25 Puett, D., 235 Pullman, B., 57

Q Que, L., 378,467(8) Que, L., Jr., 15, 18(9), 19(9), 369, 464, 478, 478(6) Quintana, C., 538,551

R Rabi, I. I., 138 Rachmilewitz, E. A., 378 Radda, G. K., 62, 90, 102(39) Rado, G. T., 426 Ragsdale, S. W., 359, 397 Raine, A.R.C., 269 Rajagopalon, K. V., 512(76), 513(76), 515

Rajarathnam, K., 4, 7(27), 14(27), 259 Ramasami, T., 512(56), 513(56), 514 Ramasamy, R., 82, 90, 90(13), 93, 94(40), 95(40), 96(40), 99(40), 103, 103(38, 40), 104(54), 105(40) Ramaswamy, D., 512(56), 513(56), 514 Ramseyer, G. O., 577, 583(102) Rance, M., 266, 267(88) Randall, E. W., 52 Randolph, M. L., 351,376 Ranjeva, R., 584 Rao, B.D.N., 274, 275(116) Rao, K. K., 224 Rao, K.V.S., 392 Rasser, B., 577, 582 Rath, A., 103 Rattle, H.W.E., 408 Rauenbueler, P., 252 Raushel, F. M., 87, 102(30) Ravi, N., 345, 478 Rawlings, J., 464 Rawls, H. S., 325 Ray, B. D., 63 Ray, W. J., Jr., 86 Rayment, I., 11,259, 261(68) Raynor, J. B., 220, 222(78) Razumas, V. J., 512(65), 513(65), 514, 518 Rechnitz, G. A., 512(83), 513(83), 515 Record, M. T., 84 Redfield, A. G., 133 Redfield, C., 271,279(103, 104) Redskin, S., 557 Reed, D. E., 501 Reed, G. H., 47-48, 78, 235, 363 Rees, D. C., 229, 320, 478 Reijerse, E. J., 137, 138(43), 167, 168(43, 74), 222(76), 223(76), 235 Reilly, C. N., 54 Reiner, C., 196 Reiner, D. F., 559 Reinhammer, B., 153,226-227, 259 Renshaw, P. F., 97 Reuben, J., 44-45, 46(15), 49, 54, 55(42), 232, 234(1), 241, 291, 297(1), 337-338, 354 Rhee, M. J., 22 Rhyu, G., 86 Rice, D. W., 62, 63(76), 66(76) Richardson, F. S., 46, 47(21), 56(21) Richter, F. W., 574

AUTHOR INDEX Rickard, L. H., 501 Riddell, F. G., 89, 90(36), 96-97, 97(43), 101(46) Riordan, J. F., 23 Ripoll, C., 559, 562, 569, 575,584-585 Rist, G. H., 126, 168(35), 190, 211,214(49), 215(49), 217,220(69), 226(4) Rivera, E., 21, 23(23), 33(23), 37(23), 269 Rivers, M. L., 556, 558 Robbins, A. H., 25 Roberge, W. G., 558 Robert, B., 328-329 Roberts, J. E., 155, 158(63), 163,226-227 Roberts, L.C.K., 282, 283(129) Robinson, M. N., 2, 74 Roder, H., 2-3, 9(40), 13(24, 40, 54), 14(24), 258, 259(55, 56), 261, 261(55, 56), 419, 421(8), 437, 450(1), 451(1); 452(1), 460(1) R6der, R., 202 Roeder, S.B.W., 205 Roffi, G., 529 Rogers, B. L., 234-235 Rogli, V. L., 551 Romans, G., 562 Romero, A., 283 Romijn, J. C., 323 Rong, Q., 101,102(50), 105 Roomans, G. M., 538,544, 551,552(36) Rooney, W. D., 84, 99(18, 19) Roos, N., 538 Rosato, N., 254, 255(49) R6sch, P., 274,275(118) Rosenzweig, A. C., 451,454(13), 455 Rosevear, P. R., 270 Ross, J.B.A., 306, 317(16), 318 Rossotti, F.J.C., 44, 55, 57(2) Rotherty, R. A., 360 Rottgardt, D., 464, 468(7), 477(7), 478(7) Rottman, G. A., 226, 229(95), 235, 239, 240(13), 243(13) Rousslang, K. W., 318 Roy, A., 419, 421(8), 450(1), 451(1), 452(1), 460(1) Ruben, D. J., 7 Rudenauer, F. G., 578, 583 Rufty, T. W., 584 Rupley, J. A., 57 Russell, D. J., 7, 10(35), 259

605

Rustici, M., 77 Ruthroff, C. L,, 204 Rutter, R., 434 Ryan, C. C., 568 S Saboowalla, F., 380 Sahlin, M., 215,216(63) Sahurai, K., 557 Saito, A., 512, 513(32) Sakkers, P.J.D., 53 Salerno, J., 361 Salerno, J. C., 342, 369 Sales, K. D., 52 Salmon, G. A., 528 Salowe, S. P., 215, 216(63) Samalius, A. S., 512(42), 513(42), 514 Sanders, C. R., 107, 114(4), 263, 266(81), 267(81) Sanders-Loehr, J., 368 Sands, R. H., 191,223(7), 225, 345 Sanghera, G. S., 512(45, 60, 62), 513(45, 60, 62), 514, 518(60, 62), 521(45) Santos, H., 2-3, 4(29), 9(29), 10(29), 13(12), 14(12, 29, 58, 61), 16(58, 61), 257 Santos, R. A., 19, 43(15) Sarasua, M. M., 115 Sarma, V. R., 57 Sates, D. J., 7 Satterlee, J. D., 1, 2(3), 4(26), 6(26), 7(35), 10(35), 14(26), 15(66), 259 Sauer, M. C., 529 Saunders, J.K.M., 44 Scarselli, M., 77 Schaafsma, T. J., 313,321-322 Schaffer, A., 268 Schapira, A.H.V., 383 Scheer, H., 328 Scheller, F., 512(72), 513(72), 515 Scherage, N. A., 8 Scherer, P.O.J., 321,325, 328, 328(89) Schierbeck, R., 64 Schiffer, M., 320 Schmalbein, D., 202 Schmid, R. D., 512(71), 513(71), 515 Schmidt, J., 302, 304(5) Schmieder, P., 250, 252 Schoemaker, W. H., 303,304(7)

606

AUTHOR INDEX

Schofield, R. M. S., 575 Scholes, C. P., 191,209(6), 223(6), 226-227 Schoot Uiterkamp, A. J. M., 17, 22, 29(I), 30 Schou, M., 78, 79(2) Schuhmacher, M., 577, 582 Schuler, R. H., 214, 525 Schulman, G. I., 104 Schultz, R., 200, 201(23) Schweiger, A., 122-123, 125, 133, 139, 176(21), 178(21), 202-203 Scopes, R. K., 63 Scott, C. P., 550 Scott, L.D.L., 514(84), 515 Scozzafava, A., 11, 13(51), 14(51, 65) Sealock, R. M., 573 Sealy, R. C., 331,354 Searle, G.F.W., 321 Seeley, P. J., 62 Seibert, M., 321 Seidel, H., 195 Sekimukai, S., 512(81), 513(81), 515 Sellers, P., 116, 118 Senda, M., 512, 512(49), 513(33, 49), 514, 521(33, 49) Seo, Y., 93, 96(42) Sevely, J., 550 Sevick, J., 204 Sgarbi, C., 562 Shain, I., 510 Shaka, A. J., 9 Shaw, D. C., 371 Shelburne, J. D., 551,583 Sherman, F., 507 Sherry, A. D., 45, 74, 76, 76(99), 89, 103(35) Shetna, Y. I., 223 Shiau, F.-Y., 7 Shidara, S., 512(58), 513(58), 514 Shimizu, R., 578 Shu, Z. X., 512, 513(36) Shulman, R. G., 104 Shuman, H., 551 Siegel, F. L., 73 Siegel, L. M., 342, 464 Sieker, L. C., 57, 472 Siemers, T. C., 89 Sievers, R. E., 44 Sigel, A., 479 Sigel, H., 479

Silberberg, J., 102, 106(52) Silvestro, I., 556 Simmonds, M. B., 423 Simon, G. T., 552-553 Simon, H., 512(40), 513(40), 514 Simonis, U., 13 Simson, J.A.V., 539 Singel, D. J., 122, 167, 235, 241, 242(15), 243(15) Singh, I., 270 Singhal, G., 328 Sishta, B. P., 283 Sivaraja, M., 343 SjOberg, B.-M., 215, 216(63) Skelton, N. J., 274, 276(115), 278(115) Skjeldal, L., 11,259, 261(68) Skotheim, T. A., 512(47), 513(47), 514 Skrzynia, C., 115 Slappendel, S., 363 Sligar, S., 358 Sliger, S. G., 14, 16(62) Slodzian, G., 579-580, 582, 582(108) Sloop, D. J., 122, 123(10), 128(10) Smith, B. E., 254,255(47) Smith, C. A., 228, 584 Smith, E. T., 492, 512, 513(35), 521(35) Smith, K. M., 7 Smith, T. D., 221,338, 354 Smith, T. W., 89, 103(32) Smith, U., 320 Snyder, G. H., 62 Sod, E. W., 538 Soffe, N., 2, 34, 37(47), 266, 267(86) Sogiura, Y., 363 Solomon, E. I., 153, 341-342 Solomonson, L. P., 512(57), 513(57), 514 Soma, M., 303,304(7) Sombyo, A. P., 551 Sommer, F., 556, 567-569, 571,575(74) Sommer, J. R., 552 Song, W., 372, 373(49) SCrensen, O. W., 32, 34(42), 247, 268 Sosnoff, C. S., 512(76), 513(76), 515 Sousa, L. R., 319 Southampton Electrochemistry Group, 508-510 Sowadski, J. M., 40 Spaeth, J.-M., 196, 197(20), 207 Spanne, P., 558 Speck, S. H., 227

AUTHOR INDEX Spee, R., 323,324(74) Spence, J. T., 337 Spera, S., 252, 276(33), 278(33) Spicer, S. S., 539 Spiller, M., 270 Springer, C. S., Jr., 80, 84, 89, 99(9, 18, 19), 103(32), 105, 106(63) Sroubek, Z., 577 Stabler, R., 235, 237, 238(12), 239(12), 240(12) Stackebrandt, E., 328 Staehelin, L. A., 323 Stam, P., 220,222(76), 223(76) Standing, K. G., 579 Staskeviciene, S. L., 512(44), 513(44), 514 Steel, R. W., 522 Steiger, W., 583 Steiner, R., 328 Stenkamp, R. E., 153 Stephens, E. M., 78, 86, 86(25) Stephens, P. J., 491 Stephens, R. L., 11 Stevens, T. H., 158, 227, 365 Stockman, B. J., 252, 280(28) Stone, E. W., 216 Stonehuerner, J. G., 512(73), 513(73), 515 Stoorvogel, W., 212 Storm, C. B., 22,254, 255(47) Stout, C. D., 25, 225 Strous, G. J., 212 Stubbe, J., 215,216(63) Sucheta, A., 485, 496(14), 497, 512(74), 513(74), 515, 521(74) Sudfeldt, C., 235, 237, 238(12), 239(12), 240(12) Sudmeier, J. L., 37, 39, 40, 41(54) Sudnick, D. R., 47 Sugiura, Y., 339 Suguira, Y., 378 Suhl, H., 426 Summer, A. T., 551 Summers, M. F., 263,267(79) Sun, S.-C., 501 Sund, H., 291 Surerus, K. K., 464,468(7), 474,477,477(7), 478(7, 11) Sutin, N., 281 Sutton, S. R., 556 Suzuki, E., 93, 96(42) Suzuki, S., 512(58), 513(58), 514

607

Suzuki, T., 3, 9(23), 11(48), 14(23, 60), 15(48) Sv~ird, M., 116 Svirmickas, G.J.S., 512(42), 513(42), 514 Swanson, M., 227 Sw~trd, M., 47, 107, 114(1), 118(1) Swartz, H. M., 331,331(3), 345(3), 351,376 Sweeney, W., 10, 14(44) Swift, T. J., 48 Swinkels, D. W., 77 Sykes, A. G., 485,528 Sykes, B. D., 31, 45, 53, 62, 67, 69(87), 7071, 72(86), 73 Szabo, A., 279-280 Szwergold, B. S., 102

T Tabata, Y., 522 Tagawa, S., 522 Taimuty, S. I., 529 Takahashi, H., 512(81), 513(81), 515 Takase, S., 512(58), 513(58), 514 Takemoto, T., 378 Talburi, S., 8 Tam, S. C., 73, 75(94) Tanaka, H., 378 Tanaka, J., 522 Tang, J., 320 Taniguchi, V. T., 512(52), 513(52), 514 Tanswell, P., 64, 65(84), 66(84), 69(84) Tapiero, H., 556 Tarasevich, M. R., 512(50, 53, 55, 72), 513(50, 53, 55, 72), 514-515,516(53) Tart, G. E., 368 Tatsuma, T., 512(64, 67, 68), 513(64, 67, 68), 514-515,518(68) Taube, H., 53 Tavares, P., 345,478 Taylor, B. N., 335 Taylor, L., 360 Tebbutt, P., 511 Teesdale, W. J., 568 Teixeira, M., 404 Teleman, A., 118 Teleman, O., 118, 274, 276(114), 278(114) Telser, J., 170, 234, 237(7), 239(7) Terner, J., 434 Tesser, G. L., 77

608

AUTHOR INDEX

Thanabal, V., 247, 250, 250(17), 252, 279(27), 280(27, 28) Thanos, J., 512(40), 513(40), 514 Theil, E. C., 233, 235 Thellier, M., 559, 561-562, 562(63), 563, 569, 575, 584-585 Thomann, H., 122-123, 137, 138(44), 140, 142, 153(44), 156(48), 160-161, 162(67), 163(67), 168(44), 171, 171(44), 173(65, 82), 176(22), 178(22), 179, 180(82), 184(82), 186, 186(82) Thomas, J.D.R., 512(62), 513(62), 514, 518(62) Thompson, A. C., 557 Thompson, J. S., 340 Thompson, W. W., 46 Thomson, A. J., 369, 379, 485, 487, 490491, 491(13, 16, 17), 492, 492(13, 16), 493,493(13), 496(14), 497 Thorpe, C., 504 Thulin, E., 17, 107, 113, 114(1), 116, 118, 118(1) Thurman, G., 235, 243 Thurnauer, M. C., 321 Tibell, L.A.E., 37 Tibris, J.C.M., 359 Tiede, D., 320 Tien, M., 14 Till, A. M., 90, 102(39) Timkovich, R., 16 Timmerhans, K. D., 448 Tindall, P. 3., 122, 155(14) Tinti, D. S., 307 Tipton, P. A., 234-235, 241(5), 242(5), 244(5) Tom8, A.J.R., 82, 90(16), 92(16), 102(16), 105(16), 106(16) Torchia, D. A., 252, 266, 267(87), 279(24), 280(24-26) Tousimis, K., 552 Toy, A. D., 221 Toyama, K., 512(82), 513(82), 515 Transwell, P., 56, 63(60), 64(60) Trapane, T. L., 83, 84(17), .86(17), 97(17), 98(17), 101, 101(17), 102(17, 51), 116 Tras, G.H.J., 556-557 Trautwein, A. X., 412, 434, 436(29), 478 Trayer, I. P., 74 Trifunac, A. D., 523 Tromp, M.G.M., 280

Truchet, M., 582 True, A. E., 228-229, 229(95) True, A. J., 343 Truper, H. G., 328 Tsai, M.-D., 107, 113, 114(4), 263,266(81), 267(81), 359 Tschudin, R., 252, 266, 267(87), 276(32) Tsibris, J.C.M., 191,223(7) Tsopanakis, A. D., 371,411 Tucker, J. A., 551 Tiimpner, J., 577 Turano, P., 7, 10(46), 14(46, 47, 59) Turner, A.P.F., 512, 512(46), 513(34, 46), 514, 514(84), 515, 518 Turner, D. L., 2-3, 4(29), 6(30), 9(29), 10(29, 30), 13(12, 30), 14(12, 29, 58, 61), 16(58, 61), 257 Tvet, A. B., 572 Tweedle, M. F., 450 Tyryshkin, A. M., 244

U Ueda, T., 522 Ugurbil, K., 254, 255(48), 318 Ullrich, J., 323-324, 324(73, 74) Underwood, J. H., 557 Unger, S. W., 16 Urry, D. W., 83, 84(17), 86(17), 97(17), 98(17), 101, 101(17), 102(17, 51), 116

v

Valentine, J. S., 342 Valentine, M., 434 Vallee, B. L., 23, 331,340 Vallini, G., 11, 13(51), 14(51, 65) van Beeumen, J., 283 van Berkel-Arts, A., 168 van Camp, H. L., 161, 165(66), 225-226 van Deenen, L.L.M., 331,354 van de Kamp, M., 247, 248(13, 15, 16), 254, 255(49), 263(13), 264(13), 277(13) van der Bent, S. J., 313 van der Vos, R., 324, 327, 329 Van der Waals, J. H., 302, 304(5, 7) van der Zwet, G. P., 321,322(57), 323 van Dijk, C., 168

AUTHOR INDEX Van Divender, J. M., 84 van Dorp, W. G., 303,304(7) van Duynhoven, J.P.M., 77 van Earle, N.A.J.M., 137, 138(43), 168(43) van Egmond, J., 306 van Gelder, B. F., 341 van Gunsteren, W. F., 289 Van Langevelde, F., 556-557,574 van Leeuwen, F. R., 341 van Mierlo, C.P.M., 247 V~inng~rd,T., 190,220, 221(74), 226(4), 377, 387,409 Van Steveninck, M.E.V., 540 Van Steveninck, R.F.M., 540 van Vleck, J. H., 414, 426(2) van Wijk, F. G., 322 van Willigen, H., 236, 239(8), 240(9), 243(8) Varfolomeev, S. D., 512(55), 513(55), 514 Varfolomev, S. D., 512(53), 513(53), 514, 516(53) Vasak, M., 32-33, 34(42, 44), 268 Vasmel, H., 323, 325,328, 328(66, 69) Vasudevan, S. G., 371 V~zquez, A. E., 491 Vedani, A., 286 Veeman, W. S., 302, 304(5) Vega, A. J., 1 Veldink, G. A., 363 Venkatachalam, C. M., 83, 84(17), 86(17), 97(17), 98(17), 101(17), 102(17), 116 Venters, R. A., 220, 221(79), 227-229, 229(95) Ver Berkmoes, A. A., 575 Vermeglio, A., 310, 311(23), 323(23) Vidal, B., 557 Vidziunaite, R. A., 512(63), 513(63), 514 Viegers, M.P.A., 556 Viezzoli, M. S., 15 Villafranka, J. J., 80, 86, 102(30), 256, 270(52), 271(52) Vinay, P., 104 Vincent, J. S., 235 Vincent, S. P., 371,411 Vingsbo, O., 544 Violette, C. A., 324 Vis, R. D., 556-557 Vives, S. S., 512(75), 513(75), 515 Vliegenthart, J.F.G., 363 Vogel, H. J., 47, 115, 118, 118(17) Vojnovic, B., 530

609

Volk, R. J., 584 von Hofsten, A., 544 von Schiitz, J. U., 306-307, 321-324, 324(73) von Wettstein, D., 321 Von Zglininicki, T., 538 Vrieze, J., 328 Vuister, G. W., 247

W Wacker, T., 133 Wagner, G., 32-34, 34(42, 44), 37(48), 247, 250, 250(17), 252, 268-269, 276, 276(30), 279(27), 280(27-29) Walker, F. A., 13, 14(62), 16(62), 361 Walker, F. W., 559 Walker, S. R., 583 Wall, J., 542 Walter, N.P.C., 62, 63(77) Walton, N. J., 501,504, 507(2), 510 Wand, A. J., 2, 9(40), 13(40, 54), 258, 259(55), 261,261(55) Wang, B. C., 25 Wang, H., 227 Wang, J. X., 568 Wang, Y. L., 581-582 Wardman, P., 523,527 Wariishi, H., 15 Warren, C. D., 11 Warren, D. C., 411 Wasserman, R. H., 73,584 Watanabe, T., 512(64, 67, 68, 82), 513(64, 67, 68, 82), 514-515,518(68) Watari, H., 93, 96(42) Watson, H. C., 62, 63(77), 64 Watt, F., 570, 573 Wayen, D. H., 579 Weatherly, G. C., 553 Weaver, J., 384 Weaver, L. H., 46 Webb, G. A., 80 Weber, C., 276 Wedd, A. G., 337 Weers, J. G., 302, 320(6) Weger, M., 165 Wei, Y. H., 161, 165(66), 226 Weiland, Th., 202 Weinberg, W. H., 504

610

AUTHOR INDEX

Weiner, J. H., 485 Weir, M., 369 Weiss, A., 418 Weiss, E., 434 Weiss, M. A., 273 Weiss, R., 434 Weissbluth, M., 424 Wells, G. B., 340 Wenk, R., 103 Wennerstr6m, H., 107, 109-110, 263, 266(78), 267(78) Wenzel, T. G., 46 Werbelow, L. G., 109 Werner, H. W., 577-580, 582-584 Werst, M. M., 181,219, 224,226, 240 Werth, M. T., 478 Wertz, J. E., 124, 212, 214(53), 217(53) West, D. J., 235 Westhead, E. W., 56, 63(60), 64, 64(60), 65(84), 66(84), 69(84) Westler, J. L., 11,259, 261(68) Westlund, P.-O., 109 Wetherburn, D., 426 Wever, R., 235, 280, 341 Wezeman, F., 103, 104(54) Whang, W., 102, 106(52) Wherland, S., 191 Wherli, F. W., 80 Whiffen, D. H., 183 Whitaker, R. G., 511 Whitehead, L. A., 313 Whitfield, G., 133 Whitford, D., 13, 271, 279(105), 282, 503, 510 Wicks, J. D., 103 Wider, G., 252 Wieghardt, K., 426, 431(24) Wieloch, T., 115 Wijmenga, S. S., 247, 248(13), 263, 263(13), 264(13), 277(13) Wikstr6, S. O., 538 Wilcott, M. R., 45 Wilcox, D. E., 341 Wilkins, R. G., 331 Willard, J., 360 Willet, R. D., 426 Williams, D. H., 44 Williams, G., 2, 74, 261,262(7) Wilfiams, J. P., 53

Williams, K. R., 22 Williams, R. J., 2 Williams, R.J.P., 1-2, 15(1, 8), 44-45, 52, 55-57, 57(2, 48, 49), 58, 58(49), 59(48), 60(49), 61(48), 62-63, 63(60), 64, 64(60), 65(81, 84), 66(84, 81), 69(84), 73-74, 74(81), 75(94), 258, 259(57, 58), 261(57, 58), 275, 281-282, 331 Williams, T. C., 67, 69(87) Williamson, M., 25, 246, 258(3), 279(3) Williamson, R. L., 306, 318 Williams-Smith, D. L., 371, 411 Wilson, G. L., 337, 348, 351 Wilson, G. S., 402, 518 Wilson, H. R., 64 Wilson, L. J., 450 Wilson, P. W., 223 Wingfield, P. T., 252-253,270(40), 278(35) Winkler, H., 412, 478 Winkler, M. E., 252 Winter, M., 434, 436(29) Wissocq, J. C., 561-562, 562(63) Withers, G., 101 Witte, A., 202 Witte, H., 418 Wittenkeller, L., 82, 90(16), 92(16), 101, 102(16, 50), 105(16), 106(16) Witzel, H., 235, 237, 238(12), 239(12), 240(12) Woehler, S. E., 283 Woessner, D. E., 101 Wokaun, A., 246, 264(4), 265(4), 266(4), 279(4) Wolf, H. C., 307, 321-324, 324(73, 74) Wolff, R. K., 522 Wollenberger, U., 512(72), 513(72), 515 Wood, H. G., 359 Woods, L.F.J., 380 Woon, T.-C., 379 W6rg6tter, E., 32, 34, 34(42), 37(48), 268 Worthington, L., 18 Wright, P. E., 247, 250(18), 252, 266, 267(88), 288(18) Wroblewski, J., 538 Wroblewski, R., 538 Wu, J., 14, 16(62) WiRhrich, K., 2, 7(32), 11(11), 32-34, 34(42, 44), 37(48, 49), 62, 77, 246, 250(2), 252-

AUTHOR INDEX 253,268, 270(36-39, 90), 276, 276(1, 30) Wyckoff, H. W., 31, 40, 40(38), 42(38) X Xavier, A. V., 1-2, 13(12), 14(12, 61), 15(1, 8), 16(61), 44, 52-53, 57(2, 48), 58(48), 59(48), 61(48), 257,404 Xu, R. X., 273 Xu, Y., 105, 106(63) Xuong, N. H., 25

Y Yacynych, A. M., 512(79), 513(79), 515 Yamada, H., 339, 363 Yamamoto, Y,, 3, 9(23), 11(48), 14(23, 60), 15(48), 363 Yamanaka, H., 512, 513(31) Yamasi, H., 557 Yang, A. S., 220 Yarmuch, D. M., 89 Yaropolov, A. I., 512(53, 55), 513(53, 55), 514,516(53) Yates, M. G., 491 Yeates, T. O., 320

611

Yeh, P., 504 Yim, M. B., 340 Yokoi, H., 226 Yonetani, T., 191,209(6), 223(6), 363 Yoshida, T., 368 Yu, L. P., 4, 7(27), 13(53), 14(27, 62), 16(62), 259 Yu, M. L., 577

Zak, O., 78, 226, 229(95) Zamenhof, R. G., 561 Zanchi, G., 550 Zanchini, C., 399 Zang, L.-H., 318-320 Zeppezauer, M., 478 Zewail, A. H., 306 Zhao, D., 252, 275(34), 278(34) Zhao, J., 512(73), 513(73), 515 Zhong, Y. C,, 389 Ziegelmiller, D., 561 Zierod, K., 538 Zimenko, A., 556 Zimmerman, R., 378 Zoski, C. G., 504 Zulich, J., 306 Zumft, W. G., 451,454(13), 455

612

SUBJECT INDEX

Subject Index

A Absorbance-detected magnetic resonance spectroscopy, 290 instrumentation, 312-315 in photosynthesis, reaction center studies, 322-324 singlet, 298-299 triplet, 299 Aconitase active site structure and mechanisms, ENDOR spectroscopy, 224-225 bovine heart mito'chondrial, iron-sulfur cluster, ENDOR spectroscopy, 224 Adenosine triphosphatase Ca 2+_

alkali metal N M R spectroscopy, 86 lanthanide-induced shift studies, 78 Na÷,K +-, alkali metal NMR spectroscopy, 84 S-Adenosylmethionine synthetase, EPR with VO 2" electron spin probes, 235 ADMR, s e e Absorbance-detected magnetic resonance spectroscopy AES, s e e Auger electron spectrometry Albumin, serum amino acid and cofactor triplet states, optically detected magnetic resonance studies, 318 EPR with VO 2÷ electron spin probes, 235 Alcohol dehydrogenase zJ3Cd NMR, chemical shifts, 20-21 coimmobilized with horseradish peroxidase, as biosensor, 518 electrochemistry, 513, 521 Fe(lI)-substituted, integer spin EPR spectroscopy, 478 Alcohol radicals, production by pulse radiolysis, 527

Alkali metals cations, binding constants to metalloenzymes and metalloproteins, calculation, 97 ion binding to cell membrane, 101 ion concentrations extra- and intracellular, calculation, 98-99 intracellular, M1R-derived, calculation, 99 ion transport ionophore-induced, 96-97 in red blood cell suspensions, 99101 monitoring, 97 in nuclear magnetic resonance, 78-106 nuclear relaxation, measurement, 98 nuclides NMR properties, 80-82 NMR receptivity, 80-82 nuclear relaxation, 82-83 relaxation rates, transmembrane differences in cell suspensions and perfused organs, 88 Alkaline phosphatase cadmium substitution, 22 N3Cd NMR H3Cd-~3C scalar coupling, 37-40 HaCd-31P scalar coupling, 39, 42 chemical exchange modulation, 29-31 chemical shifts, 20-21 T~ and NOE data for, 26 with phosphate ligand, ll3Cd NMR chemical shifts, 20-21 Tj and NOE data for, 26 Alpha particles, interactions with matter, 565-566 Amicyanin multidimensional NMR conformational equilibria studies, 275

SUBJECT INDEX oxidation state change studies, 282283 paramagnetic metal ion experiments, 263 Thiobacillus versutus, metal environment mobility, multidimensional NMR studies, 272-273 Amides, proton exchange rates, determination by multidimensional NMR, 275278 Aminoglycosides as promoters for direct electrochemistry of metalloenzymes, 516 structure, 516-517 Ammonia, 14N ENDOR with VO 2÷ electron spin probes, 239, 241 a-Amylase, lanthanide-induced shift studies, 46 Apoferritin, VO 2. electron spin probes ESEEM spectroscopy, 242-243 ~4N ENDOR spectroscopy, 239, 241 Aspartate carbamoyltransferase, 1~3Cd NMR spectroscopy, 23, 25 Auger electrons, 541-542 Auger electron spectrometry, 541,543544 Azurin Alcaligenes, metal environment, multidimensional NMR studies, 265 amino acid and cofactor triplet states, optically detected magnetic resonance studies, 318 113Cd NMR spectroscopy chemical shifts, 20-21 T~ and NOE data for, 26 electron self-exchange rate, determination, 284-285 molecular dynamics simulations, 288290 oxidation state changes, multidimensional NMR studies, 282-283 Pseudomonas aeruginosa metal environment, multidimensional NMR studies diamagnetic metal ion experiments, 265 NH exchange experiments, 277 paramagnetic metal ion experiments, 263-264 pFl titration experiments, 254-256 structure, NMR studies, 247-249

613

B Backscattering analysis, 569-570 Bacteria, see also specific bacteria photosynthetic, fluorescence-detected magnetic resonance spectroscopy, 321-324 Bacterioferritin, EPR study, 369 Bacteriophage J~, ssDNA-binding gene 5 protein interaction with Tb(DOTP) 5 , 13C NMR study, 74-76 surface, lanthanide-induced shift studies, 74-76 Bacteriophage IKe, ssDNA-binding gene 5 protein, interaction with Gd(DOTP) ~-, proton NMR study, 77 Bacteriophage M13, ssDNA-binding gene 5 protein, interaction with Gd(DOTP)5% proton NMR study, 77 Bacteriophage T4, triply point-mutated lysozyme, amino acid and cofactor triplet states, optically detected magnetic resonance studies, 318 Benzene dioxygenase, Pseudornonas putida, mixed-valence binuclear iron complex, EPR spectra, 367 Biological specimens, fixation, 535-540 Bohr magneton, nuclear, 124 Boltzmann distribution, spins in high-spin iron complexes, 378 Boltzmann formula, 334 Boltzmann relation, 130 Bragg's law, 545 Bremsstrahlung, 544-545 Bromoperoxidase, EPR/ESEEM with VO ~+ electron spin probes, 235 Bruker ENDOR accessory, 196, 200

C Cadmium .1Cd natural abundance, 16 as nuclear magnetic resonance probe, 16 lI3Cd natural abundance, 16 in NMR studies of metaUoprotems, 16-43

614

SUBJECT INDEX

nuclear relaxation, 24-29 nucleus, J coupling, 33 protein-bound, rotational correlation time rR, 27 Cd(lI), ionic radius, 17 Calbindin Dgk molecular dynamics simulations, 287 nuclear magnetic resonance spectroscopy 43Ca, 117-118 multidimensional amide proton exchange studies, 278 metal environment mobility studies, 274 Calcium binding proteins 43Ca NMR spectroscopy, 118 competition binding studies, 47 lanthanide-induced shift studies, 6773, 78 43Ca atomic and nuclear properties, 108 in NMR, 107-118 nuclear relaxation, 108 nuclear spin I, 107 replacement by lanthanide shift reagents, 46 Calmodulin EF-hand sites, 109 EPR with VO 2÷ electron spin probes, 235 lanthanide-induced shift studies, 73, 78 nuclear magnetic resonance alkali metal, 86 43Ca, 117 ~3Cd, chemical shifts, 20-21 multidimensional, NH exchange studies, 276, 278 CAMECA instruments, 542, 578-579, 582 Carbon-13, NMR properties, 80-81 Carbonate dehydratase cadmium substitution, 22 113Cd NMR chemical shifts, 19 Tt and NOE data for, 26 EPR with VO 2÷ electron spin probes, 235 Carbonate dehydratase A, bovine, complex with ~SN-labeled neoprontosil, H3Cd NMR signal, It3Cd-15N scalar coupling on, 39-41

Carbonate dehydratase C, 1~3Cd NMR signal, "3Cd-13C scalar coupling on, 38 Carbon dioxide radical anions, production by pulse radiolysis, 526-527 Carbonic anhydrase, "3Cd-substituted, ll3Cd NMR chemical shifts, 20-21 Carbon monoxide acceptor oxidoreductase, electrochemistry, 513 Carbon monoxide dehydrogenase, Rhodospirillum rubrum, electrochemistry, 513,521 y-Carboxyglutamic acid, bone, calcium affinity determination, 115 Carboxypeptidase A ~3Cd NMR spectroscopy chemical exchange modulation, 29-30 chemical shifts, 19-21 EPR with VO 2÷ electron spin probes, 235 Carnosine, 14N ENDOR with VO 2+ electron spin probes, 239, 241 Catalase, iron-sulfur clusters, EPR spectroscopy, 383 Cation shift reagents, paramagnetic, 47 Cerium C~ for, 51 spin expectation value, 51, 53 as structural probe, 58 Cesium binding constants to metalloenzymes and metalloproteins, calculation, 98 in biological samples, distribution and transport studies, 90-93 133Cs NMR properties, 81 NMR receptivity, 82 natural abundance, 78 physiological and pharmacological importance, 79 Chemical exchange measurement, 11 NOESY cross-peaks arising from, 11 nuclear magnetic resonance effects, 1314, 29-31, 47-49 Chemical shift, 13-14 113Cd NMR, 18-21 lanthanide complex, 49-54; see also Lanthanide-induced shift Chemical shift anisotropy, 266 in ~J3Cd nuclear relaxation, 24-29

SUBJECT INDEX Chloroflexus aurantiacus, reaction center,

triplet-minus-singlet absorbance difference spectroscopy, 328 Chromium(Ill), metal site accessibility studies, 271 Cliff-Lorimer relationship, 547 Cobalt(II), EPR, 339-340 Collagen, EPR with VO2+ electron spin probes, 235 Computer programs, for simulation of EPR spectra, 376 Concanavalin A, ll3Cd NMR chemical shifts, 19-21 solid-state, 43 Copper Cu(II) electron paramagnetic resonance, 340342 spin-frustrated trimer, magnetic susceptibility, 434-436 in redox-active proteins, 434-436 Copper proteins metal environment, multidimensional NMR studies, diamagnetic metal ion experiments, 263,265 with S = ½, continuous wave ENDOR studies, 225-228 Correlation spectroscopy, 2, 15,246 advantages and disadvantages, 7 applications in paramagnetic systems, 12 dispersion mode, 8-9 double quantum filter, 3, 8 heteronuclear, 9 IH-ll3Cd, 34-37 via heteronuclear multiple quantum coherence, for protein structure studies, 253 paramagnetic protein studies, 4-6, 9 heteronuclear multiple quantum, see Heteronuclear multiple quantum correlation spectroscopy magnitude and absolute value spectra, comparison, 7-8 paramagnetic metal ion studies, 259261 relayed, 8 two-dimensional tH, 23 COSY, see Correlation spectroscopy p-Cresol methylhydroxylase, P s e u d o m o n a s putida, electrochemistry, 513,518-520 Cryoembedding, 538-539

615

Cryofixation, biological specimens, 536539 Cryosectioning, 536-538 Cryosubstitution, 540 CSA, see Chemical shift anisotropy Curie behavior, 261 Curie spin, 55 CW-EPR, see Electron paramagnetic resonance, continuous wave Cytochrome b, EPR spectroscopy, 361 Cytochrome b2 in methyl viologen solution, pulse radiolysis, 532-533 ruthenium-modified, pulse radiolysis, 533-534 Cytochrome bs, complexes with cytochrome c, electrochemistry, 506-507 Cytochrome c -cytochrome b~ complexes, electrochemistry, 506-507 oxidized and reduced forms, crossassignments between, 9 -plastocyanin complexes, electrochemistry, 506-507 two-dimensional exchange spectroscopy, 261-262 Cytochrome c3, Desulfovibrio vulgaris NOESY spectra, 3 ROESY spectrum, 12 Cytochrome c55~, P s e u d o m o n a s aeruginosa, structure, NMR studies, 247, 25O Cytochrome-c oxidase copper ENDOR spectroscopy, 226-227 electrochemistry, 510-513 integer spin EPR spectroscopy, 467-468, 478 Cytochrome-c peroxidase, electrochemistry, 513,516 Cytochrome oxidase, binuclear iron-copper clusters, EPR, 369 Cytochrome P-450, EPR spectroscopy, 360-361

D Deoxyhemerythrin azide, binuclear oxygen-bridged iron clusters, EPR, 368369

616

SUBJECT INDEX

Deoxymyoglobin, integer spin EPR spectroscopy, 478 DEPT, s e e Distortionless enhancement by polarization transfer Desulfoferredoxin, integer spin EPR spectroscopy, 478 Desulforedoxin, EPR spectroscopy, 361364 Deuterons, interaction with matter, 565566 Diamagnetic ions, NMR spectroscopy, 2, 13 one-dimensional, 1-2, 6, 16, 263267 two-dimensional, 1-16, 267-269 Dibenzyl-14-crown-4, alkali metal ion transport induced by, measurement, 99-101 2,6-Dichlorophenolindophenol, as oxidant of protein redox systems, 407 Dipolar interactions, 342, 357 spectroscopic detection, 387-389 controls, 395 distance determination, 391-393 extrinsic probes, 390-391 intrinsic probes, 390 Leigh effect, 393-394 P~/2, contribution of T2 to, 394395 precautions, 395 spectral diffusion in, 394-395 theory, 385-387 Distortionless enhancement by polarization transfer, 265-267 DNA-binding proteins, cadmium substitution, 17 Double-X filter, 268 DQF-COSY, s e e Correlation spectroscopy, double quantum filter Dynamical nuclear polarization, 192 Dynamic frequency shifts, second-order, 108-109 Dysprosium Cj for, 51 spin expectation value, 51, 53 Dysprosium(III)-bis-tripolyphosphate, 47, 88-93, 103-104, 115 preparation, 89-90 Dysprosium triethylenetetraminehexaacetate, 88-93, 103-104 preparation, 89

E EDS, s e e Spectrometer, energy dispersive EDTA, nitrosyl ferrous complex, EPR spectroscopy, 365 EEDOR, s e e Electron-electron double resonance EELS, s e e Electron energy loss spectrometry EI-EPR, s e e Electron paramagnetic resonance, pulsed electron nuclear double resonance-induced Elastase, lanthanide-induced shift studies, 73 Elastin, polypentapeptide, calcium affinity determination, 116 ELDOR, s e e Electron-electron double resonance Electrochemistry direct, 501 dynamic, 479-480 metalloenzymes, 501-522 proteins electrodes for, 503-506 nonmetal, 503-504 promoters, 501-503,516 time-dependent, 506 Electrodes enzymes adsorbed on, as biosensors, 516-518 for protein electrochemistry, 503-506 nonmetal, 503-504 pyrolytic graphite in analysis of metalloprotein redoxactive centers, 486-488 surface characteristics, 504 Electron-electron double resonance, 306307 and ENDOR, combination, 178 Electron energy loss spectrometer, 550 Electron energy loss spectrometry, 541, 549-551 biological applications, 552 inelastically scattered electron spectrum, characteristics, 549-550 with parallel detection system, 550552 quantitative analysis, 550-551 and X-ray microanalysis, comparison, 551-552 Electron microscopy, analytical, 540-553

SUBJECT INDEX biological applications, 551-553 measurements performed in, 542-543 physical bases, 541-542 Electron nuclear double resonance spectroscopy, 121,357 advantages, 210 coherence transfer, 123, 128 continuous wave, 121-122, 190-231 amplitudes, 166-167 coil system, 197-202 impedance matching, 202-206 experiment spin Hamiltonian approach to, 211-212 terms from, 213-218 theory and application, 207-223 instrumentation, 191-207 metalloprotein studies with S = ½, 223-228 with S > ½, 228-231 axial symmetry, 228-230 orthorhombic symmetry, 230-231 modulation, 192-196 probes, 149 Q-band, 219 radio frequency modulation schemes, 193-196 amplitude modulation, 195-196 second field modulation, 195-196 resonators, 197-202 loop-gap, 150, 202,219 S-band, 219-220 sensitivity, enhancement at low modulation frequencies, 196-197 spectra, simulations, 220-223 temperature, 145-146 Davies hole burning in, 135 metalloproteins, 153-164 mixing period, 138-140 pulse sequences, 128-129 transfer of spin populations in, 130 double, 183,206-207 pulsed, 182-185 and electron electron double resonance, combined, 178 energy levels, 123-128 enhancement, 121-122 and EPR, comparison, 210-211 ESEEM-edited, 171-172 experimental considerations, 143-153

617

hyperfine selective, 152, 176 instruments for, 143-153 iron proteins, 359 Mims blind spots, 132, 137-138, 157 hole burning in, 135 hyperfine contrast selectivity, 137 metalloproteins, 153-164 mixing period, 138-140 preparation period, 131-132 pulse sequences, 128-129 multiple quantum, 123 orientation-dependent frequencies, 125 pulsed, 121-122 of central metal nuclei, 161-164 and continuous wave ENDOR, comparison, 164-167 detection period, 129, 132-133 electron coherence effects, 141 electron nuclear coherence effects, 141-142 orientation selectivity by electron spin echo envelope modulation, 143 and ESEEM, comparison, 167-171 microwave transmitter design, 148 mixing period, hyperfine enhancement factor, 138-140 preparation period, hyperfine contrast selectivity, 135-138 probes, 148-150 proton and nitrogen ligand nuclei, 154-161 radio frequency transmitter for, 150152 sensitivity, 147 spectra, amplitudes in, 133-143 sublevel polarization transfer temperature, 146 resolving power, 210-211 sample volume, 146-147 spectrometer, microwave operating and radio frequency range, 152-153 radio frequency range, 152-153 temperature for, 145-146 time scales, 144-145 transition frequencies, 123-128 two-dimensional, 123, 176-182 hole burning in, 176-177 hyperfine selective studies, 178-182 pulse sequence, 176-177

618

SUBJECT INDEX

vanadyl(IV) spin probes, 232, 236240 Electron nuclear electron triple resonance, 176-182 Electron nuclear multiple resonance spectroscopy, pulsed, see Pulsed electron nuclear multiple resonance spectroscopy Electron nuclear nuclear triple resonance, pulsed, 182-185 Electron paramagnetic resonance, 330-353 adiabatic rapid passage, 191 anisotropic and isotropic spectra, computer simulations, 349-351 average integrated intensity f~.ctor gpav, 409 continuous wave, 332 d j configuration, 336-337 d 5 configuration, 337-339 d 7 configuration, 339-340 d 8 configuration, 340 d 9 configuration, 340-342 dipole-dipole interactions, 357 effective spin, 335-336 exchange interactions, 357 experimental considerations, 345-348 g factor, 354-355 orientational dependence, 336 orientation selectivity, 126 hole burning in, 134-135 hyperfine interaction, 356 hyperfine splittings, 356 orientational dependence, 336 information content, 332 inhomogeneously broadened spectral lines, 119-120, 134-135, 332 instrumentation, 345-347, 369-370 integer spin, and Mrssbauer spectroscopy, combination, 463-479 iron complexes, 353-384 information content, 359-360 oxidation and reduction methods, 372-374 iron proteins, 353-384 in oivo studies, 380-384 isotope substitution technique, 347-349 in Kramers doublet, 332-333 low-temperature quantitation in, standards for, 410 magnetically coupled systems, 342-345 principle, 354

progressive power saturation, for T~i determination, 387-389 pulsed electron nuclear double resonance-induced, 185-189 rapid freeze quenching technique, 347 sample for concentration, 371-372 preparation, 347, 370-371 saturation recovery, for T~i determination, 387, 389 signal intensities, quantitation, 376-380 errors, 380 in even-spin systems, 379 in high-spin iron complexes, 378-379 in low-spin iron complexes, 377 from whole cells, 380 spectra analysis, 348-353 average spectral density, 120 characteristics, 355-357 conditions for running, 374-375 line broadening, 119-120 simulation, 376 spectrometers, 345-347, 369-370 spin echo-detected recovery, for T~/ determination, 387, 389 spin Hamiltonian, 335-336 analysis, 348-353 spin-orbit coupling, 355 spin quantitation, 351-353 subspectra, 176 superhyperfine interaction, 356-357 temperature effects and control, 375-376 transition metal ions, 332-345 vanadyl(IV) spin probes, 232-235 Zeeman interaction, 355 zero-field splitting, 355-356 Electron paramagnetic resonance spectroelectrochemical cells, 397-400 standard, 398-400 with visible capability, 401 Electron paramagnetic resonance spectroelectrochemistry, 396-41 l electrochemical aspects, 402 equilibrium criteria, 407-408 EPR quantitation procedure, 408-411 oxidation methods, 406--407 redox mediator titrants and indicators, 404-406 reduction methods, 406-407 spectroscopic aspects, 408-411

SUBJECT INDEX Electron-probe microanalyzer, 542 Electrons Auger, see Auger electrons hydrated, production by pulse radiolysis, 526 Electron self-exchange, 262,281-284 Electron spin echo envelope modulation, 137-138, 142, 357 ENDOR-edited, 172-176 experimental and instrumental considerations, 143-148 orientation selectivity, 143, 159-160 and pulsed ENDOR, comparison, 167171 vanadyl(IV) spin probes, 232,240-244 Electron spin resonance, see Electron paramagnetic resonance Electron transfer long-range, in proteins, pulse radiolysis studies, 532-534 promoters, 501-503,516 reaction, thermodynamic parameters, 396 ENDOR, see Electron nuclear double resonance spectroscopy Enoate reductase, electrochemistry, 513 Enzymes adsorbed on electrodes, as biosensors, 516-518 K+-activated, alkali metal NMR spectroscopy, 87 EPR, see Electron paramagnetic resonance Erbium Cj for, 51 spin expectation value, 51, 53 Erythrocytes, suspensions alkali metal ion transport, ionophoreinduced, 99-101 alkali metal NMR spectroscopy magnetization transfer method, 96-97 modified inversion recovery method, 93-96 shift reagent method, 90-93 lithium-free, membrane potential measurement, 103 ESE, see Electron self-exchange ESEEM, see Electron spin echo envelope modulation ESR, see Electron paramagnetic resonance Europium Cj for, 51

619

Eu(II1), as structural probe, 47 spin expectation value, 51, 53 Exchange interaction, 425-426 Exchange spectroscopy, two-dimensional, 11 cytochrome c, 261-262 EXCTSY, 2, 11 in 2(4Fe-4S) center ferredoxins, 14 in tetraheme proteins, 14 EXSY, see Exchange spectroscopy, twodimensional F Factor VIII, calcium affinity determination, 115 FDMR, see Fluorescence-detected magnetic resonance spectroscopy Fermi contact interaction, 124, 257 Fermi contact shifted resonances, heteronuclear COSY studies, 4-6, 9 Ferredoxin A z o t o b a c t e r oinelandii, redox-aetive center, voltammetry, 489-493 CIostridium pasteuranium, redox-active center, voltammetry, 490-492 Desulfovibrio africanus, redox-active center, voltammetry, 489-490, 497 2(4Fe-4S), 2D NMR spectroscopy, 14 iron-sulfur clusters in, EPR, 343-344, 365,367-368 parsley, mixed-valence binuclear iron complex, EPR spectra, 367 P s e u d o m o n a s putida, expression in Escherichia coli, in vivo quantitation, 380-381 Thermodesulfobacterium c o m m u n e ,

redox-active center, voltammetry, 490-493 two-dimensional NMR spectroscopy, 14 Ferredoxin II, Desulfovibrio gigas iron-sulfur cluster, multifield saturation ma~,nOization studies, 461-462 M6ssbauer and EPR spectroscopy, 472476 Ferricytochrome b562, 2D NMR spectroscopy, 14 Ferricytochrome c horse heteronuclear COSY spectrum, 5 lanthanide-induced shift studies, 62

620

SVBJECa" INDEX

metal site accessibility studies, 271 paramagnetic NMR shift and/or relaxation agents for, 74-75 surface, lanthanide-induced shift studies, 74-75 Ferricytochrome c', high-spin, 15 Ferricytochrome c"

Glutamine synthetase, adenylylated, lanthanide-induced shift studies, 46 Glyceraldehyde-3-phosphate dehydrogenase, lanthanide-induced shift studies, 62 GUPIXE software, 568 Gyromagnetic ratio, 333

Methylophilus methylotrophus

NOESY spectrum, 10 TOCSY spectrum, 4 proton exchange studies, 14 Ferritin electron paramagnetic resonance, 369 EPR/ENDOR/ESEEM, with VO 2+ electron spin probes, 235 FETEM, see Transmission electron microscope, filtered electron Fixation, biological specimens for localization of metal atoms, 535-541 Flash photolysis, 522-523 Flavin derivative, as solute for selective reducing radical generation, 527 Flavocytochrome b2, electrochemistry, 513 Flavocytochrome c55z, electrochemistry, 513 Fluorescence-detected magnetic resonance spectroscopy, 296, 298-302 in photosynthesis, 321-322 Fluorine-19, in NMR measurement of cell membrane potential, 103 Forward scattering analysis, 569 Freeze-drying, 538 Freeze quenching, rapid, 347

G Gadolinium Cj for, 51 Gd(III) nuclear relaxation, 55 as structural probe, 44-45, 47, 57-62 spin expectation value, 51, 53 Galactose oxidase, electrochemistry, 513 Gamma ray emission, particle-induced, 570-571 instrumentation, 573 Glucocorticoid receptors, lJ3Cd-substituted, 1~3Cd NMR chemical shifts, 2021 D-Gluconate dehydrogenase, Pseudomonas fluorescens, electrochemistry, 513,521

H Heliobacterium chlorum, reaction center,

triplet-minus-singlet absorbance difference spectroscopy, 328 Helium-3, interaction with matter, 565566 Heme axial ligands, protons in, assignment, 14 redox potential, structural control, 14 Heme cavity, proton exchange studies, 14 Heme proteins ENDOR spectroscopy, 191 oxidation state changes, multidimensional NMR studies, 282 two-dimensional NMR spectroscopy, 14 Hemoglobin, R- and T-states, EPR spectroscopy, 365-366 Hemosiderin, EPR analysis, 369 Heteronuclear multiple quantum correlation spectroscopy IH-mCd, 34-37 via heteronuclear single quantum coherence, for metal environment studies, 256 Histidine, 14N ENDOR with VO ~+ electron spin probes, 239, 241 HIV, see Human immunodeficiency virus HMQC, see Heteronuclear multiple quantum correlation spectroscopy Holmium Cj for, 51 spin expectation value, 51, 53 Homonuclear Hartmann-Hahn spectroscopy, 246 Horseradish peroxidase adsorbed on electrodes, as biosensor, 516-518 nuclear magnetic resonance multidimensional, paramagnetic metal ion studies, 259-261 two-dimensional, 15

SUBJECT INDEX HS-ENDOR, s e e Electron nuclear double resonance spectroscopy, hyperfine selective HSQC spectroscopy, s e e Correlation spectroscopy, heteronuclear, via heteronuclear single quantum coherence Human immunodeficiency virus, nucleocapsid protein, ~t3Cd-substituted, u3Cd NMR chemical shifts, 20-21 Hydrogen, s e e Deuterons; Protons Hydrogenase CIostridium p a s t e u r a n i u m

H cluster, 168 Davies ENDOR spectrum, 184 ENDOR-induced SE/EPR spectra, 186-189 pulsed double ENDOR, 184-185 iron-sulfer cluster active site, ESEEM-edited ENDOR spectroscopy, 171-172 oxidized form combined ESEEM and pulsed ENDOR spectroscopy, 168-170 ENDOR-edited ESEEM, 173-176 HS-ENDOR spectra, 180-181 electrochemistry, 513 /z-Hydroxybis(tz-carboxylato)divanadium(lll) complex, magnetic susceptibility, 432-434 Hydroxyl radicals, production by pulse radiolysis, 525 Hyperfine shifts, in paramagnetic systems, 12, 15 Hyperfine splittings, 119-120

I Imidazole, 14N ENDOR with VO 2+ electron spin probes, 239, 241 INEPT, s e e Insensitive nuclei enhancement by polarization transfer Inorganic pyrophosphatase, H3Cd-substituted, NaCd NMR chemical shifts, 2021 Insensitive nuclei enhancement by polarization transfer, 265-267 Insulin, EPR with VO 2+ electron spin probes, 235

621

Integer spin systems, M6ssbauer spectra, 464 Intersystem crossing, 291-292 Iron centers in metalloproteins binuclear oxygen-bridged, EPR properties, 367-369 redox potential, in EPR spectroscopy, 360 chemistry, relationship to magnetic properties, 357-359 coordination geometry, in EPR spectroscopy, 360 d electron energy levels and electron distributions, in octahedral and tetrahedral coordination, 357-358 electron paramagnetic resonance, 360369 isotope-enriched samples for, 359 spectra, identification, 359 Fe(IIl), electron paramagnetic resonance high-spin, 337-339, 355-356, 358-359, 361-364 low-spin, 337-339, 360-361 heine magnetic axes, orientation, 14 structure, 15 ligand type, EPR spectroscopy, 360 oxidation states, 357 polynuclear, EPR properties, 369 quantitation, in EPR spectroscopy, 359360 in redox-active proteins, 281 Iron complexes, EPR spectroscopy, 353384 Iron-oxo proteins, integer spin EPR spectroscopy, 478 Iron proteins electron paramagnetic resonance spectroscopy, 353-384 even-spin systems, 379 high-spin systems, 378-379 low-spin systems, 377 in vivo studies, 380-384 in whole cells, signal quantitation, 380 high-potential, ENDOR spectroscopy, 224 Iron-sulfur clusters antiferromagnetic coupling in, 343-344 electron paramagnetic resonance, 365368

622

SUBJECT INDEX

in ferredoxins electron paramagnetic resonance spectra, 343-344 types, 343 redox-linked activities, voltammetric studies, in metalloproteins adsorbed on electrodes, 485-486 in oivo quantitation, 380-381 Iron-sulfur proteins ENDOR spectroscopy, 191 high-potential Chromatium vinosum, EPR spectrum, 344 iron-sulfur clusters in, EPR spectrum, 365-368 two-dimensional NMR spectroscopy, 14 metal center coupling in, 343 with S = ½, continuous wave ENDOR studies, 223-225 1SC, see Intersystem crossing 1SECR-COSY, 8-9 Isoleucyl tRNA synthetase, lanthanideinduced shift studies, 46 K Kinases, lanthanide-induced shift studies, 56 L Laccase, electrochemistry, 513, 516 c~-Lactalbumin calcium-binding, 43Ca NMR spectroscopy, 118 competition binding studies, 47 lanthanide-induced shift studies, 78 Lactoferrin, EPR/ESEEM with VO 2+ electron spin probes, 235 Langevin formula, 415 Lanthanide complexes axial symmetry assumption for, 52, 5960, 77-78 as extrinsic probes, for EPR, 391 formation, kinetics, 55 isostructuralit.y, 45 Lanthanide-induced shift, 49-54 in biological macromolecules, 56-77 in Ca:+-binding proteins, 67-73, 78 contact shift, 49, 53

diamagnetic complex formation shift, 49-50 dipolar shift, 49-52 in hen egg white lysozyme, 56-62 in Mg2+-ATP-dependent phosphoglycerate kinase, 62-69 in parvalbumin, 67-73 Lanthanide ions Cj for, 51-52 ionic radii, 46 paramagnetic nuclear magnetic resonance effects, basic theory, 47-56 nuclear relaxation rates, 54-56 replacement of naturally occurring ion functional, 46 isomorphous, 46 as spectroscopic probes, applications, 46-47 spin expectation values, 51, 53 as structural probes, 45-47 Lanthanide shift reagents, 43-78; see also specific reagent

with alkali metal NMR, 88-93 applications, 44, 77-78 for "3Ca studies, 115 Lewis acid behavior, 44 non-S-state, nuclear relaxation, 55 Lanthanum(Ill), as structural probe, 47, 49 Larmor frequency electron, 124 nuclear, 124-125 Lipoxygenase EPR spectroscopy, 363 nitrosyl, EPR spectroscopy, 365 Lithium in biological samples, distribution and transport studies, 90-93 interactions with red blood cell membrane, probe for, 101-102 6Li, in NMR, 80-82, 90-93 7Li, in NMR, 81-82, 84-86, 90-96, 99103 natural abundance, 78 neutron capture radiography, 560, 562563,565 physiological and pharmacological importance, 79 Lithium salts, 6Li-enriched, source, 82 Lowicryl resin, properties, 539 Lutetium(Ill), as structural probe, 47, 49

SUBJECT INDEX

Lysozyme amino acid and cofactor triplet states, optically detected magnetic resonance studies, 318 calcium-binding, 43Ca NMR spectroscopy, 118 hen egg white 13C NMR, 61-62 lanthanide-induced shift studies, 56-62 lanthanide(III) ions in, proton shift and relaxation data for, 58, 60 physicochemical properties, 56-57 X-ray crystal structure, 57 and experimental NMR data, comparison, 58, 61 triply point-mutated, from bacteriophage T4, amino acid and cofactor triplet states, optically detected magnetic resonance studies, 318 Lysyl oxidase, electrochemistry, 513

M Magic angle spinning techniques, 18, 41, 43 Magnesium, replacement by lanthanide shift reagents, 46 Magnetic susceptibility, 412-436 alkali metal nuclei, 105-106 bulk, in superconducting magnets and electromagnets, sign and magnitude, 105-106 definition, 412-415 examples, 431-436 measurement, theoretical aspects, 424431 porphyrin radical complexes, 434-435 relaxation term, 55 spin-frustrated copper(II) trimer, 434436 St = $2 = 1 dimer, 432-434 susceptometer, 415-423 data acquisition, 421-423 magnetic property measurement system, 419-421 superconducting quantum interference device, 415-418 Magnetic susceptibility tensor, for lanthanide-protein derivatives, 50, 6972, 78

623

Manganese Mn(II), EPR, 337-339 in redox-active proteins, 280 Manganese-superoxide reductase, Thermus thermophilus, saturation magnetization, 441-446 MAS, see Magic angle spinning techniques McConneU relation, 214-216 2-Mercaptoethanol, in metal ion exchange procedures, 23 Metal atoms, location in biological systems, physical methods, 535-586 sample preparation for, 535-540 cryomethods, 536-539 precipitation methods, 539-540 Metal force fields, in molecular dynamics simulations, 285-290 Metalloenzymes active site structure, alkali metal NMR spectroscopy, 87 alkali metal NMR, 84-87 cadmium substitution, 17 electrochemistry, 501-522 direct studies, 515-521 indirect studies, 509-515 monovalent and divalent cations in, distance between, measurement, 87 phosphate-binding, ~3Cd NMR, t~3Cd3tp scalar coupling, 41-42 Metalloproteins adsorbed on electrodes, redox-active centers redox status, control of, 482 sample economy, 482 voltammetric studies, 479-500 cadmium substitution, 22 H3Cd-substituted 113Cd-113Cd scalar coupling in, 31-33 chemical shifts, 19-21 NMR spectroscopy ~13Cd, solid-state, 43 heteronuclear techniques, 33-43 electron nuclear double resonance spectroscopy, 153-164 metal environments mobility, NMR spectroscopy, 271280 structure, NMR spectroscopy studies, 254-271 paramagnetic metal ions, 256-263 pH effects, 254-256

624

SUBJECT I N D E X

monovalent and divalent cations in, distance between, measurement, 87 multifield saturation magnetization studies, 437-463 nuclear magnetic resonance spectroscopy alkali metal, 84-87 mCd, 16-43 paramagnetic, 2D NMR spectroscopy, 1-16 survey of achievements in, 13-15 redox centers, imaging, 489-492 Metallothionein Cd 2+ cluster sites, 31 H3Cd NMR spectroscopy, 25 ll3Cd-~H scalar coupling, 33, 37 with proton decoupling, 29 T~ and NOE data for, 26 mCd-substituted, mCd NMR chemical shifts, 19-21 mammalian, mCd NMR, 32-33 structure, 2D NMR spectroscopy, 267268 Metallothionein i, crab, mCd NMR, 31-32 Metal site, accessibility, NMR spectroscopy, 271 Methane monooxygenase binuclear oxygen-bridged iron clusters, EPR, 368-369 hydroxylase component, Fe3+-Fe 3÷ cluster, integer spin EPR spectroscopy, 467-468, 477-478 Methylococcus capsulatus, mixedvalence binuclear iron complex, EPR spectra, 367 Methemoglobin, EPR signal quantitation, 382-383 Methyl viologen, as solute for selective reducing radical generation, 527 Metmyoglobin electron paramagnetic resonance spectroscopy, 361, 364 in heart, EPR signal quantitation, 382383 horse heart, EPR spectroscopy, 377, 379 MIA, see Microwave-induced absorbance Microscopy, see also specific techniques Microwave-induced absorbance, 307, 317 Microwave-induced delayed luminescence, 302

Microwave-induced delayed phosphorescence, 302, 304 Microwave-induced fluorescence, 307, 317 Microwave-induced phosphorescence, 307, 317 MIR, see Modified inversion recovery MLEV sequences, 9 Modified inversion recovery, 93-96, 104105 Molecular dynamics simulations, in analysis of metalloproteins, 285-290 Molybdenum Mo(V) electron paramagnetic resonance, 336337 intermediates and model complexes, EPR spectra, 348-351 in redox-active proteins, 281 M6ssbauer spectroscopy and integer spin EPR, combination, 463479 Desulfovibrio gigas ferredoxin II, 472-476 non-Kramers systems, 465-472 spectra in limit A = 0, 476-478 multifield saturation magnetization studies after, 437-438, 463 Multifield saturation magnetization, metalloproteins, 437-463 applications, 437 background signals, 438,449-453 from ferromagnetic impurities, 438, 452 from paramagnetic molecular oxygen dissolved in air-saturated water, 43~. 451 from spin I = ½nuclei, 438, 450-451 Brillouin curves for, 438-440 Curie law slopes at high temperature, 439-440 data, 441-443 data analysis, 454-462 single spin fits, 460 two-spin fits, 460-462 equipment, 443-449 fitting software for, 449, 45~-4sa multi-instrument sample holder for, 452453 resolution, 437-438, 443-447 sensitivity, 438

SUBJECT INDEX spin Hamiltonian, 440-441 theoretical saturation magnetization curves, 441 theory, 438-440 zero-field splitting, 440-441 Myoglobin heine vinyl substituents, orientation and mobility, 14 structure, 15 Myosin light chains, lanthanide-induced shift studies, 73 subfragment S l, surface, lanthanideinduced shift studies, 74

N NADH dehydrogenase, iron-sulfur clusters, EPR spectroscopy, 383 NADPH dehydrogenase, ENDOR spectra, 190-191 1,4-Naphthosemiquinoneanion radical, EPR and ENDOR spectra, 210-211 Neodymium Cj for, 51 spin expectation value, 51, 53 as structural probe, 58 Nernst plot, spectroelectrochemical data, 403 -404 Neutron capture radiography, 559-564 detectors, 561,564 neutrons for, sources, 563 performance, 562-564 principle, 559-560 Nickel(IlL electron paramagnetic resonance, 340 Nitrate reductase, electrochemistry, 513 Nitrile hydratase, EPR spectroscopy, 363 Nitrite reductase electrochemistry, 513 EPR spectroscopy, 361,364 Nitroaryl compounds, as solutes for selective reducing radical generation, 527 Nitrogenase A z o t o b a c t e r vinelandii

integer spin EPR spectroscopy, 476, 478 molybdenum-iron cofactor, ENDOR spectroscopy, 229-230 integer spin EPR spectroscopy, 478

625

Klebsiella p n e u m o n i a e , integer spin EPR

spectroscopy, 476, 478 P cluster in state W x, M/Sssbauer and EPR spectroscopy, 465,467-468, 476-478 X a n t h o b a c t e r autotrophicus, integer spin EPR spectroscopy, 476 Nitrosyl complexes, ferrous, EPR, 363366 Nitrosyl iron-sulfur complex, EPR spectroscopy, 365-366 Nitrous oxide reductase, P s e u d o m o n a s stutzeri, multifield saturation magnetization studies, 454-460 NMRD, see Nuclear magnetic resonance dispersion NOE, see Nuclear Overhauser effect NOESY, see Nuclear Overhauser effect spectroscopy Non-Kramers systems, M6ssbauer and EPR spectroscopy, 464-472 N-type selection, 7-8 Nuclear magnetic resonance dispersion, 270 Nuclear magnetic resonance spectrometer for 43Ca studies, 1t4 Fourier transform, 80 Nuclear magnetic resonance spectroscopy alkali metal, 78-106 applications, 79-80 biological applications, 83, 88-102 precautions, 102-106 in cell suspensions and perfused organs, 88-102 chemical shifts, 82, 86 information content, 97-102 intracellular signals, double-quantum coherence transfer pulse sequence for, 97 Lorentzian lineshape, 84 magnetization transfer method, 96-97 in metalloenzymes, 84-87 in metalloproteins, 84-87 modifed inversion recovery method, 93-96, 104-105 nuclide properties, 80-82 nuclide receptivity, 80-82 relaxation rates and times, 83-84 shift reagent method, 88-93, 104 average spectral density, 120 43Ca, 107-118

626

SUBJECT INDEX

applications, 107, 115-118 chemical exchange effects, 111-113 rate determination, 117 chemical shift range, 109 correlation time, determination, 117 electric quadrupole moment effects, 108-109 macromolecular interaction studies, 116 quadrupole coupling constant determination, 117 relaxation rates in absence of chemical exchange, 109-111 sample preparation, 114 spectra acquisition, 114 spectrometers, 114 ll3Cd, in metalloproteins, 16-43 chemical shifts, 18-21 instrumentation, 22 in metalloproteins, 16-43 chemical exchange effects, 29-31 diamagnetic metal ion experiments, 263, 265 sample preparation, 21-23 sensitivity, 17-18 solid-state, in H3Cd proteins, 41, 43 chemical exchange effects, 47-49 chemical shifts, 49-54 cross-relaxation, 278-279 ]33Cs advantages, 106 applications, 84 chemical shifts, 88, 98 precautions, 102 in red blood cell suspensions, intraand extracellular resonances, 90, 92 shift reagent method, 90-93 and electron paramagnetic resonance, combination, 119 19F, in measurement of cell membrane potential, 103 four-dimensional, 247 ~H, multidimensional high-resolution, 118 39K, 102 applications, 84 properties, 81 in red blood cell suspensions, 90 shift reagent method, 90-93

6Li properties, 80-81 receptivity, 82 in red blood cell suspensions, 90 shift reagent method, 90-93 7Li, 102 advantages, 106 applications, 84-86 ionophore-induced alkali metal ion transport monitoring studies, 99101 modified inversion recovery method, 94-96 nuclear relaxation times, in alkali metal ion binding to cell membrane0 101 properties, 81 receptivity, 82 in red blood cell suspensions, 90-91 shift reagent method, 90-93, 103 metal environment studies, 254-271 metalloproteins amide proton exchange rate determination, 275-278 conformational equilibria, 274-275 extrinsic ligand studies, 269-271 heteronuclear techniques, 33-43 "3Cd-13C scalar coupling, 37-40 lI3Cd-IH scalar coupling, 33-37 N3Cd-]SN scalar coupling, 40-41 H3Cd-3tP scalar coupling, 41-42 limitations, 245 metal force fields, 285-290 molecular dynamics simulations, 285290 oxidation state change studies, 280285 saturation transfer experiment, oxidation state change studies, 281282 T~ and T~ measurements, in oxidation state change studies, 282-285 multidimensional diamagnetic metal ion studies, 263269 paramagnetic metal ion studies, 256263 with ~'s values of 10"8-10-9 sec, 261264 with ~'s values of 10-tI-10 -13 sec, 258-261

SUBJECT INDEX as probe of metal environment, 24429O 23Na, 102 ionophore-induced alkali metal ion transport monitoring studies, 99101 properties, 81 receptivity, 82 in red blood cell suspensions, 90 relaxation times, 102 shift reagent method, 90-93 one-dimensional, 245 paramagnetic metalloproteins, 1, 16 ~70, small molecules, 271 3tp, in measurement of cell membrane potential, 103 pH titration experiments, 254-256 protein structure studies, 245-253 J coupling in, 246 nuclear Overhauser effects in, 246 quadrupolar nuclei, 114 87Rb precautions, 102 properties, 81 receptivity, 82 in red blood cell suspensions, 90 shift reagent method, 90-93 relaxation rates, measurement, 278-280 small molecules, 270-271 spin I = ½nuclei, 114 three-dimensional, 245,247 two-dimensional, 245-246 advantages, 16 effective sensitivity, 6 paramagnetic metalloproteins, 1-16 time scale for, 2 and X-ray diffraction, comparison, 245 Nuclear microprobe analysis, 565-575 instrumentation, 572-573 microanalytical methods for, 566-572 nuclear reaction analysis method, 570572 particle-induced X-ray emission, 566568 scattering analysis method, 568-570 Nuclear Overhauser effect, 2, 8-9, 246, 279 detection, 10-11 one-dimensional experiment, 6 Nuclear Overhauser effect spectroscopy, 2-3, 6, 10-11, 15

627

applications in paramagnetic systems, 12-13 cross-peaks, 10-11 paramagnetic metal ion studies, 259-261 for protein structure studies, 253 protein-substrate complex, 269 two-dimensional, 246 cross-peaks for amide protons in presence of nitroxides in DzO, 77 Nuclear quadrupole interaction, 127 Nuclear reactions, 558 with charged particles, tracks, in metal detection, 564-565 nuclear microprobe analysis, 570-572 tracks, detection, 558-565 Nuclear relaxation, 278-279 O Optically detected magnetic resonance spectroscopy absorbance-detected, s e e Absorbancedetected magnetic resonance spectroscopy applications to protein research, 316-329 decay curves, simulation, 304 double resonance, 305-307 fluorescence-detected, s e e Fluorescencedetected magnetic resonance spectroscopy hole burning, 305-307 instrumentation, 312-316 line shape, 305 metalloproteins, prospects for, 329-330 microwave switching under continuous illumination, 303 phosphorescence-detected, s e e Phosphorescence-detected magnetic resonance spectroscopy phosphorescence detection, 304 in photosynthesis, 320-329 populating probabilities, 303-305 principles, 295-316 quantitative description, 297-305 pitfalls, 304-305 response to onset of illumination, 303 slow-passage, 298-299 in absence of microwaves, 298 with saturating microwaves, 298-299 time dependence, 297-298 transient, 299-303

628

SUBJECT INDEX

average decay rate, determination, 300-303 with no optical excitation, 301-303 saturating microwaves in onset, 301 recovery from, 301-302 triplet states in proteins, 290-330 amino acid and cofactor studies, 318320 energy transfer, 319-320 heavy atom effects, 319 linear dichroic triplet-minus-singlet absorbance difference spectroscopy, 309-312, 324-329 optical microwave double resonance spectra, 307-312 triplet-minus-singlet absorbance difference spectra, 308-309 Ovotransferrin, EPR with VO 2+ electron spin probes, 235 Oxygen-17, in NMR studies of small molecules, 271

P Pancreatic trypsin inhibitor basic, lanthanide-induced shift studies, 62 bovine, structure, NMR studies, 253 Parallel electron energy loss spectrometry, 550-552 Paramagnetic metal ions nuclear magnetic resonance spectroscopy, 256-264 in protein center, functional mechanism, 13 Paramagnetic probes for metal cluster investigations, 384-395 secondary, for biological metal cluster investigations, 384-395 Paramagnetism, 119, 256-264, 331-333, 354 measurement, multifield saturation magnetization technique, 437-463 Parvalbumin CD domain, 67 competition binding studies, 47 EF domain, 67-73 lanthanide-induced shift studies, 67-73 nuclear magnetic resonance spectroscopy

alkali metal, 86 43Ca, 117 .3Cd chemical shifts, 20-21 solid-state, 41, 43 physicochemical properties, 67 PDMR, see Phosphorescence-detected magnetic resonance spectroscopy PEELS, s e e Parallel electron energy loss spectrometry PENMR, s e e Pulsed electron nuclear multiple resonance spectroscopy Peroxidase, electrochemistry, 513 L-Phenylalanine phosphoramidate phenyl ester, bound to "3Cd-carboxypeptidase A, 3~p NMR, "3Cd-3~P scalar coupling, 42 Phosphoglucomutase, alkali metal NMR spectroscopy, 86 Phosphoglycerate kinase, Mg2+-ATP dependent active site probe, 62-69 ATP-binding sites, 64 competitive inhibition by lanthanides, 64 lanthanide-induced shift studies, 62-69 nucleotide binding in solution 31p NMR studies, 63 proton NMR studies, 63 surface, lanthanide-induced shift studies, 74 yeast active site, 62-63, 68-69 properties, 62 X-ray crystal structure, 62-63 Phospholipase A2 calcium affinity determination, 115 lanthanide-induced shift studies, 78 Phosphorescence-detected magnetic resonance spectroscopy, 296, 299, 3 0 1 - 3 0 2 Phosphorus-31, in NMR measurement of cell membrane potential, 103 Photolysis, flash, s e e Flash photolysis Photosynthesis, optically detected magnetic resonance studies, 320-329 PIXAN software, 568 PIXE, s e e X-ray emission, particle-induced Plastocyanin "3Cd NMR spectroscopy chemical shifts, 20-21 T~ and NOE data for, 26

SUBJECT INDEX -cytochrome c complexes, electrochemistry, 506-507 French bean, structure, NMR studies, 247, 251 molecular dynamics simulations, 288289 multidimensional NMR diamagnetic metal ion experiments, 265 oxidation state change studies, 282283 Porphyrin radical complexes, magnetic susceptibility, 434-435 Potassium 39K, in NMR, 81, 84, 90-93, 102 natural abundance, 78 physiological and pharmacological importance, 79 Potassium ferricyanide EPR signal, 372 as oxidant of protein redox systems, 407 Potentiometry, redox, s e e Redox potentiometry Praseodymium Cj for, 51 spin expectation value, 51, 53 Precipitation, metal cations, 539-540 Proteins binding sites, 113Cd at, spin-lattice relaxation times, 26 Ca:÷-binding intestinal, competition binding studies, 47 lanthanide-induced shift studies, 6773, 78 DNA-binding, cadmium substitution, 17 gene 32, 113Cd NMR spectroscopy chemical shifts, 20-21 T1 and NOE data, 25-26 iron-containing, s e e Iron proteins iron-sulfur, s e e Iron-sulfur proteins lanthanide-induced shift studies, 56 Mn2+-substituted, alkali metal NMR spectroscopy, 87 structure, nuclear magnetic resonance spectroscopy studies, 245-253 surface, lanthanide-induced shift studies, 73-77 testicular S-100-1ike, EPR with VO 2+ electron spin probes, 235

629

Proton relaxation enhancement, 270 Protons amide, exchange rates, determination by multidimensional NMR, 275-278 interactions with matter, 565-566 NMR properties, 80-81 P-type selection, 8 Pulsed electron nuclear multiple resonance spectroscopy, 118-189 advantages, 119 double resonance, s e e Electron nuclear double resonance spectroscopy experimental considerations, 143-153 instruments for, 143-153 microwave transmitter design, 148 probes, 148-150 radio frequency transmitter for, 150152 samples concentration, 147 volume, 146-147 sensitivity, 147 spectrometer, 144 temperature for, 145-146 time scales, 144-145 Pulse radiolysis, 522-534 in analysis of intermediate oxidation states of metal ions in metalloproteins, 523 dosimetry, 529-530 electron transfer studies, 523 inductive beam monitor for, 529-530 long-range electron transfer study, in proteins, 532-534 oxidizing radical production, 525526 principle, 522 radiation chemical basis, 524-525 redt~cing radical production, 526527 secondary emission chamber for, 52953O selective probe technique, 523 solution preparation and handling, 530532 technique, 527-534 time resolution, 522 Pyridine, t4N ENDOR with VO 2+ electron spin probes, 239, 241 Pyruvate kinase, alkali metal NMR spectroscopy, 84

630

SUBJECT INDEX

Q Quadrupolar splittings, 109 Quadrupole coupling constant, 109 Quadrupole interactions, 335-336 Quadrupole relaxation, 266 Quinone, as solute for selective reducing radical generation, 527

R Radiolysis, pulse, see Pulse radiolysis Rat, tissues, EPR, detection of iron proteins, 382 Reaction centers absorbance-detected magnetic resonance spectroscopy studies, 322-324 bacterial absorbance difference spectroscopy studies, 325-329 structure, 320 linear dichroic triplet-minus-singlet absorbance difference spectroscopy studies, 324-329 plant, absorbance difference spectroscopy studies, 325-329 triplet-minus-singlet absorbance difference spectroscopy studies, 324, 326-327 Redox-active centers, in metalloproteins adsorbed on electrodes, voltammetric studies, 479-500 Redox potentiometry electrochemical equations in, 402-404 and spectroscopy, see Spectroelectrochemistry Relaxation rates, in absence of chemical exchange, 109-111 Relaxation times T~, "3Cd at protein binding sites, 26 1"1 and/'2, 7Li loaded in red blood cells, 101-102 Relaxometry, 270 Resonator loop-gap, 150, 202,219 bridged, 150 for LD-ADMR and ADMR, 313-314 slotted tube, 150 Rhodobacter capsulatus, light-harvesting complexes, fluorescence-detected magnetic resonance spectroscopy, 321

Rhodobacter sphaeroides photosynthetic system, fluorescencedetected magnetic resonance spectroscopy, 321-322 triplet state, electron-electron double resonance spectrum, 306 Rhodopseudomonas viridis, reaction center fluorescence-detected magnetic resonance spectroscopy, 322-323 triplet-minus-singlet absorbance difference spectroscopy, 324-328 Ribonucleotide reductase binuclear oxygen-bridged iron clusters, EPR, 368-369 pulse radiolysis study, 528-529 Rieske clusters, EPR, 343,367-368 RNA, transfer, lanthanide-induced shift studies, 56 ROESY, 11-12 applications in paramagnetic systems, 13 tH, water, 270 cross-peaks, 11-12 for protein structure studies, 253 Rubidium natural abundance, 78 physiological and pharmacological importance, 79 Rubredoxin electron paramagnetic resonance spectroscopy, 363-364 integer spin, 478 iron-sulfur clusters, EPR, 368

S Samarium Cj for, 51 spin expectation value, 51, 53 Scanning electron microscope, 542 Scanning electron microscopy, in nuclear microanalysis, 575 Scanning transmission ion microscopy, 575 Scattering analysis, 569-570 instrumentation, 573 Secondary ion mass spectrometry microscopy analytical image quality, 580-582 biological applications, 575-576, .584-585 depth profiling, 583-584 digital imaging processing for, 579-580

SUBJECT INDEX image acquisition time, 581 image quantification, 582-583 instrumentation, 578-580 metallic elements, sputtering process, 577 minimal metal concentrations for, 580581 principle, 576 specimen preparation for, 538 three-dimensional imaging, 583-584 SEM, s e e Scanning electron microscope; Scanning electron microscopy Serum, EPR spectroscopy, 383 SIMS, s e e Secondary ion mass spectrometry microscopy Sodium interactions with red blood cell membrane, probe for, 102 23Na, in NMR, 81-82, 90-93, 99-102 natural abundance, 78 physiological and pharmacological importance, 79 Sodium dithionate as potentiometric titrant, 406 properties, 372-373 as reductant of protein redox systems, 406-407 Solomon-Bloembergen equations, 54, 87 Spectroelectrochemistry, EPR methods, 396-41 l applications, 396-397 cells for, 397-400 standard design, 398-400 with visible capability, 401 data, Nernst plot, 403-404 oxidation methods, 406-407 redox mediator titrants and indicators, 404-406 reduction (oxidation) methods, 406407 Spectrometers double focusing, 579 electron energy loss, 550 electron nuclear double resonance spectroscopy, 152-153 electron paramagnetic resonance, 345347, 369-370 energy dispersive, 542, 546-547 nuclear magnetic resonance for 43Ca studies, ll4 Fourier transform, 80

631

for pulsed electron nuclear multiple resonance spectroscopy, 144 quadrupole mass filter, 579 time-of-flight, 579 wavelength dispersive, 542, 545-547 X-ray, 545-547 Spin Hamiltonian formalism, 123-125, 211-218, 334-336, 424-425 anisotropic parameters, determination, 348-353 Heisenberg-Dirac-van Vleck, 426 for M6ssbauer and EPR spectroscopy, 465 for multifield saturation magnetization, 440-441 for St = $2 = 1 dimer, 426-432 for vanadyl(IV) EPR spectra, 232-233 in zero magnetic field, 293-295 Spin-lattice relaxation, 334 SQUID susceptometer, s e e Superconducting quantum interference device susceptometer SRS project, synchrotron radiation-induced X-ray fluorescence microprobe, 556-557 SRXRF, s e e Synchrotron radiation-induced X-ray fluorescence Stellacyanin 113Cd NMR spectroscopy chemical shifts, 20-21 T~ and NOE data for, 26 electron spin echo detected EPR, 153155 ENDOR spectra, 153-161, 190-191 Cu nuclei, 162-164 Davies, 137, 154-164 Mires, 137, 156-158 nutation of sublevel magnetization, 138-139 proton and nitrogen hyperflne coupling assignment, 154-161 transient nutation patterns showing Rabi oscillation frequencies, 139140 ENDOR spectroscopy, 226 high-pH form, copper coordination structure, 153-154 HS-ENDOR spectra, 178-180 metal environment, multidimensional NMR studies, diamagnetic metal ion experiments, 265

632

SUBJECT INDEX

paramagnetic metal ion studies, 259 physicochemical properties, 153 STEM, s e e Transmission electron microscope, scanning Sternheimer antishielding factor, 81-82 STIM, s e e Scanning transmission ion microscopy Succinate dehydrogenase, electrochemistry, 513,521 Sulfide:cytochrome-c oxidoreductase, C h r o m a t i u m v i n o s u m , electrochemistry, 521 Sulfite oxidase, electrochemistry, 513 Superconducting quantum interference device susceptometer, 415-418, 443449 magnetic property measurement system, 416 data acquisition, 421-423 experimental conditions for, 421 sample holder, 419-420 Superconducting quantum interference magnetization measurements, principles, 415-418 Superexchange coupling, 342 Superoxide dismutase H3Cd NMR spectroscopy chemical shifts, 20-21 I"1 and NOE data, 26 Cu(II)2,Zn(II)2-, oxidized form, NMR spectroscopy, 15 Synchrotron radiation, in elemental analysis, 554-556 Synchrotron radiation-induced X-ray fluorescence analysis, 553-558 microprobes, 556-558

T Tb(DOTP)5-, interaction with bacteriophage fa ssDNA-binding gene 5 protein, ~3C NMR study, 74-76 Terbium Cj for, 51 spin expectation value, 51, 53 Tb(III), as structural probe, 47 Thioredoxin, metal environment, multidimensional NMR studies, pH titration experiments, 256

Thulium Cj for, 51 spin expectation value, 51, 53 Tm(DOTP)5-, 88-93, 103 preparation, 89 TOCSY, s e e Total correlation spectroscopy, two-dimensional Total correlation spectroscopy, two-dimensional, 3-4, 9, 246 applications, in paramagnetic systems, 12-13 dispersion mode, 8-9 principle, 8 Transcription factors GAL4 Ix3Cd NMR, 33, 269 ~I3Cd-~H scalar coupling, 33, 35-37 chemical shifts, 19-21 Tt and NOE data, 25-26 Cd 2÷ clusters, 31 structure, 2D NMR spectroscopy, 269 LAC9, 113CdNMR chemical shifts, 2021 Transferred hyperfine interaction, 124 Transferrin 65Cu-substituted, copper ENDOR spectroscopy, 227-228 electron paramagnetic resonance spectroscopy, 363 ENDOR spectroscopy, 230-231 EPR/ENDOR/ESEEM with VO 2+ electron spin probes, 235 lanthanide-induced shift studies, 78 Transition metal ions, EPR, 332-345 Transition metals, sites in metalloenzymes and proteins, 119 Transmission electron microscope biological applications, 553 conventional, 542, 550 filtered electron, 543 scanning, 542, 550 Traveling wave tube amplifier, 144, 148 Triple resonance spectroscopy, 206-207 General scheme, 182-183,206-207, 217218 Special scheme, 182, 206-207, 218 Triplet-minus-singlet absorbance difference spectra, 307-309 Triplet-minus-singlet absorbance difference spectroscopy, linear dichroic, 309-312 instrumentation, 315-316

SUBJECT INDEX

Triplet states linear dichroic triplet-minus-singlet absorbance difference spectroscopy, 309-312 instrumentation, 315-316 physics, 291-295 in proteins, optically detected magnetic resonance, 290-330 spin Hamiltonian, in zero magnetic field, 293-295 Tris-HCl, in metal ion exchange procedures, 22-23 Tris-HCl-acetate, in metal ion exchange procedures, 22-23 Troponin C 43Ca NMR, 117 It3Cd NMR, chemical shifts, 20-21 lanthanide-induced shift studies, 73 Tumor cells, Friend leukemia, synchrotron radiation-induced X-ray fluorescence analysis, 555 Tungsten in redox-active proteins, 281 W(V), EPR, 336-337

U Ubiquinone reductase, iron-sulfur clusters, EPR spectroscopy, 383 Ultracryosectioning, 536 Uricase, coimmobilized with horseradish peroxidase, as biosensor, 518 Uteroferrin, NOESY spectra, 15

V Vanadium in redox-active proteins, 280 V(IV), EPR, 336-337 Vanadyl(IV) ENDOR/ESEEM spin probes, 232-244 physicochemical properties, 232 Voltammetry, metalloprotein redox-active centers advantages, 481-484 analysis and interpretation, 489-500 instrumentation, 486-488 in kinetic analysis of coupled processes, 484 kinetic data, extraction, 497-500

633

optimal conditions, 484-485 with rapid coupled reactions, 496 sensitivity to active site-exogenous reagent interactions, 484 with slow coupled reactions, 492-496 in visualization and quantitation of redox activities, 485-486 waveform analysis, 482-483 W WALTZ16 sequences, 9

X Xanthine oxidase coimmobilized with horseradish peroxidase, as biosensor, 518 dipole-dipole coupling in, 342 electrochemistry, 513 95Mo-enriched, EPR spectra, 348-351 X a n t h o b a c t e r a u t o t r o p h i c u s , nitrogenase, integer spin EPR spectroscopy, 476 X filter, 268 X-ray emission particle-induced, 566-568 instrumentation, 573 photon-induced, 553-556 X-ray emission spectrum, 544-545 X-ray fluorescence, 553-554 synchrotron radiation-induced, s e e Synchrotron radiation-induced Xray fluorescence X-ray microanalysis, 541 biological applications, 551-553 and electron energy lqss spectrometry, comparison, 551-552 X-ray microtomography, 558 X-ray spectrometer, 545-547 X-ray spectrometry, 544-549 filtered X-ray images, 548-549 quantitative analysis, 547-548 XRMA, s e e X-ray microanalysis Xylose isomerase ENDOR with VOz+ electron spin probes, 235 IH, 237-239 14N, 239, 241 EPR with VO2+ electron spin probes, 235

634

SUBJECT INDEX Y

Ytterbium Cj for, 51 spin expectation value, 51, 53 Yb(IIl), as structural probe, 45, 47, 6773

Z Zeeman effect, pseudonuclear, 228-229 Zeeman interactions, 124, 336, 355

Zeeman splitting, 208 Zero-field splitting in EPR, 355-356 in multifield saturation magnetization, 440-441 Zinc metalloenzymes 113Cd NMR, N3Cd-13C scalar coupling, 38 molecular dynamics simulations, 287288 Zinc metalloprotein, N3Cd NMR spectroscopy, 17