Biophysical Techniques in Photosynthesis (Advances in Photosynthesis and Respiration)

Biophysical Techniques in Photosynthesis

Advances in Photosynthesis

VOLUME 3

Series Editor: GOVINDJEE Department of Plant Biology University of Illinois, Urbana, Illinois, U.S.A. Consulting Editors: Jan AMESZ, Leiden, The Netherlands Eva-Mari ARO, Turku, Finland James BARBER, London, United Kingdom Robert E. BLANKENSHIP, Tempe, Arizona, U.S.A. Norio MURATA, Okazaki, Japan Donald R. ORT, Urbana, Illinois, U.S.A. Advances in Photosynthesis provides an up-to-date account of research on all aspects of photosynthesis, the most fundamental life process on earth. Photosynthesis is an area that requires, for its understanding, a multidiscipli­ nary (biochemical, biophysical, molecular biological, and physiological) approach. Its content spans from physics to agronomy, from femtosecond reactions to those that require an entire season, from photophysics of reac­ tion centers to the physiology of the whole plant, and from X-ray crystal­ lography to field measurements. The aim of this series of publications is to present to beginning researchers, advanced graduate students and even specialists a comprehensive current picture of the advances in the various aspects of photosynthesis research. Each volume focusses on a specific area in depth.

The titles to be published in this series are listed on the backcover of this volume.

Biophysical

Techniques in

Photosynthesis

Edited by

Jan Amesz and Arnold J. Hoff Department of Biophysics Huygens Laboratory, University of Leiden, 2300 RA Leiden The Netherlands

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: Print ISBN:

0-306-47960-5 0-7923-3642-9

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Contents

Preface

xi

Part One: Optical Methods 1

Developments in Classical Optical Spectroscopy Jan Amesz Summary I. Introduction II. Absorption and Absorption Difference Spectroscopy III. Fluorescence References

2

Linear and Circular Dichroism Garab

3

3

4

6

8

11

Summary I. Introduction II. Linear Dichroism III. Circular Dichroism IV. Concluding Remarks Acknowledgements References

3

3

11

12

16

24

35

35

35

Fluorescence Kenneth Sauer and Martin Debreczeny Summary I. Introduction II. Steady-State Fluorescence III. Time-Resolved Fluorescence IV. Conclusion Acknowledgements References

41

41

42

45

52

59

59

60

v

vi

4

5

6

Contents Ultrafast Spectroscopy of Photosynthetic Systems

Ralph Jimenez and Graham R. Fleming Summary

I. Introduction

II. Laser Sources

III. Fluorescence Upconversion

IV. Transient Absorption

V. Concluding Remarks

References

63 63 64 66 70 72 72

Data Analysis of Time-Resolved Measurements

Alfred R. Holzwarth

75

Summary

I. Introduction

II. Methods for Time-Resolved Data Analysis

III. Some Applications to Photosynthesis

IV. Conclusions

Acknowledgements

References

75 76 76 87 89 91 91

Photosynthetic Thermoluminescence as a Simple Probe of Photosystem II

Electron Transport

Yorinao Inoue Summary

I. Introduction

II. Origins of TL from Photosynthetic Apparatus

III. Application of TL as a Probe of PSII Photochemistry

IV. Perspective: Merits and Demerits of TL Technique

Acknowledgements

References

7

63

Accumulated Photon Echo Measurements of Excited State Dynamics in Pigment–

Protein Complexes Thijs J. Aartsma, Robert J.W. Louwe and Peter Schellenberg Summary I. Introduction II. Homogeneous and Inhomogeneous Linewidths III. Photon Echo Phenomena IV. Accumulated Photon Echo: Experimental V. Energy Transfer VI. Photon Echo Experiments on Reaction Centers VII. Conclusion and Perspectives Acknowledgements References

93 93 94 94 103 104 105 105

109

109

109

110

111

114

116

118

119

120

120

Contents

8

vii

Spectral Hole Burning: Methods and Applications to Photosynthesis N. Raja S. Reddy and Gerald J. Small Summary I. Introduction II. Experimental Methods III. Applications Acknowledgements References

9

Infrared and Fourier-Transform Infrared Spectroscopy Werner Mäntele Summary I. Introduction: Looking Back 100 Years II. What can Infrared Spectroscopy Tell us about the Processes in Photosynthetic Membranes and Reaction Centers?

III. From Bands to Bonds: Strategies for Band Assignments IV. Fourier-Transform Infrared (FTIR) Spectroscopy V. Single Wavelength IR Techniques VI. Sample Preparation for Infrared Spectroscopy VII. Conclusions and Outlook Acknowledgements References

10

Resonance Raman Studies in Photosynthesis – Chlorophyll and Carotenoid

Molecules Bruno Robert Summary I. Introduction II. Introduction to Raman and Resonance Raman Spectroscopy III. Resonance Raman Spectroscopy of Photosynthetic Pigments IV. Resonance Raman Spectroscopy as Method of Chemical Analysis in Photosynthesis V. Resonance Raman Spectroscopy as a Probe for Molecular Conformation VI. Resonance Raman Spectroscopy as a Probe for Intermolecular Interactions VII. Time-Resolved Resonance Raman Studies VIII. Resonance Raman Spectroscopy as a Probe for Studying the Nature of Electronic Transitions

IX. Perspectives Acknowledgements References

11 Stark Spectroscopy of Photosynthetic Systems Steven G. Boxer Summary I. Introduction II. Methods III. Limitations and Conceptual Issues IV. Examples of Recent Results for Photosynthetic Systems Acknowledgements References

123

123

124

126

129

134

135

137

137

138

139

139

141

152

155

157

157

157

161

161

162

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163

167

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174

174

177

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177

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188

188

viii

Contents

12 The Photoacoustic Method in Photosynthesis – Monitoring and Analysis of

Phenomena Which Lead to Pressure Changes Following Light Excitation Shmuel Malkin Summary Introduction – Historical Notes and Main Aspects I. II. Experiments and Results with the Gas-phase Coupled Microphone Time Domain with a Sample Coupled III. Experiments and Results in the

Piezoelectric Sensor IV. Applications to Physiological Studies References

191

191

192

194

202 204

204

Part Two: Magnetic Resonance 13 Magnetic Resonance: An Introduction Arnold J. Hoff 14 Time-Resolved Electron Paramagnetic Resonance Spectroscopy – Principles and

Applications Haim Levanon Summary Introduction I. II. Experimental III. Results IV. Concluding Remarks Acknowledgements References

15

Electron Spin Echo Methods in Photosynthesis Research R. David Britt Summary I. Introduction II. ESEEM III. ESE-ENDOR IV. Additional Examples of ESE Applications in Photosynthesis V. Instrumentation Acknowledgements References

16 ENDOR Spectroscopy Wolfgang Lubitz and Friedhelm Lendzian Summary I. Introduction II. Principles of Electron–Nuclear Multiple Resonance Spectroscopy III. Selected Applications of ENDOR to Photosynthesis IV. Concluding Remarks Acknowledgements References

209

211

211

212

213

218

229

229

230

235

235

235

238

243

246

249

252

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255

255

256

258

268

272

272

272

Contents

17

ix

Optically Detected Magnetic Resonance (ODMR) of Triplet States in

Photosynthesis Arnold J. Hoff

Summary I. Introduction II. The Triplet Spin Hamiltonian in Zero Magnetic Field III. Optical Detection of Magnetic Resonance, ODMR IV. Double Resonance V. ODMR in Photosynthesis VI. Concluding Remarks References

18

Magic Angle Spinning Nuclear Magnetic Resonance of Photosynthetic

Components Huub J.M. de Groot Summary I. Introduction II. Magic Angle Spinning NMR Spectroscopy III. Probing the Local Environment of M(Y)210 in Rb. sphaeroides R26 RC with MAS IV. The Configuration of the Spheroidene in the Rb. sphaeroides RC V. The Asymmetric

Binding in Rb. sphaeroides R26 VI. New Developments. CIDNP and Correlation Spectroscopy VII. Concluding Remarks Acknowledgements References

277

277

278

278

279

284

288

295

295

299

299

300

300

302

305

306 309

312

312

312

Part Three: Structure and Oxygen 19

Structure Determination of Proteins by X-Ray Diffraction Marianne Schiffer Summary I. Introduction II. Theory, Equations, and Some of the Terms Used in X-Ray Structure Determination III. Determination of Protein Structure IV. Quality of the Structure V. Comparison with Structural Information Obtained with Other Techniques Acknowledgements References

20

317

317

317

318

318

323

323

323

324

Electron Microscopy Egbert J. Boekema and Matthias Rögner

325

Summary I. Principles II. Periodic Averaging III. Single Particle Averaging IV. Concluding Remarks Acknowledgements References

325

326

330

332

335

335

335

x

Contents

21 X-Ray Absorption Spectroscopy: Determination of Transition Metal Site

Structures in Photosynthesis Vittal K. Yachandra and Melvin P. Klein Summary I. Introduction II. X-Ray Absorption Spectroscopy (XAS) III. Applications of XANES and EXAFS in Photosynthesis IV. Future Directions Acknowledgements References

22 Mössbauer Spectroscopy Peter G. Debrunner Summary Introduction I. II. Mössbauer Spectroscopy: Physics and Formalism III. Applications References

23 Characterization of Photosynthetic Supramolecular Assemblies Using Small Angle

Neutron Scattering David M. Tiede and P. Thiyagarajan Summary I. Introduction II. Small Angle Neutron Scattering III. SANS Studies of Photosynthetic Complexes IV. Concluding Remarks Acknowledgements References

337

337

338

338

345

350

351

352

355

355

356

357

365

371

375

375

376

377

379

388

388

389

24 Measurement of Photosynthetic Oxygen Evolution Hans J. van Gorkom and Peter Gast

391

Summary I. Introduction II. Polarography III. EPR Oximetry IV. Mass Spectrometry V. Photoacoustic Spectroscopy VI. Galvanic Sensors VII. Prospects Acknowledgements References

391

392

392

398

401

402

402

402

403

403

Index

407

Preface

Progress in photosynthesis research is strongly dependent on instrumentation. It is therefore not surpris­ ing that the impressive advances that have been made in recent decades are paralleled by equally impressive advances in sensitivity and sophistication of physical equipment and methods. This trend started already shortly after the war, in work by pioneers like Lou Duysens, the late Stacy French, Britton Chance, Horst Witt, George Feher and others, but it really gained momentum in the seventies and especially the eighties when pulsed lasers, pulsed EPR spectrometers and solid-state electronics acquired a more and more prominent role on the scene of scientific research. This book is different from most others because it focuses on the techniques rather than on the scientific questions involved. Its purpose is three-fold, and this purpose is reflected in each chapter: (i) to give the reader sufficient insight in the basic principles of a method to understand its applications (ii) to give information on the practical aspects of the method and (iii) to discuss some of the results obtained in photosynthesis research in order to provide insight in its potentalities. We hope that in this way the reader will obtain sufficient information for a critical assessment of the relevant literature, and, perhaps more important, will gain inspiration to tackle problems in his own field of research. The book is not intended to give a comprehensive review of photosynthesis, but nevertheless offers various views on the exciting developments that are going on. The methods discussed in the book can be roughly divided in three categories: methods of optical electronic and vibrational spectroscopy, magnetic resonance methods and methods mainly aimed at obtaining structural information. Optical methods and phenomena that are discussed include linear and circular dichroism, time resolved fluorescence and absorbance measurements, including their data analy­ sis, vibrational spectroscopy (infrared and resonance Raman) and specialized techniques, such as photon echo, hole burning, Stark and photoacoustic spectroscopy. The section on magnetic resonance is mainly devoted to electron spin resonance and the various techniques that apply: EPR, ESE, ENDOR and ODMR. One chapter is devoted to magic angle spinning NMR. Knowledge of the structure of the photosynthetic apparatus is a prerequisite for obtaining insight in its function. With one exception (oxygen measurements) the third section of the book concerns methods that are primarily aimed at obtaining such structural information. A variety of techniques is discussed: Xray diffraction, X-ray absorption, electron microscopy, Mössbauer spectroscopy and neutron scattering. Although the book does not pretend to give an exhaustive overview of all types of physical measurements in photosynthesis, we feel that it gives a fairly comprehensive picture of the most important techniques and of their applications. This book has been made possible by the help and effort of many. First of all we are indebted to the authors of the various chapters. All of them, we think, furnished us with first-rate contributions highlighting their field of specialization. Second, we would like to thank the editor-in-chief of the series, Govindjee, who engendered the idea for this book, and with incessant and unfailing enthusiasm guided us with his electronic messages. Third, we thank the secretarial staff of our department, Mrs. B.C. van Dijk and Mrs. M.J. Gouw who helped us in various ways. Finally we want to acknowledge the skill of Gilles Jonker and his staff at Kluwer in producing the book. Jan Amesz Arnold J. Hoff xi

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PART ONE

Optical methods

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Chapter 1

Developments in Classical Optical Spectroscopy

Jan Amesz Department of Biophysics, Huygens Laboratory, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands

3 3 4 6 8

Summary I. Introduction II. Absorption and Absorption Difference Spectroscopy III. Fluorescence References

Summary An overview is given of the development of optical techniques as applied to photosynthesis research during the last 50 years and their importance for present day research is discussed. The review concerns the “classical” techniques, i.e. measurements of absorbance and of light-induced changes of absorbance and fluorescence emission and excitation spectroscopy. Abbreviations: BChl – bacteriochlorophyll; Chl – chlorophyll; FMO protein – Fenna–Matthews–Olson protein of green sulfur bacteria

I. Introduction

devices and new light sources became available, including pulsed lasers for flash spectroscopy as well as computers for data processing and regis­ tration. Hand in hand with these developments optical studies of photosynthesis have acquired growing importance for gaining insight in the mol­ ecular mechanisms of phosynthesis. In the chapters that follow, accounts will be given of the present state of the art, and examples will be given of the information obtained by mod­ ern optical methods. This chapter will survey some of the developments during the last 50 years and will discuss some of the “traditional” me­ thods that still play an important role in photo­ synthesis research. Pioneers of the early days were H. Kautsky and E.C. Wassink and, at a somewhat later stage, L.N.M. Duysens, C.S. French, B. Chance, H.T. Witt and B. Kok. The first two studied the fluor­ escence properties of photosynthetic material (Kautsky and Hirsch, 1931; Kautsky and Franck,

In view of the key role of pigments in photosynth­ esis, it is not surprising that optical methods have played, and continue to play, an important role in photosynthesis research. Engelmann (1882) was the first to show, by means of action spectra of oxygen production, that chlorophyll and the so-called accessory pigments are involved in pho­ tosynthesis. Progress in optical research on pho­ tosynthesis, however, was for a long time ar­ rested, mainly because simple and sensitive techniques for measuring and recording light in­ tensities were lacking. About 50 years ago, how­ ever, a rapid development of optical techniques set on. World War II saw the development of the photomultiplier, and in the years that followed faster and more reliable and sensitive electronic Correspondence: Fax: 31-71-5275819; E-mail: [email protected]

3

J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 3–10. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

4

1943; Vermeulen et al., 1937), while spectroscopy of light-induced absorbance changes was pione­ ered by Duysens (1952). In particular, the latter technique has proved invaluable to study the components of photosynthetic electron transport. One of the early results of such studies was, around 1960, the discovery of the two photosys­ tems in plant photosynthesis (Duysens et al., 1961). The use of pulsed lasers was initiated in the sixties (DeVault and Chance, 1966; Wolff et al., 1969; Netzel et al., 1973) and has now progressed into the femtosecond region, enabling the study of early processes of energy transforma­ tions in excited pigments (see Chapter 4 by Ji­ menez and Fleming). French (French and Young, 1952) and in particular Duysens (1952) were the first to apply fluorescence spectroscopy to study energy transfer in photosynthetic organisms. French also devised various ingenious apparatus for the deconvolution of absorption spectra, the automatic recording of action spectra and for the measurement of so-called derivative spectra (French et al., 1954; French, 1955; French and Harper, 1957; Allen et al., 1960). Today, such measurements are routinely, and much more con­ veniently, performed with the aid of computer analysis. In the next two sections, we shall survey these developments in some more detail, and briefly discuss the importance of these “classical” optical techniques in modern photosynthesis research. II. Absorption and Absorption Difference Spectroscopy Measurement of the absorption spectrum is one of the basic methods to obtain information about the characteristics of photosynthetic material. As this is normally done with commercial apparatus an extensive discussion of the method should not be necessary here. Nevertheless, absorption spec­ tra of rather poor quality are being published occasionally even today, and this is mainly due to the fact that these commercial apparatus are not designed for scattering material. Light scat­ tering, if not properly corrected for, not only causes an upward shift and distortion of the ab­ sorption spectrum, but it may also decrease the amplitude of absorption bands (Amesz et al., 1961; Latimer and Eubanks, 1962). Moreover,

Jan Amesz additional distortion may occur due to selective scattering near the absorption bands (Latimer, 1959). The effects can be minimized by collecting the transmitted light over a relatively large angle. Other methods that may be applied are adjusting the refractive index of the medium and the socalled opal glass method (Shibata, 1958), the lat­ ter, however, at the expense of sensitivity. In some cases reliable data can be obtained by flu­ orescence detected absorbance (Kramer et al., 1985). Of course, the same principles apply to more specialized absorption measurements, such as linear and circular dichroism and absorption difference spectroscopy. It should be noted, how­ ever, that even a properly measured absorption spectrum is not identical to that of the same pig­ ments in solution, if the pigments are contained in particles that have a non-negligible absorption. This is the so-called “flattening effect” (Duysens, 1956). Due to the presence of different “pools” of chemically identical pigments or to excitonic in­ teractions the in vivo absorption spectra of photo­ synthetic pigments nearly always consist of strongly overlapping absorption bands which are, moreover, inhomogeneously broadened. French and coworkers (Allen et al., 1960) determined the first derivatives of the absorption spectra to distinguish the various in vivo absorption bands of chlorophyll. A more convenient and nowadays extensively used method to enhance the resol­ ution of absorption (or other) spectra is by meas­ uring the second or even fourth derivatives (Mar­ tin, 1959; Butler and Hopkins, 1970a, 1970b; see Fig. 1). Caution, however, is needed in the inter­ pretation because the resulting bands are not only sharpened, but side bands are also generated by the differentiation. Duysens (1952,1957) was the first to apply the measurement of changes of absorbance, induced by illumination, to the study of photosynthesis. Since then, this method has continuously gained importance and it is still one of the most effective methods to study molecular processes in photo­ synthesis. Although pump-probe measurements with high time resolution are now in the forefront of research (see Chapter 4 by Jimenez and Flem­ ing), the classical methods, using a continuous or semi-continuous measuring beam, are still being extensively used in photosynthesis research, and

Classical optical spectroscopy

these are still of sufficient importance to warrant a brief discussion. In the “older” apparatus, such as described by Duysens (1957) and Chance (1951), the measur­ ing beam was modulated mechanically and was either split into a measuring and a reference beam or alternatively passed two monochromators set at different wavelengths. The resulting a.c. pho­ tomultiplier signal was then fed into a lock-in amplifier to reduce effects of the non-modulated “actinic” illumination and to provide enhanced stability and sensitivity. Alternatively, modulated actinic light has been used for generating modu­ lated signals of intermediates with a sufficiently

5

short lifetime (Kok, 1959; Spruit, 1971; Nishi­ mura et al., 1969). The relatively slow modulation employed in the above mentioned apparatus precludes mea­ surements of absorbance changes faster than a few ms. In fact, the development of rapid and stable d.c. amplifiers has largely obviated the need for light modulation, and apparatus with a continuous measuring beam, employing xenon or laser flash actinic illumination have been exten­ sively used in photosynthesis research. Fluor­ escence artifacts can be corrected for, if neces­ sary, by subtracting the signal obtained without a measuring beam. Flash spectroscopy was pio­

6 neered by Porter and Norrish (see Porter, 1968) to study reactions in gases and liquids. Early ap­ paratus for use in photosynthesis research have been described by Witt et al. (1959), Wolff et al. (1969) and Ke et al. (1964). A time resolution of is easily obtained, but can be extended to about 20 ns (Wolff et al., 1969). A 2 ns resolution has been obtained by using a xenon flash as a quasi-continuous light source (van Bochove et al., 1984; Kleinherenbrink, 1992). An appar­ atus for measuring time resolved difference spec­ tra based on an array of pulsed light emitting diodes has been described by Klughammer et al. (1990).

III. Fluorescence Fluorescence from leaves and photosynthetic pig­ ments was first observed by D. Brewster and G.G. Stokes, in the mid-nineteenth century (see Rabinowitch, 1951), but it was only about a cen­ tury later when fluorescence measurements be­ came an increasingly important tool in photo­ synthesis research. An interesting quantitative survey of the various topics studied by fluor­ escence in 200 publications of the period 1978– 1983 can be found in the review of Lavorel et al. (1986). Measurements with high time resolution will not be discussed in this chapter; and neither will be those of fluorescence polarization; for a dis­ cussion of those measurements the reader is re­ ferred to Chapter 3 by Sauer and Debreczeny. But also the “classical” fluorescence techniques still provide an important tool in photosynthesis research. They may be roughly divided in three types: measurements of emission spectra, of exci­ tation spectra and measurements of (relative) fluorescence yields. All three methods require relatively simple equipment, which nevertheless have been perfected steadily during the last five or six decades, (Lavorel et al., 1986; Schreiber, 1986; Schreiber et al., 1993) with digital process­ ing being standard nowadays. A discussion of some of the pitfalls and possible errors, like those caused by false light and self-absorption of fluor­ escence can be found in Amesz (1973). An appar­ atus specially designed for photosynthesis re­ search is now commercially available from Walz, Effeltrich, Germany. Apparatus have also been

Jan Amesz devised for field studies and productivity mea­ surements (Renger and Schreiber, 1986; Öquist and Wass, 1988). The prominent emission bands in photosyn­ thetic material are normally those of the longestwavelength Chls or BChls of the system, due to energy transfer from short-wavelength to longwavelength absorbing pigments. Emissions from shorter-wavelengths absorbing pigments are usu­ ally weaker because thermal equilibrium favors emission from the pigment with the lowest en­ ergy. Conspicuous exceptions are found in the fluorescence spectra of green bacteria, red algae and cyanobacteria, where relatively strong bands are observed from the chlorosomes and the phy­ cobilisomes (Amesz and Vasmel, 1986; Fork and Mohanty, 1986), showing that the efficiency of energy transfer from these extramembranous antenna systems to the pigments in the photosyn­ thetic membranes is less than 100%. The same applies to the so-called FMO protein, so that the fluorescence spectra of green sulfur bacteria are dominated by the fluorescence bands of chloro­ somes and FMO protein, whereas emission from the BChls in the core complex is hardly observ­ able (Amesz and Vasmel, 1986; Otte et al., 1991). A quantitative determination, however, of the transfer efficiencies from such measurements re­ quires knowledge of the “intrinsic” fluorescence yield of these antenna components, i.e. the yield in the absence of energy transfer to other pig­ ments. Such knowledge is normally not available, and therefore one has to rely on fluorescence excitation spectra to obtain such information. Any phenomenon brought about by light can in principle be characterized by its excitation (ac­ tion) spectrum. Such a spectrum defines the rela­ tive efficiencies of absorbed or incident photons of various wavelengths to bring about the phe­ nomenon under study. Unfortunately, published action spectra are often poorly defined. A pro­ perly measured action spectrum gives quantitat­ ive information about the pigment or pigments that sensitize the reaction, but care must be taken to avoid errors, such as those caused by a non­ linearity of the response with light intensity and self-screening within the sample (Amesz, 1973). Quantitative information on efficiencies of en­ ergy transfer is obtained by comparison of the excitation spectrum with the absorption spec­

Classical optical spectroscopy

trum. Unavoidable imperfections and differences in the optical arrangement used for measuring these two spectra, especially with scattering samples, set a limit to the accuracy of such a comparison, and this means that an excitation spectrum will not normally give reliable infor­ mation in the range between, say, 90 and 100%

7

transfer efficiency. Fig. 2 shows fluorescene exci­ tation spectra of chloroplasts, measured at low temperature. By choosing the proper emission wavelength, the excitation spectra of Photosys­ tems I and II can be measured independently in such a preparation, and this allows a distinction between the chlorophylls associated with the two

8

photosystems (note e.g. the predominance of the Chl b bands in the Photosystem II spectrum). Corresponding absorption spectra of the two pho­ tosystems are not available, unless one resorts to fractions solubilized by detergents, but the gen­ eral impression from these and similar experi­ ments is that the transfer efficiency from shortwavelength to long wavelength Chls is close to 100% in both photosystems. This means that the excitation spectra provide us with a means to determine the in situ absorption spectra of the two photosystems, information that cannot be ob­ tained in any other way. The anoxygenic photosynthetic bacteria have only one photosystem and do not pose such prob­ lems. Fig. 3 shows a fluorescence excitation spec­ trum of a purple bacterium illustrating the lower limit of accuracy that can be obtained in transfer efficiency measurements (Kleinherenbrink et al., 1992). The absorption spectrum shows the antenna and reaction center bands in chroma­ tophores of the BChl b containing purple bac­ terium Rhodopseudomonas viridis. The reaction center bands are completely lacking in the exci­ tation spectrum of antenna fluorescence, showing that the efficiency of energy transfer from the reaction center to the antenna does not exceed 2% (Otte et al., 1993). The widely held “trap–

Jan Amesz limited” model for energy conversion clearly does not apply in this case. Measurement of absolute yields of fluor­ escence in photosynthetic material is notoriously difficult, since it requires absolute measurements of light intensities and a representative sampling of all fluorescence emitted (Weber and Teale, 1957). In fact, one may doubt if accurate numbers have ever been published for photosynthetic ma­ terial. Relative yields can be measured much more easily, and such measurements, as a func­ tion of time, of added ions and inhibitors and of light intensity, have been done extensively during the last decades. Starting with the work of Duy­ sens and Sweers (1963), studies of the so-called variable fluorescence and induction effects have yielded a wealth of information on various aspects of the mechanism of photosynthesis. A discussion is beyond the scope of this chapter; the reader may be referred to reviews of van Gorkom (1986), Renger and Schreiber (1986), Krause and Weis (1991) and Dau (1994). Historical aspects have been reviewed by Duysens (1986) and Gov­ indjee (1995). References Allen MB, French CS and Brown JS (1960) Native and ex­ tractable forms of chlorophyll in various algal groups. In: Allen MB (ed) Comparative Biochemistry of Photoreactive Systems, pp 33–51. Academic Press, New York. Amesz J (1973) Spectrophotometric methods in photobiology. In: Checcucci A and Weale RA (eds) Primary Molecular Events in Photobiology, pp 21–43. Elsevier, Amsterdam. Amesz J and Vasmel H (1986) Fluorescence properties of photosynthetic bacteria. In: Govindjee, Amesz J and Fork DC (eds) Light Emission by Plants and Bacteria, pp. 423– 450. Academic Press, Orlando, FL. Amesz J, Duysens LNM and Brandt DC (1961) Methods for measuring and correcting the absorption spectrum of scattering suspensions. J Theor Biol 1: 59–74. Butler WL and Hopkins DW (1970a) Higher derivative analy­ sis of complex absorption spectra. Photochem Photobiol 12: 439–450. Butler WL and Hopkins DW (1970b) An analysis of fourth derivative spectra. Photochem Photobiol 12: 451–456. Chance B (1951) Rapid and sensitive spectrophotometry. III. A double beam apparatus. Rev Sci Instr 22: 634–638. Dau H (1994) Short-term adaptation of plants to changing light intensities and its relation to Photosystem II photo­ chemistry and fluorescence emission. J. Photochem Photo­ biol B: Biol 26: 3–27. DeVault D and Chance B (1966) Studies of photosynthesis using a pulsed laser. I. Temperature dependence of cyto­

Classical optical spectroscopy chrome oxidation rate in Chromatium. Evidence for tunne­ ling. Biophys J 6: 825–847. Duysens LNM (1952) Transfer of Excitation Energy in Photo­ synthesis. Doctoral Thesis, University of Utrecht. Duysens LNM (1956) The flattening of the absorption spec­ trum of suspensions, as compared to that of solutions. Biochim. Biophys. Acta 19: 1–12. Duysens LNM (1957) Methods for measurement and analysis of changes in light absorption occurring upon illumination of photosynthesizing organisms. In: Gaffron H (ed) Re­ search in Photosynthesis, pp 59–61. Interscience Pub­ lishers, New York. Duysens LNM (1986) Introduction to (bacterio)chlorophyll emission: a historical perspective. In: Govindjee, Amesz J and Fork DC (eds) Light emission by Plants and Bacteria, pp 3–28. Academic Press, Orlando. Duysens LNM and Sweers HE (1963) Mechanism of the two photochemical reactions in algae as studied by means of fluorescence. In: Studies on Microalgae and Photosynthetic Bacteria, pp 353–372. Univ of Tokyo Press, Tokyo. Duysens LNM, Amesz J and Kamp BM (1961) Two photoch­ emical systems in photosynthesis. Nature 190: 510–511. Engelmann TW (1882) Über Sauerstoffausscheidung von Pflanzencellen im Mikrospectrum. Botan Z 40: 419–426. Fork DC and Mohanty P (1986) Fluorescence and other characteristics of blue-green algae (cyanobacteria), red algae, and cryptomonads. In: Govindjee, Amesz J and Fork DC (eds) Light Emission by Plants and Bacteria, pp 451–496. Academic Press, Orlando. French CS (1955) Fluorescence spectrometry of photosyn­ thetic pigments. In: Johnson FH (ed) The Luminescence of Biological Systems, pp 51–74. Am Ass for the Advance­ ment of Science, Washington DC. French CS and Harper GE (1957) Derivative spectrophotome­ try. Carnegie Institution of Washington Year Book 56: 281–283. French CS and Young VK (1952) The fluorescence spectra of red algae and the transfer of energy from phycoerythrin to phycocyanin and chlorophyll. J Gen Physiol 35: 873–890. French CS, Towner H, Bellis DR, Cook RM, Fair WR and Holt WW (1954) A curve analyser and general purpose graphical computer. Rev Sci Instr 25: 765–775. Govindjee (1995) Sixty-three years since Kautsky: chlorophyll a fluorescence. Aust J Plant Physiol 22: 131–160. Kautsky H and Franck U (1943) Chlorophyll Fluoreszenz und Kohlensäure Assimilation XII. Zusammenfassung der bisherigen Ergebnisse und ihre Bedeutung für die Kohlen­ säureassimilation. Biochem Z 315: 207–232. Kautsky H and Hirsch A (1931) Chlorophyll Fluoreszenz und Kohlensäure Assimilation. Biochem Z 274: 423–434. Ke B, Treharne RW and McKibben C (1964) Flashing light spectrophotometer for studying the fast reactions during photosynthesis. Rev Sci Instr 35: 296–300. Kleinherenbrink FAM (1992) Trapping Efficiencies and Elec­ tron Transfer in Photosynthetic Bacteria. Doctoral Thesis, University of Leiden. Kleinherenbrink FAM, Deinum G, Otte SCM, Hoff AJ and Amesz J (1991) Energy transfer from long-wavelength ab­ sorbing antenna bacteriochlorophylls to the reaction cen­ ter. Biochim Biophys Acta 1099: 175–181.

9 Klughammer C, Kolbowski J and Schreiber U (1990) LED array spectrophotometry for time resolved difference spec­ tra in the 530–600 nm wavelength region. Photosynth Res 25: 317–327. Kok B (1959) Light-induced absorption changes in photosyn­ thetic organisms. II: A split-beam difference spectrophoto­ meter. Plant Physiol 34: 184–192. Kramer HJM, Amesz J and Rijgersberg CP (1981) Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K. Biochim Biophys Acta 637: 272–277. Kramer HJM, Westerhuis WHJ and Amesz J (1985) Low temperature spectroscopy of intact algae. Physiol Végét 23: 535–543. Krause GH and Weis E (1991) Chlorophyll fluorescence and photosynthesis – the basics. Ann Rev Plant Phys Plant Mol Biol 42: 313–349. Latimer P (1959) Influence of selective light scattering on measurement of absorption spectra of Chlorella. Plant Phy­ siol 34: 193–199. Latimer P and Eubanks CAH (1962) Absorption spectropho­ tometry of turbid suspensions: a method for correcting for large systematic distortions. Arch Biochem Biophys 98: 274–285. Lavorel J, Breton J and Lutz M (1986) Methodological prin­ ciples of measurement of light emitted by photosynthetic systems. In: Govindjee, Amesz J and Fork DC (eds) Light Emission by Plants and Bacteria, pp 57–98. Academic Press, Orlando. Martin AE (1959) Multiple differentiation as a means of band sharpening. Spectrochim Acta 14: 97–103. Netzel TL, Rentzepis P and Leigh J (1973) Picosecond kinetics of reaction centers containing bacteriochlorophyll. Science 182: 238–241. Nishimura M, Legallais V and Mayer D (1969) Multipurpose phosphoroscopic instrument for the study of phosphoresc­ ence, induction of fluorescence and absorbance change of turbid biological materials. Rev Sci Instr 40: 271–273. Otte SCM, van der Heiden JC, Pfennig N and Amesz J (1991) A comparative study of the optical characteristics of intact cells of photosynthetic green sulfur bacteria containing bac­ teriochlorophyll c, d or e. Photosynth Res. 28: 77–87. Otte SCM, Kleinherenbrink FAM and Amesz J (1993) Energy transfer between the reaction center and the antenna in purple bacteria. Biochim Biophys Acta 1143: 84–90. Öquist G and Wass R (1988) A portable, microprocessor operated instrument for measuring fluorescence kinetics in stress physiology. Physiol Plantarum 73: 211–217. Porter G (1968) Flash photolysis and some of its applications. Science 160: 1299–1307. Rabinowitch EI (1951) Photosynthesis and Related Processes, Vol II, part 1, Spectroscopy and Fluorescence of Photosyn­ thetic Pigments; Kinetics of Photosynthesis. Interscience Publ, New York. Renger G and Schreiber U (1986) Practical applications of fluorometric methods to algae and higher plant research. In: Govindjee, Amesz J and Fork DC (eds) Light Emission by Plants and Bacteria, pp 587–619. Academic Press, Or­ lando. Schreiber U (1986) Detection of rapid induction kinetics with

10 a new type of high-frequency modulated chlorophyll fluor­ ometer. Photosynth Res. 9: 261–272. Schreiber U, Neubauer C and Schlina U (1993) PAM fluoro­ meter based on medium- frequency pulsed Xe- flash meas­ uring light: A highly sensitive new tool in basic and applied photosynthesis research. Photosynth Res. 36: 65–72. Shibata K (1958) Spectrophotometry of intact biological ma­ terials. Absolute and relative measurements of their trans­ mission, reflection and absorption spectra. J Biochem (Tokyo) 45: 599–623. Spruit CJP (1971) Sensitive quasi-continuous measurement of photoinduced transmission changes. Meded Landbouwho­ geschool Wageningen 71: 1–6. Van Bochove AC, Swarthoff T, Kingma H, van Grondelle R, Duysens LNM and Amesz J (1984) A study of the primary charge separation in green bacteria by means of flash spectroscopy. Biochim Biophys Acta 764: 343–346.

Jan Amesz Van Gorkom H (1986) Fluorescence measurements in the study of Photosystem II electron transport. In: Govindjee, Amesz J and Fork DC (eds) Light Emission by Plants and Bacteria, pp 267–289. Academic Press, Orlando. Vermeulen D, Wassink EC and Reman GH (1937) On the fluorescence of photosynthesizing cells. Enzymologia 4: 254–268. Weber G and Teale FWJ (1957) Determination of the abso­ lute quantum yield of fluorescent solutions. Trans Faraday Soc 53: 646–655. Witt HT, Moraw R and Müller A (1959) Blitzlichtphotome­ trie. Z Physik Chem NF 20: 193–205. Wolff C, Buchwald H-E, Rüppel H, Witt K and Witt HT (1969) Rise time of the light induced electrical field across the function membrane of photosynthesis. Z Naturforsch 24b: 1038–1041.

Chapter 2 Linear and Circular Dichroism

Garab Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, P.O. Box 521, H-6701, Hungary

Summary I. Introduction A. Polarized Light B. Absorbance of Light by Molecules II. Linear Dichroism A. Polarization of the Electronic Transitions of Chls B. Anisotropy in Protein Complexes and Membranes 1. LD of Absorbance 2. Photoacoustic Linear Dichroism 3. Polarized Fluorescence Emission C. Methods of Orientation of Membranes and Particles 1. Mechanical Orientation Techniques 2. Orientation in a Magnetic Field 3. Orientation in an Electric Field 4. Photoselection D. Determination of the Orientation Angle of Dipoles in Realistic Systems 1. Degree of Orientation, Distribution Functions 2. Membrane Curvature, Microscopic LD 3. Fluctuations E. Miscellaneous Applications of LD 1. Estimation of Shape and Size of Particles 2. Spatial Position of Organelles 3. Information on the Band Structure III. Circular Dichroism A. Physical Origins of CD Signals B. CD of Photosynthetic Pigments 1. Intrinsic CD of Isolated Molecules 2. Excitonic Interactions 3. Differential Scattering, Psi-type CD, and Differential Polarization Imaging C. Secondary Structure of Chl-Containing Proteins D. Artifacts IV. Concluding Remarks Acknowledgements References

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Summary The efficiency of photosynthetic light energy conversion depends largely on the molecular architecture of the photosynthetic membranes. Linear and cicular dichroism (LD and CD) techniques have contributed Correspondence: Fax: 36-62-433434; E-mail: [email protected]

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J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 11–40. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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Garab

significantly to our knowledge of the molecular organization of the pigment system in various complexes and membranes. Systematic LD studies have led to the recognition of an apparently universal property of pigment systems in vivo: all pigments in all photosynthetic organisms display non-random orientation with respect to each other, to the protein axes and to the membrane plane. This molecular organization plays an important role in the energy transfer between pigment molecules. CD spectroscopy is widely used for the detection of excitonic interactions, which have been found to occur in virtually all reaction center and antenna complexes. Excitonic CD carries information on the distances and orientation of the interacting pigment molecules. CD is also capable of revealing information about certain macroorganizational parameters in molecular aggregates with sizes commensurate with the wavelength of visible light. These non-invasive techniques can be used for systems in a wide range of structural complexity, from isolated pigment molecules to whole organelles. CD and LD techniques have been extended to the (sub)picosecond time range. Combined with the methods of quantitative evaluation of data, these techniques will certainly remain indispensable in elucidation of the structure and function of the photophysical and photochemical apparatus. The purpose of this chapter is to provide an introduction to the theory and practice of LD and CD methods in photosynthesis. The main emphasis will be placed on the underlying principles and the basics of the experimental procedures, complemented with a few illustrations of results. I would also like to draw attention to a few recently introduced polarization techniques which are ripe for application in photosynthesis. Abbreviations: BChl – bacteriochlorophyll; Chl – chlorophyll; CB – circular birefringence; CD – circular dichroism; CDS – circular differential scattering; CIDS – circular intensity differential scattering; CPL – circularly polarized luminescence; DPI – differential polarization imaging; DR – dichroic ratio; FDCD – fluorescence-detected circular dichroism; FMO – Fenna–Matthews–Olson [complex]; FP – fluorescence polarization ratio; LB – linear birefringence; LD – linear dichroism; LHCII – light-harvesting chlorophyll-a/b-protein complex of photosystem 2; MCD – magnetic circular dichroism; ORD – optical rotatory dispersion; PALD – photoacoustic LD; Pheo – pheophytin; PSI or II – photosystem I or II; PChl – protochlorophyll; psi – polymer and salt-induced

I. Introduction In investigations of the primary processes of photosynthesis, the ultimate goal is to understand the structure and function of the photophysical and photochemical machinery. The efficiency of the primary step in the conversion of light energy to chemical energy depends largely on the molecular architecture of the reaction centers and the antenna system. The high efficiency of the primary charge separation and stabilization is in large part due to the special organization of the reaction centers. The details of the operation of the reaction centers, however, are still not fully understood. Energy migration in the antenna is largely determined by the molecular architecture of the pigment system. An optimized antenna organization should minimize quantum losses and ensure an efficient energy supply to the reaction

centers under a wide range of environmental and physiological conditions. On the other hand, a controlled dissipation of the absorbed energy in the antenna can prevent photoinhibitory damage of the photosynthetic machinery and thereby play a protective role. This requires a highly organized molecular architecture, which should neverthe­ less be capable of structural reorganizations. For an understanding of the structure and function of the building blocks and also the mechanism of operation of the entire photosynthetic apparatus, non-invasive techniques, such as LD and CD, are of special value. For a complete understanding of the function, the structural and optical information must be combined. Full struc­ tural information can be provided only by atomic resolution crystallography. The information content of polarization spectroscopy is substantially less than that of crystallography. However, many

LD and CD

Chl-containing complexes or other constituents of the photosynthetic apparatus appear to resist crystallization. Furthermore, in complex systems, e.g. membranes or organelles, a number of struc­ tural parameters can be determined only by means of LD and CD investigations. These me­ thods are also indispensable when structural reor­ ganizations and ultrafast processes are to be monitored. Full structural information is available on the bacterial reaction center of Rhodopseudomonas viridis (Deisenhofer et al., 1984). As pointed out by Breton and Nabedryk (1987), conclusions from polarization spectroscopy are in good quali­ tative accord with the results of X-ray crystal­ lography. A recent systematic study on bovine gamma-crystallines showed the excellent agree­ ment of LD and X-ray data (Bloemendal et al., 1990). The first part of this chapter will survey the basic principles related to polarized light and the interaction of light with absorbing matter. Then, the methods of LD and CD will be overviewed, together with a few examples of their application; these merely serve for illustration and cannot sub­ stitute a systematic review. Throughout the chap­ ter, the emphasis will be placed on the concepts, and the mathematical formalism will be used sparingly. LD and CD spectroscopy have developed sig­ nificantly in the past decade. LD spectroscopy is now used routinely to monitor ultrafast processes in the reaction center complex (Kirmaier et al., 1985) and energy migration pathways in the antenna (reviewed by van Amerongen and Struve, 1995). The method of recording transient CD spectra at picosecond time resolution has also been elaborated (Xie and Simon, 1991). The “standard” techniques have recently been com­ bined with novel methods, such as photoacoustic et al., 1985), differential polariz­ LD ation microscopy (Finzi et al., 1989) and circular differential polarization scattering (Garab et al., 1988c). Some other techniques, such as vi­ brational CD, CPL, FDCD have as yet been little used in photosynthesis, but hold a promise for future applications. The pioneering work and basic conclusions on the orientation of photosynthetic pigments in vivo were reviewed by Breton and Verméglio (1982),

13

and later developments were summarized by Bre­ ton (1986) and Garab et al. (1987). LD methods have been in the focus of many textbooks and reviews (Hofrichter and Eaton, 1976; Johansson and Lindblom, 1980; Clayton, 1980; Nordén et al., 1992; Bloemendal and van Grondelle, 1993; van Amerongen and Struve, 1995). CD and MCD in different Chl-containing systems were first re­ viewed by Sauer (1972). The theory of excitonic CD and results on Chl-containing complexes were dealt with in depth by Pearlstein (1982, 1987, 1991).

A. Polarized Light Light is an electromagnetic wave which oscillates periodically in both time and space. In the wave, the electric and magnetic vectors, which are pro­ portional to each other in magnitude, are mutu­ ally perpendicular, and also perpendicular to the direction of propagation. Non-polarized light consists of vibrations in many different polarization directions. In linearly polarized light, (often called plane polarized light), the electric vector, E (“the light vector”), oscillates sinusoidally in a direction (plane) which in spectroscopy is conventionally called the polar­ ization direction (plane). In circularly polarized light, the magnitude of E remains constant, but it traces out a helix as a function of time. In accordance with the convention used in CD spec­ troscopy, in right and left circularly polarized light beams, when viewed by an observer looking toward the light source, the end-point of E would appear to rotate clockwise and counterclockwise, respectively. It is useful to apply the principle of superposi­ tions to conceptualize the state of polarization of a light beam. As shown in Fig. 1A and B, circu­ larly polarized light can be represented as the sum of two orthogonal linearly polarized beams in which the amplitudes are equal and the phases With a phaseare shifted by exactly + or retardation of another linearly polarized beam is obtained, the polarization of which is ortho­ gonal to the original polarization direction (Fig. 1C). With retardation angles different from or or if the amplitudes of the two orthognal linearly polarized components are not equal, the polarization will become elliptical, i.e. the gen­

14

eral form. (For a more elaborate treatment of polarized light and the basic principles of the classical theory of optics, the reader is referred to textbooks, e.g. by Born and Wolf, 1980.) Through the use of the principle of superposi­ tions, circularly or linearly polarized light can eas­ ily be constructed. In the following the modu­ lation method used in most dichrographs will be outlined. Let us consider a linearly polarized light produced, for example, by a birefringent crystal. Let us transmit this beam through a slab of an isotropic transparent material (e.g. fused silica) with edges oriented at 45° with respect to E. If the slab is pressed and drawn in one direction by applying a.c. voltage (V) on a piezoelectric transducer (M, modulator), LB is induced, which results in phase shifts of one of the linearly polar­ ized component beams; the phase shift is linearly

Garab

proportional to the applied voltage (Fig. 2). For samples which show anisotropy of absorbance for orthogonal circularly or linearly polarized beams, the light intensity (I) transmitted by the sample (S) varies periodically with f or 2f, respectively. I and can be measured with a photomultiplier (PMT) and an appropriate demodulation tech­ nique (DEM), and thus CD and LD signals can be recorded. (For typical block diagrams and some technical aspects, see Bloemendal and van Grondelle, 1993; Johnson, 1985.) As will be evident in section III, it is useful to envisage the linearly polarized light as the sum of right and left circularly polarized light beams of equal intensity. In Figs. 1A and B the sum of the horizontal components is zero and thus the sum of the two orthogonally circularly polarized beams indeed reduces to a linearly (vertically) polarized beam. A general light beam can also be conveniently represented by the Stokes’ parameters, a 4 × 1 column matrix, and the light-matter interaction can be described by the 4 × 4 Mueller matrix:

LD and CD

The Stokes parameters (I,Q,U,V) characterize the monochromatic light beam propagating along z axis: its intensity, degree of linear polarization in xz/yz, at ±45° and circular polarization, respec­ tively. In most samples, there are correlations be­ tween different elements of the matrix. Gen­ erally, however, all 16 elements can be indepen­ dent and yield useful structural information on the sample (Kim et al., 1987a). Although there is an impetus to measure more elements of the matrix (Tinoco et al., 1987), in most cases only LD and CD the absorption are determined, mainly because of technical dif­ ficulties and the poor understanding of the physi­ cal meaning of other elements.

B. Absorbance of Light by Molecules During an optically induced transition, the elec­ tron distribution of the molecule oscillates peri­ odically with the frequency of the absorbed light. This means a transient oscillation of the electric and magnetic moments, which can generally be regarded as transition dipole moments, and m, respectively. However, in the UV to IR spectral range, only the electric dipole transitions have significant intensities and thus the absorption can be satisfactorily described by the electric transi­ tion dipole moment, which for an optically in­ duced electronic transition between the ground and excited states, a and b, is defined by the vector integral: Here and are the wave functions of the corresponding states of the molecule, and the electric dipole operator contains the sum of the products of each charged particle (electron or nucleus), and their position vector, In classical terms, the interaction is described by the induction of an oscillating dipole by the oscillating

15

electric field vector of the light. (In order to simplify the physical interpretation, the classical and quantum mechanical pictures will be used interchangeably.) In accordance with the Born–Oppenheimer approximation, the wave functions are written as the product of the electronic (e) and nuclear (n) wave functions:

In the first approximation, the electronic trans­ itions are considered for fixed nuclear positions, i.e. for no vibronic coupling of the transition. The admixture of vibronic components may complic­ ate the interpretation of the polarization mea­ surements. The probability of absorbance is proportional to the square of the scalar product of the electric vector of the light and the transition dipole vector of the molecule:

This means that a light beam polarized parallel to the transition dipole vector has the maximum probability of absorption, whereas if it is polar­ ized perpendicular to no absorption can take place. This serves the basis for LD spectroscopy. Let us consider an ‘oriented gas’, a set of noninteracting molecules in which all molecules are aligned parallel to each other. For a concentra­ tion (c) of 1 M and a pathlength (l) of 1 cm, let the absorbance with polarization parallel to the direction of the molecular dipoles be For the other two orthogonal linear polarizations the ab­ sorbance is evidently zero. On the other hand, in a random gas, after averaging, we obtain with any direction of the polarization of the light. is the isotropic molar extinction coefficient; The length of the transition moment vector (in the dipole strength, is correlated with the molar extinction coefficient:

where is the frequency of the light, i.e. the dipole strength is determined by the integrated area of the absorbance band.

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Garab

II. Linear Dichroism Linear dichroism is the differential absorption of two orthogonal linearly polarized light beams and in a macroscopically oriented sample: There are two basic cases for LD investigations: (i) If the spatial position of the molecules is known in the laboratory coordinate system (i.e. the molecules are macroscopically aligned), the orientation of the molecular dipoles can be deter­ mined with respect to the molecular coordinates, (ii) When the orientation of a transition dipole is known with respect to the molecular coordinate system, LD carries information on the orientation of the molecule, or at least on that of the transi­ tion dipole with respect to the symmetry axis of the sample. In most applications, the orientation of dipoles with respect to the coordination system of the object is at the focus of interest. However, the depth of information provided by these studies depends on the knowledge of the nature of the electronic transitions of the molecules and on the reliability of the data concerning the orientation of the transition dipoles with respect to the mol­ ecular coordinate system. It should be noted that, for symmetry reasons, not all molecules can exhi­ bit LD (Hofrichter and Eaton, 1976) but Chls, carotenoids, cytochromes and phycobilins possess linearly polarized electronic transitions, and thus are readily accessible for LD studies (Breton and Vermeglio, 1982; Juszcak et al., 1987). On the other hand, the method of LD is not confined to optical transitions. The anisotropy properties of magnetic transitions, which have proved very use­ ful in the characterization of triplet transitions, can be studied with polarized microwaves (Hoff, 1990). X-ray LD acquires information about the orientation of specific chemical bonds (Ade and Hsiao, 1993).

A. Polarization of the Electronic Transitions of Chls The interpretation of the electronic spectra of Chls and the determination of the polarizations of the major electronic transitions with respect to the molecule-fixed coordinate system are far from

definitive. Our knowledge is based mainly on the results of LD studies of molecular Chl solutions oriented in different systems, e.g. stretched film (Breton et al., 1972) multilayers (Hoff, 1974), host crystal (Moog et al., 1984) and liquid crystals (Bauman and Wrobel, 1980; et al., 1987). Recently Langmuir Blodgett film was used to orient plastoquinone molecules (Kruk et al., 1993). The molecules in these systems must not interact with each other or with the host matrix, because interactions may lead to changes in the polarization directions. The orientation of different transition dipoles of Chls and Pheos are given with respect to the X-Y molecular framework of the tetrapyrrole plane, and is measured in degrees, clockwise from the X axis of the molecular frame. Conven­ tionally, the X passes through C7, the position of phytyl substitution, and the Y axis through the N in Fig. 3). atoms of pyrroles I and III (The axes, are selected on the basis of the electronic symmetry of the sys­ tem.) The main electronic transitions of Chls are la­ and and beled with indices X and Y, for the red and the Soret bands, respec­ and tively, to indicate that they are polarized along these axes. However, Bauman and Wrobel (1980)

LD and CD

17

showed that the red band of Chl a and BChl a have mixed X and Y character. Fragata et al. (1988) calculated the polarization of different UV–VIS transitions of Chl a and Pheo a and showed that the main transition, (0 – 0) of Chl a which absorbs at 670 nm is found at 70° (Fig. 3). Van Gurp et al. (1989) also determined about 20° for the deviation of of Chl a from the Yaxis but preferred to take the transition moment on the other side of the symmetry axis, i.e. at Furthermore, it was 109°, thereby closer to concluded that other transitions of Chl a cannot be characterized in simple terms of a transition moment in the molecular frame but must be de­ scribed in terms of averages of goniometric func­ tions.

B. Anisotropy in Protein Complexes and Membranes With proper selection of the orientation method (see below) it can be ensured that the orthogonal polarizations of the measuring beam, hereafter coincide with the preferen­ referred to as and tial alignment of the sample, e.g. the plane of membranes or the long axis of complexes or ag­ gregates. This simplifies interpretation of the data appreciably. For the general case, the Euler transformation must be applied. 1. LD of Absorbance (i) Let us consider oriented planar membranes which contain absorbing dipoles with well-defined orientation angle with respect to the membrane plane (Fig. 4A). The orientation of and its unit vector, can be characterized with the azimuthal and orientational angles, and and

where u, v and n are also unit vectors. Since inside the membrane cannot be fixed with respect to the coordinate system, averaging must be per­ formed:

(ii) For aligned protein complexes (Fig. 4B):

Hence,

and

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The quantities which are directly related to the orientation angle are the parameter S, the re­ and the dichroic ratio duced dichroism (DR):

If in membranes, S > 0 (DR > 1) it can be con­ cluded that the dipoles tend to lie in the mem­ brane plane, with with re­ spect to the normal to the plane is called the magic angle, an orientation which cannot be dis­ tinguished from random orientation in an LD ex­ periment. The dichroism of the sample is often charac­ terized by the quantity which in some papers is referred to as dichroic ratio and in others called reduced LD. However, it must be noted that, although this expression is well suited for the general case when the symme­ try of the system is not known, its use may be misleading for systems with uniaxial symmetry where 2. Photoacoustic Linear Dichroism PALD is the differential dissipation of irradiation with orthogonal linear polarizations of light. This method provides a special tool to measure the orientation dependence of the radiationless deex­ citation processes. (Contributions from the aniso­ tropic character of heat conductivity have to be determined separately.) PALD has been used for both model systems and native particles et al.,1985; et al.,1984; et al., 1990). For a recent review, see et al. (1991). 3. Polarized Fluorescence Emission In a dipolar approximation, the fluorescence em­ ission is polarized because, for the same elec­ tronic transition, the orientation of the emitting dipole is parallel to the absorbing dipole. The intensity of the emission in a given polarization direction is proportional to the squares of the scalar products. In a coordinate system as in Fig.

Garab In and a macroscopically oriented sample, the dichroic ratio of fluorescence, which is often called the fluorescence polarization ratio (FP), can be used to calculate the orientation angle:

For this correlation to be valid, FP must be ex­ cited with non-polarized light, and the system must involve perfect energy transfer. For the gen­ eral case, FP depends not only on the fluor­ escence, but also on the absorbance of the sam­ ple, i.e. photoselection may play an important role (Szitó et al., 1985). A general theoretical analysis has been presented by van Gurp et al.(1988). For steady-state fluorescence, with ex­ citation in the blue, this effect is usually small, as can be demonstrated by comparing FP spectra recorded with nonpolarized and linearly polarized excitations, respectively. The magnitude of polarization of the fluor­ escence emission of intrinsically anisotropic and oriented sample upon excitation with non-polarized light is sometimes evaluated in the form of This representation, however, may cause confusion. stands for Conventionally, the degree of polarization of fluorescence after polarized excitation of a randomly oriented sam­ ple, the indices referring to the polarization di­ rections of the observation with respect to the direction of polarization of the exciting beam (see Chapter 3). In complex systems, e.g. photosynthetic mem­ branes, it is technically difficult to separate FP from P. As recognized by Breton et al. (1973), the intrinsic anisotropy of dipoles, which in oriented can contribute sig­ samples gives rise to nificantly to the measured value of P even if macroscopically FP = 1. This “residual polariza­ tion” is due to the fact that differently oriented membranes contained in a randomly oriented suspension exhibit different P values. In order to minimize this effect, the membranes should be oriented with their planes facing the observation. (For further comment, see II.D.2). Polarized fluorescence emission measurements have recently been used to determine the degree

LD and CD

19

of orientation of LHCII in compressed gel, and aided the precise determination of the orientation angles of different absorbance transition dipoles (van Amerongen et al., 1994).

C. Methods of Orientation of Membranes and Particles Orientation of a sample can be achieved with various techniques. Which method is applied de­ pends on the experimental conditions and on the goal of the investigation, and a general recipe cannot be given. 1. Mechanical Orientation Techniques In one of the simplest cases, the gravitational field is used to align the particles during drying. With flat membranes, a high degree of orientation can be achieved. However, since membranes tend to lie face-down on a supporting quartz or glass plate, LD can be measured only at a tilt angle with respect to the plate (Breton et al., 1973). This may easily introduce artifacts due to reflec­ tions. This method is useful when the water con­ tent of the sample must be low, e.g. in polarized IR spectroscopic studies (Nabedryk et al., 1984). The spreading of oblate particles with a fine brush over a quartz plate also yields a dry sample. The particles are aligned reasonably well, with their long axis parallel to the direction of spread­ ing (Breton et al., 1973). Some artifacts may be present (Haworth et al., 1982), which are proba­ bly due to the uneven surface of the film. The method of stretching films (e.g. polyvinyl alcohol) containing the particles can be used in a wide range of sizes, from pigments (Breton et al., et al., 1972) to whole chloroplasts 1985). The main advantage of this method is that the degree of orientation can readily be evaluated as a function of the extent of stretching (Nordén, 1980). Orientation by flow, i.e. orientation in a hyd­ rodynamic gradient, is used mainly for long, cyl­ indrical molecules or particles. The shear gradient is usually formed in the annular gap between a rotating and a fixed cylinder. In flow orientation, the particles can be suspended at low concentra­ tion, and there is essentially no restriction as con­ cerns the reaction medium. (For a review, includ­

ing technical and theoretical questions, see Nordén et al., 1992.) The currently most versatile and probably most widely used method is the gel-squeezing technique. This method was invented by Abdou­ rakhmanov et al. (1979) and was described for polyacrylamide gel, which is a continuum gel, i.e. it permits the alignment of particles of different sizes and shapes. Orientation by gel squeezing combines the advantages of film stretching and flow orientation: the degree of orientation can be determined precisely, while the aqueous environ­ ment of the sample is preserved. As illustrated in Fig. 5A, flat membranes (or disc-like particles) tend to align themselves with their plane perpendicular to the unidirectional compression. Rod-like particles also tend to align in the plane perpendicular to the compression, but otherwise they remain randomly oriented. Thus, for rod-like particles it is useful to apply a second direction of squeezing (Fig. 5B). With gel-compression, deformable membrane spheres (e.g. vesicles, chromatophores and chlo­

Garab

20

roplast ‘blebs’) can be deformed into ellipsoids and the intrinsic dichroism of the sample can thereby be made apparent and quantitatively evaluated (Kiss et al., 1985; Abdourakhmanov and Erokhin, 1980). Polyacrylamide gel is transparent in the entire visible and near-IR spectral regions and with the admixture of glycerol can be used for low temper­ ature measurements. Increasing the concentra­ tion of acrylamide and increasing the ratio of bis­ acrylamide to acrylamide renders the gel more rigid, with a smaller mesh size. Rigid and soft gels are suitable for pigment-protein complexes and membranes, respectively (A.O. Ganago, per­ sonal communication). For many applications, the components of the gel do not perturb the functions of the embedded particles (Ganago et al., 1982; Vermeglio et al., 1990; Breton and Ikegami, 1989). However, this was not the case for the oxidation state of P700 (Breton and Ikegami, 1989), and for the electric properties of chloroplast thylakoid membranes (Osváth et al., 1994). Acrylamide also diminished the big CD of chloroplasts. The fact that poly ac­ rylamide significantly reduces the intensity of light scattering (Haworth et al., 1982) may be indicative of a partial disintegration of the sample due to incorporation of the gel into the mem­ brane. This can probably account for obser­ vations that in heliobacterial membranes BChl g was bleached (van Dorssen et al., 1985) and arti­ facts appeared in complexes of fucoxanthin-containing algae (Hiller and Breton, 1992). 2. Orientation in a Magnetic Field The magnetically induced orientation of a photo­ synthetic system was first reported in Chlorella by Geacintov et al. (1972). A major advantage of this method is that practically no restriction ap­ plies as concerns the composition of the medium, and the degree of alignment can be nearly 100%. However, as the method is based on the diamag­ netic susceptibility anisotropy of the sample, which is a collective property of the particle, both the shape and size of the particle may limit orientability in normally available fields of 1–2 T. (For a detailed analysis of the mechanism of orien­ tation see Knox and Davidovich, 1978; Papp and Meszéna, 1982.) In practice, this method is lim-

ited to granal chloroplasts and large aggregates of LHCII (Kiss et al., 1986), which are aligned with their membrane planes and sheets perpen­ dicular to the field vector (Geacintov et al., 1972; Breton et al., 1973; Garab et al., 1981; Kiss et al., 1986). Orientation in a magnetic field can be trapped at low temperature (Vermeglio et al., 1976) or in a gel (Finzi et al., 1989). 3. Orientation in an Electric Field An electric dipole placed in a unidirectional elec­ tric field orientates so as to minimize the total energy of the system (Charney, 1988). The mea­ surements are usually restricted to low ionic strength. The electrophoretic movement of part­ icles can be prevented by using alternating volt­ age or pulses. It has been shown that the fre­ quency and the magnitude of the voltage may influence not only the degree, but also the me­ chanism of orientation through permanent and induced dipole moments (Gagliano et al., 1977; Charney, 1988). Chloroplasts can be oriented in while an a.c. (50 Hz) field of about small particles, e.g. chromatophores or isolated complexes, can be easily oriented by millisecond A quasielectric pulses of steady-state alignment of particles can be trapped in a gel, and thus the orientation can be studied in the absence of an external electric field (Dér et al., 1986). 4. Photoselection The technique of photoselection is based on the selective excitation of molecules by linearly polar­ ized light, which induces anisotropy in the sam­ ple. This occurs alike in intrinsically isotropic samples (e.g. solutions of molecules) and in samples containing randomly oriented intrinsi­ cally anisotropic particles (e.g. a membrane susp­ ension). The anisotropy function, r, for ab­ sorbance difference is defined as

where the indices refer to the orientation of the polarization direction of the probe light with re­ spect to that of the excitation and the iso­

LD and CD

tropic absorbance change. The angle between the sensitizing molecular absorbance dipole and the detected absorbance or emission dipole can be calculated by measuring the absorbance or emis­ sion of the detected chromophore with polariz­ ation directions parallel and perpendicular to the actinic polarization (Breton and Vermeglio, 1982). This technique has become increasingly important in determination of the mutual orien­ tation of the dipoles in the reaction center pre­ parations containing small number of Chl mol­ ecules (Kwa et al., 1994). The method of photoselection is also ideally suited to the moni­ toring of energy transfer processes (van Amerongen and Struve, 1995).

D. Determination of the Orientation Angle of Dipoles in Realistic Systems In idealized systems, which were considered in the examples above, it is assumed that the degree of orientation is 100%, that the membranes are flat, that the rod- or disc-like particles or mem­ branes cannot be deformed, that there is no fluc­ tuation in the orientation angles and that both LD and absorbance can be measured with high precision. With the exception of the last con­ ditions, in realistic systems these assumptions do not hold and the conclusions from idealized sys­ tems remain qualitative. (Although LD and ab­ sorbance can be measured with high precision, when they are measured in separate instruments, minor wavelength shifts may introduce quite large distortions in S. Smaller but significant distortions may be caused if LD exhibits too large ampli­ and use tudes. Most dichrographs measure the approximation of which is not true if 1. Degree of Orientation, Distribution Functions In some systems, a large proportion of the part­ icles are found to be perfectly aligned whereas the complementary population remains at ran­ dom, thus the degree of orientation can easily be defined. For instance in an external magnetic field of about 1 T, the orientation is saturated for whole chloroplasts, whereas chloroplasts frag­ ments can not be oriented even at much higher

21

field strengths (Garab et al., 1981). In other sys­ tems, e.g. with gel-squeezing or film-stretching techniques, the random suspension is gradually shifted toward the ‘perfect’ alignment, but satur­ ation cannot be attained for finite values of the deformation parameters. These systems are characterized by distribution functions which characterize the alignment of the particles. The distribution functions for rod-shaped and disc-shaped particles can be calculated on the basis of the behavior of rigid particles in the squeezed gel. It is envisioned that rigid rods in an amorphous, uniform, continuum matrix, rotate in such a way that the ratio of the projections of their long axis changes identically to the ratio of the corresponding sample dimensions. A similar correlation is applied for the plane of discs (Ganago and Fock, 1981). For disc-shaped particles after unidirectional compression:

and for rod-shaped particles after two-directional compression:

In both cases:

where M (>1) is the compression parameter (or often called squeezing parameter) (see Fig. 5). It is easy to show that if and if and thus and vanish, and reduce to the idealized cases, respectively. (For the dis­ tribution function and calculations for the unfav­ orable cases, i.e. rod-shaped particles with unidi­ rectional compression and disc-shaped particles with two-directional compression, see Ganago and Fock, 1981.) Fig. 6 shows and as a function of the orientation angle for the idealized and real­ istic cases. In our laboratory S values in chloro­ plasts with M = 2, typically increase from – 0.01 to about + 0.23 between 650 and 690 nm; FP

22

values are found between 1.1. and 1.7 (Szitó et al., 1984, 1985). It is assumed that no friction occurs between the particle and the gel, and there is no deforma­ tion of the shape. Since, however, distortions may occur it is advisable to carry out the experiments with different known values of the squeezing par­ ameter, with cells of defined dimensions and ex­ trapolate the value of the orientation angle to the non-squeezed case, and/or carry out the calcu­ lations with different presumptions (Kiss et al., 1985). In practice, distortions are small if (Ganago et al., 1983; Kiss et al., 1985). A properly prepared sample must be homo­ genous, and cracks, which may occur during too fast cooling or too large squeezing, for instance, should be avoided. Due to the compression, strains may be induced in the cell wall, which can introduce artifacts. For a homogenous sample without cracks and strains, the color pattern be­ tween two crossed polarizers is bright and uni­ form. Although the technique of gel-squeezing has been shown to yield reliable data on the orien­ tation angles, it is difficult to prove that the basic assumptions are correct for the general case. Further complications may arise if the shape of the particle has mixed character. Thus, for a

Garab

quantitative analysis the best strategem is to apply independent orientation techniques (see e.g. van Amerongen et al., 1988). 2. Membrane Curvature, Microscopic LD Membrane curvature can be taken into account by means of simple geometrical models. For chlo­ roplasts, such model calculations led to the con­ clusion that the long-wavelength emitting di­ poles of Chl a lie essentially in the plane of the dipoles of membranes. It was also shown that Chl a span a much larger angular interval than previously thought (Garab et al., 1981). (For the estimated range of the orientation angle, see Fig. 6 and data above.) The importance of structural factors is evident in differential polarization images of chloroplasts (Finzi et al., 1989; Garab et al., 1991a). As can be seen in Fig. 7, the magnitude of the local LD depends strongly on the curvature of membranes, and the macroscopic LD evidently represents only an averaged value. LD microscopy likewise revealed that, due to the curvature of membranes, the local LD does not vanish even in face-aligned chloroplasts, de­ spite the fact that LD = 0 in a suspension and in the plane of the image (Finzi et al., 1989). The

LD and CD

fact that residual polarization due to intrinsic ani­ sotropy (Breton et al., 1973) may contribute sig­ nificantly to the degree of polarization of fluor­ escence (see II.B.3) points to the need for microscopic fluorescence polarization investi­ gations, possibly in combination with fluor­ escence lifetime imaging (Gadella et al., 1993). Such techniques would probably permit esti­ mation of the magnitude of local order in native membranes and/or in lamellar aggregates of pur­ ified complexes, provided the local order is longranged enough compared to the resolution. 3. Fluctuations The orientation of a transition dipole may fluctu­ ate in a certain angular interval. The fluctuation of the pigment dipoles can originate from (i) the pigment-protein complexes and/or (ii) the fluctu­ ation of the protein axes in the membrane. In both cases the fluctuation can be either dynamic or statistical, i.e. it can originate from a rocking type of motion or is due to statistical disorder. If the long axis of the protein is embedded in the membrane at an angle with respect to the membrane normal, and there is a fluctuation of the orientation of the protein axis in the interval between and but fluctuation of the orien­ tation angle of the dipole with respect to the protein axis is not permitted, we obtain:

23

where,

denotes averaging. This shows that spectral variations of S for the same set of dipoles can be equally ascribed to the variation in the orientation of the protein axis with respect to the membrane plane and the orientation of the dipole with respect to the pro­ tein axis. It may be speculated that major struc­ tural rearrangements in the antenna are ac­ companied by reorientation of some complexes. Such changes may be responsible for the observed LD changes due to state transitions in cyanobac­ teria (Homer-Dixon et al., 1994). Data obtained on algal mutants and on chloro­ plasts treated with linolenic acid suggested the importance of fluctuations due to the increased fluidity of membranes (Szitó et al., 1984, 1985). On the other hand, large fluctuations were not encountered in untreated wild-type chloroplasts.

E. Miscellaneous Applications of LD Besides the two basic uses of LD spectroscopy outlined above (II.A and B), LD measurements can yield further structural information on the system.

Garab

24

1. Estimation of Shape and Size of Particles

3. Information on the Band Structure

Uni- and bidirectional compressions of a gel re­ sult in a better alignment for disc- and rod-shaped particles, respectively. Thus, if the shape of the (sufficiently rigid) particle is unknown, measure­ ment of the LD as a function of the squeezing parameters can lead to discrimination between disc-shaped and rod-shaped particles. This is called the reverse problem of LD (Ganago and Fock, 1981). From an analysis of the relaxation kinetics after electric or magnetic orientation, the size of the particle can be estimated (Geacintov et al., 1972; Kiss et al., 1986). In an electric field, the kinetics of the rise of the LD signal also carries information on the mechanism of the alignment (Charney, 1988), and therefore on the electric properties of the particle (e.g. contribution and orientation of the permanent dipole vector). Van Haeringen et al. (1994) determined the electric dipole moment of PSI trimers. (They also achieved the experimental separation of LB from LD.) Via the magnetic orientability, the size of the particle and the relative order inside the mac­ rostructure can be estimated (Barzda et al., 1994).

For fully allowed, intense electronic transitions, the polarization is generally constant across an isolated absorbance or fluorescence band. (For cases involving the admixture of vibronic trans­ itions, however, see Nordén et al., 1992.) Thus, the measurements can be used to resolve overlap­ ping bands (Garab and Breton, 1976; Kramer and Amesz, 1982; Mimuro et al., 1990). Essentially the same concept was applied recently in the deconvolution of LD spectra (Zucchelli et al., 1994). This analysis showed that all of the major absorbance forms of Chl a display considerable orientational homogeneity across the band. Hemelrijk et al. (1992) used absorbance, LD and CD spectra to identify different spectral forms of Chl a and b in LHCII. A similar analysis was performed by Matsuura et al. (1993) in chloro­ somes. The knowledge of band-structure is also necessary for the determination of the orientation angles of different absorbance and fluorescence transition dipoles (e.g. Garab et al., 1981; van Amerongen et al., 1994).

III. Circular Dichroism

2. Spatial Position of Organelles

CD is the differential absorbance of left and right circularly polarized light:

If the anisotropy of the transition dipoles of a complex system is well characterized, e.g. that of a membrane or a whole chloroplast, information can be obtained on the spatial position of the system, e.g. on the position of a chloroplast in a cell, and the light-induced orientation of chloro­ plasts inside the cell can be monitored (Tlalka and Gabrys, 1993). Polarization microscopy played a special role in the early studies of the optical anisotropy of chloroplasts (reviewed by Breton and Vermeglio (1982)). With the advance of dif­ ferential polarization imaging techniques (Kim et al., 1987a) and laser scanning microscopy (Shot­ ton and White, 1989), a more refined use of polar­ ization microscopy seems possible. The fact that chloroplasts can be sliced optically during the re­ cording of LD images (Garab et al., 1991a) opens up the possibility for 3–dimensional reconstruc­ tion of the thylakoid membrane system through the use of LD.

CD arises from the intra- or intermolecular asym­ metry (helicity) of the molecular structure. The helicity (chirality) of the structure means that it cannot be superimposed on its mirror image; this property is also often called handedness. This lack of symmetry, which arises, for example, if a carbon atom in the molecule is bonded to four different residues, is the property of nearly all organic molecules synthesized in biology. As the handedness of a molecule is the same from any direction, the selective absorbance of left and right circularly polarized light can be observed in a randomly oriented sample. (For oriented sys­ tems, see below.) Although CD is the most commonly deter­ mined chiroptical quantity, it is necessary to recall the correlations between CD and the other mani­ festations of optical activity: ellipticity, ORD and CB.

LD and CD

25

Absorbance by an optically active sample in­ duces ellipticity in the linearly polarized measur­ ing beam. This can be understood if it is taken into account that linearly polarized light can be decomposed into two oppositely rotating circu­ larly polarized light components of equal ampli­ tudes (see I.A). After the selective absorbance of one of the components, the two intensities do and thus the beam not remain equal will become elliptically polarized. It is obvious that CD and ellipticity are equivalent quantities. Although modern home-built or commercially available dichrographs measure absorbance dif­ ferences, it is common practice to express CD in units of ellipticity, millidegrees (m°, mdeg; absorbance unit). (It is interesting to note that the method elaborated for the measure­ ment of time-resolved CD with nanosecond resol­ (Lewis ution is based on ellipsometry and not et al., 1992).) In ORD measurements, the rotation of the orientation of linearly polarized light is measured as a function of wavelength. In an optically active material, and the electric vectors, rotate at different speeds This results in a net rotation in the direction of of the linearly polarized measuring beam. (For a weakly absorbing sample, the elliptically polarized light is considered to be linearly polarized along the major axis of the ellipse.) The optical rotation of a sample can be measured at any wavelength, i.e. also outside the absorbance bands. This is the main advantage of ORD over CD. The relation between CD and ORD is given by the Krönig–Kramers transforms (see Born and Wolf, 1980). Thus, the information from CD and ORD is redundant. The optical activity of a chiral molecule for each electronic transition is characterized by the rotational (or rotatory) strength of the transition. This is analogous to the dipole strength (see I.B) and is measured via the area under the CD band. As concerns the physical meaning of rotational it must be stressed that this does strength of not depend solely on the electric dipole the transition, but also on the magnetic dipole (m): (19)

The magnetic transition dipole is a purely imagi­ nary vector. In the Rosenfeld equation (Eq. 19),

Im indicates that the rotational strength corre­ sponds to the imaginary part of the scalar prod­ uct, and thus it is a real number. It is evident that, for a molecule to be optically active, both and m must be non-zero, and m must have a For these to occur, the component parallel to molecule must have a non-zero absorbance and be asymmetric. To explain this latter condition, it may be recalled that the electric dipole transition moment corresponds to a linear oscillatory mo­ tion of charge induced by the electric field of light (see section I.B), whereas the magnetic transition dipole can be regarded as a light-induced current loop. In asymmetric molecules, light induces a circular motion about the direction of which corresponds to a helical displacement of charge. In molecules that contain a plane or center of symmetry, rotational strength vanishes. (E.g. for a ring, m is perpendicular to the plane of the is in the plane.) This explains the ring, while correlation between the magnitude of the CD and the helicity of molecules, which facilitates the helical flow of charges (Woody, 1985; Charney, 1979).

A. Physical Origins of CD Signals (i) In the basic case, CD arises from intrinsic asymmetry or the asymmetric perturbation of a molecule (Woody, 1985). For a single electronic transition, CD has the same band shape as the absorption, and its sign is determined by the handedness of the molecule (positive or negative Cotton effect). (ii) In molecular complexes or small aggregates, CD is generally induced by short-range, excitonic coupling between chrom­ ophores (Tinoco, 1962; DeVoe, 1965). Excitonic interactions give rise to a conservative band struc­ ture (i.e. the positive and negative bands of the split spectrum, plotted on an energy scale, are represented with equal areas). (iii) In complex systems, such as DNA aggregates, condensed chromatins, viruses, etc., very intense CD signals have been observed, with non-conservative, anomalously shaped bands accompanied by long tails outside the absorbance. The CD signals of these samples have been shown to originate from the differential absorbance and differ­ of left and right circu­ ential scattering larly polarized light:

Garab

26

Systematic studies have revealed that these sig­ nals are associated with the macro-organization of the system; they provide valid and unique structural information about large chiral objects and carry information on the long-range chiral organization of chromophores (Keller and Busta­ mante, 1986a,b; Tinoco et al., 1987). CD signals (i)–(iii) originate from different levels of structural complexity. These different types of CD will be treated in somewhat more detail in the following section. Two other types of CD signals must be added to complete the list of CD due to different physical origins. These latter CD signals can be combined with any level of structural complexity of the sample. (iv) If a chiral molecule (or complex) is lumi­ nescent, the emitted light will be partly circularly polarized (Steinberg, 1978). CPL provides a tool for studies of the chirality of the excited state. CPL data on photosynthesis are scarce. Gafni et al. (1975) conducted comparative studies of CD and CPL on Chl dimers in solution, subchloro­ plast particles and chloroplasts, and demon­ strated large differences in both magnitude and sign. Additionally, it was concluded that in chlo­ roplast the emission anisotropy did not depend on the fluorescence yield, which can probably be interpreted as an indication that CPL reflects mainly the organization of the antenna rather than that of the reaction center. Since the sensi­ tivity of CPL to scattering and sieve effects (Duy­ sens, 1956) is different from that of CD, CPL may be a complementary tool in the elucidation of the macro-organization of chromophores in complex systems. CPL should not be confused with FDCD (Tin­ oco et al., 1987). FDCD detects the difference between the fluorescence intensities due to left and right circularly polarized light. FDCD can also be used to separate CDS from CD. In photo­ synthetic systems, complications may arise from the large number of fluorescing Chl molecules, and from the intense energy transfer among Chls. (This may explain the lack of data.) FDCD tech­ nique has recently been combined with lifetime measurement (Wu et al., 1993). (v) An external magnetic field parallel to a direction of propagation of light represents a per­ turbation that induces CD in chiral or achiral

samples. Since MCD arises via a different me­ chanism, it is superimposed on the ‘natural’ CD (Sutherland and Holmquist, 1980). Its magnitude is linearly proportional to the field strength. MCD has contributed significantly to the knowledge of the fine structure of porphyrins (Sutherland, 1978)). MCD is able to detect very weak trans­ itions; hence, certain choromophores can be used as markers. Some weak absorbance and CD bands, e.g. Chl a at 580 nm, exhibit very intense MCD. Fluorescence detection of MCD is also a commonly used tool, e.g. for the separation of MCD signals originating from fluorescing chrom­ ophores from the total MCD signal. In analogy to CPL, the magnetically induced chirality of the excited state can be detected as magnetic circular emission. In magnetically orientable systems, MCD and orientation-dependent CD can be combined et al., 1982; Garab et al., 1988a). In general, the CD of oriented systems requires special attention as concerns both the possible artifacts (Davidsson et al., 1980) and the interpre­ tation of data. CD in oriented systems contains additional information due to the fact that chir­ optical effects develop along different molecular and crystal axes in specific and different ways (Charney, 1979). For instance, the orientation dependence of excitonic bands can reveal infor­ mation on the symmetry of the excitonic aggre­ gate. In photosynthesis, the conclusion reached by Charney in 1979 still applies: “This field re­ mains ripe for application”. Vibrational CD (Keiderling et al., 1993) and Raman optical activity techniques (Barron, 1982) combine CD with vibrational methods.

B. CD of Photosynthetic Pigments Different levels of molecular organizations which give rise to different CD signals by different physical mechanisms can also be recognized in Chl-containing systems (Fig. 8). (i) In monomeric solutions, the intrinsic CD of Chls, with band shapes identical to those of the absorbance bands, is very weak (Dratz et al., 1966). (ii) In pigment-protein complexes, Chls typically exhibit CD with a conservative band structure, which arises from excitonic interactions (Pearlstein, 1982). (iii) Granal thylakoid mem­

LD and CD

27

ecular structure by Houssier and Sauer (1970). The intrinsic CD of most open-chain tetrapyrroles (Scheer, 1982) is much greater than that of Chls. Carotenoids are achiral in solution, but when bound to protein they display significant optical activity (Frank et al., 1989). In BChl-containing organisms, CD can be used for the investigation of carotenoid binding (Cogdell and Scheer, 1985). CD suggests asymmetry in the binding environ­ ment, but the participation of excitonic interac­ tions cannot be ruled out (Frank and Cogdell, 1993). 2. Excitonic Interactions

branes and macroaggregates of LHCII (Gregory et al., 1980; Garab et al., 1988a) are characterized by non-conservative CD signals with extremely large amplitudes and long scattering tails, attri­ buted to the long-range coupling of chromoph­ ores in chirally organized macrodomains (Garab et al., 1988c; Finzi et al., 1989). In a hierarchic system, CD signals of different physical origins are superimposed on each other. Thus, the observed CD spectra of Chl dimers, for example, always contain the spectra of the monomers; this causes some deviation from the conservative band structure, which can be cor­ rected by subtracting the intrinsic CD from the observed signal (for details, see Houssier and Sauer, 1970). Psi-type CD bands of chloroplasts and LHCII were shown not to interfere signifi­ cantly with the excitonic bands (Garab et al., 1988a; Garab et al., 1991b; Barzda et al., 1994). Thus, the CD of complex systems, at least in principle, can be deconvoluted to the component spectra of different physical origins. 1. Intrinsic CD of Isolated Molecules The CD spectra of several Chl and PChl pigments and their Pheos in diethyl ether were recorded and their origins explained in terms of the mol­

The coupling of two or more molecular dipoles with one another leads to shifts in absorbance bands and can generate CD. Let us consider two identical pigment molecules separated by a dis­ tance vector R. If the two molecules are brought close enough to each other to interact electron­ ically, but are still sufficiently apart for the elec­ trons to remain localized on each of the mol­ ecules, the absorbance band splits into two bands. The degree of separation of the bands depends on the interaction energy between and the two dipoles

where vector

is in debye, R is in nm; with the unit

This means that the interaction energy depends not only on the dipole strength of the mol­ ecules, but also on their distance and mutual orientation. It is to be noted here that the rate of the Förster-type of resonance energy transfer (Förster, 1965) is proportional to The prefer­ dipoles with entially in-plane orientation of respect to the membrane plane (as shown by LD) largely facilitates energy migration in directions parallel to the membrane plane (Garab et al., 1981). Energy transfer interactions are dealt with in a recent review by van Grondelle et al. (1994). The rotational strength (in units of DebyeBohr magnetons) for the excitonic CD of a dimer is given as:

28

where + and – designate the lower and higher energy transitions of the two exciton states, re­ is the band center energy, and the spectively, vectors in scalar triple products are unit vectors, It is clear that for the for the monomers, dimer is independent of i.e. the CD of the couplet is and conservative. In general, an additional term can originate from electric-magnetic coupling (Cantor and Schimmel, 1980). However, in most cases the contribution from this source, also with conserv­ ative band structure, is weak. The peak positions of the excitonic CD bands coincide with those of the split absorbance bands, as illustrated by the “stick” spectra (Fig. 9), which can be “dressed” by Gaussian bands (Scherer and Fischer, 1991). In complex systems, the absorbance may exhibit numerous sub-bands

Garab

due to environmental effects, which makes identi­ fication of the different bands very difficult. In contrast, CD selectively detects the excitonic in­ teractions on a weak background of the intrinsic CD of the pigment molecules, thereby offering a much better sensitivity than absorbance for iden­ tification of the the excitonic bands . On the other hand, in some geometries no excitonic CD signal appears, e.g. if the two dipoles are coplanar or if they are oriented parallel to one another. In many conformations of the dimer and favor­ able orientation angles of the dipoles with respect to the symmetry axis of the complex, the excitonic nature of a dimer can also be recognized in LD (Pearlstein, 1982). Thus, at least for excitonic interactions, the absorbance, CD and LD spectra are correlated with each other. For excitonic in­ teractions with known geometry it is possible to calculate these spectra, as in purple bacterial re­ action centers (Scherer and Fischer, 1991). In most applications, however, LD and CD spectra

LD and CD

are recorded with the aim of determining the spatial relationship and interactions among pig­ ment dipoles, respectively. It must also be real­ ized that in complex systems intense excitonic interactions may be confined to a relatively small cluster of pigment molecules. On the other hand, the majority of pigment dipoles are non-randomly oriented with respect to the symmetry axis (Bre­ ton and Vermeglio, 1982; Garab et al., 1987). The characteristic CD spectra of different part­ icles and isolated complexes have been studied by many authors. The experimental results and their interpretation have been reviewed (Pearlstein, 1987, 1991). The brief summary below serves to illustrate most typical applications. Although the six pigments in the purple bac­ terial reaction center are in close contact, the CD of the complex is dominated by the signal of the special pair, and the remaining BChl and BPheo molecules exhibit less intense bands. As discussed in depth by Pearlstein (1991), since the diameter of the macrocycles is larger than the center to center distance of the two molecules, the point dipole approximation is not satisfactory. Further corrections are necessary if the Soret dipoles in­ dipole of a nearby molecule teract with the (Scherz and Parson, 1986) or if a charge transfer complex is formed (Parson and Warshel, 1987). Picosecond CD transients also indicated the role of non-excitonic CD contributions (Xie and Simon, 1991). Model systems, such as BChl aggregates in micelles, permit characterization of both the exci­ tonic and non-excitonic CD bands, which are similar to those in the reaction center (Scherz, 1992). The CD spectra of in vitro PChl aggregates also display many similarities to those in native systems (Böddi and Shioi, 1990). The pigment organization in the reaction cen­ ter of a green bacterium, Chloroflexus aur­ antiacus, was analyzed by using exciton theory and the structure of Rps. viridis (Deisenhofer et al., 1984). The data suggested that the arrange­ ment of the chromophores is very similar to that in purple bacteria, which explains the functional similarity of the two reaction centers (Vasmel et al., 1986). The contribution of P680 to the CD of the PSII reaction center D1-D2-cyt b559 was investigated by Braun et al. (1990): the split (+)679 nm and (–)669 nm bands were interpre­

29

ted as indicating a loosely coupled dimer of Chl a, in which the interaction is much weaker than in the purple bacterial reaction centers (cf. also van der Vos et al., 1992). In an exciton-coupled aggregate, many interac­ tions occur, which affect the absorbance, CD and LD of the aggregate (Pearlstein, 1991). The water-soluble BChl a-containing complex of the green photosynthetic bacterium, Prosthecocloris aestuarii, the Fenna–Matthews–Olson (FMO) complex was the first photosynthetic pigmentprotein complex to be structurally characterized by atomic resolution crystallography (Fenna and Matthews, 1975) and is one of the best-studied complex in photosynthesis. However, interpreta­ tion of the absorbance and CD of the FMO com­ plex turned out to be more difficult than antici­ pated, and in fact CD has been explained satisfactorily (Lu and Pearlstein, 1993). In the simulation, the distinct interaction of each of the seven BChl molecules with protein moieties, and the trimeric nature of the complex had to be taken into account (Pearlstein, 1992). Lu and Pe­ arlstein (1993) found that some cryosolvents per­ turb the structure. Zhou et al. (1994) have re­ ported the occurrence of redox sensitive structural changes in the FMO complex which could be detected by CD. For LHCII, in which the structure is known at 3.4 Å resolution (Kühlbrandt et al., 1994) an exact interpretation of the CD data has not been presented. The characteristic spectra of CP2 and LHCII were interpreted in terms of a Chl b trimer (van Metter, 1977; Gülen and Knox, 1984). How­ ever, it was later concluded (Hemelrijk et al., 1992) that the CD signal probably originates from an array of pigment molecules in which not only Chl b-Chl b, but also Chl b-Chl a interactions play an important role. For intramembrane purple bacterial com­ plexes, the lack of high-resolution structure ham­ pered analysis of the CD spectra in terms of exact structural parameters. Analyses of the CD spec­ tra and the amino acid sequences led to the con­ struction of models which satisfactorily described the structure and function of these antenna com­ plexes (Scherz and Parson, 1986; Zuber and Bru­ nischolz, 1991; Visshers et al., 1991; see also Pearlstein, 1992). The crystal structure of the B800-850 antenna complex from Rps. acidophila

30

has been determined to a resolution of 2.5 Å by McDermott et al. (1995). This opened up the possibility to use CD toward the understanding of the excitation energy transfer processes. In heliobacteria, several BChl g forms have been identified which transfer energy efficiently to the longest wavelength (808 nm) form, but CD indicates that only a relatively small number of pigments participate in excitonic interactions (van Dorssen et al., 1985), which emphasizes the im­ portance of pigment clusters in bacterial antenna complexes. CD spectra have been recorded for most subchloroplast particles and complexes (Bassi et al., 1985), but most of them have not been ana­ lyzed in terms of exciton theory. These CD spec­ tra are clearly dominated by different excitonic bands characteristic of the different pigment-protein complexes, suggesting that CD can be used for the “fingerprinting” of complexes (for an overview, see Garab et al., 1987). The Chl b-containing antenna of Prochlorotix hollandica has been shown not to contain the characteristic CD bands of LHCII, which indicates that this com­ plex is not closely related to LHCII (Matthijs et al., 1989). The similarity of the CD spectra of the antenna Chls in the native membrane and the trimeric form of the isolated PSI reaction center complex suggests that trimeric PSI pre-exists in the membrane (Shubin et al., 1993). In all the above systems, proteins provide the binding sites, which in turn ensure the appropri­ ate distances and mutual orientations of the Chl molecules. In chlorosomes, BChl c oligomers ap­ pear to be the main building blocks, thus investi­ gations of model systems of large Chl aggregates are of great interest (Scherz et al., 1991; Gottstein et al., 1993). Studies of macroaggregates of PChl also revealed many similarities to some in vivo systems (Böddi and Láng, 1984; Sundqvist et al., 1980). In chlorosomes and in macroaggregates of Chls and PChl (i) the CD is sensitive to the conditions of preparation, (ii) the size of the ag­ gregates is commensurate with the wavelength of visible light, and (iii) the pigment molecules ap­ pear to be assembled with long-range order. Thus, the CD signals may originate in part from the long-range chiral order of the pigment mol­ ecules, as suggested by Lehmann et al. (1994) who observed a giant CD signal in chlorosomes

Garab treated with protease. Identification of the origin of the CD signals in these highly-organized mac­ rosystems and determination of the possible con­ tribution of psi-type effects requires systematic investigations. 3. Differential Scattering, Psi-type CD, and Differential Polarization Imaging The theoretical framework for understanding the CD in large chiral assemblies has been extensively developed over the past two decades. Preferential scattering of one of the circular polarizations of the light by chiral samples has been interpreted within the framework of the CIDS theory (Busta­ mante et al., 1985). The theory for psi-type aggre­ gates describes the interaction of light with large inhomogenous molecular aggregates containing a high density of intensely interacting chromo­ phores (Keller and Bustamante, 1986a,b). The theory of imaging macrodomains that have differ­ ent optical properties and different molecular structures has been elaborated for CD (Keller et al., 1985) and extended to all transmisson or scattering Mueller images (Kim et al., 1987a,b). CIDS results from interference effects of wave­ lets generated at different points in the object in which the point polarizable groups are helically arranged, and the pitch and the diameter of the helix are commensurate with the wavelength of the measuring light. Since the wave­ lets maintain a well-defined polarization and phase relationships to each other, the interfer­ ence phenomenon is greatest when the wave­ length of the circularly polarized light closely matches the dimensions of the macrohelix. In the first Born approximation, each dipole is con­ sidered independent of the others. The interac­ tion of subunits can be taken into account in higher Born approximations (Tinoco et al., 1987). Theory predicts that CIDS as a function of scattering angle:

exhibit lobes of alternating sign (Bustamante et al., 1985), the profile of which is determined by the helical parameters of the chiral macrostruc­ ture. It must be stressed that the angle-depen-

LD and CD

dence of the non-polarized scattering is not re­ lated to the profile of CIDS. Non-polarized scattering carries information on the shape and size of the particle rather than on the organization of chromophores inside the scattering particle. Nevertheless, both non-polarized and differential polarization scattering signals, especially when measured as a function of the angle of obser­ vation, carry useful information on the macro­ structural parameters of large objects. The psi-type CD theory (Keller and Bustam­ ante, 1986a,b) is based on the classical theory of coupled oscillators (DeVoe, 1965). The theory of DeVoe considers that light induces oscillating (transition) dipoles in the polarizable groups of the object, and the induced dipoles interact as static dipoles, with a distance-dependence of However, in large objects, it is necessary to con­ sider not only these short-range interactions but also long-range effects. In psi-type aggregates the full electrodynamic interaction between the di­ poles must be taken into account (Keller and Bustamante, 1986a). In small aggregates, the en­ tire aggregate at any instant is in the same phase of the wave upon the interaction with the light. In contrast, in large aggregates which are com­ mensurate with the wavelength this is not true and retardation effects can play an important role. At distant points of observation the oscillat­ ing dipole can be regarded as a radiating spherical wave. Thus, the chromophores at large distances can be coupled via a radiation coupling mechan­ ism between the dipoles. In large chirally or­ ganized aggregates the significance of the radi­ ation coupling, which is essentially due to multiple scattering inside the particle, can be comparable to that of the static dipole coupling. The electric field at any point in space x, due to an oscillating electric dipole, located at can be written as: Furthermore,

which means that the electric field at any point is the superposition of the incident electric field and the sum of the fields produced by all oscillating dipoles. This shows that for the general case the quantity of interest in understanding the

31

CD (and other optical properties) of large aggre­ gates is not the coupling between individual pairs of chromophores, but the coupling between any given chromophore and the rest of the chromo­ phores in the macrodomain. The interaction tensor has been given in an explicit form (Keller and Bustamante, 1986a):

where and The first and third terms of the tensor, with dependences, describe the static dipole and coupling and the radiation term, respectively. The second term, with is called the interme­ diate coupling. The final term ensures that the self-interaction is zero. Due to static, intermediate and radiation coup­ ling between dipoles, intense “anomalous” CD signals are generated (Keller and Bustamante, 1986b). The magnitude of the signal is controlled by the volume, chromophore density and pitch of the helically organized macrodomain (Kim et al., 1986). Further, the shape of the spectra depends mostly on the pitch and the handedness, with sign-inverted mirror spectra for opposite hand­ edness. In psi-type aggregates, the theoretical prediction is that if the long-range coupling be­ tween the dipoles is strong, the excitation gen­ erated at one chromophore can delocalize for the entire aggregate. This is called the collective ab­ sorbance, which increases or decreases at a given wavelength depending on how well the light is able to produce a collective excitation in the sys­ tem. When the group polarizabilities are made weaker, the groups are moved farther apart or the density of the chromophores inside the aggre­ gate is diminished, the ability of an excitation created at a given position in the aggregate to transfer to a different part becomes less and less efficient. Finally, for the case of a small system the theory of psi-type CD (Keller and Bustam­ ante, 1986a,b) reduces to the classical theory of DeVoe (1965), which describes excitonic interac­ tions. Since psi-type aggregates also satisfy the crite­ ria for CIDS, psi-type CD is always accompanied

32

by CIDS. In CIDS alone, the static and interme­ diate coupling fields become insignificant and can be omitted from Differential polarization imaging (DPI) is a method suitable for structural investigations of large anisotropic objects. It can provide infor­ mation on the macro-organization of large mol­ ecular structures, and resolve microscopic do­ mains of distinct optical anisotropy. In DPI, the image is composed of points carrying information on a differential polarization quantity such as CD or LD or other elements of the Mueller matrix (Kim et al., 1987a,b; Kim and Bustamante, 1991). Philipson and Sauer (1973) recognized that CDS contributes to the CD of chloroplasts. Later, it was shown that scattering does not significantly distort the “true” CD bands in chloroplasts and LHCII macroaggregates, but is superimposed on the excitonic CD signals and carries distinct physi­ cal information on the macro-organization of pig­ ments (Garab et al., 1988a,c). A comparison of non-polarized scattering with the anomalous CD in thylakoid membranes subjected to different ionic strengths and osmotic pressure revealed that while CD selectively responds to structural re­ arrangements of the pigment-protein complexes, non-polarized scattering yields far less specific in­ formation (Garab et al., 1991b). Further, intense light scattering per se, e.g. in a suspension of thylakoid membranes does not noticeably affect the excitonic CD bands. of chloroplast thylakoid membranes was measured in a set-up involving an Ar-ion laser, a Pockel’s cell and goniometric detection of the scattered light. It was found that chloroplasts exhibited four CD lobes with alternating signs, which was attributed to a left-handed helix with an estimated pitch and radius of 200–400 nm (Garab et al., 1988c). The helically organized macrodomains were imaged at different wavelengths in a confocal CD microscope (Finzi et al., 1989, 1991). The images displayed huge signals emerging from discrete “is­ lands” of the chloroplasts; the diameter of which could be estimated to be between 0.3 and 0.6 Local CD spectra recorded in different pixels of individual chloroplasts exhibited broad positive and negative bands from different domains (Finzi

Garab et al., 1989). Microscopic data ruled out the inter­ pretation of the anomalous CD of chloroplast suspensions in terms of short-range interactions and lent support to the notion that the main bands originate from helically organized macrodomains of the pigment system. The chirally organized macrodomains in chloroplasts have also been shown to undergo gross (up to 80–90%) lightinduced reversible structural changes which can be detected in the major “anomalous” CD bands (Garab et al., 1988b). The significance of these structural changes is not understood exactly, but these changes are likely to be correlated with the energy dependent non-photochemical quenching which is capable to dissipate excess exci­ tation energy in the antenna (see e.g., Horton et al., 1991; Istokovics et al., 1992). In granal chloroplasts and LHCII macroaggre­ gates, the density of chromophores is high, the dipoles interact intensely with each other, and the complexes are assembled in large 3–dimensional aggregates (Barzda et al., 1994). Hence, if asym­ metry is introduced, e.g. during macroaggreg­ ation, these systems satisfy the conditions for psitype aggregates. Barzda et al. (1994) showed that, in accordance with the prediction of psi-type the­ ory, the magnitude of the major CD bands of LHCII and chloroplasts increased with the size of the macroaggregates. A similar correlation was found for the B880 antenna complex of Rps. mar­ ina (R. Meckenstock, G. Garab, R. A. Brunish­ olz and H. Zuber, unpublished.) With LHCII and B880 the size of the aggregate can be varied in a broad range. These systems offer the convenience that the measurements can be carried out in the visible and near-IR spectral regions. Furthermore, our knowledge concerning the structure of the pigment system is usually more advanced than that on the chromophores of most non-photosynthetic psi-type aggregates. Thus, photosynthetic systems appear to be ideally suited for systematic studies of psi-type CD. Without systematic studies under well defined ex­ perimental conditions and on systems permitting realistic model calculations quantitative interpre­ tation of psi-type spectra does not seem possible. It would be equally important to understand the functional significance of the psi-type organiza­ tion of the pigment system in granal chloroplasts.

LD and CD

C. Secondary Structure of Chl-containing Proteins It has been established that the CD of proteins is sensitive to the secondary structure. The signal observed in the far-UV spectral range is primarily determined by the spatial arrangement of the amide chromophores, and therefore the CD re­ flects the backbone conformation of the polypep­ tides. The amide chromophore has plane symme­ try and is itself not optically active. In a peptide, a chiral electrostatic field can be provided by the surrounding amides and other polar groups; exci­ ton interactions between chromophores also con­ tribute significantly to the CD signal (Woody, 1985; Johnson, 1990). Some of the interactions can be specifically assigned to certain conformations (Woody, 1985). conformations are characterized by The the 222 nm negative band and an excitonic coup­ let at (–)208 nm/(+)192 nm. The spectrum of the also depends on the length of the chain. are usually characterized by a (–)216 nm band and a much stronger positive band between 195 and 200 nm. Different types of which play an important role in the folding of proteins, have been shown to occur in equilibrium (Perczel et al., 1991). These are usually characterized by negative bands at 220–230 nm and 180–190 nm, and a positive band between 200 and 210 nm. Aperiodic (random coil) proteins have an unor­ dered structure and usually exhibit weak and highly variable CD spectra, often with a negative band at around 200 nm. In accordance with the principle of additivity of CD signals, the CD spectrum of a protein can be considered to be the weighted sum of the spectra of the secondary conformations. This is the basis for analysis of the spectra for prediction of the secondary structure of proteins. Semiem­ pirical methods for this analysis are based on dif­ ferent sets of CD spectra of proteins with known secondary structure (Woody, 1985; Johnson, 1990). The prediction methods, and especially those which use linear combinations of known structures and CD spectra and also apply statisti­ cal methods in order to account for variabilities (Provencher and Glöckner, 1981; Johnson, 1990), yield a highly reliable prediction of the

33

content, while those relating to and turns are less reliable (Johnson, 1990). This conclusion of Johnson (1990) has been confirmed by a syste­ matic analysis of UV-CD and Fourier transform infrared spectra (Hollósi et al., 1993; Pribic et al., 1993). Conformational analyses of the proteins for the content of purple bacterial reaction centers have yielded values comparable to those deduced from X-ray data (47–55% and 42%, re­ spectively) . Similar contents were found in other isolated complexes (Breton and Nabed­ with ryk, 1987).The orientation of the respect to the membrane normal was found to be less than 30°, as determined from IR LD mea­ surements (Nabedryk et al., 1984). Based on UV-CD measurements Paulsen et al. (1993) concluded that pigment binding plays an important role in the determination of the se­ condary structure of LHCII.

D. Artifacts In molecular solutions or small aggregates, most of the artifacts originate from the optical trail of the set-up and the cell, which contain residual strains that cannot be fully eliminated. These can be taken into account by subtracting the baseline measured with the same cell in the same orien­ tation and with a mimicked absorbance. In the presence of large particles, which “con­ centrate” the chromophores and are commensur­ ate with the wavelength of the measuring light, both the absorbance and the CD are affected by flattening and light scattering (Duysens, 1956; Bustamante and Maestre, 1988). The degree of flattening of the CD spectrum is twice as large as the flattening of the absorbance, and both are increasingly more significant toward shorter wavelengths (Bustamante and Maestre, 1988). However, in the presence of CDS contributions, the flattening effects can be more complex; cor­ rections can then be difficult and efforts must be concentrated first on correcting for CDS. In a conventional dichrograph, CD of ab­ sorbance and CDS are combined into the appar­ ent CD signal. CDS can most easily be recognized by varying the acceptance angle of the photomul­ tiplier, e.g. by changing the distance between the

34

sample and the photomultiplier (Philipson and Sauer, 1973). It is relatively simple to separate CDS from CD of absorbance. Outside the ab­ sorbance band, CD is produced by CDS and exhi­ bits a monotonously decreasing signal (“long tail”) on the long wavelength side of the ab­ sorbance band. CDS can also contribute to the CD signal inside the absorption band. (Inside the absorbance band, the anomaly can also originate from psi type CD, which is always accompanied by CDS,) As CDS can be intense in the forward direction, and as there is a ‘cross term’ between the absorbance and scattering (Bustamante and Maestre, 1988) simple corrections for scattering contributions do not provide reliable results. FDCD and photoacoustic techniques have been proposed for such corrections. FDCD, however, may not easily be adapted for the highly fluor­ escing photosynthetic systems, and to my knowl­ edge, photoacoustic CD measurements have not been attempted. In chloroplasts and LHCII macroaggregates, MCD of Chls, which originates from inside the complexes and is therefore subjected to the same alterations as natural CD, has been shown not to be distorted significantly by CDS (Garab et al., 1988a). In chloroplast suspensions, “regular” scattering (which does not discriminate between left and right circularly polarized light) does not distort the CD signal to any noticeable extent. This may be observed, for example, in turbid chloroplast suspensions in which the electrostatic conditions do not permit the formation of large aggregates (Garab et al., 1991b). CD is correlated with CB, and thus CB is often suspected to contribute to the CD signal. It has been demonstrated, however, that both in con­ ventional CD measurements and in ellipsometry CB in first order does not contribute to the signal (Björling et al., 1991; Lewis et al., 1992). LD and LB can easily cause artifacts. Since all photosynthetic membranes are intrinsically aniso­ tropic, the extent of interference to CD by LD and LB must always be investigated in oriented systems. The facts that LD is usually more than an order of magnitude stronger than CD, and that imperfections may occur in the optical trail, make CD sensitive to LD and LB. This problem of CD in oriented systems has been addressed by

Garab many authors (Davidsson et al., 1980; Shindo et al., 1985; Shindo, 1985). (In randomly oriented samples LD and LB can be induced by a linearly polarized excitation. Such excitation can cause artifacts in pico- and nanosecond CD transients.) Artifacts in CD due to LD and LB and their ‘cure’ were analyzed in detail by Björling et al. (1991) and Lewis et al. (1992). CD was found to be most sensitive to the coupling of LD with stray LB in the optical components before the sample. An artifact due to coupling between the LB of the optical system and the LD of the sample was reported by Francke et al. (1994), who observed spurious CD signals in intact cells of heliobac­ teria. The LD was caused by gravitational orien­ tation of the cells, whereas the LB originated mainly from the strain in the window of the cryo­ stat. The magnitude of the spurious signal in­ creased dramatically in response to lowering of the temperature, which suggested an additional (probably internal pressure-dependent) LB inside the sample. The effects of different contributions to the CD images from sources, such as LD, linear dif­ ferential scattering and LB were analyzed by Kim et al. (1987b). It was shown that most of these contributions can be separated on the basis of the symmetry behaviour of different Mueller matrix elements upon rotation of the optical compo­ nents. In particular, CD image artifacts due to imperfections in circular polarization can be elim­ inated by using the fact that the CD-related im­ or are invariant on the rotation ages whereas contributions from a linear anisotropic signal are sensitive to rotation. CD images of chloroplasts were tested for lin­ ear dichroic contributions in both face-aligned positions, (LD = 0) and edge-aligned and it was found that the main features of the CD images were unaffected. (The LD images changed sign upon 90° rotation of the sample or the photoelastic modulator (Finzi et al., 1989).) Similar experiments were performed on macro­ scopic samples with magnetically aligned chloro­ plasts trapped in gel. CD spectra were recorded in vertical position and at ± 45° and ± 90° with respect to the vertical direction. The contribution position) was found to be of LD to CD (in smaller than the CD signal (Garab et al., 1991a).

LD and CD

IV. Concluding Remarks The application of LD and CD spectroscopic techniques have provided a rich yield of structural information on the pigment systems in various photosynthetic complexes, membranes and or­ ganelles. For most applications, LD and CD have solid theoretical background and elaborated ex­ perimental procedures. Thus these techniques are suitable for routine applications in a wide range of studies. For special applications, however, further work is needed. In LD, earlier investigations concentrated mainly on qualitative conclusions and establishing the general rules of dipole orientations in vivo. Now, there appears a demand for a quantitative approach, and its use in monitoring the primary photophysical and photochemical processes. Con­ clusions on the orientation of the molecules inside Chl-containing complexes may, however, remain tentative because of some uncertainities in the polarization directions of the electronic trans­ itions of Chls. The importance of excitonic CD has been de­ monstrated thoroughly in almost all photosyn­ thetic complexes studied so far. As shown for the case of the FMO complex, CD contains infor­ mation on virtually all possible pigment–pigment interactions as well as the interactions of the pig­ ments with the protein moieties. This infor­ mation, with so much details, is clearly impossible to extract from the CD spectrum alone. For less characterized structural entities, the conclusion from CD may be confined to the identification of the most important excitonic interactions. This can be improved by analyzing LD, CD and ab­ sorbance spectra on the basis of the physical cor­ relations between the bands of excitonic origin, and also by using independently determined structural parameters. CD and LD studies can be extended to intact systems where unique structural information ap­ pears to be available on the macro-organization of the pigments. Signals originating from highly organized systems, however, may be combined with artifacts, which suggests a cautious ap­ proach. Our understanding of the CD features of highly organized objects with sizes commensurate with the wavelength is far from being complete

35

but progress is very likely, for these questions are in the focus of interest in many spectroscopic laboratories. It is somewhat surprising that only three out of sixteen elements of the Mueller matrix are determined routinely. In highly organized large objects which contain high density of interacting transition dipoles, and where these dipoles are distributed in a well defined anisotropic pattern, and the sample exhibits multilevel optical activi­ ties, these elements may provide insight into the molecular organization of macrodomains. Acknowledgements I thank Dr. H. van Amerongen and Prof. R. van Grondelle for critical reading of the manuscript and helpful suggestions. I am indebted to my coworkers, Virginijus Barzda and Tamás Jávorfi, for their help in the preparation of the figures. I am grateful to Profs. M. Hollósi and H. Scheer, and Dr. L. Zimányi for stimulating discussions. Thanks are due to Prof. R. Jennings and Dr. H. van Amerongen for providing their preprints. This work was supported by a grant from the Hungarian Research Fund, OTKA (IV/2999). References Abdourakhmanov I and Erokhin YE (1980) Linear dichroism of pigments associated with spherical chromatophores. Model of orientation in polyacrylamide gels. Mol Biol (USSR) 14: 539–548. (Russian edition). Abdourakhmanov I, Ganago AO, Erokhin YuE, Solov’ev A and Chugunov V (1979) Orientation and linear dichroism of the reaction centers from Rhodopseudomonas sphaero­ ides R-26. Biochim Biophys Acta 546: 183–186. Ade H and Hsiao B (1993) X-ray linear dichroism microscopy. Science 262: 1427–1429. Barron LD (1982) Molecular Light Scattering and Optical Activity. Cambridge Univ. Press, Cambridge. Barzda V, Mustárdy L and Garab G (1994) Size dependency of circular dichroism in macroaggregates of photosynthetic pigment-protein complexes. Biochemistry 33: 10837– 10841. Bassi R, Machold O and Simpson D (1985) Chlorophyllproteins of two photosystem I preparations from maize. Carlsberg Res Commun 50: 145–162. Bauman D and Wrobel D (1980) Dichroism and polarized fluorescence of chlorophyll a, chlorophyll c, and bacteri­ ochlorophyll a dissolved in liquid crystals. Biophys Chem 12: 83–91. Björling SC, Goldbeck RA, Milder SJ, Randall CE, Lewis

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Chapter 3 Fluorescence

Kenneth Sauer* and Martin Debreczeny1 Department of Chemistry and Structural Biology Division, Lawrence Berkele.y Laboratory, University of California, Berkeley, Ca 94720–1460, USA; 1 Chemistry Division, Argonne National Laboratory, Argonne, IL 60439, USA

Summary I. Introduction A. Electronic Excitation and Emission B. Excited State Formation, Relaxation and Decay II. Steady-State Fluorescence A. Fluorescence Spectrophotometer B. Excitation Spectra C. Emission Spectra D. Light Scattering Interference E. Polarization/Depolarization F. VariableFluorescence 1. Photochemical Trapping Competes with Fluorescence 2. Electric Fields Influence Fluorescence Associated with Photosynthetic Materials 3. Temperature Dependence of Fluorescence III. Time-Resolved Fluorescence A. Experimental Considerations 1. Time-Correlated Single Photon Counting (TCSPC) 2. Fluorescence Upconversion 3. Deconvolution 4. Exciton Annihilation B. Isotropic Time-Resolved Fluorescence C. Anisotropic Time-Resolved Fluorescence IV. Conclusion Acknowledgements References

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Summary

Fluorescence emission is a direct reflection of the properties of excited electronic states of molecules as they return radiatively to the ground electronic state. Fluorescence provides information about the (1) energy of the emitting state relative to the ground state, (2) lifetime of the excited state, (3) orientation of transition dipole moments and (4) symmetry properties of the ground and excited states. Fluorescence is especially valuable as a probe of photosynthetic systems, because it constitutes a sensitive competitive path to photochemical energy conversion, resulting in fluorescence quenching. Fluorescence spectra provide knowledge of the energy levels of different pigment pools in the light-harvesting antenna and reaction center complexes. Steady-state and time-resolved depolarization studies reflect the rapid excitation transfer processes that occur within these multi-pigment arrays in photosynthetic membranes. Using theoretical formulations, such as the Förster inductive resonance transfer mechanism, these *Correspondence: Fax: 1-510-4866059; E-mail: [email protected]

41

J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 41–61. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

42

Kenneth Sauer and Martin Debreczeny

transfer rates can be related directly to molecular geometries derived from X-ray crystallography and to fundamental spectroscopic properties of the molecules involved. The consequences of electric fields formed by primary charge separation across photosynthetic membranes can be seen in the influence of an applied electric field on the fluorescence intensity and relaxation kinetics. Much of our current knowledge of the primary processes of photosynthetic energy conversion has derived from fluorescence measurements. Abbreviations: Chl – chlorophyll; FWHM – full-width half-maximum; ic – internal conversion; IRF – instrument response function; is – intersystem crossing; LHC – light-harvesting complex; PC – phycocy­ anin; TCSPC – time-correlated single-photon counting

I. Introduction Fluorescence emission derives from excited elec­ tronic states of molecules. In photosynthesis the molecules of interest are associated with the antenna and the reaction centers. Fluorescence from chlorophylls, bacteriochlorophylls and phy­ cobiliproteins in whole organisms or in prepara­ tions of active membrane fragments or sub-complexes provides information about the roles of these molecules in primary photosynthetic energy conversion. Because the excited electronic states exist between the initial absorption of photons and the ultimate charge separation that completes the conversion of light energy into chemical en­ ergy, monitoring the fluorescence provides direct evidence of the mechanism and dynamics of the primary events in photosynthesis.

A. Electronic Excitation and Emission First we will look at the sequence of events in­ volved in the evolution of electronic excitation and relaxation (decay) that are monitored using fluorescence. We will begin with a summary of the “intrinsic” properties of the excited electronic states of an isolated pigment molecule, like chlorophyll or bacteriochlorophyll. Then we will examine the consequences of collecting chrom­ ophores in the pigment-protein complexes that are ubiquitous in photosynthetic membranes and their associated components. The resulting delo­ calization of the excitation is essential to the func­ tion of the antenna in collecting light. Finally, we consider the influence of the reaction centers or

photochemical traps that extract the excitation energy for conversion into chemical potential. The time scale of this sequence of events ranges from a few femtoseconds required for transforming a ground electronic state into an excited state, picoseconds for collecting the exci­ tation at the reaction centers, and picoseconds to nanoseconds in the reaction centers to accomplish the charge separation steps and occasionally the reversal of these steps to produce delayed fluor­ escence on a still longer time scale. We see from this scenario that the molecule whose fluor­ escence is detected may have been excited by photon absorption directly, may have received its excitation energy by transfer from other pigment molecules or it may be excited by the return of excitation from the traps. Each of these paths has a distinctive signature in the time dependence, wavelength dependence, depolarization, etc. of the fluorescence. In many cases the steps in the path can be elucidated using time-resolved mea­ surements; however, steady-state measurements, which average the time-dependent behavior, also provide useful guides to investigating and in­ terpreting the excited state relaxation. Both of these approaches will be explored in this chapter.

B. Excited State Formation, Relaxation and Decay The absorption of electromagnetic radiation by a molecule is properly described using quantum mechanics. Useful descriptions of how to charac­ terize this process are given in several mono­ graphs (Cantor and Schimmel, 1980; Lakowicz,

Fluorescence

1983; Struve, 1989). For large chromophoric molecules like the photosynthetic pigments in protein environments, the course of events is extremely complex, and it cannot be described precisely. It is useful, therefore, to separate the overall process into a series of stages or influences that can be considered separately. One such sequence is illustrated, in part, in Fig. 1. 1. The ground state G of the molecule that is

sampled by the incident radiation is a therm­

ally equilibrated ensemble of configurations.

2. The absorption spectrum of a molecule reflects

the energy (frequency) dependence of the

probability of achieving a particular excited

electronic state configuration, etc.

The photon energy must correspond to the difference in energy between the initial and

final states of the molecule, and the transition

dipole moment describes the quantum mech­

anical coupling between the ground and ex­

cited states.

43 3. Excited electronic states of photosynthetic pig­ ments invariably involve delocalized trons associated with the conjugated or aro­ matic bonding systems in chlorophylls, openchain tetrapyrroles or carotenoid polyenes. Because the mass of the electron is small com­ pared with the nuclear mass of the atoms in the molecule, the redistribution of the electron in the excited state orbital occurs rapidly in comparison with nuclear motion. Thus, the excited electronic state of the molecule is pro­ duced with a nuclear configuration that is in­ itially the same as that of the ground electronic state at the time of arrival of the photon (Franck–Condon Principle). 4. Inhomogeneous broadening of the absorption bands results from the large variety of micros­ tates that is present in the initial thermal distri­ bution of ground state configurations, together with the equally large variety of excited state configurations that can result from the absorp­ tion of photons of a particular energy by a large population of molecules. 5. Relaxation of the nuclear configuration in the excited electronic state is a consequence of the change in charge distribution produced by the transfer of the electron from the ground state orbital to the excited state orbital. This relax­ ation results from an exchange of energy among internal modes of motion of the chrom­ ophore, as well as interchange with other molecules in the surroundings – especially the protein matrix and other nearby chromophores. A component of this relaxation process, which is typically complete within a few picoseconds in photosynthetic pigments in condensed media, is the decay or transfer by internal con­ version, ic, from higher energy excited elec­ tronic states etc. to the lowest energy excited state having the same spin multiplic­ ity (typically singlet) as the ground state. 6. Thermal equilibration occurs as the excited state configurations attain the distribution cor­ responding to the temperature of the environ­ ment. This occurs typically within a few pico­ seconds of photon absorption and, except for molecules such as carotenoids with very short excited state lifetimes, is complete prior to most of the fluorescence emission. Where sig­

44 nificant emission occurs prior to excited state

thermalization, this is detectable using a com­

parison of absorption or excitation spectra

with fluorescence emission spectra using re­

lations derived by Stepanov (1957).

7. Several distinct fates are possible for the thermally relaxed excited electronic state. a) Fluorescence – radiative decay from the ex­

cited state back to the ground state G.

The probability for this to occur is governed

by the same quantum mechanical principles

that are involved in the absorption of radi­

ation by the ground state. The spectrum of

fluorescence is typically red-shifted (Stokes

shift) relative to the longest wavelength ab­

sorption band, because the excited elec­

tronic state of the molecule has an altered

(relaxed) nuclear configuration relative to

that of the ground state. This results in a

lower excited state energy and a slightly

higher ground state energy relative to those

involved in absorption and, because the

Franck–Condon Principle applies to fluor­

escence also, this provides the basis for the

red-shift in the fluorescence spectrum.

b) Internal conversion (ic) – radiationless

transition to the ground electronic state

manifold. In this case the excited state en­

ergy is dissipated thermally by relaxation to

the surrounding medium.

c) Intersystem crossing (is) – singlet-to-triplet

conversion. The resulting change in electron

spin multiplicity is formally forbidden quan­

tum mechanically, but for complex mol­

ecules this can nevertheless be a major route

for decay of excited singlet states.

d) Excitation transfer – migration of excitation

from donor (D) to acceptor (A) molecules.

In situations commonly encountered in pho­

tosynthetic materials this occurs by an in­

ductive resonance process described initially

by Förster (1948, 1967). The inductive res­

onance mechanism results in transfer among

molecules which may be either the same or

different chemically. The probability or rate

of transfer depends on (1) the spectral en­

ergy overlap between D and A, (2) the in­

verse sixth power of the distance between

D and A, (3) their relative orientation, and

(4) the intrinsic fluorescence lifetime of D.

Kenneth Sauer and Martin Debreczeny The important range of spatial distances in­ volved is 1.5 to 10 nm, corresponding to transfer rates of 1 to 1000 per nanosecond. e) Quenching – any process that decreases the excited state lifetime from that of the “iso­ lated” molecule. Trapping of excitation in reaction centers resulting in productive charge separation is an example of photochemical quenching. Other types of quench­ ing can occur from the proximity or addition of quencher molecules Q, which may be paramagnetic species, heavy atoms or ions, excitation transfer acceptors [section (d), above] or pigment aggregates. To the extent that these compete with natural relaxation processes such as fluorescence, they serve to decrease the fluorescence yield, thereby providing an indirect monitor of the quench­ ing. 8. Polarization is a consequence of the vectorial nature of the absorption and emission of radi­ ation. The transition dipole moments associ­ ated with these processes have both a magni­ tude (related to the allowedness or the “oscillator strength” of the transition) and a direction relative to molecular axes. For a mol­ ecule with a fixed orientation in space, the probability that it will absorb light depends on the direction of propagation of the incident radiation as well as on the relative orientation of the oscillating electric field vector. Subse­ quent emission from the excited state is polar­ ized in a manner that depends on the transition moment vector for fluorescence and on the direction of observation relative to that of ex­ citation. Depolarization of the fluorescence can result from: a) Intramolecular relaxation ic from higher ex­ cited electronic states having absorption transition moments oriented differently from that of fluorescence. b) Rotation of the molecule during the excited state lifetime. c) Excitation transfer to a differently oriented molecule, which then emits the fluor­ escence. 9. Fluorescence lifetimes are determined by the intrinsic fluorescence lifetime of the molecule as modified (decreased) by competing processes. The intrinsic lifetime (15–20 ns for

Fluorescence most chlorophylls) is governed by the transi­

tion dipole moment for spontaneous emission

(Einstein A coefficient). For molecules having

similar ground- and excited-state nuclear con­

figurations, the intrinsic lifetime can often be

calculated from the absorption properties of

the molecule.

10. The fluorescence yield describes the fraction of the excited state population that results in fluorescence. Because fluorescence is de­ creased by competing excited-state relaxation processes, the reduction of fluorescence yield is directly proportional to the decrease in the fluorescence lifetime relative to the intrinsic lifetime (the lifetime in the absence of com­ peting processes). 11. Fluorescence relaxation for simple molecules in dilute solutions and at low incident light intensities is kinetically first-order in the ex­ cited state concentration or population. This results in a simple single-exponential decay. Modifications of this simple kinetic behavior can result from a) inhomogeneous excited-state populations,

typically reflecting molecules in different

local environments giving rise to compo­

nents with different relaxation rates,

b) high local concentrations of excited states

that result in excitation annihilation (bimol­

ecular), often a consequence of high inten­

sities used in pulsed-laser experiments,

c) significant depletion of the ground-state population accompanying repeated high-intensity pulsed excitation, d) long-lived quenching species (triplets, oxidized or reduced reaction centers, etc.). In photosynthetic membranes or pigment complexes the fluorescence decay is never found to be simple single-exponential. It is, in fact, this complexity that often provides insight into the details of the excitation transfer and trapping processes associated with photosynthesis, as we shall explore in the remainder of this chapter. 12. Phosphorescence is emission associated with relaxation from the lowest energy state of (typically) a triplet manifold of states to the ground state. The spectrum of phosphorescence emission from a particular molecule is different from that of the fluorescence. Phosphorescence often appears at longer wave-

45

lengths than fluorescence and occurs with a much longer relaxation time (milliseconds to seconds), because it involves a process that is formally quantum mechanically forbidden. Although phosphorescence from Chl a has been reported, it has not played a significant role in photosynthesis research.

II. Steady-State Fluorescence

A. Fluorescence Spectrophotometer Fluorescence is commonly measured using a fluorescence spectrophotometer. Many commercial instruments are available, and a representative configuration is shown schematically in Fig. 2. The basic components are (1) a source of exciting light, (2) a sample containing a fluorescent ma­

46 terial, (3) a device for detecting the fluorescence intensity in a particular direction, and (4) elec­ tronic components for displaying, recording or storing output information in a form that is acces­ sible using computer software. The configuration shown in Fig. 2 includes monochromators in both the excitation and emission beams. For a general purpose instrument it is desirable to be able to scan either monochromator to record excitation or emission spectra. Fluorescence is emitted in all directions, but with an intensity distribution that depends strongly on the angle with respect to the excitation beam, the polarization of the light and intrinsic properties of the fluorescing species. The detection system is typically ar­ ranged to observe fluorescence emitted at an angle such as 90° to the incident intensity. This minimizes interference from the transmitted light propagated in the forward direction, which is much more intense than the fluorescence. Polariz­ ers can be inserted into the excitation or emission beam. A polarizer defines the orientation of the electric vector of the transmitted light in the plane perpendicular to its direction of propagation. The detector, which may be a vacuum-tube photomul­ tiplier or a solid state photodiode, must be sensi­ tive at the wavelengths of the fluorescence. Two important modes of treating the output signal from the detector are (1) analog detection of the output voltage or current, and (2) single-photon counting. The latter is particularly effective in suppressing the contributions of low-level noise (dark current) contributed by the detector elec­ tronics, thereby enabling the detection of weak fluorescence signals. For special purposes one or more of the design components or features shown schematically in Fig. 2 can be varied. Some of these modifications are of considerable impor­ tance for photosynthesis studies, and they will be mentioned at appropriate places in this chapter.

B. Excitation Spectra A fluorescence excitation spectrum displays the relative efficiency of different wavelengths of ex­ citing light in generating fluorescence. Light that is incident on a homogeneous, clear sample with an intensity (see Fig. 2) is partially transmitted, and partially absorbed, (We will consider presently the complications introduced

Kenneth Sauer and Martin Debreczeny by samples that scatter a significant portion of the incident light.) Light which is absorbed by the phospho­ sample may result in fluorescence, rescence, non-radiative return to the ground state or photochemistry. The fluorescence quantum yield, is the fraction of light absorbed that is emitted as fluorescence. Thus,

for a homogeneous (non-scattering) sample. For samples that are sufficiently dilute and illumi­ nated at low intensities, both and are lin­ early dependent on the incident light intensity; under these conditions the ratio is independent of light intensity. However, the quantum yield will, in general, depend on the wavelength of the incident light. Comparison of the fluorescence excitation spectrum with the absorption spectrum provides direct evidence of the excitation-wavelength de­ pendence of the quantum yield. An example of the usefulness of this property is seen in Fig. 3. Photosynthetic materials such as chloroplast thylakoids that contain several different light-harvesting pigments often exhibit efficient excitation transfer from short wavelength-absorbing pig­ ments to Chl a, which has a lower-energy excited state and hence a longer wavelength absorption band. A portion of the excitation arriving at Chl a is then emitted as fluorescence. If excitation transfer from the accessory pigments in the native photosynthetic membranes is highly efficient, then the fluorescence excitation spectrum will be superimposable on the absorption spectrum, indi­ cating that the quantum yield of fluorescence is wavelength independent. By contrast, when the pigments are extracted into organic solvents or when the attachment of phycobilisomes or chloro­ somes to the membranes is disrupted by cell breakage excitation transfer from the accessory pigments is no longer effective. Thus, wave­ lengths absorbed by the accessory pigments, mainly Chl b and carotenoids in the case of spin­ ach chloroplast thylakoids (Fig. 3), do not lead to Chl a fluorescence, and the fluorescence exci­ tation spectrum resembles only that portion of the absorption owing to the Chl a itself, as is seen in the excitation and absorption spectra of the

47

Fluorescence

C. Emission Spectra

extracted pigments (solid curves, especially in the region between 450 and 500 nm in Fig. 3). This approach has been used to explore the relative efficiencies of different accessory pigments in transferring excitation to chlorophyll in a variety of in vivo situations.

The fluorescence emission spectrum of a single substance in solution reflects radiative transitions typically from the lowest excited electronic state to the ground state. Because of relaxation of the nuclear configuration in the excited state prior to emission, the fluorescence typically undergoes a Stokes shift to longer wavelength, amounting to 4 to 7nm in the case of chlorophylls at room temperature, as seen in Fig. 3. To the extent that the transitions involving the lowest excited state show vibrational sub-structure (always incom­ pletely resolved for large chromophores in con­ densed media), the fluorescence emission spec­ trum and the absorption spectrum in the long wavelength region exhibit a mirror-image relation to one another on a scale where the spectra are plotted as a function of the frequency (energy) of the radiation. Fig. 3 shows examples of such behavior for thylakoids and for the pigment ex­ tract, comparing the emission spectra at the righthand side of Fig. 3(a) with the absorption spectra in the long-wavelength region of Fig. 3(b). Fail­ ure of this relation may indicate situations where there is (1) significant overlap of more than one electronic transition in the absorption spectrum, and transitions of Chl as is the case for the a or Chl b, (2) incomplete relaxation or thermaliz­ ation of the excited state prior to emission, or (3) excitation transfer among identical molecules in different local environments that produce differ­ ent spectral shifts, or among chromophores that are chemically distinct but have overlapping ab­ sorption spectra. A particularly sensitive test for the presence of any of these contributions is achieved using the method devised by Stepanov (1957), and which has been applied to a study of excitation equilibration in Photosystem II (H. Dau and K. Sauer, Biochim Biophys Acta, sub­ mitted). Photosynthetic membranes also contain nonfluorescing pigments. Carotenoids, for example, absorb strongly in the visible and near-UV region of the spectrum but have almost undetectable fluorescence. The cause of this behavior is a lowlying electronic state that cannot be populated by direct light absorption from the ground state but that can be reached efficiently by intersystem crossing from a higher energy excited state. Such a state which quenches the fluorescence of the

48 molecule may, in general, be a paramagnetic tri­ plet state or may have symmetry elements that cause it not to couple radiatively with the ground state. Nevertheless, molecules such as caroteno­ ids can transfer excitation to “sensitize” the flu­ orescence of nearby chlorophyll molecules in photosynthetic membranes. The fluorescence of mixtures of chromophores of different chemical types is, in general, an addi­ tive superposition of the contributions of each of the molecules involved, if the chromophores do not interact with one another and if the sample is sufficiently dilute to avoid optical distortions. However, because the fluorescence of each mol­ ecular species is also determined by its distinctive absorption properties, the measured emission spectrum from a mixture of fluorophores depends critically on the excitation wavelength that is se­ lected. Useful assays of mixtures of photosyn­ thetic pigments have been devised in this way. For example, chlorophyll b can be detected at very low levels in the presence of a much larger concentration of chlorophyll a in a pigment ex­ tract using the facts that (1) absorption (exci­ tation) by Chl b at wavelengths between 450 and 460 nm occurs in a region of the spectrum where Chl a absorbs hardly at all, and (2) emission from Chl b between 654 and 650 nm occurs in a region where Chl a emission is small. (Boardman and Thorne, 1971) For this assay to work, it is ob­ viously necessary to avoid higher pigment concen­ trations where excitation transfer from chloro­ phyll b to chlorophyll a would quench the fluorescence of the former. Self-absorption of the fluorescence may occur in the case of strongly absorbing samples. The fluorescence spectrum undergoes distortion owing to fluorescence re-absorption by the sam­ ple itself, primarily in the wavelength region where there is the greatest overlap between the absorption and the fluorescence emission of the sample. As a consequence, the fluorescence sig­ nal is suppressed at these wavelengths, and the apparent fluorescence emission maximum is shifted to longer wavelengths where the spectral overlap is less. Although conditions differ for dif­ ferent experimental set-ups, a good rule of thumb is to use samples with absorbance (optical den­ sity) less than 0.05 at both the exciting wavelength and in the region of maximum spectral overlap.

Kenneth Sauer and Martin Debreczeny It is usually impossible to avoid self-absorption distortions of the fluorescence spectra for intact leaves or even for chloroplast suspensions, be­ cause the local concentrations of pigments are quite high even for diluted suspensions. For samples such as heavily pigmented leaves, where very little light passes all the way through the sample, fluorescence can nevertheless be detected by using a front-face illumination geometry, either by moving the detector or by turning the sample so that fluorescence emitted from the di­ rectly illuminated surface is detected. If the light at the excitation wavelength is totally absorbed by the sample, then the fluorescence observed is essentially independent of the sample concentra­ tion (or leaf thickness); however, self-absorption effects are still present in the emission spectrum.

D. Light Scattering Interference Samples that are inhomogeneous (in terms of their refractive index) on a scale of the order of the wavelength of light and longer are subject to light scattering. This is a prominent property of plant leaves, cell suspensions and, to a lesser ex­ tent, of suspensions of sub-membrane complexes such as reaction centers and antenna pigmentproteins. As a consequence, some or even most of the incident light is redirected into all directions, much the same as fluorescence, although the de­ tailed dependence on angle is different. Scat­ tering can be elastic (Rayleigh scattering), with no change in wavelength of the incident light, or inelastic, where the shift is both to longer (Stokes scattering) and to shorter (anti-Stokes scattering) wavelengths. The Raman effect is an important form of inelastic scattering that results from coup­ ling to vibrational transitions in the chromophore or in the surrounding matrix. It is not necessary for the material to absorb at the wavelength of incident light for either elastic or inelastic scat­ tering to occur. The scattered light is readily de­ tected by sensitive fluorescence spectrophotomet­ ers, is usually strongly polarized, and can serve as a strong source of interference. Rayleigh scattering is sufficiently strong in any sample that it is impossible using steady-state me­ thods to measure the fluorescence at the same wavelength as the exciting light, even for homo­ geneous solutions that are “dust-free”. However,

Fluorescence when the scattering is not too intense, the fluor­ escence excitation and emission wavelengths can be within a few nanometers of one another, de­ pending on the quality of the monochromators used. Double- and even triple-monochromators, often supplemented with blocking optical filters, are required to suppress the transmission of stray light at wavelengths away from the one selected for excitation and to prevent the exciting light wavelength from reaching the detector. Another way to separate light-scattering from fluorescence signals is to use time-resolved measurements; light-scattering occurs essentially instantaneously, whereas fluorescence relaxation is measurably slower.

E. Polarization/Depolarization Because of the vectorial nature of the interaction between electromagnetic radiation and the mol­ ecular transition dipole moments for absorption and emission, fluorescence is typically polarized. The polarization occurs at two stages. (1) During the excitation process of an isotropic sample of randomly oriented chromophores, a sub-population of molecules is “photo-selected” for exci­ tation by the projection of the electric vector of the incident radiation on the transition moment of each absorbing molecule. The excited state population is therefore anisotropic, having a vec­ torial character. (2) Each excited molecule, in turn, emits radiation that is polarized parallel to its fluorescence transition dipole moment, but propagation of the fluorescence occurs predomin­ antly in directions that are not along the transition moment vector. Between the excitation and emis­ sion processes, events such as internal conver­ sion, chromophore rotation or excitation transfer [see Section I.B.8] may occur in the sample. Each of these processes leads either to an alteration of the polarization direction or to depolarization (randomization of orientation). Detailed analyses of the consequences of each of the effects noted above have been published. As indicated in Fig. 2, optical polarizers can be inserted into the excitation and/or the emission beam to test the extent of polarization and its dependence on wavelength for a particular sam­ ple. For purposes of illustration, let us suppose that the excitation and emission propagation di­

49 rections and in Fig. 2) are at 90° to one another and lie in the horizontal plane. Unpolar­ ized light incident on the sample has its electric vector oscillating in the plane perpendicular to the propagation direction. Even in the absence of inserted polarizers, photoselection will occur in the sample owing to the fact that the oscillating electric field does not have a component in the direction of propagation of the light and, hence, will be biased against exciting those molecules whose absorption transition moments happen to be oriented in that direction. For this reason the population of excited molecules in a sample il­ luminated from one direction is always aniso­ tropic. Addition of a polarizer to the excitation beam selects a polarization direction for the electric vector in a plane that includes the direction of propagation and the polarizer transmission axis. Absorption of this plane-polarized radiation by an isotropic sample in turn modifies the ani­ sotropy of the excited-state population initially produced. It is useful and sufficient for our purposes to consider only two orientations of the polarizer axis, one producing light polarized in the vertical plane and the other in the horizontal plane (obtained by rotating the polarizer 90° about an axis parallel to the propagation di­ rection). Depending on which orientation of the polarizer is chosen, a different subset of the chro­ mophores will be excited, determined by the pro­ jection of the oscillating electric field of the excit­ ing light on the absorption transition dipoles of the individual chromophores. Depending on which subset is excited, the projection of the en­ semble of molecular vectors is different in the direction of propagation of the fluorescence emis­ sion. This can be readily detected by inserting an analyzing polarizer into the fluorescence beam and orienting it so as to transmit light polarized in either the vertical or in the horizontal plane. If the polarizer in the excitation beam is oriented vertically, the degree of polarization of the sam­ ple fluorescence can be readily determined from the relative intensities of emitted light detected when the analyzer is oriented eithe parallel horizontal) to the vertical) or perpendicular direction of polarization of the exciting beam. (Because each of the instrument components in a fluorescence spectrophotometer, from the light

50 source through to the detector, introduces polar­ ization effects quite apart from those of the sam­ ple, it is necessary to correct for this instrument polarization function, including its dependence on the wavelengths of excitation and emission. A straightforward way of accomplishing this correc­ tion is described by Houssier and Sauer (1969) and by Lakowicz (1983).) For purposes of interpretation of polarization measurements for a particular sample, the mea­ sured (and corrected) polarized fluorescence in­ tensities are combined to give a value for the polarization anisotropy, A, where

Each of the quantities involved in Eq. (2) is de­ pendent on the wavelengths of excitation and em­ ission. (The reader should be aware that an alter­ native description of the polarization anisotropy is sometimes seen, especially in the earlier litera­ ture, where the factor of 2 in the denominator is not included.) Analyses of the relation between polarization/depolarization properties of particular samples have been described in detail in the literature. (Van Amerongen and Struve, 1995) Some rel­ evant properties that influence the interpretation are (1) whether the sample is truly isotropic or whether there is partial or complete ordering of the directions of the transition moments, as in a single crystal or in a sample of oriented mem­ brane fragments, (2) rotation of the chromophore during the excited state lifetime, and (3) exci­ tation transfer among the chromophores within the sample. If either (2) or (3) is extensive for an isotropic sample, this can lead to complete depolarization of the fluorescence and an ani­ sotropy value of zero. For an isotropic sample where the individual molecules are (1) effectively fixed in their orien­ tation and (2) unable to transfer excitation to their neighbors during the excited state lifetime, the anisotropy value can range between + 2/5, when the absorption and emission transition di­ poles are parallel to one another, to – 1/3, when they are mutually perpendicular. Intermediate values of the polarization anisotropy can be in­ terpreted in terms of the angle between the ab-

Kenneth Sauer and Martin Debreczeny sorption and emission transition dipoles. If exci­ tation transfer occurs among the molecules in the sample, the polarization anisotropy can be in­ terpreted in terms of the relative orientation of the absorption transition moment of the chromo­ phore initially excited, D, and the fluorescence transition moment of the acceptor chromophore, A, that emits the fluorescence. It is important for purposes of this analysis that the system of chromophores does not physically rotate to a sig­ nificant extent during the excited state lifetime. For chromophores with fluorescence lifetimes of a few nanoseconds in aqueous solution at room temperature, the effective molecular weight should be in excess of 100 kDa to avoid serious contributions from rotational diffusion (Rigler and Ehrenberg, 1976). Polarization anisotropy measurements have been used for a variety of purposes in connection with photosynthesis studies. The relative di­ rections of the absorption transition moments for transitions to different electronic excited states of pigment molecules like chlorophyll, bacteriochlo­ rophyll or protochlorophyll have been derived from experimental measurements and compared with theoretical deductions (Gouterman and Stryer, 1962). Studies of fluorescence depolariz­ ation of pigment molecules in solution as a func­ tion of concentration have provided evidence that the range of excitation transfer for chlorophylls, for example, extends to 60 to 100 Å (Knox, 1975). For multiple chromophores present in pigment proteins or photosynthetic membrane prepara­ tions, the extent of depolarization reflects both the relative orientations of the chromophores and their ability to transfer excitation. Such analyses are aided by knowledge of the structural arrange­ ment of the chromophores where such infor­ mation is available from X-ray crystallography, and from time resolved measurements of the relaxation of the fluorescence anisotropy. We shall describe an example of such a study that has been applied to C-phycocyanin (PC) in Section III.

F. Variable Fluorescence 1. Photochemical Trapping Competes with Fluorescence An important application to photosynthetic sys­

Fluorescence tems arises from the competition between fluor­ escence and photochemical trapping in reaction centers. As a consequence of this competition, fluorescence yields are complementary to the yi­ elds of productive electron transport initiated by the reaction centers (Latimer et al., 1956; Gov­ indjee et al., 1986; Krause and Weis, 1991; Dau, 1994). In Photosystem II and in purple bacteria, closing the reaction centers by strong illumi­ nation, using inhibitors of primary electron trans­ port or adding reducing agents that keep the en­ dogenous secondary electron acceptors in the reduced state, increases the yield of fluorescence from 3 to 5 fold. The maximum fluorescence yield is still less than 10%, indicating that there are other important alternative paths of excited state relaxation in these closed or blocked prepara­ tions. Special instrumentation has been developed to measure variable fluorescence yields and the as­ sociated kinetics of fluorescence induction. De­ pending on the light intensity used to close the traps and on the conditions of inhibition, the pro­ cess occurs primarily on the nanoseconds to se­ conds time scale (Mauzerall, 1972). This is the time required for electron acceptors close to the reaction centers to become reduced or electron donor pools to become exhausted. In plants, whole cells, or intact chloroplasts, longer term adjustments in the redox levels of the donor and acceptor pools occur, and slower changes can be readily seen over intervals of minutes to hours (Kautsky and Hirsch, 1931; Büchel and Wilhelm, 1993). For reasons that are not yet well under­ stood, Photosystem 1 makes little or no contribu­ tion to the variable fluorescence signals (Briantais et al., 1986). Instrument requirements for these studies dif­ fer significantly depending on the time scale in­ volved. Because light plays the dual role of stimu­ lating fluorescence and providing the mechanism for closing the photochemical traps, it is necessary for the fluorescence measurement to be able to distinguish between the two effects. One common approach is to use two different sources of illumi­ nation, one of which provides “exciting” light that is chopped or modulated at a frequency of several hundred and the other pro­ vides steady (unmodulated) “actinic” light when it is turned on. The exciting light intensity is set

51

sufficiently low that it does not significantly alter the photochemical state of the sample. Thus, the component of the fluorescence that is detectable using a lock-in amplifier tuned to the exciting light modulation frequency serves as a probe of the fluorescence efficiency or yield, regardless of the intensity of the fluorescence produced simul­ taneously by the unmodulated actinic light, even though the latter is by far the larger contribution to the overall fluorescence signal. Clearly it is necessary to pay careful attention to the design of the electronic circuitry to insure the isolation of these two signals. Commercial instrumentation has been developed for this purpose, including devices that can be used to monitor fluorescence induction in the leaves of plants growing in the field (Büchel and Wilhelm, 1993). In addition to studies of the kinetics of electron transfer reactions, the effectiveness and mode of operation of electron transport inhibitors and the role of different electron donors or acceptors, fluorescence induction has been used to analyze the heterogeneity of Photosystem II in higher plants and how this heterogeneity reflects the dis­ tribution of Photosystem II in thylakoid mem­ branes (Melis, 1991). 2. Electric Fields Influence Fluorescence Associated with Photosynthetic Materials Because the transport of electrons across photo­ synthetic membranes is vectorial, trans-membrane electric fields are generated or modulated during the photosynthetic light reactions. These electric fields provide not only a source of chemi­ cal potential for driving some of the dark energyconserving biochemical processes, but also an in­ teraction with pigment molecules in the mem­ branes that absorb or fluoresce strongly. (See chapter by S. Boxer in this volume.) The direct effect of the field produces carotenoid and chloro­ phyll absorption band shifts that are sensitive to both the magnitude and direction of the field, which in turn provides a calibration of the strength of the field at those molecules (Junge, 1982). Fluorescence yield changes´ also occur, both as a direct effect of the field and as an indirect consequence of alterations in the rates of primary charge separation and recombination in the reaction centers which are competing with the

52 fluorescence. Several studies have focused on the consequences of externally applied electric fields. Macroscopic samples containing whole cells, membranes, sub-membrane fragments or isolated complexes placed between external electrodes can be subjected, at least briefly, to applied fields (Lockhart et al., 1988). In such ex­ of periments the samples are generally spatially iso­ tropic, so that the effect of the directionality of the field is impossible to determine. A second approach makes use of trans-membrane fields produced by transient ion gradients generated be­ tween the inside and outside spaces of enclosed membrane vesicles like chloroplast thylakoids or bacterial chromatophores. The direction of the electric field can be changed by altering the rela­ tive salt concentrations of the solutions used in rapid mixing experiments (Dau and Sauer, 1991). Based on both steady-state changes in fluor­ escence intensity and time-resolved fluorescence relaxation measurements, the effect of the ap­ plied electric field on the kinetics of primary charge separation and recombination has been investigated.

Kenneth Sauer and Martin Debreczeny not most, light-harvesting complexes isolated from a wide variety of photosynthetic organisms. The absorption bands associated with these long wavelength-emitting pigments are difficult to de­ tect, indicating that they reflect only a small por­ tion of the pigment molecules present. Because the excited states responsible for the long wave­ length fluorescence are at energies close to or even lower than the excited states of the associ­ ated reaction centers, the role of these pigments in the collection of light and the funneling of excitation to the reaction centers is of consider­ able interest. Nevertheless, little is known at pre­ sent about the mechanism of the very effective quenching of this fluorescence at room tempera­ ture. It has not been possible, for example, to provide a clear link between the photochemical (open/closed) state of the reaction centers and the intensity of long wavelength fluorescence. III. Time-Resolved Fluorescence

A. Experimental Considerations

3. Temperature Dependence of Fluorescence

1. Time-Correlated Single Photon Counting (TCSPC)

For most fluorescent molecules present in homo­ geneous dilute solution, the effect of lowering the temperature of the sample is primarily to narrow the spectral widths of the components of the emis­ sion spectrum (also of the absorption and exci­ tation spectrum), thus improving the spectral res­ olution. Apart from this sharpening of the incompletely resolved spectra, there are no dram­ atic changes in fluorescence yield. Many photo­ synthetic organisms and isolated pigment-protein complexes, however, show dramatic increases in fluorescence yield upon lowering the temperature (see e.g., Butler et al., 1979; Mukerji and Sauer, 1989). The increase can be 20 fold or more be­ tween room temperature and 77 K. Furthermore, the spectrum of the fluorescence increase is typi­ cally shifted significantly to long wavelength, in comparison with the bulk of the fluorescence at room temperature. In fact, this is a reflection of the heterogeneity of the molecules giving rise to fluorescence emission in such samples; low temperature-enhanced, long wavelength-emitting pigments appear to be associated with many, if

The traditional instrument for time-resolving fluorescence is schematically the same as the steady-state apparatus shown in Fig. 2. However, the light source in a time-resolving instrument must be pulsed or modulated rather than continu­ ous in intensity. We will limit our discussion here to techniques employing pulsed light sources; for a discussion of time resolution of fluorescence by modulation of a continuous light source, see Lakowicz (1983) or Lakowicz et al. (1990). If a pulsed laser is used as the excitation source, the natural line width of the laser is usually narrow enough that it is unnecessary to use an excitation monochromator. The fluorescence induced in the sample by the excitation pulse is time-resolved by the detector. The time of arrival of a fluorescence photon at the detector is compared with the time of the excitation pulse. The intensity of photons striking the detector must be limited so that single photons can be detected without distortion from multiple photon events. Fluorescence photons ar­ riving at particular time delays relative to the excitation pulse are gated into channels of a

Fluorescence chosen temporal width and a fluorescence decay profile is collected on a multi-channel analyzer. What we have described above is known as the time-correlated single photon counting (TCSPC) technique. This method of time-resolving fluor­ escence is widely used and its practical implemen­ tation has been described in detail (O’Connor and Phillips, 1984). By the simultaneous acquisition of fluorescence photons at multiple time delays, TCSPC has the advantage of allowing for the collection of high signal-to-noise data in a rela­ tively short time. The time resolution of this tech­ nique is limited by a combination of the temporal width of the excitation pulse and the time-response of the fluorescence detector. In recent years the temporal width of lasers has rapidly decreased to a point where picosecond and subpicosecond laser pulses are readily achievable with commercially available systems. If maximal time resolution of fluorescence is desired, the lim­ iting factor for the TCSPC technique is usually the response time of the detector. Micro-channel plate detectors, a type of photomultiplier de­ signed to achieve optimal time resolution, cur­ rently have time resolution (transit-time spread) as fast as 25 ps. (Hamamatsu Photonics, R3809U series)

2. Fluorescence Upconversion A more recently developed technique of timeresolving fluorescence, known as fluorescence upconversion, has the advantage over the TCSPC technique of being theoretically limited in time resolution by the temporal pulse width of the excitation source rather than the detector re­ sponse time. In brief, a train of laser pulses is split into two beams, one of which is used to excite the sample. The subsequent fluorescence from the sample is collected and focused onto a non-linear crystal. The other laser beam is used as a variable delay gating pulse and is focused onto the same area of the crystal (see Fig. 4a). If the angle of the incoming light relative to the optical axis of the crystal is such that the phase matching requirement is satisfied, some of the light exiting from the crystal will be at a frequency equal to the sum of the laser and fluorescence frequencies. Since fluorescence upconversion will occur only when both sample fluorescence and

53

54 the laser gating pulse are present in the crystal, the time resolution of the experiment is laserpulse width limited (see Fig. 4b). A fluorescence decay can be recorded by incrementally optically delaying the gating pulse relative to the excitation pulse. Because only a narrow band of fluor­ escence is upconverted at a particular crystal orientation, a time-resolved fluorescence spec­ trum can be recorded by tuning the angle of the crystal. The practical implementation of such an instrument has been described elsewhere (Shah, 1988; Doust, 1982; Kahlow et al., 1988; Debrec­ zeny, 1994). The chief disadvantage of the upconversion technique is the low efficiency with which currently employed crystals upconvert flu­ orescence. This means that unless time resolution of a few picoseconds or better is needed, the TCSPC technique is still the method of choice to obtain high signal-to-noise time-resolved fluor­ escence data. The fluorescence upconversion technique of time-resolving fluorescence is similar to the pump-probe technique of measuring transient ab­ sorption in that it is a two-photon technique and theoretically allows for pulse-width limited time resolution. However, fluorescence upconversion has the advantage over pump-probe techniques that the two photon process occurs in a non-linear crystal, not in the sample. This means that the phenomena of stimulated emission and excitedstate absorption, which often complicate the in­ terpretation of pump-probe experiments, are avoided in the fluorescence upconversion experi­ ment. (See chapter by G Fleming in this volume.) 3. Deconvolution An experimentally observed time-resolved fluor­ escence signal consists of the molecular fluor­ escence signal of interest convoluted with the in­ strument response function (IRF) (O’Connor and Phillips, 1984). If the kinetics of interest occur on a time scale much longer than the temporal width of the IRF, the IRF can be treated as instan­ taneous and the convolution integral ignored. However, because interesting events in photo­ synthesis are known to occur on time scale of picoseconds and shorter, practitioners of the TCSPC technique in this field have frequently relied on deconvolution to extend their time res-

Kenneth Sauer and Martin Debreczeny olution. Typically, when using the TCSPC tech­ nique, the IRF is collected at the laser excitation frequency by using a scattering solution in place of the sample. It has been found experimentally that in order to achieve the best fits to data, a wavelength dependent time shift between the IRF and the decay must be introduced (O’Connor and Phillips, 1984). This effect has been attributed to a wavelength dependence of the time response of the detector, because the fluorescence decay and IRF are necessarily collected at different wave­ lengths. As a consequence there is some uncer­ tainty in the designation of time zero. Data from individual decays at different fluorescence wave­ lengths can be combined to produce time-resolved emission spectra, but at short times after the excitation pulse the time uncertainty leads to large spectral uncertainties, especially if the sample fluorescence changes rapidly at early times. In addition to providing much shorter IRFs, the upconversion technique has the advantage over the TCSPC technique of a more precise mea­ surement of time zero. The IRF can be measured with the same geometric arrangement as the flu­ orescence upconversion signal by tuning the non­ linear crystal to the optimal angle for generation of sum frequency light from the residual exciting pulse and the gating pulse. The peak of this IRF is the delay setting at which the exciting and gating pulses are exactly temporally overlapping (time zero). As with the TCSPC technique, it can be shown that the observed fluorescence upconver­ sion signal is the molecular fluorescence signal of interest convoluted with the IRF measured in the above manner (Doust, 1982). 4. Exciton Annihilation Because the upconversion signal is dependent on the square of the energy of the laser pulses, (Doust, 1982) the upconverted fluorescence power will be larger for high energy laser pulses at a low repetition rate than for low energy pulses at a high repetition rate, for a fixed level of aver­ age laser power. High pulse energies at low repetition rates have been used to study photo­ stable dye molecules. However, in multi-chromophore systems like photosynthetic proteins, high energy pulses can lead to exciton annihilation

Fluorescence (Geacintov and Breton, 1982). Exciton annihila­ tion can occur when two or more chromophores that are coupled by energy transfer each absorbs a photon. The experimental result is that the flu­ orescence decay profile shows an excitation inten­ sity dependence and a decay rate governed by bi-excitonic annihilation (Geacintov and Breton, 1982). A simple estimate of the extent of exciton annihilation can be obtained by dividing the number of photons absorbed in the beam spot per laser pulse by the number of light-harvesting complexes (LHC) in the beam spot. In this con­ text a LHC is a group of chromophores that are coupled by excitation transfer.

where is the molar decadic extinction coefficient of the entire LHC at the laser frequency, h is Planck’s constant, is the frequency of the laser, r is the radius of the laser beam waist, and is Avogadro’s number. The solution is assumed to be dilute. The average number of photons absorbed per LHC should be less than unity if exciton annihila­ tion effects are to be avoided. Using the above equation, the extent of exciton annihilation in the TCSPC technique and the upconversion tech­ nique are compared, choosing the trimeric aggre­ gate of C-phycocyanin (PC) excited at 624 nm with a repetition rate of 4 MHz as an example. In a typical TCSPC experi­ ment the laser power at the sample is 0.5 mW, (unfo­ and the beam waist is roughly cused). The average number of photons absorbed per pulse per PC timer in such an experiment is In a fluorescence upconversion ex­ periment a typical power seen by the sample is 1 mW and the laser is focused onto the sample to beam waist. The number of photons have a absorbed per pulse per PC trimer under these If much larger com­ conditions is plexes (for example, whole phycobilisomes which contain hundreds of coupled chromophores) are studied, exciton annihilation must be considered more carefully if the fluorescence upconversion

55 technique is to be employed. The amount of light pumping the sample can be reduced by diverting more power into the gating pulse. But since the upconverted power is a product of the gating and fluorescence powers, the reduction in pump power relative to gate power will ultimately lead to reduction in the signal level.

B. Isotropic Time-Resolved Fluorescence As mentioned in the section on steady-state flu­ orescence and shown below in Eq. (4), the fluor­ escence from a mixture of non-interacting and optically dilute chromophores can be described additively. The initial excited-state populations of the x different chromophore types are determined of the by the relative extinction coefficients, chromophores at the excitation wavelength, The evolution of the chromophore excited-state populations as a function of time are described by P(t), which has a value of 1 at the time of excitation and eventually decreases to 0, at which point the excited state has been entirely depleted. In the case of a non-interacting mixture of homo­ geneous chromophores, P(t) will decay as a single exponential. The contribution of each chromo­ phore to the observed fluorescence signal is weighted by its net fluorescence quantum yield, and the shape of its fluorescence spectrum, f, as a function of the emission wavelength,

From Eq. (4) we can see that, in addition to resolving mixtures of chromophores by differ­ ences in their excitation and emission spectra, the lifetime of each chromophore species can be used as a further discriminating factor. If the requirement that chromophores be noninteracting is relaxed somewhat to include weak interactions in which the exchange of excitation energy occurs but energetic coupling is not so great as to affect the individual chromophore spectra (these are the conditions under which Förster’s mechanism of inductive resonance is ap­ plicable), Eq. (4) still holds. However, the popu­ lation term, P(t), is no longer necessarily de­ scribed by a single exponential, nor will it necessarily decay monotonically. If excitation en­

56 ergy is transferred preferentially from a donor chromophore type to an acceptor, the population term of the acceptor chromophore initially in­ creases as a function of time if this transfer is rapid compared to decay of its excited state by means other than energy transfer. Electron transfer reactions can also be in­ cluded in Eq. (4) by treating the excited state and ionized product of each molecule as separate species, although the photosynthetic molecular ions are typically non-fluorescent. For this reason time-resolved fluorescence and transient absorp­ tion measurements often provide complementary information. In such situations the ability to monitor the disappearance of the fluorescence as the molecule becomes ionized can provide an un­ ambiguous measure of excited-state decay with­ out interference from product formation. Unlike dye molecules free in solution, the chromophores in photosynthetic systems are or­ ganized in association with proteins to have fixed relative orientations and distances of separation. The geometry of the chromophore interactions provides high efficiency energy and electron transfer. The fixed geometry means that the en­ ergy transfer from a donor to acceptor chromo­ phore in a light-harvesting complex can be de­ scribed by a narrow range of rate constants and is often well approximated by a single rate constant. Similarly, electron transfer reactions in photosyn­ thetic systems are often well represented by a single rate constant. This being the case, the time dependence of the chromophore excited-state or ionized-state populations can be described by Eq. (5). P is a vector describing excited-state and ionized-state populations of each chromophore type, while M is a square matrix containing the rate constants for energy transfer or electron transfer between different chromophore types. The diagonal elements of M (for which the donor and acceptor are the same chromophore type) contain the negative of the sum of the rate con­ stants for all means of decay including energy transfer and electron transfer from the excited state of a particular chromophore type.

If Eq. (5) is solved for the excited-state or ion-

state population of a particular chromophore spe-

Kenneth Sauer and Martin Debreczeny cies, the result is a sum of exponential terms described by Eq. (6):

Within a given system, all of the chromophore excited-state and ion-state populations contain the same number of exponential terms with the However, the ampli­ same rate constants, associated with each exponential term tudes, will be different for the different chromophore species. By incorporating Eq. (6) into Eq. (4) we can see that the isotropic time-resolved fluor­ escence signal is described by a sum of exponen­ tials. It is for this reason that the most common method of analysis of isotropic time-resolved flu­ orescence measurements by workers in the field of photosynthesis is to fit the data to a sum of exponentials. The fitted exponential amplitudes and rate constants are functions of the rate con­ stants for energy transfer and electron transfer between chromophores. A common analysis tech­ nique that relies on the assumption that the rate constants are independent of the probed emission wavelength is to fit data simultaneously at multi­ ple emission wavelengths with the exponential rate constants being linked at all wavelengths while the amplitudes are varied freely (global analysis) (Knorr and Harris, 1981; Knutson et al., 1983; Holzwarth et al., 1987; also, see chapter 5 by A.R. Holzwarth in this volume). Such tech­ niques can increase the number of resolved kin­ etic components. Unfortunately, without simpl­ ifying assumptions, it is often difficult to relate these experimentally observed exponential rate constants to the molecular rate constants that are of interest. Recently algorithms have been de­ veloped which allow one to directly extract rate constants of interest for a given model (Roelofs et al., 1992; also, see chapter 5 by A.R. Holz­ warth in this volume). In this case the difficulty often lies in establishing the uniqueness of a parti­ cular solution or model. When using the summed exponential model to describe time-resolved flu­ orescence one should keep in mind that the model is based on the assumption that kinetic processes can be described by discrete rate constants, and this will only be true to the extent that the chrom­

Fluorescence

ophores are rigidly held by the protein lattice during the relevant photo-physical events. In addition to the extraction of rate constants, the ability to observe the evolution of the fluor­ escence spectrum as the excited-state populations of a mixture of weakly coupled chromophores equilibrate allows one to extract information about the fluorescence spectra of the individual chromophores. Fig. 5a shows the time-resolved emission spectrum of the subunit of the lightharvesting protein C-phycocyanin at 77 K, excited at 580 nm (Debreczeny et al., 1993). The subunit contains only two chromophore types, re­ ferred to as and according to the amino acid residue to which the linear tetrapyrrole chro­ mophore is covalently attached. Two peaks are evident in the time-resolved spectra; one peak at about 620 nm showing greatest intensity at early times, and a second peak at about 650 nm increas­ ing in intensity concurrently with the decrease of the 620 nm peak. The spectra at the earliest times are representative of the chromophore excited-

57

state population induced by the 580 nm excitation pulse. It is evident that although both chromoph­ ores are excited by the laser pulse, the higher is preferentially ex­ energy chromophore cited. Energy transfer is most favorable in an energetically downhill direction (as predicted by Förster’s theory) so that with time we see an increase in the excited-state population of the with concurrent lower-energy chromophore loss of the excited-state population of the higherWith prior knowl­ energy chromophore edge of the relative extinction coefficients of the two chromophore types, the time-resolved spec­ tra at all times were modeled, and information about the rate constants for energy transfer and the fluorescence spectra of the individual chrom­ ophores were extracted. The inverse of the sum of the forward and back rate constants for energy transfer was found to be 64 ps with the ratio of the back-to-forward rate constants being <0.1 (Debreczeny et al., 1993). The extracted fluor­ and chromophores escence spectra of the are shown in Fig. 5b. The discussion above is restricted to the case of weak coupling in which individual chromophore spectra are not affected by inter-chromophore in­ teractions. At inter-chromophore separation dis­ tances of <20 Å, however, strong energetic inter­ actions between chromophores can lead to spectra which are not additive in terms of the spectra of the individual chromophores. In such cases the excitation energy becomes delocalized over the strongly coupled aggregates of chrom­ ophores and Eq. (4), which treats the net fluor­ escence signal as a sum of the fluorescence from the individual chromophores in a mixture, no longer applies. However, Eq. (4) may still be useful in systems in which chromophores are ar­ ranged in clusters within which chromophores are excitonically coupled, but between which energy transfer occurs by the Förster mechanism (Sauer, 1975).

C. Anisotropic Time-Resolved Fluorescence For a photo-physical event to be kinetically ob­ servable by isotropic time-resolved fluorescence spectroscopy, it must involve a change in the shape of the observable fluorescence spectrum or the fluorescence quantum yield. This means, for

58 example, that energy transfer between spectrally identical chromophores will be invisible by iso­ tropic fluorescence spectroscopy. If, however, the polarization of the emitted fluorescence is mea­ sured in addition to its intensity and wavelength, much more can be learned about the nature of the photo-physical event, including resolution of energy transfer rate constants among identical chromophores. Tao (1969) has shown that the time-resolved fluorescence anisotropy of a single molecule (cal­ culated according to Eq. (2)) can be interpreted as being a function of the dot product of the induced by the absorption transition dipole, excitation pulse, with the emission transition di­ pole, observed at some later time, t.

is the second Legendre polynomial in x, and represent a correlation the brackets around function that includes the rotational averaging ne­ cessary to describe an isotropic solution and an orientation evolution function for the emissive transition dipole. Eq. (7) shows that time-resolved fluorescence anisotropy is sensitive to ro­ tation of the net emission transition dipole as a function of time and that the net angle by which the emission dipole is rotated relative to the ab­ sorption transition dipole can be extracted from the residual anisotropy level. If energy transfer between chromophores is being probed, fluorescence anisotropy will be sen­ sitive to the relative orientation of the emission dipoles of the donor and acceptor chromophores. If the initially absorbing chromophore is also the emitting chromophore, the anisotropy value will remain high (at 0.4 if the absorption and emission transition dipoles are parallel, neglecting ro­ tational diffusion). If the emitting chromophore is different from the initially absorbing chromo­ phore, the fluorescence anisotropy value will de­ crease according to the extent by which the emis­ sion transition dipole is rotated away from the orientation of the absorbing transition dipole. In a typical case, the observed fluorescence spec­ trum contains contributions both from initially absorbing molecules and from molecules which received excitation through energy transfer. In

Kenneth Sauer and Martin Debreczeny this case, the anisotropic emission spectrum will dominate at early times when energy transfer has not had time to occur; then as energy transfer occurs, the anisotropy will decrease. If energy transfer occurs between chromophores of differ­ ent energies, the anisotropy in the long wave­ length region of the emission spectrum will nor­ mally decrease more rapidly than that in the short wavelength region, because energy transfer oc­ curs preferentially downhill energetically. The reader is referred elsewhere for deri­ vations of the model function used to fit timeresolved anisotropic fluorescence of photosyn­ thetic systems (Lyle and Struve, 1991; Cross et al., 1983; Van Amerongen and Struve, 1995). Instead we give an example of the utility of this technique in extracting rate constants for energy transfer in C-phycocyanin. Fig. 6a shows the time-resolved fluorescence anisotropy of PC mon­ omers and trimers isolated from a genetically en­ gineered strain of Synechococcus sp. PCC 7002 (Debreczeny et al., 1995). The mutant strain was one of the three engineered to be missing chromophore types found in PC, to simplify the kinetic analysis (Debreczeny et al., 1993). A model of the PC trimer isolated from the mutant strain, based on the known crystal structure of wild-type PC (Schirmer et al., 1987), is shown in Fig. 6b. Note that the monomer aggregates into symmetry (axis of symmetry the trimer with perpendicular to the plane of the diagram). Tri­ mers of PC stack to form the “rods” of the phyco­ bilisome, a well-characterized light-harvesting complex found in cyanobacteria and some algae. The fluorescence anisotropy of PC monomers in Fig. 6a decays from an anisotropy value of 0.4 to a value of 0.33 with a characteristic time of 200 ± 70 ps. Because only two chromophores are and chromoph­ present in this system (the ores), the decay time can be directly related to the rate of energy transfer between them, and the small amount of anisotropy decay indicates that the chromophore transition dipoles are oriented nearly parallel to each other (27° ± 1.5° from par­ allel). Using Förster theory, a decay time of 158 ± 12 ps can be predicted, in reasonable agree­ ment with the measured value (Debreczeny et al., 1995). When the monomer PC is aggregated into tri­ mers, a dramatic effect is seen in the fluorescence

Fluorescence

59 sured values are again in excellent agreement with predictions based on the Förster theory (1.4 ± 0.1 ps and 46 ± 5 ps, respectively). Also from the experimental anisotropy decay it is pos­ sible to estimate the angle between the transition dipoles of the and chromophores on adjac­ ent monomers (52°) and to restrict the allowed axis values of the angles formed between the of symmetry and the transition dipoles of the and chromophores. Further structural infor­ mation could be obtained if oriented rather than isotropic samples of PC were used in these experi­ ments. (See chapter by G. Garab in this volume.) IV. Conclusion

anisotropy decay. The PC trimer anisotropy de­ cays to a much lower value and reaches its resid­ ual anisotropy level much more quickly than does the monomer. Two distinct regions of the PC trimer anisotropy are evident, showing character­ istic decay times of 1.0 ± 0.2 ps (at the limit of the instrument time response) and 40 ± 2 ps. These two decay times, not seen in the monomer relaxation, can be assigned to energy transfer be­ and chromophores on adjacent tween the monomers and to energy transfer around the trimer ring, respectively (Fig. 6b). These mea­

Fluorescence has proved to be a valuable tool in investigating many questions related to photosyn­ thetic energy conversion. Knowledge of the ener­ gies and lifetimes of the excited states of pigment molecules provides insights into events associated with excitation transfer among antenna chrom­ ophores, trapping and charge separation in reac­ tion centers, charge recombination, quenching processes, etc. Existing quantum mechanical the­ ory is adequate, in most cases, for understanding these processes, at least on a time scale longer than a few picoseconds. Where detailed structural information about the pigment-protein complexes is available, for example, calculated and experi­ mental values for excitation transfer rates in chro­ mophore arrays are in good agreement. Fast relaxation events on the sub-picosecond time scale are less well understood, and this regime is now being actively explored. It is a challenge to both experimentalists and theorists to design studies that address the major outstanding ques­ tions relating to our fundamental knowledge of excited electronic states and how their properties can be used to inform our understanding of the photosynthetic process. Acknowledgements The authors thank their colleagues Mary Talbot, Shelly Pizarro and Dr. Theo Roelofs at UC Berkeley for very helpful comments on this con­ tribution. We are especially grateful to Mary Tal­ bot for providing the spectra shown in Fig. 3. The original research carried out in the authors'

60 laboratory was supported by the Director, Office of Energy Research, Office of Basic Energy Sci­ ences, Energy Biosciences Division, of the U.S. Department of Energy under Contract No. DEAC03–76SF00098.

References Boardman NK and Thorne SW (1971) Sensitive fluorescence method for the determination of chlorophyll a / chlorophyll b ratios. Biochim Biophys Acta 253: 222–231. Briantais J-M, Vernotte C, Krause GH and Weis E (1986) Chlorophyll a fluorescence of higher plants: Chloroplasts and leaves. In: Govindjee, Amesz J and Fork DC (eds) Light Emission by Plants and Bacteria, pp 539–583 Aca­ demic, Orlando, FL. Büchel C and Wilhelm C (1993) In vivo analysis of slow chlorophyll induction kinetics in algae: Progress, problems and perspectives. Photochem Photobiol 58: 137–148. Butler WL, Tredwell CJ, Malkin R and Barber J (1979) The relationship between the lifetime and yield of the 735 nm fluorescence of chloroplasts at low temperatures. Biochim Biophys Acta 545: 309–315. Cantor CR and Schimmel PR (1980) Biophysical Chemistry. Part II. Techniques for the Study of Biological Structure and Function. WH Freeman, San Francisco, CA. Cross AJ, Waldeck DH and Fleming GR (1983) Time re­ solved polarization spectroscopy: Level kinetics and ro­ tational diffusion. J Chem Phys 78: 6455–6467. Dau H (1994) Molecular mechanisms and quantitative models of variable Photosystem II fluorescence. Photochem Photo­ biol 60: 1–23. Dau H and Sauer K (1991) Electric field effect on chlorophyll fluorescence and its relation to Photosystem II charge sep­ aration reactions studied by a salt-jump technique. Biochim Biophys Acta 1098: 49–60. Debreczeny MP (1994) Comparison of the rate constants for energy transfer in the light-harvesting protein, C-phycocyanin, calculated from Förster’s theory and experimentally measured by time-resolved fluorescence spectroscopy. Ph.D. Thesis, University of California, Berkeley, Law­ rence Berkeley Laboratory Report LBL-35672. Debreczeny MP, Sauer K, Zhou J and Bryant DA (1993) Monomeric C-phycocyanin at room temperature and 77 K: Resolution of the absorption and fluorescence spectra of the individual chromophores and the energy-transfer rate constants. J Phys Chem 97: 9852–9862. Debreczeny MP, Sauer K, Zhou J and Bryant DA (1995) Comparison of calculated and experimentally resolved rate constants for excitation energy transfer in C-Phycocyanin. 1. Monomers. 2. Trimers. J Phys Chem 99: 8412–8419; 8420–8431. Doust TAM (1982) Fluorescence lifetime and depolarisation measurements using frequency up-conversion. In: Doust TAM and West MA (eds) Picosecond Chemistry and Bi­ ology, pp 1–34. Science Reviews, Northwood, Middlesex, England.

Kenneth Sauer and Martin Debreczeny Förster T (1948) Zwischenmoleculare Energiewanderung und Fluoreszenz. Ann Physik 2: 55–75. Förster T (1967) Mechanisms of energy transfer. In: Florkin M and Stotz (eds) Comprehensive Biochemistry, Vol 22, pp 61–80, Elsevier, Amsterdam. Geacintov NE and Breton J (1982) Exciton annihilation and other non-linear high-intensity excitation effects In: Alfano RR (ed) Biological Events Probed by Ultrafast Laser Spec­ troscopy, pp 157–191. Academic Press, New York. Gouterman M and Stryer L (1962) Fluorescence polarization of some porphyrins. J Chem Phys 37: 2260–2266. Govindjee, Amesz J and Fork DC (eds) (1986) Light Emission by Plants and Bacteria. Academic Press, Orlando, FL. Holzwarth AR, Wendler J and Suter GW (1987) Studies on chromophore coupling in isolated phycobiliproteins. II. Picosecond energy transfer kinetics and time-resolved flu­ orescence spectra of C-phycocyanin from Synechococcus 6301 as a function of the aggregation state. Biophys J 51: 1–12. Houssier C and Sauer K (1969) Optical properties of the protochlorophyll pigments. II. Electronic absorption, flu­ orescence and circular dichroism spectra. Biochim Biophys Acta 172: 492–502. Junge W (1982) Electrogenic reactions and proton pumping in green plant photosynthesis. In: Slayman CL (ed) Current Topics in Membranes and Transport, Vol 16, Electrogenic Ion Pumps, pp 431–465. Academic Press, New York. Kahlow MA, Jarzeba W, DuBruil TP and Barbara PF (1988) Ultrafast emission spectroscopy in the ultraviolet by timegated upconversion. Rev Sci Instrum 59: 1098–1109. Kautsky H and Hirsch (1931) Neue Versuche zur Kohlensäu­ reassimilation. Naturwissenschaften 19: 964. Knorr FJ and Harris JM (1981) Resolution of multicomponent fluorescence spectra by an emission wavelength-decay time data matrix. Anal Chem 53: 272–276. Knox RS (1975) Excitation energy transfer and migration: theoretical considerations. In: Govindjee (ed) Bioenerget­ ics of Photosynthesis, pp 183–221. Academic Press, New York. Knutson JR, Beechem JM and Brand L (1983) Simultaneous analysis of multiple fluorescence decay curves: A global approach. Chem Phys Lett 102: 501–507. Krause GH and Weis E (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu Rev Plant Physiol Plant Mol Biol 42: 313–349. Lakowicz JR (1983) Principles of Fluorescence Spectroscopy. Plenum, New York. Lakowicz JR, Jayaweera R, Szmacinski H and Wiczk W (1990) Resolution of multicomponent fluorescence emis­ sion using frequency-dependent phase angle and modu­ lation spectra. Anal Chem 62: 2005–2012. Latimer P, Bannister TT and Rabinowitch E (1956) Quantum yields of fluorescence of plant pigments. Science 124: 585– 586. Lockhart DJ, Goldstein RF and Boxer SG (1988) Structurebased analysis of the initial electron transfer step in bac­ terial photosynthesis: Electric field induced fluorescence anisotropy. J Chem Phys 89: 1408–1415. Lyle PA and Struve WS (1991) Dynamic linear dichroism in chromoproteins. Photochem Photobiol 53: 359–365.

Fluorescence Mauzerall D (1972) Light-induced fluorescence changes in Chlorella, and the primary photoreactions for the produc­ tion of oxygen. Proc Natl Acad Sci USA 69: 1358–1362. Melis A (1991) Dynamics of photosynthetic membrane com­ position and function. Biochim Biophys Acta 1058: 87– 106. Mukerji I and Sauer K (1989) Temperature-dependent steadystate and picosecond kinetic fluorescence measurements of a Photosystem I preparation from spinach. In: Briggs WR (ed) Photosynthesis, pp 105–122. Alan R. Liss, New York. O’Connor DV and Phillips D (1984) Time-correlated Single Photon Counting. Academic Press, London. Rigler R and Ehrenberg M (1976) Fluorescence relaxation spectroscopy in the analysis of macromolecular structure and motion. Quart Rev Biophys 9: 1–19. Roelofs TA, Lee C-H and Holzwarth AR (1992) Global target analysis of picosecond chlorophyll fluorescence kinetics from pea chloroplasts. A new approach to the characteriz­ ation of the primary processes in photosystem II a- and bunits. Biophys J 61: 1147–1163.

61 Sauer K (1975) Primary events and the trapping of energy. In: Govindjee (ed) Bioenergetics of Photosynthesis, pp 115–181. Academic Press, New York. Schirmer T, Bode W and Huber R (1987) Refined threedimensional structures of two cyanobacterial C-phycocyanins at 2.1 and 2.5 Å resolution. J Mol Biol 196: 677–695. Shah J (1988) Ultrafast luminescence spectroscopy using sum frequency generation. IEEE J Quantum Electron 24: 276– 288. Stepanov BI (1957) A universal relation between the absorp­ tion and luminescence spectra of complex molecules. Sov Phys Dokl 2: 81–84. Struve WS (1989) Fundamentals of Molecular Spectroscopy. Wiley-Interscience, New York. Tao T (1969) Time-dependent fluorescence depolarization and Brownian rotational diffusion coefficients of macromolec­ ules. Biopolymers 8: 609–632. Van Amerongen H and Struve WS (1995) Polarized optical spectroscopy of chromoproteins. In: Sauer K (ed) Methods in Enzymology, Vol 246. Biochemical Spectroscopy, pp. 259–283. Academic Press, Orlando, FL.

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Chapter 4 Ultrafast Spectroscopy of Photosynthetic Systems

Ralph Jimenez and Graham R. Fleming* Department of Chemistry and the James Franck Institute, The University of Chicago, 5735 S. Ellis Avenue, Chicago, IL 60637, USA

Summary I. Introduction II. Laser Sources A. Modelocked Ti:sapphire Laser Systems 1. Ti:sapphire Oscillators 2. Ti:sapphire Amplifiers B. Methods of Extending the Tuning Range III. Fluorescence Upconversion A. Experimental Technique B. SomeExamples IV. Transient Absorption A. Experimental Technique B. Some Examples V. Concluding Remarks References

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Summary This Chapter discusses the use of fluorescence upconversion and transient absorption techniques for the study of photosynthetic systems. A description is given of the state-of-the-art laser systems available for ultrafast studies, along with examples of some common techniques for extending the wavelength range of these lasers. The experimental techniques of fluorescence upconversion and transient absorption are introduced, with the goal of describing the implementation, versatility, and limitations of these experi­ ments. Recent experimental results are presented which illustrate applications of ultrafast spectroscopy to studies of excitation energy transfer in light harvesting complexes and electron transfer in bacterial reaction centers. Abbreviations: LH-I, LH-II – light-harvesting complexes I and II of purple bacteria; LHC-II – lightharvesting complex II of plants; P – primary electron donor; PS – photosystem; RC – reaction center; LBO – lithium borate;

I. Introduction

subpicosecond timescales. Thus ultrafast spectro­ scopy has played, and continues to play, a key role in characterizing these highly efficient processes. In this chapter we focus on the two most common spectroscopic techniques: time resolved absorption and fluorescence spectroscopies. Over the past ten years or so, a formidable array of

The elementary electron and energy transfer steps in photosynthesis occur on picosecond or *Correspondence: Fax: 1-312-7020805; E-mail: [email protected]

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J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 63–73. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

64 nonlinear spectroscopic techniques have been de­ veloped as have extensions into the infrared spec­ tral region. The reader is referred to recent litera­ ture for details of these techniques (Diller et al., 1992; Maiti et al., 1993, Durrant et al. 1994). Solid state lasers, in particular Ti:sapphire la­ sers have greatly simplified the practice of, and extended the capabilities of ultrafast spectros­ copy. We describe apparatuses based only on such lasers in the belief that dye laser-based sys­ tems will become obsolete within the next five to seven years. One aspect of ultrafast spectroscopy that con­ trasts it with longer timescale spectroscopy should be mentioned. Femtosecond pulses are shorter than the periods of some molecular vibrations; in other words, “vibrationally abrupt” (Scherer et al., 1993; Jonas and Fleming, 1994; Jonas et al., 1994). For example, a 50 fs pulse is abrupt with vibration. In these cases, respect to a vibrational quantum beats may be observed in the pump probe or spontaneous emission signals. The beat frequencies allow measurement of vi­ brational frequencies in absorption spectra that are quite devoid of structure. It may, however, be quite tricky to assign the frequencies to the ground or excited state and to be sure that the fundamental frequency is being observed. Jonas and Fleming (1994) and Scherer et al. (1993) dis­ cuss these points in detail. The rate of disappear­ ance of these beats reflects the timescale on which the environment perturbs the energy levels of the chromophore. A fascinating development in ultrafast photosynthesis research is the obser­ vation of coherent nuclear motion in bacterial reaction centers by Martin and coworkers (Vos et al., 1993). The beats are assigned to the excited state and suggest that vibrational dephasing may be incomplete on the timescale of the primary charge separation. The outline of this chapter is as follows. First, the newest laser sources are described. Various methods of extending the tuning range of these sources over the ultraviolet, visible, and nearinfrared regions are then outlined. Next, the prin­ ciples of fluorescence upconversion are described, along with a discussion of the optical arrange­ ments used for these experiments. Applications of fluorescence upconversion for monitoring the rates of energy transfer in bacterial and plant light

Ralph Jimenez, and Graham R. Fleming

harvesting systems, and measuring the rate of the primary charge separation in bacterial reaction centers are discussed. Finally, the experimental methodology of transient absorption is described, along with two applications of this technique to studies of bacterial reaction centers. II. Laser Sources

A. Modelocked Ti:sapphire Laser Systems 1. Ti:sapphire Oscillators Modelocked Ti:sapphire oscillators and ampli­ fiers represent an enormous simplification over the traditional femtosecond dye laser/amplifier arrangements. Additionally, modelocked Ti:sapphire oscillators can directly generate sub-20 fs pulses (Huang et al., 1992a,b; Asaki et al. 1993). This performance could only be achieved with a dye laser after amplification, continuum gen­ eration and pulse compression. The cavity ar­ rangement of a typical self-modelocked Ti:sapphire laser, given schematically in Fig. 1, shows the simplicity of these lasers. The oscillator is usually pumped by 5–8 Watts from a small frame argon ion laser, and modelocking is initiated by some mechanical disturbance of the cavity. Short pulse generation has been demonstrated from near 700 nm to above 1000 nm with essentially the same cavity design. These lasers produce pul­ ses with 1–6 nJ energy at a repetition rate around 80 MHz, with pulses as short as 11 fs. Cavity

Ultrafast spectroscopy in photosynthesis

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dumping of these oscillators has also been demon­ strated, giving 40–60 nJ pulse energies at tens to hundreds of kHz repetition rate (Pschenichnikov et al., 1994). No increase in pulse duration results from cavity dumping. Extremely low noise (< 0.1% RMS) and high long term stability com­ bined with these desirable pulse durations make these lasers ideal sources for the study of nearIR photosynthetic systems, e.g. purple bacteria, green bacteria, and heliobacteria. 2. Ti:sapphire Amplifiers For many applications, the tuning range of these lasers must be extended into the visible. There are several techniques for doing this, but higher pulse energies are usually necessary. Further­ more, lower repetition rate, higher pulse energy sources are often required for experiments in which the sample recovery time is longer than the modelocked frequency. Ti:sapphire amplifiers are most easily categorized by their repetition rates. It should be noted that all three types of ampli­ fiers are available commercially. The first category is that of Nd:YAG pumped amplifiers. The pump source is the second har­ monic of a Q-switched Nd:YAG laser. These amplifiers typically give hundreds of millijoules to several joules of pulse energy, at 10–20 Hz repetition rate. Amplified pulses as short as 21 fs have been obtained (Zhou et al., 1994). A pulse is selected from the oscillator, temporally stretched (in order to prevent damage to the amplifier due to high peak power) by a grating, routed into the amplifier cavity via electro-optic switching, and dumped out of the cavity in the same way after several round trips. A grating compressor is used after amplification to restore the pulse duration. The second type of Ti:sapphire amplifier is pumped by the second harmonic of a Q-switched Nd:YLF laser. These regenerative amplifiers op­ erate at 1–10 kHz repetition rates and typically utilize pre-amplification pulse stretching, electrooptic switching, and post-amplification com­ pression (Salin et al., 1991; Squier et al., 1993). These systems yield from tens of microjoules to one millijoule pulse energies, with pulse durations down to 30 fs (Wynne et al., 1994). The layout of a system designed and built in our laboratory

which does not employ a pulse stretcher is shown in Fig. 2 (Joo et al., 1995). The third type of amplifier system is the high repetition rate regenerative amplifier which is pumped with 14–15 Watts from a CW ion laser (Norris, 1992). In this amplifier, the pulse is in­ jected and ejected acousto-optically (electro-optic devices are limited to 10 kHz or lower). Pulse stretching prior to amplification may be em­ ployed, but more commonly the pulse is stretched by dispersive elements within the amplifier cavity. As usual, the pulse is recompressed after amplifi­ cation. These amplifiers typically give 1–5 microjoules pulse energies, at repetition rates from 50 kHz to 300 kHz. Pulses as short as 85 fs have been reported (Sosnowski et al., 1994).

B. Methods of Extending the Tuning Range A variety of methods may be employed for shift­ ing the frequency of ultrafast laser sources. One

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of the simplest methods is harmonic generation. Second and third harmonic generation of Ti:sapphire wavelengths is commonly done with LBO and BBO crystals. The wavelength range from 250 nm to 320 nm and 360 nm to 490 nm may be accessed in this way. Another important tech­ nique is continuum generation. When an ampli­ fied femtosecond pulse (pulsewidth <200 fs and energy >200 nJ) is focused into a transparent material (e.g. sapphire), the high intensity causes self focusing and subsequent self-phase modu­ lation which creates a stable white light con­ tinuum extending across the entire visible spec­ trum. For pulse energies less than 2 microjoules, this produces a high-quality Gaussian beam at all wavelengths throughout the visible and near infrared. Typically, 1–5 nJ energy is produced for a given 10 nm bandwidth. The color may be selected with an interference filter, providing en­ ough light to serve as the excitation pulse for a fluorescence upconversion experiment, or the probe pulse for a transient absorption experi­ ment. Selected portions of the continuum may be amplified by optical parametric amplification (Reed et al., 1994). One way of doing this in­ volves temporally and spatially overlapping a por­ tion of the continuum into a BBO crystal, along with an intense (1 microjoule or more) pulse of second harmonic from an amplified Ti:sapphire beam. With a 250 kHz amplifier, it has been de­ monstrated that tens of nanojoules per pulse can be produced across the visible spectrum. III. Fluorescence Upconversion

A. Experimental Technique The principle of the fluorescence upconversion method is that a short pulse of light excites a sample whose fluorescence is focused into a non­ linear crystal along with a variably delayed ‘gate’ pulse, and the sum frequency is detected as a function of the delay between the two pulses (Fig. 3). Rotation of the crystal determines the wave­ length of fluorescence upconverted. The advan­ tage of using this gating technique is that the time resolution of the experiment is determined by the width of the pulses (see below), not by the time resolution of the detection system. Detailed de-

scriptions of the upconversion method were given by Shah (1988) and Kahlow et al. (1988). The upconverted signal is usually detected by a photomultiplier tube used with a photon coun­ ter. A double monochromator is very helpful be­ cause despite the non-collinear geometry, the sig­ nal is contaminated with doubled gate beam and doubled excitation beam, both of which are or­ ders of magnitude more intense than the upcon­ verted signal. A prism may also be used for dis­ persing the upconverted light prior to the monochromator. The use of nonlinear crystals which require orthogonally polarized interacting beams (Type II phase-matching) is also helpful in this sense, in reducing the background due to one beam signals. Two optical layouts for the upconversion experiment are shown in Fig. 4.

Ultrafast spectroscopy in photosynthesis

The setup with the elliptical mirror is the easiest to align. However, the scheme with the parabolic mirrors is more flexible since there is enough space to insert a small cryostat. In either arrange­ ment, the excitation beam may be aligned so that it does not hit the reflector, and fluorescence is collected at a small angle off-axis from the exci­ tation beam. This technique makes it easier to measure fluorescence wavelengths near the exci­ tation wavelength without upconverting the exci­ tation beam. The time resolution of the upconversion ex­ periment is determined by the instrument re­ sponse function (IRF), which is approximately given by the cross-correlation of the excitation pulse with the gate pulse:

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where and are the intensity pro­ files of the excitation and gate pulses, respec­ tively. Operationally, the IRF is measured by angle-tuning the crystal to upconvert transmitted or scattered excitation light. Utilizing the setup of Fig. 4 (left), M. Du, X. Xie and coworkers (Du et al., 1992) have achieved a 70 fs IRF. For pulses appreciably shorter than 100 fs, crystals much thinner than 1 mm are required. The sum-frequency is generated at all points through the thickness of the nonlinear crystal where the gate pulse and fluorescence (or exci­ tation pulse) are temporally and spatially over­ lapped. But the group velocity mismatch between the fluorescence and gate beam wavelengths causes broadening of the IRF. For example, Du et al. (1992) calculate the group velocity mis­ crystal to be 115– match for their 0.4mm 130 fs when upconverting 940 nm fluorescence

68 with a 608 nm gate pulse. This broadening needs to be added to the measured cross-correlation in considering the time resolution of the measure­ ment (Shah, 1988). Thus, limitations of the upconversion tech­ nique make experiments with IRFs of signifi­ cantly less than 100 fs difficult, even with the availability of shorter pulses. Furthermore, the necessity of using thin crystals with large upcon­ verted bandwidths causes a difficulty in discrimin­ ating fluorescence from scattered or transmitted excitation beam. This contamination of the signal can be very difficult to overcome when measuring fluorescence much closer than 30–40 nm from the excitation wavelength. Fluorescence upconver­ sion measurements with high time resolution (<100 fs) are most readily performed on samples with large Stokes shifts. Unfortunately, this is not usually the case for chlorophylls in, for example, light harvesting systems. Many fluorescence upconversion measure­ ments resolve the isotropic emission from a sam­ ple in order to monitor the appearance or lifetime of an emitting species. In contrast, depolarization measurements can be used to resolve dynamics within a fluorescence band, such as energy trans­ fer. Performing these measurements requires the addition of a polarizer to each beam and a wa­ veplate for rotating the polarization of the exci­ tation beam. In addition, it must be verified that the optical arrangement preserves the anisotropy of the fluorescence by ensuring that the ani­ sotropy of a standard system (such as Rb. sphaeroides R26 reaction centers) is 0.4. A further complication in these studies is the pres­ ence of singlet–singlet and singlet–triplet annihi­ lation processes whenever high excitation den­ sities and/or high repetition rates are used (van Grondelle, 1985). These processes shorten the fluorescence lifetime of pigment-protein com­ plexes, and may affect depolarization measure­ ments when the systems are highly ordered. Low temperature fluorescence upconversion studies have not yet become common. Signal av­ eraging considerations make fluorescence upconversion most convenient with medium and high repetition rate laser sources. But biological samples are easily damaged, and the sample must be moved throughout the experiment. The chal­ lenge of refreshing the sample volume excited by

Ralph Jimenez and Graham R. Fleming the beam has not been easily met. Stanley and Boxer (1995) have measured fluorescence decays of bacterial reaction centers at 80 K by mounting a light (4 oz.) Joule–Thompson refrigerator (MMR Technologies) on an audio speaker, in an optical arrangement similar to that shown in Fig. 4 (right).

B. Some Examples The first example of a fluorescence upconversion experiment concerns the dynamics of primary charge separation in the reaction centers of pur­ ple bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides. The timescale for elec­ tron transfer from the primary donor, an exciton­ ically coupled dimer of bacteriochlorophyll a (de­ noted P*) to the acceptor bacteriopheophytin is around 3 picoseconds at room temperature. This rate was measured by a number of groups by monitoring the decay of stimulated emission from P* (for example, Martin et al., 1986). In measure­ ments of the decay of P* by stimulated emission, most workers have made measurements at or near the isosbestic point in the spectrum con­ sisting of ground state bleaching and absorption and P*. However, of the radical cation of P such a procedure makes it difficult to observe longer decay components in the stimulated emis­ sion. Fluorescence upconversion measurements performed by Du and coworkers clearly showed behavior which had been difficult to observe in pump-probe experiments. After exciting the spe­ or bands and time resolving the cial pair’s P* emission at 940 nm, M. Du, S.J. Rosenthal and coworkers (Du et al., 1992) observed nonexponential decay kinetics. An example of a P* decay is shown in Fig. 5. Attempts to explain this observation have ranged from considerations of heterogeneity in the protein environment of the reaction center to models based on protein fluc­ tuations on the time scale of the electron transfer (Gehlen et al., 1994; Jia et al., 1994). A second application of fluorescence upconv­ ersion to photosynthesis is to the study of energy transfer in light harvesting systems. Energy trans­ fer amongst differently oriented but otherwise similar or identical chromophores is conveniently monitored via fluorescence depolarization. In a series of polarized light experiments, Du and co­

Ultrafast spectroscopy in photosynthesis

workers (1993, 1994) have measured the depolar­ ization of fluorescence from membranes of PSIonly and LHC-II-only strains of Chlamydomonas reinhardtii. The average single-step energy trans­ fer in PSI was observed to occur in 200 fs. The depolarization process in LHC-II, on the other hand, was observed to be highly nonexponential, spanning 2 orders of magnitude from hundreds of femtoseconds to tens of picoseconds (see Fig. 6). As a final example of time resolved fluor­ escence studies of photosynthetic systems, we consider the energy transfer processes in bacterial light harvesting complexes. The purple photosyn­ thetic bacteria contain two types of light har­ vesting complexes which channel excitations to the photochemical reaction center. In Rb. sphaeroides, these complexes are thought to be built from dimers of bacteriochlorophyll a, along with the carotenoid spheroidene. The LH-I com­ plex is closely associated with the reaction center in a ringlike structure, and the LH-II complexes form pools which connect LH-I/RC structures.

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Recently, depolarization measurements have re­ solved the single-step hopping of electronic exci­ tation amongst the bacteriochlorophylls in LH-I (Branforth et al., 1994). The anisotropy of 940 nm fluorescence decays from a value around 0.4 to 0.1 with a 110 fs exponential time constant. The observed non-exponentiality may be a result of a distribution of energy transfer rates which result in a non-exponential depolarization pro­ cess. As an example of an unusual anisotropy decay, Fig. 7 shows the decay of anisotropy from the bacteriochlorophyll fluorescence upon exci­ tation of spheroidene in LH-I (Bradforth et al., 1994). This type of decay (starting near zero and going to a negative value) can result from energy transfer when the long axis of the spheroidene is tilted 30 degrees with respect to the normal of the plane containing the transition moments of two bacteriochlorophyll dimers. The projection of the spheroidene transition moment on the plane lies between the two dimer transition di­ poles

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Ralph Jimenez and Graham R. Fleming

IV. Transient Absorption

A. Experimental Technique Transient absorption methods are applicable to a far wider range of systems than fluorescence upconversion. The fact that several electronic states may contribute to the signal and that the temperature dependence of the signals can be rather subtle has recently been discussed by Jonas and coworkers (Jonas, 1994; Jonas et al., 1994). Conceptually, the experiment involves monitor­ ing the intensity of a weak probe beam transmit­ ted through a sample subsequent to the passage of a much stronger pump beam. The differential absorbance of the sample is monitored by a detec­ tor, and this signal is plotted as a function of delay between the pump and probe pulses. An experimental arrangement for pump-probe spectroscopy is shown in Fig. 8. The transmitted probe beam may be detected by a diode, or multi­ channel detection may be used in order to fre­ quency resolve the signal. Lock-in detection may be used with high repetition rate sources, detec­ tion being referenced to a chopper placed in the pump beam. Experiments with low repetition rate sources cannot efficiently utilize lock-in detec­ tion, so this type of measurement can suffer from

high background levels. Shot-by-shot normaliz­ ation of the signal to compensate for fluctuations in the laser power can also be employed, and is useful both for low repetition rate experiments, and for experiments in which a portion of white light continuum serves as the probe beam. Multi­ color pump probe experiments require care to ensure that the pump and probe beams are fo­ cussed at the same spot in the sample. Pumpprobe experiments can easily be performed at low temperatures (with low repetition rate lasers), and in contrast to fluorescence upconversion, many studies have been reported at cryogenic temperatures.

Ultrafast spectroscopy in photosynthesis

71

B. Some Examples Extensive measurements of the rates of primary charge separation in reaction centers from Rho­ dobacter capsulatus have been performed at room temperature, and low temperatures, by Jia et al. (1994). A series of mutants was constructed in which the amino acid residues at sites L181 and M208 were modified in order to change the free energy of the electron transfer reaction. The redox potential of each mutant was measured electrochemically, and the rates of electron trans­ fer were measured via monitoring the stimulated emission from P*. The fastest rate measured was for a mutant containing a tyrosine in place of the wild type phenylalanine at position L181 (< 3 ps) and the slowest rate was for the Thr(L181)– Thr(M208) mutant (around 30 ps). An example of their data is displayed in Fig. 9. The tempera­ ture dependence of the rates was also studied. A model based on a distribution of electron transfer rates arising from a distribution in free energy gaps was used to fit the data in order to estimate the reorganization energy arising from low-frequency protein and intermolecular modes co­ upled to the electron transfer. A remarkable series of pump-probe measure­ ments by J.L. Martin and coworkers (Vos et al., 1993) suggests the need to re-examine standard

models of electron transfer for the primary charge separation step. Using the mutant of Rhodo­ bacter capsulatus Martin and coworkers observed oscillations in the 77 K pump-probe signals (Fig. 10) which they interpret as arising from vi­ brational wavepackets in the excited state. These oscillations persist for many picoseconds, and in­ dicate that vibrational coherence persists on the timescale of the primary charge separation. The mutant does not undergo electron transfer due to the absence of the acceptor pheophyin, but in more recent studies, the same authors have shown that these oscillations are present in pumpprobe signals from Rhodobacter sphaeroides reac­ tion centers in their native membrane environ­ ment (Vos et al., 1994). Observation of an oscil­ latory contribution to the signal calls into question the conventional assumption that vi­ brational dephasing and relaxation occur on much shorter timescales than does the electron-transfer step. A full description of electron transfer in­ corporating a realistic model of the protein dy­ namics is still an active area of theoretical devel­ opment (Jean et al., 1992; Skourtis et al., 1993).

72 V. Concluding Remarks Improved precision in ultrafast spectroscopy will allow more sophisticated data analysis such as singular value decomposition (Woodbury et al., 1994) to determine the number of components in a transient spectrum. With improved precision, decays will in many cases no longer be satisfactor­ ily fit as single exponential processes and more complex models will be required to fit data and extract underlying system parameters. It seems likely that four and six wave mixing techniques such as the various echo methods (Nibbering et al., 1991; Cho and Fleming, 1993, 1994) will be increasingly applied to photosynthetic systems, to reveal dephasing timescales and the dynamics of spectral diffusion over wide dynamic ranges.

References Asaki MT, Huang CP, Garvey D, Zhou J, Kapteyn HC and Murnane MM (1993) Generation of 11-fs pulses from a self-mode-locked Ti:sapphire laser. Opt Lett 18: 977–980. Bradforth S, Jimenez R, Fidler V, Fleming GR, Nagarajan S, Norris J, van Mourik F and van Grondelle R (1994) Ultrafast energy transfer in the core light harvesting com­ plex of photosynthetic bacterium Rhodobacter sphaeroides observed by fluorescence upconversion. In: Barbara PF, Knox WH, Mourou GA and Zewall AH (eds.) Ultrafast Phenomena Vol IX, pp 441–442. Springer-Verlag, Berlin. Bradforth S, Jimenez R, van Mourik F, van Grondelle R and Fleming GR (1995) Excitation transfer in the core light harvesting complex (LH1) of Rhodobacter sphaeroides: An ultrafast fluorescence depolarization and annihilation study. J Phys Chem 99: 16179–16191. Cho M and Fleming GR (1993) Photon echo measurements in liquids: Numerical calculations with model systems. J Chem Phys 98: 2848–2859. Cho M and Fleming GR (1994) Fifth-order three pulse scat­ tering spectroscopy: Can we separate homogenous and inhomogenous contributions to optical spectra? J Phys Chem 98: 3478–3485. Diller R, Iannone M, Cowen BR, Maiti S, Bogomolni R and Hochstrasser RM (1992) Picosecond dynamics of bacterior­ hodopsin, probed by time resolved infrared spectroscopy. Biochemistry 31: 5567–5572. Du M, Rosenthal SJ, Xie X, DiMagno TJ, Schmidt M, Han­ son DK, Schiffer M, Norris JR and Fleming GR (1992) Femtosecond spontaneous emission studies of reaction cen­ ters from photosynthetic bacteria. Proc Natl Acad Sci USA 89: 8517–8521. Du M, Xie X, Jia Y, Mets L and Fleming GR (1993) Direct observation of ultrafast energy transfer processes in PS-I core antenna. Chem Phys Lett 210: 535–542.

Ralph Jimenez and Graham R. Fleming Du M, Xie X, Mets L and Fleming GR (1994) Direct obser­ vation of ultrafast energy transfer processes in light har­ vesting complex II. J Phys Chem 98: 4736–4741. Durrant JR, Knoester J and Wiersma DA (1994) Local ener­ getic disorder in molecular aggregates probed by the one exciton to 2 exciton transition. Chem Phys Lett 222: 450– 456. Gehlen JN, Marchi M and Chandler D (1994) Dynamics af­ fecting the primary charge transfer in photosynthesis. Science 263: 499–502. Huang CP, Asaki MT, Backus S, Murnane MM, Kapteyn HC and Nathel H (1992a) 17–fs pulses from a self-mode-locked Ti:sapphire laser. Opt Lett 17: 1289–1291. Huang CP, Kapteyn HC, McIntosh JW and Murnane MM (1992b) Generation of transform limited 32–fs pulses from a self-mode-locked Ti:sapphire laser. Opt Lett 17: 139– 141. Jean JM, Friesner RA and Fleming GR (1992) Application of a multilevel Redfield theory to electron transfer in con­ densed phases. J Chem Phys 96: 5827–5842. Jia Y, DiMagno TJ, Chan CK, Wang Z, Du M, Hanson DK, Schiffer M, Norris JR, Fleming GR and Popov MS (1994) Primary charge separation in mutant reaction centers of Rhodobacter capsulatus. J Phys Chem 97: 13180–13191. Jonas DM and Fleming GR (1994) Vibrationally abrupt pulses in pump-probe spectrsocopy. In: El Sayed MA (ed) Ul­ trafast Processes in Chemistry and Photobiology Blackwell Scientific Publications, Oxford. Jonas DM, Bradforth SE, Passino SA and Fleming GR (1994) Femtosecond wavepacket spectroscopy: Influence of tem­ perature, wavelength, and pulse duration. J Phys Chem, in press. Joo T, Jia Y and Fleming GR (1995) Ti:sapphire regenerative amplifier for ultrashort high power multikilohertz pulses without external stretcher. Opt Lett 20: 389–391. Kahlow MA, Jarzeba W, DuBruil TP and Barbara PF (1988) Ultrafast emission spectroscopy in the ultraviolet by timegated upconversion. Rev Sci Instrum 59: 1098–1109. Maiti S, Cowen BR, Diller R, Iannone M, Moser CC, Dutton PL and Hochstrasser RM (1993) Picosecond infrared stud­ ies of the dynamics of the photosynthetic reaction center. Proc Natl Acad Sci USA 90: 5247–5251 . Martin JL, Breton J, Hoff AJ, Migus A and Antonetti A (1986) Femtosecond spectroscopy of electron transfer in the reaction center of the photosynthetic bacterium Rho­ dopseudomonas sphaeroides R26: direct electron transfer from the dimeric bacteriochlorophyll primary donor to the bacteriopheophytin acceptor with a time constant of 2.8 ± 0.2 psec. Proc Natl Acad Sci USA 83: 957–961. Nibbering ETJ, Wiersma DA and Duppen K (1991) Femto­ second non-Markovian optical dynamics in solution. Phys Rev Lett 66: 2464–2467. Norris TB (1992) Femtosecond pulse amplification at 250 kHz with a Ti:sapphire regenerative amplifier and application to continuum generation. Opt Lett 17: 1009–1011. Proctor B and Wise F (1993) Generation of 13–fs pulses from laser with reduced third-order a mode-locked dispersion. Appl Phys Lett 62: 470–472. Pschenichnikov MS, De Boey WP and Wiersma DA (1994)

Ultrafast spectroscopy in photosynthesis Generation of 13–fs, 5–MW pulses from a cavity dumped Ti:sapphire laser. Opt Lett 19: 572–575. Reed MK, Steiner-Shepard M and Negus DK (1994) Optical parametric amplification of white light continuum compo­ nents at 250 kHz with a Ti:sapphire regenerative amplifier. In: Barbara PF, Knox WH, Mourou GA and Zewall AH (eds) Ultrafast Phenomena Vol IX, pp 215–216. SpringerVerlag, Berlin. Salin F, Squier J, Mourou G and Vaillancourt G (1991) Multikilohertz amplifier for high-power femtosecond pulses. Opt Lett 16: 1964–1966. Scherer NF, Jonas DM and Fleming GR (1993) Femtosecond wave packet and chemical reaction dynamics of iodine in solution: Tunable probe study of motion along the reaction coordinate. J Chem Phys 99: 153–168. Shah, J (1988) Ultrafast luminescence spectroscopy using sum frequency generation. IEEE J Quant Elect 24: 276–288. Skourtis SS, da Silva AJR, Bialek W, and Onuchic JN (1992) A new look at the primary charge separation in bacterial photosynthesis. J Phys Chem 96: 8034–8041. Sosnowski T, Klein PB, Norris TB, Bhargava RN and Gal­ lagher D (1994) Femtosecond blue continuum generation and its application to the time-resolved study of emission in Mn-doped ZnS nanocrystals. In: Barbara PF, Knox WH, Mourou GA and Zewall AH (eds) Ultrafast Phenomena Vol IX, pp 389–390. Springer-Verlag, Berlin. Squier J, Korn G, Mourou G, Vaillancourt G and Bouvier

73 M (1993) Amplification of femtosecond pulses at 10 kHz Opt Lett 18: 625–627. repetition rates in Stanley R and Boxer SG (1995) Oscillations in the spon­ taneous fluorescence from photosynthetic reaction centers. J Phys Chem, 99: 859–869. van Grondelle R (1985) Excitation energy transfer, trapping and annihilation in photosynthetic systems. Biochim Bi­ ophys Acta 811: 147–195. Vos MH, Rappaport F, Lambry JC, Breton J and Martin JL (1993) Visualization of coherent nuclear motion in a membrane protein by femtosecond spectroscopy. Nature 363: 320–325. Vos MH, Jones MR, Hunter CN, Breton J, Lambry JC and Martin JL (1994) Coherent dynamics during the primary electron transfer in membrane bound reaction centers of Rhodobacter sphaeroides. Biochemistry 33: 6750–6757. Woodbury NW, Peloquin JM, Alden RG, Lin X, Lin S, Taguchi AKW, Williams JC and Allen JP (1994) The re­ lationship between thermodynamics and mechanism during photoinduced charge separation in reaction centers from Rhodobacter sphaeroides. Biochemistry 33: 8101–8112. Wynne K, Reid GD and Hochstrasser RM (1994) Regen­ erative amplification of 30 fs pulses in Ti:sapphire at 5 kHz. Opt Lett 19: 895–898. Zhou J, Huang CP, Shi C, Murnane MM and Kapteyn HC (1994) Amplification of 21 fs pulses to the millijoule level. Opt Lett 19: 126–129.

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Chapter 5

Data Analysis of Time-Resolved Measurements

Alfred R. Holzwarth Max-Planck-lnstitut für Strahlenchemie; D-45470 Mülheim a.d. Ruhr, Germany

75 76 76 76 78 83 85 85 86 87 87 87 89 91 91

Summary I. Introduction II. Methods for Time-Resolved Data Analysis A. Single-Decay vs. Global Analysis Methods B. Data Fitting vs. Model Testing Methods C. Procedures for Optimization D. Importance of Proper Weighting Factors E. Importance of Error Analysis F. The Identifiability Problem III. Some Applications to Photosynthesis A. Purple Bacterial Energy Transfer B. Photosystem I Kinetics IV. Conclusions Acknowledgements References

Summary Advanced data analysis methods suitable for the analysis of kinetic spectroscopic data from complex chemical and biological systems are defined and reviewed. The aim of this chapter is on the one hand to introduce beginners dealing with complex data analysis problems into the matter. On the other hand it is intended as a relatively simple description, using only a minimum of mathematics, that should enable non-specialist readers from other fields to better understand the methods currently used to deal with complex kinetic problems. The first part introduces the basic concepts and terms by using a simple kinetic scheme as an example. The fundamentally different concepts of “mathematical data fitting” and “physical model testing” are defined and illustrated with some examples. Their capabilities and limi­ tations are then discussed in detail. Further subsections deal with the mathematical optimization proce­ dures, proper weighting of data points, and the importance of performing an error analysis. As a first­ hand illustration to the use of these methods in current research problems some recent applications in the field of photosynthesis research are given. Abbreviations: DAS – decay–associated spectrum; LHI – B875 light-harvesting complex of photosyn­ thetic bacteria; LHII – peripheral B800–850 light-harvesting complex of photosynthetic bacteria; PS – photosystem; RC – reaction center complex; SAAS – species-associated absorption spectrum; SAES – species-associated emission spectrum; SADS – species-associated absorption difference spectrum

Correspondence: Fax: 49-208-3063951; E-mail: [email protected]

75 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 75–92. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

76 I. Introduction The still increasing tendency to study more and more complex systems and/or phenomena with the help of time-resolved spectroscopic tech­ niques can be observed in all areas of science, and photosynthesis research represents no exception. Using advanced time-resolved techniques re­ searchers can now, often in combination with other methods, ask – and luckily answer in many cases – more sophisticated questions which are directed towards a better understanding of com­ plex multicomponent systems and processes on a molecular level. The spectroscopic or other data sets acquired during such studies are generally large and have to be analyzed in terms of complex kinetic and/or other mathematical and physical models. Traditional data analysis methods prove increasingly insufficient to extract the maximal amount of relevant information contained in such data. The aim of this chapter is to give a brief overview on the powerful and sophisticated me­ thods that have been developed over the recent years to deal with highly complex data analysis problems. The aim is actually twofold: Firstly, this chapter should clarify the terms and methods used in this area of research and thus provide a basis to enable non-specialist colleagues to read and better understand and judge papers on so­ phisticated kinetic measurements. Secondly, this contribution is aimed at scientists who want to learn about these methods and eventually apply them in their own research. Historically most of the methods described here have been developed in time-resolved flu­ orescence spectroscopy. All of these methods are quite general, however, and apply basically to all time-resolved and – with some appropriate minor modifications – also to all non-time-resolved spectroscopies and general data analysis problems. After the introduction and definition of terms like single decay analysis, global analysis, decay-associated and species-associated spectra, mathematical data fitting vs. physical model testing etc., some applications of these methods will be presented briefly. The examples presented have been chosen merely for their suitability to exemp­ lify the principles and the capability of the dis­ cussed methods. They stem for the most part

Alfred R. Holzwarth from the author’s own research subjects, for the simple reason that an in-depth analysis of these problems has been carried out in the author’s laboratory. Within the limited space available for this more technically oriented chapter no attempt will be made to give rigorous proofs for the state­ ments and relationships discussed. Rather the emphasis is on the principles. Numerous ex­ amples from other research groups who have ap­ plied similar methods could have been presented here. It is not possible nor intended, however, to give a complete overview of all papers where such techniques have been applied. The selected references cited should thus be taken merely as a guide for the more interested reader to go into the details of the methods.

II. Methods for Time-Resolved Data Analysis

A. Single-decay vs. Global Analysis Methods Suppose that one has measured some time depen­ dent quantity (fluorescence intensity, absorption difference etc.) vs. time at a certain detection The signal is usually the response wavelength to an optical excitation by a brief exciting light pulse of wavelength If the system under study is a multicomponent one, as almost all bio­ logical systems and photosynthetic systems in par­ ticular one, the kinetics observed will in general depend both on the detection wavelength and The time-deon the excitation wavelength pendent signal measured at one excitation/detection wavelength pair in a multicomponent system will not be sufficient to characterize the system fully. Suppose that the signal I(t) follows – which is true in many cases – a multiexponential law such that:

For a coupled N-component system or N-state system (a state could be an excited state of a chromophore or a group of chromophores, a rad­ ical pair state etc.) we have in general (without giving any proof) n = N, i.e. the number of kin­

Data analysis of time-resolved measurements

etic components (lifetimes) equals the number of (distinguishable) chemical components or states in the system. This is true if the kinetics of the system can be described in terms of a set of homo­ geneous first-order differential equations. How can we analyze the data from such a kinetics? In order to illustrate the procedures we will use in this and the following paragraphs the simple model example of a two-component system with reversible energy transfer (Fig. 1). Despite its simplicity this system is well-suited to allow us to understand the principles of the methods, define the terms used, and to understand the solutions. Extension to more complex systems is then straightforward. The solution of the kinetic equations for this system (Fig. 1) results in a biexponential decay

77

of the form given in Eq. (1) with n = 2. If we use the traditional way of analyzing each decay separately (single-decay analysis) we get a set of and two life­ four parameters, two amplitudes times, for each experiment at a particular excitation/detection wavelength pair. One such experi­ ment will not be sufficient to characterize the system. Carrying out the analysis for a set of M par­ experiments will result in ameters for the whole data set, i.e. 2*M ampli­ tudes and 2*M lifetimes. Inspection of the kinetic problem (Fig. 1) indicates that we expect only two lifetimes, however, which are expected to be the same at all excitation/detection wavelength pairs. This means that using conventional single-decay analysis we have determined more fitting parameters from the data set than required, since

78 we have ignored the fact that certain parameters (in this case the lifetimes) are connected or even constant across all individual (decay) experi­ ments. This is the instant where “global lifetime analysis” becomes relevant. In “global analysis” one attempts to extract a single parameter set for all experiments from a combined analysis carried out in a single analysis run by taking explicit ac­ count of those parameters of the system that are connected across experiments. In our simple case the relationship of the lifetime parameters across experiments is a simple identity, i.e. and the same relationship This is in fact the simplest form of a hold for parameter connection across experiments but is also the most common one in time-resolved spec­ troscopy. It is important to realize that any par­ ameter connection can in principle be achieved, provided it can be formulated in some mathema­ tical terms, either analytically or numerically. The nature of this connection does not follow from the time-resolved experiment itself but must al­ ways be derived from the physics or chemistry underlying the problem at study. In our simple system (Fig. 1) the total number of parameters P determined from the full data set using global analysis is then while P for single-decay analysis is for M > 1. The latter is always larger than Thus global analysis allows one to extract, from the same data set, the same information with a smaller number of parameters. For an N-component system the relation will be Without giving any M + N vs. proof, we may expect as a consequence that glo­ bal analysis will generally lead to i) a better accuracy in the values of the extracted parameters and thus ii) allow the analysis of more complex systems and/or more closely spaced lifetime compo­ nents. How large is the improvement resulting from global over single-decay analysis? This is a diffi­ cult question that can not be answered in general. Rather the answer depends on the very details of the measurement. The degree of improvement can range from very large to actually non-existent in a few special cases. In many cases however the improvement is such that one can clearly state that the problem could be analyzed in a meaning-

Alfred R. Holzwarth ful way only by global analysis while single decay analysis fails completely. The important point here is that once the details of the experiment are known we can get a good estimate about the actual degree of improvement of global vs. single experiment analysis. For this purpose also nu­ merical simulations of experimental data sets should be carried out taking into account the ex­ perimental details like S/N ratio, noise type on the data etc. (see below). Returning to our twocomponent system, Fig. 2 shows the improvement in the relative errors of the lifetimes. The figure shows that the degree of improvement will de­ pend on the type of noise (i.e. constant, poissonian, signal-dependent etc. noise) which in turn depends on the type of experiment (transient ab­ sorption, fluorescence etc.) and on the difference in the spectra of the components. A similar re­ lationship could be given for the error reduction in the amplitudes. The first application of global lifetime analysis to my knowledge has been reported by Knorr and Harris (1981). Later on the method has been further developed and applied to more complex problems by Brand and coworkers (Knutson et al. 1983; Beechem et al. 1984; 1985a). First appli­ cations in photosynthesis research were made soon thereafter (Wendler et al. 1986; Holzwarth et al. 1987; Wendler and Holzwarth, 1987). A general presentation of global analysis may be found in the publications of Beechem (1989) and Weidner et al. (1990), while the application of global analysis to photosynthesis problems has been discussed in part by Holzwarth (1987; 1988). Global lifetime analysis has been applied by a number of groups to photosynthesis problems, see e.g. Holzapfel et al. (1989); Finkele et al. (1992); Holzwarth et al. (1987); Roelofs et al. (1991); Roelofs et al. (1992); Mukerji and Sauer (1993). The method can be applied equally well without any essential modification to all kinds of kinetic analysis and applications to fluorescence kinetics, transient absorption kinetics, anisotropy decay analysis, to name just a few, and they have become quite popular.

B. Data Fitting vs. Model Testing Methods Global as well as single-decay analysis, as de­ scribed in the previous paragraph, aims at an

Data analysis of time-resolved measurements

79

adequate mathematical description of the mea­ sured (kinetic) data. A mathematical model con­ taining parameters is fitted to the data. Conse­ quently the parameters resulting from such kind of analysis are primarily mathematical parameters which do not as such give direct information in terms of a physical/chemical model. Rather these fitting parameters – in most cases of time-resolved spectroscopy amplitudes and lifetimes, along with their temperature, wavelength, etc. dependence – have to be used to derive the parameters of real interest to the researcher, which I will call here the “physical parameters”. This situation is illustrated in Fig. 3 on the righthand branch. The purely mathematical “data fit­ ting procedure” leads to the “mathematical par­ ameters” that describe the kinetics. This is the point where data analysis often ends. Unfortu­ nately, however, these “mathematical par­ ameters” as such are hardly ever of interest to the researcher. This fact is generally not realized sufficiently well. We could, however, chose an entirely different approach. Rather than to first fit a purely mathe­ matical function to the data and then try to in­ directly calculate from those parameters the “physical parameters” of real interest, we could try to fit a “physical model” directly to the data. This “physical model” would have to be a mathe­ matical description of the real physical/chemical behavior of the system and would contain the real “physical parameters” of interest, e.g. rate constants, activation energies, spectra, pK-values

80 and the like as fitting parameters. This procedure has several names in the literature like e.g. target analysis, compartmental analysis, kinetic model­ ling (Beechem et al., 1985b; Löfroth, 1986) de­ pending on the application and also on the prefer­ ences of the authors. This variety in nomenclature is partly given by historic reasons and partly by some (minor) differences in the various methods. The underlying principle is always the same, how­ ever: Direct derivation of the physical/chemical parameters of interest from the original (lifetime) data rather than from any intermediate mathema­ tical fitting parameters. This principle is illus­ trated in the left-hand branch of Fig. 3. What are the advantages of such methods over mere mathematical data fitting methods? These advantages are quite substantial and manifold. First, the direct method ensures that we try to extract from the original data set only the mini­ mally required number of unknown parameters that are of real interest to understand the prob­ lem. The second advantage is even more impor­ tant, however, and must be stressed strongly: It involves a drastic change in the “philosophy” of the data analysis such that mathematical data fit­ ting is replaced by real “physical/chemical model testing” . Using these methods several alternative physical/chemical (kinetic) models can be tested directly on the original data. As a result a decision can be made which of these models fits the data best and which ones can be excluded on the basis of a particular data set. In practice then more than one model may remain that fits the data well. In such a case additional experiments or tests must be carried out in order to distinguish these models. Furthermore the physical par­ ameters resulting from such model testing can then be judged using physical/chemical reasoning. For example any model, even if it fits the data formally well (see paragraph III for two ex­ amples) that would result in unrealistic values of physical parameters or violation of physical/chemical principles or laws, like e.g. negative rate constants, negative fluorescence spectra, or other unrealistic values could be excluded imme­ diately as a reasonable description of the system. This possibility to definitely exclude certain mod­ els is a tremendous step forward over mere mathematical data fitting. Furthermore, the fact that the parameters of real physical interest are

Alfred R. Holzwarth fitted directly to the original data has the effect of structuring the multidimensional parameter space within which the best solution to the data fitting has to be found. This structuring has the effect that the physical parameters derived are obtained with both the highest possible accuracy (given the quality of the input data set) and with a realistic error margin (see below for error analysis). In practice several possible physical/chemical models, preferentially as many as one can think of, should be tested against the data. All this may sound rather theoretical so far. For this reason we will return to our simple example of a twocomponent mixture. Referring to Fig. 1 we can now define the terms decay-associated spectrum (DAS) and species-associated absorption/emission spectrum (SAAS/SAES). The following equa­ tions describe the relationships between these terms and between mathematical and physical parameters of the model: For the two-component and mixture we would measure two DAS, with corresponding lifetimes and (Fig. 1B). The DAS (amplitudes of Eq. (1) as a func­ tion of emission or excitation wavelength) and the lifetimes would result from a global data fit­ ting within a sums of exponentials model. They do not represent the physical parameters of inter­ est, however. Rather the physical parameters of interest are the SAES or SAAS (Fig. 1B), which actually are the real emission or absorption spec­ tra, respectively, of the two species, and the rate The following equations show the constants relationships between these parameters (in case of a two-component system):

It follows from (Eq. (2)) that the DAS are gen­ erally linear combinations of the SAES (or SAAS if the measurement is carried out as a function of the excitation wavelength) with coefficients Furthermore, the lifetimes are functions only of the rate constants and the kinetic connecting scheme (See Fig. 1a) of the system under study. The coefficients for the SAES are complicated

Data analysis of time-resolved measurements functions of both the rate constants, the exci­ tation wavelength, and the extinction coefficients of the species involved (Eq. (3)). The fluor­ is a linear combination of escence decay the exponential terms with the DAS as weighting factors (amplitudes). These equations corre­ spondingly hold for transient absorption measure­ ments if the SAES are replaced by the corre­ sponding species-associated absorption difference spectra (SADS). The relationships defined in Eq. (3) for the example of the two-component mixture with en­ ergy transfer can be generalized in closed form in the following way for all kinetic systems that can be described by a set of homogeneous first order differential equations. Using the following defi­ nitions: X(t) = (n × 1) vector of concentrations of (ex­

cited state) species.

time derivatve of X(t)

T = (n × n) transfer matrix describes connectivity

of system.

vector of time zero absorbances of species.

l(t) = excitation function.

We can then write the following equation for

the time dependence

of fluorescence (or likewise transient absorption ) spectra:

with eigenvalue of matrix T. U = matrix of eigenvectors of T. n = number of (excited state) species k. species-associated emission (SAES) or difference spectrum (SADS) of species. These equations fully describe the relationship between the measurable decay data as a function of excitation wavelength emission or (in transient absorption) detection wavelength the kinetic scheme (transfer matrix T) and the rate constants of the system on the one hand and the species-associated spectra on the other hand.

81 Also the relationship between the DAS and life­ times (the mathematical parameters) and the physical parameters become clear from these equations. The relationship between the eigenval­ ues and the measurable lifetimes is The relation given in Eq. (3) is simply a special case of the closed and generalized form given in Eq. (5). This equation can be fitted directly to a time-resolved data sets after a specific kinetic model has been chosen. In this way the rate con­ stants, the SAES or SADS and, under certain conditions, also the species-associated absorption spectra (SAAS) can be obtained from the data. There is one important restriction, however: The so-called identifiability conditions (see below) must be analyzed and obeyed since they might limit the number of independent physical par­ ameters that can be fitted simultaneously. The methods outlined above are discussed in detail in several papers (Beechem et al. 1985b; Ameloot et al. 1986). Some applications to photo­ synthesis may be found elsewhere in (Holzwarth, 1990; Roelofs et al. 1992; Roelofs and Holzwarth, 1990; Müller et al. 1991; 1992; 1993). As an important extension to the basic kinetic or target analysis outlined above, further restric­ tions with respect to the values and range or the mutual relationships of the physical fitting par­ ameters can be implemented easily if such infor­ mation is available. It could come either from other measurements or could be introduced delib­ erately in order to test certain restricted models. The advantage is that, without any loss of the benefits of target analysis mentioned above, the general procedure is rather flexible and can be adapted easily even to highly complex situations, provided a mathematical description can be for­ mulated. This mathematical description does not have to be given in a closed analytical form but can even be applied in a numerical form. It also follows easily that the formulation of the physical/chemical model to be tested against the data in addition allows one to put in all available other information on the system. This additional infor­ mation may be spectroscopic, structural, thermo­ dynamic or other in nature. In this sense the physical model testing represents a truly holistic approach. Fig. 4 shows an approach often used in the socalled compartmental analysis of data repre­

82

senting antenna energy transfer/charge separation processes. Due to the complexity of an antenna/reaction center complex certain simplifying as­ sumption must be made. The level of approxi­ mation can be chosen, depending on the level of detail (time-resolution and completeness) present in the data. At one extreme level all chromoph-

Alfred R. Holzwarth ores and reaction center states could be taken into account explicitly. This would involve the full kinetic description of the system. However the measurable data hardly ever allow, except in the most simple systems like e.g. phycobiliprote­ ins (Suter and Holzwarth, 1987; Holzwarth et al. 1987) such detail of analysis. One is thus forced to make simplifying assumptions in the definition of how many distinguishable compartments (a compartment is a “quasi-state”) should be or need to be taken into account in the model. Within certain limits it is up to the researcher how many compartments he wants to define and consider in the analysis. How complex or simple a “compartment”, which in the optimal case should encompass a group of chromophores which show similar kinetics and spectra, can be defined, will depend mostly on the amount and precision of data that is available (see e.g. Holzwarth et al. 1987). The proper “compartmentalization” of the problem at hand has to be found out by testing various kinetic models on the data and involves subsequent checking of the physical parameters for their internal consistency and physically reasonable behavior. The well-known exciton/radical pair equilibrium model for photosystem II and purple bacterial antenna/RC complexes (Schatz et al., 1988) is a typical case of a compart­ ment model and has been used extensively in the past (see van Grondelle et al., 1994 for a review). It is important to note that in all cases where a full kinetic treatment (taking into account all chromophores or states separately) is not pos­ sible, the proper or “reasonable” compartmental­ ization of the system tells us a great deal about the physical nature and behavior of the system. Since any compartmentalization of a system re­ presents an approximation, a compartment model can only answer a limited number of questions about a system. Thus as the most stringent restric­ tion, no information can be obtained directly on the processes going on within a compartment, which in essence is considered a “black box” with input and output only. For example in the case of the exciton/radical pair equilibrium model, no information is obtained on the energy transfer rates between the antenna chromophores. Rather it is assumed that these rates, and thus the antenna excited state equilibration within a com­ partment, must be fast in relation to the other

Data analysis of time-resolved measurements rates in the system. If this assumption is not valid the model is not applicable and must be extended by increasing the number of compartments. In this discussion in has already been assumed im­ plicitely that usually a physical model testing ap­ proach also involves and implies a global analysis, i.e. the physical model is fitted to a large set of decay data rather than a single decay, although the latter is not entirely excluded. However the amount of information contained in a single decay curve is usually not sufficient to solve more than the most simple kinetic models. It has become clear that any “target” or “com­ partment” analysis requires a detailed physical/chemical (kinetic) model before the analysis can start, in contrast to global or single-decay “lifetime fitting”. It is thus often erroneously as­ sumed that the outcome of the analysis is already determined by the assumptions that have been put into the physical model. This is fortunately not true, however. As pointed out above, the model is tested against the data, rather than as­ sumed as being correct. The testing of course has to be done with several different and alternative models if meaningful results are to be expected. This procedure then answers the question whether a certain model fits the data or not. It can not positively prove whether a model is the correct one. In practice several different models may turn out to provide an equally good fit to the data. It is important to realize that the “model testing approach” delivers qualitatively different answers than the mathematical fitting approach. In mathematical fitting one can usually always find a good fit, provided one allows for a large enough number of parameters. In physical model testing this is not the case.

C. Procedures for Optimization Nothing has been said so far about the mathema­ tical procedures suitable to find the “optimal” values for the parameters of a given mathematical or physical model. For all kinetic problems the model equations are non-linear. Thus we require non-linear optimization methods for the optimal parameter estimation. Such optimization proce­ dures are the subject of an extended area of nu­ merical mathematics and no attempt will be made here for an extensive in-depth presentation. The

83 principal procedures may be found in monog­ raphs on numerical data analysis (see e.g. Beving­ ton, 1969). Rather some general aspects will be discussed here. First of all it is not necessary to make any distinction between mathematical data fitting and physical/chemical model testing, since the mathematical procedures for parameter opti­ mization are the same for both approaches. The first question to be answered is what we define as an “optimal” value for a fitting par­ ameter. The objective is to find the parameter value that has the maximum likelihood of being correct in a statistical sense for the data set given. For practical purposes of interest we can say that the maximum likelihood of a parameter value can be obtained by minimizing the quadratic devia­ tions of the fitting function from the data. Taking into account the non-linear nature of the prob­ lems this is called non-linear least squares minim­ ization. Although in a strict mathematical sense the assumption that the maximum likelihood value of a parameter is found by a least squares minimization is not generally valid, this neverthe­ less is a useful and practical definition. We thus define a function (called the reduced which has to be minimized using appropriate me­ thods in order to determine the optimal values for the elements of the parameter set

In this equation the double summation runs over and all data points of all experiments an individual experiment. The function denotes the value of the fitting function for a particular parameter set at the position qi while denotes the standard deviation from the mean of the particular data point N is the total number of data points in the fit, while m denotes the total number of independent fit parameters. Numerous algorithms exist for the minimiz­ ation of the least squares function and the reader is referred again to numerical mathematics and data analysis text books (Bevington, 1969). These methods are usually iterative methods, i.e. one starts with some initial estimate for a parameter

84

value and computes in a next step corrections this decreases. parameter set such that the This operation in repeated until the minimal value has been found, i.e. until the method con­ verges. The most common procedure is the Levenberg–Marquardt procedure (Marquardt, 1963) which involves a combination of the well-known Gauss–Newton method and the steepest-descent method. The steepest descent method only determines the slope of the with respect to Thus it has the the uncorrelated parameters advantage of converging quickly if the parameter estimate is far from the optimum where the slope of the surface is large. Coming closer to the mini­ mum, this procedure either fails or at least shows very poor convergence. The Gauss–Newton me­ thod on the other hand takes into account the correlation between the different parameters. It converges well if close to the minimum, but has difficulties if the solution is still far from the mini­ mum. Combining the two methods and smoothly switching from steepest descent to Gauss–Newton as the minimum is approached is achieved by the Levenberg–Marquardt method. Thus it combines the advantages of both simpler methods while avoiding their difficulties. An intricate problem involved in most minimization methods is illustrated in Fig. 5 for a singleparameter case. Starting at some point on the surface the minimization procedure should allow one to find the optimal parameter value at the

Alfred R. Holzwarth absolute (global) minimum of the (re­ in general is a hyper­ member that the surface of dimension p + 1 when p is the number is smooth, of parameters). If the the minimal value can usually be found without difficulties. If there are local minima on the way however (see Fig. 5), the minimization algorithm might well converge into such a local minimum from where it can not recover. This is true for essentially all non-linear minimization algorithms that are based on derivatives of the or the fitting function. Worst of all, for a complex multidimensional the chances of falling into a local minimum may be quite high. Fig. 5 also illustrates that falling into a local minimum might be prevented by the choice of different starting points for the iteration. This usually helps for fitting problems with a relatively small number of parameters but becomes increasingly difficult and computer time-consuming for a large par­ ameter set. One has to realize that in practice one can not have the guarantee to find the local by using any minimum of a complex of the iterative methods and one has to be well aware of that problem. A vast literature exists on the use of methods other than the Levenberg–Marquardt method or even non-least-squares methods in the analysis of kinetic data (Jennrich and Ralston, 1979; Livesey et al., 1988). Despite many claims to the contrary it is however fair to say that, with the exception of the local minimum problem, which is however inherent to the other methods as well, in the overwhelming number of cases the Levenberg– Marquardt represents a well-suitable and reliable method for non-linear parameter optimization. Thus no really good justification exists to apply other methods except in cases where the local minimum problem becomes severe. If this is the case iterative minimization schemes based on de­ or other fitting func­ rivatives of the tions have to be abandoned entirely. A new and powerful method has been developed that does not suffer from the local minimum problem. This is the so-called group of “genetic code algo­ rithms” (Goldberg and Richardson, 1987; Gold­ berg et al., 1989). To my knowledge so far hardly any applications of these algorithms to data analy­ sis in time-resolved spectroscopy exist (see e.g. Trinkunas and Holzwarth, 1994b) but an increas­

Data analysis of time-resolved measurements ing number of applications are anticipated in the near future.

D. Importance of Proper Weighting Factors Eq. (6) contains the standard deviation of the data point qi. The inverse standard deviation in this equation provides the weighting factor with which the particular data point qi contributes The standard deviation is a to the measure of the noise in the data. Often in non­ linear fitting problems this weighting factor is set to one, resulting in an equal weighting of all data points and thus ignoring the fact that experi­ mental points in general have different accuracy. It is highly important to note that by doing so one deliberately introduces a systematic error into the non-linear parameter optimization resulting in poor convergence, poor accuracy and in many cases even wrong results. In any case the cap­ ability to resolve complex fitting problems is im­ peded. Therefore the use of proper weighting (Eq. factors in the definition of the (6)) is absolutely required for a correct data analysis. The problem is serious already for a single decay but becomes even more severe if multiple experiments are being combined in a global analysis scheme. The interested reader is referred to the article by di Cera (1992). For practical purposes of fluorescence and transient absorption spectroscopy the standard deviations are often easily defined. In a singlephoton counting experiment the noise is of Pois­ sonian nature and thus the standard deviation is given by the square root of the number of counts in a channel. In a transient absorption experi­ ment measuring small absorption differences, the standard deviation is usually the same for all data points in a single experiment (constant noise inde­ pendent of the signal). In the latter case the weighting could be ignored if analyzing a single experiment only. However if several experiments are being combined in a global analysis each of them might have a different standard deviation and the proper weighting factors must again be applied. For other kinds of experiment often the noise on the data is not so easily known. In order to get the proper weighting factors one must try to get as close an estimate of the standard devi­ ation of the measured signals however.

85 Finally it is noted without proof here that the type of noise present in a signal (poissonian, con­ stant noise, signal-dependent etc.) determines and limits the degree of improvement that can theoretically be achieved by the use of global analysis (including global physical model testing approaches) (see Fig. 2). This is quite generally the case and is independent of the amount of noise that is present. In any case, however, the maximal improvement can only be expected if a proper weighting scheme is applied.

E. Importance of Error Analysis Surprisingly, even in the most sophisticated data analysis the error analysis is often ignored entirely or is carried out on a minimal and thus often useless level. This happens despite the fact that it is well-known that a value of a measured or derived quantity is only as good as the knowledge about its confidence interval. The reason for this negligence is that in most cases the estimation of a reliable and realistic confidence interval of a parameter in a complex multiparameter model can easily consume more manpower and com­ puter resources than the actual parameter optim­ ization itself. Given this situation any systematic attempt of getting even a sub-optimal estimate of a confidence interval is better than none. Very reliable methods exist, among which the Monte Carlo method is the most prominent (Straume et al., 1992). However the tremendous amount of computer time that it requires usually makes it a non-method for the error analysis of complex models. Without spending very much additional computing time the matrix procedures involved in the Levenberg–Marquardt method allow one to calculate the so-called variance–covariance matrix (Johnson et al., 1992) and get the confi­ dence intervals from its diagonal elements. The problem is, however, that the confidence intervals estimated in this way are usually by far too small since the correlation between different par­ ameters is not taken into account properly. Fur­ thermore the confidence interval is assumed to be symmetric to both sides of a parameter value which is usually not the case in a non-linear model. Quite on the contrary, the confidence in­ terval will in general be highly non-symmetric about the most likely value (Johnson, 1983). A

86 much better, and from the point of computer time still acceptable, method is the exhaustive search method. This method searches the form of the surface in the immediate vicinity of the optimal values of all parameters. This is done by stepwise carrying away a parameter from its optimal value and, using the same algorithm as has been used for the optimization, to minimize the by letting the other parameters compensate the disturbance introduced by carrying away one par­ ameter from its optimal value. This procedure is repeated several times for different values of a particular parameter, making deviation to both sides of the optimal value. As a criterion for the maximal deviation one defines a certain absolute increase in the value of the This procedure is carried out successively for each par­ ameter. The estimates to both sides of the optimal value where the has increased by the same pre-defined amount then define the confi­ dence interval for that parameter. The advantage of this procedure is that is can be implemented easily without writing much new code and that it takes full account of the correlations between parameters. Such an analysis has been carried out by Roelofs et al. (1992) for the case of a target analysis of a fluorescence lifetime experiment on higher plant thylakoids. The outcome showed that the confidence interval for certain par­ ameters was by orders of magnitude larger than the error estimate from the variance–covariance matrix and that it was in fact highly non-symmetric. The analysis also showed large differences in percentage error between different par­ ameters. This may serve as an example that error analysis is indeed important and essential for dis­ tinguishing different models on the basis of a given set of experiments. Additional methods are described by Johnson et al. (1992).

F. The Identifiability Problem The advantages of physical/chemical model test­ ing vs. mathematical data fitting have been po­ inted out in paragraph II.B of this chapter. There is one important limitation however, that has not been discussed sufficiently so far, i.e. the socalled identifiability problem. This deals wilth the possibility that a given set of experimental data might not contain enough information to extract

Alfred R. Holzwarth all the physical parameters that are contained in a physical model to be tested. It is important to realize that this has nothing to do with the accur­ acy of the data set (a low accuracy would simply lead to a larger confidence interval). Rather this is an inherent mathematical or information theo­ retical problem. Let us take our simple example of a two-component mixture with energy transfer. Suppose we have measured the fluorescence kin­ etics as a function of emission wavelength. We now want to extract the rate constants and the SAES by kinetic modelling. The model contains four rate constants and two SAES. Even in this simple case not all four rate constants can be determined but only three of them. The other one must be fixed on some assumed or otherwise determined value. It is easy to see even for the mathematically less experienced reader why this limitation exists. The experiment determines only two lifetimes and, at each detection wavelength, two amplitudes. Since the fluorescence ampli­ tudes are usually not measured as absolute values but only as relative numbers (due to the presence of unknown instrumental proportionality fac­ tors), for each detection wavelength there exist only three independent parameters, two lifetimes and one (relative) amplitude. The other ampli­ tude is a dependent one, i.e. it is the difference from 100%. Thus not enough linearly indepen­ dent equations exist in order to determine four rate constants. If one were to allow for freely adjustable rate constants in the fit, a mathema­ tical solution (good fit) would definitely result. However, the fit would not be unique and three of the rate constants would be parametrically de­ pendent on the fourth one. This means that the system is underdetermined and the fitting result in the rate constants would be to a large part arbitrary (though entirely within a mathema­ tically good description of the data set). This may serve as an example that it is abso­ lutely necessary in all cases of model testing to carefully investigate the identifiability conditions and to avoid carrying out a target analysis with an underdetermined model. For more complex models the identifiability analysis may be a tedi­ ous and not easily accomplished task. The space available here is not sufficient however to discuss this problem in more detail and the reader is

Data analysis of time-resolved measurements referred to the papers by Ameloot et al. (1986; Ameloot and Hendrickx, 1982) instead. III. Some Applications to Photosynthesis We will now give some practical examples from photosynthetic systems that are meant to provide more insight into the capability of the procedures and the kind of answers that may be expected. The data sets underlying the two examples given here have been analyzed thoroughly using both global lifetime analysis as well as global target analysis methods. Both examples have in com­ mon furthermore that only target analysis was capable of determining the underlying mechan­ isms and physical models properly. In the first example on the basis of global lifetime analysis alone one would have arrived at a completely wrong mechanism.

A. Purple Bacterial Energy Transfer The antenna systems of many purple bacteria consists of two pools, i.e. the inner core antenna pool B875 (LHI) and the peripheral antenna pool B800–850 (LHII) (van Grondelle et al., 1994) . Based mainly on transient absorption studies a sequential scheme of energy transfer (see Fig. 6 scheme I) had been proposed in the literature (Sundström et al., 1986). A key feature of this model is a quite slow energy transfer of about 35–40 ps between the LHII and the LHI pools. The global lifetime analysis of fluorescence life­ time data was in good agreement with such a model (Müller et al., 1993). Interestingly the glo­ bal target analysis, based on this model, gave a perfect fit to the data as well (Müller et al., 1993). However, inspection of the resulting rate con­ stants for the sequential model (Scheme I, Fig. 6) revealed that this kinetic model was entirely meaningless in physical terms since the ratio This ratio would have violated strongly the Boltzmann principle, given the fact that there are about 24 B875 BChls per P860. A ratio of about 1/30 was expected instead on the basis of the Boltzmann factor, i.e. about three orders of magnitude less than found in the target analysis of scheme I (Müller et al., 1993). We thus have the interesting situation that the target analysis of one particular model indicates perfect

87 fit with the data. Nevertheless the model is wrong. This conclusion could be arrived at only upon careful consideration of physical principles not explicitly imposed in the kinetic model (other models were also tested but were formally not in good agreement within the limitations of the tar­ get analysis). Thus a different kinetic scheme (shown in Fig. 6, scheme II) had to be searched for which would describe the data set well. Scheme II represents a heterogeneous model, as­ suming that there are two types of differently behaving reaction centers but the same antenna system. One such possibility was that the two types of reaction center would undergo charge separation with different rates, i.e. open and reduced closed reaction centers. This model (Müller et al., 1993) explained the data perfectly well without leading to any conflicts with other physical principles. Furthermore it is also in good agreement with other knowledge about this sys­ tem. Note that in addition the two parts of the parallel scheme II are linked by assuming equa­ transfer (rate tion rate constants for constants This is also reasonable since any change in the redox state of the RC is not expected to influence the antenna energy transfer rates. This application of target analysis and critical checking of its predictions were essen­ tial to exclude the wrong model.

B. Photosystem I Kinetics Fig. 7 shows different compartment models (com­ partments are antenna pools 1–3) for the core antenna (RC complex of photosystem I from cy­ anobacteria (the same schemes should hold in principle for the corresponding higher plants core antenna complex) (Holzwarth et al. 1993). The kinetic models 7A and 7C contain two antenna pools each. However the RC and thus charge separation is coupled to these pools in two differ­ ent ways: in 7A the RC is strongly coupled with (and is in fact part of) the small pool of longwave (red) Chl a, while in model 7C it is coupled to the main pool. Model 7B contains 3 antenna pools. This model, although theoretically reason­ able, was already too complex given the details and resolution of the experimental data sets (two lifetime components were found by global life­ time analysis in fluorescence and three in transi­

88

ent absorption). For this reason model 7B had to be discarded from the analysis since it predicts at least 3 lifetime components for fluorescence and 4 components for transient absorption. However at a somewhat higher time resolution of the ex­ perimental data one could try to test this model again. Both models 7A and 7C were tested exten­ sively on the fluorescence and the transient ab­ sorption data set. Again both models gave a (mathematically) perfect fit to the data. The re­ sulting SAES (from the fluorescence data) and the SADS (from the transient absorption data) are shown in Fig. 8 for each model. The fluor­ escence data result in two SAES, one each for each antenna pool, and the transient absorption data in three SADS, one for each antenna pool and an additional one for the primary radical pair. The latter cannot be observed in fluorescence. Clearly only one of these models could be cor­ rect. Further analysis of the SAES and SADS revealed that, despite the good fit for both mod­ els, in fact only model 7A was physically reason­ able while model 7C had to be discarded. The

Alfred R. Holzwarth

reason for this is that in the definition given in Eq. (5) the area under an SAES is directly pro­ portional to the radiative rate constant of the corresponding excited state. Since in PS I only Chls a emit, the areas should be quite similar. Likewise the SADS give directly the excitedground state difference extinction coefficients. Again these should be roughly similar in strength for the various excited Chl pools. These con­ ditions are fulfilled only for model 7A but not for 7C, however, as can be seen from Fig. 8. Further analysis also revealed that model 7C would viol­ ate the Boltzmann principle. In further analysis data about the pool sizes could be estimated from the target data which were in very good agree­ ment with a decomposition of the absorption spectrum (Holzwarth et al., 1993; Trinkunas and Holzwarth, 1994a). We are thus again faced with a situation where more than one model gives a perfect fit even in target analysis. However only one of the models is physically reasonable. Such a situation is quite common in such kind of analysis. In contrast glo­

Data analysis of time-resolved measurements

bal lifetime analysis could not distinguish between these cases. This demonstrates clearly the strength of global target analysis. IV. Conclusions The principle and some applications of various kinds and levels of data analysis of time-resolved data have been presented and some of the pitfalls have been pointed out. It has been demonstrated that the physical interpretation of a given data set may very well depend on the proper appli­ cation of sophisticated data analysis procedures. The importance of such methods will definitely grow with the complexity of the systems under study and the increasing detail at the molecular level at which answers are being sought.

89 Questions are often asked by novices in the field of data analysis: How many lifetime compo­ nents can be separated, where is the limit, are you not trying to resolve too many components? These questions often imply that any analysis in terms of more than two or, in rare cases, perhaps three lifetime components is either impossible or would involve even some kind of unreliable “guessing”. The number of lifetime components that can be separated depends on many par­ ameters and details of both the system under study as well as on the details of the experiment. Thus a general answer to that question can not be given! Rather any specific answer would be limited to a particular case and can only be given after a careful consideration of the details of the experiment and the knowledge of which different kind of models one would like to or ought to be able to distinguish. One important consequence of this situation is that it is advantageous to use the knowledge about the details of various data analysis methods already in the planning of an experiment in order to ensure optimal results. Furthermore it is always wise in case of a complex system to perform numerical simulations for vari­ ous situations, thus trying to match as closely as possible the simulated data to the experimental ones. Then the same analysis methodology should be used to estimate the parameters and (equally important) their errors for both the experimental and the simulated data sets. Doing so one gains a detailed knowledge about the capabilities and limitation(s) of the applied procedures for that particular case. There is of course absolutely no guessing in­ volved in these procedures, as is occasionally im­ plied by novices. If the principles and rules are followed properly, knowing the limitations, the results are absolutely reliable. It is furthermore fair to note that the principal procedures de­ scribed here are applicable as well to data analysis problems outside kinetic spectroscopy. Finally I should like to mention a couple of important ad­ ditional papers from research areas outside pho­ tosynthesis where similar analysis procedures as treated here have been described and/or applied in a highly fruitful fashion (Nagle, 1991; Be­ echem, 1988; Lakowicz et al. 1984; Beechem et al. 1991; Christian et al. 1981; Halvorson 1981; Hessling 1992). The interested reader is referred

90

Alfred R. Holzwarth

Data analysis of time-resolved measurements to these papers if he/she wants to get more deeply involved in global data analysis procedures.

Acknowledgements I would like to thank all my colleagues who have over the past years worked with me and who have been involved in the development and the application of the data analysis procedures de­ scribed here. Of those I would particularly like to mention Mrs. Iris Martin, a mathematician and computer programmer who has worked closely with me in the development of particular data analysis code, and Dr. Marc Müller who has spent much time on the computer developing and test­ ing many complex data analysis cases and finding out about their capabilities and limitations.

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91 W (1992) Primary electron transfer kinetics in bacterial reaction centers with modified bacteriochlorophylls at the monomeric sites BA,B. Proc Natl Acad Sci USA, 89:9514– 9518. Goldberg DE and Richardson J (1987) Genetic algorithms with sharing for multimodal function optimization. Genetic algorithms and their applications, Proc 2 Int Conf on Gen­ etic Algorithms: 41–49. Goldberg DE, Korb B, and Deb K (1989) Messy genetic algorithms: Motivation, analysis, and first results. Complex Systems 3:493–530. Halvorson, HR (1981) Determining the number of interacting species: Significant factor analysis. Biophys Chem 14:177– 184. Hessling, B, Souvignier, G, and Gerwert, K (1992) A New Approach to Analyse Kinetic Data of Bacteriorhodopsin, Factor Analysis and Decomposition. In: Structures and Functions of Retinal Proteins 221:155–158 . Holzapfel W, Finkele U, Kaiser W, Oesterhelt D, Scheer H, Stilz HU, and Zinth W (1989) Observation of a bacterioch­ lorophyll anion radical during the primary charge separ­ ation in a reaction center. Chem Phys Lett 160:1–7. Holzwarth AR (1987) A model for the functional antenna organization and energy distribution in the photosynthetic apparatus of higher plants and green algae. In: Biggins J (ed) Progress in Photosynthesis Research. 1, Nijhoff Pub­ lishers, Dordrecht, pp 53–60. Holzwarth AR (1988) Time resolved chlorophyll fluorescence. What kind of information on photosynthetic systems does it provide?. In: Lichtenthaler HK (ed) Applications of Chlorophyll Fluorescence, Kluwer Academic Publishers, Dordrecht, pp 21–31. Holzwarth AR (1990) The functional organization of the antenna systems in higher plants and green algae as studied by time-resolved fluorescence techniques. In: Baltscheffsky M (ed) Current Research in Photosynthesis. II, Kluwer Academic Publishers, Dordrecht, pp 223–230. Holzwarth AR, Wendler J, and Suter GW (1987) Studies on chromophore coupling in isolated phycobiliproteins. II Picosecond energy transfer kinetics and time-resolved flu­ orescence spectra of C-phycocyanin from Synechococcus 6301 as a function of the aggregation state. Biophys J 51:1– 12. Holzwarth AR, Schatz G, Brock H, and Bittersmann E (1993) Energy transfer and charge separation kinetics in photosys­ tem I: 1 Picosecond transient absorption and fluorescence study of cyanobacterial photosystem I particles. Biophys J 64:1813–1826. Jennrich RI and Ralston ML (1979) Fitting nonlinear models to data. Ann Rev Biophys Bioeng 8:195–238. Johnson ML (1983) Evaluation and propagation of confidence intervals in nonlinear, asymmetrical variance spaces. Analysis of ligand-binding data. Biophys J 44:101–106. Johnson ML and Faunt LM (1992) Parameter estimation by least-squares methods. In: Brand L and Johnson ML (eds) Methods in Enzymology 210 Numerical Computer Me­ thods. Academic Press, San Diego, pp 1–37. Knorr FJ and Harris JM (1981) Resolution of multicomponent fluorescence spectra by an emission wavelength-decay time data matrix. Anal Chem 53:272–276.

92 Knutson JR, Beechem JM, and Brand L (1983) Simultaneous analysis of multiple fluorescence decay curves: A global approach. Chem Phys Lett 102:501–507. Lakowicz JR, Laczko G, Cherek H, Gratton E, and Limke­ man M (1984) Analysis of fluorescence decay kinetics from variable frequency phase shift and modulation data. Bi­ ophys J 46:463–477. Livesey AK and Brochon J-C (1988) Maximum entropy data analysis of dynamic parameters from pulsed-fluorescent de­ cays. In: Harding SE and Rowe AJ (eds) Dynamic Proper­ ties of Biomolecular Assemblies, Royal Society of Chemis­ try, pp 135–147. Löfroth J-E (1986) Time-resolved emission spectra, decayassociated spectra, and species-associated spectra. J Phys Chem 90:1160–1168. Marquardt DW (1963) An algorithm for least-squares esti­ mation of nonlinear parameters. J Soc Ind Appl Math 11:431–441. Mukerji I and Sauer K (1993) Energy transfer dynamics of an isolated light harvesting complex of photosystem I from spinach: Time-resolved fluorescence measurement at 295K and 77K. Biochim Biophys Acta 1142:311–320. Müller MG, Griebenow K, and Holzwarth AR (1991) Primary processes in isolated photosynthetic bacterial reaction cen­ ters from Chloroflexus aurantiacus studied by picosecond fluorescence spectroscopy. Biochim Biophys Acta 1098:1– 12. Müller MG, Griebenow K, and Holzwarth AR (1992) Primary processes in isolated bacterial reaction centers from Rhodo­ bacter sphaeroides studied by picosecond fluorescence kin­ etics. Chem Phys Lett 199:465–469. Müller MG, Drews G, and Holzwarth AR (1993) Excitation transfer and charge separation kinetics in purple bacteria: 1. Picosecond fluorescence of chromatophores from Rho­ dobacter capsulatus wild type. Biochim Biophys Acta 1142:49–58. Nagle JF (1991) Solving complex photocycle kinetics. Theory and direct method. Biophys J 59:476– 487. Roelofs TA and Holzwarth AR (1990) In search of a putative long-lived relaxed radical pair state in closed photosystem II. Kinetic modeling of picosecond fluorescence data. Bi­ ophys J 57:1141–1153. Roelofs TA, Gilbert M, Shuvalov VA, and Holzwarth AR (1991) Picosecond fluorescence kinetics of the D1–D2– cyt-b559 photosystem II reaction center complex. Energy

Alfred R. Holzwarth transfer and primary charge separation processes. Biochim Biophys Acta 1060:237–244. Roelofs TA, Lee C-H, and Holzwarth AR (1992) Global target analysis of picosecond chlorophyll fluorescence kin­ etics from pea chloroplasts. A new approach to the charac­ terization of the primary processes in photosystem II alfaand beta-units. Biophys J 61:1147–1163. Schatz GH, Brock H, and Holzwarth AR (1988) A kinetic and energetic model for the primary processes in photosys­ tem II. Biophys J 54:397–405. Straume M and Johnson ML (1992) Monte Carlo method for determining complete confidence probability distributions of estimated model parameters. In: Brand L and Johnson ML (eds) Methods in Enzymology. 210 Numerical Com­ puter Methods, Academic Press, San Diego, pp 117–129. Sundström V, van Grondelle R, Bergström H, Akesson E, and Gillbro T (1986) Excitation-energy transport in the bacteriochlorophyll antenna systems of Rhodospirillum ru­ brum and Rhodobacter sphaeroides, studied by low-intensity picosecond absorption spectroscopy. Biochim Biophys Acta 851:431–446. Suter GW and Holzwarth AR (1987) A kinetic model for the energy transfer in phycobilisomes. Biophys J 52:673 – 683. Trinkunas G and Holzwarth AR (1994a) Kinetic modeling of exciton migration in photosynthetic systems. 2 Simulations of excitation dynamics in two-dimensional photosystem I core antenna/reaction center complexes. Biophys J 66:415– 429. Trinkunas G and Holzwarth AR (1994b) Modelling of energy transfer in photosystem I using genetic algorithm. Liet Fiz Zurn 34:287–292. van Grondelle R, Dekker JP, Gillbro T, and Sundström V (1994) Energy transfer and trapping in photosynthesis. Bi­ ochim Biophys Acta 1187:1– 65. Weidner R and Georghiou S (1990) Global methods for timeresolved and steady-state fluorescence and steady-state ab­ sorption spectroscopy. In: Lakowicz JR, (ed) Time-Resolved Laser Spectroscopy in Biochemistry II, Proc of SPIE, SPIE, Bellingham, pp 717–726. Wendler J and Holzwarth AR (1987) State transitions in the green alga Scenedesmus obliquus probed by time-resolved chlorophyll fluorescence spectroscopy and global data analysis. Biophys J, 52:717–728. Wendler J, John W, Scheer H, and Holzwarth AR (1986) Energy transfer kinetics in trimeric C-phycocyanin studied by picosecond fluorescence kinetics. Photochem Photobiol 44:79–85.

Chapter 6

Photosynthetic Thermoluminescence as a Simple Probe of

Photosystem II Electron Transport

Yorinao Inoue

Solar Energy Research Group, The Institute of Physical and Chemical Research (RIKEN),

Wako, Saitama 351–01, Japan

Summary I. Introduction II. Origins of TL from Photosynthetic Apparatus A. Processes Involved in Photosynthetic TL 1. Historical Aspects and Basic Phenomenology 2. Formation and Stabilization of Charge Pairs in PSII 3. Emission of TL by Thermally Activated Recombination (Relationships with Delayed Fluorescence) 4. Measurements and Analyses a. TL Setup and Measurements b. Simulation of Glow Curves c. Analysis of Oscillations in TL Intensity B. Assignments of TL Bands to Charge Pairs 1. The B-band 2. The Q-band 3. The A-band 4. The

5. The

6. The C-band 7. The Z-band 8. The TL-bands at Temperatures Below 77 K III. Application of TL as a Probe of PSII Photochemistry A. Acceptor Side of PSII B. Donor Side of PSII IV. Perspective: Merits and Demerits of TL Technique Acknowledgements References

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Summary The measument of photosynthetic thermoluminesence (TL) is a low-cost high-performance technique. A minimum TL setup consisting of a cooled, red-sensitive head on-type photomultiplier, high-voltage power supply, X-Y recorder, heater-installed sample holder, Dewar flask, constant voltage power supply, thermocouple and a small flash device provides one with unique and suggestive data concerning the electron transport on both the donor and acceptor sides of photosystem II. If necessary, the setup can be graded up by use of a photon counter, digital thermometer and a computer-assisted data acquisition/analysis system. The technique is not always good at determining the absolute values of physical parameters, but is Correspondence: Fax: 81-48-4624685; E-mail: r19600%rkna50.riken.go.jp

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J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 93–107. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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Yorinao Inoue

good at detecting weak alterations in PSII functioning induced by some treatment or mutation, especially those manifest only in higher S-states. Measurements can usually be done with small amounts of algal whole cells, implying its useful application in screening or preliminary characterization of genetically engineered mutants. The chapter deals with its recent applications together with its basic phenomenology including assignments of respective emission bands. Abbreviations: D1 – the 32 kDa protein of PSII reaction center; D2 – the 34 kDa protein of PSII reaction center; DCMU – 3-(3,4-dichlorophenyl)-1,1-dimethylurea; LHC – light-harvesting chlorophyll a/b complex; P680 – the reaction center chlorophyll of PSII; Phe – Pheophytin, the primary electron – the primary acceptor of PSII; PQ – plastoquinone; PSI –.photosystem I; PSII – photosystem II; – the secondary acceptor quinone of PSII; – – redox states of the acceptor quinone of PSII; tetranuclear-Mn in water-oxidizing enzyme; TL – thermoluminescence; – auxiliary electron donor to P680, Tyr 160 residue of D2 protein; – regular electron donor to P680, Tyr 161 residue of D1 protein.

I. Introduction Thermoluminescence (TL) is an outburst of light emission occurring at characteristic temperatures when organic or inorganic materials preillumi­ nated at low temperatures are warmed gradually in darkness. This phenomenon is common for light-responsive semiconductors, and is known to arise from thermally activated recombination of the electrons and positive holes that are gen­ erated by photoreactions and trapped or stabil­ ized in frozen states by low temperature. In plant materials, the outburst of light occurs at several temperatures, showing variously shaped, complicated glow curves consisting of several emission bands depending on the con­ dition of preillumination. Through extensive studies during 20 years, many of these photosyn­ thetic TL components were separated and charac­ terized in detail. It has now been firmly estab­ lished that most of the photosynthetic TL components arise from the reversal of light-driven charge separation in photosystem II (PSII) through a thermally activated recombination be­ tween the positive charges accumulated in the intermediates of the water oxidation system on its donor side and the negative charges stabilized or secondary quinone ac­ on primary ceptors on its acceptor side (for reviews see Inoue and Shibata, 1982; Sane and Rutherford, 1986; Vass and Inoue, 1992). The assignments of charge pairs responsible

for respective TL components have now enabled us to utilize TL as a simple but effective probe of PSII electron transport that provides unique and valuable information on both sides of PSII. In this chapter, we will review the current knowl­ edge about photosynthetic TL and discuss some possible new research areas to which TL tech­ nique can be applied.

II. Origins of TL from Photosynthetic Apparatus

A. Processes Involved in Photosynthetic TL 1. Historical Aspects and Basic Phenomenol­ ogy TL from plant material was first observed by Ar­ nold and Sherwood (1957) subsequent to the dis­ covery of delayed luminescence from chloroplasts by Strehler and Arnold (1951) (see also Arnold, 1991). In their early studies, they have already indicated that the glow curve from plant materials consists of several TL components having differ­ ent emission temperatures, indicative of particip­ ation of more than two species of charge pairs in photosynthetic TL. They have also indicated that these TL components originate mostly from PSII, but not from PSI, although detailed characteriz­ ation of the phenomena had to await assignment

Thermoluminescence, a specific probe of PSII of respective charge pair species responsible for each TL component. The fact that the positive charges accumulating in the oxygen-evolving system of PSII are in­ volved in the phenomenon of photosynthetic TL was first suggested by observations that darkgrown gymnosperm leaves, angiosperm leaves greened under widely-spaced intermittent flashes or algal cells grown in Mn-deficient medium do not exhibit major TL bands, unless their latent water-oxidation systems are photoactivated by exposure to continuous or shortly-spaced sequen­ tial flashes (Ichikawa et al., 1975; Inoue, 1976; Inoue et al., 1976). This suggestion was further confirmed by the finding that the intensity of major TL band exhibits a period-four oscillation (Inoue and Shibata, 1977a). This finding subse­ quently led to assignment of specific S-state inter­ mediates as the positive charge carriers responsi­ ble for major TL bands (Rutherford et al., 1982), and consequently opened up recent applications of TL for studying the roles of extrinsic proteins and other cofactors in normal or modified turn­ overs of the S-state system. As to the negative charge carriers, the first indication of the involvement of two acceptor quinones of PSII was obtained in an early work by Rubin and Venediktov (1969), who observed a clear interconversion between the two TL bands by treatment with DCMU. This observation was subsequently extended by Demeter et al. (1985a) by studying herbicide-resistant mutant plants. They observed a clear downshift of TL peak tem­ perature in the mutant plants, indicating that a properties oc­ significant alteration in curs when some amino acids are replaced in the D1 protein. Supported by these as basic findings, photosyn­ thetic TL has now become widely used as a simple but efficient probe of PSII functioning not only for biophysical purposes but also for assisting gen­ etical and environmental studies. 2. Formation and Stabilization of Charge Pairs in PSII PSII reaction center consists of four membrane proteins, D1, D2, cytochrome b-559 and the product of psbI gene, in which the functional

95 prosthetic chromophores and acceptor quinone molecules are believed to be housed in a sym­ metric way like in the reaction center of nonsulfur purple bacteria. However, in order to re­ tain the functional Mn-cluster and thereby the ability of water oxidation, several more mem­ brane proteins and one extrinsic protein are re­ quired to be associated with the reaction center (Fig. 1). Absorption of a photon by chlorophyll in PSII results in excitation of the primary electron donor, P680, which elicits fast charge separation between P680 and Phe, the primary acceptor of PSII, within a few ps. The charge separated state is then stabilized in about 300 ps by rapid electron the primary one-electransfer from to tron acceptor quinone of PSII, which is then fol­ lowed by further transfer of the electron to the secondary two-electron acceptor quinone of Reduced is stable PSII in for tens of seconds or even to hours at room temperature and constitutes the major trap of negative charges in photosynthetic TL phenom­ does not, because of its short life, ena, while unless the electron transport between and is blocked by some means, e.g. by herbicides. Upon the second photochemistry in the same re­ action center, is further reduced to but this species is readily replaced by a plastoquinone (PQ) of the pool and does not participate in pho­ tosynthetic TL under normal conditions. Stabilization of the charge separated state oc­ curs also on donor side of PSII. The cation radical is reduced by Yz, the Tyr 161 of P680, residue of D1 protein, and is then reduced by one electron transport from the Mn-cluster of the water-oxidizing apparatus, which is believed to consist of four Mn atoms. The Mn-cluster is known to assume five redox states denoted Si (i = and as the lowest and highest 0–4) with oxidation states, respectively. Repeated photoch­ emistry in PSII reaction center advances the Sstates by one step each, and abstraction of four electrons from two molecules of water finally re­ Among sults in release of one molecule of the five intermediate S-states, and are stable in dark-adapted condition, but they do not parti­ cipate in TL because they do not carry any posi­ and carry a positive tive charge, whereas equivalent (see later) and are stable for tens of

96

seconds at room temperature, so that they consti­ tute the major source of positive charges for pho­ tosynthetic TL. The proton release pattern coupled with the cycling of the S-state system has been postulated to be 1,0,1,2 based on early works (Fowler, 1977; Saphon and Crofts, 1977; Forster and Junge, 1985), but this has recently been disputed. As far as the photosynthetic TL is concerned, however, the above classical pattern well explains varieties of oscillatory phenomena of photosynthetic TL so far known. Based upon this proton release pattern, the stable S-state species that carry a positive equivalent and contribute to photosyn­ thetic TL are believed to be the and Beside Yz, another auxiliary secondary donor to P680 is known; the redox active Tyr 160 re­ Its oxidized sidue of D2 protein denoted as form known as EPR Signal IIs is stable in dark­ ness at room temperature, and has been shown recently to participate in TL (Demeter et al.,

Yorinao Inoue

1993; Krieger et al., 1993; Johnson et al., 1994). It has not been clearly established, on the other hand, whether or not can be stabilized to participate in photosynthetic TL. Cytochrome b­ 559 and some of the chlorophyll molecules sur­ rounding the reaction center of PSII are known to be photo-oxidized under some conditions. However, their oxidation products do not seem to constitute the trap of positive charges for TL, although their formation by reaction center pho­ tochemistry can provide electrons to reduce (or instead of the Mn-cluster, and thereby indirectly modulate the TL phenomenon. 3. Emission of TL by Thermally Activated Recombination (Relationships with Delayed Fluorescence) From the first report of the phenomenon, TL and delayed fluorescence have been considered to share a common origin (Arnold, 1991), a radi­

Thermoluminescence, a specific probe of PSII

ative release of stored energy upon recombi­ nation between stabilized donor and acceptor at a photochemical reaction center. In PSII, the pri­ mary product of light-induced charge separation is a singlet radical pair, which rap­ idly proceeds to stabilization of positive and nega­ tive equivalents as forms of oxidized donor and regenerates a and reduced acceptor ground state reaction center. During this course, the major part of the energy captured by the light reaction is stored in the system in the form of redox potential difference, whereas a part of the energy is lost (stabilization energy), which results in trapping of the separated charge pair being provided with a barrier of activation energy against recombination. When this free energy loss is supplemented by thermal activation, the charge pair becomes capable of recombination to re-excite P680, resulting in photosynthetic TL emission from P680* or chlorophyll molecules present nearby (Fig. 2).

97

The process of recombination of is not always understood in detail, but it is generally considered to proceed through a series of equilib­ rations among various intermediate charge sepa­ rated states (DeVault et al., 1983; DeVault and Govindjee, 1990) to finally generate radical pair in singlet or triplet configuration. Among the radical pairs with two different spin configurations, the triplet ones recombine non­ radiatively to yield ground state P680, whereas the singlet ones are capable of generating singlet excited P680* (Van Gorkom, 1985), so that they can contribute to TL or delayed fluorescence. We may thus consider that delayed fluorescence (slow components) and TL are the two different ex­ pressions of the same process: the former is due to spontaneous recombination at a constant (usu­ ally low) frequency that is limited by the rate of thermal energy supply from the environment at a given temperature, while the latter is due to recombination at higher frequencies being accel­

98 erated with temperature rise during artificial heat­ ing. In fact, more or less the same period-four oscillation has been observed for both phenom­ ena, the intensity of 30-s component of room temperature delayed fluorescence and the height of TL B-band, when leaves are exposed to a series of short flash illumination (Rutherford et al., 1984). This has also been confirmed by analyses of emission spectra of delayed fluorescence (Hideg et al., 1991) and TL (Sonoike et al., 1991). 4. Measurements and Analyses

a. TL Setup and Measurements Photosynthetic TL can be recorded with a simple setup. As opposed to the measurement of delayed fluorescence, it requires no timing control system, neither a photomultiplier gating nor a transient recorder. The most essential factor is cooling the photomultiplier in order to increase the signal-tonoise ratio by eliminating the dark current. Use of a photon counter, a digital thermometer equipped with a linearizing ROM system and a personal computer for data acquisition/analysis are preferable, but they are not always essential. A small sample holder having small heat capacity is recommended to afford quick cooling after flash illumination. A dark-relaxed leaf disc or a piece of filter paper soaked with an aliquot of dark-relaxed sample suspension (ca. chlorophyll) is fixed on a sample holder in which a small heater (ca. 50 W) is installed, illuminated with a short duration at a constant tem­ flash(es) of a few perature (usually –40 to +25°C) and then quickly cooled by immersing the sample holder in liquid nitrogen. The cooled sample (with the holder) is then placed in a transparent Dewar flask, through which the image of the sample is focused onto the photomultiplier surface through a strong lens. The sample is then warmed gradu­ ally at a constant rate of 0.5 to by use of the heater installed in the sample holder, and the TL emission is measured with the photomul­ tiplier while recording the sample temperature with a thermocouple. TL intensity vs. sample temperature is recorded on an X-Y recorder, or if necessary, through a computer. With a sample

Yorinao Inoue holder of small heat capacity, it is not easy to obtain a constant heating rate, due mainly to heat energy supply from the inner wall of the Dewar flask. It is preferable to keep the wall temperature constant by some means. Due to this difficulty and others (see Devault et al., 1983), the peak temperatures reported from various laboratories are variant. However, detection of a few degrees shift in peak temperature is not difficult if a series of measurements are done on the same setup whose operation is carefully controlled to retain reproducibility. Single flash illumination or continuous light illumination at a low temperature gives only one TL component, whereas continuous illumination during cooling the sample in liquid nitrogen gives several components. Sometimes two illumi­ nations at two different temperatures (e.g. one at room temperature and another at 77 K) are useful in order to generate positive charges and negative charges separately (for this technique see sections II.B.3,7.).

b. Simulation of Glow Curves The kinetics of photosynthetic TL process has been described in various ways (Vass et al., 1981; DeVault et al., 1983; DeVault and Govindjee, 1990), basically according to the model conceived earlier for TL phenomena in solid state system. Among them, the one developed by Vass et al. (1981) is useful for practical applications, in which the complicated multiple equilibrations are tre­ ated as a single step first order process.

where T, k and B are measured parameters or a constant; TL intensity, absolute temperature, Boltzmann’s constant and heating rate, respec­ tively. By changing c (proportionality constant), (activation energy) and s (frequency factor) as fitting parameters, we can simulate a glow curve with sufficient accuracy to resolve the over­ lapping TL bands and also to determine the acti­ vation energy for each TL components. An ef­ ficient personal computer software for graphical and numerical analysis of TL glow curves has been developed (Ducruet and Miranda, 1992). These glow curve simulations provide us with the

Thermoluminescence, a specific probe of PSII

99 quinone acceptors on the acceptor side of PSII, we can compare the effect of a treatment on the TL peak temperature between and charge pairs. If the same or similar extent of shift is detected for both TL bands, we may conclude that the shift is due to a change in the redox potential of the positively charged donor species with no or slight effects on their negative counter­ part on the acceptor side (see section III).

parameters needed to calculate the half-life of a charge pair based on the equation below, which enables us to confirm the validity of these analy­ ses (Table 1).

Among these parameters derived from TL measurements, the peak temperature gives us the most useful information. However, the peak tem­ perature data so far reported for a TL component from various laboratories differ significantly. This is firstly due to a theoretical reason, the difference in heating rate used (DeVault et al., 1983), and secondly due to a technical reason, e.g. the differ­ ence in positioning of the thermocouple around the sample or sample holder. Despite the signifi­ cant differences, a shift in peak temperature in­ duced by treating the sample can easily and cor­ rectly be detected by comparing with an untreated control, when the same TL setup is used. An upshift in peak temperature of a TL band indicates that a larger activation energy is required for the charge pair to undergo recombi­ nation, indicative of induction of a deeper trap by the treatment, while a downshift indicates in­ duction of a shallower trap (Fig. 2). If we assume that the treatment employed has a selective effect on either of the donor or acceptor side of PSII, a shift in peak temperature of TL can be directly interpreted as indicating a change in redox poten­ tial of the positively or negatively charged spe­ cies, respectively: deeper and shallower traps on the donor side correspond to higher and lower redox potentials of the positively charged species, and those on acceptor side to lower and higher redox potentials, respectively. As a matter of fact, it is not always easy to restrict the effect of a treatment to one side of PSII. However, by taking advantage of the two

c. Analysis of Oscillations in TL Intensity In dark-adapted PSII, the redox pair is usu­ ally the most dominant species among the cen­ ters. When this center is exposed to a series of short flashes, the redox pairs expected after 1, 2, 3 and 4 flashes are and respectively. Since and are the positively charged species, as discussed in the previous sec­ tion, the probability of positive charge stabiliza­ tion on the donor side oscillates in a 1,1,0,0 pat­ tern, whereas the probability of negative charge stabilization on the acceptor side oscillates in 1,0,1,0, a binary pattern, due to the two electron quinone. Thus the gate mechanism of the probability of stabilization of both positive and negative charges in one PSII reaction center, which is a prerequisite for emission of TL, oscil­ lates in a 1,0,0,0 pattern. In actual PSII preparations, this pattern is per­ turbed by several factors: (i) initial distribution (ii) initial distribution of (iii) the of probability of misses and double hits and (iv) the ratio of TL yield between and charge recombinations. Of these factors, the factors (ii) to (iv) are mostly constant: misses and double hits are around 10% and 5%, respec­ tively, and the TL yield ratio of In contrast, the initial ratio varies depending on the sample and of relaxation conditions: 50:50 in thylakoids after brief dark-relaxation or in well dark-adapted in­ tact chloroplasts, whereas it is 25:75 in well darkrelaxed thylakoids or in PSII membrane frag­ ments, so-called BBY-type particles. Table 2 describes the expected changes in the number of centers capable of emitting TL, when dark-adapted PSII preparations having two typi­ ratios are illuminated with a cal initial to the centers series of flashes. Distribution of

100

in and is assumed even, and misses and double hits are neglected. The number of TLemitting centers shows maxima after the 1st and 5th flashes if the initial or after the 2nd and 6th flashes if the initial When misses and double hits and the difference in luminescence yield are taken into account, the oscillatory patterns depicted in Fig. 3 are ob­ tained. These patterns agree well with observed ones, supporting the mechanisms of TL oscil­ lation and in turn the assignments of the B-bands (Inoue, 1983; Demeter and Vass, 1984). Upon inhibitory treatment, depletion of some essential cofactors or mutations in PSII, the oscillation de­ viates from these standard patterns, which gives information about the functioning of the S-state electron gate in inhibited cycle and the or modified PSII.

B. Assignments of TL bands to charge pairs The TL components are distinguished by their respective emission bands on the glow curve that exhibit characteristic temperatures for maximum emission. For several of these components, re­ sponsible charge pairs have been identified (Table

Yorinao Inoue

3). In the following we discuss information cur­ rently available about the properties of the re­ spective TL bands. 1. The B-band The B-band that appears at around +30 °C is the best characterized TL component. This band is clearly correlated with the water-oxidizing en­ zyme (Inoue and Shibata, 1977a), in particular with the presence of functionally active Mn (Inoue, 1976; Rozsa and Demeter, 1982), and has and charge recombi­ been assigned to nation (Rutherford et al., 1982, 1985). When ex­ cited by a series of saturating flashes, the intensity of the B-band exhibits a period-four oscillation in thylakoids (Inoue and Shibata, 1977a; 1977b; Demeter and Vass, 1984; Demeter et al., 1984), intact leaves and PSII-enriched membrane frag­ ments (Rutherford et al., 1984). Analysis of the and oscillation pattern indicates that both the are involved in generation of this TL component (Rutherford et al., 1982; Demeter as the nega­ and Vass, 1984). Participation of tive charge trap has been evidenced by modu­ ratio by chemical or lation of the initial

Thermoluminescence, a specific probe of PSII

101 shape, but below pH 6.0, they exhibit two distin­ and guishable components denoted respectively, with a few degrees difference in peak temperature (Inoue, 1981). The TL yield from the latter recombination is higher than that from the former by a factor of 1.7–2.0 due to some unknown reasons (Rutherford et al., 1985; Demeter et al., 1985b). The emission spectrum of the B-band(s) has a maximum at around 690 nm (Sonoike et al., 1991) in accordance with the hypothesis that the recombination re-excites P680 as a reversal of charge separation.

2. The Q-band Upon treating PSII with DCMU or other PSII herbicides, the B-band is abolished with a con­ comitant appearance of a new TL band between 0 and +10 °C (Rubin and Venediktov, 1969). This band is called the Q-band and arises from charge recombination (Demeter and Vass, 1984; Rutherford et al., 1982). The conversion of the B-band to the Q-band is due to inhibition by DCMU of the electron transfer between and which enables stabilization of as a nega­ The tive charge detectable by TL instead of Q-band is often used to analyze the features of stabilization of the

3. The A-band

light pretreatment or by removal and reconsti­ (Rutherford et al., 1982; Demeter tution of and Vass, 1984; Wydrzynski and Inoue, 1987). At or above pH 7.0-7.5, both and recombinations give rise to the same TL band with respect to the peak temperature and band

When PSII is preilluminated with two flashes at room temperature, then cooled to 77 K, and then further illuminated with continuous light at 77 K, a clear TL band denoted the A-band, appears at around –10°C (Läufer et al., 1978; Inoue, 1981; Demeter et al., 1985b). This band has been as­ signed to arise from charge recombination based on the following considerations: illumi­ nation with two flashes at room temperature gen­ state which is incapable of TL due erates an to the absence of negative charge (see Table 2), but the 77 K illumination oxidizes cytochromeb­ 559 or chlorophyll instead of the Mn-cluster (in and generates which recombines with the previously formed by illumi­ nation with two flashes and stabilized by cooling and unaffected during the 77 K illumination (Koike et al., 1986).

102 4. The Tris-treated PSII depleted of the functional Mncluster emits a TL band at around –10°C (Inoue et al., 1977; Rosza and Demeter, 1982). This to distinguish it from band is called the the A-band having the same emission tempera­ ture (Koike et al., 1986). The origin of this com­ ponent was not clear, but it was later proposed to arise from charge recombination between and a photooxidized histidine residue of a PSII reaction center protein by use of chemically modi­ fied PSII (Ono and Inoue, 1991a). This hypothesis was addressed in more detail by use of mutated PSII, and it was revealed that substitution of both His195 and His190 affects the emission of this TL component (Kramer et al., 1994). The positive equivalent on the photooxidized histidine exhibits a high affinity for exogenous and is proposed to be involved in photoligation of ions in the process of photoactivation of a latent oxygen-evolving system (Ono and Inoue, 1991b). 5. The A minor TL component denoted as the appears between –80 and –30°C. The emission temperature of this TL band varies depending on the excitation temperature (Ichikawa et al., 1975), being usually higher by 10 to 20 °C than the excitation temperature. This band is abolished by treatment with 1% ethanol and exhibits a periodfour oscillation dependent on S-state turnovers (Demeter et al., 1985b), although the band is reported to be emitted also from the D1/D2/cytochrome b-559 PSII reaction center preparation depleted of the Mn-cluster (Vass et al., 1989). Although it is proposed to arise from charge pair whose trapping depth is modulated by the conformation of the reaction center, its as origin is not yet clear. The participation of the negative counterpart has recently been con­ firmed by reconstitution of artificial in pur­ ified reaction center preparation by use of plastoquinone-9 (Chapman et al., 1991). 6. The C-band A TL component denoted as the C-band appears at around +50 °C on glow curves from DCMU-

Yorinao Inoue treated plant materials (Desai et al., 1975). The as the negative counterpart participation of is likely, judging from enhancement of its inten­ sity in the presence of high concentration of DCMU. The C-band is reported to exhibit a periand od-four oscillation with maxima in the states, suggesting that this band arises from charge recombination (Demeter et al., 1984). (Tyr 160 residue of D2 protein) has been proposed as a candidate for the positive equiva­ and Demeter et al. (1993), lent in Krieger et al. (1993) and Johnson et al. (1994) reported that this component arises from charge and in normal recombination between PSII as well. and

7. The Z-band A strong and broad TL band appears at around 110K when various plant materials are illumi­ nated at 77 K with continuous light. This compo­ nent, denoted as the Z-band, exhibits an emission maximum at around 730 nm, in contrast to 690 nm of the B-band arising from PSII reaction center photochemistry, and is excited with blue light at a higher yield than with red light (Arnold and Azzi, 1968). Upon raising the excitation tem­ perature above 77 K, only the higher temperature part of the Z-band appears, indicating that the trap for this component has a broad distribution of stabilization free energies. It turned out re­ cently that LHCI and LHCII, that have no photo­ chemical reaction center, or purified chlorophyll, particularly its aggregated form, emit this band more strongly than PSI or PSII (Sonoike et al., 1991), indicating that the Z-band is correlated with neither PSI nor PSII photochemistries. It has recently been found that this band is preferen­ tially charged by blue light, and the trapped charges are detrapped by illumination with red light, due probably to a local heating effect (Hargen et al., 1994). This effect might be related with the mechanism of non-photochemical quenching which is postulated to result from con­ formational changes in LHC concomitant with the accumulation of zeaxanthin via the xantho­ phyll cycle.

Thermoluminescence, a specific probe of PSII

103

8. The TL Bands at Temperatures Below 77 K

the water-oxidizing enzyme, Y) effect of these chemicals affecting the properties of the state on donor side of PSII. Such effects on TL are used for characterizing the functioning of new herbicidal compounds (Asami et al., 1988; Koike et al., 1989). Herbicide resistant mutants exhibit a remark­ ably downshifted B-band. In triazine resistant mutants of Eligeron canadensis and Synechocystis PCC 6714, the emission temperature of the Bband is downshifted by 15 °C as compared with that of the wild types (Demeter et al., 1985a). This indicates that the redox potential difference and is decreased from between 70 to 30 mV due to a structural alteration of the site by the mutation. Analysis of cy­ anobacterial mutants reveals that the emission temperature of the B-band is strongly sensitive to the replacement of Ser264 of D1 by Ala or Gly that concomitantly induces strong resistance to triazines, but is rather immune to the replacement of Phe255 by Tyr that induces resistance to phe­ nylureas, suggesting a more important role of as compared with Phe255 Ser264 in (Etienne et al., 1990; Gleiter et al., 1990, 1992).

Three new TL components emitting at around 20, 50 and 70 K have been resolved in the glow curve at temperatures below 77 K, and denoted and respectively (Noguchi et al., as 1992). The glow curve excited at liquid helium temperatures exhibits another component at around 90 K, but this turned out to be a different expression of the well-known Z-band that emits above 77 K. The properties of the three compo­ nents are essentially the same as those of the Zband: being excited preferentially by blue light and emitted more strongly from LHCs and aggre­ gated chlorophyll, suggesting that they originate from charge storage in a chlorophyll molecule interacting with another chlorophyll or solvent molecule(s). III. Application of TL as a Probe of PSII Photochemistry

A. Acceptor Side of PSII The redox couples and differ in redox potential by 50–70 mV, and this difference manifests on TL glow curves as a 25–30 °C differ­ ence in emission temperature between the Band band and Q-band, which arise from charge pairs, respectively. The energetic stability of reduced quinones can easily be studied by monitoring TL (Demeter et al., 1985a). These properties enable one to use TL for studies of herbicide resistance (for other applications see Vass and Inoue, 1992). Upon treatment of PSII with herbicides the Bband is converted to the Q-band due to inhibition of the electron transfer between and Not­ ably, however, the emission temperature of the resulting Q-band differs depending on the chemi­ cal structure of the herbicide employed: phenolic type herbicides induce a Q-band emitting be­ tween –15 and 0°C, whereas urea/triazine type herbicides induce a Q-band between 0 and +10°C (Vass and Demeter, 1982). The much lower Q-band peak temperature in the presence of phenolic type herbicides may be ascribed either or to the to a lowered redox potential of ADRY (acceleration of deactivation reactions in

B. Donor side of PSII The generation of the B-bands and the and so that the A-band involves the emission temperature and oscillatory behavior of those TL bands provide useful information re­ garding the properties of the S-states and the role of various cofactors involved in S-state trans­ itions. These properties were first used to determine the temperature dependence of S-state transition. Upon lowering the ambient temperature, the Sstate transitions become inhibited. From the os­ cillation of the B-band under flash excitation, and transitions were shown to be blocked at –35 and –20 °C, respectively, while the transition was inhibited at –65 °C (Inoue and Shibata, 1977b). Further precise stud­ and ies reveal that transitions are completely blocked at –160, –65 and –40 °C, and half-blocked at –95, –45 and –23 °C, respectively (Demeter and Vass, 1984; Demeter et al., 1985b; Koike and Inoue, 1987). Through a sophisticated analysis of the tempera­

104 ture dependence by use of two different protocols for low temperature flash excitation, “last flash at low temperature” and “all flashes at low tem­ perature”, a low-temperature sensitive intermedi­ ate state has been proposed to exist between and which might be a precursor in which proton release is not completed (Koike and Inoue, 1987). Another typical application of TL for studies of the donor side of PSII is the effect of removal of extrinsic proteins. Higher plants’ PSII contains a set of three extrinsic proteins of molecular masses of 33, 24 and 18 kDa, which are electro­ statically associated with the lumenal surface of PSII and involved in regulating the requirement for inorganic cofactors and stability of the Mncluster. By a suitable salt washing followed by mixing with concentrated extract of the three ex­ trinsic proteins, these proteins can be reversibly removed and reconstituted with concomitant in­ activation and reactivation of oxygen-evolving ac­ tivity. Upon removal of the set of proteins, ox­ ygen evolution is almost lost whereas the TL Bband can still be fully induced. Although PSII depleted of the extrinsic proteins exhibits normal the final oscillation up to the transition to release molecular oxygen is blocked (Ono and Inoue, 1985). Upon removal of the extrinsic proteins, the peak position of the Bband remains at the normal temperature, suggest­ ing no appreciable change in the stability of the charge pair. In contrast, however, the peak temperature of the Q-band is remarkably up­ shifted by 20–25 °C indicative of deeper stabiliza­ tion of the state in the absence of the extrinsic proteins (Vass et al., 1987a). The different stabili­ zation between and charge pairs may be due to a secondary but specific effect on the site, in addition to the major effect on the More or less the same TL data have been ob­ tained for genetically engineered cyanobacterium mutant cells in which the psb0 gene encoding the 33 kDa extrinsic protein is deleted (Burnap et al., 1992), indicating that the TL methods can successfully characterize the properties of PSII donor side in mutant cells without isolating thy­ lakoids. TL data implicating an incomplete as­ sociation of the 33 kDa protein with PSII mem­ branes have also been collected for cyanobactrial

Yorinao Inoue mutants in which a few amino acids on the lu­ menal loop of the CP47 protein are deleted (Gle­ iter et al., 1994). By analogy to these, TL has now been exten­ sively applied in probing redox properties of the PSII (Ono and Inoue, donor side of 1989, 1990) or PSII (Vass et al., 1987b). These applications are discussed in more detail in Vass and Inoue (1992).

IV. Perspective: Merits and Demerits of TL Technique The first stage of studies on photosynthetic TL was the 10 years following the discovery of this phenomenon by Arnold and Sherwood in 1957, during which the basic phenomenology and fund­ amental concept about this phenomenon were es­ tablished. The second stage would be the last 20 years, during which the origins of most of photosynthetic TL components were clarified subsequent to the finding that photosynthetic TL involves the so-called “charge accumulator” of the photosynthetic oxygen-evolving system. The second stage has now opened up a possibility to utilize photosynthetic TL as a simple but efficient probe for characterizing electron transport in PSII on both the donor and acceptor sides. Fol­ lowing are examples of promising areas to which photosynthetic TL can be efficiently applied: (i) Studies on structure and function of the Mncluster, particularly coupled with the use of gen­ etically engineered mutants having some defects in water-oxidation activity. By simple TL analyses we will be able to characterize apparently small but functionally serious alterations occurring on the Mn-cluster of mutant cells, particulary those manifest only in higher S-states. (ii) Studies on structural determinants of the or herbicide-binding site, particularly co­ upled, also in this case, with genetically engine­ ered mutants. Alterations in proper­ ties upon acquirement of herbicide resistance can easily be detected by the TL technique. (iii) Studies on environmental stresses including acclimation to low or high temperature and pho­ toinhibition that affect the functioning of PSII. Taking all these into consideration, the advan­ tages and disadvantages of the TL technique in photosynthetic studies can be summarized as fol­

Thermoluminescence, a specific probe of PSII lows: Advantages are: (i) Low cost setup, (ii) Simple operation requiring no experience, (iii) Simple sample preparation. Measurements with whole cells are sometimes essential in studies using algal mutants, (iv) Easy application for characterization of PSII functioning for physio­ logical or environmental purposes, (v) Selective probing of PSII by ignoring PSI. Disadvantages are: (i) The phenomenon is restricted to PSII. It cannot be applied to PSI and bacterial reaction centers. The reason for this remains to be theoret­ ically clarified, (ii) The TL data do not directly provide physical parameters. They are usually given as deviation from the control measurement, (iii) Alternative interpretations: e.g. a shift in peak temperature can be attributed to a change either on donor side or acceptor side.

Acknowledgements The author acknowledges the Science and Tech­ nology Agency of Japan (STA) which has been financially supporting the TL studies during the last 15 years under the title of Solar Energy Con­ version by Means of Photosynthesis. The author is also grateful to Drs. Mamiko Kimimura-Taguchi and Han Kab-Cho for their technical assis­ tance in preparing the manuscript, and also to Professor J. Amesz for his valuable suggestions.

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105 bination in photosystem II studied by thermoluminescence. I: Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts. Biochim Biophys Acta 764: 24–32 Demeter S, Vass I, Horvath G and Läufer A (1984) Charge accumulation and recombination in photosystem II studied by thermoluminescence. II: Oscillation of the C band by flash excitation. Biochim Biophys Acta 764: 33–39 Demeter S, Vass I, Hideg E and Sallai A (1985a) Comparative thermoluminescence study of triazine-resistant and -suscep­ tible biotypes of Erigeron canadensis L. Biochim Biophys Acta 806: 16–24 Demeter S, Rozsa Z, Vass I and Hideg E (1985b) Thermolum­ inescence study of charge recombination in photosystem II at low temperature II: Oscillatory properties of the Z and A thermoluminescence bands in chloroplasts dark- adapted for various time periods. Biochim Biophys Acta 809: 379– 387 Demeter S, Goussias C, Bernet G, Kovacs L and Petrouleas V (1993) Participation of the g = 1.9 and g = 1.82 EPR of photos­ forms of the semiquinone-iron complex, ystem II in the generation of the Q and C thermoluminesc­ ence bands, respectively. FEBS Lett 336: 352–356 Desai TS, Sane PV and Tatake VG (1975) Thermoluminesc­ ence studies on spinach leaves and Euglena. Photochem Photobiol 21: 345–350 DeVault D and Govindjee (1990) Photosynthetic glow peaks and their relationship with the free energy changes. Photosynth Res 24: 175–181 DeVault D, Govindjee and Arnold WA (1983) Energetics of photosynthetic glow peaks. Proc Natl Acad Sci USA 80: 983–987 Ducruet J-M and Miranda T (1992) Graphical and numerical analysis of thermoluminescence and fluorescence Fo emis­ sion in photosynthetic material. Photosynth Res 33: 15–27 Etienne A-L, Ducruet J-M, Ajlani G and Vernotte C (1990) Comparative studies on electron transfer in photosystem II of herbicide-resistant mutants from different organisms. Biochim Biophys Acta 1015: 435–440 Förster W and Junge W (1985) Stoichiometry and kinetics of proton release upon photosynthetic water oxidation. Photochem Photobiol 41: 183–190 Fowler CF (1977) Proton evolution from photosystem II. Sto­ ichiometry and mechanistic considerations. Biochim Bi­ ophys Acta 462: 414–421 Gleiter H, Ohad N, Hirschberg J, Fromme R, Renger G, Koike H and Inoue Y (1990) An application of thermolum­ inescence to herbicide studies. Z Naturforsch 45c: 353–358 Gleiter H, Ohad N, Koike H, Hirschberg J, Renger G and Inoue, Y. (1992) Thermoluminescence and flash-induced oxygen yield in herbicide resistant mutants of the D1 pro­ tein in Synechococcus PCC7942. Biochim Biophys Acta 1140: 135–143 Gleiter H, Haag E, Shen J-R, Eaton-Rye J, Inoue Y, Ver­ maas WFJ and Renger G (1994) Functional characteriz­ ation of mutant strains of the cyanobacterium Synechocystis sp. PCC6803 lacking short domain within the large, lumenexposed loop of the chlorophyll-protein CP47 in photosys­ tem II. Biochemistry 33 12063–12071 Hagen C, Pascal A, Horton P and Inoue Y (1995) Influence

106 of changes in the photon protective energy dissipation on red light-induced detrapping of the thermoluminescence Zband. Photochem Photobiol 62: 514–521 Hideg E, Scott RQ and Inaba H (1991) Spectral resolution of long term (0.5–50 s) delayed fluorescence from spinach chloroplasts. Arch Biochem Biophys 285: 371–372 Ichikawa T, Inoue Y and Shibata K (1975) Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the water-splitting activity in photosynthesis. Biochim Biophys Acta 408: 228–239 Inoue Y (1976) Manganese catalyst as a possible cation carrier in thermoluminescence. FEBS Lett 72: 279-282 Inoue Y (1981) Charging of the A band of thermoluminesc­ in isolated chloroplasts. ence dependent on the Biochim Biophys Acta 634: 309–320 Inoue Y (1983) Recent advances in the studies of thermolumi­ nescence of photosystem II. In: Inoue Y, Crofts AR, Gov­ indjee, Murata N, Renger G and Satoh K (eds) The Ox­ ygen Evolving System of Photosynthesis, pp 439–450. Academic Press Japan, Tokyo Inoue Y and Shibata K (1977a) Thermoluminescence bands of chloroplasts as characterized by flash excitation. In: Hall DO, Coombs J and Goodwin TW (eds) Proc Fourth Intl Congr on Photosynthesis, pp 211–221.The Biochemical So­ ciety, London Inoue Y and Shibata K. (1977b) Oscillation of thermolumi­ nescence at medium low temperature. FEBS Lett 85: 19– 197 Inoue Y and Shibata K (1982) Thermoluminescence from photosynthetic apparatus. In: Govindjee (ed) Photosynth­ esis; Energy Conversion by Plants and Bacteria, Vol. pp 507–533. Academic Press, New York Inoue Y, Ichikawa T and Shibata K (1976) Development of thermoluminescence bands during greening of wheat leaves under continuous and intermittent illumination. Photochem Photobiol 23: 125–130 Inoue Y, Yuashita T, Kobayashi Y and Shibata K (1977) Thermoluminescence changes during inactivation and reac­ tivation of the oxygen-evolving system in isolated chloro­ plasts. FEBS Lett 82: 303–306 Johnson GN, Boussac A and Rutherford AW (1994) The origin of 40–50 °C thermoluminescence bands in photosys­ tem II. Biochim Biophys Acta 1184: 85–92 Koike H and Inoue Y (1987) A low temperature sensitive and in photosynthetic intermediate state between water oxidation deduced by means of thermoluminescence measurement. Biochim Biophys Acta 894: 573–577 Koike H, Siderer Y, Ono T and Inoue Y (1986) Assignment charge recombi­ of thermoluminescence A band to by a two-step nation: sequential stabilization of and illumination at different temperatures. Biochim Biophys Acta 850: 80–89 Koike H, Asami T, Yoshida S, T Kahashi N and Inoue Y (1989) A new-type photosystem II inhibitor which blocks electron transport in water-oxidation system. Z Natur­ forsch 44c: 271–279 Kramer DM, Roffey RA, Govindjee and Sayre RT (1994) The AT thermoluminescence band from Chlamydomonas reinhardtii and the effects of mutagenesis of histidine resi-

Yorinao Inoue dues on the donor side of the photosystem II D1 polypep­ tide. Biochim Biophys Acta 1185: 228–237 Krieger A, Weis E and Demeter S (1993) Low-pH-induced ion release in the water-splitting system is ac­ companied by a shift in the midpoint redox potential of the Biochim Biophys Acta 1144: primary quinone acceptor 411–418 Läufer A, Inoue Y and Shibata K (1978) Enhanced charging of thermoluminescence A band by a combination of flash and continuous light excitation. In: Akoyunoglou (ed) Proc Intl Symp on Chloroplast Development, pp 379–387. Elsevier, Amsterdam Noguchi T, Inoue Y and Sonoike K (1992) Thermoluminesc­ ence emission at liquid helium temperature from photosyn­ thetic apparatus and purified pigments. Biochim Biophys Acta 1141: 18–24 Ono T and Inoue Y (1985) S-state turnover in the system of photosystem II particles depleted of three peripheral proteins as measured by thermolumi­ nescence: Removal of 33 kDa protein inhibits to transition. Biochim Biophys Acta 806: 331–340 Ono T and Inoue Y (1989) Removal of Ca by pH 3.0 treat­ ment inhibits to transition in photosynthetic oxygen evolution system. Biochim Biophys Acta 973: 443–449 Ono T and Inoue Y (1990) Abnormal redox reactions in photosynthetic centers in NaCl/EDTA-washed PSII. A dark-stable EPR multiline signal and a new un­ known positive charge accumulator. Biochim Biophys Acta 1020: 269–277. Ono T and Inoue Y (1991a) Biochemical evidence for histi­ dine oxidation in photosystem II depleted of the Mn-cluster for FEBS Lett 278: 183–186 Ono T and Inoue Y (1991b) A possible role of redox active histidine in photoligation of Mn into photosynthetic evolving enzyme. Biochemistry 30: 6183–6188 Rozsa Z and Demeter S (1982) Effect of inactivation of the oxygen evolving system on the thermoluminescence of iso­ lated chloroplasts. Photochem Photobiol 36: 705–708 Rubin AB and Venediktov PS (1969) Storage of the light energy by photosynthesizing organisms at low temperature. Biofizika 14: 105–109 Rutherford WA, Crofts AR and Inoue Y (1982) Thermolumi­ nescence as a probe of photosystem II photochemistry; the origin of the flash-induced glow peaks. Biochim Biophys Acta 682: 457–465 Rutherford WA, Govindjee and Inoue Y (1984) Charge ac­ cumulation and photochemistry in leaves studied by therm­ oluminescence and delayed light emission. Proc Natl Acad Sci USA 81: 1107–1111 Rutherford WA, Renger G, Koike H and Inoue Y (1985) Thermoluminescence as a probe of PSII: The redox and protonation sate of the secondary acceptor quinone and evolving enzyme. Biochim Biophys Acta 767: 548– the 556 Sane PV and Rutherford WA (1986) Thermoluminescence from photosynthetic membranes. In: Govindjee, Ames J and Fork DC (eds) Light Emission by Plants and Bacteria, pp 329–360. Academic Press, Orlando Saphon S and Crofts AR (1977) Protolytic reactions in photo­ system II. A new model for the release of protons ac­

Thermoluminescence, a specific probe of PSII companying the photooxidation of water. Z Naturforsch 32c: 617–626 Sonoike K, Koike H, Enami I and Inoue Y (1991) The emis­ sion spectra of thermoluminescence from photosynthetic apparatus. Biochim Biophys Acta 1058: 121–130 Strehler BL and Arnold WA (1951) Light production by green plants. J Gen Physiol 34: 59–73 Van Gorkom HJ (1985) Electron transfer in photosystem II. Photosynth Res 6: 97–1158 Vass I and Demeter S (1982) Classification of photosystem II inhibitors by thermodynamic characterization of the therm­ oluminescence of inhibitor-treated chloroplasts. Biochim Biophys Acta 682: 496–499 Vass I and Inoue Y (1992) The moluminescence in the study of photosystem II. In: Barber J (ed) The Photosystems: Structure, Function and Molecular Biology. Topics in Pho­ tosynthesis, Vol. 11 pp 259–294. Elsevier, Amsterdam Vass I, Horvath G, Herczeg T and Demeter S (1981) Photo­

107 synthetic energy conservation investigated by thermolumi­ nescence. Activation energies and half-lives of thermolumi­ nescence bands of chloroplasts determined by mathematical resolution of glow curves. Biochim Biophys Acta 634: 140–152 Vass I, Ono T and Inoue Y (1987a) Removal of 33 kDa extrinsic protein specifically stabilizes the charge pair in photosystem II. FEBS Lett 211: 71–76 Vass I, Ono T and Inoue Y (1987b) Stability and oscillation properties of thermoluminescence charge pairs in the evolving system depleted of or the 33 kDa extrinsic protein. Biochim Biophys Acta 892: 224–235 Vass I, Chapman DJ and Baber J (1989) Thermoluminescence properties of the isolated photosystem two reaction center. Photosynth Res 22: 295–301 Wydrzynski T and Inoue Y (1987) Modified photosystem II acceptor side properties upon replacement of the quinone site with 2,5-dimethyl-p-benzoquinone and pheat the nyl-p-benzoquinone. Biochim Biophys Acta 893: 33–42

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Chapter 7 Accumulated Photon Echo Measurements of Excited

State Dynamics in Pigment–Protein Complexes

Thijs J. Aartsma*, Robert J.W. Louwe and Peter Schellenberg Department of Biophysics, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands

Summary I. Introduction II. Homogeneous and Inhomogeneous Linewidths III. Photon Echo Phenomena A. Two-pulse Photon Echo B. Three-pulse Stimulated Photon Echo C. Accumulated Photon Echo IV. Accumulated Photon Echo: Experimental V. Energy Transfer VI. Photon Echo Experiments on Reaction Centers VII. Conclusion and Perspectives Acknowledgements References

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Summary The homogeneous lineshape of optical transitions in molecular systems contains information about the intrinsic excited state dynamics. The lineshape parameters can be obtained from accumulated photon echo measurements. This technique has been applied to investigate the excited state dynamics of isolated photosynthetic pigment-protein complexes at low temperature. In antenna systems it is found that the coherent lifetimes of the initally excited exciton state can be hundreds of picoseconds. This means that the optical excitation is intrinsically delocalized, and that a hopping-model for energy transfer is less appropriate at low temperature. In reaction centers, the accumulated photon echo decay is determined by the rate of primary charge separation. The multiexponential decay of the echo-signal is most likely due to dispersive kinetics. Abbreviations: AOM – acousto-optic modulator; APE – accumulated photon echo; BChl – bacteriochlo­ rophyll; EOM – electro-optic modulator; FMO – Fenna–Matthews–Olson; I – primary acceptor; P – primary donor; RC – reaction center; 2PE – two-pulse photon echo; 3PSE – three-pulse stimulated echo; Q – quinone

I. Introduction

*Correspondence: Fax: 31-71-5275819; E-mail:[email protected]

The primary function of photosynthetic pigmentprotein complexes is to collect energy by absorp­ tion of light and to make this energy available for biochemical metabolic processes. This occurs through a sequence of energy-stabilizing steps in­ 109

J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 109–122. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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volving energy transfer and charge separation. These primary steps are extremely fast and com­ pete very effectively with intrinsic molecular relaxation processes such as fluorescence, in­ tersystem crossing and internal conversion. For a full understanding and description of the molecu­ lar mechanism, it is necessary to investigate in detail the excited state dynamics of the complexes involved. Such information will be essential to test models for energy transfer and charge separ­ ation, and for understanding the underlying inter­ molecular interactions in relation to the molecu­ lar structure. This chapter describes accumulated photon echo experiments as a means to investigate the excited state dynamics of photosynthetic systems at low temperature. In the first part we discuss the formation and decay of the accumulated pho­ ton echo in relation to the homogeneous linewidth, consider the conditions under which an accumulated photon echo can be observed, and describe a typical experimental setup for the mea­ surements. In the second part we discuss the re­ sults which have been obtained by measurements on isolated photosynthetic pigment–protein com­ plexes. It should be emphasized that the accumulated photon echo decay is determined by the loss of excited state coherence, and is not necessarily identical to results obtained by pump-probe mea­ surements. Data from both types of measure­ ments should be considered to be complemen­ tary, and as such will be important for the correct interpretation of the excited state dynamics in­ volved. II. Homogeneous and Inhomogeneous Linewidths Optical transitions of a molecule are charac­ terized by a finite bandwidth, usually referred to as the homogeneous linewidth, which is deter­ mined by the finite lifetime of the energy levels involved in the electronic transition. Considering the homogeneous broadening mechanism, it is customary to distinguish between population relaxation and pure optical dephasing, charac­ respectively. terized by time constants and Population relaxation involves transitions be­ tween discrete energy levels within the molecule,

Thijs J. Aartsma et al. and determines the lower limit of the optical linewidth. Pure optical dephasing is due to a modu­ lation of the optical transition frequency caused by guest–host interactions such as phonon-scattering and scattering on disorder modes. Thus the homogeneous linewidth carries information about the dynamics of the molecular system. The homo­ is related to the inverse of geneous line width the overall dephasing time

This equation for the homogeneous linewidth is essentially a manifestation of the Heisenberg-uncertainty principle. At sufficiently low tempera­ tures, such that bath fluctuations are completely quenched, the homogeneous linewidth is deter­ For an ‘isolated’ molecule in a host mined by will in general be equal to the fluor­ matrix, escence lifetime. Intermolecular interactions be­ tween the guest-molecules may give rise to ad­ ditional relaxation processes which contribute to population relaxation. This is typically the case in photosynthetic systems where energy transfer and charge separation play an essential role in the conversion of photons into useful chemical energy. Therefore, it may be expected that a study of the intrinsic homogeneous linewidth may be used to characterize such processes and the associated intermolecular interactions in more de­ tail. The experimentally observed linewidth, how­ ever, is often much broader than the homogene­ ous linewidth. This is due to the effects of in­ homogeneous broadening associated with the dependence of the transition frequency on the local electrostatic field. Because of disorder, the local field may differ considerably from one site to the other, with a corresponding change in the transition frequency through interaction of this field with the molecular polarizability and/or per­ manent dipole moment. To uncover the homo­ geneous linewidth parameters from the inhomo­ geneously broadened line, special methods are required. They can be divided in two main cate­ gories: those that operate in the frequency do­ main such as transient and permanent hole burn­ ing spectroscopy, and those that operate in the time domain. Hole burning spectroscopy is re­

Accumulated photon echo measurements viewed in chapter 8 by N.R.S. Reddy and G.J. Small. III. Photon Echo Phenomena In the time domain, is measured in a photon echo experiment (Abel et al., 1966; Aartsma and Wiersma, 1976; Hesslink and Wiersma, 1978) which involves coherent excitation of the sample with short laser pulses. As a result of coherent excitation, the oscillating electric dipoles of the excited molecules will have a well-defined phase relationship. At room temperature this phase re­ lationship will be destroyed very rapidly because of thermally induced scattering processes, but at low temperature it may persist for considerable lengths of time. The coherence of the excited molecular system gives rise to a macroscopic pola­ rization in the medium, which is oscillating at the driving optical frequency. The amplitude of this the number of polarization is proportional to excited molecules. Hence, the intensity of the radiation emitted by a coherent ensemble of di­ pole oscillators will be proportional to (Dicke, 1954), whereas in an incoherent emission process However, this intensity is proportional to even in the absence of scattering processes, the coherent superposition is lost very rapidly be­ cause of the dephasing which arises from the dif­ ferent resonance frequencies in the case of in­ homogeneous broadening. A. Two-Pulse Photon Echo In the absence of phase-perturbing scattering pro­ cesses, the time development of the relative phases of the oscillators in an inhomogeneously broadened system is well-defined despite the vari­ ation in resonance frequency. The magic of the two-pulse photon echo arises from the fact that this phase development can be reversed by apply­ ing a second pulse with the proper intensity at a suitable time delay (Abella et al., 1966). Since the rephasing of the oscillators proceeds at the same rate as the dephasing, determined by their individual detuning from resonance, the coherent after the superposition is restored at a time second excitation pulse. This state gives rise to an intense burst of coherent radiation, pro­ which is known as the photon portional to

111

echo. The photon echo intensity will have a maxi­ mum when the pulse areas, defined as and for the are equal to first and second excitation pulse, respectively is the elec­ (Allen and Eberly, 1975). Here, tric field amplitude, and the pulse area is a mea­ sure for the interaction strength of the field with the transition moment The intensity of the two-pulse photon echo, will diminish as the time delay between the excitation pulses is increased because of the irre­ versible loss of coherence through excited state decay and dephasing. More explicitly,

Thus can be obtained by measuring the photon echo intensity as a function of the time delay between the excitation pulses. The dimensions of the sample are typically much larger than the wavelength of excitation. Therefore, constructive interference of the coher­ ent radiation which gives rise to the photon echo, is only observed in a very specific direction which is defined by the phase-matching condition:

where are the wave vectors of the photon echo, and the second and first excitation pulses, respectively. For a more comprehensive description of the photon echo phenomenon we refer to Allen and Eberly (1975) and Levenson and Kano (1988). B. Three-Pulse Stimulated Photon Echo The time-development of the induced polariz­ ation can be manipulated by other pulse se­ quences (Allen and Eberly, 1975). The most fa­ miliar variant is the three-pulse or stimulated photon echo which involves the application of three excitation pulses. The formation and characteristics of the stimulated photon echo can be described by considering the effect of coherent optical excitation in the frequency domain. The power spectrum of a pair of identical, coherent light pulses propagating in the same direction at a relative time delay is characterized by a peri­ odically varying amplitude with a fringe-spacing over a frequency range deter­ given by

112

Thijs J. Aartsma et al.

mined by the optical bandwidth of the light pul­ ses. If this pulse-pair excites an inhomogeneously broadened molecular system, this pattern gives rise to a similarly periodic variation of the popula­ tion density as a function of transition frequency in the ground as well as in the excited state. This is referred to as a frequency grating (Hesselink and Wiersma, 1979; Duppen and Wiersma, 1986). The population density of the excited state will have a maximum, and the ground state popu­ lation a minimum, at the peaks in the power spectrum. The frequency grating in the ground state will be complementary to that in the excited state. The modulation depth of the frequency grating is a measure of the homogeneous dephasing that has occurred during the interval This is equivalent to saying that the frequency grating can only be formed when the homogeneous linewidth is less than the fringe spacing in the power spectrum: The frequency grating is described by the diagonal elements of the density matrix. If we assume negligible population relax­ these elements can be written as ation, (Hesselink, 1980):

In this equation, and are the wave is the vectors and phases of the excitations, relative detuning of the optical excitation, and and are the pulse areas. The maximum modulation depth is achieved when For this condition the resulting modulation of the ground and excited states is depicted in Fig. 1A. A third pulse, at a time after the second, will induce the so-called stimulated echo at time The maximum amplitude is again pulse. The effect of this obtained if this is a third pulse is to transfer the phase information that was ‘stored’ in the population grating in fre­ quency space to the off-diagonal elements of the density matrix (Duppen and Wiersma, 1986). As a result a rephasing process sets in, similar to that which gives rise to the two-pulse photon echo. At after the third pulse the macroscopic a time

polarization is reestablished, resulting in the em­ ission of the stimulated echo. For a two-level system the stimulated echo intensity and direction are given by:

The first exponential factor is due to the homo­ geneous dephasing which occurs during the in­ between the first two excitation pulses, terval while the second exponential factor reflects eradi­ cation of the frequency grating through depopula­ tion of the excited state. It can be seen that by measuring the stimulated photon echo intensity for a fixed as a function of the waiting time it is possible to measure the lifetime of can be the excited state. On the other hand, measured by varying while keeping fixed. An excited molecular system will ultimately

Accumulated photon echo measurements decay to the ground state, either directly by flu­ orescence or radiationless relaxation, or via relax­ ation through some long-lived intermediate state, such as the lowest triplet state. In the former case, the frequency grating will disappear on a but in the latter case the fre­ time scale of quency grating in the ground state may persist for a considerable length of time, determined by the lifetime of the intermediate state. This third level, if it is efficiently populated, serves as a bottleneck reservoir for the excited state population (Fig. 1). It is now possible to stimulate a long-lived or anomalous photon echo from the ground state grating at a time scale corresponding to the bottleneck lifetime (Morsink et al., 1979). In the limit that the lifetime of the bottleneck state is much larger than and the echo intensity from a three-level system excited by pulses can be written as (Morsink et al., 1982):

with rate constants as defined in Fig. 1B. The term in the square bracket is the contribution from the frequency grating in the ground state The ampli­ which persists on a time scale of tude of this persistent grating, and therefore the intensity of the anomalous photon echo, is deter­ mined by the yield of the bottleneck state, given by the factor In the case of triplet states, this is the same as the intersystem crossing yield. C. Accumulated Photon Echo Two- and three-pulse photon echo measurements require relatively intense pulses so that it is neces­ sary to amplify the output of the mode-locked dye-lasers typically used in these experiments. In addition, some form of time-gating is often used to selectively monitor the echo intensity, for ex­ ample by frequency up-conversion (Hesselink and Wiersma, 1978; Meijers and Wiersma, 1992). Therefore, such experiments present a significant

113

experimental challenge. A much simpler variant of photon echo measurements is the accumulated photon echo (Hesselink and Wiersma, 1979) which can be observed if a long-lived bottleneck state exists in the molecular system (cf. Fig. 1B). Successive excitation of the sample with pulsepairs within the lifetime of the bottleneck state will result in the accumulation of a frequency grating. Such pulse-pairs are readily generated at a repeti­ tion rate of about 80 MHz, while the lifetime of the bottleneck state can easily be of the order of 1 ms or longer as is the case for a triplet state. The accumulation effect can extend over hundreds of excitation pulses, and it is possible even with weak excitation pulses to obtain a large modu­ lation depth of the frequency grating. The contri­ bution of each pulse pair to the frequency grating is given by Eq. (4). The generation of an accumulated grating has been verified experimentally in the case of a bottleneck state with infinite lifetime (Rebane et al., 1983), i.e. a photoinduced chemical transfor­ mation upon optical excitation. After the sample – a polymerized porphyrazine styrol solution – was excited with a train of pulse-pairs, the fre­ quency grating could be observed by scanning the transmission of the sample as a function of wavelength. An essential requirement for accumulation of the grating is that the phase difference (cf. Eq. (4)) must be maintained for all pulse pairs, at least for the lifetime of the bottleneck state. This condition is not difficult to achieve for typical CW mode-locked dye lasers. The relation­ and ship between the phase difference the frequency grating has been examined exper­ imentally by Jefferson and Meixner (1992). Each of the pulses in the train of pulse pairs incident on the sample, while sustaining the ac­ cumulated frequency grating, simultaneously gives rise to a stimulated echo. The intensity of this echo is strongly enhanced by the accumu­ lation effect, and the echo phenomenon under these conditions is referred to as the accumulated photon echo. Of course, the direction in which the accumulated photon echo is observed is deter­ mined by the phase-matching condition given in Eq. (6). Of particular interest is the phase-matching configuration given in Fig. 2A. Here, and are the wave vectors of the first and second

114

pulse (1 and 2 in Fig. 2B) in a pulse pair, which We examine are separated by a time delay the accumulated photon echo generated by the first pulse (3 in Fig. 2B) of a subsequent pulse According to the pair with a wave vector phase-matching condition, the accumulated photon echo is generated with a wave vector at a time delay of with respect to pulse 3. Note that the accumulated photon echo coincides in direction and time with the second pulse of the pulse pair, while it can be shown that they also have the same phase. Therefore, constructive in­ terference occurs between the accumulated pho­ ton echo and this second pulse, giving rise to a homodyne component in the measured intensity of the transmitted beam in the direction of This component is measured as an increase of the transmitted intensity, and is proportional to the amplitude of the photon echo. This means that the homodyne detected accumulated photon echo will decay as instead of the factor in Eq. (5). More importantly, this detec­ tion scheme provides a greatly enhanced sensitiv­ ity.

Thijs J. Aartsma et al. So far we have assumed that the inhomogene­ ous broadening is static and much larger than the homogeneous broadening. However, the first assumption is generally not valid for disordered systems like glasses and proteins. In these sys­ tems, low energy disorder modes cause spectral diffusion on all time scales from picoseconds to infinity, even at very low temperatures (Breinl et al., 1984; Meijers and Wiersma, 1991; Saikan et al., 1992; Thorn-Leeson et al., 1994). As a conse­ quence the distinction between homogeneous and inhomogeneous broadening becomes ill-defined. More importantly, the photon echo decay time and the linewidth as measured in hole burning spectroscopy depend on the time scale of the ex­ periment (Narasimhan et al., 1990). In accumu­ lated photon echo measurements, this time scale is of the order of microseconds to milliseconds, while in hole burning it extends from milliseconds to seconds or even minutes. The concept of the frequency grating has a strong analogy with hole burning spectroscopy. Indeed, it may be shown that the accumulated photon echo decay and the homogeneous line shape are related by a Fourier transformation (Saikan et al., 1988). Therefore, if effects of spec­ tral diffusion can be neglected, the information derived from both types of experiments is ex­ pected to be the same. Finally we note that it is not absolutely neces­ sary to use transform-limited pulses for the gen­ eration of accumulated photon echoes (Asaka et al., 1984). This can be easily understood if it is realized that the contribution to the buildup of the frequency grating is determined by the corre­ lation function of the excitation pulses. Exper­ imentally, stochastic pulses which are not fully transform-limited can be used to enhance the time resolution of the measurements (Meech et al., 1986). However, the enhanced time resol­ ution is obtained at the expense of spectral resol­ ution. IV. Accumulated PhotonEcho: Experimental In a typical accumulated photon echo experiment the sample is irradiated by two excitation beams which originate from a single mode-locked dye laser. The experimental set-up is schematically

Accumulated photon echo measurements

presented in Fig. 3. The two parallel beams are focussed into the sample, resulting in a noncollin­ ear geometry as in Fig. 2. One of the beams is delayed with respect to the other by reflection from a retroreflector mounted on a variable trans­ lation stage. In this configuration, the sample is excited by a succession of pulse pairs at a repeti­ tion rate determined by the characteristics of the mode-locked laser, typically about 80 MHz. The accumulated photon echo is detected in the direction of the delayed beam. To enhance the signal-to-noise ratio, both beams are amplitude-modulated and the signal is measured by phase-synchronous detection. The first beam is modulated by an electro-optic modulator (EOM) at a frequency which is much higher than the inverse of the lifetime of the bottleneck state. The intensity of the accumulated photon echo will be modulated at the same frequency, while the build-up of the frequency grating is not signifi­ cantly affected. The delayed beam is modulated by a mechanical chopper at a frequency which is (slightly) lower than the inverse of the bottleneck lifetime. The detection system (Fig. 4) consists of a pho­ todiode in combination with a tuned amplifier, the frequency being determined by that applied to the EOM, which is 8 MHz. The output of the amplifier is demodulated by an 8 MHz amplitude demodulation circuit. This demodulated signal,

115

carrying the 2.5 kHz modulation frequency of the delayed beam, is fed into a lock-in amplifier, the output of which is proportional to the intensity of the accumulated photon echo. This double modulation scheme provides a high sensitivity for measurements of intensity changes. Equally important for accumulated pho­ ton echo measurements, however, is the rejection of the background to the accumulated photon echo signal. The background arises from the change in transmission of the delayed beam upon

116 population of the bottleneck state. There may also be an absorbance change due to the popula­ tion of the excited state, but this is typically sev­ eral orders of magnitude smaller than the inten­ sity of the accumulated photon echo. Note that the diagram in Fig. 3 is actually identical to that of a pump-probe setup for mea­ surements of absorbance changes (van Noort, 1994), but in the latter the EOM is replaced by an acousto-optic modulator (AOM). The diffracted beam of the AOM is subject to a small frequencyshift (equal to the acoustic frequency of the AOM) which effectively destroys the phase re­ lationship between successive pulse pairs incident on the sample (de Boer, 1991). This prevents the accumulation of a frequency grating. V. Energy Transfer The accumulated photon echo technique provides a method to directly measure the coherent life­ time of the initially excited state in pigment–protein complexes. Therefore, such measurements can provide information about the mechanism of energy transfer in photosynthetic antenna systems at low temperatures, where pure optical dephas­ ing can be neglected. Note, that in the weakcoupling limit excitations are localized on single pigment molecules, and these excitations jump from one molecule to another at a rate which is given by the well-known Förster-equation (För­ ster, 1948). In the strong coupling limit, the ex­ cited states of the system are more properly de­ scribed in terms of exciton states. Such states are formally described by diagonalizing the Hamil­ tonian matrix including the dipolar coupling. Since the corresponding wavefunctions are linear combinations of the molecular wavefunctions of the interacting chromophores, exciton states are intrinsically delocalized. In this formalism, energy dissipation corresponds to (vibronically induced) transitions between exciton states. At room tem­ perature, exciton scattering processes may be very fast, and under such conditions energy trans­ fer is essentially an incoherent hopping process except, possibly, at very short times (tens of fem­ toseconds) after exitation. At sufficiently low temperature, however, one would expect a much longer lifetime of the exciton coherence, and a

Thijs J. Aartsma et al. corresponding difference in the nature of energy transfer. Accumulated photon echo experiments were performed on the water-soluble, BChl a contain­ ing Fenna–Matthews–Olson (FMO) complex from green sulfur bacteria (Louwe and Aartsma, 1994) in the wavelength range of 810 to 830 nm. This complex is well characterized in terms of structure, intermolecular interactions, and spec­ troscopic properties (Olson, 1978; Vasmel et al., 1983; Swarthoff et al., 1980; Swarthoff et al., 1981; van Mourik et al., 1994). The structure of the FMO complex has been elucidated by X-ray crystallography with near atomic resolution (Mat­ thews and Fenna, 1980; Tronrud et al., 1986) and consists of three subunits in symmetry, each containing seven BChl a molecules. The nearestneighbour distance between the BChls within each subunit is around 12 Å and that between BChls belonging to different subunits is approxi­ mately 24 Å, yielding dipolar interactions be­ tween the BChls with a maximum of respectively.

and The absorption and circular dichroism spec­ tra at cryogenic temperatures can be simulated reasonably well using an exciton model including all 21 BChls (Pearlstein, 1992; Lu and Pearlstein, 1993). Time-resolved spectroscopy has mostly been applied at room temperature (Causgrove et al., 1988; Lyle and Struve, 1990; Savikhin et al., 1994; Gillbro, 1988; Zhou et al., 1994), and showed that localization of excitations occurs in less than 1 ps. Fluorescence lifetime measure­ ments at 77 K (Zhou et al., 1994) revealed a dominant time constant of 2 ns. The experimental results of the photon echo measurements at 1.4 K show an increasing rate of dephasing as the wavelength of excitation de­ creases, with time constants varying from subpicoseconds at the shortest to hundreds of pico­ second at the longest wavelengths (Louwe and Aartsma, 1994). At all wavelenghts the photon echo decay is strongly multiexponential. How­ ever, all decays between 816 and 830 nm can be fitted within experimental error with the same set of four time constants, given by 430, 140, 50 and 8 ps. The corresponding values of are obtained by multiplying the time constants with a factor of two. We assign these time constants to the dephasing of discrete electronic energy states of

Accumulated photon echo measurements the FMO complex. The spectral distribution of these four components form distinct bands with a width of approximately The transitions are centered around 826, 825, 822 and 818 nm, respectively. Experiments at 5 K showed a similar distribution, but with time constants of 250, 75, 12.5 and 8 ps. At wavelengths shorter than 816 nm even faster components appear, down to less than a few hundred fs at 790 nm. A straightforward explanation for the discrete energy levels observed in the decay associated spectra is that they correspond to the lowest en­ ergy states in the exciton manifold of the FMO complex. In total there are 21 exciton states, 14 of which are pair-wise degenerate due the symmetry of the complex. At very low tempera­ ture, where thermally induced processes are quenched, excited state decay can only occur to lower energy levels in the system. This involves not only decay to the ground state, but also trans­ itions within the exciton manifold induced by some form of vibronic coupling. According to this picture, the accumulated photon echo decay at 1.4 K represents the loss of exciton coherence either by decay to the ground state or by downward exciton scattering. The dephasing time for a given wavelength of excitation is expected to decrease according to the number of lower lying states, in agreement with the ex­ perimental observation. In addition, the relax­ ation time will also be affected by the phonon density of states as a function of frequency. With increasing temperature, we expect that thermally activated transitions within the exciton manifold will occur. Fig. 5 shows the homogene­ ous linewidths of the lowest exciton states of the FMO complex calculated from the echo decay at 821 and 826 nm, as a function of temperature between 1.4 and 30 K. The temperature depen­ dence of the two longest time constants for the dephasing could be fitted with a power law (Völker, 1989), combined with an exponential term (Jackson and Silbey, 1983) representing thermally activated population of higher exciton states (R.J.W. Louwe and T.J. Aartsma, to be published). Persistent hole burning experiments at 4.2 K (Johnson and Small, 1991) revealed eight trans­ itions in the manifold. Relatively narrow holes width, limited by spectral resolution)

117

were only observed in the longest wavelength re­ gion of the absorption band of the FMO complex. At shorter wavelengths broad holes were ob­ tained with a width of This large width was attributed to rapid energy relaxation, with a time constant of 0.1 ps, to the lowest energy state in the system. These results differ substantially from those obtained from the accumulated pho­ ton echo. At present, the origin of this discrep­ ancy is not clear. Presumably, it is related either to the mechanism of hole burning in this system, or to the effect of spectral diffusion which would affect hole burning and accumulated photon echo measurements to a different degree considering the different experimental time scales involved. Accumulated photon echo experiments have also been performed on the light-harvesting com­ plex LHCII of green plants, which has a ab­ sorption band extending from 640 to 680 nm. The structure of this complex has been resolved to 3.8 Å resolution, and contains chlorophyll a as well as chlorophyll b. The accumulated photon echo decays are very similar to those of the FMO com­ plex in the sense that we obtain a set of time constants with a distinct spectral distribution, and

118 comparable in magnitude to those of the FMO complex (R.J.W. Louwe and T.J. Aartsma, to be published). Therefore, we believe that the results of the FMO-complex are representative of the excited state dynamics in antenna complexes at low temperature. VI. Photon Echo Experiments On Reaction Centers The kinetics of the primary electron transfer reac­ tions that occur in photosynthetic reaction centers (RCs) have been the subject of numerous investi­ gations (reviewed in Kirmaier and Holten, 1993; Zinth and Kaiser, 1993; Shuvalov, 1993). Most mea­ of these investigations involved surements to provide information about the populations dynamics of the states involved. In the accumulated photon echo experiments de­ scribed here, the optical dephasing of the excited state of the primary donor P at low temperature is measured directly. By a comparison of the dephasing rate with the population dynamics, further insight can be gained about the primary step of charge separation. The first photon echo experiments on RCs of purple bacteria have been performed by Meech et al. (1985). From their experiments, they con­ cluded that the excited state of the primary donor decayed within less than 100 fs, supposedly to an intra-dimer charge transfer state. Furthermore, they concluded from hole burning experiments, that the absorption band of the primary donor is largely homogeneously broadened. Contrary to these results, Johnson et al. (1989) later observed holes with a FWHM of several wavenumbers in transient hole burning experi­ ments, which is in contradiction to the abovementioned very fast electronic decay. The electron-phonon coupling in these systems is quite strong, in contrast to what is commonly observed in monomeric chlorophylls in organic glasses (van der Laan et al., 1991). The accumulated photon echo decay at 1.5 K in the reaction center of the photosynthetic bac­ terium Rb. sphaeroides R26 was recently reexam­ ined, and extended to measurements on the mu­ tant (M)Y210W (P. Schellenberg, R.J.W. Louwe, S. Shochat, P. Gast, A.J. Hoff and T.J. Aartsma, to be published). This mutant exhibits

Thijs J. Aartsma et al. an extraordinarily long electron transfer time (Shochat et al., 1994; Nagarajan et al., 1993). The experiments were performed under con­ ditions that the triplet state of the special pair, with a lifetime of about (Chidsey et al., 1985), functions as the bottleneck state. Fig. 6A shows the echo amplitude decay of RCs of Rb. sphaeroides R26. The decay is charac­ terized by a very sharp feature around t = 0, and a longer-lived component with a much smaller amplitude. This decay pattern is in good agree­ ment with the shape of the homogeneous line obtained by hole burning measurements (Jan­ kowiak and Small, 1993). Considering the Fourier-transform relationship, the sharp feature in the photon echo decay can be attributed to a contri­ bution of the phonon sideband (Saikan et al., 1988) and reflects the extremely rapid dephasing which is associated with vibronic relaxation. The longer decay component is correlated with the width of the zero-phonon line, and is presumably determined by electron transfer. This component is only observed in the wavelength range of 908– 920 nm, the same region where the zero-phonon hole is found in hole burning experiments (John­ son et al., 1989). Closer inspection of the longer decay compo­ nent shows that it is biexponential, with time con­ stants of 1.2 and 8 ps. The biexponential kinetics has also been observed in pump-probe and fluor­ escence lifetime measurements at room tempera­ ture as well as at cryogenic temperatures (Kirma­ ier and Holten, 1993; Müller et al., 1992; Nagarajan et al., 1990). These low temperature data agree reasonably well with the values ob­ tained from the accumulated photon echo mea­ surements. Fig. 6B shows the photon echo decay of RCs from the (M)Y210W mutant of Rb. sphaeroides at 918 nm. The fast component can again be attributed to the phonon contribution to the elec­ tronic transition. Considering the longer lived components, it can be concluded that the primary charge transfer in this mutant at low temperature is much slower than in the native species, similar to results obtained at room temperature (Shochat et al., 1994; Nagarajan et al., 1993). However, whereas the rate of charge separation in the native strain increases towards low temperature

Accumulated photon echo measurements

(Lauterwasser et al., 1991), that of the mutant decreases significantly (van Noort, 1994). Thus, both the native and the mutant RCs show a biexponential accumulated photon echo decay which presumably corresponds to the in­ trinsic dynamics of the primary step of charge separation. Several explanations have been put forward to account for this nonexponential kin­ etics which is also observed in pump-probe and fluorescence decay experiments. From our mea­ surements, the so-called parking state model (Müller et al., 1992; Hamm et al., 1993) can clearly be excluded, because the dephasing of the P* state would then be monoexponential. The biexponential decay is more consistent with the dispersion model (Kirmaier and Holten, 1990; Müller et al., 1992) which assumes that the free energy difference between P* and I being the primary acceptor, is characterized by a more or less continuous distribution. This is based on the conjecture that, in many respects, proteins behave like glasses and that the intrinsic disorder gives rise to a broad distribution of energy levels. In the dispersion model, the appropriate fit-function would be a stretched exponential, whereas in the substate model, a multiexponential function would have to be used. It is difficult to distinguish

119

between these two cases on the basis of the ac­ cumulated photon echo decay measurements. VII. Conclusion and Perspectives The accumulated photon echo technique has been used to study excited state dynamics of molecular systems, and its range of applications now in­ cludes photosynthetic pigment–protein com­ plexes. A prerequisite for the observation of the accumulated photon echo is the efficient popula­ tion of a long-lived intermediate state in the relax­ ation of the excited state to the ground state. Such an intermediate state functions as a bottleneck in the relaxation process. In photosynthetic antenna complexes the bottleneck consists of the triplet state, while in reaction center complexes either the triplet state or the charge-separated state may be involved depending on experimental con­ ditions. At sufficiently low temperatures, pure optical dephasing is negligible, and the accumulated pho­ ton echo decay is dominated by population relax­ ation. In photosynthetic reaction centers this decay is determined by primary charge separ­ ation. Thus it has been possible to measure the charge separation rate at low temperature in the

120 reaction center of Rhodobacter sphaeroides R26 and in that of the (M)Y210W mutant of the same species. The results are consistent with pumpprobe measurements. Is is also concluded that there is no reason to assume an early intermediate in the primary charge transfer step, as was sug­ gested by Meech et al. (1985). In isolated antenna complexes, the echo decay is strongly multiexponential and dependent on the wavelength of excitation. The different time constants appear to be associated with distinct bands in the absorption spectrum. These bands are attributed to transitions to discrete exciton states, which implies that the excitations are in­ trinsically delocalized. The coherent lifetimes of the exciton states are surprisingly long, and this would suggest that a hopping model for energy transfer at low temperature may not be appropri­ ate. It may be concluded that accumulated photon echo measurements have proven to be very useful in the study of excited state dynamics in pigment– protein complexes, although the full potential of this technique has not been realized yet in this field of research. The results sofar provided a new perspective on the dynamics of exciton states in such complexes, and this warrants further investi­ gations. Acknowledgements This work was supported by the Life Sciences Foundation (SLW), which is subsidized by the Netherlands Organization for Scientific Research (NWO) and by the European Union. (Contract ER8CHBGCT 930361). References Aartsma TJ and Wiersma DA (1976) Photon echo relaxation in molecular mixed crystals. Chem Phys Lett 42: 520–524. Abella ID, Kurnit and Hartmann (1966) Photon echoes. Phys Rev 141: 391–406. Allen L and Eberly JH (1975) Optical Resonance and TwoLevel Atoms. Wiley, New York. Asaka S, Nakatsuka H, Fujiwara M and Matsuoka M (1984) Accumulated photon echoes with incoherent light in doped silicate glass. Phys Rev A 29: 2286–2289. Breinl W, Friedrich J and Haarer D (1984) Spectral diffusion of a photochemical proton transfer system in an amorph­ ous organic host: Quinizarin in alcohol glass. J Chem Phys 81: 3915–3921.

Thijs J. Aartsma et al. Causgrove TP, Yang S and Struve WS (1988) Polarized pumpprobe spectroscopy of exciton transport in bacteriochloro­ phyll a protein from Prosthecochloris aestuarii. J Phys Chem 92: 6790–6795. Chidsey CED, Takiff L, Goldstein RA and Boxer SG (1985) Effect of magnetic fields on the triplet state lifetime in photosynthetic reaction centers: Evidence for thermal re­ population of the initial radical pair. Proc Natl Acad Sci USA 82: 6850–6854. de Boer S (1991) Optical Dynamics of Molecular Aggregates. Doctoral thesis, University of Groningen, The Nether­ lands. Dicke RH (1954) Coherence in spontaneous radiation pro­ cesses. Phys Rev 93: 99–110. Duppen K and Wiersma DA (1986) Picosecond multiple-pulse experiments involving spatial and frequency gratings: a uni­ fying nonperturbational approach. J Opt Soc Am B 3: 614– 621. Förster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann Phys 6: 55–75. Gillbro T (1988) Picosecond energy transfer kinetics in chloro­ somes and bacteriochlorophyll a-proteins of Chlorobium limicola In: Olson JM, Ormerod JG, Amesz J, Stackebrand E and Truper HG (eds) Green Photosynthetic Bacteria, pp 91–96. Plenum Press, New York. Hamm P, Gray KA, Oesterhelt D, Feick R, Scheer H and Zinth W (1993) Subpicosecond emission studies of bac­ terial reaction centers. Biochim Biophys Acta 1142: 99– 105. Hesselink WH (1980) Picosecond Dephasing andRelaxation in Molecular Mixed Crystals. Doctoral thesis, University of Groningen, The Netherlands. Hesselink W and Wiersma DA (1978) Picosecond photon echoes detected by optical mixing. Chem Phys Lett 56: 227–230. Hesselink WH and Wiersma DA (1979) Picosecond photon echoes from an accumulated grating. Phys Rev Lett 43: 1991–1994. Jackson B and Silbey R (1983) Theoretical description of photochemical hole burning in soft glasses. Chem Phys Lett 99: 331–334. Jankowiak R and Small GJ (1993) Spectral hole burning: A window on excited state electronic structure, heterogene­ ity, electron-phonon coupling, and transport dynamics of photosynthetic units. In: Deisenhofer J and Norris JR (eds) The Photosynthetic Reaction Center Vol II, pp 133– 177. Academic Press, San Diego. Jefferson CM and Meixner AJ (1992) Frequency-domain mea­ surements of spectral hole patterns burned with phasecoherent pulses. Chem Phys Lett 189: 60–66. Johnson SG and Small GJ (1991) Excited state structure and energy-transfer dynamics of the bacteriochlorophyll a antenna complex from Prosthecochloris aestuarii. J Phys Chem 95: 471–479. Johnson SG, Tang D, Jankowiak R, Hayes JM and Small GJ (1989) Structure and marker mode of the primary donor state absorption of photosynthetic bacteria: Hole-burned spectra. J Phys Chem 93: 5953–5957. Kirmaier C and Holten D (1990) Evidence that a distribution of bacterial reaction centers underlies the temperature and

Accumulated photon echo measurements detection-wavelength dependence of the rates of the pri­ mary electron-transfer reactions. Proc Natl Acad Sci USA 87; 3552–3556. Kirmaier C and Holten D (1993) Electron transfer and charge recombination reactions in wild-type and mutant bacterial reation centers. In: Deisenhofer J and Norris JR (eds) The Photosynthetic Reaction Center Vol II, pp 49–70. Academic Press, San Diego. Lauterwasser C, Finkele U, Scheer H and Zinth W (1991) Temperature dependence of the primary electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides. Chem Phys Lett 183: 471–477. Levenson MD and Kano SS (1988) Introduction to Nonlinear Spectroscopy. Academic Press, San Diego. Louwe RJW and Aartsma TJ (1994) Optical dephasing and excited state dynamics in photosynthetic pigment-protein complexes. J Luminescence 58: 154–157. Lu X and Pearlstein RM (1993) Simulations of Prosthecoch­ loris bacteriochlorophyll a-protein optical spectra improved by parametric computer search. Photochem Photobiol 57: 86–91. Lyle PA and Struve WS (1990) Evidence for ultrafast localiz­ ation in the band of bacteriochlorophyll a-protein from Prosthecochloris aestuarii. J Phys Chem 94: 7338–7339. Matthews B and Fenna R (1980) Structure of a green bacteri­ ochlorophyll protein. Acc Chem Res 13: 309–317. Meech SR, Hoff AJ and Wiersma DA (1985) Evidence for a early intermediate in bacterial photosynthesis.A photonecho and hole-burning study of the primary donor band in Rhodopseudomonas sphaeroides. Chem Phys Lett 121: 287–292. Meech SR, Hoff AJ and Wiersma DA (1986) Role of chargetransfer states in bacterial photosynthesis. Proc Natl Acad Sci USA 83: 9464–9468. Meijers HC and Wiersma DA (1991) Spectral diffusion in glasses: a photon-echo study of zincporphin in ethanol. Chem Phys Lett 181: 312–318. Meijers H and Wiersma DA (1992) Glass dynamics probed by the long-lived stimulated photon echo. Phys Rev Lett 68: 381–384. Morsink JBW, Hesselink WH and Wiersma DA (1979) Pho­ ton echo stimulated from optically induced nuclear spin polarization. Chem Phys Lett 64: 1–4. Morsink JBW, Hesselink WH and Wiersma DA (1982) Pho­ ton echoes stimulated from long-lived ordered populations in multi-level systems. The effect of intersystem crossing and optical branching. Chem Phys 71: 289–294. Müller MG, Griebenow K and Holzwarth AR (1992) Primary processes in isolated bacterial reaction centers from Rhodo­ bacter sphaeroides studied by picosecond fluorescence kin­ etics. Chem Phys Lett 199: 465–469. Nagarajan V, Parson WW, Gaul D and Schenk CC (1990) Effects of specific mutations of tyrosine-M210 on the pri­ mary photosynthtic electron-transfer process in Rhodob­ acter sphaeroides Proc Natl Acad Sci USA 87: 7888–7892. Nagarajan V, Parson WW, Davis D and Schenk CC (1993) Kinetics and free energy gaps of electron-transfer reactions in Rhodobacter sphaeroides reaction centers. Biochemistry 32: 12324–12336. Narasimhan LR, Littau KA, Pack DW, Bai YS, Elschner

121 A and Fayer MD (1990) Probing organic glasses at low temperature with variable time scale optical dephasing measurements. Chem Rev 90: 439–457. Olson JM (1978) Bacteriochlorophyll a-proteins from green bacteria. In: Clayton RK and Sistrom WF (eds) The Photo­ synthetic Bacteria, pp 161–178. Plenum Press, New York. Pearlstein RM (1992) Theory of the optical spectra of the bacteriochlorophyll a-antenna protein trimer from Pro­ sthecochloris aestuarii, Photosynth Res 31: 213–226. Rebane A, Kaarli R, Saari P, Anijalg A and Timpmann K (1983) Photochemical time-domain holography of weak picosecond pulses. Opt Comm 47: 173–176. Saikan S, Nakabayashi T, Kanematsu Y and Tato N (1988) Fourier-transform spectroscopy in dye-doped polymers using the femtosecond accumulated photon echo. Phys Rev B 38: 7777–7781. Saikan S, Lin JW and Nemoto H (1992) Non-Markovian relaxation observed in photon echoes of iron-free myoglo­ bin. Phys Rev B 46: 11125–11128. Savikhin S, Zhou W, Blankenship RE and Struve WS (1994) Femtosecond energy transfer and spectral equilibration in bacteriochlorophyll a-protein antenna trimers from the green bacterium Chlorobium tepidum. Biophys J 66: 110– 114. Shochat S, Arlt T, Francke C, Gast P, van Noort PI, Otte SCM, Schelvis HPM, Schmidt S, Vijgenboom E, Vrieze J, Zinth W and Hoff AJ (1994) Spectroscopic characteriz­ ation of reaction centers of the (M)Y210W mutant of the photosynthetic bacterium Rhodobacter sphaeroides. Photosynth Res 40: 55–66. Shuvalov VA (1993) Time and frequency domain study of different electron transfer processes in bacterial reaction centers. In: Deisenhofer J and Norris JR (eds) The Photo­ synthetic Reaction Center Vol II, pp 89–103. Academic Press, San Diego. Swarthoff T, de Grooth BG, Meiburg RF, Rijgersberg CP and Amesz J (1980) Orientation of pigments and pigmentprotein complexes in the green photosynthetic bacterium Prosthecochloris aestuarii. Biochim Biophys Acta 593: 51– 59. Swarthoff T, Amesz J, Kramer HJM and Rijgersberg CP (1981) The reaction center and antenna pigments of green photosynthetic bacteria. Israel J Chem 21: 332–337. Thorn-Leeson D, Berg O and Wiersma DA (1994) Low-temperature protein dynamics studied by long-lived stimulated photon echo. J Phys Chem 98: 3913–3916. Tronrud DE, Schmidt MF and Matthews BW (1986) Structure and X-ray amino acid sequence of a bacteriochlorophyll a-protein from Prosthecochloris aestuarii refined at 1.9 Å resolution. J Mol Biol 188: 443–454. van der Laan H, Smorenburg HE, Schmidt T and Völker S (1991) Permanent hole burning with a diode laser: excitedstate dynamics of bacteriochlorophyll in glasses and mi­ celles. J Opt Soc Am B 9: 931–940. van Mourik F, Verwijst RR, Mulder JM and van Grondelle R (1994) Singlet-triplet spectroscopy of the light-harvesting Bchl a complex of Prosthecochloris aestuarii. The nature of the low-energy 825 nm transition. J Phys Chem 98: 10307–10312. van Noort PI (1994) Energy Transfer and Primary Photochem­

122 istry in Photosynthetic Bacteria. A Picosecond Time-Resolved Study. Doctoral thesis, Leiden University, The Netherlands. Vasmel H, Swarthoff T, Kramer HJM and Amesz J (1983) Isolation and properties of a pigment-protein complex associated with the reaction center of the green photosyn­ thetic sulfur bacterium Prosthecochloris aestuarii. Biochim Biophys Acta 725: 361–367. Völker S (1989) Spectral hole-burning in crystalline and amorphous organic solids. Optical relaxation processes at low temperature. In: Fünfschilling J (ed) Relaxation

Thijs J. Aartsma et al. Processes in Molecular Excited States, pp 113–242. Kluwer Academic Publishers, Dordrecht. Zhou W, LoBrutto R, Lin S and Blankenship RE (1994) Redox effects on the bacteriochlorophyll a-containing Fenna-Matthews-Olson protein from Chlorobium tepidum. Photosynth Res 41: 89–96. Zinth W and Kaiser W (1993) Time-resolved spectroscopy of the primary electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodopseudomonas viridis In: Deisenhofer J and Norris JR (eds) The Photosynthetic Reaction Center Vol II, pp 71–88. Academic Press, San Diego.

Chapter 8

Spectral Hole Burning: Methods and Applications to

Photosynthesis

N. Raja S. Reddy* and Gerald J. Small Ames Laboratory, Department of Chemistry, Iowa State University, Ames IA 50011, USA

Summary I. Introduction II. Experimental Methods III. Applications A. Bacterial Reaction Centers B. Hole Burning at High Pressures C. Antenna Complexes D. Constant Fluence Hole Burning Spectroscopy Acknowledgements References

123 124 126 129 129 131 133 134 134 135

Summary Spectral hole burning Spectroscopy (SHB) is a high resolution technique that has been fruitfully applied to the study of photosynthetic protein complexes as well as electronic transitions in amorphous systems. It overcomes the limitations from inhomogeneous broadening and provides an improvement in spectral resolution of 2–3 orders of magnitude. The experimental aspects of SHB, including an apparatus for the hole burning studies at high pressures are discussed in considerable detail. For several reaction center and antenna complexes, the dependence of hole profiles on the burn wavelength has been used to identify the site inhomogeneous and homogeneous contributions to the -region absorption profiles. It has also been used to determine the excited state linear electron–phonon coupling parameters. Such data is crucial for understanding the energy and electron transfer dynamics. For example, the invariance of zero phonon hole width of P870* on burn wavelength strongly suggests that the recently observed non-single exponential decay kinetics in primary charge separation are not due to a distribution of values for electronic coupling matrix elements. The observation of nonphotochemical hole burning (NPHB) for reaction center of Rhodopseudomonos viridis has led to the identification for the first time, of all six states (including the special pair upper dimer component) of a bacterial RC. Recent results of hole burning in FMO antenna complex at high pressures have identified the need for modifying existing theories of pressure dependence of hole burned spectra. Absorption spectra for reaction center of Rhodopseudomonas viridis at high pressures are also presented. Novel constant fluence hole burning Spectroscopy used in the identification of BChl 870 and BChl 896 bands in the antenna complex of Rhodobacter sphaeroides as well as the lowest energy state of LHC II complex of Photosystem II is dismissed. Abbreviations: BChl – Bacteriochlorophyll; Chl – Chlorophyll; CP47 – A 47 kDa core antenna complex of Photosystem II; FMO complex – Fenna–Matthews–Olson complex of green sulfur bacteria; LH 1, LH 2 – Antenna complexes of purple bacteria; LHC II – Antenna complex of plants; NPHB – *Correspondence: Fax: 1-515-2941699; E-mail: [email protected]

123 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 123–136. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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Nonphotochemical hole burning; P, P870, P960 – Primary electron donor; PHB – Photochemical hole burning; PSBH – Phonon sideband holes; Q – (First) acceptor quinone; RC – Reaction center; SHB – Spectral hole burning; THB – Transient hole burning; ZPH – Zero phonon hole

I. Introduction 1994 marked the twentieth anniversary of spectral hole burning (SHB) spectroscopies as developed by R.I. Personov and L.A. Rebane and their coworkers (Gorokhovskii et al., 1974; Kharlamov et al., 1974) for application to the electronic transitions of chromophores embedded in amorphous or crystalline hosts at low tempera­ tures. For about ten years now, SHB has been fruitfully applied to photosynthetic protein com­ plexes. There are several attributes of SHB which ensure that such an application will (or should) receive greater attention. Which of these are most important depends on the problem being ad­ dressed, be it the role of phonons and structural heterogeneity in energy or electron transfer, exci­ ton level structure in complexes containing sev­ eral strongly interacting chlorophylls (Chls) or the frequencies and Franck–Condon factors of the optically active modes of the cofactors. From an analytical chemistry or materials characterization point of view, SHB can also be a useful technique as illustrated, for example, by the work of Chang et al. (1994) on the CP47 antenna complex of Photosystem II. The attributes of SHB important to the study of the excited state electronic structure and dy­ namics of photosynthetic protein complexes have been the subject of reviews by Reddy et al. (1992a), Jankowiak and Small (1993) and Jan­ kowiak et al. (1993). These reviews provide broad coverage of the complexes which have been stud­ ied and discussion of the importance of the results obtained. Also included are discussions of the basic principles of SHB and the theory of spectral hole profiles which one needs for a complete analysis of hole spectra. (The theory has been recently extended to arbitrary temperature by Hayes et al. (1994).) However, little attention in the above reviews is given to the experimental aspects of SHB. For this reason, these aspects are the primary focus of this chapter. The currently

available approaches to the recording (reading) of hole spectra are discussed along with their limi­ tations in application to photosynthetic com­ plexes. In addition, an apparatus for the study of the effects of high pressure (~1.5 GPa) on the excited state electronic structure and transport properties is described and unpublished results are presented. Other unpublished or recently published results which highlight the attributes of SHB are also given. We assume that the reader is familiar with the basic principles of SHB. For coverage of “non-photosynthetic” applications of SHB, both basic and technological, the reader is referred to the book edited by Moerner (1987) and a review article by Jankowiak et al. (1993). The latter also includes a section on photosyn­ thetic complexes. Those interested in the connec­ of the nonlinear optical suscepti­ tion (via bility) between SHB and photon-echo spectro­ scopies are referred to the review by M.D. Fayer and coworkers (Narasimhan et al., 1990) and a book by Mukamel (1995). Briefly, SHB involves the excitation of a nar­ row isochromat of chromophores within an inhomogeneously broadened absorption band. SHB occurs when the decay of the excited isoch­ romat to the ground state is either blocked (per­ sistent hole burning) or delayed (transient hole burning). Blocking of the decay to the ground state can be due to a photochemical reaction in­ itiated in the excited state such as the inner proton-tautomerization of the porphyrins or due to a rearrangement of the host environment (typi­ cally a glass or protein). These two mechanisms are commonly referred to as photochemical (PHB) and nonphotochemical (NPHB) hole burning. In transient hole burning (THB), an in­ termediate state or a shortlived product (that re­ verts to the initial material) in a photochemical reaction is used to store (temporarily) the de­ pleted ground state population. In addition to the zero-phonon holes (ZPH) at the burn laser frequency, all these mechanisms give rise, due

Spectral hole burning to coupling of the chromophore to the matrix phonons, to real- and pseudo-phonon sideband holes (PSBH) at energies higher and lower than the ZPH. In a similar manner, real- and pseudo­ vibronic holes appear due to loss of absorption at the excited state vibrational frequencies of the chromophore. To end this introduction, we briefly discuss the types of information on protein complexes SHB can provide and why they are important. Because it is a line-narrowing technique, SHB eliminates the contribution of inhomogeneous broadening to the widths of origin absorption bands. The most important contribution (at least for a good glass forming solvent and mild deterg­ stems from the fact that the members ent) to of an ensemble of a given complex have slightly different structures (conformations), a natural consequence of the “glass-like “ nature of prote­ ins. By determining the dependence of the hole structure (ZPH and its PSBH) on the burn fre­ within the origin band, one can not quency but also the center frequency only determine of the inhomogeneous distribution of zerophonon line (ZPL) transition frequencies. It has depending been found that is on the complex. At the same time one obtains the frequency distribution of the low frequency protein phonons which couple (are Franck–Condon active) to the optical transition as well as their coupling strength S (Huang–Rhys factor). For all antenna protein complexes which have been studied it has been observed that (Gillie et al., 1989; Johnson and Small, 1991; Reddy et al., is ~20– 1991) the mean phonon frequency, the width of the one-phonon profile is and that the coupling is weak, can be viewed as the S < ~0.6. The quantity would be the optical reorganization energy; Stokes shift (when the state being probed is flu­ orescent). To illustrate the importance of such results for transport dynamics we consider Förster energy transfer from one Chl (2) to another Chl (1) under the condition that the mean electronic energy gap ~ ~ For this not un­ common situation, the dominant accepting modes are the protein phonons. Furthermore, the usual overlap between the donor (2) and the acceptor (1) fluorescence and absorption bands cannot be

125 used to calculate the transfer rates at tempera­ tures much below room temperature (~200 K) because of the inhomogeneous broadening. Re­ quired for a correct calculation of the tempera­ ture dependence of the rate is the single complex spectrum, comprised of the ZPL and phonon sid­ eband, in the low temperature limit. (The spec­ trum for higher temperature can then be calcu­ lated.) Also required is the distribution of values. Fortunately, it has been shown that, if and are the inhomogeneous is broadenings, the width of the Thus, all ingredients are at hand for a calculation of the temperature depen­ dence of the rate and extent to which the kinetics are dispersive (non-exponential) (Hayes et al., 1994). We note that the physics just described has been used to investigate whether the non-single exponential decay of the primary electron donor state, P870*, of Rb. sphaeroides is due to disper­ sive kinetics stemming from a static distribution of energy gap values (Lyle et al., 1993). As re­ viewed by G.S. Small and coworkers (Reddy et al., 1992a; Jankowiak and Small, 1993; Jankow­ iak et al., 1993), the width of the ZPH burned can also yield into the origin band of a of that state in the low tempera­ the lifetime ture limit, where dephasing is dominated by population decay. In fact, the width (FWHM) of the ZPH in is given by For ps, the width is Although ultrafast time domain spectroscopy is generally a more powerful and versatile approach to transport dynamics, it should be noted that hole burning is truly state selective in that it measures decay from the total vibrational zero-point level. This turns out to be important in consideration of whether or not ther­ malization of chromophore and bath modes pre­ electron and energy trans­ cedes ultra-fast fer as first discussed by Tang et al. (1989) and Johnson et al. (1990). Finally, we emphasize that the superior resol­ ution afforded by SHB can often lead to resol­ This has proven ution of closely spaced to be most useful for the problem of exciton level structure in antenna complexes which possess a structural unit with several strongly coupled Chl molecules (Zuber and Brunisholz, 1991; Lu and Pearlstein, 1993). A very nice example of this is

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N. Raja S. Reddy and Gerald J. Small

from the work of Johnson and Small (1991) on the Fenna–Matthews–Olson BChl a complex of Prosthecochloris aestuarii. In this work it was also revealed that downward cascading between exci­ ton levels occurs in ~100 fs at 4.2 K. This result was later confirmed by the femtosecond experi­ ments. II. Experimental Methods The basic experimental setup for a hole burning is relatively simple. It consists of a laser (burn) for exciting a narrow isochromat of an inhomo­ geneously broadened absorption band and a means for probing the absorption prior to and after the burning. Modifications to this basic setup are dictated by the types of information sought and whether the type of hole burning is persistent or transient, vide infra. Absorption changes are monitored via either the transmission of a probe beam or fluorescence excitation (pro­ vided the sample is fluorescent) in any one of several ways. When one is concerned only with the ZPH, the same laser as used for burning (after suitable attenuation) can be tuned to measure the ZPH in the transmission mode. However, in the study of photosynthetic systems, the probe laser is often replaced by light from a monochromator and modulation techniques are used to improve the signal to noise ratio. A schematic diagram of an experimental set up in our laboratory for persistent hole burning is shown in Fig. 1. Both fluorescence excitation mode as well as in laser transmission mode can be employed. A Coherent ring dye laser (CR 699–29) pumped by an argon ion laser provides the required radiation for burning and scanning the holes. Sample S is located in a liquid helium cryostat with four optical windows. Two photo­ multipliers PMTS and PMTR provide the sample and reference signal. When used in transmission mode, the photomultiplier PMTS is moved to position T and absorbance is obtained from the signals from PMTS and PMTR. In the fluor­ escence excitation mode, the signal from PMTR is used to normalize the fluorescence signal from PMTS. For high resolution scans (<~20 MHz) of sufficiently narrow ZPH, the laser frequency by scanning the intracavity is scanned etalon assembly (ICA). Wider scans (~50 nm)

are achieved by removing the ICA and scanning the Lyot filter (via a stepper motor). The photon counting set up is useful when the sample is weakly fluorescent. Transient SHB can also be performed with this setup provided the bottle­ neck state has a sufficiently long lifetime (<~100 Wannemacher et al. (1993) developed the ex­ perimental setup shown in Fig. 2. It is based on burn-scan technique (Cone et al., 1984) and em­ ploys diode lasers. This setup can be used for both persistent and transient HB spectra and is capable of scanning wider frequency ranges at much faster rates The laser frequency is scanned either by adjusting the temperature of the diode or by modulating the current flow through it. A variable voltage source, triggered by a pulse generator, controls the laser current (hence the laser frequency). The pulse generator also provides synchronizing pul­

Spectral hole burning

ses for a pair of acousto-optic modulators which are used to provide light pulses of the required temporal width (for burning holes). A Fabry– Perot etalon allows for laser beam diagnostics and measures the laser frequency during a scan. Additional electronics, such as the optical shutter and oscilloscope, are also synchronized by the pulse generator. Holes are burned with a constant laser diode current (i.e. fixed burn frequency) and holes are scanned by ramping the voltage applied to the diode. Hole detection in the trans­ mission mode (using a photomultiplier and signal averaging) is accomplished with a digital oscillo­ scope and a personal computer. The time be­ tween hole burning and detection can be as short as The above two setups are limited in their scan ranges to a few tens of nm which limits the useful­ ness of SHB in studies of energy/electron transfer and excited state structure in photosynthetic com­ plexes. This problem can be overcome by em­ ploying a Fourier transform (FT) spectrometer that replaces everything after the light attenuating

127

filters NDF in Fig. 1. It is capable of fast (100 scans/minute), high resolution scans over a broad wavelength region (400–1100 nm). In another setup in our laboratory, an FT spec­ trometer operating in the visible and near-infrared region has been used in combination with a Coherent CR899–21 Ti:sapphire laser. Holographic detection of holes, based on laser induced gratings, utilizes two beams overlapped at the sample in the hole burning process (Renn et al., 1985). The spatially modulated light inten­ sity at the crossing of the two beams forms a spatial grating in the medium. The spatial grating is due to spatially modulated absorption and re­ fractive index coefficients. For readout, one tun­ able beam is used and diffraction of that beam by the spatial grating is detected. Holographic detection is a highly sensitive zero-background technique. Its application to the study of biologi­ cal samples is however, limited since the diffrac­ tion efficiency is a function of read frequency and the stringent requirement that the optical quality of the sample be high.

128

For SHB studies in antenna and reaction cen­ ter complexes at variable pressure and tempera­ ture (4.2–300 K), we have utilized a specially designed (Unipress Equipment, Poland) high pressure system (Buinowski et al., 1973). The high pressure setup consists of a three stage gas compressor and a demountable high pressure cell, containing the sample, that is located in a liquid helium cryostat Fig. 3(a). Helium gas is used as the pressure transmitting medium. High pressures generated at the compressor are transmitted to the pressure cell by a flexible beryllium–copper alloy capillary tube. The three stages of the com­ pressor are oil/gas pressure intensifiers that are interconnected, with the first stage also connected to a helium gas cylinder. The gas flow between the compressor stages is regulated by use of needle valves. At the heart of the compressor operation is a pump capable of generating oil pressures of up to 80 MPa with manually con­

N. Raja S. Reddy and Gerald J. Small

trolled output. High initial compressibility of he­ lium gas requires that the compression ratio in the first stage is close to unity. The maximum gas pressure that can be achieved in the first two stages is about 400 MPa. Gas compression in the final stage is different from that in the earlier stages because it occurs at constant mass, i.e. the amount of gas in the volume defined by the third stage (the capillary connector and the high pres­ sure cell) is kept constant. The maximum pres­ sure achievable in the third stage is about 1.5 GPa (~15,000 atm.). Fig. 3(b) shows a section of the cylindrical high pressure cell, made of beryllium–copper alloy, in the plane of the windows. Four (leuco)sapphire windows (shown in black) one of which can be replaced by a pressure gauge provide optical ac­ cess to the sample. Indium-coated brass O-rings are placed at the end of the conical plugs (P). The conical plug is held in place by a annular

Spectral hole burning

brass plug (B). Tightening the brass plugs crushes the O-rings between the conical plug and the cell body, thus isolating the high pressure sample re­ gion from outside. The capillary tube connection to the compressor is made from the top of the high pressure cell. We conclude this section by noting that the recording or reading of hole spectra in the trans­ mission mode has been, by far, the preferred method for photosynthetic protein complexes. (The advantages of using a FT spectrometer for reading have been emphasized.) There are sev­ eral reasons for this, including simplicity and that for the maximum benefits from hole burning one needs to acquire the entire hole spectrum, not just the ZPH. III. Applications

A. Bacterial Reaction Centers As reviewed by Jankowiak et al. (1993) photoch­ emical hole burning has been extensively applied to the RC complexes of Rhodobocter sphaeroides and Rhodopseudomonas viridis and, in particular, to their primary electron donor states P870* and P960*, respectively. Lyle et al. (1993) and Reddy et al. (1993b) have published PHB spectra with a vastly improved signal/noise ratio. Theoretical analyses of the burn frequency dependence of the hole spectra led to an improved set of values for the quantities that determine the P870 and P960 absorption profiles (Table I). Included in this Table are the values of inhomogeneous broaden­ ing and electron–phonon coupling parameters.

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The coupling is dictated by phonons with a mean frequency of and a special pair “marker” mode with a frequency of 115 and for P870 and P960, respectively. The correspond­ ing S-values establish that the electron–phonon coupling associated with the optical transition is strong in striking contrast with the of antenna protein complexes (cf. Introduction). Thus, P* is also “special” in that its electron–phonon coupling is strong. How this strong coupling is connected with the charge transfer character of P* is discussed in the review by Jankowiak and Small (1993). Interestingly, the coupling for the other of the RC is weak. Fig. 4 shows the burn frequency depen­ dence of the P870 hole spectrum from Lyle et al. (1993) for the deuterated RC of Rhodobacter sphaeroides. The weakness of the ZPH coincident with the burn frequency is the result of strong electron–phonon coupling. Small et al. (1992) developed a theory for non adiabatic electron-transfer and used the hole burning and other results to argue that the in­ triguing, non-single exponential decays of P*, vide supra, is not due to a distribution of P*acceptor state values. The finding of Lyle et al. (1993) that the ZPH-width of P870* is invariant to the value of the burn frequency within the inhomogeneous distribution of ZPL transition frequencies strongly suggests that the non-single exponential decays are not due to distribution of values for the electronic coupling matrix elements associated with the initial phase of charge separation. At present, a convincing explanation for the non-single exponential decay of P870* does not exist.

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DiMagno et al. (1992) reported on the nonsingle exponential decay kinetics for P870* of a series of mutants from Rhodobacter capsulatus. We have studied that mutant in which the phenyl­ alanine and tyrosine residues at sites L181 and M208 respectively are both replaced by histidine. Both amino acid residues are in the near vicinity of the special pair and accessory BChl molecules. Jia et al. (1993) reported that the decay kinetics for this mutant can be fit reasonably well with a double exponential (1.14 ps (54.4%) and 10.3 ps (45.6%)). It was the long lifetime (relative to the wild type) that attracted our attention since, all other things being equal, the ZPH of this mutant should be more pronounced than in the wild-type RC, cf. Fig. 4. The burn frequency dependent spectra for the mutant are shown in Fig. 5. Surprisingly, the ZPH is not observed for any burn frequency. Analysis of the dependence of the hole profile on burn frequency led to the electron–phonon coupling parameter values given in Table I. The key finding (from simulations) is that is significantly larger than for P870 (wild type and R-26) and P960. This explains why the ZPH is not observed. Despite the stronger coupling and, by inference,

N. Raja S. Reddy and Gerald J. Small

greater charge-transfer character of P870* for the mutant, the primary charge separation kinetics are slower. Whether or not this finding, together with others from hole burning studies, are com­ mensurate with the standard diabatic state model for charge separation is currently under investi­ gation in our laboratory. A photochemical hole burned spectrum for the RC of Rhodopseudomonas viridis is shown as the middle spectrum of Fig. 6. Again the population In bottleneck state for hole burning is the region of P960 one observes a weak ZPH coincident with the burn frequency as well as the progression associated with the aforementioned The responses of special pair marker mode, the higher energy to formation of are more difficult to interpret although it has been long suggested that feature 1 is the hole associated with band A (of the absorption spec­ trum) which is the upper excitonic dimer component of the special pair. Reddy et al. (1993a) succeeded in obtaining nonphotochem­ ical spectra for the RC, an example of which is shown in Fig. 6 (top spectrum) which provided definitive proof for this assignment (Reddy et al., 1993b). Remarkably, the anti-hole (solid arrow)

Spectral hole burning

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B. Hole Burning at High Pressures

of the P960 hole is red-shifted by rela­ tive to the origin component of the P960 absorption band, i.e. the photoinduced structural The largest transformation stabilizes P960* response from hole burning directly into P960 is hole 1 associated with absorption band A (due to The anti-hole of hole 1 is indicated by the dashed arrow and is blue-shifted by Interestingly, the equal and opposite shifting of anti-holes can be explained in terms the of the simple excitonic dimer model by assuming a reduction of ~0.5 A in Mg … Mg distance. Reddy et al. (1993b) also concluded that absorp­ tion band B is predominantly due to the out-ofof the phase linear combination of the two accessory BChl molecules while band C is due mainly to the in-phase linear combination but with a significant contribution form the two monomers of the special pair. Their work identi­ fied, for the first time, all six of a bac­ terial RC (bands D and E are primarily associated with the two bacteriopheophytin molecules).

It has been known for some time that pressure is an important tool for studying the structure and dynamics of proteins (Frauenfelder et al., 1990). In recent years, hole burning at elevated pres­ sures has been used with systems that exhibit very sharp ZPH to measure compressibilities, solvent shifts etc. in amorphous (protein) hosts (Zollf­ rank and Friedrich, 1992a, 1992b). Only modest values of high pressure (< ~10 MPa) were re­ quired in these studies because of the narrow ZPHs (due to long excited state lifetimes). To date applications of high pressures to the study of photosynthetic systems are limited (Foguel et al., 1991; Redline et al., 1991; Redline and Windsor, 1992). A simple theory, which predicted a pressure dependence of the frequency shift of the spectral holes and their broadening data was described by D. Haarer and coworkers (Sesselmann et al., 1987). A fully statistical microscopic theory of pressure effects on spectral holes and of the in­ homogeneous line shape itself, has been de­ veloped by Laird and Skinner (1989). This theory assumes that a) the transition frequency of a so­ lute molecule can be described by a sum of pair­ wise interactions with each solvent and b) each solvent molecule occupies a position that is inde­ pendent of other solvent molecules. Their theory predicted that the hole shift with pressure is burn frequency dependent (color effect) i.e. it depends on where in the inhomogeneous line the hole is burned. This dependence is utilized to obtain the compressibility values of the host matrix. Similar frequency dependence was predicted for hole width. Comparison of the experimental results (Zollfrank and Friedrich, 1992a, 1992b) with theoretical calculations for hole shift and width demonstrates that the theory of Laird and Skin­ ner is general enough to predict the results of the pressure tuning experiments, as well as the inhomogeneous line shape itself. We now briefly describe the results of our high pressure NPHB experiments on the FMO antenna complex for the green sulfur bacterium Chlorobium tepidum. Strong excitonic interac­ tions exhibited by BChl a molecules in the FMO complex have been studied in detail by Johnson et al. (1989, 1991). The FMO complex comprises

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of a trimer of subunits (each subunit consists 7 symmetry inequivalent BChl a molecules). Johnson et al. (1991) identified eight excitonic states establishing that inter-subunit interactions must be taken into account for a proper descrip­ tion of the spectroscopic data. The low energy band at 825 nm, in fact results from the two symmetry related lowest energy states at 824 nm and 827 nm. Narrow zero phonon holes can be burned only in the lowest energy state (Fig. 7). The pressure shift rates obtained using both broad absorption bands as well as the narrow zero phonon holes are similar to those observed in other proteins However, no color effect is observed, i.e. all holes burned in the 825 nm band shift, within experimental error, at the same rate. It should however be noted that in contrast to chromophores embedded in glassy hosts, a high degree of (spatial) correlation exists among the various BChl molecules in the antenna and RC complexes. This correlation violates the assumption of mutual solute position indepen­ dence in Laird–Skinner theory. The theory by Laird and Skinner should, therefore, be modified

N. Raja S. Reddy and Gerald J. Small

to take into account the strong (excitonic) interac­ tions present among the solute molecules. We have recently also obtained data on the pressure dependence of the absorption spectra of the reaction center of Rhodopseudomonas viridis in PVOH, at both 290 K (data not shown) and 4.2 K (Fig. 8). While the pressure shift rate for the accessory BChl band at ~835 nm changes from (290 K) to (4.2 K), the corresponding value for P960 is rela­ tively unchanged to The pressure coefficient for the upper dimer state is only about one third of that for P960 That the pressure coefficients for the upper dimer component and P960 differ by a factor of 3 can be qualitatively understood by considering that the frequencies of the upper and lower dimer components are affected not only by the changes in excitonic interaction but also the solvent shift for the BChl molecules of the special pair. Using the above data, the pressure rate of increase in and that for exci­ solvent shift is tonic splitting is However, an­ other possible explanation is that charge-resonance states of the special pair, which contribute to P960* and the upper dimer component, are responsible for the asymmetry of the shifts. Freib­ erg et al. (1993) have determined the pressure

Spectral hole burning coefficients for the accessory BChl band (0.26 and the P870 band of the Rhodobacter sphaeroides reaction center which is structurally very similar to that of Rho­ dopseudomonas viridis. Increase in the shift rate for P870 is surprising because the average BChl macrocycle separation is larger in P870 (3.5 Å) than in P960 (3.3 Å). From our data it appears that changes in interactions with nearby amino acid residues is a more likely cause for the differ­ ences in shift rates. It should also be noted that the two weaker bands in Fig. 8 at 808 nm and 795 nm are due to the two bacteriopheophytin molecules of the reaction center and that they exhibit only a weak pressure dependence. These results are very encouraging in the sense that they establish that energy gaps between the six states of the reaction center exhibit a significant pressure dependence. Thus, one can expect that the energy and electron transfer dynamics should also be affected. We will be continuing our inves­ tigations on both P870 and P960 at high pressures. An important question in the study of the func­ tion of transport proteins (e.g. horse radish per­ oxidase and myoglobin) is whether or not there is correlation between the tautamer configuration and the structure of the apoprotein. i.e. can a small structural change of a prosthetic group trigger a rearrangement of protein structure. Re­ cently, this aspect was investigated for mesopor­ phyrin substituted (for heme) horse radish peroxi­ dase (Friedrich et al., 1994) using high pressure hole burning. Low temperature absorption spec­ trum shows three major bands which are shown to be associated with at least four tautomeric states. Compressibility values estimated using hole shift with pressure data for two of the tautomers differ by a factor of 3. This change in compressibility of apoprotein (binding the different tautomers) corresponds to a transition from solid state phase to an almost liquid like phase, i.e. large scale structural changes in proteins can in fact be in­ duced by small changes in the prosthetic groups. Similar correlation was observed for protopor­ phyrin substituted myoglobin (Zollfrank et al., 1992).

C. Antenna Complexes Initial hole burning experiments (van der Laan et al., 1990) in B800 – 850 antenna complex of

133

Rhodobacter sphaeroides observed persistent nar­ row ZPH in the B800 band that were ascribed to B800–B850 energy transfer. However, these studies that concentrated only on ZPH widths failed to detect any holes in the BChl 850 band. Reddy et al. (1991) using an FT spectrometer based set up showed that hole burning in BChl 850 is as facile as in BChl 800 band (Fig. 9). That narrow ZPHs can be burned in the BChl 800 band establishes that its is due to inhomo­ width geneous broadening. The situation however, is different for BChl 850 band The width and position of the broad holes was independent of the burn wavelength. In addition to the broad holes, higher energy satellite holes (one which is coinci­ dent with BChl 800) were observed. Analysis of this data showed that the BChl 850 band is predominantly homogeneously broadened and the mode was identified as the dominant mode for BChl 800–BChl 850 energy transfer. The large (compared to kT at room temperature) homogeneous width of BChl 850 band explained the weak temperature dependence of the BChl 800–BChl 850 energy transfer (Reddy et al., 1991).

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D. Constant Fluence Hole Burning Spectroscopy Reddy et al. (1992b, 1993c, 1994) have shown that when a broad absorption band contains more than one underlying bands with differing hole burning properties, then hole burned spectra (burned with constant fluence) obtained as a func­ tion of burn wavelength can be used to resolve the underlying bands. This approach is especially useful in the study of excitonically coupled antenna systems (Reddy et al., 1992, 1994) where narrower ZPHs can be burned in the lowest exci­ tonic state. The idea behind this approach is that if the hole burning efficiency is constant across a band, the depths of ZPHs reflect the profile of the absorption being burned into. The BChl 870 and BChl 896 bands in the antenna complex of Rhodobacter sphaeroides are examples where this approach has been used very successfully. Based on these results BChl 870 and BChl 896 states were identified as the “shuttle” states between LH2 and LH1 complexes and LH1 and the RC.

N. Raja S. Reddy and Gerald J. Small

Similar constant fluence spectra (Fig. 10) ob­ tained for the LHC II complex of photosystem II (Reddy et al., 1994) were crucial in identifying the lowest energy state of this antenna complex at 680 nm, 4 nm below the most intense absorption band at 676 nm. That this state is the lowest state has been confirmed using fluorescence spectra and the linear electron phonon coupling par­ ameters obtained using SHB. Acknowledgements Research at the Ames Laboratory was supported by the Division of Chemical Sciences, Office of Basic Energy Science, and the U.S. Department of Energy. Ames Laboratory is operated for the U.S. Department of Energy by Iowa State Uni­ versity under contract no. W-7405–Eng-82. We would also like to thank R. Jankowiak and H.-C. Chang for help with the high pressure experiments and Deborah Hanson for providing us the Rb. capsulatus samples.

Spectral hole burning References Buinowski W, Porowski A and Laisaar AI (1973) Installation for optical investigations under high pressure at liquidnitrogen temperatures. Instrum Exp Tech (USSR) 16:274 – 278 Chang HC, Jankowiak R, Reddy NRS, Yocum CF, Picorel R, Seibert M and Small GJ (1994) On the question of chlorophyll a content of the Photosystem II reaction center. J Phys Chem 98:7725 –7735 Cone RL, Harley RT and Leask MJM (1984) Nuclear quadru­ pole optical hole-burning in stoichiometric J Phys C (Solid State Physics) 17:3101–3111 DiMagno TJ, Rosenthal SJ, Xie X, Du M, Chan CK, Hanson DK, Schiffer M, Norris JR and Fleming GR (1992) Recent experimental results for the initial step of bacterial photo­ synthesis. In:Breton J and Vermeglio A (eds) Photosyn­ thetic Bacterial Reaction Center II: Structure, Spectros­ copy, and Dynamics, pp209–217, Plenum Press, New York Foguel D, Chaloub RM, Silva JL, Crofts AR and Weber G (1992) Pressure and low temperature effects on the fluor­ escence emission spectra and lifetimes of the photosyn­ thetic components of cyanobacteria. Biophys J 63:1613– 1622 and references therein Frauenfelder H, Aldering NA, Ansari A, Braunstein D, Cowen BR, Hong MK, Iben IET, Johnson JB, Luck S, Marden MC, Mourant JR, Ormos P, Reimsch L, Scholl R, Schulte A, Shyamsunder E, Sorensen LB, Steinbach PJ, Xie A, Young R and Yue KT (1990) Proteins and pressure, J Phys Chem 94:1024 –1037 Freiberg A, Ellervee A, Kukk P, Laisaar A, Tars M and Timpmann K (1993) Pressure effects on spectra of photo­ synthetic light-harvesting protein-pigment complexes. Chem Phys Lett 214:10 –16 Friedrich J, Gafert J, Zollfrank, Vanderkooi JM and J Fidy J (1994) Spectral hole burning and selection of confor­ mational substates in chromoproteins. Proc Natl Acad Sci (USA) 91:1029–1033 Gillie JK, Small GJ and Golbeck JH (1989) Nonphotochem­ ical hole burning of the native complex of Photosystem I (PS I–200). J Phys Chem 93:1620 –1627 Gorokhovskii AA, Kaarli RK and Rebane LA (1974) Hole burning in the contour of a pure electronic line in Shpolskii system. JETP Lett 20:216 – 220 Hayes JM, Lyle PA and Small GJ (1994) A theory for the temperature dependence of hole-burned spectra. J Phys Chem 98:7337–7341 Jankowiak R and Small GJ (1993) Spectra hole-burning: A window on excited state electronic structure, heterogene­ ity, electron–phonon coupling, and transport dynamic of photosynthetic units, In: Deisenhofer J and Norris J (eds) Photosynthetic Reaction Centers, pp133 –177, Academic press, Boston Jankowiak R, Hayes JM and Small GJ (1993) Spectral holeburning spectroscopy in amorphous molecular solids and proteins. Chem Rev 93:1471–1502 Jia Y, DiMagno TJ, Chan C-K, Wang Z, Du M, Hanson DK, Schiffer M, Norris JR, Fleming GR and Popov MS (1993) Primary charge separation in mutant reaction centers of Rb. capsulatus. J Phys Chem 97:13180–13191

135 Johnson SG and Small GJ (1991) Excited state structure and energy transfer dynamics of the bacteriochlorophyll a antenna complex form Prosthecochloris aestuarii. J Phys Chem 95:471– 479 Johnson SG, Tang D, Jankowiak R, Hayes JM, Small GJ and Tiede DM (1990) Primary donor state mode structure and energy transfer in bacterial reaction centers, J Phys Chem 94:5849–5855 Kharlamov BM, Personov RI and Bykovskaya LA (1974) Stable gap in absorption spectra of solid solutions of or­ ganic molecules by laser irradiation. Opt Commun 12:191– 194 Laird B and Skinner JL (1989) Microscopic theory of revers­ ible pressure broadening in hole-burning spectra of im­ purities in glasses. J Chem Phys. 90:3274 –3281 Lu X and Pearlstein RM (1993). Simulations of Prostheco­ chloris bacteriochlorophyll a protein optical spectra im­ proved by parametric computer search. Photochem Photo­ biol 57:86 – 91 Lyle PA, Kolaczkowski SV and Small GJ (1993) Photochem­ ical hole-burned spectra of protonated and deuterated reac­ tion centers of Rb. sphaeroldes. J Phys Chem 97:6924–6933 Moerner WE (ed.) (1987) Topics in Current Physics, Persist­ ent Spectral Hole Burning: Science and Applications, Vol. 44, Springer-Verlag, New York Mukamel S (1995) Principles of Nonlinear Spectroscopy, Ox­ ford University Press, New York Narasimhan LR, Littau KA, Pack DW, Bai YS, Elschner A and Fayer MD (1990) Probing organic glasses at low temperature with variable time scale optical dephasing ex­ periments. Chem Rev 90, 439 – 457 Reddy NRS, Small GJ, Seibert M and Picorel R (1991) En­ ergy transfer dynamics of the B800– 850 antenna complex of Rb. sphaeroides: A hole burning study. Chem Phys Lett 181:391–399 Reddy NRS, Lyle PA and Small GJ (1992a) Applications of spectral hole burning spectroscopies to antenna and reac­ tion center complexes. Photosyn Res 31:167–194 Reddy NRS, Small GJ, Seibert M and Picorel R (1992b) B896 and B870 components of the Rb. sphaeroides antenna: A hole burning study. J Phys Chem 96:6458–6464 Reddy NRS, Kolaczkowski SV and Small GJ (1993a) A pho­ toinduced persistent structural transformation of the special pair of a bacterial reaction center. Science 260:68 –71 Reddy NRS, Kolaczkowski SV and Small GJ (1993b) Nonphotochemical hole burning of the reaction center of Rps. viridis. J Phys Chem 97:6934 – 6940 Reddy NRS, Cogdell RJ, Zhao L and Small GJ (1993c) Nonphotochemical hole burning of the B800 –B850 antenna complex of Rps. acidophila. Photochem. Photobiol 57:35– 39 Reddy NRS, van Amerongen H, Kwa SLS, van Grondelle R and Small GJ (1994) Low-energy exciton level structure and dynamics in Light Harvesting Complex II trimers from the Chl a/b complex of Photosystem II. J Phys Chem 98:4729 – 4735 Redline N and Windsor MW (1992) The effect of pressure on charge separation in photosynthetic bacterial reaction centers of Rps. viridis. Chem Phys Lett 198:334–340 Redline N, Windsor MW and Menzel R (1991) The effect of

136 pressure on the secondary (200 ps) charge transfer step in bacterial reaction centers of Rb. sphaeroides. Chem Phys Lett 186:204 –209 Renn A, Meixner AJ, Wild UP, Burkhalter FA (1985) Holo­ graphic detection of photochemical holes. Chem Phys 93:157–162 Sesselmann Th., Richter W, Haarer D and Morawitz H (1987) Spectroscopic studies of guest–host interactions in dyedoped polymers: hydrostatic pressure effects versus tem­ perature effects. Phys Rev B 36:7601–7611 Small GJ, Hayes JM and Silbey RJ (1992) The question of dispersive kinetics for the Initial phase of charge separation in bacterial reaction centers. J Phys Chem 96:7499 –7501 Tang D, Jankowiak R, Small GJ and Tiede DM (1989) Struc­ tured hole burned spectra of the primary donor state ab­ sorption region of Rps. viridis Chem Phys 131:99–113 van der Laan H, Schmidt Th, Visschers RW, Visscher KJ, van Grondelle R and Volker S (1990) Energy transfer in

N. Raja S. Reddy and Gerald J. Small B800 – 850 complex of purple bacteria Rb. sphaeroides: a study by spectral holeburning Chem Phys Lett 170:231– 238 Wannemacher R, Koedijk and Völker S (1993) Spectra dif­ fusion in organic glasses. Temperature dependence of per­ manent and transient holes. Chem Phys Lett 206:1–8 Zollfrank J and Friedrich J (1992a) Pressure shift and solvent shift: A hole-burning study of resorufin-doped glasses. J Phys Chem 96:7889 –7895 Zollfrank J and Friedrich J (1992b) Spectral holes under pres­ sure: Proteins and glasses. J Opt Soc. Am B 9:956 – 961 Zollfrank J, Friedrich J and Parak F (1992) Spectral hole burning study of protoporphyrin IX substituted myoglobin Biophysical J 61:716 – 724 Zuber H and Brunisholz RN (1991) Structure and function of antenna polypeptides and chlorophyll-protein complexes: Principles and variability. In: Scheer H (ed.) Chlorophylls, pp 627– 641, CRC Press, Boca Raton, FL

Chapter 9 Infrared and Fourier-Transform Infrared Spectroscopy

Werner Mäntele

Institut für Physikalische und Theoretische Chemie, Universität Erlangen-Nürnberg,

Egerlandstrasse 3, 91058 Erlangen, Germany

Summary I. Introduction: Looking Back 100 Years II. What Can Infrared Spectroscopy Tell us about the Processes in Photosynthetic Membranes and Reaction Centers?

III. From Bands to Bonds: Strategies for Band Assignments IV. Fourier-Transform Infrared (FTIR) Spectroscopy A. Basic Principles B. FTIR Spectroscopy used to Probe Protein Structures C. Techniques for Reaction-Modulated Difference Spectroscopy 1. Light-Induced Difference Spectroscopy 2. Electrochemically-lnduced Difference Spectroscopy 3. Photo-Chemo-Triggering: The Use of Caged Compounds D. Time-Resolved FTIR Spectroscopy 1. Rapid-Scan FTIR Spectroscopy 2. Stroboscope FTIR Spectroscopy 3. Step-Scan FTIR Spectroscopy V. Single Wavelength IR Techniques A. Dispersive Spectrometers B. Tunable IR Lasers C. Picosecond Pump-Probe Techniques VI. Sample Preparation for Infrared Spectroscopy VII. Conclusions and Outlook Acknowledgements References

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During the last decade, infrared spectroscopic techniques have entered the field of photosynthesis and they are meanwhile established methods for probing chlorophyll or quinone molecules in their binding sites and for the investigation of molecular processes in the protein upon electron transfer or proton transfer. This advancement has mainly become possible with the rapid development and availability of Fourier transform infrared (FTIR) spectrophotometers, but also with the development of sensitive infrared semiconducor detectors and tunable IR lasers or picosecond IR laser systems. This chapter introduces the basic concepts for using IR Spectroscopy to study such complex systems as a reaction center or even complete photosynthetic membranes. The basic principles of FTIR spectroscopy for steady-state or time-resolved investigations will be described, and examples from the study of primary electron transfer in reaction centers will be given. Single-wavelength time-resolved techniques for the Correspondence: Fax: 49-9131-858307; E-mail: [email protected]

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J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 137–160. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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Werner Mäntele

millisecond-to-nanosecond and pump-probe techniques for the picosecond time domain are reported. A separate section deals with the strategies for the assignment of IR bands, an essential issue for the use of IR techniques in studies of macromolecules. Furthermore, techniques for the preparation of IR samples from photosynthetic membranes or reaction centers are described. Abbreviations: B – accessory bacteriochlorophyll; BChl – bacteriochlorophyll; CW – continuous wave; – primary electron accepter (pheophytin); MCT – mercuryFTIR – Fourier transform infrared; cadmium-telleride; NIR – near infrared; P – primary electron demor; Q – acceptor quinone; – first and second acceptor quinones; RC – reaction center

I.

Introduction: Looking Back 100 Years

In the first volume of Physical Reviews in 1893, E.F. Nichols reported “a study of the transmis­ sion spectra of certain substances in the infrared”, using a spectrophotometer occupying two rooms, with a prism mounted in the wall between the two labs, and an assistant in the detector room taking the galvanometer reading with and without the substance in the beam (Nichols, 1893). Nich­ ols noted that “the diathermancy of chlorophyll solutions depends on the type of solvent” he used, and thus was the first one to discover spec­ tral shifts of chlorophyll absorption due to aggre­ gation states. Although we would nowadays con­ sider this as a near-infrared (NIR) experiment, it is the first study using long-wavelength radiation in photosynthesis. With increasing knowledge of the chemistry of chlorophylls and the advance­ ment of infrared instrumentation, vibrational spectroscopy in the mid-infrared region became interesting for structural studies of the photosyn­ thetic pigments, and a paper by Stair and Co­ blentz (1933) reported the infrared absorption spectra of some plant pigments. In the sixties and seventies, vibrational spectroscopic studies, mainly from the JJ Katz school, were extensively used for the investigation of chlorophyll aggre­ gation and hydration (Katz et al., 1966, 1978; Ballschmiter and Katz, 1968, 1969). Although all these studies were on isolated pigments in sol­ vents, it is obvious that they were undertaken in order to explain certain properties of the pig­ ments in their native protein environment. As for the protein side, history goes back to the 50’s when Elliot and Ambrose (1950), in a

Nature article on the structure of synthetic poly­ peptides, pointed out the specific IR absorption of polypepide secondary structures, long before crystallography could provide us with atomic in­ formation on protein structures. This spectro­ scopic approach has become well-established with a theoretical background and numerous appli­ cations for the analysis of secondary structures of proteins (for a review, see Arrondo et al., 1993). In order to apply the vibrational spectroscopy of pigments together with those of polypeptides to the analysis of pigment-protein complexes, not necessarily in photosynthesis, technical develop­ ments had to be awaited. A breakthrough has been the development, the availability, and the “affordability” of Fourier transform IR spectro­ photometers, which, for reasons given below, can provide the sensitivity needed to monitor vi­ brational modes in situ, either from a pigment in its native environment or from the protein. In this review, we shall briefly discuss the use of IR spectroscopy in the convenient sense for amide-I band analysis of photosynthetic pigmentprotein complexes. The major part will be dedi­ cated to the discussion of FTIR techniques which provide the sensitivity to monitor changes at indi­ vidual bonds of, say, a photosynthetic reaction center. The perturbation techniques needed to achieve this sensitivity lead to difference spectra, either between the stable states of a reaction or for metastable intermediates. We shall extend these FTIR techniques to time-resolved spectros­ copy, although only very few applications have been attempted up to now in photosynthesis. Single-wavelength IR techniques will be discussed in more detail, in particular since recent develop­

Infrared and Fourier-transform infrared spectroscopy ments have led to vibrational IR studies with picosecond time resolution on photosynthetic re­ action centers. A section on sample preparation for infrared spectroscopy and one on the sisy­ phean task of IR band assignment will provide the reader with the information that no photosyn­ thetic protein is too complicated to be analyzed, and with the warning that to obtain nice spectra is relatively easy as compared with the task to interpret them. II. What Can Infrared Spectroscopy Tell us about the Processes in Photosynthetic Membranes and Reaction Centers? Infrared spectroscopy analyzes vibrational pro­ perties of a molecule and can thus address other properties of the RC than those monitored by optical spectroscopy in the UV, visible, and nearinfrared region. The vibrational spectrum of the RC is composed of individual and coupled modes of the numerous bonds of the protein, the pig­ ments, the quinones, lipids, and water constitut­ ing the entire complex, and thus contains abun­ dant information on structural details and functional properties. In this chapter we shall deal with the information which can be drawn from this “messy” overlap of multiple IR bands. This subject is also covered in reviews by Hoff (1992) and Mäntele (1993a,b, 1995). In infrared spectroscopy, photons with ener­ gies which match those of vibrational sublevels of the molecule (typically in the 0.5–0.05 eV range) are directly absorbed. These low energy photons do not cause photochemistry and thus can be used at high intensities. A large variety of techniques in infrared spectroscopy has emerged meanwhile, and most of them can be applied to obtain useful information on structural and functional proper­ ties of the RC. Infrared spectroscopy probes vibrational modes of the pigments, the quinones, the poly­ peptide backbone and amino acid side chains without any selectivity and may thus be used to address questions on pigment and quinone in situ properties and interactions, on the role of the protein mediating electron transfer and coupling proton transfer to electron transfer. This is com­ plementary to Raman spectroscopy, where reson­

139

ance enhancement can yield spectra of individual pigments even in a complex photosynthetic mem­ brane (Lutz and Mäntele, 1991). A problem of the non-selectivity of IR spectroscopy is the back­ ground absorption of the bulk part of the RC, of liquids, water, and buffer, which necessitates difference techniques. Since the size of the RC protein does not allow “classical” difference spec­ troscopies where two different samples are com­ pared, “reaction-induced” IR difference tech­ niques on a single sample have to be used. The “trigger” inducing the reaction should provide a minimum disturbance of the sample, but specifi­ cally and quantitatively start the desired reaction. The result are highly structured difference spectra which represent the sum of all molecular changes associated with the induced reaction, and which are selective for the functionally important parts rather for the global structure. At present, in­ trinsic photochemical reactions, electrochemical reactions in a spectroelectrochemical cell, and in­ direct photochemical reactions releasing an effec­ tor molecule which then starts the desired reac­ tion, have been applied (for a review, see Mäntele, 1993a). Another possibibilty for a chemical triggering of a reaction using an ATR (attenuated total reflection) flow cell has been demonstrated (Baenziger et al., 1993). IR spectroscopy relies on physical processes with short intrinsic litetimes and thus allows very high time resolution for the study of intermediate states in electron transfer as well as of the dy­ namic processes in the protein. The past years have seen developments especially in time-resolved IR spectroscopy which have given access to all steps of electron transfer, from picoseconds to several seconds. To date, quite a few processes in RC have been detected in real-time in this entire time range which are functionally related to the electron transfer and proton transfer phe­ nomena, but kinetically uncoupled from these. Vibrational spectroscopy can thus provide us with a detailed molecular view of the role of the pro­ tein in mediating electron transfer. III. From Bands to Bonds: Strategies for Band Assignments Ideally, the bands in an IR spectrum are assigned to the vibrational modes of the molecule by per­

140 forming a normal mode analysis and matching the force field parameters by replacing all atoms with stable isotopes (for the details of this procedure, see Colthup et al., 1990). Although normal mode analyses have been performed for small proteins, reliable calculations are far out of reach for the RC. Even without the limitation of computer ca­ pacity, a force field yielding consistent modes would be difficult to obtain. In the case of proteins, the concept of group frequencies, i.e. of vibrational modes uncoupled from others, has proven to be very useful. This concept assumes the vibrational spectrum of a protein to be composed of independent vi­ brational modes from the backbone, from ami no acid side chain groups, from cofactors, lipids, de­ tergent and buffer molecules, and water. Coup­ ling of such modes is possible, but will only result in small shifts depending on the interactions. For each of these group frequencies, an empirical as­ signment is then attempted. One of the strategies for an empirical assign­ ment consists of isotope labelling of particular bonds and comparison of the observed with the calculated frequency shifts. In the case of a by C=O stretching mode, replacement of should result in a ca. downshift of the frequency according to (F: force constant; reduced mass). The most simple isotope labelling experiment is substitution of by easily done by resuspending concentrated RC or membrane sus­ pensions in a buffer and equilibrating. The resulting exchange of accessible N-, O-, or Sbound protons leads to shifts in the spectrum, which can be over for stretching modes. Exchange may happen at many sites and may thus lead to spectra in completely dif­ ferent from those in with the result that a clear assignment is not possible either. The prob­ ability for a given group to exchange depends on the global accessibility for water to the protein domain, on the local environment, and on the pK of the group. The extent of exchange can be monitored at the relative intensities of the amide II and the amide II' modes (the amide II mode arises from a coupling of the N–H and the C–N modes of the peptide bond, the corresponding mode in is termed amide II' ) modes. As a further complication, substitution of by

Werner Mäntele does not only act as simple mass label, but will also lead to changes of the pKs of groups and to kinetic isotope effects which can mess up an enzyme reaction. There is thus no guarantee that this isotope substitution leads to a conclusive as­ signment. Other mass labels, like or can be introduced biosynthetically by growing mic­ roorganisms on isotope-labeled nutrients, but will end unspecifically. Ideal, but still in its infancy, is site-directed isotope labeling, i.e. the place­ ment of a non-natural amino acid in an expression system to a specific position in the polypeptide chain. Although the feasibility has been demon­ strated for bacteriorhodopsin (Sonar et al., 1994), its application in photosynthesis still needs to be demonstrated. At present, isotope-labelled amino acids which are introduced into the RC via the nutrient, in combination with site-directed mutations, present a tedious yet sufficiently clear tool to identify and to assign the vibrational modes of a specific amino acid. In general, the remarkable potential of sitedirected mutagenesis of the RC is a tempting tool for IR assignments. However, any mutant resulting in severe blocking of the function renders reaction-induced IR difference spectros­ copy useless. A further problem of site-directed mutagenesis, in the case of charged residues, is the perturbation of entire charge patterns, for which IR difference spectroscopy (which moni­ tors the changes of dipole moments) is extremely sensitive. The balance seems to require gen­ eration of mutants with sufficiently small per­ turbations, but with retained general functional properties. In this line, natural variants can be considered of a similar relevance. In the RC, the exchange of cofactors by anal­ ogs is possible for the secondary and the primary quinone acceptor (Gunner and Dutton, 1989) as well as for the monomeric bacteriochlorophylls and the bacteriopheophytins (Struck and Scheer, 1990; Struck et al., 1990). The replacement of quinones by structurally different isotopically la­ belled analogs has become a useful tool for the assignment (Breton et al., 1994a–c; Brudler et al., 1994). In addition, the comparison of light or redox-induced FTIR difference spectra of reac­ tion centers with the spectra of the isolated cofac­ tors, i.e. bacteriochlorophylls, bacteriopheophy­

Infrared and Fourier-transform infrared spectroscopy tins, and quinones and all their numerous analogs, all in their neutral and relevant ion rad­ ical, dianion, or protonated forms, has created a basic data set of vibrational modes (Bauscher and Mäntele, 1992). Altogether, numerous vibrational modes from pigments, quinones, hemes, the polypeptide backbone and the amino acid side chain groups can be expected. Whether these contribute sig­ nificantly to IR difference spectra depends on the (differential) extinction coefficients. In general, molar extinction coefficients of vibrational modes are at least an order of magnitude smaller than even moderate extinction coefficients of elec­ tronic transitions and may at most reach 2000– for bonds. For most of the modes contributing to the spectra of reaction centers and other photosynthetic proteins, smaller extinction coefficients are valid: around for a peptide C=O mode

between 250 and

involved in an

for the COOH C=O mode of

aspartic acid or the antisymmetric and symmetric

modes of aspartates, and < 501

for the His mode. The water

H–O–H bending mode, which peaks at ca. 1650

has only a small (extinction coefficient)

However, since the of around

concentration of water is 55 mol/l, even a layer will lead to an absorbance of l. In order to cope with this high background absorbance of water, concentrated protein solutions or suspen­ sions or hydrated films have to be used. Table 1 summarizes the vibrational modes

which can be expected to contribute to the infra­

red absorbance or difference spectra of a reaction

center. Only the dominant modes in the

to region are given, and we

pretend, for the sake of simplicity, that they are

all well localized group frequencies. Although

many useful modes can be found at lower fre­

very little use

quencies (i.e. below

has been made of these, since decreasing detector

sensitivity and window transmission problems

present experimental difficulties. On the other

hand, the C–H, O–H, N–H or S–H frequencies

above

might well be used for diagnos­

tic purposes, but this region is in general too

congested for a detailed analysis.

141

IV. Fourier-Transform Infrared (FTIR) Spectroscopy

A.

Basic Principles

In a conventional infrared spectrophotometer, the infrared beam from a source, after passing the sample, is dispersed by a prism or a grating. A slit, which determines the width of the spectral element measured at a time is then used to block all other wavelengths, and the intensities of all spectral elements are successively measured by rotating the grating or the prism and correlating detector intensity with the position of the disper­ sive element. This results in slow acquisition of the spectrum since (1) all spectral elements are successively “scanned” and (2) the low energy in one spectral element necessitates damping with a high time constant. One possibility to reduce acquisition time would be the use of array detec­ tors similar to an optical multichannel detecting system or a diode array, but unfortunately multi­ ple IR detector elements from semiconductors (e.g., HgCdTe, InSb) are extremely costly and can hardly be fabricated with constant specifi­ cations as compared to silicon diodes. A Fourier transform spectrophotometer makes use of an interferometer, rather than scanning the individual wavelengths successively. An interfer­ ometer (typically of the Michelson type) produces an interference modulated beam which is passed through the sample and then reaches the detec­ tor. The detector intensity as a function of the interferometer position is called the interfero­ gram. It is measured for a given mechanical mirror movement and digitally stored; its Fourier transform yields the IR spectrum. For the basic principles of Fourier transform infrared (FTIR) spectroscopy, the reader is referred to some re­ views and textbooks (for example Griffiths and DeHaseth, 1986). A schematic representation of the optics used in an FTIR spectrophotometer is shown in fig. 1. The output of the detector–provided that no sample is present–is a function of the moving mirror position in the interferometer, but also of the spectral density of the source, of the transmis­ sion or reflection properties of all optical compo­ nents, and of the spectral sensitivity of the detec­ tor. The exact position of the moving mirror is

142

determined by a He–Ne laser reference interfero­ gram which is generated with the same interfero­ meter and which controls, by detection of con­ structive and destructive interference pattern of the He–Ne laser, the timing of the data acqui­ sition and digitization. The resolution of an FTIR instrument is deter­ mined by the distance d which the moving mirror travels from the point (“0”) where both branches

Werner Mäntele

of the interferometer exhibit the same optical pathlength. Typically, the mirror is moved from –d to +d, with the resolution being approxi­ mately 1/d wavenumbers. Each half interferog­ ram (–d to 0 and 0 to +d) contains full spectral information, although both halves, even for a well-adjusted interferometer, are not necessarily symmetrical. Approximations that are made for the calcu­

Infrared and Fourier-transform infrared spectroscopy

lation of the spectrum from the interferogram include the limited movement of the mirror, which is not over an infinite distance as required for the procedure of Fourier transformation; thus, the interferogram is convoluted with an apodisation function. The details of data acqui­ sion, Fourier transformation, spectrum calcu­ lation, and some possible artefacts are discussed in a very useful paper by Gronholz and Herres (1985). FTIR spectroscopy results in a number of ad­ vantages over dispersive spectroscopy. The major improvement is the simultaneous recording of all spectral elements (“multiplex spectroscopy”) analogous to measurements in the visible using a diode array. This advantage is frequently termed Felgett’s advantage. Acquisition of an interfero­ gram consisting of a few thousand data points (each digitized to at least 16 bit accuracy) takes less than a second even for a simple FTIR spec­ trophotometer, and only a few milliseconds for sophisticated “rapid scan” interferometers. We shall discuss below the relevance of this digitiz­ ation time for time-resolved measurements in the rapid-scan mode. If the same total acquisition

143 time for a spectrum is allowed as for a dispersive instrument (i.e. at least several minutes), Felgett’s advantage can be used towards a gain in signal-tonoise-ratio by accumulating and averaging many interferometer scans before performing the Four­ ier transformation. On the other hand, in the rapid-scan mode, time-resolved studies in the msec domain are possible using series of single interferometer scans stored and transformed sep­ arately. The Felgett advantage of an FTIR instru­ ment over a dispersive instrument depends on the width of the spectral range under consideration; as a rule of thumb, it is approximately given by the spectral range measured divided by the resol­ ution, i.e. around 250 for the recording of the spectral region from 1000 to at resolution. A second advantage results from the absence of resolution-determining slits in an FTIR instru­ ment. In a dispersive instrument, narrow rec­ tangular slits are used to define resolution, result­ ing in a limited throughput even for moderate resolution. In an FTIR instrument, resolution is determined by the distance d the movable mirror travels, and only circular apertures are present to purposely limit energy flow and to collimate the beam. This higher energy throughput of an FTIR instrument, allowing higher sensitivity and the measurement of very low transmitting (“black”) samples, is often referred to as Jacquinot’s advan­ tage. A third advantage of an FTIR instrument is the high wavelength accuracy because of the He– Ne laser reference interferometer present. Be­ cause of the known laser frequency, the wavelength accuracy may be as high as far more than needed for biological samples. This advantage is frequently referred to as Conne’s advantage. The usefulness of FTIR spectroscopy would be incomplete without the use of sensitive and rapid semiconductor detectors. For dispersive instru­ ments, mostly thermocouples or the pneumatic Golay cell were used. FTIR instruments, in the standard versions, are mostly equipped with pyro­ electric deuterated triglycine sulfate (DTGS) de­ tectors, which have the advantage of a wide spec­ tral sensitivity from the near IR down to a few hundred wavenumbers and which have a reason­ ably linear response. However, their responsivity

144 decreases with increasing modulation frequency, a fact which limits their application to rapid scan processes and fast data acquisition. Cooled semiconductor detectors from binary (liquid and tertiary semiconductors, either as photocond­ uctors, as photovoltaic photodiodes or as biased photoconducting diodes, have limited spectral re­ sponse, gradually increasing from short wave­ lengths to the maximum near the band edge. 1 Their detectivity may range up to Their response time can be in the order of nanoseconds, and even the standard mercury-cadmium-telluride (MCT) photocond­ ucting detectors, selected by most FTIR users, domain. These have a response time in the detectors, in combination with fast digitizers (conversion rates around 100 to 200 kHz), allow to move the interferometer mirror at a high speed, close to the mechanical limits (see below in the section on rapid-scan FTIR spectroscopy). Apart from MCT detectors, which can be used for the to region, and which can be optimized for a frequency range of interest by varying the semiconductor composition, InSb to or doped detetors Ge detectors are occasionally used. A specific problem arising with semiconductor detectors is a nonlinear relation between the radiation intensity and the detector output at high intensities,one of the disadvantages of the multiplex detection. In order to avoid photometric inaccuracies or Four­ ier transform artefacts, it is recommended to use edge filters which cut out unnecessary frequency ranges. These filters mostly consist of coated Ger­ manium; a long-wave pass filter mounted in front of the sample which cuts out, for example, the frequency range and passes the to range, will not only protect the sample from unnecessary heating, but also from photochemical reactions caused by the He-Ne beam which is coaxial to the IR beam, and will increase linearity as well as sensitivity (since the amplifier gain may be optimized). 1

The detectivity is a quantity defined from the responsivity (in Volts or amperes output per Watt of incoming radiation) and the noise voltage or current at a certain frequency and within a defined frequency interval, it allows to compare essentially different detector types and materials.

Werner Mäntele

B. FTIR Spectroscopy Used to Probe Protein Structures The IR spectrum of a protein is mainly charac­ terized by vibrational modes from the peptide building units. A total of nine predominant modes of the CONH group (“amide modes”) are found in the region between and corresponding to in-plane and out-of plane modes with very different distribution of potential energy on the individual bonds (for de­ tails, see Arrondo et al., 1993). The highest frequency modes, “amide A” and “amide B”, at approx. and respectively, are almost purely NH stretching. In the region and the protein between IR spectrum is formed by the amide I mode be­ and (predominantly tween C=O stretching) and by the amide II mode (a mixture of NH bending and around CN stretching). The amide I absorption band is suitable for the analysis of protein structures, since its position depends on the type of se­ condary structure elements. Due to different Hbonding and environment in the various types of secondary structures, the amide I mode of an helix will absorb around , that of an antiparallel sheet around and of a parallel sheet around and of unordered structure around (for a discussion of the range of each mode, see a review by Arrondo et al., 1993). These numbers are based on one side on normal mode calculations, on the other side on the IR spectra of model peptides and small proteins with known structures. While a polypeptide of a single known se­ condary structure (assumed to be infinitely long in the case of helices and sheets) should give rise to a clear-cut band structure for the amide I mode, the inverse step–i.e. deduction of the secondary structure from the amide I band pro­ file – is not always unequivocal for several rea­ sons, even if the spectrum can be recorded at almost arbitrary precision using FTIR spectros­ copy. First, the intrinsic bandwidth of the amide I band is typically higher than the separation of its constituents, assuming that different types of secondary structure lead to superposition of bands. Second, even if the decomposition of the

Infrared and Fourier-transform infrared spectroscopy amide I band is achieved, assignment of the type and extent of secondary structure to its constitu­ ents is not always unambiguous and needs ad­ ditional support by other spectroscopic or bio­ chemical data. Ideally, one would like to record a spectrum of the amide I region and obtain, via some kind of mathematical procedure, percentage numbers for helix, sheet contents, and so on. Unfortu­ nately, the availability of corresponding software has considerably encouraged this type of “black­ box” use of IR spectroscopy, and occasional offthe-shelf assignments of secondary structures have discredited the substantial amount of serious spectroscopic work in this field (for a critical as­ sessment of the method, see Surewiczs et al., 1993). Information on the type and composition of secondary structure and how it may change with some reaction is obtained by (1) recording highquality spectra of the sample in the range from to (2) subtraction approx. of the buffer, (3) application of resolution en­ hancement procedures such as derivation, decon­ volution, Fourier self deconvolution or others, and (4) band fitting or band synthesis from pos­ sible constituents. It is obvious that either of these steps can bear a number of pitfalls: Photometric accuracy and detector nonlinearities enter in the first step, incorrect subtractions in the second, artefacts of resolution enhancements in the third and fitting ambiguities in the fourth. Finally, the contribution of amino acid side chain absorbance in the amide I region is substantial, but difficult to estimate. Another experimental difficulty is aggregation, possible orientation, and thus dichroism of the sample which leads to misjudge­ ment of the intensities for secondary structure elements. Orientation of membranes and vesicles on an IR window tilted with respect to the meas­ uring beam, on the other side, can be used to deduce the orientation of helices with respect to the membrane normal.

C. Techniques for Reaction-Modulated Difference Spectroscopy Although the sensitivity of FTIR spectroscopy is high enough to detect the changes of absorption brought above by alterations at one individual

145 bond even in a protein with 100 kDa and more, the straightforward approach to calculate a differ­ ence spectrum from two spectra of two different protein samples in two different states fails in most cases. The limits of precision for the adjust­ ment of sample preparations and concentrations, cell pathlength, etc. leads to the frustrating in­ sight that this approach is only appropriate for low-molecular weight proteins, for large spectral differences between the samples, or in spectral windows with low background absorbance such as the to range. Alternative techniques, in which the spectra of one single sample in two different states (for example, in the initial and final state of a reaction) are recorded in rapid succession, with a reaction induced in between, are more appropriate. A difference spectrum is thus obtained, which is selective for the functionally relevant parts of the protein rather than for the global structure. Since a suit­ able reaction is the trigger for the resulting differ­ ence spectra, the term reaction-induced or reaction-modulated IR difference spectroscopy has been coined (Mäntele, 1993a). The “trigger” em­ ployed should provide a minimum disturbance of the sample, but quantitatively and specifically start the desired reaction. Figure 2a illustrates the principle setup used for reaction-induced FTIR difference spectroscopy. Different triggers can act on the sample in this type of experiment and modify its state, which is then characterized by FTIR as well as by UV/VIS spectroscopy. 1. Light-Induced Difference Spectroscopy Light-minus-dark IR difference spectroscopy is the most straightforward version of reactionmodulated IR techniques, and can be used for many intrinsic photoreactions of photosynthetic pigment-protein complexes. FTIR difference spectra are obtained between dark and illumi­ nated samples, either with the actinic light used to start the reaction and recording spectra before and after illumination, or under illumination creating different photostationary states. Exci­ tation can be by continuous light or by a flash as outlined in Fig. 2a. The photostationary state of an RC sample in the FTIR spectrophotometer beam can be easily modified by additional bleaching light, which

146

Werner Mäntele

Infrared and Fourier-transform infrared spectroscopy does not perturb the measurement, since the spectral regions (VIS/NIR for excitation, mid-IR for detection) are well-separated. Light-induced FTIR difference spectra reflecting charge-separor are easily obtained ation between P and at moderate bleaching intensities. The first spec­ tra of this kind date back to 1985 (Mäntele et al. ,1985) and light-induced techniques of this kind have since then been used for the study of the molecular processes concomitant with primary charge separation for many different sample forms and RC and membrane preparations (for reviews, see Hoff (1992), Mäntele (1993a,b, 1995)). In the case of bacterial photosynthetic reaction centers, these spectra reflect the differences for transition and are com­ the or differ­ monly termed ence spectra, with the understanding that the bands of the disappearing neutral state, are negative, and those of the appearing chargeseparated state, are positive. Quite anal­ ogous to bacterial RCs, spectra reflecting charge separation in Photosystem I have been obtained (Nabedryk et al., 1989). However, care need to be taken with higher intensities of the bleaching light, and correct filtering is recommended, since even minor heating of the sample results in changes of hydration, leading to unspecific signals which can be much larger than those caused by light-induced charge separation. In general, it is advantageous to add a number of light-dark dif­ ference cycles rather than to accumulate a high number of scans for the dark-adapted or the lightadapted state. Needless to say that thorough ther­ mostating of the samples is a prerequisite for high-quality spectra. Light-induced difference spectroscopy com­ bined with the use of exogeneous donors can be used to obtain difference spectra between further at low states of the RC. Rapid rereduction of redox potential conditions leads to formation of a procedure which has been used for Rhodopseudomonas viridis RC and Chromatium vinosum membrane particles, and, recently, for Rhodobacter sphaeroides RCs (Nabedryk et al., 1986, 1995). At moderately reducing conditions, but in the presence of a redox mediator to shuttle electrons rapidly to the photooxidized reduc­ tion of or has been achieved without oxid­

147 izing a detectable fraction of P (Breton et al., 1991a,b). In RCs with bound cytochromes, play­ ing with the redox potential for the IR samples has led to light-induced FTIR difference spectra where charge separation between the hemes and the quinone acceptors can be observed (Nabed­ ryk et al., 1991). At low temperature, under re­ ducing conditions, and at high-intensity illumi­ nation, the triplet difference spectrum has been obtained for Rb. sphaeroides RCs (Breton and Nabedryk, 1993). 2. Electrochemically-lnduced Difference Spectroscopy Steady-state FTIR difference spectra which have been obtained by use of the intrinsic photochem­ ical reactions, however, present signals from a mixture of states of the donor side and the ac­ ceptor side. In the case of bacterial photosyn­ thetic reaction centers, these are contributions from P, Q, and in a single spectrum reflecting charge separation. Difference spectra in which an exogeneous donor has been used seem to change only one cofactor’s redox state. However, the difference spectra due to the redox reaction of the exogeneous donor adds to that of the respective RC cofactor, although convincing evidence that the difference bands of the exog­ eneous donor are broad and featureless or simply in a different spectral region comes from control experiments. This limitation has been successfully overcome with the development of ultra-thin-layer electro­ chemical cells (Moss et al., 1990) which allow redox reactions in photosynthetic pigment-protein complexes to be triggered at a transparent electrode in combination with UV-VIS-NIR and FTIR spectroscopy. The design of this electro­ chemical cell is shown in Fig. 2b. Different types of working electrodes have been applied over the years, and the general idea is that a transparent layer of gold or platinum, a semiconductor elec­ trode, or a gold grid electrode (see Fig. 2b) is chemically modified with bifunctional reagents following the idea of Armstrong et al. (1986). Ideally, this reagent adsorbs in a single layer on the electrode and presents to the solution a charge or a charge pattern which corresponds to that of the natural reaction partner of the protein

148 under investigation, thus allowing reversible docking of the protein close enough to the elec­ trode to allow rapid electron transfer. Using this “direct” electron transfer, the addition of small redox-active molecules (“mediators”) can be avoided. Addition of mediators in micromolar concentrations with the appropriate redox midpo­ int potential, however, can considerably speed up equilibration. No spectral contributions from mediator redox reactions are observed for small enough concentration. Electron transfer at an electrode is more speci­ fic than light as a trigger, and different redoxactive cofactors present in a protein can be se­ lected by the choice of the appropriate potential (“dial-a-cofactor”). These “redox-induced” dif­ ference spectra are as detailed and sensitive as are light-induced difference spectra; moreover, individual bands can be titrated and thus assigned to a specific redox transition. In the case of bacterial RCs, FTIR difference and of the ac­ spectra of the donor side ceptor side have been obtained (Leon­ hard and Mäntele, 1992; Mäntele et al., 1990a; Bauscher et al., 1993). The electrochemical oxid­ ation of the electron donor of Photosystem I, P700, could be initiated electrochemically, and the hemes of the Rps. viridis and the Chloroflexus aurantiacus cytochrome subunit of the RC could be individually titrated (Hamacher et al., 1993; Fritz et al., 1992, 1993; Fritz, 1995). It should be mentioned that both redox-induced and lightinduced difference techniques can be combined in many cases on one sample to start photochemical reactions from a well-defined redox clamp. 3. Photo-chemo-triggering : The Use of Caged Compounds

Werner Mäntele “caged compounds”, has been reviewed by Cor­ rie and Trentham (1993). For the inactivation of an effector molecule, chemical modification or sterical “caging” are conceivable. Figure 2c shows the structure and the net reaction of the archetype of “caged com­ pounds”, caged ATP. With these “caged com­ pounds”, time resolved studies of protein reac­ tions are possible, with the time resolution only limited by the intrinsic reaction rates of the cage and diffusion to the protein. In this case, the experiment follows the scheme in Fig 2a with an intense UV flash initiating the release of the effector molecule. A general problem for the use of caged com­ pounds for IR spectroscopy is the intrinsic IR difference spectrum from the triggering reaction itself, i.e. from the photolytic release of the cage, which comprises a primary phototransformation with subsequent dark reactions. However, most cages provide spectral “windows” where differ­ ence bands from the protein reaction are not masked by bands from the triggering reaction. These windows can be enlarged using isotopically-modified cages. There are many possible applications of “cages” for the study of photosynthetic proteins. Work performed up to now comprises a rapid and per­ manent change of pH induced by a “caged pro­ ton” (Fogel et al., 1993) and the release of from caged with subsequent binding to an ion-sensitive light-harvesting complex from Rb. sulfidophilus (Beck, 1992). It appears that further applications are only limited by the imagina­ tiveness of the organic chemist who synthesizes the cage and combines biochemical requirements with requirements of vibrational spectroscopy.

D. Time-Resolved FTIR Spectroscopy The idea of reaction-modulated IR difference spectroscopy has been carried beyond the reac­ tions which can be induced by intrinsic photo­ chemical reactions or by electrochemical reac­ tions. The rapid and uniform introduction of a substrate or an effector molecule by UV photo­ lysis of an inactive but photolabile precursor mol­ ecule allows protein reactions to be triggered in an IR sample. The synthesis, the reactions, and the applications of such inactive photolabile pre­ cursor molecules, which have been termed

Actually, three variants of time-resolved FTIR spectroscopy have proven to be usetul to obtain time-resolved vibrational spectra of proteins, and (at least) two of them have been successfully ap­ plied to photosynthetic systems. For all three me­ thods, the high energy throughput and the wave­ length accuracy (termed Jacquinot and Conne advantage in section IV. A) can be fully exploited; however, they differ in the use of the Felgett (Multiplex) advantage. In addition, their applica­

Infrared and Fourier-transform infrared spectroscopy bility to reaction center studies stringently de­ pends on the number of repeatedly excited pro­ cesses needed for averaging to obtain a good signal-to-noise-ratio. Figure 3 illustrates the prin­ ciples of time-resolved FTIR spectroscopy. 1. Rapid-Scan FTIR Spectroscopy In rapid-scan-FTIR spectroscopy illustrated in Fig. 3a (Braiman et al., 1987), the interferometer mirror is scanned back and forth in cycles as fast as possible and successive interferograms are stored in the computer memory (transformation of the interferograms can be performed later). A trigger, typically a flash or a short interval of illumination from a lamp defined by a shutter, is then fired between or during interferogram re­ cording after sufficient interferograms have been stored to calculate a low-noise spectrum of the unperturbed sample (i.e. of the state before the flash). Difference spectra evolving with time are then calculated between the pre-flash spectrum and the spectra calculated from successive inter­ ferograms after the flash. A “blank” difference spectrum calculated from two sets of spectra of the unperturbed sample before the flash usually serves as a control for sample stability and defines a “level of confidence” for the later spectra. At first sight, mechanical problems in rapidly scanning a solid mirror driven by an electromag­ netic linear motor on an air bearing at more than 20 cycles/second over approx. 1 cm amplitude – while interferometric accuracy of the mirror (i.e. its surface rectangular to the beam) must be maintained – seems limiting, but a serious bottle­ neck is given by the digitization of the detector signal, too. We keep in mind that 2000–8000 data points need to be digitized for the interferogram, each with at least 16 bit accuracy. Improved hard­ ware and sophisticated software now allow to re­ cord interferograms during the forward and the reverse movement of the mirror (see Fig. 3a); these interferograms should in principle be identi­ cal, but in practice need a normalization proce­ dure to overlap. Moreover, software procedures have been developed to use the four “half-interferograms” of a full mechanical forward-reverse interferometer cycle in order to provide more points on the time axis (see Fig. 3a). Principally, both “half-interferograms” (–d to 0 and 0 to +d)

149 contain full spectral information, and thus only need to be complemented by their mirror image to serve as a basis for the calculation of a spec­ trum. Again, only with an ideal interferometer the two halves of an interferogram are identical, and correction factors are needed to make identi­ cal spectra for the mirror moving from to –d to 0 and from 0 to +d. Using all these procedures, four spectra non-equidistant in time may be calcu­ lated from a single forward/reverse interferogram cycle. The duration of a cycle depends on the spectral resolution needed. After reversal of the move­ ment at the extreme point of the mirror move­ ment, a short way is needed before a uniform movement is reached and data can be recorded at –d or +d in order to avoid mirror vibrations. Rapid-scan FTIR spectroscopy is particularly suitable for slow reactions (msec to sec) or reac­ tions which can only be triggered once (in case of irreversible bleaching of the sample or trapping of a state), or when back reactions are so slow that repetitive excitation becomes unattractive. It has been used successfully to obtain difference and states of spectra between the bacterial RC, by recording spectra immediately after a short illumination of RC containing ap­ (resulting in a mixture of prox. 50% difference spectra) and succcessively and after hundreds of milliseconds (resulting in state. spectra after full decay of the The normalized subtraction of both spectra gave difference spectrum (Thibodeau a et al., 1990a,b). Mechanical mirror movement and digitization of the interferogram presently set a limit for the time resolution, which is a compromise with the desired spectral resolution. At the spectral resol­ ution needed for the studies of photosynthetic systems, which is mostly spectra are pos­ sible every 20–30 ms with a routine rapid-scan interferometer and software. In our laboratory, a setup making use of a Bruker Type 66 interfero­ meter records spectra spaced on the average ap­ prox. 25ms for resolution, and approx. every 12 ms for resolution. Upon intro­ duction of an appropriate delay between the start of the interferogram and the flash, the first spec­ trum can be recorded within 1–2 ms after the flash and thus be used for quite rapid reactions.

150

Werner Mäntele

Infrared and Fourier-transform infrared spectroscopy

The number of the spectra recorded this fast only depends on the computer memory, and typically coaddition of interferograms at later time in­ tervals is used to improve signal-to-noise-ratio and to avoid extensive data floods. It can be fore­ seen that the time resolution for this version of rapid-scan FTIR spectroscopy can only mar­ ginally be improved by more rapid mechanical scanners and faster analog-to-digital converters. 2.

Stroboscope FTIR Spectroscopy

One of the bottlenecks of rapid-scan FTIR spec­ troscopy clearly is the limited time interval needed for the acquisition of a spectrum (in the order of some msec, see previous section). A transient spectral change with an intrinsic rise or decay time in the order of or faster than the acquisition time needed for the interferogram is thus convoluted in a complicated way over this time interval and a spectrum thus obtained will show a somehow averaged spectrum of the transi­ ent change. A clear attribution of band ampli­ tudes to the transient product at a given time, however, is not easily possible. Nevertheless, with a known delay between the firing of the flash and the start of the interferogram, there is a clear relation between the amplitude at each interfero­ gram data point and the delay time. “Stroboscope FTIR spectroscopy” (illustrated in Fig. 3b) makes use of this relation by recording many interferograms for an event which can be repeatedly excited and cycled rapidly. The actinic flash is fired at different delays with respect to the start of the data acqusition. If the time intervals are chosen sufficiently narrow, the interferogram amplitude for each optical delay between –d and +d can be obtained for each time delay. A complete “array of data” thus con­ sists of several thousand interferograms recorded with different fixed delays. Data points from dif­ ferent optical delays corresponding to identical delay in time can now be reconstructed to inter­ ferograms each reflecting a fixed time interval after the flash. This set can be transformed to obtain spectra at fixed time intervals after the flash. “Stroboscope FTIR spectroscopy” does not need great effort on the hardware side of the FTIR spectrophotometer, but needs additional

151

software to rebuild interferogram data. The time resolution depends on the response time of the detector, the delay time spacing, the number of data points taken, and can be in the domain. Typically, for each time delay many interfero­ grams are averaged to improve signal-to-noise ratio. In connection with the variation of time delay, recording of 10,000 to 100,000 interfero­ grams may be necessary. In addition, the multi­ plex advantage is, at least partially, lost, since multiple recording of data points cannot be avoided. This technique is thus particularly suited for protein photoreactions which can be arbi­ trarily repeated, yielding identical response for each flash event. The need for 10,000 or much more repetitive cycles also requires rapidly cyc­ ling reactions. While the technique was success­ fully applied to bacteriorhodopsin (Braiman et al., 1991), application for photosynthetic reaction centers was not yet reported. An estimation of the IR signal amplitude ex­ pected for charge separation in reaction of the absorbance) centers (on the order of indicates that at least 50,000 interferograms would be needed for a full time-dependent series of spectra. Assuming that a dead time of at least one second is needed between two flashes to allow complete relaxation, this experiment will take about one day. It is clear that the same type of experiment for the study of charge se­ paration will be extremely critical and take days to be completed, since a dead time of at least 10– 20 s between two flashes would be needed in order to allow full relaxation of the charge-separated state. Apart from the time needed for the experiment, it is questionable whether sample stability would be sufficient, i.e. whether the background absorbance could be kept constant (changing less than over this period, and whether photochemical deterioration of the sam­ ple (even if small) would not lead to artefacts because of the changing response of the protein to flash excitation. 3.

Step-Scan FTIR Spectroscopy

The major drawback of Stroboscope FTIR spec­ troscopy is the (partial) loss of multiplex advan­ tage due to multiple recording of data points and the limitation of the time resolution by the

152 matched combination of optical delay/time delay. These drawbacks are avoided by the third timeresolved FTIR technique termed stop-and-go or step-scan FTIR spectroscopy (Uhmann et al., 1991), which is illustrated in Fig. 3c. With this technique, the interferometer path (–d to +d) is divided into many steps (in the order of 500 to 2000), and instead of moving continuously, the mirror is now moved stepwise. At each step, the moving mirror is stopped and the flash-induced signal is triggered. A transient absorbance change in the sample will lead to a change of intensity for this particular point of the interferogram, which is recorded in a transient recorder. Repetitive excitation may be used to improve the signal-to noise ratio for the time-dependence of the inter­ ferogram intensity. The mirror is then moved to the next step, and the full time-dependence of the interferogram is recorded by acquiring the transient changes at all steps. This “array of data” can be transformed to time-depedent spectra after rebuilding the time-dependent interferog­ rams from the transient intensity changes. The step-scan technique, in contrast to the stroboscope technique, makes full use of the mul­ tiplex advantage, in that, except for averaging the transient changes of the interferogram intensity, no data point of the array (detector intensity as a function of time and optical delay) is recorded more than once. Thus, transient spectra can be recorded with much less repetition. However, the effort on the hardware side of the interferometer is considerably higher than with the stroboscope technique. The interferometer mirror is much less stable at a fixed position than it is during a uni­ form movement. In fact, if a step-scan spectrum is recorded at resolution, and the path of the interferometer is subdivided into 2000 in­ tervals, the increment from step to step is in the order of one and the required minimum devi­ ation from the parallel position corresponds to fluctuations in position of a few nanometer. An active stabilization of the movable mirror by mounting it onto piezo elements has been succes­ fully used. In this case, the piezo elements are driven by a control unit which uses interference signals from auxiliary lasers to maintain the lat­ eral and the angular position of the mirror. Need­ less to say that a working step-scan interferometer is extremely sensitive to vibrations and thus needs

Werner Mäntele very stable optical benches and components. It is most successfully used in evacuated FTIR spec­ trophotometers, since purging of the instrument may already introduce too many vibrations. As for time-resolution, step-scan FTIR spectroscopy is only limited by the rise time of the detector and of the transient recorder, and nanosecond applications have been reported (for a review, see Palmer et al., 1993). The arguments on sample stability discussed above for stroboscope FTIR spectroscopy also hold for the step-scan technique: Although less flashes may be needed for a complete time-dependent spectrum because of the multiplex advan­ tage, the response of the photoreactive protein to the trigger needs to be identical for each flash. It is probably for this reason that applications up to now were for bacteriorhodopsin (Weidlich and Siebert, 1993) and CO poisened myoglobin (Rödig et al., 1994), and only one application was reported for reaction centers of photosynthetic bacteria (for a review of other, nonbiological ap­ plications see: Palmer et al., 1993). Breton and coworkers (Burie et al., 1993) have used stepscan FTIR spectroscopy to investigate electron transfer in RCs at low temperature (90 K), col­ lecting data at 457 mirror positions (averaging 20 laser flashes at each position). With 20 of these cycles averaged, around 180,000 flashes were needed. Burie et al. (1993) obtained difference spectra at after a laser flash which show the essential features of the steady-state difference spectra obtained under continuous il­ lumination. Using RC poised at low redox poten­ tial, they were able to generate a flash-induced difference spectrum averaged over the time and Major bands of window between difference spectrum were found to decay the with a half-time of While this experiment took only about one day with 180,000 flashes fired at approx. 6 per second, it is clear that an investi­ gation of a photoreaction which needs longer relaxation time is much more difficult to perform. V. Single Wavelength IR Techniques Single wavelength techniques can present an alternative to FTIR techniques whenever only a small spectral range is considered or when high time resolution is needed. In addition, cyclic reac­

Infrared and Fourier-transform infrared spectroscopy tions with long relaxation times (such as, for ex­ and further ample, electron transfer from P to to could make a stroboscope or a step-scan FTIR experiment difficult if not impossible, but would still allow kinetic measurements in a lim­ ited range. Furthermore, kinetic measurements made “point by point” would even be possible for a less stable system which shows changes of kinetic parameters with time, while a stroboscope or step-scan experiment under these conditions would reflect these changes distributed through­ out the spectrum. Single wavelength techniques either use light from thermal sources or from continuous wave or pulsed lasers. A variety of techniques have been developed, covering the mid-IR range and giving access to the time domain in milli- and micro­ seconds as early as 1978 and picoseconds in the 90 s. In fact, the first IR difference spectrum of a photosynthetic membrane was obtained in 1984 using a modified IR spectrophotometer equipped with a fast chopper and a fast and sensitive MCT detector (Bartel et al., 1985). Time resolution was dominated by the duration of xenon flash to around

A. Dispersive Spectrometers A first version of a dispersive kinetic IR photo­ meter was based on a conventional IR spectro­ photometer (Perkin-Elmer Type 180) equipped with a fast MCT detector and a xenon flash for sample excitation (Mäntele, 1978; Siebert et al. 1980; Siebert et al., 1981; Mäntele et al., 1982). A fast chopper was used for modulation to reduce the low-frequency noise of the detector (1/f noise). With this setup, kinetic IR signals in the range to could be recorded at a time resolution from seconds to about 1 ms (un(chopped) and between 1 ms and some chopped). A similar setup using a gated detection for the MCT was described by Iwata and Hamag­ uchi (1989) and by Hauser (1994) with microse­ cond time resolution. These setups use a thermal source (“globar”: a silicon carbide rod heated to 600–800 °C) and a monochromator, which is typically mounted between the sample and the detector in order to reduce flash artefacts. Exci­ tation of the sample by a light pulse and the subsequent photochemistry will result in some

153 fraction of the light energy deposited as heat, which will lead to broad signals (“heat signal”) overlapping the changes of intensities and of the frequency of individual modes. Placing the mono­ chromator between sample and detector will re­ duce this artefact. The problem limiting the application of disper­ sive IR spectroscopy is the low spectral energy density. The half-width of IR bands and the over­ lap of IR signals necessitates a resolution of or better to resolve structures in the differ­ ence spectra. At this resolution, the radiation in­ tensity in a spectral element at with a given by a typical thermal source width of and cannot be increased is less than easily. The low number of photons and the detec­ tor noise limit the time resolution to the ms do­ main or necessitate excessive averaging. On the other hand, the noise of semiconductor IR detec­ tors is governed by intensity-independent compo­ nents, and would allow to increase the intensity by orders of magnitude without increasing the noise. Only one application of dispersive IR spectro­ scopy in photosynthesis has been reported up to now, mainly because of the lack of sensitivity. Bartel et al. (1985) investigated the transient IR difference spectra of thylakoids and chromato­ phore membranes and found signals in the ms time domain which were insensitive to inhibitors of electron transfer. They ascribed them to en­ ergy dissipation processes at the pigments in the light-harvesting complexes. For bacterial mem­ branes with functional reaction centers, slow ad­ ditional components were found and assigned to the photooxidation of the primary electron donor bacteriochlorophylls in the reaction center.

B.

Tunable IR Lasers

An alternative to using thermal sources and monochromators is the use of infrared lasers, which need to be tunable in order to cover at least a small part of the difference spectrum in the to region. Ideally, highly stable continuous wave lasers should be used, but applications with pulsed lasers are also conceivable. For the spectral region between ca. and ca. CO lasers with high intensity can be used. They provide tunability

154 over at least with high-intensity modes every However, they show intensity fluctuations in the ms-to-ns time domain due to mode-hopping processes, and thus cannot be used for continuous wave applications in this time do­ main2. This restriction does not apply for CO lasers as a source of IR light for ultrashort pulse generation in upconversion experiments (see next section). Probably the most convenient tunable IR la­ sers for time-resolved IR spectroscopy are diode lasers. These diode lasers are made from lead salt semiconductors (europium doped lead selenide) and can be made for the frequency range between Their frequency range can 2000 and be chosen by varying the semiconductor composi­ tion, and, within that range, they can be tuned by varying the temperature of the semiconductor chip. This can be either reached by varying the heat sink temperature or by adjusting the laser current. Operating temperature is between a few K and 150 K at the highest, and stability of laser radiation requires temperature fluctuations to be smaller than approx. 1 mK. The tuning range can be up to for one laser, and modes are found within this range every with an output power of up to 1–2 mW. A kinetic IR photometer using these lasers has been described (Mäntele et al., 1990b) which uses several of these lasers in a cryostat, each at a different frequency range. The lasers, pread­ justed in temperature, can be moved into the focus of an off-axis paraboloid which couples the beam into the photometer. A monochromator can be switched into the beam to determine the precise frequency, but also to cut out unwanted side modes. The IR probing beam is then focused onto the sample on a spot of around 1 mm 2 , which allows microsampling or lateral sample scanning for bleaching samples. The transmitted light is collected and focused on a MCT detector of se­ lected sensitivity. The time resolution is only de­ termined by the risetime of the detector, which can be 20ns for a photovoltaic MCT detector. With a laser output power of up to a few mW, 2 Preliminary experiments performed by the author in col­ laboration with H. Pascher, Department of Physics, Univer­ sity of Bayreuth, using an actively stabilized CO laser at cryo­ genic temperatures, indicate that the stability would be sufficient for its use as a source of cw measuring light for timeresolved IR experiments.

Werner Mäntele signal-to-noise is high enough to allow detection of absorbance changes in the order of to with a single flash experiment. This setup has been successfully used to monitor transient signals associated with electron transfer in photo­ synthetic reaction centers (Hienerwadel et al., 1992, 1995). With the time resolution available, as electron transfer from to well as all molecular relaxations concomitant with this step could be followed in real-time. Signals from quinone modes upon formation of the se­ miquinone anion could be identified, but also sig­ nals from the protein host site indicating microconformational changes. Most of these rearrangements of groups or polarization of di­ poles occur in a concerted action and with the electron kinetic parameters of transfer. In the to range, however, signals with kinetic parameters un­ coupled from electron transfer were observed, which correspond to proton uptake by Asp and Glu side chains. In particular, proton uptake by pocket could be demon­ Glu L212 in the strated, and evidence was obtained for ionizable residues forming a cluster of partial charges around

C. Picoseond Pump-Probe Techniques Several laboratories have applied picosecond techniques for ultrashort IR studies on RC. Two basically different experimental approaches have been used. In the first one mainly put forward by R.M. Hochstrasser’s group in Philadelphia, a continuous wave CO laser (or a tunable diode laser) which defines the probing wavelength is mixed in a nonlinear optics experiment with an ultrashort light pulse in the visible, which is de­ layed with respect to the pump beam (Diller et al., 1991a,b; Hochstrasser et al., 1992; Maiti et al., 1993). The shifted frequency pulse resulting from the mixing of the IR light and the visible light pulse is detected in the visible region (“up­ converted”), with the timing done by the ultra­ short light pulse. This technique allows easier de­ tection of the light pulse with high sensitivity in the visible; however, the signal-to-noise ratio de­ pends on the stability of the continuous wave IR source. In the second approach put forward by W. Zinth’s group in Munich, an ultrashort IR pulse is

Infrared and Fourier-transform infrared spectroscopy generated from visible light pulses by subtractive mixing techniques (Hamm et al., 1995). This short pulse of IR light is then delayed with respect to the visible light pump pulse and sent to the sample as a probe pulse. The transmitted IR light is detected with a MCT IR detector. The authors elegantly used the spectral width of their IR pulse, which is approx for multiwav­ elength detection. The IR pulses were dispersed in a grating spectrometer and measured with a 10-element MCT IR detector with a spectral resolution of Apart from faster data ac­ quisition, this simultaneous detection allowed a direct comparison of band intensities in this range. The authors obtained IR differ­ ence spectra from Rb. sphaeroides RCs at 1 psec and at 10 and 1000 ps. The latter spectrum, which charge separation, represents the state of FTIR dif­ closely corresponds to the ference spectrum obtained under steady-state il­ lumination. The 10 ps difference spectrum mainly charge separation, and represents the shows essential features of FTIR differ­ ence spectra previously obtained for Rps. viridis (Nabedryk et al., 1986; Mäntele et al., 1988) and Rb. sphaeroides RC (Nabedryk et al., 1995). The 1 psec spectrum, however, corresponds to the transition, and shows a broad background of increased absorption in addition to changes of intensity and frequency of localized modes, such as the 9-keto mode of the primary donor bacteri­ ochlorophylls. One of the expectations of picosecond spectro­ scopy was to obtain information on the contribu­ tions of the protein moiety to the primary steps of electron transfer, but unambiguous assignment of peptide C=O groups or amino acid side chains has not yet been possible. In addition, neither of the picosecond work provided clear evidence for or against electron transfer with an intermediate and isotope-labelled or chemically-modified bacteriochlorphyll incorporated at the monomer bacteriochlorophyll site will be needed for a clear decision. VI. Sample Preparation for Infrared Spectroscopy For the preparation of IR samples, the wave­ length range to be investigated defines the pri­ mary strategy. As discussed above, of the fre­

155 quency range accessible with most FTIR spectrophotometers which spans from >4000 to the range is the most attractive one (and the most investigated), since the majority of bands with diagnostic rel­ evance is located there. In this range, and over­ lapping most of the peptide C=O modes which can be used for structural studies, the strong water H–O–H bending mode is centered around Although its extinction coefficient is it necessitates thin lay­ only ca. ers for aqueous samples, since the absorbance from water at this frequency is around 1 for a As an additional problem, pathlength of the extinction coefficients for most of the protein modes (at most are one or two orders of magnitude smaller than the electronic transition moments of pigments, quinones, or hemes. RC samples for IR spectroscopy thus need to be of much higher concentration than samples used for optical transmission spectro­ scopies. Samples for spectroscopic investigations and approx. form between an exception, since this “IR window region” al­ or more. For the lows pathlengths up to to range, the design of IR samples follows a compromise between the water content needed for unperturbed function, con­ centration, and stability. IR cell windows used for studies on photosyn­ thetic protein complexes are almost always a compromise between transmission (from 190 nm to stability against aqueous solu­ tions, and price. IR cells with windows allow spectroscopic investigations of the same samples from the UV to the mid-IR, or excitation in the UV/VIS/NIR range and detection in the mid-IR. As an alternative, or win­ dows could be used: both offer slightly better but exhibit access to the region below less stability against aqueous solutions. Windows made from Ge or ZnSe crystals have only rarely been used, although they offer wide access to the low-frequency range. Their high refractive index, however, resulting in loss of light by reflection, and their cutoff at short wavelengths (Ge at ZnSe at 550 nm), limit their use for reaction-modulated IR difference techniques. Proba­ bly for funding limits, the routine use of diamond windows, which would be ideal for the visible and the IR, has not been reported.

156 For investigations of chlorophyll-protein com­ plexes, the extinction coefficients for the pig­ ments in the NIR (for example for the monomeric BChls of the RC) and for the RC protein (the and amide I band) differ considerably resp.). However, the high number of amide groups contributing to the amide I mode lead to comparable absorbance at for a RC sam­ 800 nm and at approx. ple. This facilitates excitation by visible or NIR light coaxially with the IR beam, or simultaneous VIS/NIR and mid-IR spectroscopic investigations such as described in a review by Mäntele (1993b) and shown in Fig. 2a. Two major types of samples have been used for FTIR and kinetic IR investigations. The first type are thin, semihydrated films dried on IR windows from solutions or suspensions of photo­ synthetic pigment-protein complexes in deterg­ ent, reconstituted in lipid vesicles, or from mem­ braneous particles. For the preparation of these dried films, salt, buffer, and detergent concentra­ tions in the starting suspensions need to be re­ duced considerably to avoid excessively high salt concentrations or detergent enrichment in the films upon drying. In order to maintain full ac­ tivity of the pigment-protein complexes, con­ trolled hydration of the films is essential. How­ ever, well-defined pH and ionic strength are almost impossible to maintain, and there has been a continuous concern that the properties of the protein in these films at reduced water content might not correspond to those of the protein di­ lute solutions. In addition, pigment-protein com­ plexes can become oriented upon drying, leading to polarization effects even for samples perpend­ icular to the IR beam. In the case of films from photosynthetic RCs, careful control of the struc­ tural and functional integrity by measuring the electron transfer rates was performed, and has provided evidence that the electron transfer ac­ tivity of the RC at high hydration (for example at equilibrium with 98% relative humidity) is in­ distinguishable from that in diluted solution. The second type of IR samples used are con­ centrated solutions or suspensions. Detergent-solubilized RCs can be concentrated to 1–2 mM in ultrafiltration cells. Of these concentrated protein solutions, pelleted membrane fractions, or pel­ leted lipid vesicles containing the protein only a

Werner Mäntele few are needed. Ionic strength can be easily controlled this way, and orientation does not occur. However, the pH may be offset toward the isoelectric point of the RC if the concentration of the buffer is too low with respect to that of the RC which has multiple buffering groups. This type of sample has been routinely used for elec­ trochemical investigations (see section IV.C.2), where diffusibility of the RC is a prerequisite. IR microcells are typically formed from two IR windows(typically separated by an annular spacer which defines the pathlength. These cells, which are mostly of local design, are necessarily demountable in order to be filled with the concen­ trated solutions or suspensions and to be cleaned. A serious disadvantage of these cells is that capil­ lary forces suck the water out of the central part (typically a few mm in diameter) lead to slow drying of the samples. We have solved this prob­ lem by cutting a circular groove in one of the windows around the sample area which, with a depth of at least 0.2 mm, breaks the capillary forces brought in by the spacer. A further im­ provement could be achieved by grinding a flat depression at the sample area which defines the pathlength (for a description of these cells, see Hienerwadel, 1993). These sample cuvettes are just formed by two windows (one with a depres­ sion and a circular groove around) and a flat cover window, with no spacer needed. Microcells with pathlengths between 4 reproducible have been obtained this way which and sample for days. When concentrated seal a solutions or suspensions are transferred to these microcells and the cover window is closed, excess material is pressed into the circular groove where it maintains humidity. Although the high protein concentration may seem frightening for biochemists, the quantities of protein needed for either sample type have frequently been overestimated (this may be a mis­ judgement from the “old days” of IR spectros­ of a 0.5–1 mM copy). Quantities of ca. solution are sufficient for a film or concentrated suspension, and correspond to what is con­ veniently used at higher dilution for UV and VIS spectroscopy. Only slightly higher quantities (5– of a 0.5–1 mM solution) are needed to fill the spectroelectrochemical cell.

Infrared and Fourier-transform infrared spectroscopy VII. Conclusions and Outlook IR and FTIR spectroscopy for the investigation of photosynthetic pigment-protein complexes, in the past decade, has received increasing interest3 and is now routinely used by a growing number of research groups. It was accepted that IR spec­ troscopy can be used to probe the relaxation of the cofactor and of the protein in electron transfer processes (not only in photosynthetic RCs). Even within these ten years, the progress from easy-toobtain light-induced FTIR difference spectra to the more sophisticated IR difference spectra of single cofactors or to transient spectra showing slow or even ultrafast electron transfer steps in real-time was breathtaking (at least for me). The major lesson we have learnt from IR spec­ troscopy is that no large conformational changes occur with electron transfer. It taught us to look closer and search for microconformational changes in the vicinity of cofactors, which have been identified for some cofactors and are most pronounced (and best characterized) for the quin­ and ones. The IR signals characterized for reduction illustrate why the protein is sometimes called an “optimized solvent”. The total of the changes in the vibrational spectrum of a cofactor upon its redox reaction presents a clue to internal and external reorganization energy. Ultrafast IR spectroscopy of RC is just at its beginnings, and yet the present data range from several hundred femtoseconds to nanoseconds. The possibility to follow the role of the protein in the primary steps of photosynthetic electron transfer is fascinating. The weight of these experi­ ments is still on the technical highlights, but the search for protein signals in primary charge separ­ ation has started. The deficit of IR spectroscopy of photosyn­ thetic pigment-protein complexes is in the part of the band assignment. It is clear that the combined effort of site-directed mutagenesis and isotope labelling can help to assign the detailed band structures in FTIR difference spectra. Whether 3

This is characterized by the way the techniques are classi­ fied on meetings: In 1985, FTIR spectroscopy of RC was listed among “. . . new and potentially interesting techniques”, while in 1995, a light-induced FTIR difference spectrum of RC was already presented as a “. . . . classical FTIR difference spectrum”.

157 site-directed isotope-labelling can become appli­ cable in photosynthesis needs to be demon­ strated. However, I doubt that we shall ever be able to fully exploit the wealth of information contained in an IR difference spectrum. Yet, fo­ cusing on “hot” spectral regions like the Asp and Glu side chain carboxyl group range between and is most promising, and I am optimistic that a full mapping of this range for the bacterial RC is within reach. Altogether, the use of IR spectroscopy on the RC over a wide wavelength range and over more than twelve orders of magnitude in the time do­ main will help us to understand the role of the protein matrix in charge separation, stabilization and photosynthetic energy conversion. Acknowledgements I thank my colleagues working in the field of IR and FTIR spectroscopy of proteins for valuable discussion and for providing information on un­ published work and on work in press. I appreciate the comments and suggestions from my cowork­ ers, and thank S. Grzybek for the preparation of Fig 3. Our own work in this field has only been possible because of generous financial support from the Deutsche Forschungsgemeinschaft through several grants and a Heisenberg fellow­ ship. References Armstrong FA, Hill HAO and Walton NJ (1986) Reactions of electron-transfer at electrodes. Quart Rev Biophys 18: 261–322. Arrondo JLR, Muga A, Castresana J and FM (1993) Quantitative studies of the structure of proteins in solution by Fourier-transform infrared spectroscopy. Prog Biophys Molec Biol 59: 23–56. Baenziger JE, Miller KW and Rothschild KJ (1993). Fourier transform infrared difference spectroscopy of the nicotinic acetylcholine receptor: Evidence for specific protein struc­ tural changes upon desensitization. Biochemistry 32: 5448– 5454. Ballschmiter K and Katz JJ (1968) Long wavelength forms of chlorophyll. Nature 220: 1231–1233. Ballschmiter K and Katz JJ (1969) An infrared study of chlorophyll-chlorophyll and chlorophyll-water interaction. J Am Chem Soc 91: 2661–2677. Bartel K, Mäntele W, Siebert F and Kreutz W (1985) Timeresolved infrared studies of light-induced processes in thy­ lakoids and bacterial chromatophores. Evidence for the

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Chapter 10

Resonance Raman Studies in Photosynthesis –

Chlorophyll and Carotenoid Molecules

Bruno Robert

Section de Biophysique des Protéines et des Membranes, DBCM / CEA and URA 1290/CNRS,

CE. Saclay, 91191 Gif l Yvette, France

Summary I. Introduction II. Introduction to Raman and Resonance Raman Spectroscopy III. Resonance Raman Spectroscopy of Photosynthetic Pigments A. Chlorophyll and Related Molecules 1. Carbonyl Stretching Modes 2. Vinyl Stretching Modes and Other Contributions in the High Frequency Range 3. Marker Bands for Coordination State of the Central Mg Atom 4. Activity of the Different Modes According to the Excitation Wavelength B. Carotenoid Molecules C. Technical Aspects – Recent Advances IV. Resonance Raman Spectroscopy as Method of Chemical Analysis in Photosynthesis V. Resonance Raman Spectroscopy as a Probe for Molecular Conformation VI. Resonance Raman Spectroscopy as a Probe for Intermolecular Interactions A. The Binding Sites of the Primary Electron Acceptors in Different Photosystems B. The Molecular Structure of the Primary Electron Donor in Bacterial Reaction Centers C. The Amino Acids Interacting with the Long-Wavelength-Absorbing BChl Pair in Light-Harvesting Complexes from Purple Bacteria VII. Time-Resolved Resonance Raman Studies VIII. Resonance Raman Spectroscopy as a Probe for Studying the Nature of Electronic Transitions IX. Perspectives Acknowledgements References

161 162 162 163 163 164 164 165 166 166 166 167 168 169 169 169 170 171 172 173 174 174

Summary After a short introduction to the physical principles that govern resonance Raman spectroscopy, the various possibilities of this technique for studying (bacterio)chlorophyll and carotenoid molecules within photosynthetic systems are briefly discussed. Marker bands for coordination state of the central Mg atom and for the interaction state of the various carbonyl of (bacterio)chlorophyll molecules are described. A series of examples are given, illustrating how resonance Raman spectroscopy may help in: (1) assessing the molecular structure of a chlorin-type electron carrier; (2) precisely determining the configuration of a protein-bound carotenoid molecule; (3) measuring the interaction strength of the different carbonyl substituents of (bacterio)chlorophyll in a given proteic binding site; and (4) obtaining information on the electronic excited states involved in the photosynthetic process. Literature concerning the application of time-resolved resonance Raman to bacteriochlorophyll and carotenoid molecules in photosynthetic systems is also reviewed. Correspondence: Fax: 33-1-69084389; E-mail: [email protected]

161 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 161–176. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

162

Bruno Robert

Abbreviations: (B)Chl – (bacterio)chlorophyll; (B)Pheo – (bacterio)pheophytin; FT – Fourier-transform; THF – tetrahydrofuran; P – primary electron donor; PS – photosystem; RC – reaction center; RR –resonance Raman

I. Introduction For more than 20 years (Lutz, 1972), resonance Raman (RR) spectroscopy has developed in the field of photosynthesis, and its application has led to a number of significant contributions to this field. Although this spectroscopy is now applied on most of the molecular entities participating in the initial events of the photosynthetic process, in a wide variety of organisms, it is still routinely performed in few laboratories only in the world. This situation probably results from the many technical constraints and difficulties of the me­ thod, some of which have been solved recently by technological breakthroughs such as the intro­ duction of Fourier-transform techniques to Raman spectrometers. The aim of this chapter is not to exhaustively review the contribution of resonance Raman spectroscopy to the under­ standing of the photosynthetic process, but rather to highlight its various possibilities through parti­ cular examples. Further, the limitations of this technique, either intrinsic, or experimental, will be discussed throughout the text, so as to illus­ trate when and why resonance Raman spectros­ copy can (or cannot) yield useful information and the nature of this information. In order to keep this chapter as clear and con­ cise as possible, I will focus mainly on the use of Raman spectroscopy for studying chlorophyll and carotenoid molecules. Although this choice might appear quite arbitrary, it can be justified as fol­ lows. The amount and nature of information ob­ tained by a vibrational technique such as reson­ ance Raman spectroscopy is strongly dependant on the nature of the molecules considered, and every example involving a study of a new mol­ ecule (such as e.g. a cytochrome or an iron–suphur protein) should be accompanied by a com­ plete introduction of RR spectroscopy on this particular type of molecule. Moreover, and this is especially true for the two particular examples quoted above, the use of resonance Raman has developed for these molecules outside of the field

of photosynthesis, and a comprehensive introduc­ tion of their Raman spectra should mostly include references to these other fields.

II. Introduction to Raman and Resonance Raman Spectroscopy The Raman effect is the phenomenon of a change of frequency of the light, when it is scattered by polyatomic molecules. If the frequency of the and that of the scattered light incident light is the frequency shift will corre­ is spond, in wavenumbers to the energy exchanged between the incident photon and the considered molecule during their inelastic colli­ sion. This energy, gained or lost by the photon, must be equal to that of a transition between molecular energy levels, in the following uniquely vibrational energy levels of the scattering mol­ ecules. Performing Raman spectroscopy thus es­ sentially consists in measuring the vibrational en­ ergy levels of a given electronic state (the groundstate in classical Raman spectroscopy, but it can be an excited state if it is populated) by measuring the different Raman frequencies contained in the light scattered by a given type of molecule. From simple physical considerations (for a general in­ troduction to the Raman effect, see Carey (1982), it can be deduced that the molecular vibrations which will be Raman-active will be those involv­ ing a change in molecular polarizability. One of the major consequence of this situation, at least for biochemical studies, is that the Raman spec­ trum of water is weak, and thus seldom interferes with that of the biological molecules, and this is an important advantage for the Raman technique over infra-red absorption spectroscopy. The vibrational levels of a particular molecule depends on its structure (i.e. the nature of its constituent atoms and the bonds between these atoms). Raman spectroscopy can thus be used as an analytical method for determining the chemi­ cal structure of the molecule. It also depends on the conformation of the molecule considered, as

Resonance Raman spectroscopy well as on the intra- and intermolecular interac­ tions this molecule is involved in. These par­ ameters may thus also be deduced from Raman spectroscopy. Moreover, although any vi­ brational mode formally involves the coordinates of every atom of the vibrating molecule, in the case of large molecules such as chlorophyll, some of these modes mainly involve the coordinates of particular chemical groups (such a vinyl or a carbonyl group). In this particular case, the other atoms of the molecule may be treated, to some extent, as a perturbation of this ‘localized’ vi­ brational mode, and the resolution of the method becomes submolecular. However, this information would be clearly dif­ ficult to extract from biological material as com­ plex as the photosynthetic membranes if Raman spectroscopy did not exhibit a very specific, as well as convenient, property: a way of selecting the chemical nature of the scattering molecule, through the resonance Raman effect. What is called the resonance Raman effect is the enhance­ ment of the Raman effect when the exciting fre­ quency matches an electronic transition of the irradiated molecule. This effect, which may cause an enhancement by a factor of allows the selective observation of resonance Raman spectra of the absorbing molecule in a complex medium, provided that this molecule is the only one pos­ sessing an absorption transition. This resonance phenomenon gives to resonance Raman spectros­ copy the ability to probe the interaction state and conformation of chromophores within proteins, and sometimes even when these remain embed­ ded in a membrane, with little if any interference from signals due to the protein itself. In reson­ ance conditions, the enhancement concerns a fraction only of the vibrational modes of the mol­ ecule. Most often, when only one electronic state is involved in the resonance phenomenon, the Raman bands arise from those modes in which variations of nuclei positions corresponds to dis­ tortions experienced by the molecule upon the electronic transition between the ground- and the excited state used for inducing the resonance. Because of this property, in resonance Raman, although the position of peaks depends solely on the electronic ground-state, their intensities yield information about the electronic excited state in­

163 volved in the resonance process, and, in parti­ cular, about the nature of the vibrational modes coupled to this electronic transition. III. Resonance Raman Spectroscopy of Photosynthetic Pigments

A.

Chlorophyll and Related Molecules

Detailed reviews on the nature of the modes ac­ tive in resonance Raman spectroscopy of chloro­ phyll compounds have already been published (Lutz, 1984; Lutz and Robert, 1988; Lutz and Mäntele, 1991). Briefly, most of the bands ob­ served in RR spectra of chlorophyll a and related molecules (i.e. ca. 30) appear to arise from inplane modes (Lutz, 1979). This can easily be understood as most of the electronic transitions of Chl a and related molecules are composed of transitions polarized parallel to the molecu­ lar plane. This still holds for vibrational modes involving coordinates from peripheral substitu­ ents, and thus the conformation of these groups may be evaluated on the basis of the RR activity of their stretching modes (Lutz, 1984). Most of these modes are complex, involving mixing of several internal coordinates. However, there is a small number of more localized modes, which have been assigned on the basis of isotopic substi­ tutions, and comparisons between IR, Shpol’skii and RR spectra of various chlorophyll and por­ phyrin derivatives (Lutz, 1979, 1984). Indeed, only a few normal mode calculations have been performed for chlorophyll molecules, as these are particularly complex, due to the low molecular symmetry of these compounds (Boldt et al., 1987; Donohoe et al., 1988). For this reason, most of the conclusions drawn about biochemically-relevant samples are supported by comparisons with chemical models, from which the sensitivity of the different Raman bands is experimentally de­ termined. It must be noted that resonance Raman spectroscopy will yield information on only those modes which are coupled with the electronic transitions of the (bacterio)chlorin molecules. It will thus yield information only on those chemical groups which may play a role in tuning the physic­ ochemical properties involved in the energy/electron transfer between these molecules. In this

164 respect, the additional mode selection which oc­ curs during the resonance process in Raman spec­ troscopy does not constitute a limitation, but rather an advantage for photosynthetic studies. 1. Carbonyl Stretching Modes In the high frequency range of(bacterio)chlorophyll RR spectra bands arise from near to pure stretching modes of the car­ bonyl groups of these molecules (i.e. 9-keto for Chl a and BChl c, 9-keto and 2-acetyl for BChl a and b, 9-keto and 3-formyl for Chl b and BChl e) (Lutz, 1972, 1974). The stretching frequency of the 9-keto carbonyl modes is observed at ca in the absence of intermolecular inter­ actions, in an apolar environment. In (bacterio)pheophytins, their frequency is slightly higher and for Pheo a and BPheo a, respectively) (Mattioli et al., 1993). The fre­ quencies of these modes is rather insensitive to the presence of another carbonyl group attached to the macrocycle as no more than a shift is observed between the frequency of the 9-keto stretching modes of Chl a and 2-acetyl Chl a (Feiler et al., 1994a). However, the 9-keto stretching mode of BChl c is observed at i.e. ca lower than in Chl a, although these molecules mainly differ by the presence of the residue at position 2 (a hydroxy and a vinyl group in BChl c and Chl a, respec­ tively) (Feiler et al., 1994b). The stretching modes of the 2-acetyl and 3-formyl carbonyl in the absence groups are observed at of intermolecular interactions (Lutz, 1984). Sche­ matically, the frequency of these modes is primar­ ily sensitive to the intermolecular interactions in which they are involved, and, to a lesser extent to the permittivity of their environment. Upon entering intermolecular interactions, such as Hbonding, downshifts as large as are fre­ quently observed, while the range of frequency variation due to solvent effect is ca (Lutz, 1984; Koyama et al., 1986). The free enthalpy of the H-bonds established between the (B)Chl carbonyl groups and their partner molecule may be evaluated, using a Badger-type calculation (Zadorozhnyi and Ishchenko, 1965). Considering the position of these substituents on the macrocycle rings, the stretching modes of

Bruno Robert the carbonyl in position 2 and 9 are expected to be more active in resonance with Y-polarized transitions. Consistently, excitation of BChl a in the transition did not yield observable band in the carbonyl frequency range (Lutz, 1984). By contrast, a formyl carbonyl at position 3 is ex­ pected to be more intense upon X-polarized exci­ tations. These properties have recently been used to attribute the bands present in BChl e contain­ ing chlorosomes to either the 9-keto or the 3­ formyl stretching modes (Feiler et al., 1994c). In transition of preresonance conditions with the (bacterio)chlorin molecules, very weak contribu­ tions from the ester carbonyl can be observed at (Mattioli et al., 1991). Until now, ca these very weak bands have been seldom used for determining the interaction state of these groups. As these groups are expected to be poorly conjugated with the (B)Chl macrocycle, bands arising from their stretching modes should experi­ ence relatively smaller shifts upon H-bonding, and, moreover, these H-bonds should have lim­ ited influence on the physicochemical properties of the chlorophyll molecules. 2. Vinyl Stretching Modes and Other Contributions in the High Frequency Range As carbonyl stretching modes have been exten­ sively used for assessing the interaction pattern of (bacterio)chlorophyll molecules in vitro as well as in vivo, it is of importance to identify the other possible contributions in the same spectral range. Early Raman studies of Chl a did not reveal any region, intense band in the which might primarily arise from the stretching mode of the vinyl C = C bond at position 2 (Lutz and Kleo, 1974). Similar conclusions had been reached by comparing RR spectra of nickel-methylpheophorbide and of nickel-mesopyropheophorbide (Boldt et al., 1987). By comparing the RR spectra of Chl a and 2-acetyl Chl a, or those of BChl a and 2-vinyl BChl a, Feiler et al. (1994b) observed the contribution of this mode at in vinyl-containing derivatives (Fig. 1). In Soret resonance, this contribution is very weak and partially hidden by those arising from the methine bridges stretching modes. From normal mode calculations, as well as from experiments performed on reaction center­

Resonance Raman spectroscopy

165

samples unambiguously indicates the presence of intermolecularly interacting 2-acetyl carbonyl groups.

3. Marker Bands for Coordination State of the Central Mg Atom

bound BChl a, it was proposed that CaCm and/or CbCb modes could contribute in the 1620– range (Boldt et al., 1987; Boldt et al., 1993). Experiments conducted on isolated BChl a in various conditions of excitation did not reveal the presence of any such modes (Lutz, 1984; Lutz and Robert, 1988; Mattioli et al., 1993). The same conclusion was reached for Chl a, after the replacement of the vinyl group at position 2 by an acetyl group (Fig. 1) (Feiler et al., 1993b). These predicted CaCm or CbCb modes must ne­ cessarily give rise to extremely weak bands, or should be slightly less up-shifted than predicted by the calculations (Feiler et al., 1993b). It can thus be safely concluded that the presence of a band in BChl a-containing

Bands arising from two types of modes have been used for diagnosing the coordination state of the central Mg atom of chlorophyll derivatives. First, the bands arising from modes involving this atom, in the frequency range (Lutz et al., 1975), constitute a reliable marker of the Mg atom coordination for Chl a, b and BChl a mol­ ecules (Lutz, 1977, 1984; Fujiwara et al., 1988). It must be noted that these bands do not involve the coordinates of the coordinating atom, i.e. that they cannot be used for. diagnosing the chemical nature of the axial ligand. They have been used in vivo for determining the interaction state of protein-bound Chl molecules (Lutz, 1977). They are particularly weak in BChl a RR spectra, and their use may prove difficult in biological studies. The band arising from the methine bridges stretching modes, at ca has also been used for diagnosing the coordination state of the central Mg atom, as well as, in Chl molecules, bands in the frequency range, arising from complex modes of the conjugated ring. These bands exhibit a definite, although in­ direct, sensitivity to the coordination state of the central Mg atom They constitute convenient mar­ kers because of their very high activity in reson­ ance with the Soret electronic transition. The band arising from the stretching mode of the mewhen the thine bridges is located at central Mg atom binds two external ligands, and at frequencies higher than when it in­ teracts with one axial ligand only (Cotton and van Duyne, 1981). The frequency of this band also slightly depends on the temperature and on the excitation conditions (Mattioli et al., 1992). For Chl a molecules, a doublet of bands is observed when the central Mg is fiveat when it is coordinated, and at six-coordinated (Fujiwara and Tasumi, 1986; Tas­ umi and Fujiwara, 1987).

166

4. Activity of the Different Modes According to the Excitation Wavelength The activity of resonance Raman bands depends on the coupling between the vibrational modes and the electronic transition used for inducing the resonance effect. High activities of the methine bridge stretching modes may be achieved by ex­ citing BChl a under Soret resonance conditions (Lutz, 1979, 1984). In these conditions, the band arising from these modes when the central Mg atom is five-coordinated) dominates the RR spectra, and it may partially hide the 2­ acetyl carbonyl stretching modes, which are usu­ ally weak at Soret resonance. Some carbonyl stretching modes have been reported missing in BChl spectra recorded in these conditions of exci­ tation. This was the case in particular for the RR spectra of the primary donor in Rhodobacter sphaeroides reaction centers (Robert and Lutz, 1986; Mattioli et al., 1991), and for those of the 800 nm-absorbing BChl in peripheral light-harvesting complexes from Rhodopseudomonas pa­ lustris (Robert and Lutz, 1985; Sturgis et al., 1994). Excitations in preresonance with the transition of these molecules is definitively more favorable for the observation of bands arising from the carbonyl stretching modes. By contrast, inferring the number of axial ligands on the cen­ tral Mg of these molecules is often easier in reson­ ance conditions with the Soret electronic transi­ tion.

B. Carotenoid Molecules Carotenoid molecules are extremely efficient Raman scatterers and they have been the subject of RR studies as early as 1932 (Euler and Hellström, 1932). Their RR spectra mainly con­ 1120– sist of four groups of bands at ca. 1530 1200 1000 and Systematic studies of different isomers of (Saito et al., 1983 Koyama et al., 1983), as well as nor­ mal mode calculations performed on these mol­ ecules in different configurations, have allowed the evaluation of the sensitivity of these different bands to the molecular conformation, as well as the precise attribution of each of these (Saito and Tasumi, 1983). The presence of particular bands could be safely connected to specific molecular

Bruno Robert structure, e.g. the presence of a band is diagnostic for a 15–15' cis conformation. Most of the different conformations of carotenoids may be distinguished on the basis of their Raman spec­ tra (Koyama et al., 1988). As the signal arising from these molecules is particularly intense, they have been the subject of most of the time-resolved resonance Raman spectroscopy studies in photosynthesis (see below).

C. Technical Aspects – Recent Advances RR scattering is a low-probability, low yield pro­ cess. It thus requires quite high irradiance on the sample, generally at wavelengths matching with the absorption transitions of the molecules being studied. Photodegradation of the pigments may be avoided either by cooling the samples to cryo­ genic temperatures or by using spinning cells or flowing samples. This crucial problem may be much more easily overcome by inducing the res­ onance phenomenon with the lowest energy transitions of the pigments, i.e. with red or infra­ red excitations. Up to five years ago, this was extremely difficult if not impossible, as under these conditions of excitation the intrinsic fluor­ escence of the pigments results in signal back­ grounds which can be many orders of magnitude more intense than the Raman signal itself. New detectors, namely charge-coupled devices, have recently appeared, which allow the recording of RR signals of chlorophyll molecules excited in the lowest singlet transition. These detectors have practically no dark noise, a very high quantum efficiency and extended dynamics, which is abso­ lutely necessary for recording the Raman signal over the intense fluorescence background. How­ ever, although their response in the 600–900 nm range is excellent, their sensitivity dies rapidly beyond 950 nm. Nevertheless, recording Raman spectra in these conditions must be performed with the most extreme care, in order to avoid artifactual signals. A second technological break­ through has occurred with the development of Fourier-transform Raman spectrometers. In these spectrometers, the signal scattered by the sample is sent, after the filtering of the Rayleigh line to the interferometer of classical FT-IR spec­ trometers. At the moment, this technique allows only a single excitation wavelength, 1064 nm.

Resonance Raman spectroscopy

167

However, the recording of all spectral elements at one time counterbalances the fact that the Raman signal is much weaker in these conditions (the scattering effect depending on Although this technique is much more demanding in terms of sample concentration is often necessary) it allows the recording of RR spectra at room temperature and it possesses many different ad­ vantages which will be described amongst the ex­ amples chosen. IV. Resonance Raman Spectroscopy as Method of Chemical Analysis in Photosynthesis Resonance Raman spectroscopy may provide de­ tailed information on the chemical nature of the molecules studied. This property, combined with the possibility of selective excitation of these mol­ ecules in a complex medium was recently used for determining the chemical nature of the primary electron acceptor in bacterial reaction centers of the green sulfur bacterium Chlorobium limicola. Several different pigments had been proposed for this particular cofactor, including BPheo c (van Bochove, 1984), BChl c-like pigment (Braumaun et al., 1986; Shuvalov et al., 1986) and Chl aisomer (van de Meent et al., 1992). Selective res­ onance Raman information on chlorin molecules in these photosystems was achieved by using a 441.6 nm excitation. From the absence of finger­ print contributions of BPheo at Feiler et al. (1994) concluded that the preparation was devoid of BPheo c. In BChl c the vinyl group in position of ring I of Chl a is replaced by a hydroxy-ethyl group. Accordingly, the band at arising from the vinyl stretching modes is absent from its RR spectra (Feiler et al., 1994a). Raman experiments performed on pig­ ment extracts from Chlorobium reaction centers revealed the presence of this band, and it was concluded that the chlorin molecule present in Chlorobium RC contains a vinyl group i.e. that this pigment is a Chl a-type molecule (Feiler et al., 1994a). Resonance Raman spectroscopy may also be used for assessing more subtle structural features, such as the charge distribution over an assembly of (B)Chl molecules. Mattioli et al. (1991) showed that FT-Raman spectra excited at 1064

nm of reaction centers from purple bacteria con­ tain primarily preresonant contributions from their primary electron donor, which is a dimer of BChl (P) (Fig. 2). When P is oxidised, exciting at the same wavelength results in resonance con­ ditions with the weak 1250 nm electronic trans­ itions of the state. FT-Raman spectra of pur­ ple bacterial reaction centers containing P in either its neutral or oxidized form are thus dram­ atically different from each other (Fig. 2). From state, the charge dis­ the Raman study of the tribution over each bacteriochlorophyll in the cat­ ion dimer could be addressed. In the FT-Raman a band is observed at spectra of which arises from the 9-keto carbonyl stretching mode of one BChl molecule, up-shifted by the presence of the positive charge. In the FT-Raman of P in its neutral state, this band contributes at (Fig. 2) (Mattioli et al,, 1991; Mattioli et al., 1994). Upon formation, the stretching mode of this 9-keto carbonyl stretching mode thus

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experiences a upshift, as compared to the induced by one-electron oxidation of BChl in aprotic solvents (Mäntele et al., 1988). The charge on P is thus mainly borne by one of the BChl molecules. This BChl was identified as being the one located on the L side of the RC (Mattioli et al., 1991). The different factors in­ fluencing this charge distribution were studied by FT-Raman, and it was shown that it is influenced by the presence of H-bonds between the BChl constituting the primary donor and the protein (Wachtveitl et al., 1993; Mattioli et al., 1994) and not by site-selected mutations at the M210 locus (Jones et al., 1994). V. Resonance Raman Spectroscopy as a Probe for Molecular Conformation Although it would be certainly of interest, until now there have been no resonance Raman bands which could be precisely linked to particular con­ formations of the conjugated macrocycle. By con­ trast, RR Spectroscopy has brought a number of conclusions concerning the conformation and configuration of carotenoid molecules embedded in photosynthetic proteins. In reaction centers of purple bacteria, it was reported in 1976 that the spheroidene bound to the protein had an unusual RR signal (Lutz et al., 1976). The presence of a band at was considered as evidence of a 15–15' cis configuration for this molecule (Koyama et al., 1982). Nuclear magnetic reson­ ance experiments performed on carotenoid ex­ tracted from the reaction centers of various bac­ teria confirmed this configuration (Lutz et al., 1987). However, it was shown by RR spectros­ copy that, during the extraction procedure, the conformation of the molecule was modified (Lutz et al., 1987) and it was thus concluded that an additional twist of the polyene chain exists in the structure of the RC-bound carotenoid (Fig. 3). Relying on calculations performed by Saito and Tasumi (1983), Lutz et al. proposed that the twisted parts of the carotenoid bound to the reac­ tion centers should be the and/or regions. These studies have been extended to the neurosporene conformation in reaction centers from Rb. sphaeroides, strain G1C, and

similar results were obtained (Koyama et al., 1988). In antenna complexes, it has been shown by RR spectroscopy that the bound carotenoid mol­ ecules are generally in all trans configuration (Ro­ bert, 1983; Robert and Lutz, 1985), although these molecules might be slightly distorted (Iwata et al., 1985).

Resonance Raman spectroscopy VI. Resonance Raman Spectroscopy as a Probe for Intermolecular Interactions

It is possible to determine, by RR spectroscopy, the strength of the interactions assumed by the conjugated carbonyl groups of the (bacterio)chlorin pigments, as well as the number of external ligands on their central Mg atom, when these molecules are embedded in a protein (Lutz, 1984; Lutz and Robert, 1988). This unique property of Raman spectroscopy has been extensively used in many different types of photosynthetic proteins. Three examples of such studies will be detailed in this section, so as to illustrate the diversity of results which can be obtained in this way.

A. The Binding Sites of the Primary Electron Acceptors in Different Photosystems In reaction centers from purple photosynthetic bacteria, the primary electron acceptor is the BPheo molecule mainly surrounded by the L su­ bunit of the protein (Kirmaier and Holten, 1987). The electronic transition of this pigment, at low temperature, is located at 545 nm, whilst that of the inactive (or accessory) BPheo is located near 535 nm. Because of this small difference, selective Raman excitation of each of these pig­ ments could be performed. In Rb. sphaeroides reaction centers, the stretching frequencies of the keto carbonyl groups of the acceptor and access­ respectively ory BPheo are 1678 and (Lutz, 1980, Robert, 1990). The 2-acetyl of the accessory BPheo is thus free, whilst that of the acceptor BPheo is involved in a ca. 4 kCal/M intermolecular interaction, likely with the Glu L 104 residue (Michel et al., 1986). These interac­ tions are interspecifically conserved in purple bac­ teria (Zhou et al., 1987, 1989; Zhou, 1989). Michel and Deisenhofer (1988) proposed the structures of the purple bacterial reaction centers and PS II to be related, and according to this model, this particular Glu L 104 residue is aligned with the Glu D1 131 in the D1 subunit of PS II. In the latter photosystem, the primary acceptor is a pheophytin, and the Soret electronic transi­ tion is expected to be slightly blue-shifted relative to that of the various Chl molecules present in this type of preparation. Exciting D1D2 particles

169

(i.e. isolated reaction centers of PS II) at 406 or 413 nm indeed yielded Pheo contributions in the carbonyl stretching region of the RR spectra, located at 1680 and near (Moënne-Loccoz et al., 1989). Selective reduction of the ac­ ceptor Pheo molecule allowed the assignment of the band to the keto carbonyl group of this molecule. The strength of the interaction in which this group is involved is thus nearly iden­ tical to that observed in bacterial reaction centers (Moënne-Loccoz et al., 1989). It was thus con­ cluded that the acceptor Pheo molecule in PS II particles interacts with the D1 131 Glu amino acid, and the geometry of this interaction is con­ served between reaction centers from purple bac­ teria and PS II. In Photosystem I, the study of the specific inter­ actions assumed by the primary electron acceptor may prove difficult, as the latter is a Chl a, and thus is not chemically different from the bulk pigments. From an extremely weak sequence homology, Robert and Möenne-Loccoz (1989, 1990) predicted that these interactions should be similar to those observed in PS II and bacterial reaction centers. Recently, Feiler et al. (1994a) studied the interactions assumed by the Chl a molecule which is the primary electron acceptor in the PSI-related reaction centers of C. limicola. The stretching mode of its keto carbonyl group is located at Relative to the frequency of this group when free from interactions corresponds to downshift of ca. . It was thus concluded that this group is involved in intermolecular interactions, the strength of which is very close to that mea­ sured in PS II and reaction centers from purple bacteria. These authors thus proposed that the binding site of the primary acceptors in all photo systems share common features, possibly inher­ ited from a common ancestor (Feiler et al., 1994a).

B. The Molecular Structure of the Primary Electron Donor in Bacterial Reaction Centers In reaction centers from Rps. viridis, the primary electron donor consists of two BChl molecules in van der Waals contact at the level of the ring I, which bears the 2-acetyl carbonyl substituent

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(Deisenhofer et al., 1984). It was initially pro­ posed that the Mg atoms of these two molecules would each interact with the imidazole sidechain of neighboring histidine residues, and also with the 2-acetyl carbonyl group of the neighboring BChl molecule (Zinth et al., 1985). Selective ob­ servation of the contributions of the primary elec­ tron donor in protein could be obtained, under resonance conditions at 363.8 nm, by difference techniques. In these spectra, the methine bridge stretching band was located at It was thus concluded that both Mg atoms of the BChl constituting P were binding a single axial ligand. The stretching modes of the 2-acetyl carbonyl groups indicated that one of these group was in­ termolecularly bound, whilst the other one was free from interactions. A picture of the primary electron donor was thus drawn, different from the interpretation of the crystallographers (Rob­ ert and Lutz, 1986), in which the central Mg of each of the two BChl molecules was interacting with the neighboring histidine residues only. Upon the refinement of the structures derived from X-ray crystallography, it appeared that the structure of P was that deduced from the RR spectroscopy studies (Deisenhofer and Michel 1989). It is worth noting that, whereas most often RR spectroscopy was used for discarding struc­ tural models involving direct (B)Chl-(B)Chl in­ teractions, it was also extensively used for demon­ strating this type of interaction between BChl c molecules in chlorosomes from green photosyn­ thetic bacteria (Lutz and van Brakel, 1988; Hilde­ brandt et al., 1991; Feiler et al., 1994d).

C. The Amino Acids Interacting with the Long-Wavelength-Absorbing BChl Pair in Light-Harvesting Complexes from Purple Bacteria Resonance Raman spectroscopy, when used in conjugation with site-selected mutagenesis, is a promising strategy for determining which amino acids are interacting with (bacterio)chlorophyll(s) in a proteic binding site, especially in the absence of structural results derived from crystallography. In RR spectra from the core antenna of purple bacteria, two bands are present at 1641 and in the carbonyl stretching frequency

range. The former was attributed to both the 2­ acetyl carbonyl stretching modes of the two BChl molecules present in these complexes, and the latter to both their two 9-keto carbonyl stretching modes (Robert and Lutz, 1985). In site-selected mutants from Rb. sphaeroides in which the tryp­ tophane 43 of the subunit was replaced by a phenylalanine, the intensity of the band decreased by a factor of two, whilst that of the increased (Fig. 4) (Olson et al., 1994). This experiment shows that the Trp governs the H-bonding state of one of the 2-acetyl carbonyl groups in these complexes. When this residue was further mutated to a tyrosine, the

171

Resonance Raman spectroscopy

thus the function of these light-harvesting com­ plexes (Fowler et al., 1994). VII. Time Resolved Resonance Raman Studies

band at

in wild-type complexes splits (Fig.

into two components at 1633 and 4). As

is a frequency that has been observed for 2-acetyl carbonyl groups of BChl molecules interacting with the phenol sidechain of a tyrosine (Wachtveitl et al., 1993), this result Trp is located led to the conclusion that the in the binding site of one of the BChl interacting directly with its 2-acetyl carbonyl (Fig. 5). Similar experiments, performed on peripheral light-harvesting complexes from Rb. sphaeroides, showed that the tyrosines and were governing the H-bonding state of each of the two 2-acetyl carbonyl groups of the 850 nm-absorbing pair of BChl molecule. It was further suggested that these H-bonds finely tune the absorption, and

As the lifetime of the Raman process is extremely short s or shorter), Raman experiments may be performed with time resolution as short as the picosecond time scale. In photosynthesis, however, although much is anticipated from such experiments, only a few time-resolved resonance Raman experiments have been reported. This is due to two different types of technical difficulty, (i) (B)Chl Raman signals are weak, and care must be taken for limiting the instantaneous irradiance on the (B)Chl-containing sample so as not to de­ stroy them, and (ii) the cation and/or anion and/or triplet states of (B)Chl pigments which are formed during or after the first steps of the elec­ tron transfer possess even smaller resonance Raman cross-sections than the neutral pigments. Only FT-Raman techniques, with excitation at 1064 nm, have yielded selective contributions of the cation state of the primary electron donor in RCs of purple bacteria (Mattioli et al., 1991), and of the green bacterium C. limicola (Feiler et al., 1994d). Most time-resolved studies on (B)Chl pigments have concerned the reaction centers of purple bacteria. To record resonance Raman spectra of transient states, pulsed lasers are often required. However, if one transient state has a lifetime longer than all others, then, when the number of photons per reaction center per second during the experiment is higher than the inverse of the lifetime of this transient state, it is possible to populate it sizeably, while the other transient states will be just populated according to the ratio between their lifetime and the lifetime of the longer excited state. By adjusting the intensity of the laser beam used for analysing the Raman signal, spectra of Rb. sphaeroides R 26 RCs con­ taining small and large amounts of the transient state were recorded using Soret excitation condition (Robert and Lutz, 1988). The transfer results in a local of an electron from P to conformational change in the binding site of the accessory BChl located between P and the access­

172

ory BPheo and the 9-keto carbonyl group of this pigment becomes involved in a stronger H-bond with its environment than when the reaction cen­ state (Robert and ters are in their resting Lutz, 1988). It was, moreover, shown by chemi­ cally oxidizing P, that this change in the H-bond pattern of this group is directly correlated to the Recent refinements of the Rps. formation of viridis structure indicate that a water molecule is present near this keto carbonyl group, being Hbonded on one side to the imidazole ring of the histidine involved in liganding the central Mg ion of one of the two BChls constituting P (Deisen­ hofer and Michel, 1989) and on the other at a reasonable distance for interacting with the keto molecule. A small carbonyl group of the motion of this water molecule during P oxidation could modify its interactions with this carbonyl group. Recent molecular dynamics simulations indicate that, indeed, the distance between this water molecule and the 9-keto carbonyl of the accessory BChl nearby is sensitive to the positive charge borne by P (M. Marchi, unpublished data). Three groups have recorded RR spectra of re­ action centers from purple bacteria in their transi­ ent state, by making use of nanosecondor picosecond-pulsed excitations (Mattioli et al., 1989; Atkinson et al. 1990; Koyama et al., 1992). RR spectra (excited at or near the maximum of the Soret transition of the neutral bacteriochlorin molecules) obtained by these three groups are quite similar to each other. The main change ob­ served in these spectra is a net decrease of the neutral BPheo contributions. In addition to the disappearance of neutral BPheo contributions, the above-mentioned shift of a carbonyl vibrator from a BChl molecule could be present in these spectra, but this latter fact could not up to now be more clearly confirmed (Mattioli et al., 1989). Picosecond time-resolved experiments have been performed on both chromatophores from purple bacteria and chloroplasts from higher plants, under conditions of excitation favoring the contributions of the excited states of carotenoid molecules (Hayashi et al., 1991; Hashimoto and Koyama, 1990; Kuki et al., 1990). There seems to be a general consensus that the resonance pro­ perties of these molecules in their excited states reveal the existence of their long-postulated

Bruno Robert state, which has been proposed to be involved in the carotenoid to chlorophyll energy transfer. However, the effect of the proteic environment on the structure of this excited states is far from being clear. For example, the stretching mode in the state of spheroidene has been in Rb. sphaero­ reported to shift down by ides chromatophores, relative to isolated sphero­ idene (Kuki et al., 1990), while the same mode of the same state of spirilloxanthin has been ob­ served, in chromatophores of Chromatium vinosum and in organic solvents, completely unshifted (Hayashi et al., 1991). Resonance Raman contributions arising from the triplet state of carotenoid molecules have also been observed in vivo, in chromatophores of Chromatium vinosum, using an apparatus with picosecond time resolution (Hayashi et al. 1991), and in the RC from Rb. sphaeroides, at microse­ cond resolution (Lutz et al., 1983). In the latter case, it was thus concluded that no cis–trans iso­ merisation occurs during the ground to triplet state transition. Furthermore, the presence of a very intense band in these spectra indi­ cates that the molecule also retains the twisted conformation it possesses in its ground-state. Ex­ periments conducted with a continuous pumpprobe excitation at 530 nm, have revealed the presence of a very active low frequency mode near in these spectra, which has not yet been assigned (Robert et al., 1985). VIII. Resonance Raman Spectroscopy as a Probe for Studying the Nature of Electronic Transitions While the position of the different resonance Raman bands yields information concerning the ground electronic state of the molecule studied, their intensities may provide information on the nature of the excited state used to induce the resonance phenomenon. During the photosyn­ thetic process, some very important steps of the energy transduction (namely light-harvesting and primary charge separation) involve excited states of (bacterio)chlorophylls. A precise description of the structure of these excited states, which often concern (B)Chl dimers, is needed for our understanding of the physicochemistry of these reactions. Until now only very few groups have

Resonance Raman spectroscopy attempted to get this type of information from RR spectroscopy, mainly because the excited states involved in the energy conversion are the lowest (B)Chl singlet states, the strong fluor­ escence of which has for long precluded any RR measurement. Two groups have reported RR spectra of the primary electron donor in RC from purple bacteria, using near infra-red excitations provided by a Titanium-Sapphire continuous laser (Shreve et al., 1991; Palaniappan et al., 1992). Shreve et al. (1991) extracted the Raman contributions from the intense fluorescence back­ ground using shifted excitation difference. Ac­ cording to this technique, the Raman signal and the fluorescence background are recorded at two excitation wavelengths differing by a few waven­ umbers. In the computed difference between these two excitation only Raman contributions are expected to shift according to the excitation used. Palaniappan et al. (1992) designed an ex­ periment at low temperature in which RCs are mixed with ethylene glycol to decrease the level of the light scattered by the sample. Fluorescence backgrounds were subtracted after having been fitted by a polynomial curve. Results obtained by these teams are not fully compatible. Spectra obtained by Shreve et al. (1991) are dominated by a small number of rela­ tively intense low-frequency modes, located be­ and three bands in the tween 38 and medium frequency range at 685, 730 and These features depend on the precise position of the excitation, and low-frequency modes typical from the primary electron donor were assigned. Spectra reported by Palaniappan et al. (1992) are composed of a very large number of bands of similar intensities from ca. and few bands only appear at the to same frequencies as those reported by Shreve et al. (1991). They do however exhibit a common feature, in the sense that they contain many intense bands in the low frequency region (30– which appear to be typical of the pri­ mary electron donor contributions. However, considering how much these two sets of results differ from each other, one may question whether the few similarities they exhibit have any signifi­ cance. It would of course be of enormous interest, once the origin of the discrepancies between these

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results have been understood, to know whether or not the modes observed by these teams, which must be coupled to the P to transition, are transition. Low indeed coupled with the frequency modes have been up to now poorly studied for BChl molecules. Careful comparison with spectra obtained from monomer and dimer model systems would be of great help in interpre­ ting these spectra. It has for example been re­ ported that, at some excitation conditions, the low frequency modes may be quite intense even for monomeric BChl (Lutz, 1979), and some of the frequencies observed are very near to those reported in the work of Shreve et al. (1990). In the absence of such work on model system, any conclusion has to be drawn with the most extreme care. However, it is clear that this type of experi­ ments have opened a whole new field of appli­ cation for RR spectroscopy in photosynthesis, and that they might give a unique opportunity to understand, in the long run, the physicochemistry of the charge separation mechanism. IX. Perspectives From this short overview of the different pos­ sibilities of resonance Raman spectroscopy, it is clear from the examples chosen that most of the work performed has concerned bacterial photo­ synthesis, although it is relatively more difficult to record RR spectra of BChl than those of Chl molecules. In this particular field of biology, RR spectroscopy is now a mature technique. Photo­ synthetic proteins from higher plants are more complex, and it is consequently more difficult to characterize the contributions of a selected pig­ ment in their RR spectra. However, it is most likely that the forthcoming years should see an increasing number of contributions from reson­ ance Raman in this particular field of photosynth­ esis. It is also clear that the full potential of reson­ ance Raman has still not been explored, in particular with regard to the information which can be obtained by time-resolved resonance Raman and/or on the excited electronic states of (B)Chl molecules. On those two latter subjects, RR spectroscopy should develop from the pre­ liminary, but particularly exciting data which have already been reported.

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Acknowledgements The author thanks Delphine Albouy, James N. Sturgis, Tony A. Mattioli and Diane Spiedel for their skillful help and their many suggestions dur­ ing the writing of this manuscript. References Atkinson GH, Hayashi H, Tasumi M and Kolaczkowski S (1990) Picosecond resonance Raman spectroscopy of Rho­ dobacter sphaeroides reaction centers. In: Michel-Beyerle ME (ed) Reaction Centers of Photosynthetic Bacteria, pp 140–146. Springer-Verlag, Berlin Boldt NJ, Donohoe RI, Birge RR and Bocian DF (1987) Chlorophyll model compounds: Effects of low symmetry on the resonance Raman spectra and normal mode descrip­ tions of Nickel(II) porphyrins. J Am Chem Soc 109: 2284– 2298 Braumann T, Vasmel H, Grimme LH and Amesz J (1986) Pigment composition of the photosynthetic membrane and reaction centers of the green bacterium Prosthecochloris aestuarii. Biochim Biophys Acta 848: 83–91 Carey PR (1982) Biochemical Applications of Raman and Resonance Raman Spectroscopies. Academic Press, New York. Cotton TM and van Duyne RP (1981) Characterization of bacteriochlorophyll interactions in vitro by resonance Raman spectroscopy. J Am Chem Soc 103: 6020–6026 Deisenhofer J and Michel H (1989) The photosynthetic reac­ tion center from the purple bacterium Rhodopseudomonas viridis. EMBO J. 8: 2149–2169 Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1984) X-ray structure analysis of a membrane protein complex: electron density map at 3 Å resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis. J Mol Biol 180: 385–398 Donohoe RI, Frank HA and Bocian DF (1988) Resonance Raman spectra and normal mode descriptions of a bacteri­ ochlorophyll a model complex. Photochem Photobiol. 48 531–537 Feiler U, Mattioli TA, Katheder I, Scheer H, Lutz M and Robert B (1994a) Effects of vinyl substitutions on reson­ ance Raman spectra of (bacterio)chlorophylls. J. Raman Spectrosc 25: 365–370 Feiler U, Albouy D, Pourcet C, Mattioli TA, Lutz M and Robert B (1994b) Structure and binding site of the primary electron acceptor in the reaction center of Chlorobium. Biochemistry 33: 7594–7599 Feiler U, Albouy D, Lutz M and Robert B (1994c) Pigment interactions in chlorosomes of various green bacteria: a resonance Raman study. Photosynth Res 41:175–180 Feiler U, Albouy D, Mattioli TA and Robert B (1994d) Res­ onance Raman studies of the primary electron donor in the reaction centre of Chlorobium limicola. Biochemistry (submitted) Fowler GJS, Sockalingum GD, Robert B and Hunter CN (1994) Blue shifts in bacteriochlorophyll absorbance corre-

Bruno Robert late with changed hydrogen bonding patterns in light-harvesting 2 mutants of Rhodobacter sphaeroides with alter­ ations at Tyr 44 and 45 Biochem J 299: 695–700 Fujiwara M and Tasumi M (1986) Resonance Raman and infrared studies on axial coordination to chlorophylls a and b in vitro. J Phys Chem 90: 250–255 Fujiwara M, Hayashi H, and Tasumi M (1988) Low-frequency vibrational spectra of chlorophylls a and b in solution: effects of axial ligation. Croatica Chem Acta 61: 435–446 Hashimoto H and Koyama Y (1990) The state of a carot­ enoid bound to spinach chloroplast as revealed by picose­ cond transient Raman spectroscopy. Biochim Biophys Acta 1017: 181–186 Hayashi H, Noguchi T, Tasumi M and Atkinson GH (1991) Vibrational spectroscopy of excited electronic states in car­ otenoids in vivo. Biophys J 60: 252–260 Hildebrandt P, Griebenow K, Holzwarth A and Schaffner K (1991) Resonance Raman spectroscopic evidence for the identity of the bacteriochlorophyll c organization in protein-free and protein containing chlorosomes from Chloro­ flexus aurantiacus. Z Naturforsch 46c: 228–232 Iwata K, Hayashi H and Tasumi M (1985) Resonance Raman studies of the conformations of all-trans carotenoids in light-harvesting systems of photosynthetic bacteria. Bi­ ochim Biophys Acta 810: 269–273 Jones M, Dawson MH, Mattioli TA, Hunter CN and Robert B (1994) Mutations at the M 210 Tyr level in bacterial reaction centers do not affect charge localization on the primary donor. FEBS Lett 339:18–24 Kirmaier C and Holten D (1987) Primary photochemistry of reaction centers from the photosynthetic purple bacteria. Photosynth Res 13: 225–260 Koyama Y, Takii T, Saiki K, Tsukida K and Yamashita KJ (1982) Configuration of the carotenoid in the reaction cen­ ters of photosynthetic bacteria. Comparison of the reson­ ance Raman spectrum of the reaction centers of Rhodop­ seudomas sphaeroides G1C with those of cis–trans isomers from Biochim Biophys Acta 680:109–118 Koyama Y, Takii T, Saiki K and Tsukida K (1983) Configur­ ation of the carotenoid in the reaction centers of photosyn­ thetic bacteria. 2) Comparison of the resonance Raman lines of the reaction centers with those of the 14 different Photobiochem Photobi­ cis–trans isomers of ophys 5:139–150 Koyama Y, Umemoto Y and Akamatsu A (1986) Raman spectra of chlorophyll forms. J Molecular Struct 146: 273– 287 Koyama Y, Takatsuka I, Nakata M and Tasumi, M (1988) Raman and infra-red spectra of the all-trans, 7-cis, 9-cis, key bands distin­ 13-cis and 15-cis isomers of guishing stretched or terminal bent configurations from central-bent configurations. J Raman Spectrosc 19: 37–49 Kuki M, Hashimoto H and Koyama Y (1990) The state of a carotenoid bound to the chromatophore membrane of Rhodobacter sphaeroides 2.4.1. as revealed by transient resonance Raman spectroscopy. Chem Phys Lett 165: 417– 422 Lutz M, (1972) Resonance Raman spectroscopy of plant pig­ ments in solution and included in chloroplast layers. Compt Rend Acad Sci Ser. B, 275: 97–504

Resonance Raman spectroscopy Lutz M (1974) Resonance Raman spectra of chlorophyll in solution. J. Raman Spectrosc 2: 497–516 Lutz M (1977) Antenna chlorophyll in photosynthetic mem­ branes: a study by resonance Raman spectroscopy. Bi­ ochim Biophys Acta 460: 408–430 Lutz M (1979) Application de la diffusion Raman de réson­ ance aux pigments chlorophylliens. Thesis, Université Pi­ erre et Marie Curie, Paris Lutz M. (1980) Resonance Raman studies of the bacterial photosynthetic reaction center. In: Murphy WF (ed) Proc. VIIth Intern. Conf on Raman Spectroscopy, pp 520–521. North Holland, Amsterdam Lutz M (1984) Resonance Raman Studies in Photosynthesis. In: Clark RJH and Hester RE (eds) Advances in IR and Raman Spectroscopy. Vol 11, pp 21 1–300. John Wiley and Sons, New York Lutz M and Kléo J (1974) Diffusion Raman de resonance de la chlorophylle d. Compt Rend Acad Sci Paris 279:1413– 1416 Lutz M and Mäntele W (1991) Vibrational spectroscopy of chlorophylls. In: Scheer H (ed) The Chlorophylls, pp 855– 902. CRC press, Boca Raton, Florida Lutz M and Robert B (1988) Chlorophylls and the photosyn­ thetic membrane. In: Spiro TG (ed) Biological Appli­ cations of Raman Spectroscopy. Vol 3, pp 47–411. John Wiley and Sons, New York Lutz M and van Brakel G (1988) Ground-state molecular interactions of bacteriochlorophyll c in chlorosomes of green bacteria and in model system: a resonance Raman study. In: Olson JM, Ormerod JG, Amesz J, Stackebrandt E and Trüper HG (eds) Green Photosynthetic Bacteria, pp 23–34, Plenum Press, New York and London Lutz M, Kléo J, Gilet R, Henry M, Plus R and Leicknam JP (1975) Vibrational spectra of chlorophyll a and b labelled with and In: Klein ER and Klein PD (eds) Proc 2nd International Conference on Stable Isotopes. OakBrook, Ill, pp 462–468. US Dept of Commerce, Spring­ field, Va. Lutz M, K1éo J and Reiss-Husson F (1976) Resonance Raman scattering of bacteriochlorophyll, bacteriopheophytin and spheroidene in reaction centers of Rhodopseudomonas spheroides. Biochem Biophys Res Comm 69: 711–717 Lutz M, Chinsky L and Turpin PY (1983) Triplet states of carotenoid bound to the reaction centers of photosynthetic bacteria. Time resolved resonance Raman spectroscopy, Photochem Photobiol 36: 503–513 Lutz M, Szponarski W, Berger G, Robert B and Neumann J­ M (1987) The stereoisomerism of bacterial, reaction centerbound carotenoids revisited: an electronic absorption, res­ onance Raman and 1H-NMR study. Biochim Biophys Acta 894: 423–433 Mäntele WG, Wollenweber AM, Nabedryk E and Breton J (1988) Infrared spectroelectrochemistry of bacteriochloro­ phylls and bacteriopheophytins: implications for the bind­ ing of the pigments in the reaction center from photosyn­ thetic bacteria. Proc Natl Acad Sci USA 85: 8468–8472 Mattioli TA, Robert B and Lutz M (1989) Time-resolved resonance Raman spectroscopy of the in bacterial reaction centers. In: Bertoluzza A, Fagnano C

175 and Monti P (eds) Spectroscopy of Biological Molecules. State of the Art, pp 303–304) Esculapio, Bologna Mattioli TA, Hoffmann A, Robert B, Schrader B and Lutz M (1991) Primary donor structure and interactions in bacterial reaction centers from near-infrared Fourier-transform res­ onance Raman spectroscopy. Biochemistry 30: 4648–4654 Mattioli T, Sockalingum D, Lutz M, Robert B (1992) Low temperature Fourier-transform Raman studies on bacterial reaction centers. In: Murata N (ed) Research in Photo­ synthesis, Vol. 1, pp 403–408. Kluwer Academic Pub­ lishers, Dordrecht Mattioli TA, Hoffmann A, Sockalingum DO, Schrader B, Robert B, and Lutz M (1993) Application of near IR Four­ ier transform resonance Raman spectroscopy to the study of photosynthetic proteins. Spectrochim Acta 49A: 785– 799 Mattioli TA, Williams JA, Allen W and Robert B (1994) Changes in primary donor hydrogen bonding interactions in mutant reaction centers from Rhodobacter sphaeroides. Identifications of the Vibrational frequencies of all the con­ jugated carbonyl groups Biochemistry 33: 1636–1643 Michel H and Deisenhofer J (1988) Relevance of the photo­ synthetic reaction center from purple bacteria to the struc­ ture of Photosystem II. Biochemistry 27: 1–7 Michel H, Epp O and Deisenhofer J (1986) Pigment-protein interactions in the photosynthetic reaction center from Rhodopseudomonas viridis. EMBO J 5: 2445–2451 Möenne-Loccoz P, Robert B and Lutz M (1989) A resonance Raman characterization of the primary electron acceptor in Photosystem II. Biochemistry 28: 3641–3645 Möenne-Loccoz P, Robert B, Ikegami I and Lutz M (1990) Structure of the primary electron donor in Photosystem I: a resonance Raman study. Biochemistry 29: 4740–4746 Olsen JD, Sockalingum GD, Robert B and Hunter CN (1994) Modification of a hydrogen bond between a bound chromo­ phore and the subunit of the light-harvesting I antenna of Rhodobacter sphaeroides. Proc Natl Acad Sci U.S.A. 91: 7124–7128 Palaniappan V, Aldema MA, Frank HA and Bocian DF (1992) Qy excitation resonance Raman scattering from the special pair in Rhodobacter sphaeroides reaction centers. Implication for primary charge separation. Biochemistry 31: 11050–11058 Palaniappan V, Martin PC, Chinwat V, Frank HA and Bocian DF (1993) Comprehensive resonance Raman study of photo synthetic reaction centers from Rhodobacter sphaeroides. Implication for pigment structure and pigment-protein interactions. J Am Chem Soc 115: 12035–12049 Robert B (1983) Etude par diffusion Raman de resonance de complexes proteine-pigment antennes des Rhodospirillales. Thesis, Université Pierre et Marie Curie, Paris Robert B (1990) Resonance Raman studies of bacterial reac­ tion centers. Biochim Biophys Acta 1017: 99–111 Robert B and Lutz M (1985) Structures of antenna complexes of several Rhodospirillales from their resonance Raman spectra. Biochim Biophys Acta 807: 10–23 Robert B and Lutz M (1986) Structure of the primary donor of Rhodopseudomonas sphaeroides: difference resonance Raman spectroscopy of reaction centers. Biochemistry 25: 2303–2309

176 Robert B and Lutz M (1988) Proteic events following charge separation in the bacterial reaction center: resonance Raman spectroscopy. Biochemistry 27: 5108–5114 Robert B and Möenne-Loccoz P (1989) Un site possible pour l’accepteur primaire d’électrons du photosystème I. Compt Rend Acad. Sci. Paris Série III, 308: 407–409 Robert B and Möenne-Loccoz P (1990) Is there a proteic substructure common to all photosynthetic reaction cen­ ters? In: Baltcheffski M (ed) Current Research in Photo­ synthesis, Vol 1, pp 65–68. Kluwer, Dordrecht Robert B, Szponarski W and Lutz M (1985) Resonance Raman studies of transient states in bacterial reaction cen­ ters. Springer Proc Phys 4: 220–224 Saito S. and Tasumi M (1983) Normal-coordinate analysis of carotene isomers and assignments of the Raman and infrared bands. J. Raman Spectrosc 14: 310–321 Saito S, Tasumi M and Eugster CH (1983) Resonance Raman spectra of all-trans and 15-cis isomers of carotene in the solid state and in solution, measurements with various laser lines from ultraviolet to red. J Raman Spectrosc 14: 299–309 Shreve AP, Cherepy NJ, Franzen S, Boxer SG and Mathies RA (1991) Rapid-flow resonance Raman spectroscopy of bacterial photosynthetic centers. Proc Natl Acad Sci USA 88:11207–11211 Shuvalov VA, Amesz J and Duysens LNM (1986) Picosecond spectroscopy of isolated membranes of the photosynthetic green sulfur bacterium Prosthecochloris aestuarii upon se­ lective excitation of the primary donor. Biochim Biophys Acta 851: 1–5 Sturgis JN, Cogdell RJ, Jirsakova W, Reiss-Husson F and Robert B (1994) The structure and properties of the bac­ teriochlorophyll binding site in peripheral light-harvesting complexes from purple bacteria, Biochemistry (submitted) Tasumi M and Fujiwara M (1987) Vibrational spectra of

Bruno Robert chlorophylls, in: Clark RJH and Hester RE (eds) Advances in Spectroscopy. Vol. 14, Ch 6, pp 407–429. Wiley, London Van Bochove AC, Swarthoff T, Kingma H, Hof RM, van Grondelle R, Duysens LNM and Amesz J (1984) A study of the primary charge separation in green bacteria by means of flash spectroscopy. Biochim Biophys Acta 764: 343–346 Van de Meent EJ, Kobayashi M, Erkelens C, van Veelen PA, Otte SCM, Inoue K, Watanabe T and Amesz J (1992) The nature of the primary electron acceptor in green sulfur bacteria. Biochim Biophys Acta 1102: 371–378 Wachtweitl J, Farchaus JW, Dos R, Lutz M, Robert B and Mattioli TA (1993) Structure, spectroscopic and redox pro­ perties of Rhodobacter sphaeroides reaction centers bearing point mutations near the primary electron donor. Biochem­ istry 32: 12875–12886 Zadorozhnyi BA, and Ishchenko IK (1965) Hydrogen bond energies and shifts of the stretching vibration bands of groups. Opt Spectrosc (Engl. transl. 19: 306–308 Zhou Q (1989) Relations structure-fonction au sein du centre réactionnel bactérien: études par spectrométrie Raman de resonance, Thesis, Université Pierre et Marie Curie, Paris Zhou Q, Robert B and Lutz M (1987) Intergeneric structural variability of the primary donor of photosynthetic bacteria: resonance Raman spectroscopy of reaction centers from two Rhodospirillum and Rhodobacter species. Biochim Bi­ ophys Acta 890: 368–376 Zhou Q, Robert B and Lutz M (1989) Protein-prosthetic group interactions in bacterial reaction centers: resonance Raman spectroscopy of the reaction center of Rhodop­ seudomonas viridis. Biochim Biophys Acta 977: 10–18 Zinth W, Knapp EW, Fischer SF, Kaiser W, Deisenhofer J and Michel H (1985) Correlation of structural and spectro­ scopic properties of a photosynthetic reaction center. Chem Phys Lett 119: 1–4

Chapter 11 Stark Spectroscopy of Photosynthetic Systems

Steven G. Boxer Department of Chemistry, Stanford University, Stanford, CA 94305–5080, USA

Summary I. Introduction II. Methods A. Analytical Methods B. Sample Preparation and Dielectric Breakdown C. Light Sources, Detectors and Power Supplies III. Limitations and Conceptual Issues A. Experimental Limitations B. Analytical Limitations C. Local Field Correction IV. Examples of Recent Results for Photosynthetic Systems A. The Special Pair B. Vibrational Stark Spectroscopy C. Unidirectional Electron Transfer in the RC Acknowledgements References

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Summary The effects of applied electric fields on the absorption or emission spectrum of a molecule is known as Stark Spectroscopy. This method probes the movement of charge associated with optical excitation, and is thus sensitive to features which are important for chromophore systems that carry out charge separ­ ation, such as photosynthetic systems. This chapter updates a review written about 2 years ago which also described electric field effects on reaction dynamics, notably electron transfer reactions (Boxer, 1993). Several recent extensions of the method such as higher order Stark Spectroscopy and vibrational Stark Spectroscopy are discussed along with examples. Experimental methods and methods of analyzing spectra are discussed in great detail, along with a discussion of experimental and conceptual issues which complicate the analysis of Stark spectra. Abbreviations: HOSS – Higher order Stark Spectroscopy; LDAO – Lauryldimethylamine-N-oxide; RC – Reaction center; VSE – Vibrational Stark Spectroscopy I.

Introduction

The effect of an applied electric field on an absorption or emission spectrum is known as the Stark effect. This term is used interchangeably with electroabsorption or electrochromism, both Correspondence: Fax: 1-415-7234817; E-mail: [email protected]

of which have been widely used in the literature. The basic approach goes back to early in this century when electric fields were first used to perturb the energy levels of atoms in the gas phase. Extensive work was published on simple molecules, often as guests in host crystals, primar­ ily as a test of the predictions of molecular orbital calculations (Hochstrasser, 1973). Recent developments in this area were reviewed by me two 177

J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 177–189. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

178 years ago with a specific emphasis on photosyn­ thetic systems (Boxer, 1993). That review also described experiments on the use of applied elec­ tric fields to perturb reaction dynamics, notably the rate of electron transfer reactions in photo­ synthetic reaction centers (RCs), measured either directly (Lockhart et al., 1990; Franzen et al., 1990; Franzen and Boxer, 1993) or indirectly via the effect of the electric field on the fluorescence which competes with electron transfer (Lockhart and Boxer, 1988; Lockhart et al., 1988). The em­ phasis in the following is on recent developments of absorption Stark spectroscopy and its appli­ cation to understanding the excited states of pho­ tosynthetic pigments. I have particularly stressed experimental and conceptual complications, as well as the successes. The change in transition frequency due to an externally applied field F, is given by: where and are the change in dipole mo­ ment and polarizability, respectively, between the states involved. The most desirable method for obtaining Stark effect spectra is for uniaxially ori­ ented molecules. If the first term dominates, as is often the case, this interaction gives rise to a linear shift in the absorption as the applied field strength is increased (the so-called linear Stark effect); the second term depends on the square of the applied field (the so-called quadratic Stark effect). In principle, it is possible to obtain infor­ mation on the components of the difference pola­ rizability tensor by varying the orientation of the molecule in the applied electric field. Unfortu­ nately, uniaxial orientation is only rarely achieved in practice for complex molecules, and I am not aware of an example of a biological molecule where the Stark spectrum has been analyzed quantitatively. For membrane proteins, the sample can be uniaxially oriented across a lipid bilayer (no orientation in the plane of the bilayer). It is then possible to create an electric field by varying the ionic strength on the two sides of the bilayer. This gives rise to spectral shifts, known historically as electrochromic shifts, and, at least at a qualitative level, these shifts are very widely used to probe changes in transmembrane potential using voltage sensitive dyes. A somewhat related effect in-

Steven G. Boxer volves creating the field internally, rather than externally. For example, if charge is separated in response to light, as in the RC, the electric field due to these charges causes shifts in both the electronic absorption and vibrational bands of other spectator chromophores within the RC. This internal, transient electric field has a fixed spatial relationship with the spectator chromoph­ ores, so the observed Stark or electrochromic bandshifts can be treated as if the sample were completely oriented. Of course in this case it is not possible to vary the applied electric field strength systematically. Another possibility is that the protein containing a chromophore can be bi­ axially oriented, e.g., the protein is oriented ac­ ross the bilayer, but the orientation does not dis­ tinguish one side of the bilayer from the other. If the absorption spectrum of a chromophore in such a system were very narrow, then the appli­ cation of a transmembrane electric field would split the absorption due to the interaction of the fixed field direction with the two antiparallel pro­ jections of the chromophore difference dipole moments on the membrane normal. In practice, the absorption spectrum of biological chromoph­ ores is invariably inhomogeneously broadened, and the linewidth is typically several hundred which is considerably larger than the inter­ and the applied field action energy between (for reasonable values of either). In this case, the effect of the field is to broaden the absorption spectrum, and, so long as the interaction energy is small relative to the inhomogeneous linewidth, the effect depends quadratically on applied elec­ tric field. In this case the distinction between ef­ and can not be made on the fects due to basis of the field dependence, but must rather be made by analyzing the lineshape of the Stark ef­ fect spectrum. The same situation applies to nonoriented, immobilized samples which are simple to prepare and are the only cases to be discussed further in the next sections.

II. Methods

A. Analytical Methods It is straightforward to show that for an isolated absorption band in a non-oriented, immobilized sample when is smaller than the inhomogene­

Stark spectroscopy

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ous line width, the change in absorption upon application of a electric field, F, is given by (Lip­ tay, 1974; Mathies, 1974):

where depends on the transition polarizability and hyperpolarizability; +

(neglecting the contribution from the cross term of the transi­ tion polarizability and and is the molecular angle between and the transition moment p. is obtained from the second deriva­ tive coefficients of the Stark spectra obtained at different values of the experimental angle be­ tween F and the electric vector of the linearly f is the local polarized light used to probe field correction which is discussed further below. In order to obtain information on the desired electro-optical properties, the coefficients A, B, and C in Eq. (2), the observed change in absorp­ tion is analyzed in terms of contributions from the zeroth, first and second derivatives of the absorption band. We have recently extended the conventional method to include the higher harmonic responses of the absorption to the applied AC field, a tech­ nique we have called higher-order Stark spectros­ copy (HOSS) (Lao et al., 1995a). The field-induced change in absorbance by an externally applied sinusoidal electric field is given by:

Changes

in

absorbance, etc. are recorded using lock-in detection at the 2nd, 4th and 6th­ harmonic frequencies, respectively, of the field modulation frequency The nth-order spectrum depends on the nth power of the applied field, Like the conventional or Stark spectrum, the higher order Stark spectrum can be fit to sums of derivatives of the absorption lineshape. For spectrum, example, in the case of the is fit to the sum of up to the 4th derivative of the absorption lineshape. Addition­ ally, for each nth-order Stark spectrum, the nth

derivative component depends only upon and Thus, if the nth-order Stark spectrum is domi­ nated by the nth derivative of the absorption domi­ spectrum, it is immediately evident that An even simpler diagnostic for nates this case is that the (n + 2)-order Stark spectrum is the second derivative of the nth-order spec­ trum. As seen below this simple diagnostic is quite powerful because it is possible to obtain Stark spectra with good signal-to-noise, whereas it is quite difficult to obtain good quality higher derivatives of the absorption spectrum.

B. Sample Preparation and Dielectric Breakdown The conventional Stark effect depends quad­ ratically on applied field strength (Eq. 2) and the higher order Stark spectrum on higher even powers of the field (Eq. (3)). Because the higher order terms have coefficients that become (usu­ ally) much smaller with term order, it is very desirable to obtain the highest possible applied electric field strength. The ultimate limit is deter­ mined by dielectric breakdown. Several factors determine the field at which dielectric breakdown occurs. (1) Low temperatures are essential for achieving the highest applied field strengths. We typically perform our experiments with samples immersed in liquid nitrogen in a strain-free optical dewar bubbling is minimized by blowing a thin stream of gaseous helium over the surface). In other cases, we have cooled samples by blow­ ing cooled gaseous nitrogen or helium over the sample in an optical dewar, or by contacting the sample with a cold-finger. Heat transport is less efficient in these cases than when the sample is immersed in liquid nitrogen, so it is difficult to obtain the highest fields. Recently, in collabor­ ation with MMR Technologies, Inc. (Mountain View, CA), we have adapted a miniature Joule– Thompson refrigerator for electric field experi­ ments (Stanley and Boxer, 1995). The MMR re­ frigerator is so small that it can easily be trans­ lated during an experiment, e.g., when the sample is degraded by prolonged exposure to a high peak power laser. In this device the sample is in contact with a cold finger which is cooled by the expansion of pure nitrogen through a network of capillaries. The problem with this system, and

180 many others that use refrigerators, is that the samples are often liquids at room temperature, the cold-fingers operate under vacuum, and the sample cell is not vacuum tight The entire MMR refrigerator can be put in a – 20°C or – 80°C freezer until the liquid sample (typically contain­ ing 50–75% glycerol) is very viscous. At that point the refrigerator can be evacuated without causing sample bubbling. Alternatively the same can be very rapidly cooled using methane. Ob­ viously this problem is not present for polymer film samples (e.g. in polyvinylalchohol); how­ ever, we have found that much better results can be obtained with frozen glass samples because the inhomogeneous broadening is typically less, the sample is not dehydrated, and it is possible to work with extremely small quantities by avoiding the sample waste associated with the preparation of films. In some cases it is useful to work at pumped liquid helium temperatures because this produces the narrowest spectral lines. This is achieved using a standard helium cryostat. (2) By working with the thinnest possible sample it is possible to obtain the best glasses and the highest fields. Of course this requires that the sample be very concentrated in order to achieve sufficient optical density. There are various approaches to making thin samples. We typically use precision spacers made of Teflon which are available in thicknesses down to about 10 microns. (3) We have found that the highest fields can be obtained by reducing the concentration of detergents as much as possible. In the case of photosynthetic RCs, we typically work at the absolute minimum detergent concentration compatible with main­ taining good solubility (about 0.01% LDAO). (4) We use a power supply which has a current fuse which quickly cuts off the applied field as soon as current flow is sensed. Dielectric breakdown is a cumulative effect, and the sample can be preser­ ved if breakdown is halted as soon as it begins. This is also advantageous because valuable samples or samples in difficult conditions (e.g. pumped He) are not destroyed. It is also an im­ portant safety precaution. We can routinely ob­ tain applied electric fields (AC modulation) of approximately 1 MV/cm on frozen aqueous glasses immersed in liquid nitrogen for 10–25 mic­ ron cells. This can be achieved even at room temperature in polymer films, and fields as high

Steven G. Boxer as 3 MV/cm have been achieved for very thin polymer films (several micron) using pulsed elec­ tric fields (Lao et al., 1993).

C. Light Sources, Detectors and Power Supplies For most photosynthetic pigments, the absorption of interest occurs in the visible and near-infrared regions of the spectrum. The change in absorp­ tion can be probed using a conventional high pressure xenon or tungsten-halogen lamp whose output is dispersed through a monochromator. Si photodiodes are excellent detectors from about 400 to 1100 nm and have extremely low noise. Because they are only moderately sensitive, rela­ tively high probe light intensities are needed. This can create interesting complications for pigment systems like the RCs which undergo charge separ­ ation with high quantum efficiency. Thus, electric field effects on the kinetics (Lockhart et al., 1990; Franzen et al., 1990; Franzen et al., 1992; Franzen et al., 1993) and steady-state population of intermediates (Franzen and Boxer, 1993) may be combined or may compete with the Stark ef­ fect on absorption (Lao et al., 1995b) or emission (Lockhart et al., 1991) giving rise to lineshapes which bear little relationship with the sum-ofderivatives form of Eq. (2). Si avalanche photodi­ odes have the advantage of integrated amplifi­ cation. Most Si detectors are overcoated to min­ imize degradation by UV light and cannot be used effectively in the near UV. We have found that a photomultiplier with good near-UV sensi­ tivity, wired so that only a few stages of gain are used, offers a reasonable compromise between signal-to-noise and spectral sensitivity. This has recently been used in a detailed study of the Stark effect of tryptophan (Pierce and Boxer, 1995). The infra-red region has not been explored very much thus far. We have used a germanium photo­ diode for Stark effect measurements on weak mixed-valence transitions in the Creutz–Taube ion in the 1–2 micron range (Oh and Boxer, 1990; Oh et al., 1991) and on the 1250 nm absorption in Rb. sphaero­ band which is characteristic of ides RCs (Stocker et al., 1993). Recently, we have performed the first conventional Stark experi­ ments in the 2–4 micron range on simple organic nitriles using an InSb detector (Chattopadhyay

Stark spectroscopy and Boxer, 1995). Vibrational Stark effects (VSE) have only rarely been measured in con­ densed phases; however, this should be a rich area of investigation in the future. These mea­ surements have recently been extended into the mid-IR using a mercury–cadmium–telluride (MCT) detector (Chattopadhyay and Boxer, to be published). A Nernst glower source is ad­ equate for vibrational Stark measurements. In most cases the signal from the detector is processed in a lock-in amplifier. Although any conventional lock-in works well for most cases, recently introduced digital lock-in amplifiers offer many advantages. In particular, these lock-ins have exceptional dynamic reserve so that very small signals can be reliably extracted. This is especially important for HOSS and VSE experi­ ments. The power supply used to generate the applied field is a critical component in the set­ up. A number of suppliers manufacture voltage amplifiers which can be used with a standard waveform generator to produce the desired out­ put. In collaboration with Joe Rolfe in the Stan­ ford Chemistry Department electronics shop we have developed several generations of power supplies which offer many practical advantages (convenient variation of amplitude and modu­ lation frequency, DC offset, fast shut-off if cur­ rent flows, etc.). HOSS experiments are espe­ cially sensitive to harmonic distortion in the power supply output as this can introduce higherharmonic signals which are not related to the desired signals described in Eq. (3). We currently minimize this problem by using a digitally syn­ thesized sine wave from the lockin amplifier which is then amplified. Detailed schematics and power supplies are available through the author. III. Limitations and Conceptual Issues

A. Experimental Limitations The sample thickness is one of the primary sources of uncertainty, as the field strength is obtained by measuring the thickness and the ap­ plied voltage (the latter can be obtained with high precision with a calibrated high voltage probe). There have been suggestions that the Stark spec­ trum itself is a useful method for calibrating the field. Of course this cannot be used as a primary

181 standard because it depends on knowing the field strength. Once the Stark effect has been mea­ sured using an independently calibrated field, then the Stark effect for a particular transition can be used as a secondary standard, so long as there are no other sources of uncertainty. We have found that small sample-to-sample varia­ tions in the linewidth (associated with different amounts of detergent, buffer, sample imperfec­ tions, etc.) can lead to variations in the magnitude of the change in absorption for a given field. Thus, it is hazardous to use the Stark effect itself as a field calibration. Another poor method is to use the sample capacitance to measure thickness. This not only depends on independent infor­ mation about the dielectric properties of the sam­ ple (generally not known very accurately), but it is also a bulk measurement and can give deceptive results for samples with thickness variations. It is, however, very useful to measure the sample capacitance during an experiment as a monitor of sample integrity. For frozen glass samples, the cell thickness can usually be accurately measured at room tempera­ ture, either by measuring interference fringes or by using solutions of known concentration. How­ ever, because the Stark effect is usually measured at low temperature, the absorption spectra of standard solutions may change, the sample may contract, reducing the thickness, and the contrac­ tion may not be homogeneous across the portion of the sample which is probed, especially if thin windows are used to constuct the cell. Under the most favorable conditions, when the sample is thin and freezes homogeneously, interference fringes are observed at low temperature. Al­ though these fringes interfere with the analysis of the absorption spectrum, they are entirely absent in the Stark spectrum which is obtained using lock-in detection at some multiple of the field modulation frequency. The sample absorption, needed for lineshape analysis, can be measured on a separate sample. It is possible to obtain very precise thickness measurements on very thin films which are prepared by spin-coating. We have used a device manufactured by Dektak which is commonly used in the semi-conductor industry for measuring the thickness of thin films. This device works by scratching the film through to the substrate and then passing a stylus across the

182 sample surface and into the scratch to determine the thickness. Using this device, thin films can be measured with an accuracy of about 1000 Å, and it is possible to obtain quantitative information on film flatness over large areas. Some groups have used polymer films pressed between glass plates. This is an especially unsatisfactory method in our experience because there are surface varia­ tions on the film, and it is difficult to avoid air gaps.

B. Analytical Limitations The absorption spectrum from which the deriva­ tives are obtained must have extremely good signal-to-noise, especially in the wings of the absorp­ tion. Because the derivatives are typically much noisier than the Stark effect data, simply using the derivatives to fit the data introduces considerable uncertainty into the analysis. A better approach is to fit the absorption to an arbitrary lineshape, e.g. a sum of Gaussians (with no physical mean­ ing to the individual components), and then ob­ tain the analytical derivatives of this best fit. Al­ though an improvement, the best-fit to the absorption tends to be least accurate in the wings of the absorption. This is not a problem for fitting the absorption; however, it is a problem for fitting the Stark data as the wings of the absorption are important in the derivatives. The best approach we have developed to date is to simultaneously fit the absorption and the Stark data (Middendorf et al., 1993). A fundamental assumption underlying the ap­ plication of Eq. (2) is that the electro-optic par­ and are constant across ameters such as the absorption band. If the Stark spectrum can not be fit to a sum of derivatives, it may be that the electro-optical parameters vary across the in­ homogeneous band width. A case of great rel­ evance to some photosynthetic pigments (e.g. ca­ rotenoids (Gottfried et al., 1991a, b) and the dimeric special pair primary electron donor (Mid­ dendorf et al., 1993)) is when the chromophore but a large When intrinsically has a small such a chromophore is inserted into an ordered environment, e.g. a protein matrix, then the in­ ternal matrix electric field due to the constellation of polar, charged and polarizable groups in the protein can induce a dipole moment in the chrom-

Steven G. Boxer ophore. There will always be small variations in these fields from protein to protein (inhomogene­ ous broadening), and thus a distribution of in­ duced dipole moments. An interesting nonbio­ logical example of this has been studied in detail by V.P. Wild and co-workers who examined the induced dipole moment in a centrosymmetric or­ ganic dye at different wavelengths within the in­ homogeneous absorption by measuring the Stark effect on holes burned at different wavelengths at 1.5 K (Vauthey et al., 1994). They found that in a moderately polar the value of the induced polymer matrix varied by a factor of 3 across the absorption band. Stimulated by this work, we measured the conventional and higher order Stark effect spectra for the same dye in the same matrix (Moore, Bublitz and Boxer, unpublished results). As expected, it was not possible to fit the conventional Stark effect spectrum with any combination of derivatives of the absorption. Al­ though such a failure is not automatically diagnos­ tic that this is physically what is occuring, it is one possibility. The higher-order Stark spectra can provide further insight and constraint on the fitting process. Another problem is that even a simple elec­ tronic transition is almost always accompanied by some vibronic structure, often poorly resolved. Because the intensity of these vibronic features depends on coupling with other states whose electro-optic properties may be different, it is possible that the Stark effect for these features will be intrinsically different from that of the (typically) dominant 0–0 transition. This has not been exam­ ined systematically in any detail. In one case, the carotenoid spheroidene in the B800–850 antenna complex from Rb. sphaeroides, a prominent vib­ ronic progression is observed. The entire progres­ sion could be quite well fit to the second deriva­ tive of the absorption band (Gottfried et al., 1991a,b). Multiple, overlapping electronic absorption bands, sometimes from the same molecular spe­ cies, or, as is the case of RCs, from different chromophores are another difficulty. The obvious approach to this problem is to deconvolve the absorption features. Although this is a standard procedure for the analysis of complex absorption bands, the requirements for the analysis of the Stark spectrum are much more stringent because

Stark spectroscopy the electrooptic properties of the bands may be quite different from each other and the wing of the Stark effect of a transition with a large dipole moment may, for example, swamp the Stark ef­ fect for a nearby absorption with a small dipole moment. One of the most insidious examples arises when there are partially overlapping ab­ sorption features which have very different Stark effects, as in photosystem II RCs. Even in the purest preparations, there is substantial overlap among all the absorption bands of different chro­ region. In an early attempt mophores in the to analyze this spectrum, Lösche et al. (1988) concluded that P680 has a small comparable to or smaller than a monomeric chlorophyll. This might indicate that P680 is not dimeric. We ana­ lyzed the Stark spectrum of PSII RCs, and at­ tempted to fit the data more accurately. Our best fit suggested that for P680 was at least as large or larger than for a monomeric chlorophyll (Steffen, 1994); however, because the band over­ lap was so great, we never felt confident enough to draw any further conclusion. Finally, there is the possibility that the basic Liptay formalism underlying Eq. (2) is inappro­ priate. This may occur when the observed transi­ tion is strongly coupled to or degenerate with a dark state. General treatments have been de­ veloped by Reimers and Hush (1991) and Sacra et al. (1995), the latter for the special case of semi-conductor nanocrystals. To date, there is no evidence that a breakdown of the fundamental assumptions inherent in the Liptay formalism plays a significant role in the Stark spectra of photosynthetic pigments; however, this may well prove to be wrong, especially as the analyses be­ come more refined.

C. Local Field Correction The local field correction accounts for the differ­ ence between the actual field felt at the position of the chromophore whose Stark spectrum is being probed and the precisely known applied electric field. Lack of knowledge of the local field correction is a long-standing limitation in the quantitative comparison between observed Stark effects and theory. The local field correction is discussed in depth in classical texts, such as Böttcher (1973). The local field correction is not

183 to be confused with the matrix electrostatic fields due to the protein. The latter are always present and may be very large in an ordered medium such as a protein, or they can change in time, as in the case of the electric field due the charges formed following light-induced electron transfer. The local field correction accounts for the difference in the field felt at the probe chromophore upon application of the external applied field. In gen­ eral the local field correction is a tensor quantity. Taking the local field correction to be a scalar, The local field correction is not the dielectric constant. Various models for the local field correction, such as the Lorentz model (Böttcher, 1973), give expressions for f in terms of the dielectric constant, but the dependence is rather weak, e.g., for a spherical cavity the Thus, for typical val­ dependence is: ues of the dielectric constant of frozen solutions, f is not much greater than unity. Irrespective of the form chosen for the cavity, the value of f is greater than 1. Thus, the internal field actually felt at the position of the chromophores is greater than the externally applied field, though not by very much for typical dielectrics at low tempera­ ture. Expressions for the local field correction be­ come still more complex for a chromophore in­ side a protein which is itself a dilute solute in a frozen glass (Lösche et al., 1988). However, because the great majority of the sample to which the external field is applied is the bulk frozen solvent and this is the same for different chrom­ ophores within a protein complex (e.g. the RC) or among different chromophore-protein com­ plexes embedded in the same frozen solvent, dif­ ferences in the local field correction for different chromophores in different local environments are likely to be small. Thus, although the absolute value of the electro-optic parameters may be sys­ tematically in error (likely by a small amount), the relative values are likely to be reliable. We have taken the approach of reporting electrooptic parameters in terms of the local field correc­ tion as a formality. Finally, the angle between the change in dipole moment and the transition dipole moment used to probe the Stark effect does not depend on the local field correction fac­ tor (see Eq. 2). This angle is often of greater

184 interest than the precise magnitude of the change in dipole moment itself. There are approaches to obtain quantitative information on the local field correction. For ex­ ample, in the RC, the driving force for the recom­ is quite well bination reaction known. Upon application of a large external elec­ tric field, it should be possible to perform the reverse reaction, namely charge separation with­ out photoexcitation, as the energy of the state is tuned below that of ground state By measuring the concentration of at equilibrium in an accurately calibrated external electric field, and comparing this with the equilib­ rium concentration expected given the magnitude dipole moment (whose magnitude of the does not depend to any appreciable extent on the details of the locations of the charges on either radical ion), it should be possible to obtain the dipole in the RC. actual field felt by the We attempted this experiment, and at the highest fields available were unable to detect any appreci­ formation in Rb. sphaeroides RCs able in a PVA film. Based upon what is known about these RCs in the absence of an external field, we could conclude that f in this system could be no greater than 1.3, i.e., if it had been greater, the field external would have produced a measurable concentration of (Franzen and Boxer, 1993). A possibly related effect involving charge separation between P and cytochrome has been reported in Rps. viridis RCs (Alegria et al., 1993). Extensions of this approach to other sys­ tems where the driving force is smaller are in progress. IV. Examples of Recent Results for Photosynthetic Systems As mentioned at the outset, results until late 1992 were summarized in detail in an earlier review (Boxer, 1993) and these will not be repeated. A few recent results are discussed briefly in the following.

A. The Special Pair In the original papers describing the Stark effect for the special pair, it was obvious that for

Steven G. Boxer its transition was larger than for a monomeric BChl, either the B bands in the RC or the pure isolated monomeric chromophores (Lockhart and Boxer, 1987; 1988; Lösche et al., 1987, 1988a,b). The quantitative analysis assumed that only the second derivative contributed to the lineshape, although it was clear that this was not rigorously true. By working at 1.5 K in a frozen glass, where the special pair absorption is narrower and some substructure is evident, it was possible to test the lineshape analysis with much greater precision (Middendorf, 1992). The Stark lineshape at 1.5 K is not well fit by the second derivative of the absorption, rather there appears to be a signifi­ cant contribution from the first derivative and some zeroth derivative. The interpretation of this result, no matter how good the data and the fit, is predicated on the validity of the Liptay formal­ ism, which may break down for this system. As­ suming the validity of the Liptay formalism, we for the special pair is huge compared find that to that of a monomer. The estimated magnitude is little affected by this more refined analy­ of sis, though increases from about 38° to about is several 45°. Although it is still the case that times larger for the dimer than the monomer, the is much larger really striking finding is that for the dimer. This can be interpreted as being a natural consequence of mixing intra-dimer charge-transfer character into the lower exciton state of P (Middendorf et al., 1993). If this model is correct, then it might be expected that as a large field is applied, radically shifting the energies of the CT states, then the Stark lineshape should change. Model calculations demonstrate that this should be observed if the CT states are within about of the exciton states (the precise value is model dependent). We have measured the lineshape in PVA films for fields up to nearly 3 MV/cm and find an excellent fit to the expected quadratic field dependence of the amplitude, with no change in lineshape. Thus, these data suggest that the CT states are several thousand higher (or, in principle, lower) than the exciton states. The value of extracted from the lineshape analysis is even larger than for long polyenes which are known to be highly polarizable. Of course it is not possible to remove the special pair

Stark spectroscopy from the RC while maintaining its structure and study its electro-optic properties in a simpler ma­ trix, as is possible for polyenes, though we have studied the Stark spectra of a number of coval­ ently linked special pair model systems (Midden­ dorf, 1992). If we make the assumption that the of the special pair is intrinsic (gas phase) small, and the observed large for the special pair is induced by interaction between the matrix (protein) electrostatic field in the RC and the large of the special pair, then we can estimate the matrix electric field to be at least several MV/cm. Given the absence of a dependence of the lineshape on applied field, this internal matrix field may be even larger. Furthermore, as dis­ cussed in detail in Middendorf et al. (1993), it is likely that is highly anisotropic, with the dominant component along the long axis of the special pair. If the matrix electric field pointed along this direction, then a much larger value of would be observed. Therefore, we concluded that the matrix field is large and points roughly perpendicular to the long axis of the special pair. This is roughly along the local axis of the RC. Some recent electrostatics calculations support this suggestion (M. Gunner, D. Chandler, per­ sonal communication). Higher-order Stark spectra have been obtained for monomeric BChl and BPheo, the special pair at 77 and 1.5K, the heterodimer mutant ((M)H202L), and for the carotenoid spheroidene both pure in an organic glass and in the B800– 850 antenna complex (Lao et al., 1995a). A key result is shown in Fig. 1 which compares the se­ cond and fourth order Stark spectra of the wildtype homodimer and mutant (M)H202L heterodimer. The absorption spectrum of the heterodimer consists of two features whose total oscil­ lator strength is comparable to that of the homodimer (because the bands are so spread out, it appears to be weaker). In earlier work (Hammes et al., 1990), it was evident that there was a huge Stark effect for the lower energy band compared to that of the homodimer, and this is evident in Fig. 1. However, it proved difficult to analyze the Stark spectrum in terms of contribu­ and because it is difficult to obtain tions of precise information on the absorption lineshape of the transition corresponding to the large Stark

185

effect. As seen in Fig. 1, the fourth order Stark effect on this lowest energy transition is likewise very strong, and it is very close to the second derivative of the 2nd-order conventional Stark spectrum. Thus, for this transition, dominates The contrast between this result and the com­ parable data for the homodimer is striking. It is obvious by inspection that the fourth order Stark spectrum is not the second derivative of the 2nd order Stark spectrum for the native homodimer. This confirms the earlier conclusion that the Stark lineshape for the special pair is not dominated but rather that makes the dominant by contribution (Middendorf et al., 1993). A general theoretical treatment of the higher order lineshape is in hand, and we are currently attempting to extract further information on the components of the polarizability tensor from this data.

B. Vibrational Stark Spectroscopy Chromophores which absorb in the visible and near IR are not that common in most proteins; however, molecular vibrations are associated with every residue. Recently, with the advent of better FTIRs and time resolved IR methodology, there has been considerable interest in assigning some of the vibrational features of RCs (as well as many other proteins) and using changes in the vibrational frequencies or intensities to monitor the response of the protein to electron transfer events (e.g. Maiti et al., 1993). In order to evalu­ ate these changes quantitatively, it would be use­ ful to have an independent experimental cali­ bration of the sensitivity of the vibrational frequencies to electric fields. Towards this end, we have performed the first measurements of the VSE for anisonitrile (chosen largely because it is a strong transition in a convenient spectral region) (Chattopadhyay and Boxer, 1995). The VSE spectrum is shown in Fig. 2. From this we obtain and i.e., is collinear with the transition moment, as expected. If an electric field were aligned with this bond di­ rection, the Stark tuning rate is: Extensions to a wider variety of vibrational transitions are in progress. These data provide fundamental information on vibrational anharmonicity, as well as calibrating the sensitiv­ ity of vibrational spectra to local electric fields.

186

C. Unidirectional Electron Transfer in the RC One useful application of the electronic Stark ef­ fect is to provide a calibration for electrochromic shifts due to the transient internal electric field produced by charge separation (Steffen et al., 1994). In particular, when charge is separated the resulting electric field is between P and felt by the monomeric bacteriochlorophyll and bacteriopheophytin monomers (referred to as B and H, respectively). In order to calibrate these shifts, information on the sensitivity of monom­ eric pigments to electric fields is first obtained from conventional Stark spectroscopy. The B and H bands consist of overlapping absorptions from the monomeric BChls and BPheos, respectively, on the functional L and non-functional M sides. This limits the accuracy of the determination of

Steven G. Boxer

both the magnitude and direction of for these chromophores; however, from an analysis of the are Stark spectrum the magnitude of the roughly the same on the functional and non-funcis more prob­ tional sides. The direction of lematic because the measurement of the angle dependence is compromised when bands overlap. We have no reason to believe that the direction of for the monomeric chromophores is appre­ ciably different within the RC than when the pig­ ments are extracted from the RC and studied in a simple organic solvent. Unfortunately, the angle on the transition defines two projections of moment direction, i.e. two antiparallel cones. the B bands are ob­ Upon formation of served to shift to higher energy and the H bands shift to lower energy. At a qualitative level this result immediately restricts the absolute direction of on each chromophore type to one of the

Stark spectroscopy

two cones. Thus, both the magnitude and abso­ lute direction of for all four spectator chrom­ ophores are known reasonably well. The electrochromic bandshifts can be obtained by subtracting an absorption spectrum of a sam­ ple in the state from one in the ground state, normalized for the amount of bleach of P at its absorption maximum. The data at 1.5 K are shown in Fig. 3. Here too the overlap of the absorption bands causes some uncertainty; how­

187

ever, it is evident by inspection that and shift by approximately the same amount, shown expanded in Fig. 4. Because the Stark effect spec­ trum in an external field demonstrates that their absorption bands are comparably sensitive to an electric field, this result suggests that the field at the sites due to the transient internal and electric field is about the same. However, it is obvious by inspection of the X-ray structure that the internal field due to should be than at because substantially larger at is is much closer to the charges on and than is Thus, we are forced to conclude that the dielectric screening on the L-side is consider­ ably greater than on the M side. A quantitative analysis suggests that it is approximately three times greater on the functional side (Steffen et

188

Steven G. Boxer band region of the spectrum, we believe that its intensity is quite small and the band is likely broad. These ideas are supported by more de­ tailed analyses of the absorption spectra of modi­ fied RCs, including the Stark spectra (Moore and Boxer, to be published). Acknowledgements This work was supported in part by grants from the NSF Biophysics and Chemistry programs. The author gratefully acknowledges contributions to the work from his lab from Drs. David Lockh­ art, Dennis Oh, Thomas Middendorf, David Gottfried, Sharon Hammes, Martin Steffen, Steffen Franzen, Arun Chattopadhyay, and Kaiqin Lao, and from Laura Moore, Laura Maz­ zola, and Huilin Zhou. References

al., 1994). This dielectric asymmetry, probed functionally, may be a significant contributor to unidirectional electron transfer in the RC. Its molecular origin is not known; however, it is likely the result of long-range interactions from many amino acids. A similar analysis of the B bands gives nearly identical results for the dielec­ tric asymmetry, even though some might question are en­ whether the B-band shifts upon tirely electrochromic in origin (there is no evi­ dence for appreciable exciton interactions be­ tween the P or B chromophores and the H chromophores). Our view is that the exciton in­ teraction between P and B is considerably smaller formthan the shifts which occur upon ation. Furthermore, although the upper exciton band of P contributes to the absorption in the B-

Alegria G, Moser CC, and Dutton PL (1992) Pulsed electric field induced reversed electron transfer from ground state to the cytochrome c heme in Rps. viridis. In: Breton J and Verméglio A (eds) The Photosynthetic Bacterial Reaction Center II, pp 313–319. Plenum Press, New York. Böttcher CJF (1973) Theory of Dielectric Polarization, Vol 1, Elsevier, Amsterdam. Boxer SG (1993) Photosynthetic reaction center spectroscopy and electron transfer dynamics in applied electric fields. In: Deisenhofer J and Norris JR (eds) The Photosynthetic Reaction Center Vol 2 pp 179–220. Academic Press, New York. Boxer SG, Goldstein RA, Lockhart DJ, Middendorf TR and Takiff L (1989) Excited states, electron transfer reactions, and intermediates in bacterial photosynthesis. J Phys Chem 93: 8280–8294. Chattopadhyay A and Boxer SG (1995) Vibrational Stark spectroscopy. J Amer Chem Soc 117: 1449–1450. Franzen S and Boxer SG (1993) Temperature dependence of the electric field modulation of electron transfer rates: Charge recombination in photosynthetic reaction centers. J Phys Chem 97: 6304–6318. Franzen S, Goldstein R and Boxer SG (1990) Electric field modulation of electron transfer reaction rates in isotropic systems: Long-distance charge recombination in photosyn­ thetic reaction centers. J Phys Chem 94: 5135–5149. Franzen S, Lao K and Boxer SG (1992) Electric field effects on kinetics of electron transfer reactions: Connection be­ tween experiment and theory. Chem Phys Lett 197: 380. Gottfried DS, Steffen MA and Boxer SG (1991a) Large protein-induced dipoles for a symmetric carotenoid in a photo­ synthetic antenna complex. Science 251: 662–665. Gottfried DS, Steffen MA and Boxer SG (1991b) Stark effect spectroscopy of carotenoids in reaction center and antenna

Stark spectroscopy complexes from photosynthetic bacteria. Biochim Biophys Acta 1059: 76–90. Hammes S, Mazzola L, Boxer SG, Gaul D. and Schenck C (1990) Stark Effect spectroscopy of the heterodimer mutant of Rb. sphaeroides. Proc Nat Acad-Sci 87: 5682–5686. Hochstrasser R (1973) Electric field effects on oriented mol­ ecules and molecular crystals. Acc Chemical Research 6: 263–269. Lao K, Franzen S, Stanley R, Lambright D and Boxer SG (1993) Effects of applied electric fields on the quantum yields of initial electron transfer steps in bacterial photo­ synthesis: I Quantum yield failure. J Phys Chem 97: 3165– 13171. Lao K, Moore LJ, Zhao H and Boxer SG (1995a) Higherorder Stark spectroscopy: Polarizability of photosynthetic pigments. A new method for probing the electronic proper­ ties of biological chromophores. J Phys Chem, 99: 496– 500. Lao K, Franzen S, Stanley R, Steffen MA, Lambright D and Boxer SG (1995b) Effects of applied electric fields on the quantum yields of initial electron transfer steps in bacterial photosynthesis: II Field induced absorption anisotropy and quantitative analysis. Chem Phys 197: 259–275. Liptay W (1974) in Excited states, EC Lim, (ed), Vol 1, pp 129–229, Academic Press, New York. Lockhart DJ and Boxer SG (1987) Magnitude and direction of the change in dipole moment associated with excitation of the primary electron donor in Rb. sphaeroides reaction centers. Biochemistry 26: 664–668. Lockhart DJ and Boxer SG (1988a) Stark effect spectroscopy of Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers. Proc Natl Acad Sci USA 85: 107–111. Lockhart DJ and Boxer SG (1988b) Electric field modulation of the fluorescence spectrum in Rhodobacter sphaeroides reaction centers. Chem Phys Lett 144: 243–250. Lockhart DJ, Goldstein R and Boxer SG (1988) Structurebased analysis of the initial charge separation step in bac­ terial photosynthesis: Electric field induced fluorescence anisotropy. J Chem Phys 89: 1408–1415. Lockhart DJ, Kirmaier C, Holten D and Boxer SG (1990) Electric field effects on the initial electron transfer kinetics in bacterial photosynthetic reaction centers. J Phys Chem 94: 6987–6995. Lockhart DJ, Hammes SL, Franzen S and Boxer SG (1991) Electric field effect on emission lineshapes when electron transfer competes with emission: an example from photo­ synthetic reaction centers. J Phys Chem 95: 2217–2226. Lösche M, Feher G and Okamura MY (1987) The Stark effect in reaction centers from Rhodobacter spheroides R-26 and Rhodopseudomonas viridis. Proc Natl Acad Sci USA 84: 7537–41. Lösche M, Feher G and Okamura MY (1988) The Stark effect in photosynthetic reaction centers from Rhodobacter sphaeroides R-26, Rhodopseudomonas viridis and the complex of photosystem II from spinach. In: Breton J and Verméglio A (eds) The Photosynthetic Bacterial Reaction Center — Structure and Dynamics, pp 151–164, Plenum, New York.

189 Mathies RA (1974) Experimental and Theoretical Studies of the Excited Electronic States of Some Aromatic Hydrocar­ bons Through Electric Field Perturbation and Through Chemical Substituents. PhD Thesis, Cornell University, Ithaca, New York. Middendorf TR (1992) Photochemical Holeburning and Stark Effect Spectroscopy of Photosynthetic Reaction Centers and Model Compounds. PhD Thesis, Stanford University. Middendorf TR, Mazzola LT, Lao K, Steffen MA and Boxer SG (1993) Stark effect (electroabsorption) spectroscopy of photosynthetic reaction centers at 1.5K: Evidence that the special pair has a large excited-state polarizability. Biochim Biophys Acta 1144: 223–234. Oh D and Boxer SG (1990) Electrochromism of the near-IR absorption spectra of bridged ruthenium mixed valence complexes. J Amer Chem Soc 112: 8161–8162. Oh DH, Sano M and Boxer SG (1991) Electro-absorption (Stark effect) spectroscopy of mono and bi-ruthenium charge transfer complexes: Measurements of changes in dipole moments and other electro-optic properties. J Amer Chem Soc 113: 6880–6890 (1991). Pierce DW and Boxer SG (1995) Stark effect spectroscopy of tryptophan. Biophys J 68: 1583–1591. Reimers JR and Hush NS (1991) Electric field perturbation of electronic vibronic absorption envelopes: Application to characterization of mixed valence states In: Prassides K (ed) Mixed-valence systems: Applications in chemistry, physics and biology, pp 29–39, Kluwer Academic Pub­ lishers, Dordrecht. Sacra A, Norris DJ, Murray CB and Bawendi MG (1995) Stark spectroscopy of systems with overlapping interacting transitions: Application to CdSe nanocrystallites. J Chem Phys, 103: 5236–5245. Stanley RA and Boxer SG (1995) Oscillations in the spon­ taneous emission from photosynthetic reaction centers. J Phys Chem, 99: 859–863. Steffen MA, (1994) Electrostatic Interactions in Photosyn­ thetic Reaction Centers. PhD Thesis, Stanford University, 1994. Steffen MA, Lao K and Boxer SG (1994) Dielectric asym­ metry and the origin of unidirectional electron transfer in photosynthetic reaction centers. Science 264: 810–816. Stocker JW, Hug S and Boxer SG (1993) Stark effect spectros­ band of Rb. sphaeroides reaction copy of the 1250 nm centers and related model compounds. Biochim Biophys Acta 1144: 325–330. Vauthey E, Voss J, deCaro C, Renn A and Wild UP (1994) Spectral hole-burning and Stark effect: frequency depen­ dence of the induced dipole moment of a squaraine dye in polymers. Chem Phys 184:347–356. Maiti S, Cowen BR, Diller R, Iannone M, Moser CC, Dutton PL and Hochstrasser RM (1993) Picoseond infrared studies of the dynamics of the photosynthetic reaction center. Proc Natl Acad Sci USA 90: 5247–51. Walker GC, Maiti S, Cowen BR, Moser CC, Dutton PL and Hochstrasser RM (1994) Time resolution of electronic transitions of photosynthetic reaction centers in the infra­ red. J Phys Chem 98: 5778–5783

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Chapter 12 The Photoacoustic Method in Photosynthesis –

Monitoring and Analysis of Phenomena Which Lead to

Pressure Changes Following Light Excitation

Shmuel Malkin Biochemistry Department, The Weizmann Institute of Science, Rehovot, 76100, Israel

Summary I. Introduction — Historical Notes and Main Aspects II. Experiments and Results with the Gas-Phase Coupled Microphone A. Experimental Technicalities B. The Photothermal Signal – Observations, Properties and Interpretation 1. Measurement of Energy Storage (ES) 2. ES Dependence on Experimental Parameters Dependence on the Modulation Frequency f a. b. ES as a Function of the Wavelength

c. ES as a Function of the Light Intensity I d. The Effect of Fluorescence – Determination of its Absolute Quantum Yield e. Time Domain Measurements f. Some Numerical Estimates 3. Acoustic Signals Obtained with Magnetic Field Modulation (Magnetophotoacoustic Effect) C. The Photobaric Signal – Observations, Properties and Interpretation 1. The Photothermal/Photobaric Composite Signal 2. Separation of the Photobaric Signal 3. Quantitation of the Photobaric Signal a. The Diffusion Model b. O/T as a Measure of the Quantum Yield 4. The Limiting Rate in the Oxygen Evolution Process 5. Uptake Signals III. Experiments and Results in the Time Domain with a Sample Coupled Piezoelectric Sensor IV. Applications to Physiological Studies References

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Summary Following light excitation and relaxation of the excited state, pressure changes are developed in an investigated sample or in the gas phase around the sample. Commonly, these changes result from the conversion of the light energy to heat, leading to temperature rise and hence to pressure increase (photothermal effect). Although quite small and rapid, the pressure changes are detectable by suitable fast sensors. These are either piezoelectric transducers, used in a flash photolysis mode with time domain measurement or microphones, used commonly in conjunction with periodically modulated light excitation (yielding periodic pressure perturbations) but also in a flash photolysis mode. Correspondence: Fax: 972-8-9344118; E-mail: [email protected]

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J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 191–206. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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Shmuel Malkin

Photosynthetic samples are quite unique iª that their photochemical activities give rise to additional mechanisms which contribute appreciably to pressure changes. These are: (1) gas evolution and uptake, most notably oxygen evolution, detectable in leaves and lichens with a sensor put in the gas phase (photobaric effect), (2) molecular volume changes, resulting from photosynthetic charge separation and proton transfer to and from the buffered medium. The methodology which deals with the measurement and analysis of these pressure changes is commonly known as photoacoustics (or optoacoustics) and the resulting signals are referred to as photoacoustic (optoacoustic) signals. Although implied by the name, sound production as such, is important only at sufficiently high frequencies (or short times) where the sound wavelength is small relative to the sample dimensions. Otherwise, the pressure changes are practically uniform over the entire sample. This chapter outlines methods to measure, separate and analyze each of the contributions to the photoacoustic signals in photosynthetic samples and the value of the information obtained by this analysis. Broadly speaking, the photothermal contribution allows to deduce the magnitude of energy storage with the capability to specifically assign energy and decay kinetics to particular electron transfer, intermediates; the photobaric contribution serves as a sensitive analytical tool for photosystem II activity and electron transport in general, in complex structures such as leaves; molecular volume changes are related to molecular structural changes within the reaction center. The use of periodically modulated light offers various other advantages: high sensitivity, separation from dark processes and possibility of conditioning by background (non-modulated) lights without interfering with the measurements. Applications include straightforward analysis of the interaction between the two photosystems, charac­ terization and dynamics of physiological changes and stress conditions, possible detection of cyclic electron flow in-vivo, among others. Abbreviation: ES – Energy storage; f – Modulation frequency; P870 – Primary electron donor (in – First purple bacteria); PA – Photoacoustic; PL – Photochemical loss; PS – Photosystem; and second acceptor quinones I. Introduction — Historical Notes and Main Aspects The fate of excited states is usually monitored by their decay, the formation of new state or chemi­ cal species and by light emission. A more special­ ized task is to measure the energy lost as heat during each step of the relaxation. This is because the involved temperature changes are very rapid and small, in the order of millidegree Celsius with a nearly saturating flash for ordinary concentra­ tions of a photochemical substrate, and perhaps three orders of magnitude less for a photosyn­ thetic sample that may contain hundreds of ab­ sorbing pigments per one reaction center. Never­ theless, it is possible, utilizing a number of consequent physical effects, to measure tempera­ ture changes with very sensitive sensors. One way, among others, is the photoacoustic (or op­ toacoustic) method (abbreviated PA), utilizing

microphones or piezoelectric transducers to mea­ sure the prompt pressure changes caused by the temperature changes (Callis et al., 1969; Ro­ sencwaig, 1980; Patel and Tam, 1981; Tam 1986; Braslavsky and Heioff, 1989). PA studies concerning photophysical relax­ ation in the gas phase were already carried out in the forties, using periodically modulated light and a microphone which was inserted in the sam­ ple gas. The rate of heat release is modulated and is phase shifted, depending on the modulation frequency, due to the finite relaxation times. This results in corresponding phase shifts in the pres­ sure modulation. From the frequency depen­ dence of the amplitude and phase of the micro­ phone signal (relative to a proper reference, where the relaxation to the ground state is suffi­ ciently rapid and heat release is prompt) it was possible to deduce the energy and first order decay constants of the relaxation intermediates

The photoacoustic method (Hunter and Stock, 1974; Hunter et al., 1974, and references therein). Callis et al. (1969) constructed a flash photo­ lysis microphone calorimeter for the liquid phase, using a special microphone that was inserted into the liquid. A flash produced an increase in the pressure following the increase in the tempera­ ture, which was recorded as a function of time. The excess pressure was released at longer times than the experiment time, by a constructed slow leak valve and by the slow heat release to the surrounding. Initial studies aimed at triplet states of organic dyes. In these experiments there was an initial “prompt” pressure increase (on the time scale of the experiment), due to the prompt heat release in the fast transitions from the excited singlet. This was followed by a slower measurable pressure increase due to the relaxation of the triplet, allowing to determine its yield, energy and relaxation kinetics. The same mode of PA measurement was later applied to photosynthetic bacterial samples: chromatophores (Callis et al. 1972) and reaction centers (Arata and Parson 1981). In these systems there was an additional contribution to the microphone response, which was assigned to a change in the molecular volume (at constant pressure). This was clearly demon­ strated at ca. 4°C in the aqueous suspension (where the thermal expansion is zero) by the ap­ preciable microphone response, which was ob­ viously absent from a photochemically inert refer­ ence. Prompt molecular volume changes, assigned to the electron transfer from P870 to were negative (i.e. signaling a contrac­ or tion). A slower kinetic phase of expansion was assigned to molecular volume change associated with proton transfer. At a higher temperature, the photothermal contribution could be isolated, by subtraction of the molecular volume changes, resulting in an estimate for the energies of and Similar studies were conducted on bacteriorhodopsin containing mem­ branes (Ort and Parson 1979 a–c). A different PA experimental approach, appli­ cable for thin, optically dense, or strongly light scattering layered samples, uses a microphone in the gas phase surrounding the sample. With peri­ odically modulated or pulsed light excitation, the resulting modulated or pulsed heat is first conduc­

193 ted to the surface, before the transduction to pressure changes. A thin gas layer adjacent to the surface, whose thickness is in the order of a “thermal diffusion length” (cf. below for a defi­ nition) undergoes expansion and relaxation cycles, acting as a piston on the bulk gas and resulting in bulk pressure changes (Rosencwaig and Gersho, 1978; Rosencwaig, 1980). The main limiting factor, in determining the strength of the signal, is the efficiency of the modulated heat diffusion to the sample’s surface. The oscillatory part in the temperature field within the sample is mathematically a superposition of waves, propa­ gating from point sources where modulated heat is evolved, with amplitudes which are pro­ portional to the extent of light conversion to heat. These thermal waves are however peculiar in that their amplitude decreases exponentially along the distance of propagation (a result of the heat con­ duction differential equation, being first order in the time). This property is characterized by a “thermal diffusion length” parameter, equal to the distance over which the amplitude is attenuated by a factor e, where f is is the the modulation frequency, the heat diffusivity, the heat conductivity, the specific heat (at constant pres­ density and sure). Besides its significance as an attenuation is also equal to the thermal wave­ factor, is an average effective length. Roughly, range for the thermal waves; only that part of the light absorbed along a distance of a very small number of thermal diffusion lengths below the surface will be effective in producing a measur­ able PA signal. For most non-conductors of heat is equal to just a few microns (e.g. ca. at 100 Hz for water), so that this mode of PA probes essentially the part of the sample adjacent to the surface. Layered suspensions of photosynthetic ma­ terial (Carpentier et al., 1985, 1989; Owens et al., 1990) as well as water soaked marine algal thalli (Fork et al., 1991; Malkin et al., 1990) gave PA signals which represent photothermal conver­ sion only and reflect significant photochemical en­ ergy storage. When PA measurements were carried out in leaves, the PA signals could not be interpreted by the photothermal mechanism alone. It was

194 concluded that a large fraction of the signal arises from modulated oxygen evolution (Bults et al., 1981), obviously arising from the modulated change in the number of moles released into a confined volume of the gas phase. This mechan­ ism was tentatively named “photobaric”. Regard­ ing the significance of assimilation and pho­ torespiratory oxygen uptake in producing (uptake) PA signals, it is inferred that their modulation is heavily damped (at the range of frequencies used – higher than ca. 10 Hz) and therefore their contribution to the PA signal is negligible. An analogous transmission mechanism, as with thermal waves, probably exists also in the photob­ aric effect (Bults et al., 1981, Poulet et al., 1983 Korpiun et al., 1992). The modulated flux of the newly formed oxygen generates concentration waves in the aqueous phase, which propagate to the surface, where the gas is released. In com­ plete analogy to thermal waves, the concentration waves are damped exponentially as they propa­ equal to gate, with a characteristic distance (over which the wave amplitude de­ is the oxygen creases by a factor e), where at com­ diffusion constant. Typical values for mon frequencies (around 30 Hz) is roughly near which is also the closest distance from the chloroplasts to the inner air phase in leaves. The transduction of oxygen evolution to increase of pressure must therefore be located only at the inner air phase of the leaf. Therefore, the photob­ aric signal is observed in leaves, also in lichens (Canaani et al., 1984), less prominently in half dried layered microalgae (Canaani, 1986) but not from in suspensions of photosynthetic material, water soaked sea weeds, and water infiltrated leaves. The name photoacoustic implies sound. The original photoacoustic effect, discovered in the last century (Bell, 1881 cited in Rosencwaig, 1980) involved indeed the production of audible sound by the absorption of periodically modu­ lated light. However, in the usual low frequency PA experiments, the dimensions of the sample compartment are smaller than the sound wavelength so that the pressure variations are actually uniform. Nevertheless, the name photoacoustics (or optoacoustics) is used for all cases.

Shmuel Malkin A more recent experimental mode, which uses a pulsed laser beam transversing the sample, is capable of measurement of high frequencies and involves true acoustic waves. Light induced ther­ mal and molecular volume changes, which occur in the small confined region of the laser beam path, act as a piston on the adjacent fluid body to generate an acoustic wave, which propagates to the wall of the cuvette and then sensed by a piezoelectric transducer, attached to the wall from the outside (Patel and Tam, 1981; Tam, 1986; Braslavsky and Heioff, 1989). The intensity of the resulting signal is directly related to the volume change, whether thermal or molecular. However, the shape of the signal is a complicated function involving both the relaxation kinetics in the sample, the sound propagation and the re­ sponse properties of the sensor, and it is not a trivial matter to extract exact relaxation infor­ mation from the signal (Puchenkov, 1994). Vari­ ous photochemical and photobiological systems were investigated by this PA mode (Peters and Synders, 1988; Braslavsky and Heioff, 1989, Churio and Braslavsky, 1994; Nitsch et al., 1988, 1989). In summary, PA signals in photosynthetic samples involve: (1) photothermal conversion (2) molecular volume changes, and (3) gas evolution. These effects are often superposed and proce­ dures are sought to separate each, for its different information content and as a general or specific marker for activity. This will be the essence of this chapter. Due to the limited space most of the older literature, except for some leading papers, is not included in the references list. These are listed as references in the newer literature and are re­ viewed elsewhere (cf. Moore, 1983; Malkin, 1986; Buschman and Prehn, 1990; N’SoukpoéKossi and Leblanc, 1990; Fork and Herbert, 1993; Braslavsky and Heioff, 1989; Malkin and Canaani, 1994). II. Experiments and Results with the Gasphase Coupled Microphone

A. Experimental Technicalities Work with gas-phase coupled microphone is most simple and common (Moore, 1983), requiring a

The photoacoustic method miniature microphone, communicating with a small volume of a gas phase around a tightly enclosed flat thin sample: a leaf disc, a lichen piece, an algal thallus, a filter paper impregnated with photosynthetically active suspension. The apparatus must be well insulated from ground vibrations. Background PA signals, arising from other cell parts, are minimized by correct light collimation and use of good polished window (preferably quartz). The usable frequency range is usually below about 1000 Hz, as the PA signal strongly decreases as f increases and there is in­ creased proportion of background signals from non-photosynthetic pigments (e.g. from the leaf epidermis, also cell parts). Modulated light is ob­ tained by using either a chopper, electronic modulation (e.g. with light emitting diodes (Snel et al., 1990; Kolbowsky et al., 1990) or using flash sources (Mauzerall, 1990). Additional nonmodulated (termed “background”) lights, which obviously do not produce signal of their own, are used to “bias” the photosynthetic system into any desired physiological or electron transfer state. With periodic modulation the microphone output is analyzed by a lock-in amplifier, yielding either the amplitude and phase, or the two amplitudes of the sinusoidal (“inphase”) and cosinusoidal (“quadrature”) components, which define the sig­ nal as a mathematical two-dimensional vector. The separate contributions to the signal must ob­ viously combine as vectors (Poulet et al., 1983). With flash excitation a transient signal is ob­ tained, which is studied as a function of time (Mauzerall, 1990; Kolbowsky et al., 1990; Reising and Schreiber, 1992; Cha and Mauzeral, 1993). Apparently, a leaf disc sample confined to a small isolated volume is unsuitable for studying is very rapidly its photosynthesis, because replenishment depleted during illumination. by continuous gas streaming through the cell pro­ duces too much background noise. This problem is somewhat circumvented by a construction which allows gas streaming between consecutive measurements (Reising and Schreiber, 1992) or by diffusion through sintered glass (Herbert and Fork, 1992) or through a small orifice (T. Pun­ net – private communication). Without such gas control, photosynthesis is running at a compen­ sation point, at which photorespiration may be dominant. However, this does not make much

195 difference to the interpretation of the PA mea­ surements, which are related to electron trans­ port, as such. Also, it is possible to work with fully expanded leaf, and bring only a small central section into the PA cell. In this case, there is very probably an equilibration by lateral diffusion of the inner air composition between the exposed and unexposed parts, shown by the immediate concentration in the gas effect of increased phase above the exposed part on the PA signal (S. Malkin, unpublished), which acts to inverse the signal sense by adding an uptake component (Reising and Schreiber, 1992, cf. below).

B. The Photothermal Signal – Observations, Properties and Interpretation A pure photothermal signal is observed when the modulated gas evolution and molecular volume changes are negligible. The second is always insig­ nificant with the gas-phase microphone. The first is usually negligible in sufficiently thick aqueous samples and in leaves at sufficiently high modu­ lation frequencies, because of the damping of the oxygen concentration waves. Suitable samples for study of photothermal conversion are algal and subcellular suspensions, thalli of marine algae and water infiltrated leaves. Ordinary leaves and lich­ ens must be used at sufficiently high frequencies (typically above ca. 300 Hz for a leaf and 50 Hz for a lichen, depending on the species). 1. Measurement of Energy Storage (ES) Figure 1 represents a schematic typical experi­ mental record for the photothermal PA signal, excited by periodically modulated light. The en­ ergy storage is evaluated by comparing the signal to that of a reference where all light energy is promptly converted to heat. Since the PA signal is extremely sensitive to the thermal/optical par­ ameters of the sample, its surface and the sur­ rounding gas phase (Rosencwaig and Gersho, 1977), the reference must have exactly the same parameters. A physical substitution of the sample with a reference, still keeping exactly the same parameters, is impractical. However, a reference state is easily and reversibly achieved in photo­ synthetic samples by adding sufficiently strong background light, which saturates photosynthesis

196

and drives its quantum yield, close to zero. This results in a reversible increase of the PA photothermal signal from a level S to the refer­ ence level R. The relative energy storage (i.e. the average fraction of the absorbed photon energy (hv) stored as chemical energy), neglecting flu­ orescence changes, is defined by ES = (R – S)/R. The increase from S to R is typical of viable photosynthetic system and is used as a marker of activity. 2. ES Dependence on Experimental Parameters Energy strategy (ES), is a function of the modu­ lation frequency f, of the wavelength of the average light intensity I and of the sample’s con­ dition. To see this we may write approximately, neglecting fluorescence:

Shmuel Malkin

where R is reference level, is the energy con­ tent of the formed intermediate(s) per successful photochemical event, hv is the photon energy and depends on f and possibly c is the light speed. depends on I and the sample’s con­ on dition.

a. Dependence on the Modulation Frequency f The modulated signal reflects heat which is prompt, relative to the modulation cycle time (1/f). Slower heat evolution is not modulated. is assigned to intermediate(s) formed Hence in a time much shorter than 1/f and decay in a time much longer. Also, S and R have the same phase angle in those ranges of f where these con­ ditions are satisfied (e.g. as in Fig. 1). Between

The photoacoustic method

these frequency regions there are transition seg­ ments where S is phase shifted with respect to R, with an in-phase amplitude (relative to R) which grows with f and connects between adjacent pla­ teaus, and a quadrature amplitude (relative to R) which is maximized at half transition points (Fig. 2). The frequencies at the half transition points where is the lifetime of are equal to the shorter lifetime intermediate. In the above analysis (cf. Malkin and Cahen, 1979) S is always measured relative to R, to eliminate the strong dependence on f caused by the heat conduction mechanism and the transduction to pressure.

b. ES as a Function of the Wavelength ES depends explicitly on (Eq. (1)), but may vary too. Assuming that is constant, is proportional to and a scan through gives a relative quantum yield spectrum (Fig. 3). This procedure has the obvious advantage that light intensity or absorption measurements are un­ necessary. However, one should consider that may vary, due to the presence of more than one photochemical pathways (e.g. a combination of linear electron flow and PS I cyclic electron trans­ port – the last one favored in far-red wave­ lengths). Indeed, a comparison between energy storage activity and linear electron transport ac­ tivity, led to a conclusion about the presence of

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an in-vivo cyclic PS I electron flow at far-red wavelengths (Herbert et al., 1990), which later was characterized in more detail (Ravenel et al., 1994).

c. ES as a Function of the Light Intensity I decreases as I increases, finally tending to zero at light saturation. Normally, it is desirable to perform ES measurements at sufficiently low light is maximum. To study ES intensities where vs. I, which is a relative measure of vs. I, it is most convenient to add background light of vary­ ing intensities. ES vs. I dependence conforms often to the customary hyperbolic relation be­ tween rate and light intensity (Fig. 4), allowing to extrapolate and find the idealized maximum ES at zero intensity (Carpentier et al., 1985, 1989; Owens et al., 1990) and also to deduce the satur­ ation behavior of the reaction rate.

d. The Effect of Fluorescence – Determination of its Absolute Quantum Yield If fluorescence is not neglected, Eq. (1) takes a more extended form:

where

and

are the mean electromagnetic

198 frequencies of the (modulated) exciting light and the fluorescence emission, respectively, is the fluorescence yield of the measured sample. – at sufficiently low light intensities, where the fluorescence yield attains its mini­ mum is the maximum fluorescence yield, obtained at light saturation. Eq. (2) results from the obvious proportionality between and between S and R and The ratio (R – S)/R expresses the relative de­ ficit of the signal due to both photochemistry and fluorescence and was termed “photochemical loss” (abbreviated PL). Eq. (2) allows to calcu­ late ES more rigorously as:

Shmuel Malkin When a good reference state is not available for the PA measurement (e.g. in the absence of photochemistry), an alternative analysis then considers a comparison between the change in PA, normalized for light absorption, vs. the wavelength (called “thermal deactivation spec­ trum”) and the relative fluorescence yield vs. the exciting wavelength. From the variations in these parameters it may be possible to adjust these spectra to an absolute quantum yield scale. This is useful in analyzing energy transfer yields for a multichromophore antenna preparations and was utilized to study photosynthetic systems oriented in streched polyvinylalcohol films. et al., 1986, 1991; Wróbel and Hendrich, 1989).

e. Time Domain Measurements Inserting accepted estimates for the fluorescence yields (cf. also below), the use of Eq. (1) instead of the more complicated Eq. (3) leads to a small error only (a few per cent) in ES (Malkin and Canaani, 1994). The absolute fluorescence yield can be esti­ mated from a combined PA and relative fluor­ escence measurements when there is no photoch­ emistry, if both change as a function of some 1990; Dau and Hansen, parameter 1990). One might utilize the transient change in the relative fluorescence measured at light saturation, known as “non-photochemical quen­ ching”, occuring in whole oxygenic organisms, most prominently in leaves (Krause and Weis, 1991), which consist of a drop in from its maximal initial value, to a significantly lower value The PA signal is expected to increase correspondingly from to since the only competing reactions under light saturation are heat emission and fluorescence. Writing and (where a is a proportionality coefficient), it follows that:

can be computed from and the ratio of the relative fluorescence values corresponding to and Preliminary estimations are available for leaves. From the data in Dau and Hansen (1990) and

A microphone transient signal was obtained for layered Chlorella cells, following brief flash exci­ tation (Cha and Mauzerall, 1992). The signal rise and decay transient reflects heat release kinetics, heat diffusion and sensor time response. The overall signal from a sample is smaller than that of a reference, indicating energy storage, but the time characteristics are broadly similar, indicating that the heat release kinetics is not discerned and that the following heat conduction process limits the time response. Thus, ES measurement at a particular transient signal time point (7 ms in this work) is probably related to an earlier time in the true heat release kinetics.

f. Some Numerical Estimates Most optimal in vivo measurements of ES, near the absorption edge (ca. 680 m), in leaves and algae, center around 0.3, showing a weak or no f dependence in the amenable range (10–500 Hz) with no or small phase angle shift between S and R (Malkin et al., 1992, cf. Fig. 1). This indicates that not much energy dissipation occurs between about 0.3 – 20 ms after the photoact and with it the conventional estimate turns out that kCal per mole electron transfer, nearly corresponding to the combustion heat of 1/24 mole glucose (ca. 28 kCal). The flash experiments of Cha and Mauzerall (1993, cf. above) identified ES (at less than 7 ms, cf. above) for different partial reactions, that could be iso­

The photoacoustic method

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lated in different conditions: PS I cyclic electron = 15 kCal per mole electron transfered flow PS II + PS I whole at 695 m, assuming electron flow and PS II electron transport from the Mn complex to 3. Acoustic Signals Obtained with Magnetic Field Modulation (Magnetophotoacoustic Effect) Following light induced primary electron transfer in quinone depleted bacterial reaction centers, the triplet state of the primary donor is obtained by charge recombination from the charged donor acceptor pair, which, during its life time, can cycle between singlet and triplet states. In the presence of a magnetic field (in a range below 1 kGauss) the probability to obtain the triplet decreases, due to the energy splitting of the triplet sub-levels, of which two become inaccessible (Hoff, 1981). Using constant illumination and pe­ riodically modulated magnetic field, the triplet state concentration is modulated at twice the fre­ quency of the field modulation (since the mag­ netic field effect is direction independent), which is picked up by a number of methods (Hoff et al., 1993). Using a suitably constructed PA cell to work at cryogenic temperatures, pressure modulation signals were recently detected from such quinone depleted bacterial reaction centers when these were exposed to modulated magnetic field and illuminated. It was proved, by a number of criteria, that this effect reflects the modulated heat release, which accompanies the return of the triplet to the ground state. Calibration of this “magneto photoacoustic” signal was done, by comparison to an ordinary PA measurement, de­ monstrating the ability to estimate the energy or yield or lifetime of the triplet state when the two others are known (Malkin and Hoff, 1994).

C. The Photobaric Signal – Observations, Properties and Interpretation 1. The Photothermal / Photobaric Composite Signal When oxygenic photosynthetic membranes lie sufficiently close to a gaseous phase, the photo­ thermal signal is usually accompanied by the pho­

tobaric one. This mechanism had to be invoked to explain the “abnormal” behavior of the PA signal in leaves at low modulation frequencies (less than about 100 Hz), which becomes very prominent at the lowest available f (e.g. 10 Hz): when an intense background light was added there was, instead of the typical increase, charac­ teristic of a photothermal signal, a rather marked decrease in the signal amplitude, accompanied by a large phase shift (Fig. 5; cf. also Bults et al., 1981; Poulet et al., 1983). Such effect indicated an additional contribution to the PA signal, which was eliminated at light saturation. The modulated photothermal signal (which is always present) could be measured separately, by photothermal

200

Shmuel Malkin

radiometry, monitoring the modulated part in the thermal infra-red radiation emitted from the leaf. This disclosed a normal behavior of the photo­ thermal conversion (Kanstad et al., 1983). It was clear that the additional contribution to the PA signal is not photothermal. Its assignment to ox­ ygen evolution is supported by: (1) its elimination at light saturation (no modulation in oxygen evol­ ution occurs then); (2) the absolute requirement of a gaseous phase in close proximity to the pho­ tosynthetic membranes (Bults et al., 1981; Malkin et al., 1992), indicating the involvement of gas evolution into the inner air phase; (3) its strong damping as f increases, consistent with a diffusion mechanism (cf. below). (4) the parallel behavior of the photoboric signal and the directly mea­ sured oxygen evolution, with regard to phenom­ ena such as photosynthetic induction (Bults et al., 1981), notably the initial oxygen gush (Malkin, 1987), wavelength dependence (e.g. the “far-red drop”; Bults et al., 1981) and Emerson enhance­ ment with addition of background far-red light (Canaani and Malkin, 1984). 2. Separation of the Photobaric Signal In a crude approximation, the PA level in the presence of a strong saturating background light serves as a base-line for the photobaric signal. However, since the photothermal signal itself in­ creases by the background light, the true base­ line is usually lower (Fig. 5). Its position may be found by an independent estimate of the PL (e.g. from high frequency measurements; infrared pho­ tothermal measurements; measurements on the same leaf after water infiltration). However, it is possible to obtain the photobaric signal alone as one of its vectorial components, which is perpen­ dicular to the photothermal signal vector. For this, the phase of the lock-in amplifier is adjusted in the presence of a strong saturating background light until one signal component (say, the quadra­ ture) is nullified. No photothermal contribution occurs in the quadrature channel and the quadra­ ture signal that appears upon switching off the background light is purely photobaric (Fig. 5). Experience showed that the phase of the photo­ thermal contribution is stable, so that a single phase adjustment is usually sufficient even for lengthy (hours) experiments. This photobaric

component, perpendicular to the photothermal signal vector, may alternatively be computed for any arbitrary setting of the phase angle, using simple vector analysis. It is equal to: where subscripts Q and I represent quadrature and in­ phase components of the signal, S, and super­ scripts(+) and (–) represent the presence and absence of background light, respectively. Fig. 6 exemplifies the time domain PA re­ sponses to exciting flashes, in absence and pres­ ence of background light. A particular nice time

The photoacoustic method separation between photothermal and photobaric signals is seen with weak light pulses (Kolbowsky et al., 1990; Reising and Schreiber, 1994). In very short and strong flashes the photobaric signal transient is saturated, limited by the internal elec­ tron carriers pool, while the photothermal signal increases approximately linearly with the light in­ tensity. Under such conditions. the photobaric signal becomes small, relative to the photother­ mal signal, and the time discrimination between them is more diffuse (Canaani et al., 1988; Mauz­ erall, 1990). Still, it was possible to discriminate the photobaric signal by proper substraction and observe its dependence on the flash number and the intensity of the last flash, in a series of con­ secutive saturating flashes, which opens a way to study S-state phenomena in the oxygen evolution from leaves (Canaani et al., 1988). 3. Quantitation of the Photobaric Signal

a. The Diffusion Model As a first approach to the quantitative treatment of the photobaric signal, it is necessary to over­ simplify the complex anatomical structure of the leaf. Since the mesophyll cell has its chloroplasts arranged on the periphery, close to the boundary (at an average distance L to the inner air phase), the simplest model is that of a single sheet of a photosynthetic membrane in an aqueous phase, parallel to the air boundary, at a fixed distance L. L is much smaller than the membrane surface dimensions, allowing a one-dimensional diffusion model, where oxygen concentration waves propa­ gate in a direction perpendicular to the boundary. With the above model, it was possible to derive a simple relation between the ratio O/T, where O and T are the amplitudes of the photobaric and the maximum photothermal signals, respectively, and the modulation frequency, f, based on the attenuation of the concentration waves, as they propagate to the surface. This relation takes the following linear form:

is the limiting value for O/T as The plot of Ln (O/T) vs. was indeed linear in

201 many cases (Malkin et al., 1992). In assessing the effects of stress on leaf photosynthesis it was found that not only O/T at a particular f changed by the treatment but possibly also the slope of the above plot, indicating changes in the anatomical structure of the leaf (Havaux et al., 1986, 1987). Therefore, such effects should be better quant­ ified by which is diffusion independent. For completion, the diffusion model should include also possible sinks, where oxygen may be absorbed (by respiration and photorespiration) on its way to the inner air phase. Probably there is no place for such sinks in the small space be­ tween the photosynthetic membrane and the cell boundary. However, in at least one isolated case (maple tree leaves) this possibility was raised to explain the slow transient decrease of the photob­ aric signal with time, at low light intensity, ob­ served uniquely for this species (Charland et al., 1992).

b.

O/T as a Measure of the Quantum Yield

If the entire PA signal is generated in the inner air phase, the contributions from each source point inside the leaf to the photothermal and photob­ aric signals have the same ratio. The photobaric signal (O) is proportional to and the maxi­ mum photothermal signal (T) is proportional to and hv I. Their ratio, O/T, is proportional to Its is therefore a relative measure of scan over gives a quantum yield spectrum (with no need to measure light intensity or absorption). Keeping small amplitudes of the light intensity modulation, changing the average light intensity by adding background light at various intensities, O/T may be considered as proportional to the derivative of the rate vs. intensity – which one may call a “differential” quantum yield. By inte­ gration of O/T vs. I curves the relative rate vs. intensity dependence is reconstructed (Poulet et al., 1983). An absolute calibration for the oxygen evol­ ution signal was suggested, but not yet im­ plemented, by measuring the relative oxygen gush signal which occurs as a pulse in the begin­ ning of the induction period, and comparing to the size of the plastoquinone pool (Malkin, 1987; Malkin and Canaani, 1994).

202 4. The Limiting Rate in the Oxygen Evolution Process The photobaric signal should be influenced also by the rates of the partial processes which lead to oxygen evolution, leading expectedly to a devi­ ation from the linear plot of Eq. (5), at modu­ lation frequencies comparable or higher than the rate limiting constant, k. While earlier work indi­ cated such a deviation, corresponding to a reac­ tion time of about a ms (Poulet et al., 1983; Canaani et al., 1984), a careful analysis indicated that this deviation was probably an artefact, caused by underestimation of the PL in the photo­ thermal signal (Malkin et al., 1992). Thus, the limiting reaction time may even be smaller than a ms. This conflicts with conclusions from recent oxygen electrode measurements at low electrode polarization (Plijter et al., 1988), concluding that the reaction time is very much longer (cf. also H.J. van Gorkom, this volume). Since PA signals monitor the gas evolution directly, by the pres­ sure variation, they must be regarded as very reliable informant compared to the oxygen elec­ trode, which depends on the process of oxygen reduction by the polarized cathode, and is sensi­ tive to imperfections in its surface. Time domain PA signal profiles (Mauzerall, 1990; Kolbowsky et al., 1990; Reising and Schreiber, 1994 – cf. Fig. 6) show even more clearly that oxygen evolution does not delay more than about a ms, probably just reflecting diffusion lag. 5. Uptake Signals While there is an increasing phase angle shift between the photobaric and the photothermal sig­ nals as f increases (Poulet et al., 1984) the two phase angles tend to approach each other as There are situations where at low frequenc­ ies the direction of the photobaric signal tends to be 180° out of phase, (i.e. with a negative sense, compared to the photothermal modulation), indi­ cating uptake rather than evolution. In a time domain measurement this was seen clearly as a negatively going transient, when the direction of the photothermal signal is defined as positive. Uptake signals were seen after heat shock (Havaux et al., 1987b), after dark adaptation and during photosynthetic induction (Malkin, 1987;

Shmuel Malkin Mauzerall, 1990; Reising and Schreiber, 1992). Particularly noticeable are uptake signals ob­ level (Reising tained upon increase of the and Schreiber, 1992). The simplest interpretation is that the uptake reflects oxygen photoreduction through PS I, as it is also induced by modulated far-red light, which normally does not produce a modulated oxygen evolution signal. However, Reising and Schreiber (1994) concluded, from the effect of a Carbonic anhydrase inhibitor, that at levels least the uptake signal seen at elevated arises from modulated solubilization of caused by the light induced modulation in the pH of the stroma. Accordingly, the uptake seen after dark adaptation was explained by the dark accumulation (by respiration) in the confined sample compartment (Reising and Schreiber, 1992). III. Experiments and Results in the Time Domain with a Sample Coupled Piezoelectric Sensor This experimental mode, involving laser exci­ tation, was reviewed in detail (Tam, 1986; Bras­ lavsky and Heioff, 1989) and is sometimes re­ ferred to as LIOAS (laser induced optoacoustic spectroscopy). As was mentioned in the Introduc­ tion, the signal time profile, (Fig. 7) is complex, requiring special methods of its diagnostics, hence for routine measurements only the amplitude of the first peak (or the difference between the first and second peaks) is often tentatively measured, and taken to indicate the heat release in a time scale shorter than the time scale of the appear­ ance of the signal peaks (about a Measure­ ments are performed in bulk aqueous suspensions (sometimes with the addition of glycerol, to in­ crease the thermal expansion coefficient), through which a laser pulse is sent. Referencing for maximum heat production is here possible by replacing the sample with a photochemically inert reference, dissolved in the same buffer, keeping the same light absorption coefficient. The thermal and elastic properties are kept the same, being mainly due to the aqueous medium. Initial results were collected for PS I and PS II particles (Nitsch et al., 1988), purple bacterial cells (Nitsch et al., 1989) and a leaf submerged in water (Jabben and Schaffner, 1985). In these,

The photoacoustic method

203 mental proportionality coefficient. Eq. (6) can be used in a variety of ways. For example, an inert reference solution will yield at a given tempera­ a reference signal ture hence:

(subscript T1 refers to values of the parameters is the prompt maximum heat, equal at to the absorbed laser pulse energy). Inserting the and we obtain values for 4°C

no consideration was made on the presence of molecular volume changes, which were found in later experiments, and the signal was totally as­ cribed to photothermal conversion. Although the significance of the numbers obtained for energy storage is therefore indecisive, it was later argued (Malkin et al., 1994), that the use of 30% glycerol in the medium, which increases the thermal ex­ pansion coefficient significantly, makes the mol­ ecular volume changes less significant, relative to the photothermal ones. Recent experiments with purple bacterial reac­ tion centers (Malkin et al., 1994), and with part­ icles of PS I and PS II, chloroplasts and algal cells (Delosme et al., 1994) disclosed indeed significant molecular volume changes, observed separately at ca. 4°C (Fig. 7). The signal amplitude, S, at any other temperature is a composite of photothermal and molecular volume changes. Thus:

where C and are the thermal expansion coef­ ficient, specific heat and specific density of the sample, respectively, the heat released and the molecular volume change. a is an instru­

If and are temperature independent (at constant laser energy) S/R would be a linear func­ with a slope tion of from which the energy storage can be found, and an intercept from which is calculated. Such plot was found to be indeed linear, which supports the above assumptions (Malkin et al., 1994). The results indicated significant energy storage, assigned to the state in contrast to the previous slower (ms) PA measurement, which yielded a very small energy storage, from which it was con­ cluded that the free energy change was mainly entropic (Callis et al., 1972; Arata and Parson, 1981). Arata and Parson expressed their concern that something was wrong with their PA results, but could not trace the real reason. Perhaps there is a hidden relaxation process in a time scale and ms, involving the entire protein between moiety, which converts enthalpic free energy into entropic free energy. So far, this dilemma still waits for an answer. Delosme et al. (1994) used LIOAS measure­ ments for spectral measurements of the quantum yield in oxygenic organisms, in which significant molecular volume changes were also observed and were used to calculate relative quantum yi­ elds. The quantum yield varied as a function of the wavelength in chloroplasts, but not in PS I or PS II particles. The quantum yield changes reflect probably the variations of the light distribution between the two photosystems.

204

IV. Applications to Physiological Studies PA measurements are particularly suited to get physiological information, being non invasive, sensitive, and with the capacity to reach various physiological states by background lights (Malkin and Canaani, 1994; Fork and Herbert, 1993). Ex­ amples are: quantitative information on light dis­ tribution between the two photosystems and its changes in different states (Canaani and its Malkin, 1984; Canaani, 1986; Malkin et al., 1990; Fork et al., 1991; Veeranjaneyulu et al., 1991a,b; Mullinaux et al., 1991; Bruce and Saleihan, 1992); the relation between the quantum yield of photochemistry and non-photochemical quench­ ing of fluorescence (Snel et al., 1990a). Charac­ terization of cyclic electron transport, by energy storage activity (Malkin et al., 1990; Herbert et al., 1991; Ravenel et al., 1994); characterization of photosynthesis induction (Malkin, 1987; Havaux 1988; Snel et al., 1990b; Reising and Schreiber, 1992); characterization of stress effects (Havaux et al., 1986, 1987a,b; Havaux, 1988). For a more complete list, see Fork and Herbert, 1993; Malkin and Canaani, 1994). The advantage of the PA measurements, for this analysis, is in that they are fast and that they measure true (gross) photosynthesis, compared to the custom­ ary measurements of net photosynthesis, which include all losses. In future work, particularly in PA work with leaves, it would be advantageous to take the ex­ ample of Snel et al. (1992) and develop a PA system that can simultaneously measure at least at two frequencies and two wavelengths (by pro­ viding two measuring light channels and two lockin amplifiers), allowing simultaneous mea­ surements of energy storage and oxygen evol­ ution (low and high frequencies) and of separate measurement of PS I activity (far-red vs. shortwavelength modulated lights). This is necessary in view of the impossibility to repeat exactly the same experiment in a leaf. References Arata H and Parson WW (1981) Enthalpy and volume changes accompanying electron transfer from P-870 to quinones in Rps. spheroides reaction centers. Biochim Bi­ ophys Acta 636: 70–81.

Shmuel Malkin Braslavsky SE and Heihoff K (1989) Photothermal methods. In: Scaiano JC (Ed) CRC Handbook of Organic Photoch­ emistry. Vol 1, pp 327–355, CRC Press, Boca Raton. Bruce D and Salehian O (1992) The efficiency of primary photosynthetic processes in state 1 and state 2. Biochim Biophys Acta 1100: 242–250. Bults G, Horwitz BA, Malkin S and Cahen D (1981) Photoac­ oustic measurements of photosynthetic activities in whole leaves – photochemistry and gas exchange. Biochim Bi­ ophys Acta 679: 452–465. Buschmann C and Prehn H (1990) Photoacoustic spectros­ copy – photoacoustic and photothermal effects. In: Lin­ skens H-F and Jackson JF (eds) Modern Methods in Plant Analysis Vol 11, pp 148–180, Springer-Verlag, Berlin. Callis JB, Gouterman M and Danielson JDS (1969) Flash calorimeter for measuring triplet yields. Rev Sci Instr 40: 1599–1605. Callis JB, Parson WW and Gouterman M (1972) Fast changes of enthalpy and volume on flash excitation of chromatium chromatophores. Biochim Biophys Acta 267: 348–362. Canaani O (1986) Photoacoustic detection of oxygen evol­ ution and state 1 – state 2 transitions in cyanobacteria. Biochim Biophys Acta 852: 74–80. Canaani O and Malkin S (1984) Distribution of light excitation in an intact leaf between the two photosystems of photo­ synthesis – changes in absorption cross-sections following state 1 – state 2 transitions. Biochim Biophys Acta 766: 513–524. Canaani O, Ronen R, Garty J, Cahen D, Malkin, S and Galun M (1984) Photoacoustic study of the green alga Trebouxia in the lichen Ramalina duriaei in-vivo. Photosynth Res 5: 297–306. Canaani O, Malkin, S and Mauzerall D (1988) Pulsed pho­ toacoustic detection of flash-induced oxygen evolution from intact leaves and its oscillation. Proc Nat Acad Sci (US) 85: 4725–29. Carpentier R, Nakatani H and Leblanc RM (1985) Photoac­ oustic detection of energy conversion in a Photosystem II submembrane preparation from spinach. Biochim Biophys Acta 808: 470–473. Carpentier R, Leblanc RM and Mimeault M (1989) Photoac­ oustic detection of photosynthetic energy storage in Photo­ system II submembrane fractions. Biochim Biophys Acta 975: 370–376. Cha Y and Mauzerall D (1992) Energy storage of linear and cyclic electron flows in photosynthesis. Plant Physiol 100: 1869–1877. Charland M, Veeranjaneyulu K, Charlebois D and Leblanc RM (1992) Photoacoustic signal generation in leaves: Are O2–consuming processes involved? Biochim Biophys Acta 1098: 261–265. Churio MS, Angermund KP and Braslavsky SE (1994) Combi­ nation of laser induced optoacoustic spectroscopy and sem­ iempirical calculations for the determination of molecular volume changes. The photoisomerization of carbocyam­ ines. J Phys Chem 96: 1776–1782. Dau H and Hansen U-P (1990) A study on the energy-dependent quenching of chlorophyll fluorescence by means of photoacoustic measurements. Photosynth Res 25: 269– 278.

The photoacoustic method Delosme R, Béal D and Joliot P (1994) Photoacoustic detec­ tion of flash-induced charge separation in photosynthetic systems. Spectral dependence of the quantum yield. Bi­ ochim Biophys Acta 1185: 56–64. Fork DC and Herbert SK (1991) A gas-permeable photoac­ oustic cell. Photosynth Res 27: 151–156. Fork DC and Herbert SK (1993) The application of photoac­ oustic techniques to studies of photosynthesis. Photochem Photobiol 57: 207–220. Fork DC, Herbert SK and Malkin S (1991) Light energy distribution in the brown alga Macrocystis Pyrifera (Giant kelp). Plant Physiol 95: 731–739. D (1990) Joint applications of fluorescence and photoacoustic methods in photobiology. Appl Fluor Techn 2: 11–14. D, Erochina LG, Balter A, Lorrain L, Szurkow­ ski J and Szych B (1986) Polarized absorption, fluorescence and photoacoustic spectra of phycobilisomes embedded in poly(vinylalcohol) films. Biochim Biophys Acta 851: 173– 180. D, Cegielski R and Abdurakhmanov IA (1991) Excitation energy transfer from carotenoids to bacterioch­ lorophyll in the bacterium Chromatium minutissimum. Photosynthetica 25: 621–629. Havaux M (1988) Induction of photosynthesis in intact leaves under normal and stressing conditions followed simulta­ neously by transients in chlorophyll fluorescence and pho­ evolution. Plant Physiol Bi­ toacoustically monitored ochem 26: 695–704. Havaux M, Canaani O and Malkin S (1986) Photosynthetic responses of leaves to water stress, expressed by photoac­ oustics and related methods I. Probing the photoacoustic method as an indicator for water stress in vivo. II. The effect of rapid drought on the electron transport and the relative activities of the two photosystems. Plant Physiol 82: 827–839. Havaux M, Canaani O and Malkin S (1987a) Inhibition of photosynthetic activities under slow water stress measured in vivo by the photoacoustic method. Physiol Plantarum 70: 503–510. Havaux M, Canaani O and Malkin S (1987b) Oxygen uptake by tobacco leaves after heat shock. Plant Cell Environ 10: 677–683. Herbert SK, Fork DC and Malkin S (1990) Photoacoustic measurements in-vivo of energy storage by cyclic electron flow in algae and higher plants. Plant Physiol 94: 926–934. Hoff AJ (1981) Magnetic field effects on photosynthetic reac­ tions. Quart Rev Biophys 14: 599–665. Hoff AJ, Gast P, van der Vos R, Vrieze J, Franken EM and Lous EY (1993) Magnetic field effects: MARY, MIMS and MODS. Z Physik Chem 180: 175–192. Hunter TF and Stock MG (1974) Photophysical processes in the vapour-phase measured by the optic-acoustic effect. Part 2 – Triplet state yield and life time in high pressure acetyl vapour. Part 3 – Radiactionless processes from the lowest singlet and triplet states of and benzene. J Chem Soc Faraday Trans II 70: 1022–1039. Hunter TF, Rumbles D and Stock MG (1974) Photophysical processes in the vapour-phase measured by the optic-acoustic effect. Part 1 – The model and apparatus for the study

205 of rediactionless processes. J Chem Soc Faraday Trans II 70: 1010–1021. Jabben M and Schaffner K (1985) Pulsed laser induced op­ toacoustic spectroscopy of intact leaves. Biochim Biophys Acta 809: 445–451. Kanstad SO, Cahen D and Malkin S (1983) Simultaneous detection of photosynthetic energy storage and oxygen evolution in leaves by photothermal radiometry and pho­ toacoustics. Biochim Biophys Acta 722:182–189. Kolbowski J, Reising H and Schreiber U (1990) Computercontrolled pulse modulation system for analysis of photoac­ oustic signals in the time domain. Photosynth Res 25: 309– 316. Korpiun P, Malkin S and Cahen D (1992) Interpretation of the photoacoustic effect in leaves by evolution and transport of heat and oxygen. In: Bicanic D (Ed) Photoacoustic and photothermal phenomena, pp 59–61, Springer-Verlag, Berlin. Krause GH and Weis E (1991) Chlorophyll fluorescence and photosynthesis: the basics. Ann Rev Plant Physiol Plant Mol Biol 42: 413–449. Malkin S (1986) Photoacoustic probing of energy storage and gas exchange in the photosynthesis of leaves. J Chem Soc Farad Trans II 82: 2233–2235. Malkin S (1987) Fast photoacoustic transients from dark ad­ apted intact leaves — I. Oxygen evolution and uptake pulses. A phenomenological record. Planta 171: 65–72. Malkin S and Cahen D (1979) Photoacoustic spectroscopy and radiant energy conversion: Theory of the effect with special emphasis on photosynthesis. Photochem Photobiol 29: 803–813. Malkin S and Canaani O (1994) The photoacoustic method and its characteristics in use for the study of photosynthesis. Ann Rev Plant Physiol Plant Mol Biol 45: 493–526. Malkin S and Hoff AJ (1994) Acoustic detection of triplet states formed by radical pair recombination in quinonedepleted photosynthetic reaction centers, by magnetic field modulation (magnetophotoacoustic effect) – a feasibility study. Photochem Photobiol 59: 670–676. Malkin S, Herbert SK and Fork DC (1990) Light distribution, transfer and utilization in the marine red alga Porphyra perforata from photoacoustic energy storage measurement. Biochim Biophys Acta 1016: 177–189. Malkin S, Charland M and Leblanc RM (1992) A photoacous­ tic study of water infiltrated leaves. Photosynth Res 33: 37–50. Malkin S, Churio M, Shochat S and Braslavsky S (1994) Photochmical energy storage and volume changes in the microsecond time range in bacterial photosynthesis – a laser induced optoacoustic study. J Photochem Photobiol B 23: 79–86. Mauzerall D (1990) Determination of oxygen emission and uptake in leaves by pulsed, time resolved, photoacoustics. Plant Physiol 94: 278–283. Moore TA (1983) Photoacoustic spectroscopy and related techniques applied to biological materials. In: Smith KC (ed) Photochemical and photobiological reviews Vol 7 (pp 187–221) Plenum, New York. Mullineaux CW, Griebenow S and Braslavsky SE (1991) Pho­ tosynthetic energy storage in cyanobacterial cells adapted

206 to light-states 1 and 2. A laser-induced optoacoustic study. Biochim Biophys Acta 1060: 315–318. Nitsch C, Braslavsky SE and Schatz GH (1988) Laser-induced optoacoustic calorimetry of primary processes in isolated photosystem I and photosystem II particles. Biochim Bi­ ophys Acta 934: 201–212. Nitsch C, Schatz GH and Braslavsky SE (1989) Laser-induced optoacoustic calorimetry of primary processes in cells of Rhodospirillum rubrum. Biochim Biophys Acta 975: 88– 95. N’Soukpoé-Kossi CN and Leblanc RM (1990) Application of photoacoustic spectroscopy in photosynthesis research. J Mol Structure 217:69–84. Ort DR and Parson WW (1979a) Flash-induced volume changes of bacteriorhodopsin. J Biol Chem 253:6158–6164. Ort DR and Parson WW (1979b) The quantum yield of flashinduced proton release by bacteriorhodopsin-containing membrane fragments. Biophys J 25: 341–354. Ort DR and Parson WW (1979c) Enthalpy changes during the photochemical cycle of bacteriorhodopsin. Biophys J 25: 355–364. Owens TG, Carpentier R and Leblanc RM (1990) Detection of photosynthetic energy storage in a photosystem I reac­ tion center preparation by photoacoustic spectroscopy. Photosyn Res 24: 201–208. Patel CKN and Tam AC (1981) Pulsed optoacoustic spectros­ copy of condesned matter. Rev. Mod Phys 53: 517–550. Peters KS and Synder GJ (1988) Time resolved photoacoustic calorimetry: probing the energetics and dynamics of fast chemical and biochemical reactions. Science 241: 1053– 1057. Plijter JJ, Aalbers SE, Barends JPF, Vos MH and Van Gor­ kom HJ (1988) Oxygen release may limit the rate of photo­ synthetic electron transport: the use of a weakly polarized cathode. Biochim Biophys Acta 935: 299–311. Poulet P, Cahen D and Malkin S (1983) Photoacoustic detec­ tion of photosynthetic oxygen evolution from leaves – Quantitative analysis by phase and amplitude measure­ ments. Biochim Biophys Acta 724: 433–446. Puchenkov O (1994) Restoration of laser-induced process kin­ etics from photoacoustic measurements. Proc 8th Int Top­ ical Meeting on Photoacoustic and Photothermal Phenom­ ena pp 161–162.

Shmuel Malkin Ravenel J, Peltier G and Havaux M (1994) The cyclic electron pathways around photosystem I in Chlamydomonas rein­ hardtii as determined in vivo by photoacoustic measure­ ments of energy storage. Planta 193:251–259. Reising H and Schreiber U (1992) Pulse–modulated photoac­ oustic measurements reveal strong gas-uptake component at high concentration. Photosynth Res 31:227–238. Reising H and Schreiber U (1994) Inhibition by ethoxyzolam­ ide of a photoacoustic gas uptake: Evidence for carbonic Photosyn Res 42: anhydrase catalyzed 65–73. Rosencwaig A (1980) Photoacoustics and photoacoustic spec­ troscopy. Wiley, New York. Rosencwaig A and Gersho A (1977) Theory of the photoac­ oustic effect with solids. J Appl Phys 47: 64–69. Snel JFH, van Leperen W and Vredenberg WJ (1990a). Com­ plete suppression of oxygen evolution in open PS 2 centers by non-photochemical fluorescence quenching? In: Balts­ chevsky M (ed) Current Research in Photosynthesis Vol 2, pp 911–914, Kluwer Academic Publishers, Dordrecht. Snel JFH, Kooijman M and Vredenberg WJ (1990b) Corre­ lation between chlorophyll fluorescence and photoacoustic signal transients in spinach leaves. Photosyn Res 25: 259– 268. Snel JFH, Polm MW, Buurmeijer WF and Vredenberg WJ (1992) Deconvolution of photobaric and photothermal sig­ nals from spinach leaves. In: Bicanic D (ed) Photoacoustic and photothermal phenomena III. Springer series in optical sciences. Vol 69, pp 65–68, Springer-Verlag, Heidelberg. Tam A C (1986) Applications of photoacoustic sensing tech­ niques. Rev Mod Phys 58: 381–431. Veeranjaneyulu K, Charland M, Charlebois DCN and Le­ blanc RM (1991a) Photosynthetic energy storage of photo­ systems I and II in the spectral range of photosynthetically active radiation in intact sugar maple leaves. Photosynth Res 30: 131–138. Veeranjaneyulu K, Charland M, Charlebois DCN and Lebl­ anc RM (1991b) Photoacoustic study of changes in energy storage of PS I and PS II during state 1 – state 2 transition. Plant Physiol 97: 330–334. Wróbel D and Hendrich W (1989) Thermal deactivation and energy transfer in isolated photosystem II and light-harvesting complexes in polyvinyl alcohol film. J Photochem Photobiol B 3: 319–332

PART TWO

Magnetic resonance

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Chapter 13

Magnetic Resonance: An Introduction

Arnold J. Hoff Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands

Abbreviations: CP-MAS – cross polarization-magic angle spinning; cw – continuous wave; ENDOR – electron-nuclear double resonance; EPR – electron paramagnetic resonance; ESE – electron spin echo; ESP – electron spin polarization; hfi – hyperfine interactions; NMR –nuclear magnetic resonance; ODMR – optically detected magnetic resonance; RC – reaction center

Photosynthetic charge separation results in two ionized cofactors: the primary donor cation and the primary acceptor anion Both are radicals possessing an unpaired spin, which are subsequently stabilized by dark electron transport. The stabilized radicals can be studied by electron paramagnetic resonance, EPR, and its multiple resonance extensions, as electron-nuclear double and triple resonance, ENDOR and TRIPLE. With EPR one looks at the valence electrons, in contrast to X-ray diffraction, which probes the core electrons. Thus, with EPR information can be obtained on the electronic wavefunctions directly involved in the electron transport properties of the cofactor system. The major sources of information offered by cw EPR spectra of doublet states (S = 1/2) are the g-factor (or g-matrix for anisotropic systems) and the hyperfine interactions (hfi). The former is an integral property of the radical, probing the average valence electron density distribution, and serves as a fingerprint for the identification of paramagnetic entities. The latter is a local probe that reflects the density distribution of the unpaired spin at individual nuclei of the radical and its immediate environment. The EPR signals of photosynthetic material are usually too little resolved to permit analysis of hyperfine splittings

and a broad, unstructured line shape results. This inhomogeneous broadening makes it difficult or impossible to measure the hfi directly. Here, mul­ tiple resonance techniques are necessary, and have been widely employed, first in frozen solu­ tion at cryogenic temperatures, later predomin­ antly in liquid solution. In the past few years, single crystals (the dream of every EPR spectro­ scopist!) of bacterial RCs have become available, giving new life to solid state ENDOR. In the past, EPR of doublet states has contri­ buted to the identification of numerous reactants in primary electron transport in bacterial and plant photosystems. This application is still im­ portant, especially in the study of the acceptors in lesser known bacterial systems, such as the green photosynthetic bacteria. There is a trend to enhance resolution by working at higher fre­ quency, and we may expect mm EPR spectro­ scopy (at 95, 135 or even 230 GHz) to become increasingly more important. In addition to the above-described radical doublet states, a paramagnetic excited triplet state may be generated in RCs in which forward elec­ tron transport is blocked, e.g. by prereduction or removal of the secondary acceptor. The photothen must induced radical pair state decay via recombination, which at cryogenic temperatures may yield for almost 100% the triplet state of the primary donor, EPR and optically detected magnetic resonance, ODMR, and

Correspondence: Fax 31-71-5275819; E-mail: [email protected]

209 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 209–210.

© 1996 Kluwer Academic Publishers. Printed in the Netherlands.

210 ENDOR of this triplet state gives information on the molecular structure of the primary donor and its environment. When photolytically produced radicals are studied by EPR one often finds that the relative population of the spin levels is not in Boltzmann equilibrium. Thus, some hyperfine lines or parts of an inhomogeneously broadened line are in em­ ission, or in enhanced absorption. Often both these effects are seen in a single EPR spectrum. This phenomenon is called Electron Spin Polariz­ ation, ESP. It is best seen employing time-resolved EPR techniques, but in some cases the lifetime of the polarization is sufficiently long to make it possible to observe ESP spectra using cw EPR. Analysis of the ESP spectrum gives infor­ mation on the magnetic interactions between the cofactors, which is difficult to obtain in any other way. These interactions in turn report on the elec­ tronic interactions responsible for electron trans­ port. In recent years two variants of time-resolved EPR have come to the fore: direct-detection EPR with a time resolution now approaching a few tens of ns, and pulsed EPR, or Electron Spin Echo (ESE) spectroscopy, with a time resolution of a few hundred ns. ESE spectroscopy offers in addition the possibility to determine small hyper­ fine and dipolar couplings and, in favorable cases, quadrupole couplings of and nuclei, through the so-called Electron Spin Echo Envel­ ope Modulation (ESEEM) phenomenon. With the advent of commercially available instrumen­ tation pulsed EPR is now within reach of a grow-

Arnold J. Hoff ing number of laboratories, and finds increasing­ ly wider application in biophysical research in photosynthesis. Until recently applications of magnetic reson­ ance concerned almost exclusively EPR and its offshoots, as ENDOR, ODMR and reactionyield detected magnetic resonance (RYDMR), with incidental use of NMR for the measurement of relaxation times. With the advent of selective spin-isotope labeling techniques, however, it is now also possible to carry out high resolution, solid state NMR of reaction centers. The appli­ cation of cross polarization magic angle spinning (CP-MAS) solid state NMR to frozen bacterial RCs enriched selectively in and amino acids or cofactors promises to complement and extend structure determination with X-ray analysis. With the above-described array of magnetic resonance spectroscopies an impressive body of data and new insights in the fundamental mechan­ ism of photosynthetic energy conversion has been accumulated, and magnetic resonance now rivals optical spectroscopy as a tool for penetrating the structure-function relationships of the photosyn­ thetic reaction center. In the following chapters (14–18) compact treatments are presented of time-resolved EPR (Levanon), pulsed EPR (Britt), ENDOR (Lubitz), ODMR (Hoff), and solid-state NMR (De Groot). The presentations succintly cover theory and instrumentation, whereas the applications to research in photo­ synthesis are high-lighted with some recent re­ sults.

Chapter 14 Time-Resolved Electron Paramagnetic Resonance

Spectroscopy – Principles and Applications

Haim Levanon Department of Physical Chemistry and the Farkas Center for Light Induced Processes, the Hebrew University of Jerusalem, Jerusalem 91904, Israel

Summary I. Introduction II. Experimental A. TREPR Spectroscopy: General B. Methods of TREPR Detection 1. Light Modulation-Field Modulation (LFM) 2. Continuous Wave Direct-Detection (DDEPR) 3. Pulsed Microwave Detection, Fourier Transform EPR (FTEPR) 4. Comparison between Two TREPR Methods: DDEPR vs. FTEPR III. Results A. Introduction B. Triplet Detection in Anisotropic: Solid and Fluid Phases 1. Introduction 2. Liquid Crystalline Hosts 3. Examples a. Mesogenic Residues Attached to Porphyrins b. Sign Determination of the ZFS Parameter and Triplet Dynamics of Nonplanar Porphyrins C. Electron Transfer 1. Introduction 2. Examples a. Donors and Acceptors with Coulombic Interaction b. Multistep Electron Transfer in Covalently-Linked Assemblies c. Mixed Triplet- and Singlet-Initiated Intramolecular ET in LCs d. Why Liquid Crystals Attenuate Electron Transfer Rates e. Quantum Beats in Correlated Radical Pairs IV. Concluding Remarks Acknowledgements References

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Summary The complexity of natural photosynthesis has prompted intensive studies of simpler model systems that might reproduce essential features of the in vivo apparatus. This is the so-called mechanistic approach, intended to solve some of the pertinent problems still open in primary photosynthesis. In this survey we concentrate on the application of time-resolved electron paramagnetic resonance (TREPR) Spectros­ copy in studying photoinduced processes in model systems. The model systems consist of expanded electron systems as donors, and of covalently as well as non-covalently linked (electrostatically bound) Correspondence: Fax: 972-2-618033; E-mail: [email protected]

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donor–acceptor systems. The required details of the electronic structure and interactions can be deter­ mined by advanced TREPR (and related spectroscopies described in this book) which combines high spectral resolution and time resolution in the 10 –100 ns domain. One of the most important features in TREPR is the electron spin polarization (ESP) effects associated with the reaction products of photoexcited donor–acceptor systems. Therefore, by controlling the environmental conditions, such as solvent polarity, viscosity, temperature, and medium anisotropy, the ESP pattern of such reactions is quite unambiguous. For example, the different line shape patterns reflect the variation of the molecular architecture, namely the relative orientation of the donor–acceptor and the nature of the spacer. The differences in molecular structures are manifested by the TREPR spectra through the magnitude of the spin–spin coupling (J) and the dipolar interaction (D), thus leading to different electron spin polarization mechanisms. In that respect, TREPR has a clear advantage over optical spectroscopy, which lacks adequate energy resolution. It is shown that TREPR applied to donor–acceptor systems, in isotropic and anisotropic liquid crystal (LC) media, allows a better understanding of the role of the microenviron­ ment in long-range ET, a subject directly related to the in vivo protein–chromophore interaction. Abbreviations: CIDEP – chemically induced dynamic electron polarization; CRP – correlated radical pair; CW – continuous wave; DDEPR – direct detection EPR; diff – diffusion; DsA – donor–spacer– acceptor; EPR – electron paramagnetic resonance; ESP – electron spin polarization; ET – electron transfer; EnT – energy transfer; FTEPR – fourier transform EPR; ISC – intersystem crossing; LCs – liquid crystals; LFM – light modulation-field modulation; PSI – photosystem I; PcQ – porphyrincyclohexylene-quinone; PpQ – porphyrin-phenyl-quinone; RPM – radical pair mechanism; RCs – reaction centers; SO – spin orbit; THF – tetrahydrofuran; TM – triplet mechanism; TREPR – timeresolved EPR; ZQC – zero-quantum beats I. Introduction Fifty years have passed since Zavoisky (1945) came out with his first report on electron para­ magnetic resonance (EPR) detection “of liquid solutions” (Zavoisky, 1945a, 1945b). Neverthe­ less, as Zavoisky pointed out, the first discovery of paramagnetic absorption in “solid solutions” (doped crystals of iron and chromium) by calori­ metric methods was made by Gorter in 1936 (Gorter, 1936a, 1936b). In the past 50 years, EPR spectroscopy has been rapidly developing with emerging sub-fields of interest and thousands of publications. Obviously, this chapter is not in­ tended to cover all areas of research associated with EPR spectroscopy. Here, we shall cover bri­ efly the main features of EPR spectroscopy, fo­ cussing on the basic principles of time-resolved EPR (TREPR) and its application in photoind­ uced processes, of which photosynthesis is the most celebrated one. Other interdisciplinary areas are covered separately in this book and in recent reviews (Trifunac et al., 1986; Bixon et al., 1992).

Many photophysical and photochemical reac­ tions in solids and liquids, which involve para­ magnetic intermediates, exhibit electron spin pol­ arization (ESP) effects, indicating deviation from thermal spin equilibrium. Departure from ther­ mal equilibrium may be due to symmetry-driven molecular selection rules, such as selective singlet–triplet intersystem crossing (ISC) or other spin-state mixing mechanisms. Sufficiently high time-resolution, compared to spin relaxation rates, allows for the observation of ESP effects in three different modes: 1) Photoexcited triplet detection and ESP of chromophores (D) (Levanon and Norris, 1978, 1982; Budil and Thurnauer, 1991).

2) Doublet detection and ESP of uncorrelated donor (D) and acceptor (A) pairs, that are photolytically produced by the respective pro­ cesses (Eqs. (2) and (3)), for singlet and triplet precursors. Chemically induced dynamic elec­ tron polarization (CIDEP) in terms of radical

Time-resolved EPR pair mechanism (RPM) or triplet mechanism (TM) is often associated with diffusing radicals (Salikhov et al., 1984; Trifunac et al., 1986).

or

3) Detection of correlated radical-ion pairs was first reported in photosynthetic RCs (Thur­ nauer and Norris, 1980), and was treated quantitatively in later studies (Buckley et al., 1987; Closs et al., 1987; Hore, 1989; Stehlik et al., 1989; Morris et al., 1990). Correlated radical pair (CRP) polarization, observed in radical pairs held together in micelles (DA) (Closs et al., 1987), or by Coulombic interac­ (Hugerat et al., 1991; Zilber tions et al., 1992; Rozenshtein et al., 1993), or by a spacer s at a fixed distance, (DsA). Similar to the previous case, CRP can also be formed either via a singlet or a triplet precursor:

or

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the spin-correlated biradical state, representing coupled donor–acceptor pairs (Eqs. (4), (5)). The chemical assemblies (representative struc­ tures are shown in Fig. 1), which are engaged in photoinduced energy- and electron-transfer (ET and EnT, respectively) reactions and which are of interest to model photosynthesis, can be gro­ uped as: 1) traditional porphyrin and chlorophyll systems; 2) porphyrin-quinone systems (Fig. 1, 1– 3), which are linked together either covalently via a spacer, DsA (Bixon et al., 1992; Wasielewski, 1992), or via hydrogen bonds (Fig. 1, 4) (Sessler et al., 1993) or electrostatically bound porphyrins (Hugerat et al., 1991); 3) nonconventional por­ phyrinoids, e.g., the expanded porphyrins, such as sapphyrin (Bauer et al., 1983), the texaphyrins (Sessler et al., 1988, 1989), the porphycenes and stretched porphycenes (Fig. 1, 5) (Berman et al., 1993); and 4) non-porphyrinoid systems which consist of ion-pairs of doubly charged organic –systems and alkali metals (Zilber et al., 1992; Rozenshtein et al., 1993, 1994) and the novel carbon clusters, the fullerenes (Levanon et al., 1993, 1994; Regev et al., 1993; Michaeli et al., 1994). The fullerenes will not be covered here, and the reader is referred to a review (Levanon et al., 1994). Finally, specific environments in which these assemblies are embedded in will also be dis­ cussed; in particular, liquid crystal (LC) hosts, where the interacting species are confined to an anisotropic medium with reduced rotational and translational degrees of freedom (Levanon, 1987). II. Experimental

A. TREPR Spectroscopy: General In this chapter we confine ourselves to systems that follow reactions (1–5) and can be studied by either one, or two modes of TREPR, i.e., (CW) continuous wave and/or pulsed-TREPR. Since several electronic states are involved in photophysical and photochemical processes, the sys­ tems will be described in their different paramag­ netic states. These include: 1) photoexcited triplet state of chromophores (Eq. (1)); 2) doublet-state radicals, representing in-cage and charge-separated intermediates (Eqs. (2) and (3)); and 3)

Studying photoinduced ET or EnT processes, re­ quires time-resolved spectroscopic techniques. Traditionally, optical methods are used to study ET events, and new methods in the pico- and even femto-second time scale are one path toward understanding primary charge-separation process in photochemical events (Wasielewski, 1992; Jra et al., 1993; Martin, JL et al., 1993). Neverthe­ less, any increase in time resolution is ac­ companied by a decrease in reliable spectral res­

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olution because of the uncertainty principle, leading to a loss of vibrational, fine, and hyperfine structures (Bixon et al., 1992). Magnetic reson­ ance has the potential for much higher spectral resolution (KHz for EPR, and less than 1 Hz for NMR), which is compensated by a reduction in time resolution that is on the edge of the typical formation kinetics of a paramagnetic species. With ESP present, TREPR spectroscopy satisfies the need for accurate data of good sensitivity on kinetic rates and also provides spectra of the transient species (Levanon and Norris, 1978, 1982; McLauchlan, 1990; Budil and Thurnauer, 1991; Bixon et al., 1992). Moreover, develop­ ments in microwave technology resulted in the construction of high-frequency (high-field) EPR spectrometers. High-field studies, in particular combined with light excitation, are promising in

Haim Levanon

structure determination, a subject which is of prime interest in photosynthesis. Indeed, there are already reports at 250 GHz G for g = 2) (Budil et al., 1989), at (Lebedev, 1990), at 140 150 GHz GHz (Prisner et al., 1993), at 94–95 GHz mm) (Burghaus et al., 1991, 1992; Klette et al., 1993; Van der Est et al., 1993; Wang,Wet al., (Box et al., 1994), or at 70 GHz 1979). In this chapter we confine ourselves to TREPR studies of photoinduced processes, with a few examples described, employing 3 basic methods of TREPR, in the X-band region (~9.5 GHz).

Time-resolved EPR

B. Methods of TREPR Detection 1.

Light Modulation-Field Modulation (LFM)

This earliest method of TREPR combines light modulation and magnetic field modulation (LFM) with phase-sensitive detection (Levanon and Weissman, 1971; Levanon, 1979), as depicted schematically in Fig. 2. The light sources in LFM experiments may consist of either continuous ex­ citation being chopped by a mechanical sector, or a modulated light source (e.g., xenon arcs) with a varying frequency between 1–1000 Hz. This method suffers from relatively poor time resol­ dictated by the specution of about 20 trometer’s band width of 10 KHz, governed by the field modulation frequency of 100 KHz, com­ mon to most commercial EPR spectrometers (Hoff et al., 1977; Poole, 1983). The relatively low time resolution of LFMEPR, together with the significant advancements in EPR detection (see below), makes this EPR detection method less common in time-resolved experiments. 2. Continuous Wave Direct-Detection (DDEPR) In this experimental setup (Fig. 2), referred to as CW direct-detection EPR (DDEPR), the timedependent signal is produced via wavelength se­ lective laser excitation (Kim and Weissman, 1979; Furrer et al., 1981; Weissman, 1982; Gonen and Levanon, 1985; Budil and Thurnauer, 1991). The signal, generated under CW microwave exci­ tation, is taken from the preamplifier which is connected directly to the microwave diode detec­ tor, with care to avoid any interference from the 100-KHz filters. This signal is then fed into an automatic back-off amplifier triggered prior to the laser pulse, ensuring the transient signal is on a dc level. The output signal is fed into a digitizer interfaced to a computer on-line with the experi­ ment. Kinetic traces, that is the magnetization, for each field position are acquired and stored. A series of complete TREPR spectra, at are re­ different times after the laser pulse constructed from these traces by slowly stepping the magnetic field (only one point in the EPR spectrum is followed as a function of time after each laser pulse). In this experimental setup the

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earliest detectable signal is at about 50–150 ns after the laser pulse. These time resolutions allow the detection of Torrey oscillations (Torrey, 1949) as well as quantum beats (Kothe et al., 1994) (see below). 3. Pulsed Microwave Detection, Fourier Transform EPR (FTEPR) While pulsed techniques represent the bulk of modern NMR spectroscopy, technical limitations associated with the considerably faster time re­ gime and high microwave power delayed a paral­ lel development in EPR spectroscopy. These technological difficulties have been overcome within the last decade, leading the way to con­ siderable progress which is described in recent books and review articles (Budil et al., 1989; Bowman, 1990; Schweiger, 1991; van Willigen et al., 1993). We will restrict the discussion here to the application of those techniques which are of relevance to photosynthesis. Pulsed EPR spectroscopy combined with laser excitation has been used with either electron spin echo (ESE) detection (Norris et al., 1978; Thur­ nauer et al., 1979) or Fourier transform (FTEPR) detection with time resolution of about 10 ns (An­ gerhofer et al., 1988a, 1988b; Bowman et al., 1988; Prisner et al., 1988; Bowman, 1990; van Willigen et al., 1993). In a typical FTEPR experiment (Fig. 2), the magnetization is created by a short laser pulse (the same as in DDEPR). At selected delay times after the laser pulse, a short and intense microwave pulse (typically, 16–24 ns, 1–2 KW) is applied to the spin system. The resulting free induction decay (FID) signal is digitized and Fourier transformed to obtain the spectrum. The spectrometer response time, called the dead time limits the time resolution and causes phase distortions in the spectrum. Linear prediction, (Barkhuijsen et al., 1985, 1986) may be used to reconstruct the FIDs within the dead time regime. In this way, both the spectral and time resolutions are improved, i.e., it becomes possible to gather information in the time slice where the laser and microwave pulses overlap with a ~ 10 ns time resolution between time slices. For sufficiently strong signals one can ob­ serve the first-order kinetics of the magnetization

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Time-resolved EPR decay (via spin lattice and chemical processes) corresponding to half-lives of ns (Norris et al., 1980). 4. Comparison Between Two TREPR Methods: DDEPR Vs. FTEPR These two methods avoid the constraints of field modulation, resulting in time resolution enhance­ ment (compared to LFM) by three orders of mag­ nitude, down to less than 100 ns. The combi­ nation of laser excitation with either DDEPR or FTEPR provides a good match between time resolution and the time scale of electron- and energy-transfer reactions, with a positive identi­ fication of the species detected. Also, the two techniques are complementary to each other; DDEPR works well with broad EPR lines and large spectral widths, while FTEPR is best with narrow spectra having sharp lines. In that respect, the main advantage of using FTEPR is the possi­ bility of detecting transient radicals within a few nanoseconds (~10 ns) after laser excitation, and conducting sophisticated pulse experiments (Schweiger, 1991) combined with light excitation. Thus, the time window between 10 and 200 ns becomes available by the employment of DDEPR and FTEPR. In typical experiments, the laser pulse initiates a reaction producing paramagnetic species, free radicals, radical pairs, or triplets. In most cases, the paramagnetic species having only magnetiz­ along the applied magnetic field, can ation, be completely characterized by the populations in the different spin levels. The time dependence of the populations is sufficient for determining the reactions and rates involved. In FTEPR, a microwave pulse converts which is not readily measurable, into and which forms the observable FID signal. Any additional triplets or radicals, bom after the microwave pulse, gen­ and do not contribute to the FID. erate new Any chemical process destroying radicals or tri­ plets produces line broadening and affects line shapes, but will not change the integrated inten­ sity of the lines in the spectrum (Bartels, 1988; Bowman, 1990). Thus, FTEPR gives a spectrum of the originally present at the time of the microwave pulse with little complication from re­ actions occurring after the pulse.

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In DDEPR, a weak CW microwave field con­ into tinuously converts a small portion of which are then detected. The and and persist for a time approximately equal to the inverse of the linewidth. Thus, the DDEPR signal over a time window represents the integrated A second feature of DDEPR is that of about the signal at early times is distorted by the finite response time of the spin system (Hasharoni et al., 1991; Levanon and Bowman, 1993). Regard­ ing CW spectrometers, a serious drawback of DDEPR method may be associated with the pos­ sible misinterpretation of the spectra, taken im­ mediately after the laser pulse, of small hyperfine splittings and narrow linewidth (Hore and McLauchlan, 1979; Basu et al., 1984; Hasharoni et al., 1991), due to the appearance of Torrey oscillations at off-resonance fields. At short times, the spins precess, as described by the Bloch equations, about the effective magnetic field. This precession produces transient signals for a time on either side of each hyperfine line. Thus, at this effect produces spectra times shorter than which have a very different appearance than spec­ tra of the same radicals at longer times. We em­ phasize this point since the experimental results which exemplify the theoretical predictions are scarce although a relevant example has been dis­ cussed (Hasharoni et al., 1991). It is noteworthy that Torrey oscillations have been detected and analyzed while investigating the spin dynamics of triplet fullerenes (Regev et al., 1993). Within the context of photoinduced intermol­ ecular ET reactions involving triplet porphyrins (or chlorophylls) as precursors (donors), and con­ ventional quinones (acceptors), both method of detection are essential and complementary (Levanon and Bowman, 1993). In reactions with radical-ion products of small hyperfine coupling con­ stants and narrow line widths (e.g., quinones), it has been demonstrated that the pulsed microwave method is more advantageous (Hasharoni et al., 1990, 1991; van Willigen et al., 1993). Neverthe­ less, one has to consider also that the excitation bandwidth of pulsed EPR is presently limited to about 100 MHz, insufficient for many EPR appli­ cations, particularly in the slow motion regime, typical for reaction centers (RCs) investigations. In summary, DDEPR and FTEPR applications

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exist in most photosynthetic and model system studies. III. Results

A. Introduction In recent years, several groups have been in­ volved in pursuing the mechanistic approach in model photosynthesis, that is relevant to primary photosynthesis on the molecular level (Bixon et al., 1992). This approach seeks to reproduce the states associated with basic features of energy transfer and charge separation in photosynthesis. While simple porphyrins fail to mimic natural photosynthesis, they are important in im­ plementing novel techniques in time-resolved spectroscopy. In that respect, the covalentlylinked porphyrin-quinone systems are successful in many respects in the mechanistic approach (singlet-initiated ET and long-lived charge-separated states) but, nevertheless, also fail to mimic natural photosynthesis, because the in vivo chro­ mophores are not covalently linked via spacer groups. The base-paired porphyrin-quinone sys­ tem (4 in Fig. 1) are more closely related to the in vivo donor acceptor species (Sessler et al., 1993). Thus, with regard to the mechanistic ap­ proach, although the porphyrin moiety is the leading structure in these studies, we do not ex­ clude other chemical systems which may be perti­ nent to the mechanistic point of view.

B. Triplet Detection in Anisotropic: Solid and Fluid Phases 1. Introduction In Fig. 3 we show the two mechanisms of triplet formation via ISC routes, i.e., spin-orbit (SO) vs. radical pair (RP). By differentiating these me­ chanisms, the triplet state becomes a powerful diagnostic probe for structure determination (Le­ vanon and Norris, 1978, 1982; Budil and Thur­ nauer, 1991). In fact, the triplet state is appropri­ ate for probing the photoexcited singlet state, which is not accessible to EPR techniques. Parameters that affect a triplet EPR spectrum are: 1) values and signs of the zero-field splitting (ZFS) parameters, D and E; 2) selective ISC rates

of triplet sub-level formation, that are related to ESP effects; and 3) anisotropy of the environment in which the triplet chromophores are embedded. The former two aspects were dealt extensively in the literature cited above. Here, we shall focus on LCs. Rigid isotropic matrices lack the free molecular motion essential for diffusion in energy and electron-transfer reactions, while LCs can maintain their anisotropic properties in the fluid phase in accommodating and orienting guest chromophores (Fessmann et al., 1988; Regev et al., 1990, 1991a; Van der Est et al., 1990). 2. Liquid Crystalline Hosts The utilization of LCs as host matrices for large molecules facilitates the triplet EPR spectral analysis due to the partial orientation of the guest solute in the LC (Levanon, 1987). In a uniaxial nematic phase characterized by a positive aniso­ tropic diamagnetic susceptibility, the long axes of the LC molecules are aligned along a preferred orientation, defined by a macroscopic quantity called the director, L. A common LC is E-7, which is a mixture of biphe­ nyls (Levanon, 1987), whose phase transitions are:

In such a uniaxial LC the partial orientation im­

Time-resolved EPR

poses a cylindrical distribution of the solute about L having the following effects on the EPR spec­ trum (Fig. 4). (a) case: since the chromophore planes are distributed axially about L, there is a vanishing probability for the out-of-plane axis to be parallel to B, leading to the total exclusion of the Z lines from the spectrum. (b) case: by rotating the frozen sample by in an axis perpendicular to B, there is a

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non-vanishing probability of finding any one of the three canonical orientations parallel to B. The overall effect from such organization is an im­ proved S/N ratio and suppression, or enhance­ ment, of various line in the EPR spectrum due to alteration of the probability of certain molecu­ lar axis to be parallel to B. A different class of LCs (e.g., ZLI-1167 (Pohl and Eidenschink, 1978; Regev et al., 1991a)) are characterized by a negative anisotropic diamag­

Haim Levanon

220 netic susceptibility, i.e., A implies that all phases consist of do­ negative mains having directors spanned in a plane normal to the external magnetic field (Fig. 4). In such a case the triplet EPR spectra exhibit spectral fea­ tures which are reversed from those found with These properties are of extreme impor­ tance when EnT and ET are monitored by TREPR in the fluid phases of these LCs. While triplet EPR of aromatic with typical ZFS are difficult to detect in isotropic flu­ ids (Weissman, 1958; McLauchlen et al., 1994), the reduction in rotational degrees of freedom in fluid LCs makes triplet EPR detection feasible (Fessmann et al., 1988; Regev et al., 1990, 1991a; Van der Est et al., 1990). Inhibition of spin-rotational correlation times in the organized photo­ synthetic apparatus is the reason why the “special pair” triplet is EPR detected at 296 K (Hoff and Proskuryakov, 1985). Hence, EPR spectroscopy may, in many respects, compete with optical me­ thods for dynamic processes. This approach was implemented by studying the molecular dynamics of photoexcited triplet states of chlorophylls and related chromophores, incorporated into LC matrices, over a wide temperature range. Triplet EPR spectra of porphyrins, porphycenes, and texaphyrins, oriented in the frozen nematic phase of uniaxial symmetry, indicate that the director, L, is aligned in the plane of the porphyrin ring (Levanon, 1987) (see also Fig. 4). This yields unique probabilities, dictated by the LC symme­ try, for each of the principal axes of the ZFS tensor to be parallel to the external magnetic field. The resulting EPR spectra are highly aniso­ tropic showing the greatest differences when the director, L, is either parallel or perpendicular to the external magnetic field, B. The relation between order properties of the host matrix (LC) and the molecular structure of the guest-oriented chromophore is reflected in the triplet magnetic parameters (e.g., ZFS and spin dynamics) deter­ mined by line shape analysis. Such analysis allows to determine energytransfer processes in coval­ ently linked dimers (Gonen and Levanon, 1986; Regev et al., 1986) and, most importantly, relates molecular structure to magnetic dipolar coordin­ ate system (Gonen and Levanon, 1986; Regev et al., 1989). Let us examine a few examples.

3.

Examples

a. Mesogenic Residues Attached to Porphyrins Attaching mesogenic residues or “arms” ortho­ gonal to the porphyrin ring (Fig. 5) yields porphy­ rins and ZnMesogenP) compatible with standard liquid crystal compounds (Neum­ ann et al., 1991; Michaeli et al., 1992). This chemical modification is sufficient to change the orientation of the porphyrin ring from parallel to perpendicular to the director, L, as shown by the EPR line shape of the triplet spectrum. For the hypothetical case, where the mesogenic arms are infinitely long, the out-of-plane magnetic axis of the porphyrin is expected to be collinear with the director, L. For the other extreme case, where the mesogenic arms are infinitely short, i.e., con­ ventional porphyrins, the LC director lies in the porphyrin plane. These two cases are illustrated in Fig. 5, where the triplet spectrum of clearly exhib­ enP is compared to that of iting out-of-phase relationship between the two triplets.

b. Sign Determination of the ZFS Parameter and Triplet Dynamics of Nonplanar Porphyrins It is well established that the ZFS parameter, D, that characterizes the photoexcited triplet, is an important diagnostic probe, in terms of electron distribution and spin alignment, as was demon­ strated on sapphyrin dication in ethanol and the nematic E-7 (Levanon et al., 1990; Regev et al., 1991b). Although the absolute value of D is easily deduced from EPR spectra, an unambiguous sign determination at relatively high temperatures (~100 K) is not straightforward and thus requires experiments to be carried out at liquid helium temperatures (Hornig and Hyde, 1963) or by per­ forming magnetophotoselection measurements (Thurnauer and Norris, 1976). This method has been implemented for the absolute sign determi­ nation of D in triplets of sapphyrin dication mon­ omers and dimers (Regev et al., 1991b). The anisotropic LC properties and the triplet spectra associated with them can also be utilized by the sign determination of D, as demonstrated

Time-resolved EPR

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with several chromophores (Levanon et al., 1990; Berman et al., 1993). The principles of sign deter­ and mination are demonstrated below. acetylene-cumulene porphycenes (“stretched porphycenes”) represent coherent with respect to molecular dimensions and symme­ try (Jux et al., 1990; Vogel et al., 1990; Martire et al., 1993; Vogel, 1993) (5 in Fig. 1 and Fig. 6). TREPR experiments of their photoexcited triplet state were carried out in isotropic (toluene) and anisotropic (a nematic LC) matrices (Berman et al., 1993). While “stretched porphycenes” exhibit a red-shift in their absorption spectra, as a func­ tion of their molecular length, the magnitude of the ZFS parameter, D, does not exhibit such a dependence. This apparent anomaly was in­ terpreted in terms of Eq. (6), where the sign and magnitude of the ZFS parameter, D, should be determined not only by the molecular length, but also by the triplet spin alignment, with respect to the molecular plane. From the point dipole– dipole approximation, the dipolar interaction is given by Levanon and Norris (1978, 1982). where and r are the relative orientation and distance between the dipoles, respectively. In the case of nearly square porphyrins and related chro­ mophores, the side-by-side triplet spin alignment i.e., D>0. The headcorresponds to to-tail spin alignment, which is the case in the “stretched porphycenes” (Fig. 6) and sapphyrin i.e., (Levanon et al., 1990), corresponds to D < 0. These two limiting cases can be easily differentiated by the EPR spectra taken in the LC matrix, at the parallel, and perpendicu­ lar, configurations (Berman et al., 1993). A related research area is associated with the triplet dynamics, as studied by TREPR, of nonplanar porphyrins. It is noteworthy that unlike the traditional planar porphyrins (e.g., triplet state can probe the consequences of nonplanarity on the photophysics of porphyrins. Such a study has been reported recently on the nonplanar free base and zincoctaethylporphyrins, and oriented in a ne­ matic LC (Regev et al., 1994). Again, the use of the LC has a two-fold purpose: as demonstrated earlier, triplet spin dynamics can be studied over a wide range of temperatures not possible with

Haim Levanon conventional chromophores in isotropic solvents (Regev et al., 1990, 1991, 1993); and it serves as a primitive model of the effects organized protein environments may have on nonplanar chromoph­ ores in vivo. Analysis of the EPR spectra of and in the LC, as a func­ tion of temperature, suggests fast exchange pro­ cesses between different conformers attributable to variable excursions from the canonical X-ray structures of the porphyrins in their ground state. These types of studies are relevant to the mech­ anistic approach, where TREPR spectroscopy al­ lows to follow conformational changes of porphy­ rins a process that, most likely, occurs in the photosynthetic apparatus, i.e., the chromophores packing in the proteins.

C. Electron Transfer 1. Introduction Charge separation and ET processes are funda­ mentally important in photochemistry and photo­ biology with a substantial number of applications. Numerous donor–acceptor systems have been studied by fast and ultrafast optical methods as well as by fast time-domain EPR spectroscopy. The first observation by Tollin and Green (1962) of the EPR signal from the benzoquinone anion radical in illuminated solutions of chlorophyll and benzoquinone (Tollin and Green, 1962) started a series of TREPR studies of porphyrin-based chromophores. The first applications of FTEPR to light-driven intermolecular ET reactions were carried out by Bowman and Massoth (1987) (Massoth, 1987) fol­ lowed by Prisner et al. (1988) and Angerhofer et al. (1988a). Prisner et al. (1988) reported first on the ET reaction between and duroqui­ none, exhibiting strong polarized EPR signals at in Fig. very early times after the laser pulse 2c) that decayed over the course of a few micro­ seconds. Currently, many FTEPR experiments, utilizing photoexcited porphyrin or porphyrinlike donors and quinones acceptors, are well de­ scribed in the literature (Levanon and Bowman, 1993; van Willigen et al., 1993). The ET reactions, as depicted by Eqs. (2)– (5), represent the different approach of utilizing covalently linked donor–spacer–acceptor rigid

Time-resolved EPR

systems. Covalently linked chromophores have been used by Closs and Miller (1988) to study the ET in system of well defined geometry and energy (Closs and Miller, 1988). These landmark studies have contributed significantly to the under­ standing of ET in general and thereby serve as a

223

basis for understanding many of the chemical events in natural and model photosynthesis. Although numerous rigid model systems exist in the literature, only a few studies report spin polarized TREPR-spectra following light exci­ tation (Hasharoni et al., 1990, 1991, 1993; Wasie­

224

lewski et al., 1990; Lendzian and von Maltzan, 1991). The main reason for the scarce data is that charge separation and recombination are too fast (ps to a few ns) for EPR detection. Attempts to circumvent these constrains have been pursued by modifying the chemical architecture of the co­ valently linked compounds, and/or by controlling the environmental conditions, such as solvent polarity, viscosity and temperature. In those cases where these goals have been achieved, DD and FTEPR are well suited for characterizing the spin state and spin dynamics of charge-separated spe­ cies. As expected, triplet initiated ET reactions are most commonly observed, whereas singlet in­ itiated ET reactions are often too fast to be di­ rectly monitored by EPR. They can, however, be deduced from the spin dynamics of the transient radical pairs involved. 2. Examples

a. Donors and Acceptors with Coulombic Interaction While all the above-mentioned experiments in­ volve neutral photoexcited states, which are in­ volved in the ET reactions, only a few experi­ mental (Depew and Wan, 1986) and theoretical (Pedersen and Freed, 1974) studies are related to ESP in systems with Coulombic interaction of charged species. In principle, an attractive interradical potential can strongly affect the lifetime of the RP in the cage and, thus, effectively dimin­ ish a rate of diffusion from the cage. It is expected that this type of ion-pairing should result in a reduction of diffusion rates, which can be mani­ fested as specific CIDEP and CRP patterns. To achieve these purposes an classic system of basic importance (Paul et al., 1956; Hoijtink et al., 1961) was chosen to be investigated (Zilber et al., 1992; Rozenshtein et al., 1993). The system consists of ion-pairs of doubly charged pyrene (Py) and alkali metals (M = Li, Na, K, Rb, Cs) in tetrahydrofuran (THF) solutions, Py/M/THF. The basic experiments utilize FTEPR spectros­ copy combined with pulsed laser excitation (Fig. 2c). The reduction of Py by alkali metals in THF predominantly results in the formation of two ion-paired complexes in equilibrium (Friedenberg and Levanon, 1976; Eliav et al., 1981):

Haim Levanon

Depending upon the nature of the metal cation, these ion-paired complexes either exist as solventseparated ion-pairs, or as contact ion-pairs. Counter ions with large atomic radii, like are likely to form contact ion pairs, where the average metal–metal distance in the complex is calculated to be ap­ proximately 12 Å. For metal ions with smaller and the metal–metal dis­ radii, like tances are assumed to be increased by the in­ clusion of two solvent molecules, one intervening between each metal atom and Py (Rozenshtein et al., 1993). The formation of solvent-separated ion-pairs can also be enforced with large metal by the use of chelates, such as ions like cryptands that bind selectively alkali-metal cat­ ions (Friedenberg and Levanon, 1976; Rozensh­ tein et al., 1993). Photoexcitation of results in the formation of an excited singlet state that decays by ISC to a longer-lived polarized triplet,

The polarized triplet state is the precursor for subsequent ET resulting in paramagnetic states that obey different ESP mechanisms (Zilber et al., 1992; Rozenshtein et al., 1993):

The RP-cage size, is in the order of a few Å, with a characteristic diffusion time of where D is the mutual diffusion coef­ ficient. Thus, the experimental setup allows to investigate in-cage processes (e.g., CRP), which are characterized by Recalling that for normal liquids we expect that nanosecond EPR detection allows to observe directly the RP in fluids with nearly

Time-resolved EPR

normal viscosity. The observed spin dynamics in these systems covers different CIDEP and CRP mechanisms, and by proper tuning of the Coulombic-solvent interactions via specific alkali metal and solvation conditions, the various ESP effects can be controlled and differentiated. The resulting FTEPR spectra are displayed in Fig. 7. Except for the Py/K/THF system, all other sys­ tems exhibit practically the same CIDEP effects. The dynamic behavior of these systems are un­ ique examples that combine together, within the same chemical system, properties that bear a di­ rect relation to primary photosynthesis: 1) a fixed donor–acceptor distance due to the restricted dif­ fusion, as implemented by the CRPM; and 2) time evolution of the RP constituents developing normal CIDEP effects. The remarkably similar

225

CIDEP effects observed with Li, Na, Rb, and Cs (triplet-initiated), and the exceptionally different effects observed with K (singlet-initiated), strongly suggest that these alkali-metal systems are new examples of the inverted region in the Marcus theory (Marcus, 1964; Marcus and Sutin, 1985).

b. Multistep Electron Transfer in Covalently-Linked Assemblies Time-resolved optical studies of a carotenoid-free base porphyrin-diquinone tetrad (1 in Fig. 1) and related compounds (Gust and Moore, 1989, 1991; Gust et al., 1990; Wasielew­ ski, 1992) suggest that optical excitation of the porphyrin moiety produces a series of ET steps.

226

An electron is transferred from the porphyrin in its lowest excited singlet state to the adjacent naphthoquinone moiety, with a rate of to produce a radical-ion pair, This initial state is thought ultimately to produce on the basis of the charac­ teristic optical absorption of the carotenoid rad­ ical cation. The quinone anion was not detected optically and no direct evidence of the spin mul­ tiplicity was obtained. TREPR in liquid solution should remove the ambiguity concerning which quinone the unpaired electron resides on, prior to the back reaction. For an unambiguous charac­ terization of the radical pair, pulsed-and CW­ EPR studies were employed (Hasharoni et al., 1990, 1991). Indeed, EPR evidence (assignment of the hyperfine splittings) indicates that the charge-separated product involves the end-quinThe two detection methods confirm the one experimental validity and the origin of the derivative-like spectra assigned to the end quinone radical interacting with the carotenoid cation radical This interaction produces the derivative-like spectra via a CRP mechanism. Although both methods detect and identify the radical anion in the FTEPR detects both end radicals and and, more impor­ tantly, confirms the lifetime of the state and its singlet-state genesis. Another example is the singlet-initiated charge separation in a rigid triad, TAPD–ZnP–NQ, where the porphyrin is situated between the di­ amine and naphthoquinone (Wasielewski et al., 1990). Light excitation of ZnP is followed by pri­ mary charge separation to singlet radical-pair with subsequent hole transfer on a sub-nanosecond time scale to form the CRP, with a center-to-ceni.e., ter distance of 23 Å between TAPD and NQ, very similar to the reaction center case. The ob­ served ESP pattern at X-band, as well as the temperature-independent EPR spectra (in the range of 5–77 K), resemble strikingly that of bac­ terial RC and PS I (Hoff et al., 1977; Petersen et al., 1987).

c. Mixed Triplet- and Singlet-Initiated Intramolecular ET in LCs The original study by TREPR (Lendzian and von Maltzan, 1991) showed that photoinduced ET,

Haim Levanon in a porphyrin linked to benzoquinone via an aromatic phenoxy spacer (diade, P–Q), is tem­ perature and solvent viscosity dependent. In low viscous solvents at relatively high temperatures, singlet-initiated ET is the dominant mechanism to produce the charge-separated state, whereas by increasing the solvent viscosity and by lower­ ing the temperature, triplet state population be­ gins to compete with the singlet state. The ener­ getics associated with the switched-ET processes is depicted in Fig. 3. At high temperatures, where the singlet precursor dominates, the reactions are too fast for EPR detection, while in the soft glass region the triplet channel is operative (i.e., ISC and subsequent triplet ET can compete with sing­ let ET), thus allowing EPR detection. The TREPR experiment, carried out in the soft glass, results in spectra exhibiting two fea­ tures, the first is the broad triplet spectrum of the porphyrin moiety; and the other is a narrow signal at g ~ 2, which was identified as the triplet state spectrum of the RP. Replacing the aromatic spacer with an aliphatic one (cyclohexylene) (Lendzian et al., 1991) did not change signifi­ cantly the interactions present within the RP. These observation were interpreted in terms of a cross-over of the ET from the nonadiabatic limit to the solvent controlled adiabatic limit. This is made possible by slowing down the dielectric relaxation time of the solvent via temperature decrease and viscosity increase. Evidently, EPR studies of ET in model systems embedded in iso­ tropic media are limited to a small range of tem­ peratures, in which both triplet and ET products can be observed concurrently. EPR triplet detection in fluid phases of LCs (Regev et al., 1990) was the impetus of employing these matrices in studying ET reactions, under the influence of: 1) the directionality of the mol­ ecular entity imposed by the LC director, with respect to the external magnetic field; and 2) the different dielectric properties of the LC matrix. This approach was practiced with different DsA molecules oriented in LC solutions (Hasharoni et al., 1995). These molecules are the cis and trans isomers of zincporphyrin-cyclohexylene-quinone (t-PcQ and c-PcQ, the cis isomer is shown in Fig. 1 (3) and the trans is shown in Fig. 8) (Hasharoni et al., 1993); the para and meta isomers of zincporphyrin-phenyl-quinone (p-PpQ and m-

Time-resolved EPR

PpQ), and zincporphyrin-amide-lumiflavin (3 in Fig. 1) (Hasharoni et al., 1995). The results ob­ tained employing LCs are different from those in the isotropic ethanol, with respect to spin dynam­ ics and solvent-solute interactions. In fact, the latter three compounds do not exhibit any TREPR spectra in ethanol. In Fig. 8 we present the time evolution of the EPR spectra at different temperatures. Below 200 K, only one spectrum is observed and is attributed to the triplet of the porphyrin, with no apparent mixing of additional spectra. Upon increasing the temperature a relatively narrow g ~ 2.00 signal starts to emerge and superimposes on the broad triplet spectrum of the porphyrin moiety. The narrow spectrum is that of and its time evolution corresponds to the disappearance of the triplet spectrum. This

227

behavior, where mixed spectra are detected, is noticed over a wide temperature range of 210– 330 K, covering the entire nematic (i.e., fluid) phase of the LC, for this particular LC. In the case of t-PcQ and p-PpQ the experiments clearly show that above 280 K the RP spectrum changes phase, implying a switch from triplet-initiated to singlet-initiated ET. The TREPR spectra describ­ ing this dramatic effect are shown in Fig. 8. Unfortunately, these model systems fail to mimic the photosynthetic reaction center with re­ spect to temperature dependence of the ET rates. Nevertheless, despite this discrepancy the model systems are important, when considering the me­ chanistic point of view, as the spin dynamics of charge separation in the model and the in vivo cases are very close.

228

d. Why Liquid Crystals Attenuate Electron Transfer Rates Numerous intramolecular electron transfer (ET) reactions, in solutions at room temperature, pro­ ceed with rates of and are moni­ tored by transient optical spectroscopies in the picosecond time scale (Wasielewski, 1992; Jra et al., 1993; Martin, JL et al., 1993). Although op­ tical methods allow one to extract the actual rate their ability to iden­ constants of the process, tify unambiguously the constituents in a multi­ component ET process is rather limited. Contrary to optical spectroscopy, as was demonstrated above, TREPR is unique in its ability to identify transient radicals (Bixon et al., 1992; Levanon and Bowman, 1993). However, its time resolution is in the 50 –100 ns range, thus restricting its appli­ cation in ultrafast photochemical ET processes. Evidently, should be reduced by 4–6 orders of magnitude so that EPR detection will be feas­ ible. This is the main reason why TREPR results of ET in fluids are quite scarce. The inability of isotropic solvents to be em­ ployed in TREPR studies of intramolecular ET is removed by using LCs. The main reasons ac­ counting for this are two fold: 1) the anisotropic properties of LCs; and 2) the temperature range of TREPR detection covers the whole nematic phase of the LC (solid and liquid), thus extending the ET detection to a temperature range that may exceed 100 degrees (see phase transition tempera­ tures above). These advantageous properties of LCs, over isotropic solvents, was explained re­ cently (K. Hasharoni and H. Levanon, 1995), by linking together the external field effect on the dielectric properties of LCs (Carr, 1962; Meier and Saupe, 1966; Martin, AJ et al., 1971), and the internal field change imposed by the ET process (Sumi and Marcus, 1986). Such a relationship can be understood in terms of the coupling between the molecular modes (the origin of the ET) and the solvent modes. During the ET process, a nonequilibrium charge distribution is formed around the molecule. To acquire a new charge distribu­ tion about the newly formed charge-separated state, the solvent dipoles have to change their orientation about the charge. This dielectric relaxation is characterized by the solvent longi­ that is retudinal dielectric relaxation time,

Haim Levanon lated to the Debye relaxation time. In the solventcontrolled adiabatic limit of the ET, is the rate-determining step of the ET process. Thus, to detect a fast EPR response, the reaction dynamics should be brought into that limit which can be controlled by varying the viscosity and tempera­ ture. This is where isotropic solvents and LCs acts as a rate-determindiffer. In the former, ing step only in a narrow temperature range (ap­ proximately 20–30 degrees). Beyond this range, the reaction becomes nonadiabatic, i.e., it de­ pends only weakly on the solvent characteristics. With LCs, due to the existence of a potential barrier, the nematic potential hinders an isotropic rotation (Meier and Saupe, 1966; Martin, AJ et al., 1971). The variation of upon increasing of the temperature is very slow. When an electric field is applied to the LC, for example if an ET process occurs within the solute molecules, the is slow because the LC dipoles do change of not rotate freely as in the isotropic case. This is the origin of the rate attenuation in LCs. Eq. (13) shows how the Debye dielectric relaxation time in a LC, is reduced with respect to its value in an isotropic solvent, (Martin, AJ et al., is closely related to via 1971) (note that the dielectric constants):

where g is the retardation factor and q is the nematic potential, that depends on the LC order parameter. Fortunately, various donor–acceptor systems, DsA, obey this expectation as experimentally confirmed (Hasharoni et al., 1995; Berman et al., 1995). In all these systems, TREPR spectra were detected and analyzed in the entire soft-glass and nematic range.

e. Quantum Beats in Correlated Radical Pairs Spin-correlated radical pairs (CRP) can be de­ scribed by four spin states: and mixing is operative and Usually, only it results in the formation of two new states, that are not natural eigenstates of the spin Hamilton­ ian. This gives rise to a coherent superposition of these two states, i.e., a zero-quantum coherence

Time-resolved EPR (ZQC) (Salikhov et al., 1990; Bittl and Kothe, 1991; Wang, Z et al., 1992; Zwanenburg and Hore, 1993). The oscillatory behavior of the probability of finding the system in one of the newly formed two eigenstates produces oscil­ lations of the magnetization, namely, quantum beats. These can be manifested in the TREPR spectra, if the ZQC can be converted into a single-quantum coherence by the micro­ wave field of the EPR experiment. This phenom­ enon is similar to the well-known Torrey oscil­ lations (Torrey, 1949) The latter may appear concurrently with the quantum beats. However, in the Torrey oscillation case, the system is pre­ pared with no coherence among its eigenstates, at t = 0, while in the quantum beats case the RP formation process creates the coherence at t = 0. Although it is not a trivial experiment, the detection and analysis of quantum beats allows to assign values to some spin Hamiltonian par­ ameters and lifetimes, which cannot be deter­ mined unambiguously from the spectra because of limited spectral resolution (Bittl and Kothe, 1991) To detect quantum beats by EPR, a time-resolved experiment has to be set up. The transient CW-EPR method is preferred over pulse tech­ niques because the dead time problem can be eliminated. However, EPR observations of quan­ tum beats in CRPs are scarce (Kothe et al., 1991, 1994), due to the difficulty in achieving the high time resolution required (~10–20 ns) in the DDEPR experiment. The reaction so-far studied is the primary ET in plant photosystem I (Kothe et al., 1991, 1994):

229

beats because of hyperfine interactions. The oscil­ The lations vary as a function of both and were variations of the beat frequency with attributed to the anisotropic nature of the trans­ ients. This anisotropy might be used to calculate orientational parameters of the RP under study (Bittl and Kothe, 1991; Kothe et al., 1991, 1994). From simulations and fitting of the signals, the lifetime of the secondary RP in Eq. (14) was found to be ~250 ns (Kothe et al., 1991, 1994). Reproducing the quantum beats by the simula­ tions indicates that the second RP is formed in an initial pure singlet state, i.e., any triplet mixing present during the lifetime of the first RP can be neglected due to its short lifetime. This result strongly favors the ESP scheme where the life­ time of the first RP is too short to allow for any polarization to build. IV. Concluding Remarks TREPR spectroscopy, in its current time resol­ ution, is an important method for the detailed study of photoinduced EnT and ET processes, which, within the context of this chapter, bears a direct relation to primary photosynthesis. In some studies, TREPR is employed as a better “detec­ tor” for conventional laser photolysis experi­ ments, while implementation of this spectroscopy with other systems (e.g., the alkali-metal or the fullerenes) is unique in addressing questions that have no direct analog in other spectroscopic me­ thods currently used. Acknowledgements

where

is the primary chlorophyll donor and and (FeS) are the electron acceptors. In this system, a spin-polarized EPR signal was de­ tected and attributed to the radical pair The transverse magnetization of the various transitions in the spin-polarized EPR spectrum exhibit oscillatory behavior at short times (t < ~130 ns) following the laser pulse. This short time is due to rapid averaging of the quantum

I am indebted to my students Kobi Hasharoni and Assia Berman for their assistance, comments and helpful suggestions in preparing this manu­ script. The inspiring and fruitful discussions with Prof. J. R. Norris are highly appreciated. I thank Dr. M. C. Thurnauer for her constant interest and to Dr. A. Shain for reading the manuscript and his helpful comments. The Farkas Center is supported by the Bundenministerium für For­ schung und Technologie and the Minerva Gesel­ schaft für die Forschung GmbH. The research described herein was supported by the U.S.– Israel BSF, the German–Israel Foundation,

230

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231 Jux N, Koch P, Schmickler H, Lex J and Vogel E (1990) Acetylene-cumulene porphyrinoids. Angew Chem Int Ed Engl 29: 1385–1387 Kim SS and Weissman SI (1979) Transient magnetization fol­ lowing photoexcitation. Rev Chem Intermed 3: 107–120 Klette R, Törring JT, Plato M, Möbius K, Bönigk B and Lubitz W (1993) determination of the g tensor of the pri­ mary donor cation radical in single crystals of Rhodobacter sphaeroides R-26 reaction centers by 3-mm high-field EPR. J Phys Chem 97: 2015–2020 Kothe G, Weber S, Bittl R, Ohmes E, Thurnauer MC and Norris JR (1991) Transient EPR of light-induced radical pairs in plant photosystem I: observation of quantum beats. Chem Phys Lett 186: 474–480 Kothe G, Weber S, Ohmes E, Thurnauer MC and Norris JR (1994) Transient EPR of light-induced spin correlated radical pairs: manifestation of zero-quantum coherence. J Phys Chem 98: 2706–2712 Lebedev YS (1990) High-frequency continuous wave electron spin resonance. In: Kevan L and Bowman MK (eds) Mod­ ern Pulsed and Continuous-Wave Electron Spin Reson­ ance, pp 365–404, John Wiley and Sons, New York Lendzian F and von Maltzan B (1991) Triplet electron transfer and time-resolved EPR: detection of radical-pair state of a covalently linked porphyrin-quinone. Chem Phys Lett 180: 191–197 Lendzian F, Schlüpmann J, von Gersdorff J, Möbius K and Kurreck H (1991) Investigation of the light-induced charge transfer between covalently linked porphyrin and quinone units by time-resolved EPR spectroscopy. Angew Chem Int Ed Engl 30: 1461–1463 Levanon H (1979) Principles and application of optical perturbation-electron paramagnetic resonance (OPEPR) spec­ troscopy. In: Dorio M and Freed JH (eds) Multiple Elec­ tronic Resonance Spectroscopy, pp 437–474, Plenum, New York Levanon H (1987) Spin polarized Triplets Oriented in Liquid Crystals. Rev Chem Intermed 8: 287–320 Levanon H and Bowman MK (1993) Time-domain EPR spec­ troscopy of energy and electron transfer. In: Deisenhofer J and Norris JR (eds) Photosynthetic Reaction Centers, pp 387–418, Academic Press Inc, New York Levanon H and Norris JR (1978) The photoexcited Triplet State and Photosynthesis. Chem Rev 78: 185–198 Levanon H and Norris JR (1982) Triplet state and chloro­ phylls. In: Fong FK (ed) Light Reaction Path of Photo­ synthesis, pp 152–195, Springer-Verlag, Berlin Levanon H and Weissman SI (1971) Transient effects in exci­ tation of triplet state. J Am Chem Soc 93: 4309–4310 Levanon H, Regev A, Michaeli S, Galili T, Cyr M and Sessler JL (1990) The photoexcited triplet state of sapphyrin dicat­ ion. Unusual spin alignment in monomers and spin delo­ calizaion in dimers. Chem Phys Lett 174: 235–240 Levanon H, Meiklyar V, Michaeli S and Gamliel D (1993) Triplet state and dynamics of photoexcited C70. J Am Chem Soc 115: 8722–8727 Levanon H, Michaeli S and Meiklyar V (1994) Triplet dynam­ and its participation in electron transfer ics of and reactions. Time-resolved electron paramagnetic resonance. In: Kadish K and Ruoff RS (eds) Recent Advances in the

232 Chemistry and Physics of Fullerenes and Related Ma­ terials, pp 894–908, The Electrochemical Society, Penning­ ton, NJ Marcus RA (1964) Chemical and electron transfer theory. Annu Rev Phys Chem 15: 155–196 Marcus RA and Sutin N (1985) Electron Transfers in Chemis­ try and Biology. Biochim Biophys Acta 811: 265–322 Martin AJ, Meier G and Saupe A (1971) Extended debye thoery for the dielectric relaxation in nematic liquid crys­ tals. Symp Faraday Soc 5: 119–133 Martin JL, Migus A, Mourou GA and Zewail AH (1993) Ultrafast Phenomena VIII, Springer-Verlag, Berlin Martire DO, Jux N, Aramendia PF, Negri M, Lex J, Braslav­ sky SE, Schaffner K and Vogel E (1993) Photophysics acetylene cumulene and photochemistry of and porphyrinoids. J Am Chem Soc 114: 9969–9978 Massoth RJ (1987): Time resolved Fourier transform EPR studies in solutions of vitamins and antioxidants. PhD Dis­ sertation, Kansas State University McLauchlan KA (1990) Continuous-wave electron spin reson­ ance. In: Kevan L and Bowman MK (eds) Modern Pulsed and Continuous-Wave Electron Spin Resonance, (pp 285– 363) John Wiley and Sons, New York McLauchlan KA, Shkrob IA and Yeung MT (1994) Roomtemperature observations of spin-polarized triplet anthra­ cene. Chem Phys Lett 217: 157–162 Meier G and Saupe A (1966) Dielectric Relaxation in Nematic Liquid Crystals. Molecular Crystals 1: 515–525 Michaeli S, Hugerat M, Levanon H, Bernitz M, Natt A and Neumann R (1992) Control of porphyrin orientation in thermotropic liquid crystal by molecular design. A timeresolved EPR study. J Am Chem Soc 114: 3612–3618 Michaeli S, Meiklyar V, Schulz M, Möbius K and Levanon H (1994) Photoinduced electron transfer from triplet fuller­ ene, to tetracyanoethylene. Fourier transform elec­ tron paramagnetic resonance study. J Phys Chem 98: 7444– 7447 Morris AL, Norris JR and Thurnauer MC (1990) Reaction centers of photosynthetic bacteria. In: Michel-Beyerle ME (ed) Biophysics. Vol 6, pp 423–434, Springer-Verlag, Berlin. Neumann R, Hugerat M, Michaeli S, Natt A, Bernitz M and Levanon H (1991) Controlled orientation of porphyrins by molecular design in thermotropic liquid crystals: a time resolved EPR study. Chem Phys Lett 182: 249–252 Norris JR, Thurnauer MC, Bowman MK and Trifunac AD (1978) Electron spin echo and photosynthesis. In: Dutton PL, Leigh JS and Scrapa A (eds) Frontiers of Biological Energetics. Vol 1, pp 581–591, Academic Press, New York Norris JR, Thurnauer MC and Bowman MK (1980) Electron spin echo spectroscopy and the study of biological structure and function. In: Lawrence JH, GofmanJW and Hayes TL (eds) Advances in Biological and Medical Physics. Vol 17, pp 365–416, Academic Press, New York Paul DE, Lipkin D and Weissman SI (1956) Reaction of sodium metal with aromatic hydrocarbons. J Am Chem Soc 78: 116–120 Pedersen JB and Freed JH (1974) Some theoretical aspects

Haim Levanon of chemically induced dynamic nuclear polarization. J Chem Phys 61: 1517–1525 Petersen J, Stehlik D, Gast P and Thurnauer MC (1987) Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron depleted bacterial reac­ tion centers with time-resolved K-band EPR; evidence the photosystem I acceptor is a quinone. Photosynth Res 14: 15–29 Pohl L and Eidenschink R (1978) High resolution nuclear magnetic resonance spectrometry in thermotropic nematic liquid crystals with negative diamagnetic anisotropy. Anal Chem 50: 1934–1935 Poole PP Jr (1983) Electron Spin Resonance, 2nd edition Wiley-Interscience, New York Prisner T, Dobbert O, Dinse KP and van Willigen H (1988) FT ESR study of photoinduced electron transfer. J Am Chem Soc 110: 1622–1623 Prisner TF, McDermott AE, Un S, Norris JR, Thurnauer MC and Griffin RG (1993) Measurements of the g-tensor of the signal from deuterated cyanobacterial photosystem I particles. Proc Natl Acad Sci USA 90: 9485–9488 Regev A, Galili T, Levanon H and Harriman A (1986) The effect of mutual orientation on the triplet state magnetiz­ ation of covalently linked hybrid porphyrin dimers. Chem Phys Lett 131: 140–146 Regev A, Berman A, Levanon H, Murai T and Sessler JL (1989) Principal ZFS tensor axes versus optical transition measurements of novel expanded porphyrins (texaphyr­ ins). Time-resolved triplet EPR spectroscopy. Chem Phys Lett 160: 401–406 Regev A, Levanon H, Murai T and Sessler JL (1990) Triplet EPR detection of porphyrinoids in fluid liquid crystals at elevated temperatures. J Chem Phys 92: 4718–4723 Regev A, Galili T and Levanon H (1991a) The photoexcited triplet state as a probe for dynamics and phase memory in a multiphase liquid crystal. Time-resolved EPR spectros­ copy. J Chem Phys 95: 7907–7916 Regev A, Michaeli S, Levanon H, Cyr M and Sessler JL (1991b) Solvent effect in randomly and partially oriented triplets of the sapphyrin cation. Optical and fast EPR­ magnetophotoselection measurements. J Phys Chem 95: 9121–9129 Regev A, Gamliel D, Meiklyar V, Michaeli S and Levanon H (1993) Dynamics of probed by electron paramagnetic resonance. Motional analysis in isotropic and liquid crystal­ line matrices. J Phys Chem 97: 3671–3679 Regev A, Galili T, Medforth CJ, Smith KM, Barkigia KM, Fajer J and Levanon H (1994) Triplet dynamics of confor­ mationally distorted porphyrins: Time resolved electron paramagnetic resonance. J Phys Chem 98: 2520–2526 Rozenshtein V, Zilber G, Rabinovitz M and Levanon H (1993) Irregular photoinduced electron transfer between doubly-charged pyrene and alkali metal cations studied by Fourier-transform electron paramagnetic resonance. J Am Chem Soc 115: 5193–5203 Rozenshtein V, Zilber G and Levanon H (1994) Dynamics of spin polarized photoelectrons in rubidium-THF solutions. J Phys Chem 98: 4236–4242

Time-resolved EPR Salikhov KM, Molin YN, Segdeev RZ and Buchachenko AL (1984) Spin Polarization and Magnetic Effects in Radical Reactions, Elsevier, Amsterdam Salikhov KM, Bock LH and Stehlik D (1990) Time develop­ ment of electron spin polarization in magnetically coupled, spin correlated radical pairs. Appl Magn Res 1. 195–211 Schweiger A (1991) Pulsed electron spin resonance spectros­ copy: basic principles, techniques, and examples of appli­ cations. Angew. Chem Int Ed Engl 30: 265–292 Sessler JL, Murai T, Lynch V and Cyr M (1988) An “ex­ panded porphyrin”: the synthesis and structure of a new aromatic pentadentate ligand. J Am Chem Soc 110: 5586– 5588 Sessler JL, Murai T and Lynch V (1989) Binding of pyridine and benzimidazole to a cadmium “expanded porphyrin”: solution and x-ray structural studies. Inorg Chem 28:1333– 1341 Sessler JL, Wang B and Harriman A (1993) Long-range pho­ toinduced electron transfer in an associated but not coval­ ently linked photosynthetic model system. J Am Chem Soc, 115: 10418–10419 Stehlik D, Bock CH and Petersen J (1989) Amsotropic elec­ tron spin polarization of correlated spin pairs in photosyn­ thetic reaction centers. J Phys Chem 93: 1612–1619 Sumi H and Marcus RA (1986) Dielectric relaxation and intra­ molecular electron transfer. J Chem Phys 84: 4272–4276 Thurnauer MC and Norris JR (1976) Magnetophotoselection applied to the triplet state observed by EPR in photosyn­ thetic bacteria. Biochem Biophys Res Commun 73: 501– 504 Thurnauer MC and Norris JR (1980) An electron spin echo phase shift observed in photosynthetic bacteria algae. Pos­ sible evidence for dynamic radical pair interactions. Chem Phys Lett 76: 557–561 Thurnauer MC, Bowman MK and Norris JR (1979) Time resolved electron spin echo spectroscopy applied to the study of photosynthesis. FEBS Lett 100: 309–312 Tollin G and Green G (1962) Light induced single electron transfer between chlorophyll a and quinones in solution. Biochim Biophys Acta 60: 524–538 Torrey H (1949) Transient nutations in nuclear magnetic res­ onance. Phys Rev 76: 1059–1068 Trifunac AD, Lawler RG, Bartels DM and Thurnauer MC (1986) Magnetic resonance studies of paramagnetic trans­ ients in liquids. Prog Reaction Kinetics 14: 43–156 Van der Est AJ, Steidl P, Schulz M, Bock CH, Vieth HM and Stehlik D (1990), The application of time–resolved

233 EPR to the study of photoreactive molecules in solid and liquid crystalline environments. Appl Magn Res 1: 54–69 van der Est A, Bittl R, Abresch EC, Lubitz W and Stehlik D (1993) transient EPR spectroscopy of perduterated Zn­ substituted reaction centers of Rhodobacter sphaeroides R­ 26. Chem Phys Lett 212: 561–568 van Willigen H, Levstein PR and Ebersole MH (1993) Appli­ cation of Fourier transform electron papramagnetic reson­ ance in the study of photochemical reactions. Chem Rev 93: 173–197 Vogel E (1993) The porphyrins from the “annulene chemist’s” perspective. Pure and Appl Chem 65: 143–152 Vogel E, Jux N, Rodriguez-Val E, Lex J and Schmickler H (1990) Porphyrin homologs. [22]porphyrin(2.2.2.2), an extended porphycene. Angew Chem Int Ed Engl 29:1387– 1390 Wang W, Belford RL, Clarkson PH, Forrer J, Nilges MJ, Timken MD, Walczak T, Thurnauer MC, Norris JR, Morris AL and Zhang Y (1994) Very high frequency EPR­ 94 GHz instrument and applications to primary reaction centers from photosynthetic red bacteria and to other dis­ ordered systems. Appl Magn Res 6: 195–215 Wang Z, Tang J and Norris JR (1992) The time development of the magnetic moment of correlated radical pairs. J Magn Reson 97: 322–334 Wasielewski MR (1992) Photoinduced electron transfer in supramolecular systems for artificial photosynthesis. Chem Rev 92: 435–461 Wasielewski MR, Gaines GL, O’Neil MP, Svec WA and Ni­ emczyk MP (1990) Spin-polarized radical ion pair forma­ tion in a fixed-distance photosynthetic model system. J Am Chem Soc 112: 4559–4565 Weissman SI (1958) On detection of triplet molecules in solu­ tion by electron spin resonance. J Chem Phys 29: 1189– 1190 Weissman SI (1982) Recent developments in electron para­ magnetic resonance transient methods. Annu Rev Phys Chem 33: 301–318 Zavoisky E (1945a) Paramagnetic relaxation of liquid solu­ tions for perpendicular fields. J Phys USRR, 9: 211–216. Zavoisky E (1945b) Spin-magnetic resonance in paramag­ netics. J Phys USRR 9: 245–245 Zilber G, Rozenshtein V, Levanon H and Rabinovitz M (1992) Photoinduced electron transfer between doublycharged pyrene and alkali metals. Fourier transform EPR study. Chem Phys Lett 196: 255–260 Zwanenburg G and Hore PJ (1993) EPR of spin-correlated radical pairs. Analytical treatment of selective excitation including zero-quantum coherence. Chem Phys Lett 203: 65–74

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Chapter 15 Electron Spin Echo Methods in Photosynthesis Research

R. David Britt Department of Chemistry, University of California, Davis, Davis, CA 95616, USA Summary I. Introduction A. Inhomogeneous Broadening B. Electron Spin Echoes II. ESEEM A. 2-Pulse ESEEM B. 3-Pulse ESEEM III. ESE-ENDOR A. Davies ESE–ENDOR B. Mims ESE–ENDOR IV. Additional Examples of ESE Applications in Photosynthesis A.

Exchange about the PS II Mn Cluster B. Davies ESE–ENDOR of C. Davies

ESE–ENDOR of Mn Clusters V. Instrumentation Acknowledgements References

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Summary Electron spin echo methods allow photosynthesis researchers to probe the local structure of paramagnetic transition metal and organic radical centers. In particular the electron spin echo methods of electron spin echo envelope modulation (ESEEM) and electron spin echo – electron nuclear double resonance (ESE–ENDOR) are used to measure nuclear spin transitions of magnetic nuclei of, and in the vicinity of, paramagnetic species. The resulting magnetic hyperfine and electric quadrupolar parameters can be interpreted to give insights into the structure of a complex and its interaction with the neighboring protein matrix and molecules bound as substrates or inhibitors. Abbreviations: CW – continuous wave; ENDOR – electron nuclear double resonance; EPR – electron paramagnetic resonance; ESE – electron spin echo; ESEEM – ESE envelope modulation; PSII – acceptor quinone; RF – radio frequency photosystem II; transfer systems, photosynthetic reaction centers contain a variety of organic cofactors and transition metal complexes that can be found or poised in paramagnetic states. Thus electron paramagnetic resonance (EPR) has been a very powerful tool in photosynthetic research. However conven­ tional continuous-wave (CW) EPR spectroscopy has limitations that can be overcome by performing pulsed EPR experiments. This chapter fo­

I. Introduction

A. Inhomogeneous Broadening By their very nature of being light-driven electron

Correspondence: Fax: 1-916-7528995; E-mail: [email protected]

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J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 235–253. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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cuses on those pulsed EPR techniques based on multipulse electron spin echo (ESE) sequences. The ESE methods are useful because EPR lineshapes of photosynthetic paramagnetic entities are typically dominated by inhomogeneous broadening as illustrated in Fig. 1. The inhomo­ geneously broadened lineshape results from the overlap of resonances (shown as “spin packets”) resulting from a near continuum of magnetic en­ vironments. One such origin of inhomogeneous broadening is the unresolved overlap of hyperfine lines from many coupled nuclei, because the number of hyperfine lines increases multiplic­ atively with the number of classes of coupled nu­ clei. For example, for the case of a spin S = 1/2 complex coupled to k different classes of equivalent spin nuclei with coupling constants the number of EPR lines and the EPR spec­ tral density are given by the following expressions (Kurreck et al., 1988):

R. David Britt

If the spectral density becomes sufficiently high, the spacing between individual hyperfine lines will become less than the intrinsic lifetime-broadened linewidth of each spin packet, and the EPR lineshape will become dominated by the resulting Gaussian lineshape. Other sources of inhomo­ geneous broadening include hyperfine and g an­ isotropies in non-crystalline samples and site-tosite “strain” of such parameters (Mims, 1972a).

Electron spin echo methods

B. Electron Spin Echoes Fortunately, the inhomogeneous broadening can be negated by the use of multipulse magnetic resonance sequences to generate spin echoes (Mims, 1972a; Mims and Peisach, 1981; Slichter, 1990). The sequence and underlying mechanism

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of the simplest spin echo sequence, that of the 2­ pulse Hahn echo (Hahn, 1950), is illustrated in Fig. 2. At point (a), illustrated in the sequence and in the underlying rotating frame vector pic­ field is applied along the xture, a resonant axis to rotate magnetization from its initial equili­ brium direction (along the z-axis coincident with

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the static field).. By point (b) the magnetiz­ ation has been rotated by onto the rotating frame y-axis and the field is removed. In the subsequent interval individual spin packets, of which a select set of five are displayed, will pre­ cess at different frequencies due to the effect of inhomogeneous broadening. Spin packet 0 has a resonance frequency equal to the rotating frame frequency relative to the laboratory frame, and therefore remains fixed along the y-axis. The re­ maining spin packets have higher or lower res­ onant frequencies, and therefore precess away from the y-axis during the free induction (c) fol­ pulse. At time (d) a pulse is lowing the applied to the spin system. The spin packets are rotated into the positions displayed at point (e). Subsequent to this second pulse, each spin con­ tinues to precess with its original sense and rate relative to the y-axis, and at a time after this pulse all spin packets refocus simultaneously onto the –y-axis. This evanescent magnetization co­ herence is referred to as a spin echo, and it mani­ fests itself as a burst of radiation that can be detected with the spectrometer. The detected echo can be used to generate a field swept EPR spectrum analogous to the con­ ventional CW EPR spectrum. Field modulation is not employed, so the spectrum displayed is a measure of the direct absorption rather than the field derivative. This can be beneficial for the detection of signals with broad, relatively feature­ less lineshapes where the field derivative is every­ where small (Nishi et al., 1980; Britt et al., 1992; Gilchrist et al., 1992; Zimmermann et al., 1993). Moreover since the spin echo experiment is in­ trinsically a time-domain measurement, its time resolved character can be fruitfully exploited. Thus ESE spectroscopy can be utilized to study kinetics of electron transfer and spin polarization (Thurnauer and Clark, 1984; Bosch et al., 1992; Moenne-Loccoz et al., 1994) as well as to accu­ rately measure relaxation times of electron spin systems (Bosch et al., 1991; Lorigan and Britt, 1994). However the primary focus of this chapter is on applications of ESE spectroscopy designed to overcome the deleterious effects of inhomo­ geneous broadening obscuring hyperfine interac­ tions with magnetic nuclei. The techniques of electron spin echo envelope modulation (ESEEM) and electron spin echo – electron nu-

R. David Britt clear double resonance (ESE-ENDOR) are util­ ized to detect the nuclear spin transitions of such nuclei that are in magnetic contact with electron spins. In these experiments the spin echo is the carrier onto which nuclear spin information is encoded, either by time domain interference (ESEEM) or through RF-driven magnetization transfer (ESE-ENDOR). Both techniques will be illustrated with experiments on a representative photosynthetic paramagnetic center: the semiqui­ generated by dithionite none PS II radical reduction in PS II particles where the non-heme Fe(II) has been converted to a diamagnetic lowspin state by treatment with cyanide (Sanakis et al., 1994). II. ESEEM In the ESEEM experiment, electron spin echoes are formed by the application of two or more resonant microwave pulses. In addition to in­ ducing the electron spin transitions, the micro­ wave pulses may also induce “semi-forbidden” transitions of nuclear spins magnetically coupled to the electron spins, resulting in quantum mech­ anical coherences in the nuclear spin sublevels associated with the electron spin levels. These coherences create interference effects which can be measured by varying the electron spin echo pulse timing. Fourier analysis of the resulting time-domain electron spin echo envelope modu­ lation pattern reveals the frequencies of the nu­ clear spin transitions. The frequencies and ampli­ tudes of the Fourier peaks can be interpreted to determine hyperfine and electric quadrupolar interactions of the coupled nuclei. Typical ESEEM experiments utilize two-pulse Hahn echo (Fig. 3a) or three-pulse ‘stimulated’ echo (Fig. 3b) sequences (Mims, 1972a, 1972b; Kevan, 1979; Mims and Peisach, 1981, 1989; Thomann and Bernardo, 1993).

A. 2-Pulse ESEEM In the two-pulse ESEEM experiment the electron spin echo amplitude is measured as a function of the interpulse spacing (Fig. 3a). A qualitative picture of how hyperfine information is encoded onto the echo amplitude as a function of is provided by Fig. 4, which parallels the final three

Electron spin echo methods

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steps of Fig. 2. We focus on a single spin packet (1) starting at the onset of the pulse (d). Sup­ pose that during the pulse there is a partially allowed spin transition for a class of I = 1/2 nuclei hyperfine-coupled to the electrons of the spin packet. Those electrons whose nuclear partners do not flip remain in spin packet 1 after the pulse (e). However those electrons whose coupled nuclei do flip are shifted into a different spin packet 1´ whose precession frequency differs from that of packet 1 by the hyperfine frequency The magnetization vectors of the two packets are coincident initially, but will beat in and out of phase due to the frequency difference between the two packets. Packet 1 converges with other packets on the –y-axis to form the echo at time after the pulse. The projection of the 1´ packet magnetization onto the –y-axis at time affects the amplitude of the echo. Thus the echo amplitude is modulated by the hyperfine fre­ quency. A quantum mechanical treatment such as the density matrix approach used by Mims (1972a, 1972b) is required to properly describe the ESEEM phenomenon, including the amplitude of the modulation and the effects of other nuclear spin Hamiltonian terms such as the nuclear Zee­ man and electric quadrupolar interactions. One specific result of the density matrix analysis is that in addition to the fundamental nuclear spin transition frequencies, sum and difference fre­ quencies also appear in the 2-pulse ESEEM ex­ periment. Figure 5 shows 2-pulse ESEEM results for the anion radical of PS II. The time domain modulation pattern is shown in Fig. 5a. The fre­ quency domain spectrum shown in Fig. 5b is gen­ erated as the Fourier Transform of the time do­ main pattern. The peak at 14.1 MHz arises from weakly coupled protons. The strongly coupled protons of the radical do not produce measur­ able modulation at this magnetic field (3316 G), and these are generally best studied with ESE– ENDOR (vide infra). However there is much information in the lower frequency range of the spectrum. The pattern of three sharp low fre­ quency peaks and a broader peak at higher fre­ modulation when quency are characteristic of the hyperfine and external magnetic fields are of approximately the same amplitude (Mims and

R. David Britt Peisach, 1978; Flanagan and Singel, 1987). Under this “exact cancellation” condition the local mag­ netic field at the is essentially nulled for one electron spin orientation, and three sharp peaks arise because of the electric quadrupole interac­ tion that splits the three levels of the I = 1 nucleus in the absence of a magnetic field (Das and Hahn, 1958). The higher frequency “double­ quantum” peak arises from the transition be­ tween the outer energy levels of the for the other electron spin orientation where the hyper­ fine and external magnetic fields add. The corre­ sponding transitions involving the inner level are typically too broad to observe in non-crystalline samples. Analysis of the frequencies and lineshapes of the quadrupolar peaks and the double quantum peak provides a full determination of the electric quadrupole and hyperfine interac­ tions. As in other forms of magnetic resonance, iso­ topic substitution provides one of the most powerful assignment tools in pulsed EPR spectro­ scopy (for example: Hoff et al., 1985; De Groot et al., 1985; Evelo et al., 1989; Britt et al., 1989; Tang et al., 1994; Warncke et al., 1994). Figure 5c shows the frequency domain ESEEM results for in globally PS II particles. The low frequency region of the spectrum is com­ pletely altered, confirming our assignment of the nucleus coupled to previous transitions to a the radical. For the I = 1/2 nucleus we resolve a single transition at 2.78 MHz, a value very close to twice the Larmor frequency of 1.43 MHz at the applied field of 3316 G. This confirms that we are indeed in the “exact cancel­ lation” limit for this nitrogen. We do not observe a low frequency peak from the nucleus for the electron spin orientation where the two fields cancel because it is of too low a frequency to observe with the 2-pulse ESEEM sequence. This explicitly brings up an important drawback to the 2-pulse sequence. The 2-pulse echo amplitude de­ creases rapidly with increased due to spin-spin relaxation processes. In the representation of Fig. 2 this corresponds to the magnitude of the magnetization vector of each spin packet decreas­ ing over time due to random spin-spin dephasing. The effect of this limited “phase-memory” is dra­ matically seen in Fig. 5a, where the echo ampli­ tude has dropped to almost nothing after 3

Electron spin echo methods

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This loss of the echo carrier with time leads to a loss of resolution of low frequency modulation components. Fortunately, this problem can be circumvented by using the 3-pulse ESEEM me­ thod based on the stimulated echo sequence (Mims, 1972a).

B. 3-Pulse ESEEM The stimulated echo sequence can be seen as the first (solid) pulse train of the 3-pulse ESEEM pulse rotates the diagram (Fig. 3b). The first magnetization onto the y-axis. A second pulse, applied after the magnetization has dephased for time rotates this magnetization pat­ tern into the xz-plane. A spin packet with a fre­ quency that matches the rotating frame frequency will be shifted onto the –z-axis, as will spin pack­ ets that have rotated back onto the y-axis during the dephasing time On the other hand, spin packets which have rotated onto the –y-axis by time will be rotated onto the z-axis. This gives rise to a sinusoidal non-equilibrium magnetiz­ ation pattern encoded along the z-axis as illus­ trated in Fig. 6. This magnetization pattern de­ cays on the order of the spin-lattice relaxation which is typically much longer than the time pulse at time T phase memory time. A final

R. David Britt

generates the stimulated echo a time later. This stimulated echo is essentially the free induction decay of the sinusoidal magnetization pattern im­ posed by the first two pulses. In the 3-pulse ESEEM experiment, the stimulated echo is mea­ sured as a function of the time T. The great ad­ vantage is that the carrier echo now lasts a much longer time, allowing better spectral resolution of the modulation frequencies. A disadvantage is that the echo is only one half the initial intensity of the 2-pulse echo because magnetization com­ ponents remaining along the ±x axis after the first two pulses are lost on the timescale of the phase memory. Another difference that appears from the density matrix theory is that only funda­ mental nuclear spin transition frequencies appear in the 3-pulse ESEEM experiment, although their amplitudes depend on the specific value of the the first interpulse time used in the experiment, and certain frequency components may be com­ pletely suppressed for certain values (Mims, 1972b, 1972c). An investigator typically performs the two-pulse ESEEM experiment along with a set of three-pulse ESEEM experiments with dif­ ferent values to completely characterize the ESEEM effects. Also, additional information correlating hyperfine transitions can be gained using the 2-dimensional ESEEM-derived method

Electron spin echo methods of HYSCORE (Hofer et al., 1986) which has recently been introduced to photosynthesis re­ search by Käss et al. (1995). Figure 7a shows the longer time range that can be exploited in the 3-pulse ESEEM experiment for the radical. The carrier stimulated echoes show little diminution out to a time of 16 where the modulation is essentially damped by the intrinsic linewidth of the nuclear transitions. The frequency resolution in the Fourier Trans­ form ESEEM spectrum (Fig. 7b) is much greater than in the corresponding 2-pulse spectrum (Fig. 5b). The weakly coupled proton modulation in these data sets is suppressed by working at a multiple of the proton Larmor frequency, and the frequency domain data are therefore plotted only to 6 MHz to expand the low frequency region of the spectra. The three peaks at 0.75, 2.06, and 2.85 MHz arise from the nucleus with electric quadrupole coupling parameters MHz and These quadrupole values are characteristic of a in a peptide bond (Ed­ monds, 1977), so we postulate that this modu­ lation arises from a peptide nitrogen hydrogenbonded to the quinone. The 5.0 MHz frequency of the double quantum transition allows us to calculate a hyperfine coupling of 2.0 MHz to this nitrogen. The 3-pulse ESEEM spectrum of the sample (Fig. 7c) shows a very sharp at 0.268 MHz. The low frequency presence of such exceedingly sharp ESEEM lines is predicted by Lai et al. (1988) for I = 1/2 nuclei such as near the exact cancellation limit. A dipolar coupling of 0.3 MHz would give rise to such a line-narrowed peak under these experi­ mental conditions. The 2.81 MHz transition seen in the 2-pulse spectrum (Fig. 5c) is suppressed at the value of 213 ns used in this data set. We also observe a contribution at the Larmor frequency of 1.43 MHz arising from additional weakly coupled nuclei. III. ESE ENDOR Unlike ESEEM, the ESE–ENDOR experiments do not rely on semi-forbidden nuclear spin trans­ itions during the microwave pulses. Rather, the nuclear transitions are driven directly with sepa­ rate high-power radio frequency (RF) pulses. In an ESE–ENDOR experiment, an alteration of

243

the initial electron spin magnetization is created by one or more high-power resonant microwave pulses. Application of the radio frequency pulse further perturbs the electron magnetization if the RF pulse induces spin transitions of nuclei mag­ netically coupled to the electron spins. The nu­ clear spin transition frequencies are measured by varying the radio frequency while monitoring the effect of the RF pulse on a subsequent electron spin echo (Hoffman et al., 1993; Thomann and Bernardo, 1993). Pulse sequences introduced by Davies (1974) and by Mims (1965) are typically utilized (Fig. 8).

A. Davies ESE–ENDOR The Davies ESE–ENDOR sequence is shown in Fig. 8a. The inverting microwave pulse inverts the electron magnetization in a narrow resonant bandwidth within the inhomogeneously broad­ ened line (Fig. 9a). When resonant with a nuclear spin transition, the RF pulse transfers magnetiz­ ation between an unperturbed region of the spec­ trum and the resulting “hole”, decreasing the ex­ tent of magnetization inversion in the hole. This results in an altered magnitude of the spin echo induced by a final two-pulse ESE sequence. The Davies sequence works best for relatively strongly coupled nuclei. For weakly coupled nuclei the nuclear spin flips simply transfer magnetization within the hole, giving a negligible change in echo amplitude. For nuclei with a modest coupling this effect can be mitigated by using long microwave pulses to burn narrow holes in the inhomogene­ ously broadened line. Figure 10a shows the Davies ESE–ENDOR of sample. The broad overlap­ the ping powder patterns centered about the Larmor frequency of 15.4 MHz arise from pro­ radical and its immediate environ­ tons of the ment. The powder patterns are obtained with very good signal intensity. Detailed assignments have not been made. The 2.8 MHz transition is also observed with good intensity, though down appreciably from the proton signals due to the smaller gyromagnetic ratio for the nucleus. Larmor frequency peak observed in the The 3-pulse ESEEM is not detected. Parallel ex­ periments show poor signals in the low frequency region (data not shown), most likely due to a

244

R. David Britt

Electron spin echo methods

combination of quadrupolar broadening and a smaller gyromagnetic ratio for the nucleus.

B. Mims ESE–ENDOR Figure 8b displays the Mims ESE–ENDOR se­ quence, in which the RF pulse is provided follow­ ing the generation of the sinusoidal magnetization pulses of a pattern produced by the first two stimulated echo sequence (Fig. 6). The amplitude of this sinusoidal magnetization pattern is re­ duced by RF-induced nuclear spin transitions, re­ sulting in reduced stimulated echoes when the radio frequency is resonant with nuclear spin transitions. The Mims ENDOR transitions are thus inverted; driving the nuclear resonance re­ duces the echo size. In principle, the Mims se­ quence gives larger ENDOR effects than the Davies sequence, but suffers from “blind-spots” arising from the for couplings at multiples of sinusoidal nature of the induced magnetization

245

pattern; transferring magnetization from one trough to another has no effect on the echo ampli­ tude. However, for weakly coupled nuclei, i.e. nuclei with hyperfine interactions of a few MHz or less, this is not a problem as long as the value is kept small (minimum values of approxi­ mately 100 to 300 ns are typical depending on the ‘dead-time’ of the specific instrument). In fact, the value can be adjusted to give maximal sensi­ tivity for a given hyperfine coupling value. Figure 10b shows the Mims ESE–ENDOR of the sample. The proton region was not scanned because we knew from experience that the broad proton powder patterns would be distorted because of the blind spots intrinsic to this method. In the nitrogen region of the spectrum the 2.8 MHz feature is clearly resolved. Unlike in the Davies ENDOR spec­ trum, there is also some intensity observed at the Larmor frequency of 1.56 MHz.

To summarize, we have obtained pulsed EPR

246

results on the radical using both ESEEM and and ni­ ESE–ENDOR methods. For the trogen nuclei near the exact cancellation limit the ESEEM sequences give superb results. Measur­ ing spin transitions of nitrogen nuclei in this re­ gime is one of the classic applications of ESEEM (Mims and Peisach, 1981). Good results for are obtained with both Davies and Mims ESE– results are ENDOR sequences though the less favorable. For studying the broad proton powder patterns the Davies ESE–ENDOR method gives the best results.

R. David Britt

IV. Additional Examples of ESE Applications in Photosynthesis

A. Exchange about the PS II Mn Cluster In the preceding experiments we observed the strength of ESEEM is measuring spin transitions of weakly coupled nitrogens. Another classic ESEEM application is measuring hyperfine inter­ actions to weakly coupled exchangeable deut­ erons (Kevan, 1979). The modulation from the nucleus is approximately three times gre­ ater than from a proton in an equivalent site. Figure 11 shows the 2-pulse ESEEM results for the multiline signal of the oxygen evolv­

Electron spin echo methods

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Electron spin echo methods

ing complex (Dismukes and Siderer, 1981; Brudvig, 1989) following exchange. The timedomain pattern is simulated with contributions from proximal deuterons. The simulation is not unique, but we do require multiple deuterons at dipolar-coupled distances on the order of 2.5–2.7 Å to simulate the depth and rapid modulation damping of the time domain data. These close distances support water or hydroxo ligation to the Mn cluster by the of the Kok cycle. Further and ENDOR experiments will help to refine these results. B. Davies

ESE–ENDOR of

The tyrosine radical of Photosystem II (Barry and Babcock, 1987; Debus et al., 1988) has been studied with ESEEM in globally (Evelo et al., 1989) and selectively deuterated (Warncke et al., 1994) samples. ESEEM methodology for examin­ ing such radicals has recently been advanced (Warncke and McCracken, 1994). In our laboratory, Davies ENDOR has yielded very nice results on the and tyrosine radicals (Gilchrist et al., 1995). Figure 12 shows the Dav­ ies ESE–ENDOR and corresponding simulations radical. The ENDOR of the broad of the depleted PS II (Boussac radical observed in tyrosine are each very et al., 1990) and of the similar except for a slight increase in frequency of the more-strongly coupled proton, corre­ sponding to a decrease in the dihedral angle between the CH bond and the tyrosine ring normal from 47 to 43° (data not shown). We consider the strong similarity of the and pleted radical ENDOR spectra to be strong evi­ dence that the radical signal orig­ inates from C. Davies

ESE–ENDOR of Mn Clusters

The EPR spectrum of the PS II Mn cluster is complex because of the overlap of approximately 1300 hyperfine lines from four 100% natural abundance nuclei (Eq. (1)). We are using the Davies ESE–ENDOR method to measure the hyperfine and quadrupole couplings of these I = 5/2 nuclei. The couplings are quite large, so the Davies ESE–ENDOR sequence clearly is best for this application. The data analy­

249

sis for the tetranuclear cluster is complicated by the many overlapping EPR transitions that make up the overall signal, so we have started detailed analysis on a simpler system: an antiferromag­ netically exchange-coupled Mn(III)Mn(IV) dinu­ clear cluster (Cooper et al., 1978). The EPR spec­ trum of the S = 1/2 ground state of this cluster shows 16 resolved lines resulting from the overlap of the 36 powder patterns resulting from the two inequivalent nuclei. The top trace of Fig. 13 shows the field-swept ESE spectrum of the first four lines and a simulation of the contributing hyperfine powder patterns. The first two lines arise from unique powder patterns, the second two from two overlapping powder patterns, and the central transitions (not shown) each result from 3 EPR powder patterns. Except when per­ forming ENDOR in the outermost EPR lines, one must take in account multiple initial magnetic quantum numbers for the two nuclei when performing the simulations. The experimental ESE–ENDOR results obtained at a number of the EPR peaks across the spectrum are shown in the lower traces of Fig. 13 along with simulations. The simulations are seen to match reasonably well across the spectrum. Parameters used to simulate the ENDOR of the Mn(IV) ion are MHz and

hyperfine parameters 211 MHz and quadrupole parameters

26.7 MHz and For the higher frequency transitions for the Mn(III) ion the employed MHz and parameters are MHz and quadrupole parameters and V. Instrumentation

The principal requirement for performing pulsed EPR experiments such as described in this chap­ ter are high microwave pulse power (~1 kW) and electronics to provide for control of pulse timings on the nanosecond time scale. Traditionally, pulsed EPR instrumentation has been laboratorybuilt (Norris et al., 1980; Nishi et al., 1980; Britt et al., 1989; Sturgeon and Britt, 1992; McCracken et al., 1992). However, the commercial instru­ ment provided by Bruker Instruments is now being used by a number of photosynthesis re­ search groups (Astashkin et al., 1994; Käss et al.,

250

1995; Davis et al., 1993; Zimmermann et al., 1993). Over the past few years the advantage of per­ forming ESEEM experiments over a wide range of magnetic field values has become clearly recog­ nized, and for a fixed g-value, this translates to performing experiments over a wide microwave frequency range (Singel, 1989). For example, for nuclei with weak dipolar couplings, the modu­

R. David Britt

lation depth is inversely proportional to the square of the external field strength (Kevan, 1979). Moreover, as previously described, for nu­ clei with moderate hyperfine couplings, it is ne­ cessary for optimal results to be able to operate at external field values that cancel the external hyperfine fields (Flanagan and Singel, 1987; Lai et al., 1988). Therefore it is very important to have a spectrometer (or spectrometers) with a

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wide frequency range. For example, the pulsed EPR instrument that we recently constructed op­ erates over a 10 GHz frequency range from 8 to 18 GHz (Sturgeon and Britt, 1992). It is also very useful to have a wide range of frequencies available in the ESE–ENDOR experiment in order to track the movement of transition fre­ quencies with field and to separate overlapping transitions from different nuclear species. In par­ ticular, at X band frequencies the ubiquitous pro­ ton signals often overlap with signals from other nuclei such as and If for some reason an ESE–ENDOR instrument were de­ signed to work at a single microwave frequency, it would be preferable if the frequency were 20 GHz or higher in order to spread the ENDOR signals over a wider frequency range and to move the proton signals to a frequency range less popul­ ated with transitions from other nuclear species. One direction being actively pursued is in the area of ultra-high frequency pulsed EPR using superconducting magnets (Prisner et al., 1992, 1994; Weber et al., 1989; Disselhorst et al., 1995). Acknowledgements Research in my laboratory is supported by a grant from the National Institutes of Health, and by a NRICGP grant from the United States Depart­ ment of Agriculture. I thank Professor William Armstrong, Dr. James A. Ball, Dr. Bruce A. Diner, M. Lane Gilchrist Jr., Gary A. Lorigan, Dr. Jeffrey M. Peloquin, David Randall, Dr. Bradley E. Sturgeon, and Dr. Xiao-Song Tang for contributing unpublished data for this chap­ ter. References Astashkin AV, Kodera Y and Kawamori A (1994) Pulsed EPR study of manganese g = 4.1 signal in plant photosystem II. J Magn Reson 105: 113–119. Barry BA and Babcock GT (1987) Tyrosine radicals are in­ volved in the photosynthetic oxygen-evolving system. Proc Natl Acad USA 84: 7099–7103. Bosch MK, Evelo RG, Styring S, Rutherford AW and Hoff AJ (1991) ESE relaxation measurements in the photosys­ tem II. The influence of the reaction center non-heme iron on the spin-lattice relaxation of Tyr D. FEBS Lett 292: 279–283. Bosch MK, Gast P and Hoff AJ (1992) Applications of ESE spectroscopy in the study of electron spin polarization in bacterial photosynthesis. Pure Appl Chem 64: 847–857.

R. David Britt Britt RD. Zimmermann JL, Sauer K and Klein MP (1989) Ammonia binds to the catalytic Mn of the oxygen evolving complex of photosystem II: evidence by electron spin echo envelope modulation spectroscopy. J Am Chem Soc 111: 3522–3532. Britt RD, Lorigan GA, Sauer K, Klein MP and Zimmermann JL (1992) The g = 2 multiline EPR signal of the state of the photosynthetic oxygen-evolving complex originates from a ground spin state. Biochim Biophys Acta 1140: 95– 101. Brudvig GW (1989) EPR spectroscopy of manganese en­ zymes. In: Hoff AJ (ed) Advanced EPR: Applications In Biology and Biochemistry, pp 839–863. Elsevier, Amster­ dam. Boussac A, Zimmermann JL, Rutherford AW and Lavergne J (1990) Histidine oxidation of the oxygen-evolving photosystem-II enzyme. Nature 347: 303–306. Cooper SR, Dismukes GC, Klein MP and Calvin M (1978) Mixed valence interactions in bridged manganese complexes. Electron paramagnetic resonance and magnetic susceptibility studies. J Am Chem Soc 100: 7248–7252. Das TP and Hahn EL (1958) Nuclear Quadrupole Resonance Spectroscopy. Academic Press, New York. Davies ER (1974) A new pulse ENDOR technique. Phys Lett 47A: 1–2. Davis IH, Heathcote P, MacLachlan DJ and Evans MCW (1993) Modulation analysis of the electron spin echo signals of in vivo oxidized primary donor nitrogen-15 chlorophyll centers in bacterial, P870 and P960, and plant photosystem I, P700, reaction centers. Biochim Biophys Acta 1143: 183– 189. Debus RJ, Barry BA, Babcock GT and McIntosh L (1988) Site-directed mutagenesis identifies a tyrosine radical in­ volved in the photosynthetic oxygen-evolving system. Proc Natl Acad USA 85: 427–430. De Groot A, Evelo R, Hoff AJ, De Beer R and Scheer H (1985) Electron spin echo envelope modulation (ESEEM) spectroscopy of the triplet state of the primary donor of and bacterial photosynthetic reaction centers and of and bacteriochlorophyll a. Chem Phys Lett 118: 48–54. Dismukes CG and Siderer Y (1981) Intermediates of a polynu­ clear manganese center involved in photosynthetic oxid­ ation of water. Proc Natl Acad USA 78: 274–278. Disselhorst JAJM, van der Meer H, Poluektov OG and Schmidt J (1995) J Magn Res (to be published). Edmonds DT (1977) Nuclear quadrupole double resonance. Phys Rep 4: 233–290. Evelo RG, Hoff AJ, Dikanov SA and Tyryshkin AM (1989) An ESEEM study of the oxidized electron donor of plant photosystem II: evidence that D is a neutral tyrosine rad­ ical. Chem Phys Lett 161: 479–484. Flanagan HL and Singel DJ (1987) Analysis of ESEEM patterns of randomly oriented solids. J Chem Phys 87: 5606–5616. Gilchrist ML, Lorigan GA and Britt RD (1992) Pulsed elec­ tron paramagnetic resonance studies of calcium-depleted photosystem II membranes. In: Murata N (ed) Research in Photosynthesis, pp 317–320. Kluwer, Dordrecht. Gilchrist ML, Ball JA, Randall DW and Britt RD (1995)

Electron spin echo methods Proximity of the manganese cluster of photosystem II to the redox active tyrosine Proc Natl Acad Sci USA 92: 9545–9549. Hahn EL (1950) Spin echoes. Phys Rev 80: 580–594. Hofer P, Grupp A, Nebenführ H and Mehring M (1986) Hyperfine sublevel correlation HYSCORE spectroscopy: a 2D ESR investigation of the squaric acid radical. Chem Phys Lett 132: 279–282. Hoff AJ, De Groot A, Dikanov SA, Astashkin AV and Tsvet­ kov YD (1985) Electron spin echo envelope modulation and spectroscopy (ESEEM) of the radical cations of bacteriochlorophyll a. Chem Phys Lett 118: 40–47. Hoffman BM, DeRose VJ, Doan PE, Gurbiel RJ, Houseman ALP and Telser J (1993) Metalloenzyme active-site struc­ ture and function through multifrequency CW and pulsed ENDOR. In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance, Vol 13, pp 151–218. Plenum, New York. Käss H, Rautter J, Bönigk B, Hofer P and Lubitz, W (1995) 2D ESEEM of the radical cations of bacteri­ ochlorophyll a and of the primary donor in reaction centers of Rhodobacter sphaeroides. J Phys Chem 99: 436–448. Kevan L (1979) Modulation of electron spin-echo decay in solids. In: Kevan L and Schwartz RN (eds) Time Domain Electron Spin Resonance, pp 279–341. John Wiley and Sons, New York. Kurreck H, Kirset B and Lubitz W (1988) Electron Nuclear Double Resonance Spectroscopy of Radicals in Solution. VCH Verlag, Weinheim. Lai A, Flanagan HL and Singel DJ (1988) Multifrequency electron spin echo envelope modulation in S = 1/2, I = 1/2 systems: Analysis of the spectral amplitudes, line shapes, and linewidths. J Chem Phys 89: 7161–7166. Lorigan GA and Britt RD (1994) Temperature dependent state multiline pulsed EPR relaxation studies of the signal of the photosynthetic oxygen-evolving complex. Bio­ chemistry 33: 12072–12076. McCracken J, Shin DH and Dye JL (1992) Pulsed EPR studies of polycrystalline cesium hexamethyl hexacyclen sodide. Appl Magn Reson 3: 305–316. Mims WB (1965) Pulsed ENDOR experiments. Proc Royal Soc London 283: 452–457. Mims WB (1972a) Electron spin echoes. In: Geschwind S (ed) Electron Paramagnetic Resonance, pp 263–351. Plenum Press, New York. Mims WB (1972b) Envelope modulation in spin-echo experi­ ments. Phys Rev B 5: 2409–2419. Mims WB (1972c) Amplitudes of superhyperfine frequencies displayed in the electron-spin-echo envelope. Phys Rev B 6: 3543–3545. Mims WB and Peisach J (1978) The nuclear modulation effect in electron spin echoes for complexes of and imidaz­ and J Chem Phys 69: 4921–4930. ole with Mims WB and Peisach J (1981) Electron spin echo spectros­ copy and the study of metalloproteins. In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance, Vol 3, pp. 213–263. Plenum, New York. Mims WB and Peisach J (1989) ESEEM and LEFE of metallo­ proteins and model compounds. In: Hoff AJ (ed) Ad­ vanced EPR: Applications in Biology and Biochemistry, pp 1–57. Elsevier, Amsterdam.

253 Moenne-Loccoz P, Heathcote P, Maclachlan DJ, Berry MC, Davis IH and Evans MCW. (1994) Path of electron transfer in photosystem 1: Direct evidence of forward electron Biochemistry 33: 10037–10042. transfer from to Nishi N, Hoff AJ and van der Waals JH (1980) Electron spin echo studies on chloroplasts. Spectral characteristics of electron transport components and light-induced trans­ ients. Biochim Biophys Acta 590: 74–88. Norris JR, Thurnauer MC and Bowman MK (1980) Advances in Biological and Medical Physics 17: 365–416. Prisner TF, Un S and Griffin RG (1992) Pulsed ESR at 140 GHz. Isr J Chem 32: 357–363. Prisner TF, Rohrer M and Mobius K (1994) Pulsed 95 GHz, high-field EPR heterodyne spectrometer with high spectral and time resolution. Appl Magn Reson 7: 167–183. Rigby SEJ, Nugent JHA and O’Malley PJ (1994) The dark stable tyrosine radical of photosystem 2 studied in three species using ENDOR and EPR spectroscopies. Biochem­ istry 33: 1734–1742. Sanakis Y, Petrouleas V and Diner BA (1994) Cyanide bind­ ing at the non-heme of the iron-quinone complex of photosystem II: at high concentrations, cyanide converts the from high (S = 2) to low (S = 0) spin. Biochemis­ try 33: 9922–9928. Singel DJ (1989) Multifrequency ESEEM. In: Hoff AJ (ed) Advanced EPR: Applications in Biology and Biochemistry, pp 119–133. Elsevier, Amsterdam. Slichter CP (1990) Principles of Magnetic Resonance, 3rd ed. Springer-Verlag, Berlin. Sturgeon BE and Britt RD (1992) Design of a sensitive pulsed EPR spectrometer with an 8 to 18 GHz frequency range. Rev Sci Instrum 63: 2187–2192. Tang XS, Diner BA, Larsen BS, Gilchrist ML, Lorigan GA, and Britt RD (1994) Identification of histidine at the cata­ lytic site of the photosynthetic oxygen-evolving complex. Proc Natl Acad USA 91: 704–708. Thomann H and Bernardo M (1993) Pulsed electron nuclear double and multiple resonance spectroscopy of metals in proteins and enzymes. In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance, Vol 13, pp 275–322. Ple­ num, New York. Thurnauer MC and Clark C (1984) Electron spin echo envel­ ope modulation of the transient EPR signals observed in photosynthetic algae and chloroplasts Photochem Photo­ biol 40: 381–386. Warncke K and McCracken J (1994) electron spin echo envelope modulation spectroscopy of strong, hyperfine coupling in randomly oriented paramagnetic sys­ tems. J Chem Phys 101: 1832–1841. Warncke K, McCracken J and Babcock GT (1994) Structure of the tyrosine radical in photosystem II as revealed by electron spin echo envelope modulation (ESEEM) spectroscopic analysis of hydrogen hyperfine interactions. J Am Chem Soc 116: 7332–7340. Weber RT, Disselhorst JAJM, Prevo LJ, Schmidt J and Wen­ kebach WT (1989) Electron spin echo spectroscopy at 95 GHz. J Magn Res 81: 129–144. Zimmermann JL, Boussac A and Rutherford AW (1993) The manganese center of oxygen-evolving and calcium-depleted photosystem II: a pulsed EPR spectroscopy study. Bio­ chemistry 32: 4831–4841.

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Chapter 16 ENDOR Spectroscopy

Wolfgang Lubitz* and Friedhelm Lendzian Max-Volmer-lnstitut für Biophysikalische und Physikalische Chemie, Technische Universität Berlin, Straße des 17. Juni 135, D-10623 Berlin, Germany

Summary I. Introduction II. Principles of Electron–Nuclear Multiple Resonance Spectroscopy A. ENDOR in Liquid Solution 1. Spin Hamiltonian, Energies and Transition Frequencies 2. Phenomenological Description of the ENDOR Experiment 3. TRIPLE Resonance 4. ENDOR-lnduced EPR B. ENDOR in the Solid State 1. ENDOR in Frozen Solutions a. Isotropic g b. Anisotropic G-Tensor (Orientation Selection) 2. ENDOR in Single Crystals C. ENDOR of Triplet State Molecules D. New Techniques III. Selected Applications of ENDOR to Photosynthesis A. Pigment Radicals in Vitro B. Radical Ions in Photosynthetic Reaction Centers C. ENDOR of Triplet States IV. Concluding Remarks Acknowledgements References

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Summary The basic principles of electron-nuclear multiple resonance techniques, like ENDOR and TRIPLE resonance, are described as applied to paramagnetic molecules in liquid and frozen solutions and in single crystals. The advantages of these techniques as compared with conventional EPR are discussed. ENDOR and TRIPLE resonance allows the detection of the electron-nuclear hyperfine coupling con­ stants (hfcs) of large paramagnetic molecules in a complex surrounding, even in cases where the EPR spectrum is completely unresolved. From the assigned hfcs a map of the valence electron spin distribution over the molecule is obtained. When applied to solid state samples – and in particular to single crystals – the full hyperfine tensors of the various magnetic nuclei can be determined. Such measurements yield additional information about the spatial structure of a paramagnetic center. New developments with respect to very high frequencies (high field ENDOR), time resolution (pulsed EPR/ENDOR), and new detection schemes (stochastic ENDOR) are briefly described. Selected applications of ENDOR to photosynthesis are discussed, which focus on isolated pigment radicals and the radical ions and triplet states created during the charge separation process in bacterial and plant reaction centers. *Correspondence: Fax: 49-30-31421122; E-mail: lubitz @echo.chem.tu-berlin.de

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J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 255–275. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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Wolfgang Lubitz and Friedhelm Lendzian

Recent applications of the technique to reaction center single crystals and to the structural characteriz­

ation of genetically modified reaction centers are highlighted.

Abbreviations: (B)Chl – (bacterio)chlorophyll; (B)Ph – (bacterio)pheophytin; cw – continuous wave;

ENDOR – electron nuclear double resonance; EPR – electron paramagnetic resonance; ESE – electron

spin echo; ESEEM – electron spin echo envelope modulation; hfc – hyperfine coupling constant; hfs –

hyperfine structure; HOMO – highest occupied molecular orbital; LUMO – lowest unoccupied molecu­

lar orbital; MO – molecular orbital; mw – microwave; P – primary donor (BChl-dimer);

of the dimer bound to the protein L-subunit; of the dimer absorbing at 865 nm;

bound to the protein M-subunit; RC – reaction center; rf – radio frequency; RHF-INDO/SP – restricted Hartree Fock-intermediate neglect of differential overlap/spin polarization; SCF – self consistent field; TRIPLE – electron nuclear nuclear triple resonance

I. Introduction In reaction centers of plants, algae and photosyn­ thetic bacteria light-induced charge separation generates cation and anion radicals of the cofac­ tors involved in this process. The method of choice to identify and characterize these species is electron paramagnetic resonance (EPR). In the past, this technique has been used extensively to identify various paramagnetic states in vivo sim­ ply by comparison of their spectral parameters (mainly g factors and linewidths) with those of model compounds in vitro (Norris et al., 1971; Feher and Okamura, 1978). The application of EPR techniques to bacterial reaction centers has been reviewed by Hoff (1993). EPR has also been used to study other paramagnetic species in pho­ tosynthetic systems like triplet states (Budil and Thurnauer, 1991) or transition metal complexes (Miller and Brudvig, 1991; Debus, 1992) and even protein-derived radicals (Barry, 1993; Ho­ ganson and Babcock, 1992; Boussac et al., 1990). Many of the organic radicals can be classified which have the unpaired electron as delocalized in the of the conjugated system. The electron spin interacts with the vari­ ous magnetic nuclei in the radical by magnetic dipole–dipole and contact interactions leading to the hyperfine structure (hfs) of the spectra. After spectroscopic assignment of the measured hyper­ fine coupling constants (hfcs) to the various nuclei a map of the electron spin density distribution over the molecule is obtained. This can then be compared with calculations of the spin density

distribution by advanced molecular orbital (MO) methods (Plato et al., 1991). These data are the basis for a profound understanding of the electron transfer processes and for the function of the re­ action center complex in photosynthesis. However, EPR spectroscopy of biological sys­ tems has some serious problems: (i) very often the hfs patterns are not resolved due to interac­ tion of the electron spin with too many different nuclei. From such spectra only the g-factor and the Gaussian envelope linewidth are available for the characterization of the radical. (ii) Several radical species are frequently present in the sam­ ple which lead to overlapping spectra. (iii) Seri­ ous line broadening occurs due to immobilization of the radical through binding to a large biopo­ lymer. To overcome at least some of the aforemen­ tioned limitations, more advanced methods must be applied, in particular multiple resonance tech­ niques. A typical example is Electron Nuclear DOuble Resonance (ENDOR), which was intro­ duced by Feher (1956) in the solid state and al­ most a decade later extended to radicals in solu­ tion by Hyde and Maki (1964). In ENDOR spectroscopy the NMR transitions of magnetic nuclei in paramagnetic systems are detected via intensity changes of a simultaneously irradiated EPR line. The inherently higher resolution of ENDOR yields detailed information about the hyperfine interactions, even when the EPR spec­ trum is completely unresolved. An example is given in Fig. 1, which shows both EPR and ENDOR spectra of the bacteriochlorophyll a cat­

ENDOR spectroscopy

257

258 ion radical in isotropic solution. The hfs is not resolved in EPR, whereas the ENDOR spectrum directly yields the hfcs of the proton and nitrogen nuclei. The electron spin density distribution of a rad­ ical is most easily obtained in liquid solution via determination of the isotropic hfcs, i.e., by liquid state ENDOR. Unfortunately, many biological systems can only be studied in frozen solutions (powders or glasses), since the radical intermedi­ ates often have to be stabilized by freeze trap­ ping. Furthermore, the systems are often so large that anisotropic interactions are not averaged out by tumbling in solution, which leads to powder spectra even at ambient temperature. In prin­ ciple, powder ENDOR allows the determination of anisotropic and isotropic hfcs, although the spectral analysis is much more elaborate than in the liquid phase. If single crystals are available, the full hyperfine (hf) tensors of the various nu­ clei can be determined by ENDOR. ENDOR has been extended to electron–nuclear–nuclear TRIPLE resonance (Möbius and Biehl, 1979) in order to improve the sensitivity and resolution, and to measure the relative number of nuclei contributing to a resonance and the relative signs of the hfcs. In this chapter we want to describe continuous wave (cw) ENDOR and related techniques in some detail and illustrate the applications of the methods by selected examples from the field of photosynthesis. Here, we shall focus on the rad­ ical ions generated in the charge separation pro­ cess. The basic principles of EPR spectroscopy which are not described here can be found in Chapter 14 (Levanon) and several monographs (Atherton, 1993; Weil et al., 1994, Poole, 1983). For a more elaborate description of ENDOR spectroscopies the reader is referred to some re­ view articles (Möbius and Lubitz, 1987; Möbius et al., 1989; Hüttermann, 1993; Hoffmann et al., 1993) and books (Kevan and Kispert, 1976; Kur­ reck et al., 1988; Poole and Farach, 1994) which also present applications to other systems, e.g. to transition metal complexes in biological systems. Extensions of EPR and ENDOR to higher mw­ frequency bands are reviewed by Möbius (1993). The important application of electron spin echo (ESE) techniques like ESEEM and Pulsed ENDOR to determine hyperfine interactions in

Wolfgang Lubitz and Friedhelm Lendzian paramagnetic species related to photosynthesis are described by Britt (Chapter 15) and other time-resolved EPR methods by Levanon (Chap­ ter 14) in this monograph. II. Principles of Electron–Nuclear Multiple Resonance Spectroscopy

A. ENDOR in Liquid Solution 1. Spin Hamiltonian, Energies and Transi­ tion Frequencies For a paramagnetic molecule in fluid (isotropic) solution the static spin Hamiltonian contains the electron and nuclear Zeeman terms and the iso­ tropic hyperfine coupling term. In a strong mag­ it is given to first order by: netic field,

where g is the electronic g-factor; the Bohr magneton; and are the nuclear g-factors and are the zand magneton, respectively; components of the electron and nuclear spin op­ erators along the direction of the magnetic field is the respective isotropic hyperfine coup­ ling constant (in frequency units), and h is Planck’s constant. The sum i runs over all nuclei in the radical. with The corresponding energy eigenvalues to first order, classified by the electronic and nuclear and are given magnetic quantum numbers, by

where and –I + 1 , . . . + I ) . The resulting first order spin energy level sys­ tem for the simplest case of one electron coupled to one nucleus is shown in Fig. 2. According to the selection rules and two EPR transitions are obtained at frequencies centered around the electron Zeeman frequency, In a conventional cw-EPR experiment, the microwave

ENDOR spectroscopy

frequency, conditions

is kept constant and the resonance

are adjusted by sweeping the magnetic field Consequently, the magnitudes of hyperfine coup­ ling constants, a, obtained from EPR are usually given in magnetic field units (millitesla, mT), which may be converted to frequency units by use of the relation: where is the hyperfine coupling in mT. In an NMR experiment on this spin system (selection rule and also two transitions are obtained with frequencies which are now centered around the nuclear Zee­ man frequency In this simple spin system of Fig. 2 no resol­ ution enhancement of the NMR is obtained as compared with EPR. However, the number of allowed EPR transitions increases multiplic­ atively with the number of magnetically inequiva­ lent nuclei coupled to the electron; whereas, each set of magnetically equivalent nuclei contributes only two lines to the NMR spectrum (Kurreck et al., 1988). Here, lines of different nuclei (e.g.

259

and are grouped around their respective nuclear Larmor frequencies, which further im­ proves the resolution and helps with the assign­ ment. In radicals derived from large biomolecules this leads to a significant resolution enhancement of NMR (or ENDOR) as compared with EPR. From the ENDOR spectrum of the BChl a cation radical (Fig. 1C) 12 proton hyperfine coupling constants (hfcs) and four hfcs can be obtained; the corresponding number of ENDOR lines is 24 for the and 8 for the From these hfcs more than EPR lines are calculated. The resulting EPR spectrum (Fig. 1A) therefore consists only of a Gaussian envelope from which the hyperfine information cannot be obtained. 2. Phenomenological Description of the ENDOR Experiment The sensitivity of conventional NMR experiments on paramagnetic molecules in fluid solution is rather low due to the small population differences of nuclear Zeeman levels and the large linewidth resulting from the interaction with the unpaired electron(s). In an ENDOR experi­ ment the NMR transitions are therefore detected via intensity changes of a partially saturated EPR see Fig. 2). The intensity transition (e.g.

260 of this “pumped” transition is limited by all elec­ tron and nuclear relaxation rates which connect these two levels (directly or via levels 4 and 2, Fig. 2). If now one NMR transition (e.g. is pumped also with saturating power, the observed is effectively desaturated, EPR transition which leads to an increase of the EPR signal intensity. The magnitude of the observed ENDOR effect is typically only a few percent of the EPR signal intensity. ENDOR is, however, several orders of magnitude more sensitive than NMR on a paramagnetic system. The ENDOR response depends on the delicate interplay of the various electron and nuclear in­ duced rates and relaxation rates. For the case of liquid solution, this has been extensively investi­ gated for various magnetic nuclei both exper­ imentally and theoretically, considering all rel­ evant electron and nuclear relaxation mechanisms (Freed, 1979; Plato et al., 1981, see also Kurreck et al., 1988). In liquid solution, besides internal dynamics, Brownian rotational diffusion of the molecules modulates the various interactions in the radical and is, therefore, responsible for the relaxation processes. It enters into the equations for the relaxation rates in form of the rotational correlation time obtained from the Debye– Einstein relation in which is the effective molecular volume, and T are the viscosity and temperature of the solvent and k is the Boltzmann constant. Optimum ENDOR signals are obtained when no “relaxation bottleneck” exists in the spin sys­ tem, i.e., (neglecting cross relaxation rates see Fig. 2). For organic radicals in liquid solution one often finds and (Plato et al., 1981). Therefore, the rates may be equalized by changing the temperature or viscosity of the solvent. However, the different nuclei in the molecule generally have different optimum values, since they have different iso­ tropic and anisotropic hyperfine couplings which determine the relaxation rates (Plato et al., 1981; Kurreck et al., 1988). Therefore, the obtained ENDOR effect is different for each nucleus and does not reflect the number of contributing equivalent nuclei but rather reflects the different relaxation behavior.

Wolfgang Lubitz and Friedhelm Lendzian Isotropic EPR and ENDOR spectra are only obtained in the limit (Redfield, 1965) where denotes the amplitude of the timedependent anisotropic magnetic interaction (g or hyperfine anisotropy in energy units). For organic is in the order of radicals in common solvents to seconds and relation (7) is valid in most cases. However, for a large protein like the photosynthetic reaction center (RC) with an estimated molecular weight of daltons, the s at room temperature in water value is and only small hf anisotropies MHz) are effectively averaged out (Lendzian et al., 1981). For large proteins or highly aggregated species solid state EPR and ENDOR spectra are expected even in liquid aqueous solution at ambi­ ent temperature due to the slow tumbling motion. For further information and experimental de­ tails of ENDOR, the reader is referred to Kevan and Kispert (1976), Kurreck et al. (1988), Möbius et al. (1989), Atherton (1993), Poole and Farach (1994). 3. TRIPLE Resonance For biological samples, the relaxation rates often cannot be adjusted and leading to a very small ENDOR effect. In this situation, one can short-circuit the “nuclear relaxation bottle­ neck” by a second strong rf field. In this electron– nuclear–nuclear triple resonance experiment (called “Special TRIPLE”), the two rf fields are tuned to drive both transitions, and of the same nucleus simultaneously (see Fig. 2), thereby enhancing the efficiency of the relaxation bypass and hence the signal inten­ sity (Möbius and Biehl, 1979). The second advan­ tage of Special TRIPLE is that the EPR desatu­ ration becomes independent of nuclear relaxation, i.e., the line intensities now reflect the number of nuclei involved in a transition. TRIPLE lines can therefore be assigned more easily than ENDOR lines. Special TRIPLE also has the advantage of somewhat higher resolution than ENDOR. In Fig. 3, a Special TRIPLE spectrum is com­ pared schematically with an ENDOR spectrum. Both spectra are typically recorded in the first­

ENDOR spectroscopy

derivative mode using frequency modulation of the rf field (Kurreck et al., 1988). The Special TRIPLE looks like an “ENDOR half spectrum” with its origin at the free proton frequency, The intensity is, however, higher (the exact factor depends on experimental conditions) and the line intensities reflect approximately the relative number of contributing protons. Electron–nuclear–nuclear triple resonance can be generalized to include more than one nucleus, for example, two inequivalent protons (Fig. 3). In this “General TRIPLE” experiment, the ENDOR spectrum is swept with the first modu­

261

lated rf field while a second, fixed (unmodulated) rf field, is applied to pump a specific NMR transi­ tion. In general, all NMR transitions in the same electron spin manifold (characterized by the same value) as the additionally irradiated one will decrease in intensity, whereas those in the other manifold will increase. Since the assignment of a particular high- or low-frequency transition to an value is determined by the sign of the hfc, the observed intensity changes in the General TRIPLE spectrum can be used to determine the relative signs of the hfcs in the radical. In a het­ eronuclear TRIPLE experiment, the sign of

262 must be known (Kurreck et al., 1988). In Fig. 3 (top), such a General TRIPLE spectrum is dis­ played schematically in which line 2 is addition­ ally pumped. This leads to an enhancement of the respective low-frequency line 2' (Special TRIPLE effect). For the line pair 1,1', the high-frequency line is increased and the low-frequency line is decreased in intensity. Thus, the respective hfcs and have opposite signs. Absolute signs can be obtained from theory or measured directly in NMR experiments (De Boer and MacLean, 1966; see also Lubitz, 1991). The signs of the hfcs are important for theoretical reasons and are helpful for assigning the couplings to molecular positions. A more thorough theoretical descrip­ tion of TRIPLE resonance can be found in (Mö­ bius and Biehl, 1979; Möbius et al., 1989; Kur­ reck et al., 1988).

Wolfgang Lubitz and Friedhelm Lendzian ecules. In the case of transition metal ions cryo­ genic temperatures are usually required for ENDOR detection due to the short electron spin relaxation times. There exist several excellent re­ cent review articles covering this field which de­ monstrate the ability of ENDOR to gain infor­ mation in particular about the ligand structure (Schweiger, 1982; Hoffmann et al., 1993; Hütter­ mann, 1993; Thomann and Bernardo, 1993). Here, we concentrate on paramagnetic organic doublet state molecules in frozen solutions and in single crystals; triplet states are handled in a subsequent section. In the solid state, the static spin Hamiltonian of a radical contains, in addition to the terms in Eq. (1), all angular dependent traceless interactions and is given by:

4. ENDOR-lnduced EPR Occasionally different radical species may be pre­ sent in the same sample. The EPR of such a mixture, especially when the g values are similar, often consists of a superposition of different spec­ tra which are difficult or impossible to separate. Since the density of spectral lines is much smaller in ENDOR than in EPR, it is very likely that the different species have non-overlapping ENDOR lines. In this case the different EPR spectra can be recorded separately using ENDOR-induced EPR (EIE), first reported by Hyde (1965). In this experiment the intensity of an ENDOR line belonging to one species is monitored while sweeping the magnetic field over the EPR spec­ trum. Thereby, it is often necessary to sweep also the rf frequency, since the nuclear Zeeman is proportional to Some ex­ frequency, amples for EIE are given by Kurreck et al. (1988). A special application of EIE is in single crystals, where it offers the possibility to separate overlapping EPR spectra of different sites.

B. ENDOR in the Solid State In the solid state ENDOR spectroscopy has a much larger field of applications than in liquids. This includes practically all paramagnetic states ranging from transition metal complexes to im­ mobilized organic radicals and triplet state mol-

Here, G is the g-tensor and is the hyperfine (hf) tensor of nucleus i containing the isotropic (i = x,y,z) and the traceless part dipolar part with elements and is the quadrupole (i = x,y,z). coupling tensor which describes the interaction of the nuclear electric quadrupole moment (for with the electric field gradient at the nu­ cleus (Atherton, 1993). Even to first order, the expressions for the energy eigenvalues and EPR and NMR transition energies are rather com­ plicated for the general case. This results mainly from the fact that the nuclear spin quantization is in the resultant of the applied and hyperfine is in general no longer a magnetic field and “good quantum number” for the various eigenstates. A detailed treatment of the angular depen­ dent energy eigenvalues and the EPR and ENDOR transition frequencies is given by Ather­ ton (1993). For the case of isotropic g-factor and no nu­ clear quadupole interaction, the first order energy eigenvalues for the spin system are given by:

ENDOR spectroscopy where A is the hyperfine coupling tensor (fre­ and are the electron and quency units), nuclear spin quantum numbers and is the unit vector along the field, with components in the reference axis the direction cosines of system. The first order NMR frequencies for this case are given for by:

with the high-and low-frequency NMR trans­ itions. These two NMR (ENDOR) frequencies are in general no longer symmetric about Only in the limit of isotropic A or for small anisotropy this symmetry is re­ of A tained and Eq. (10) reduces to

It is important to note that this equation holds also exactly when the field is along one of the principal axes of A (Atherton, 1993). This means that the turning points in a powder ENDOR spec­ trum directly yield the true principal values of A, provided g is isotropic. This is a good approxi­ mation for most organic radicals. In general, however, g-anisotropy has to be considered. The NMR frequencies for are then given by (Atherton, 1993)

and the G-tensor principal values and eigenvec­ tors have to be known for an analysis of solid state ENDOR spectra. Only for coaxial tensors G and A, and for along one of the principal axes of G and A, can the respective true principal values of A be obtained directly from the ENDOR spectrum.

263 neglecting nuclear quadrupole interaction. For more details the reader is referred to the literature (Atherton, 1993; Hoffmann et al., 1993; Hüttermann, 1993; Thomann and Bernardo, 1993).

a. Isotropic g This is usually a good approximation for organic radicals studied in X-band (microwave frequency 9.5 GHz). In large molecules the hyperfine inter­ action with many nuclei often leads to an unre­ solved Gaussian envelope EPR spectrum. This case is shown schematically in Fig. 4A. Molecules of all orientations, with respect to the magnetic field, contribute to the ENDOR spectrum ob­ tained at the center of the EPR line. The turning points of this powder ENDOR spectrum yield directly the principal values of the respective nu­ clear hyperfine tensors (Fig. 4A, bottom). A spe­ cial case is of purely dipolar hf tensors, which are often obtained from matrix protons of the surrounding medium, e.g. in hydrogen bonds. In the point dipole approximation the angular de­ pendent dipolar hyperfine coupling is given by:

where is a constant depending on the respective nuclear magnetic moment, is the electron spin density at the contact position, r is the magnitude of the distance vector between electron and nuclear spin and is the angle be­ tween this vector and the direction of the mag­ netic field. From Eq. (13) an axially symmetric from tensor is obtained with which the distance r can be derived. For short and extended this distances simple approach is, however, insufficient and iso­ tropic contributions to A have to be considered.

1. ENDOR in Frozen Solutions In frozen solutions all the angular dependent spectral contributions sum up to a so-called pow­ der spectrum (Atherton, 1993; Weil et al., 1994). Here, we discuss qualitatively two limiting cases in order to demonstrate the resolution enhance­ ment of ENDOR obtainable in frozen solutions. Thereby, we restrict ourselves to the case

b. Anisotropic G-Tensor (Orientation Selection) If the g-anisotropy is larger than the hyperfine couplings in the spin system, it will dominate the EPR spectrum and can be used to select specific orientations of the molecules with respect to the magnetic field. This is demonstrated sche­

264

matically in Fig. 4B for the case of an axially symmetric G tensor. If ENDOR spectra are re­ corded at the outer turning points of the EPR and “single crystal-like” spectra spectrum are obtained (Rist and Hyde, 1970). This leads to a greately enhanced spectral resolution and for the case of coaxial G and A tensors the separation of the ENDOR lines corresponds directly to respectively (Fig. 4B, bottom). and For any other position in the EPR spectrum usually many different orientations contribute to the ENDOR response and the spectra become

Wolfgang Lubitz and Friedhelm Lendzian

more complicated. With the help of simulation programs (Hüttermann, 1993; Hoffmann et al., 1993) it is, however, possible to elucidate princi­ pal values and relative orientations of principal axes of G and A from combined EPR and ENDOR epxeriments in frozen solutions. There are many reports in the literature in which, for example, the ligand structure of transition metal complexes and metalloenzymes has been deter­ mined in this way (Schweiger, 1982; Hoffmann et al., 1993; Hüttermann, 1993; Thomann and Bernardo, 1993).

ENDOR spectroscopy 2. ENDOR in Single Crystals The most detailed information about the hyper­ fine structure is obtained from single crystal stud­ ies. In the case of small molecular systems (or­ ganic radicals, transition metal complexes) it is usually necessary to incorporate the paramagnetic species in a diamagnetic host crystal in order to avoid spin–spin interactions between neigboring centers. In crystals of proteins or metal enzymes the paramagnetic centers are often far enough separated by the protein, and such interactions become sufficiently small to be neglected. In general, ENDOR spectra have to be ob­ tained for rotation of the magnetic field in the crystallographic planes of the crystal. The “ro­ and tation diagrams” of the NMR transitions, must be analyzed according to Eq. (12) using the g-tensor previously determined by EPR. The analysis yields the hyperfine tensor A in the crys­ tal axis system. Diagonalization of A then gives the principal values and the orientations of the tensor axes in the crystal axis system. If G can be approximated to be isotropic and the hyperfine anisotropy is small Eq. (12) reduces to Eq. (11). Here, the two NMR transitions and are centered around for any values of (orientation of in the chosen axis system). In this case, the symmetric and about allows the displacement of application of Special TRIPLE resonance (like in the case of an isotropic hfc, a). The Special TRI­ from is then PLE frequency (deviation of simply given by

for rotation of in the crystal i–j plane. Such a case is presented below in Fig. 5 (Lendzian et al., 1993). Different sites of the paramagnetic centers in the crystal unit cell may complicate the ENDOR spectra (“site splitting”) and may lead in some cases to ambiguities for the orientations of the principal axes of A (Atherton, 1993). In general, EPR and ENDOR in single crystals for all nu­ yield the G and hyperfine tensors, clei i, including the respective eigenvectors (di­

265 rection cosines of the principal axes). These con­ tain information about the spatial structure of the molecule which may then be compared with the structure obtained, for example, by X-ray analy­ sis (Scholes et al., 1982; Hutchinson 1992).

C. ENDOR of Triplet State Molecules The spin Hamiltonian for an electronic triplet state including hyperfine interaction is given by (Atherton, 1993)

where D is the zero field splitting tensor. In or­ ganic triplet states this term describes the dipolar interaction between the two electron spins which leads to an angular dependent splitting of the two EPR transitions: (i) and (ii) (see Chapter 14). In the high field approximation the first order energy levels show hyperfine splitting only for the and levels. The level is only split by the nuclear Zeeman interaction. Usually, the elements of D are much larger than the hyperfine terms. This can be used to obtain orientation selection of the molecules in frozen disordered solution via EPR in the same way as described above for anisotropic g. In the case of isotropic g, which is a good approximation for most organic triplet states, the NMR fre­ quencies are given by Eq. (12). Considering the simple case where D and A are coaxial and the magnetic field is parallel to the principal axis of the z component of the zero field splitting tensor D the NMR frequencies in the high-field appproximation are: (i) for the EPR transition and (ii) for the EPR transition The two EPR transitions are separated by where D is the zero field splitting constant (see Chapter 14). All NMR lines corresponding to positive appear at frequencies below whereas, lines arising from negative lie above for the EPR transition (i), and vice versa for EPR transition (ii). Hence, when the sign of D is

266

Wolfgang Lubitz and Friedhelm Lendzian

ENDOR spectroscopy known, the absolute signs of the hfcs are obtained directly from the ENDOR spectra. There is also an NMR transition at the Larmor frequency for both EPR transitions. This behavior is demon­ strated in ENDOR spectra for naphthalene triplet states in frozen solution (Kirste and van Willigen, 1982) and enables the determination of the rela­ tive signs of D and

D. New Techniques A crucial factor which determines the magnitude of the observed ENDOR effect in continuous wave (cw) spectroscopy is the balance of electron and nuclear relaxation rates and the rates induced by the coherently irradiated mw and rf fields. This is a serious drawback for many spin systems, where, for example, the relaxation rates cannot be easily adjusted by variation of the temperature or mw and/or rf saturation is a problem. In these cases pulsed techniques are clearly superior to cw-spectroscopy. An alternative experimental technique for re­ solving the hyperfine structure of inhomogene­ ously broadened EPR spectra is pulsed EPR, es­ pecially electron spin echo envelope amplitude modulation (ESEEM), which is described in Chapter 15. The observed modulation effects re­ sult from coherent excitation of “allowed” and “forbidden” EPR transitions by a strong micro­ wave pulse, and the observed echo amplitude modulations depend on off-diagonal elements of see e.g. Dikanov the respective hf tensors, and Tsvetkov (1992) and Schweiger (1990). Large effects are observed, if these off-diagonal ele­ ments are comparable in magnitude to the nu­ clear Zeeman splitting, whereas small or even no modulation effects are observed in the case of values. Therefore, this technique is small mostly applied to nuclei with small magnetic moments (e.g. which in turn are diffi­ cult to detect in cw-ENDOR (Käß et al., 1995). This restriction does not exist if pulsed EPR is extended to Pulsed ENDOR. Several pulse schemes have been employed in this technique. In all cases no match of electron and nuclear relaxation rates is required, since the whole pulse sequence is employed in a time shorter than the relaxation times. In general, one (or two) rf pul­ ses are applied in the preparation or mixing pe­

267 riod between a series of microwave (mw) pulses and the amplitude of the finally detected electron spin echo is observed as a function of the rf fre­ quency. The frequencies of the NMR (ENDOR) transitions are the same as those for cw-ENDOR. There are several recent reviews about techniques and applications of Pulsed ENDOR (Dinse, 1989; Grupp and Mehring, 1990; Schweiger, 1991; Thomann and Bernardo, 1993). A more detailed de­ scription is given in chapter 15. For the study of transition metal complexes is often and metal enzymes the case met even at low temperatures, i.e., the electron and nuclear relaxation rates cannot be matched leading to a strongly reduced ENDOR effect. In order to avoid fast passage effects, which lead to distorted spectra and strongly reduced ENDOR intensities (Kevan and Kispert, 1976), the rf modulation frequency, used for detection, For rad/s has to be smaller than this reduces additionally the apparent signal in­ tensities due to the noise contribution of the microwave spectrometer (Poole, 1983). This disadvantage can be overcome, if the NMR fre­ quency is not stepped sequentially through the spectrum, but if frequency values for each step are selected stochastically from the entire range of the ENDOR spectrum. Thereby discrete NMR frequencies are assigned each to a separate chan­ nel and the ENDOR signal is accumulated for each channel. In this “stochastic ENDOR” ex­ periment the NMR frequency channels can be in a selected with a much higher rate than conventional cw-ENDOR experiment which, in general, leads to a signficantly increased sensitiv­ ity. Details of this technique and some appli­ cations are reviewed by Brüggemann and Niklas (1994). Recently, EPR- and also ENDOR-spectrometers operating at 95 GHz (W band) have been developed in a few laboratories. These spectrometers offer a ten times better resolution of G components compared with the standard X band (9 GHz). In addition to other advantages, e.g. for pulsed EPR, the operation at higher fields enables the detection of orientationally selected ENDOR spectra also from frozen solutions of organic radicals (Rohrer et al., 1995). An over­ view of recent developments and applications of

268

high-field EPR and ENDOR has been given by Möbius (1993). III. Selected Applications of ENDOR to Photosynthesis

A. Pigment Radicals in Vitro EPR, ENDOR and TRIPLE resonance has been extensively used to study the electron spin density distribution of the radical cations and radical anions of bacteriochlorophylls, bacteriopheophy­ tins, chlorophylls and pheophytins, which occur as electron carriers in the reaction centers of pho­ tosynthetic bacteria and plants. An overview of the field has been given by Fajer and Davis (1979) and more recently by Lubitz (1991). These studies yielded detailed maps of the electron distribution in the frontier orbitals (HOMO and LUMO) of these species which can also be calculated quite reliably by all valence electron SCF methods like RHF-INDO/SP and related MO methods (for a review see Plato et al., 1991). Such studies on the isolated pigment radical ions are important for a comparison with the same species occurring in the reaction centers of plants and bacteria. As an example, Fig. 1 shows the EPR and ENDOR spectra of the BChl a cation radical in liquid solution, which was first studied by Borg et al. (1976). In this species the unpaired electron (HOMO) and in­ is delocalized in the teracts with the various magnetic nuclei in the molecule etc., see Fig. 1B). This leads to a very complex EPR spectrum (A) with almost no resolved hfs. The ENDOR spectrum of this species (C) contains much fewer resonance lines and directly yields 12 hfcs and 3 hfcs. The ENDOR lines are grouped around (near 14 MHz) and (1 MHz), respectively. All 4 nitrogen hfcs could be clearly resolved in a (Lubitz et al., 1984; Käß et al., 1995). BChl The relative signs of all hfcs were obtained by General TRIPLE resonance. Assignments of the hfcs to their specific molecular positions were achieved by several experimental approaches, in­ cluding a comparison with results from related NMR methods, structurally similar radicals and, in particular, partially deuterated species using biosynthetic labeling and H/D exchange experi­ ments (Lubitz, 1991; and references therein). The

Wolfgang Lubitz and Friedhelm Lendzian ENDOR spectrum of selectively deuterated BChl that carries protons only at the methyl groups (position 1a, 5a and 2b) and in positions to is shown as an example in Fig. 1D. the ENDOR The 3 line pairs remaining in the were assigned accordingly. The observed ENDOR spectrum was also detected, and ENDOR lines could be dis­ criminated by the different temperature depen­ dence of their intensities. The optimum temperature for the deuterons was ~ 230K, whereas the nuclei require much higher tem­ peratures for their detection (270K). By use of the measured and assigned hfcs the slightly structured EPR line of BChl in solution could be perfectly simulated (see Fig. 1A).

B. Radical Ions in Photosynthetic Reaction Centers The application of magnetic resonance to photo­ synthetic reaction centers has been reviewed by Hoff (1993). Here, we focus on the radical ions generated in the RCs as studied by cw EPR, ENDOR, and TRIPLE resonance techniques. The EPR signal detected in illuminated bacterial RCs near g = 2.0026 is due to the primary electron donor cation radical The narrowing of as compared with that the EPR linewidth of of monomeric BChl formed the basis of the “special pair” hypothesis for this species (Norris et al., 1971) which proved to be correct in the RC crystal structure analysis of two bacteria (De­ isenhofer and Michel, 1989; Feher et al., 1989). were The details of the electronic structure of unraveled entirely by ENDOR techniques. Here, in particular Special TRIPLE resonance was used because of its inherently higher sensitivity and resolution. A typical liquid solution spectrum is shown in Fig. 5 (left, bottom), for which 10 hfcs were obtained by spectral deconvolution. These hfcs are smaller than those detected in monom­ eric BChl (Fig. 1C), indicating a delocaliz­ ation of the unpaired electron in a dimeric species (supermolecule). Selective deuteration of the macrocycles was again instrumental in determining that in RCs of Rb. sphaeroides (Lendzian et in RCs of Rps. viridis (Lendz­ al., 1992) and ian et al., 1988) is indeed a BChl dimer and that the unpaired electron is asymmetrically distri­

ENDOR spectroscopy buted over the dimer halves. It is interesting that a symmetric dimer has been reported very re­ cently for the primary donor in the green bacterium Chlorobium limicola based on EPR/ENDOR measurements (Rigby et al., 1994b). In several purple bacteria containing a BChl adimer the asymmetry is, however, highly con­ served (Rautter et al., 1994). For the primary donor cation radical in PS I, (Käß et al., (Rigby et al., 1994a), 1994) and in PS II, chlorophyll dimers have been recently postulated from ENDOR data with a strongly asymmetric spin density distribution over the dimer halves. ENDOR-in-solution on allows the sensi­ tive detection of even small changes in the sur­ rounding of the primary donor via changes of the spin density distribution of the dimer. An example is shown in Fig. 6, which compares Spe­ in RCs of the wild cial TRIPLE spectra of type and of two mutants of Rb. sphaeroides. In these mutants hydrogen bonds are introduced either to the 9-keto group of the L-side of the or to the related 9-keto group on the dimer M-side see Fig. 6, top. The hydrogen bonds influence the orbital energies of the two halves of the dimer and thereby the distribution of the unpaired electron. This leads to a predominant localization of the spin either on the L or the M side which is clearly seen in the spectra (Fig. 6, bottom). The effect is even more pronounced in several double mutants of the same type (Rautter et al., 1995). ENDOR and TRIPLE resonance have become important tools in the characteriz­ ation of the effects of such mutations on structure and function of the bacterial and plant RCs. More detailed information concerning the electronic structure of can be obtained by studying this species not only in liquid and frozen solutions but also in RC single crystals (Lendzian et al., 1993). Fig. 5 (left) shows such Special TRI­ in Rb. sphaeroides obtained PLE spectra of with the external magnetic field, oriented along either of the 3 principal crystallographic axes a, b, and c. In the given crystal system (space group all four in the four RCs of the unit cell are magnetically equivalent for these 3 orientations. If the crystal is rotated in one crys­ tallographic plane, e.g. in the ac-plane, a splitting in 2 sites (I and II) is observed. For this plane the respective angular dependence of the hfcs is

269

shown in Fig. 5 (right). Experiments in the other two planes (ab, bc) finally yield a complete set of hf tensors for 8 nuclei from which not only the magnitude of the anisotropic hfcs are obtained but also the orientations of the tensor principal axes with respect to the crystal axes system. The latter contain detailed information about the spa­ tial structure of the radical, since the tensor axes are related to the electron spin distribution and this is, in turn, related to the bonding structure of the molecule. In the axes information has been used advantageously to assign the hfcs to the various nuclei on the L and the M halves of the dimer (Lendzian et al., 1993). ENDOR stud­ in single crystals of mutant RCs eluci­ ies on date electronic and structural changes induced by specific mutations (Huber et al., 1994). For other systems with unknown spatial structure this me­ thod offers the possibility to determine the orien­ tation of the spin carrying molecule in the pro­ tein. This has been shown for the primary donor in single crystals of photosys­ cation radical tem I (Käß et al., 1994) and for the primary in Zn-substituted RC sin­ quinone acceptor, gle crystals of Rb. sphaeroides (Isaacson et al., 1995a, 1995b). In addition to the primary donor and the stable quinone acceptor the anion radicals of the inter­ mediate electron acceptors, have also been ex­ tensively studied by ENDOR in several bacteria and in plant photosystem II (reviewed in Lubitz, 1991). In these systems, the observed species is the anion radical of either bacteriopheophytin or pheophytin, respectively. In Fig. 7 the frozen so­ lution ENDOR spectrum of trapped in Photos­ ystem II is compared with that of the anion rad­ ical of pheophytin a (Lubitz et al., 1989). The intense and narrow lines are assigned to methyl groups in positions 1a (line pairs and and 5a (line pairs and The large inten­ sity of these lines is attributed to the free rotation of the methyl groups even in frozen matrices and the small anisotropy of the hfcs. The strong line at the free proton frequency is mainly due to weakly dipolar coupled protons from the sur­ rounding protein matrix. An analysis of these ma­ trix ENDOR lines can yield information about distances and orientations of these protons in the environment of a paramagnetic center (Kevan and Kispert, 1976). In the ENDOR spec­

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Wolfgang Lubitz and Friedhelm Lendzian

ENDOR spectroscopy

271

trum of H/D exchange experiments (Lubitz et al., 1989) revealed hfcs belonging to exchange­ able protons which can be assigned to a hydrogen bond between an amino acid (glutamate) and the 9-keto group of Ph a (Fig. 7, top). A point-dipole analysis of the respective hf tensor (Eq. (13)) Such yielded an H-bond length of a strong H bond could be one of the possible reasons for the observed differences of the methyl and in Ph (see Fig. 7). Such hf tensors in effects have also been observed for RCs of Rb. sphaeroides and Rps. viridis (for a review see Lubitz, 1991). Fairly strong and asymmetrical H bonds have in Zn­ also been detected by ENDOR for containing RCs of Rb. sphaeroides in frozen solu­ tion (Feher et al., 1985). The electron spin den­ sity distribution of this species deviates signifi­ and of the ubiquinone cantly from that of anion radical in frozen alcoholic solution. ENDOR allows the sensitive detection of these differences in the electronic structure which are related to the different functions of the two quin­ ones in the electron transfer process in the RC.

C. ENDOR of Triplet States Magnetic resonance experiments on triplet states are generally performed in the solid state (single crystals or frozen solutions) mostly at cryogenic temperatures because of the large zero field splittings giving rise to fast relaxation rates in liquid solutions. There are several transient EPR studies of photochemically generated triplet states of chlorophylls and porphyrins utilizing the large spin polarization effects usually generated by the intersystem crossing from the excited singlet state to the excited triplet state of the molecule (Levanon and Bowman, 1993). Triplet states of chlorophylls occur also in all bacterial photosys­ tems and in plant PS I and PS II, if the secondary electron acceptors are pre-reduced before illumi­ nation (Budil and Thurnauer, 1991; Miller and Brudvig, 1991). These triplet states have been characterized by optically detected magnetic res­ onance (ODMR) yielding the zero field splitting parameters (Hoff, 1989). However, this tech­ nique is usually applied in zero field where hyper­ fine interactions (except the nuclear quadrupole interaction) occur only in second order (Chapter 17).

272 There are several cw-ENDOR studies of the photoexcited triplet state of porphyrins in frozen solutions performed in high field (X-band) de­ monstrating the ability of this method to resolve hyperfine structure using orientation selection via the large zero field splitting (see e.g. Hamacher et al., 1993). Triplet states of the primary donors in photosynthesis have been studied extensively by X-band EPR in single crystals yielding detailed information about the orientation of the zero field splitting tensor axes with respect to the molecular structure (see e.g. Norris et al., 1989). However, no hyperfine information was obtained. In the only ENDOR study reported so far (Lendzian et al., 1985b) several proton hfcs of in Rb. sphaeroides R-26 could be resolved, but no defi­ nite assignment to specific nuclei was made. For the same species the nitrogen hfcs have been de­ RCs (de termined by ESEEM on Groot et al., 1985). In both papers it was con­ cluded that the triplet excitation is delocalized over the two BChl a molecules constituting the Determination of the spin primary donor density distribution in the triplet state of the pri­ mary donor in all photosystems would yield im­ portant information about the electron distribu­ tion in the LUMO of P. This is important for understanding the forward electron transfer reac­ tions in the RCs.

Wolfgang Lubitz and Friedhelm Lendzian relative orientations of ligand atoms with respect to a metal center. In organic radicals the orien­ tations of principal axes of hf tensors are often related to directions of covalent bonds. Very de­ tailed information is obtained from single crystals and this type of spectroscopy has been termed “ENDOR-crystallography” (Hutchinson, 1992). The field of cw-ENDOR, in particular on or­ ganic radicals in liquid solution, is well estab­ lished, certainly also because of the commercial availability of cw-ENDOR spectrometers for al­ most two decades. New developments and appli­ cations are expected for the time domain ENDOR techniques. In particular, in combi­ nation with pulsed laser excitation, in which pho­ toreactions create spin polarization, ENDOR of­ fers the possibility to resolve the hyperfine structure of transient species. This has been de­ monstrated by transient cw ENDOR experiments on organic radicals in liquid solution and photoex­ cited triplet states (Lendzian et al., 1985a; Kay et al., 1995). In the solid state, pulsed ENDOR techniques are expected to be advantageous. Ap­ plied to photosystems, these techniques offer the possiblity to resolve the hyperfine structure of short-lived radical ions and triplet states on a microsecond time scale. Thereby light-induced structural changes and their influence on the pro­ cess of charge separation could directly be fol­ lowed.

IV. Concluding Remarks In this chapter, we have described the basic prin­ ciples of ENDOR and some applications of this technique to photosynthesis. Thereby, we have concentrated on continuous wave (cw) spectros­ copy. Basically, two different types of infor­ mation can be obtained from the measured hyper­ fine interactions: (i) Isotropic hfcs obtained from ENDOR yield, after proper assignment to mol­ ecular positions, a map of the spin density distri­ bution in the orbital(s) occupied by the unpaired electron(s). This offers experimental values for wave function coefficients at the position of speci­ fic nuclei in the molecule. These are important values, e.g. for calculating rates in electron trans­ fer reactions. This information is most easily ob­ tained for radicals in liquid solution. (ii) Aniso­ tropic hf tensors obtained from ENDOR contain structural information, e.g. about distances and

Acknowledgements The authors are grateful to Prof. K. Möbius (Freie Universität Berlin) and Prof. G. Feher (UC San Diego) and their former and present coworkers for many helpful discussions and for their contributions to the work reviewed in this chapter. Furthermore, we want to thank all mem­ bers of our group at the TU Berlin who were involved in the presented work and who are cited in the references. Financial support from the De­ utsche Forschungsgemeinschaft (Sfb 312) and Fonds der Chemischen Industrie (W.L.) is grate­ fully acknowledged. References Allen JP, Feher G, Yeates TO, Komiya H and Rees DC (1987) Structure of the reaction center from Rb. sphaero­

ENDOR spectroscopy ides R-26: The cofactors. Proc Natl Acad Sci USA 84: 5730–5734. Atherton NM (1993) Principles of Electron Spin Resonance. Horwood, Chichester. Barry BA (1993) The role of redox-active amino acids in the photosynthetic water-oxidizing complex. Photochem Pho­ tobiol 57: 179–188. Borg DC, Forman A and Fajer J (1976) ESR and ENDOR studies of the cation radical of bacteriochlorophyll. J Am Chem Soc 98: 6889–6893. Boussac A, Zimmermann J-L, Rutherford AW and Lavergne J (1990) Histidine oxidation in the oxygen-evolving photosystem-II enzyme. Nature (London) 347: 303–306. Brüggemann W and Niklas JR (1994) Stochastic ENDOR. J Magn Reson Series A 108: 25–29. Budil DE and Thurnauer MC (1991) The chlorophyll triplet state as a probe of structure and function in photosynthesis. Biochim Biophys Acta 1057: 1–41. de Boer E and MacLean C (1966) NMR study of electron transfer rates and spin densities in p-xylene and p-diethylbenzene anions. J Chem Phys 44: 1334–1342. de Groot A, Evelo R, Hoff AJ, de Beer R and Scheer H (1985) Electron spin echo envelope modulation (ESEEM) spectroscopy of the triplet state of the primary donor of and bacterial photosynthetic reaction centers and and bacteriochlorophyll a. Chem Phys Lett 118: of 48–54. Debus RJ (1992) The manganese and calcium ions of photo­ synthetic oxygen evolution. Biochim Biophys Acta 1102: 269–352. Deisenhofer J and Michel H (1989) The photosynthetic reac­ tion centre from the purple bacterium Rhodopseudomonas viridis. EMBO J 8: 2149–2170. Dikanov SA and Tsvetkov YD (1992) Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. CRC Press, Boca Raton. Dinse K-P (1989) Pulsed ENDOR In: Hoff AJ (ed) Advanced EPR. Applications in Biology and Biochemistry, pp 615– 631. Elsevier, Amsterdam. Fajer J and Davis MS (1979) Electron spin resonance of por­ phyrin cations and anions. In: Dolphin D (ed) The Por­ phyrins. Vol IV, pp 197–256. Academic Press, New York. Feher G (1956) Observation of nuclear magnetic resonances via the electron spin resonance line. Phys Rev 103: 834– 835. Feher G and Okamura MY (1978) Chemical composition and properties of reaction centers. In: Clayton RK and Sistrom WR (eds) The Photosynthetic Bacteria, pp 349–386. Ple­ num Press, New York. Feher G, Isaacson RA, Okamura MY and Lubitz W (1985) ENDOR of semiquinones in RC’s from Rhodopseudo­ monas sphaeroides. In: Springer Series in Chemical Physics 42: 175–189. Springer-Verlag, Berlin. Feher G, Allen JP, Okamura MY and Rees DC (1989) Struc­ ture and function of bacterial photosynthetic reaction cen­ ters. Nature (London) 339: 111–116. Freed JH (1979) Theory of multiple resonance and ESR satur­ ation in liquids and related media. In: Dorio MM and Freed JH (eds) Multiple Electron Spin Resonance Spectro­ scopy, pp 73–142. Plenum Press, New York.

273 Grupp A and Mehring M (1990) Pulsed ENDOR spectroscopy in solids. In: Kevan L and Bowman MK (eds) Modern Pulsed and Continuous-Wave Electron Spin Resonance, pp 195–229. John Wiley and Sons, New York. Hamacher V, Wrachtrap J, von Maltzan B, Plato M and Möbius K (1993) EPR and ENDOR study of porphyrins and their covalently linked dimers in the photoexcited tri­ plet state. Appl Magn Reson 4: 297–319. Hoff AJ (1989) Optically detected magnetic resonance of tri­ plet states. In: Hoff AJ (ed) Advanced EPR. Applications in Biology and Biochemistry, pp 633–684. Elsevier, Am­ sterdam. Hoff AJ (1993) Magnetic resonance of bacterial photosyn­ thetic reaction centers. In: Deisenhofer J and Norris JR (eds) The Photosynthetic Reaction Center. Vol II, pp 331– 382. Academic Press, San Diego. Hoffmann BM, DeRose VJ, Doan PE, Gurbiel RJ, Houseman ALP and Telser J (1993) Metalloenzyme activesite structure and function through multifrequency cw and pulsed ENDOR. In: Berliner LJ and Reuben J (eds) Bio­ logical Magnetic Resonance, EMR of Paramagnetic Mol­ ecules. Vol 13, pp 151–214. Plenum Press, New York. Hoganson CW and Babcock GT (1992) Protein-tyrosyl radical interactions in photosystem II studied by electron spin res­ onance and electron nuclear double resonance spectros­ copy: Comparison with ribonucleotide reductase and in vitro tyrosine. Biochemistry 31: 11874–11880. Huber M, Isaacson RA, Abresch EC, Gaul D, Schenck CC and Feher G (1996) Electronic structure of the oxidized primary electron donor of the HL(M202) and HL(L173) heterodimer mutants of the photosynthetic bacterium Rho­ dobacter sphaeroides: ENDOR on single crystals of reac­ tion centers, Biochim Biophys. Acta, in press. Hutchinson CA (1992) The study of structure by electron magnetic resonance methods. A brief history. Appl Magn Res 3: 219–255. Hüttermann J (1993) ENDOR of randomly oriented mononu­ clear metalloproteins: toward structural determinations of the prosthetic group. In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance, EMR of Paramagnetic Molecules. Vol 13, pp 219–250. Plenum Press, New York. Hyde JS and Maki AH (1964) ENDOR of a free radical in solution. J Chem Phys 40: 3117–3118. Hyde JS (1965) ENDOR of free radicals in solution. J Chem Phys 43: 1806–1818. Isaacson, RA, Abresch EC, Feher G and Lubitz W (1995a) ENDOR studies of in single crystals of reaction centers from Rb. sphaeroides. Biophys J 68(2): A246. Isaacson RA, Lendzian F, Abresch EC, Lubitz W and Feher G (1995b) Electronic structure of in reaction centers from Rhodobacter sphaeroides. I. Electron Paramagnetic Resonance in single crystals. Biophys J 69: 311–322. Käß H, Fromme P, Witt HT and Lubitz W (1994) and ESEEM of in single crystals of photosystem I from Synechococcus elongatus. Biophys J 66(2): A228. Käß H, Rautter J, Bönigk B, Höfer P, and Lubitz W (1995) radical cations of bacteri­ 2D ESEEM of the ochlorophyll a and of the primary donor in reaction centers of Rhodobacter sphaeroides. J Phys Chem 99: 436–448. Kay CWM, Di Valentin M and Möbius K (1995) A time­

274 resolved Electron Nuclear Double Resonance (ENDOR) study of the photoexcited triplet state of free-base tetra­ phenylporphyrin. Solar Energy Materials and Solar Cells 38: 111–118. Kevan L and Kispert LD (1976) Electron spin double reson­ ance spectroscopy. John Wiley and Sons, New York. Kirste B and van Willigen H (1982) ENDOR on photoexcited triplets randomly oriented in solid solution. Chem Phys Lett 92: 339–342. Kurreck H, Kirste B and Lubitz W (1988) Electron nuclear double resonance spectroscopy of radicals in solution. Ap­ plication to organic and biological chemistry. In: Marchand AP (ed) Methods in stereochemical analysis, VCH, Weinheim. Lendzian F, Lubitz W, Scheer H, Bubenzer C and Möbius K (1981) In vivo liquid solution ENDOR and TRIPLE resonance of bacterial photosynthetic reaction centers of Rhodopseudomonas sphaeroides R-26. J Am Chem Soc 103: 4635–4637. Lendzian F, Jaegermann P and Möbius K (1985a) Time-resolved CIDEP-enhanced ENDOR in short-lived radicals. Chem Phys Lett 120: 195–200. Lendzian F, van Willigen H, Sastry S, Möbius K, Scheer H and Feick R (1985b) Proton ENDOR study of the photoex­ cited triplet state in Rb. sphaeroides R-26 photosynthetic reaction centers. Chem Phys Lett 118: 145–150. Lendzian F, Lubitz W, Scheer H, Hoff AJ, Plato M, Tränkle E and Möbius K (1988) ESR, ENDOR and TRIPLE reson­ ance studies of the primary donor cation radical in the photosynthetic bacterium Rps. viridis Chem Phys Lett 148: 377–385. Lendzian F, Geßner C, Bönigk B, Plato M, Möbius K and and resonance Lubitz W (1992) in isotopically of the primary donor cation radical labeled reaction centers of Rb. sphaeroides In: Murata N (ed) Research in Photosynthesis, Vol I, pp 433–436. Kluwer Academic Publishers, Dordrecht. Lendzian F, Huber M, Isaacson RA, Endeward B, Plato M, Bönigk B, Möbius K, Lubitz W and Feher G (1993) The electronic structure of the primary donor cation radical in Rb. sphaeroides R-26: ENDOR and TRIPLE resonance studies in single crystals of reaction centers. Biochim Bi­ ophys Acta 1183: 139–160. Levanon H and Bowman MK (1993) Time-domain EPR spec­ troscopy of energy and electron transfer. In: Deisenhofer J and Norris JR (eds) The Photosynthetic Reaction Center, Vol II, pp 387–418. Academic Press, San Diego. Lubitz W (1991) EPR and ENDOR studies of chlorophyll cation and anion radicals. In: Scheer H (ed) Chlorophylls, pp 903–944. CRC Press, Boca Raton. Lubitz W, Isaacson RA, Abresch EC and Feher G (1984) Electron Nuclear Double Resonance of the primary donor cation radical in reaction centers of Rhodopseudo­ monas sphaeroides: additional evidence for the dimer model. Proc Natl Acad Sci USA 81: 7792–7796. Lubitz W, Isaacson RA, Okamura MY, Abresch EC, Plato M and Feher G (1989) ENDOR studies of the intermediate electron acceptor radical anion in Photosystem II reac­ tion centers. Biochim Biophys Acta 977: 227–232. Miller A-F and Brudvig GW (1991) A guide to electron para-

Wolfgang Lubitz and Friedhelm Lendzian magnetic resonance spectroscopy of Photosystem II mem­ branes. Biochim Biophys Acta 1056: 1–18. Möbius K (1993) High-field EPR and ENDOR on bioorganic systems. In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance, EMR of Paramagnetic Molecules, Vol 13, pp 253–271. Plenum Press, New York. Möbius K and Biehl R (1979) Electron–nuclear–nuclear TRI­ PLE resonance of radicals in solutions. In: Dorio MM and Freed JH (eds) Multiple Electron Spin Resonance Spectroscopy, pp 475–507. Plenum Press, New York. Möbius K and Lubitz W (1987) ENDOR spectroscopy in photobiology and biochemistry. In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance, Vol 7, pp 129–247. Plenum Press, New York. Möbius K, Lubitz W and Plato M (1989) Liquid-state ENDOR and TRIPLE Resonance. In: Hoff AJ (ed) Ad­ vanced EPR, Applications in Biology and Biochemistry, pp 441–494. Elsevier, Amsterdam. Norris JR, Uphaus RA, Crespi HL and Katz JJ (1971) Elec­ tron spin resonance of chlorophyll and the origin of signal I in photosynthesis. Proc Natl Acad Sci (USA) 68: 625– 628. Norris JR, Budil DE, Gast P, Chang C-H, El-Kabbani O and Schiffer M (1989) Correlation of paramagnetic states and molecular structure in bacterial photosynthetic reaction centers: The symmetry of the primary electron donor in Rhodopseudomonas viridis and Rhodobacter sphaeroides R-26. Proc Natl Acad Sci USA 86: 4335–4339. Plato M, Lubitz W and Möbius K (1981) A solution ENDOR sensitivity study of various nuclei in organic radicals. J Phys Chem 85: 1202–1219. Plato M, Möbius K and Lubitz W (1991) Molecular orbital calculations on chlorophyll radical ions. In: Scheer H (ed) Chlorophylls, pp 1015–1046. CRC Press, Boca Raton. Poole CP (1983) Electron Spin Resonance. A Comprehensive Treatise on Experimental Techniques, John Wiley and Sons, New York. Poole CP, Farach HA (eds) (1994) Handbook of Electron Spin Resonance. Data Sources, Computer Technology, Relaxation, and ENDOR, AIP Press, New York. Rautter J, Lendzian F, Lubitz W, Wang S and Allen JP (1994) Comparative study of reaction centers from photosynthetic purple bacteria. Electron paramagnetic resonance and elec­ tron nuclear double resonance spectroscopy. Biochemistry 33: 12077–12084. Rautter J, Lendzian F, Schulz C, Kuhn M, Lin X, Williams JC, Allen JP and Lubitz W (1995) ENDOR-studies of the primary donor cation radical in mutant reaction centers of Rhodobacter sphaeroides with altered hydrogen-bond interactions. Biochemistry 34: 8130–8143. Redfield AG (1965) The theory of relaxation processes. In: Waugh JS (ed) Advances in Magnetic Resonance, Vol 1, pp 1–32. Academic Press, New York. Rigby SEJ, Nugent JHA and O’Malley PJ (1994a) ENDOR and Special Triple Resonance studies of chlorophyll cation radicals in photosystem 2. Biochemistry 33: 10043–10050. Rigby SEJ, Thapar R, Evans MCW and Heathcote P (1994b) The primary donor of The electronic structure of the Chlorobium limicola fsp thiosulphatophilum photosyn­ thetic reaction center. FEBS Lett 350: 24–28.

ENDOR spectroscopy Rist GH and Hyde JS (1970) Ligand ENDOR of Metal Com­ plexes in Powders. J Chem Phys 52: 4633–4643. Rohrer M, Plato M, MacMillan F, Grishin Y, Lubitz W and Möbius K (1995) Orientation selected 95 GHz high-field ENDOR spectroscopy of randomly oriented plastoquinone anion radicals. J Magn Res Series A, 116: 59–66. Scholes CP, Lapidot A, Mascarenhas R, Inubushi T, Isaacson RA and Feher G (1982) Electron Nuclear Double Reson­ ance (ENDOR) from heme and histidine nitrogens in single crystals of aquometmyoglobin. J Am Chem Soc 104: 2724– 2735. Schweiger A (1982) Electron Nuclear Double Resonance of Transition Metal Complexes with Organic Ligands. Struc­ ture and Bonding, Vol 51, pp 1–121. Springer-Verlag, Berlin.

275 Schweiger A (1990) New trends in pulsed electron spin-resonance methodology. In: Kevan L and Bowman MK (eds) Modern Pulsed and Continuous-Wave Electron Spin Res­ onance, Chapter 2, pp 43–118. John Wiley and Sons, New York. Schweiger A (1991) Pulsed electron spin resonance spectros­ copy: Basic principles, techniques, and examples of appli­ cations. Angew Chem Int Ed Engl 30: 265–292. Thomann H and Bernardo M (1993) Pulsed electron nuclear double and multiple resonance spectroscopy of metals in proteins and enzymes. In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance, EMR of Paramagnetic Molecules. Vol 13, pp 275–320. Plenum Press, New York. Weil JA, Bolton JR and Wertz JE (1994) Electron Paramag­ netic Resonance. Elementary Theory and Practical Appli­ cations. John Wiley and Sons, New York.

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Chapter 17

Optically Detected Magnetic Resonance (ODMR) of Triplet

States in Photosynthesis

Arnold J. Hoff

Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504,

2300 RA Leiden, The Netherlands

Summary I. Introduction II. The Triplet Spin Hamiltonian in Zero Magnetic Field III. Optical Detection of Magnetic Resonance, ODMR A. Principles 1. Time-Resolved ODMR a. Pulsed Microwaves b. Determination of the Average Decay Rate B. Instrumentation IV. Double Resonance A. Triplet-Minus-Singlet (T–S) Absorbance Difference Spectra B. Linear Dichroic T–S Spectroscopy 1. Instrumentation for LD-(T – S) Spectroscopy V. ODMR in Photosynthesis A. ADMR Spectroscopy of Reaction Centers B. T–S Spectroscopy of Reaction Centers C. Linear-Dichroic T–S Spectroscopy 1. Bacterial Reaction Centers 2. Plant Reaction Centers a. Photosystem I b. Photosystem II VI. Concluding Remarks References

277 278 278 279 279 281 281 281 282 284 285 286 287 288 288 289 290 290 292 293 293 295 295

Summary The triplet state of aromatic molecules and of polyenes is a versatile probe of molecular structure and of the interactions with the environment, through the zero-field splitting (ZFS) parameters and the sublevel decay rates. These triplet properties can be determined accurately with magnetic resonance. Optical detection of magnetic resonance (ODMR) is often advantageous because it pairs the frequency resolution of magnetic resonance with the sensitivity of optical Spectroscopy. It is preferentially carried out in zero magnetic field, where magnetic-field dependent anisotropies are absent, thus considerably enhancing the resolution and sensitivity for disordered systems. In photosynthesis, the optical parameters used for the detection of ODMR are the fluorescence (FDMR) and the absorbance (ADMR). Especially the latter is of interest, because it can be used for samples with low fluorescence yield, and allows an optical-microwave double resonance technique, in which the wavelength of detection is scanned, keeping the microwaves at a resonant frequency. Thus, Correspondence: Fax: 31-71-5275819; E-mail: [email protected]

277 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 277–298. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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Arnold J. Hoff

triplet-singlet absorbance difference (T–S) spectra can be recorded with superior accuracy and resol­ ution. In this Chapter, the physical background of ODMR is outlined, followed by a few highlights of its application to the study of photosynthetic reaction centers. Recent studies employing ADMR-recorded linear-dichroic T–S spectroscopy are emphasized. Key results are the determination of the orientation of several transition moments in reaction centers of several bacteria and of the two plant photosystems. This has allowed insight in the configuration of the various primary donors, and has aided in the interpretation of the reaction center absorbance spectrum. A further important finding is that isolated reaction centers generally exhibit a heterogeneity of the optical and magnetic resonance parameters of the primary donor, which is attributed to a distribution of conformations of the reaction center pigments. Abbreviations: ADMR – absorbance-detected magnetic resonance; BChl – bacteriochlorophyll; BPh – bacteriopheophytin; Chl –chlorophyll; CT – charge transfer; D – primary electron donor; monomeric BChl associated with the active and non-active electron transport chain, respectively; FDMR – fluorescence-detected magnetic resonance; LD – linear dichroic; MI – microwave-induced; MIA – microwave-induced absorbance; MIF – microwave-induced fluorescence; MIP – microwaveinduced phosphorescence; ODMR – optically detected magnetic resonance; PDMR – phosphorescencedetected magnetic resonance; Pheo – pheophytin; PS – photosystem; P680 – primary donor of Photosys­ tem II; P700 – primary donor of Photosystem I; RC – reaction center; T–S – triplet-minus-singlet; ZFS – zero-field splitting I. Introduction Optical detection of magnetic resonance (ODMR) is often advantageous because it pairs the frequency resolution of magnetic resonance with the sensitivity of optical spectroscopy. It is primarily used for magnetic resonance of the tri­ plet state, as the resonant transfer of population between the triplet sublevels generally gives rise to a concomitant change in the optical properties (phosphorescence, fluorescence, absorption) of the sample, because of the different decay charac­ teristics of each sublevel. It is preferentially car­ ried out in zero magnetic field, where the triplet wave functions are not mixed by the Zeeman interaction and the triplet sublevels are split by the dipolar interaction between the two unpaired electrons (and to a lesser extent by electron – nuclear hyperfine interactions with nuclei of This enhances considerably the resolution and sensitivity for disordered systems. In photosynthesis, phosphorescence detection of ODMR is not feasible because of the very low quantum yield of phosphorescence. Detection by the fluorescence and especially by the absorption, however, is possible with quite good sensitivity. The information obtained is twofold: Firstly, the

zero-field splitting (ZFS) parameters and the sub­ level decay rates can be determined with much better accuracy than with regular EPR. These parameters reflect the structure of the primary donor and its environment. Secondly, keeping the microwave frequency constant at one of the resonant values (there are generally three ODMR resonances corresponding to the three ways one can connect two out of three sublevels with res­ onant microwaves), one can scan the wavelength at which the resonance is detected. This gives the microwave-induced fluorescence or absorption spectrum. The latter corresponds to the optical triplet–singlet (T–S) difference spectrum, which in principle carries much information on the con­ figuration of the cofactors. In this Chapter, the physical background of ODMR will be sketched briefly, followed by a few highlights of its application to the study of photosynthetic reaction centers (RCs). II. The 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

ODMR of triplet states

two-fold: spin–spin coupling and spin–orbit coup­ ling. The main contribution to the spin–spin coupling operator, is the classical magnetic dipole–dipole interaction between two electrons:

with g the electronic g-value, the electronic the magnetic moments of Bohr magneton, the the two electrons, r their distance vector, permeability in vacuum, D a tensor operator whose elements consist of integrals over the coordinates of the electrons, the total spin angular momentum operator X, Y and Z the principal values of D and (u = x, y, z) the components of along the principal axes of D. Often, these axes coincide with the molecular symmetry axes. In a two-electron approximation the triplet wave functions can be written in symmetry-adapted form

where and are the eigenfunctions of the com­ ponent of the spin operator along the z di­ rection, The functions are eigen­ with eigenvalues X, Y and Z. functions of have the property The

Thus, there is no net magnetic dipole moment associated with any of the triplet substates in zero magnetic field: but there is a transition dipole moment present between any two of the triplet substates where is the gyromagnetic ratio of the electron and is Planck’s constant divided by Thus, in

279

zero magnetic field, population can be transferred from one triplet sublevel to another by applying a resonant electromagnetic field. From Eq. (4b) it follows that the transition probability is pro­ portional to where is the amplitude of the magnetic compo­ From (5) nent of the driving field, it follows that the microwave transition (u,s = x,y,z) is polarized with transition moment along w = u × s. This allows us to perform microwave-selection spectroscopy, to which we will turn later. The resonance frequencies follow from the namely X, Y and Z. Be­ eigenenergies of cause 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: with by convention The zero-field splitting parameters represent averages over the spatial coordinates x', y', z' of the distance vector r of the two unpaired electrons, E being a mea­ sure 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 mol­ ecule 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 For a rod-like molecule, such as a bi-radical, D will be negative. III. Optical Detection of Magnetic Resonance, ODMR

A. Principles Continuous illumination will generate an equilib­ rium population of the triplet sublevels given for light levels that are not too high by is the probability to transit from the singlet excited state to the u-th triplet sublevel with the decay rate that governs de­

280

Arnold J. Hoff

excitation from the u-th sublevel back to the sing­ let ground state, K the overall rate of populating the population of its u-th the triplet state, sublevel, its total population and N the number of photoexcitable molecules. Both and are determined by molecular symmetry. When a microwave field of a frequency corresponding to a transition between the y- and the z-level is switched on, the field will transfer population from the heavily-populated, slowly decaying zlevel to the much less populated, fast decaying ylevel. A new equilibrium will be established that for a strong enough microwave field is given by with and If we take it is then immediately seen that Al­ because for though 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 triplet population will lead to a change in phosphoresc­ ence or fluorescence intensity, or in the singlet ground state absorbance. For saturating micro­ waves it is given by

In the above we have the essence of ODMR. A sample is continuously illuminated at liquid he­ lium temperatures, preferably below 2.1 K, and simultaneously irradiated by microwaves of a fre­ quency v not far from that corresponding to one of the triplet sublevel spacings: and (Fig. 1). The frequency v is slowly scanned across one of the frequencies while either the phosphorescence, (delayed) flu­ orescence or absorbance of the sample is moni­ tored. When v is close to or precisely equal to the ensemble of triplet state is in reson­ ance with the microwave field and the fluor­ escence (FDMR), phosphorescence (PDMR) or absorbance (ADMR) will be enhanced, or dimin­ ished, depending on the relative values of and Note that the FDMR or ADMR signal inten­ sity, is proportional to the square of the incident light intensity because one photon is used for producing the triplet state and a second for

probing the microwave-induced change in the op­ tical parameter. 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 micro­ wave quantum. This enhances detector sensitivity enormously. Secondly, since the modes of detec­ tion and excitation are decoupled, one is insensi­ tive to noise sources due to the microwaves (as e.g. amplitude fluctuation), especially when the transition is saturated. (Note that the optical sig­ nal does not disappear upon saturation as does the microwave absorption in microwave detec­ tion; this is another advantage of optical detec­ tion.) Thirdly, compared to high-field EPR of triplet states, in zero-field resonance one has much narrower lines and a concomitant increase in sensitivity, since the anisotropy in resonance condition associated with an applied magnetic field is absent. Finally, the possibility to probe the resonance at various wavelengths gives espe­ cially for ADMR much new information.

ODMR of triplet states 1. Time-Resolved ODMR The time dependence of the ODMR signal in response to a change in the condition of micro­ wave irradiation (switching them on or off, or applying pulses) is given by the solution of the differential equations describing the time devel­ simpli­ opment of the four-level system fied by the neglect of the population. The general analytic solution is given by Hoff and Cornelissen (1982); for the present discussion it suffices to note that the response of the system on switching on or off resonant microwaves con­ or or both is given by the sum necting of three exponentials

where and are rather cumbrous functions of the rates of decay, spin-lattice relaxation W and microwave-induced transitions, and the rate K (proportional to the light flux) of populating and and in the triplet state. For the absence of microwaves, the triplet sublevels are uncoupled and each sublevel decays after some perturbation according to

a. Pulsed Microwaves If one perturbs the four-level system slightly by a pulse of microwaves resonant between two triplet sublevels, u and v, the return to equilibrium is governed by an equation similar to Eq. (9) (see Hoff and Cornelissen (1982) for explicit re­ lations). In the absence of spin-lattice relaxation and for low light fluxes and a pulse duration that is much shorter than the fastest triplet sublevel decay time, Eq. (9) reduces to a good approxi­ mation to two exponentials with characteristic rates given by

and with amplitudes of opposite sign. Provided K

281

is low enough, the third sublevel is not perturbed. Note that the extrapolation for is non­ trivial: Relations 11a,b are valid only for really low light intensities, for which the signal-to-noise ratio is poor, especially for fluorescence detec­ tion. They are therefore not suitable to determine these probabilities can be better determined by measuring the relative amplitude of the ODMR signal when connecting two of the three triplet sublevels by saturating microwaves (see e.g. Hoff, 1989). The amplitude of the pulse response to first order in K follows from Eq. (10) for the appropri­ ate boundary condition: and where f is a parameter describing the effect of the pulse on the population of sublevels u and v: for saturation (high power and/or long pulse) ,f= 1 for inversion of and (the maximal effect). With the Normally, and thus the signal amplitude change S(FDMR,ADMR), is to first order in K given by

b. Determination of the Average Decay Rate When saturating microwaves are applied simulta­ neously to two of the three ODMR transitions, the triplet collapses to one level with average decay rate The set of differential equa­ tions describing the simplified four-level system then reduces to those for an equilibrium reaction, which immediately yields

for the triplet built-up after the onset of illumi­ nation at t = 0. Eq. (13) permits the evaluation Obviously, the of k by extrapolating to same equation applies when the triplet levels are coupled not by microwaves but by spin-lattice relaxation at higher temperatures. In fact, 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.

282

B. Instrumentation The minimal requirements for an ODMR experi­ ment are a source of light, a source of micro­ waves, a cryostat and a detector. Up-to-date dis­ cussions of these parts and the relevant electronics are given by Maki (1984) and Hoff (1989,1990,1993). Here we will limit ourselves to a few notes and a discussion of special arrange­ ments for ADMR and LD-ADMR. A schematic diagram of our present ADMR set-up is shown in Fig. 2a. With small modifications it can be used for PDMR and FDMR as well. Crucial for high-sensitivity ADMR is a highintensity, low-noise light source. To this end the

Arnold J. Hoff

high-power tungsten-halogen lamp powered by a current-stabilized DC power supply, introduced by den Blanken et al. (1982), proved very suit­ able, and is now an essential part of all ADMR spectrometers. 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 or worse. The same holds for laser excitation. Adequate filtering of infra-red radi­ ation should be provided to avoid heating of the sample. When the excitation beam is broadbanded, it may also serve as probe beam at vari­ ous wavelengths. Microwaves are supplied preferably by a sweep unit (available with a range of 0.1–27 GHz) with

ODMR of triplet states

283

284 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 tem­ peratures without amplification. For measuring the ODMR lines the microwaves are fed into a broad-band 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 reflec­ tions of the microwaves (due primarily to mis­ match between helix and conductor), which may field intensity inside considerably affect the 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 dif­ ferent physical characteristics should be used for comparison. If one is interested in the (probe) wavelength dependence in MI spectroscopy, the microwave frequency is set exactly at resonance and a narrow-band cavity may be used. We have used a loop-gap resonator (Hardy and White­ head, 1981) with slots for optical access (Fig. 2b). Such a cavity has a much higher Q-value (ratio of stored energy and loss per cycle), and conse­ quently a much higher field at the same inci­ dent power, than the helix. This is an important advantage when measuring kinetics with pulsed and thus the signal ODMR, because then amplitude S(FDMR,ADMR), is much increased. Alternatively, a microwave power amplifier may be used. A further advantage of the loop-gap resonator is that it provides a well-polarized mi­ crowave field, of which good use is made in LD-ADMR (Section IV B 1). The cryostat is preferably a double-walled he­ lium 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 to below 2.1 K, the so-called lambda 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 (van der Bent et al., 1976). Such a light pipe may also used in a bath cryostat (Chan, 1982).

Arnold J. Hoff 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 exci­ tation beam may serve as such), providing a suf­ ficiently high number of transmitted quanta that a photodiode may be used (up to 1100 nm, silicium; germanium). To observe the 1100 nm – 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 en­ hanced by modulating the amplitude of the micro­ waves and applying frequency-selective amplifi­ cation of the photodetector signal combined with lock-in detection. Noise is then reduced to that corresponding with the passband of the amplifierlock-in detector combination, leading to an in­ crease in the signal-to-noise ratio of several or­ ders of magnitude. For example, in our ADMR spectrometer a ratio of better than is routinely achieved. Obviously, the modulating frequency has to be less than the slowest sublevel decay rate. For slowly decaying triplets 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. Finally, as in all modern spectrometers the in­ strument is interfaced to a small, dedicated com­ puter that handles monochromator setting, data collection and storage, and carries out simple op­ ratio (important when erations as taking the recording the probe wavelength dependence). IV. Double Resonance Optical. Once the ODMR lines of a triplet have been determined, the resonance frequencies are known precisely, and one can investigate the de­ pendence of the intensity of a particular reson­ ance line on the probing wavelength. Thus, one irradiates the sample with (amplitude-modulated) resonant microwaves of sufficient, preferably sat­ urating, intensity, and monitors the (lock-in de­ tected) photodetector output as a function of the

ODMR of triplet states 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 these spectra does not depend on the selection of the resonance frequency, i.e. or Obviously, if more than one triplet state is present, the MI spectra provide another means to sort out which resonances belong to the same triplet state. Conversely, MI spectroscopy allows the unraveling of complex optical spectra. MIA spectra are a case apart, since they provide much more information than the MIP or MIF spectra. As will be discussed in the next section, they represent the difference of the singlet ground state, ‘normal’, absorbance spectrum and the spectrum for the system when a triplet state is present. They have therefore been labeled triplet-minus-singlet absorbance difference (T–S) spectra, rather than MIA spectra (den Blanken and Hoff, 1982). Normally, the T–S spectrum is recorded with a single-beam optical spectrometer and the ADMR signal is normalized by the intensity of the incident light, I (den Blanken and Hoff, 1982). It can be shown that for small the normalized change in transmitted light intensity is proportional to the change in absorbance so that for nonAs mentioned above, saturating light intensities, the amplitude of the T–S spectrum is proportional to I. Microwaves. Another form of double resonance is irradiating the sample with microwaves of two different frequencies, one kept constant at a particular resonance line and one scanned over a certain frequency interval. There are two varieties of such a double-resonance experiment: either the microwaves of variable frequency or those at fixed frequency are amplitude-modulated; see e.g. Maki (1984) and Hoff (1989, 1990, 1993). If in the first case the microwaves are scanned over the resonance excited by the fixed frequency, then one observes the resonance line with a “hole”, centered at the “burn” frequency. Such a hole indicates that the resonance line is inhomogeneously broadened; its width recorded for low microwave power, and narrow-banded excitation and probing, is twice the homogeneous width of the individual resonances making up the broad line. Alternatively, one may scan a different resonance, for example the line. For

285 purple bacteria, this line is very weak owing to about equal steady-state population of the x- and y-sublevels connected by the microwaves. Burning at one of the transitions transfers population from the z-level to either the x- or the y-sublevel, so that their population difference is considerably enhanced and the line becomes easily observable as an “anti-hole”. For photosynthetic preparations, the two examples of microwave double resonance were demonstrated early-on (Hoff, 1976). In the second case, the normal resonance line is not observed, and only the hole shows up having a baseline at the intensity level of the ADMR signal corresponding to the modulated microwaves, dipping to a level close to zero intensity when the frequency of burn and probe microwaves coincide. This mode makes it somewhat easier to study variations in line shape as a function of burn power, etc. (see below). Both methods of carrying out a microwave double-resonance experiment are useful for assigning ODMR lines to a particular triplet state when several triplets contribute to the ODMR spectrum. If one of the lines is ‘tickled’ with microwaves, then from all other lines only those will be affected that represent either transitions within the triplet sublevel manifold to which the ‘tickled’ line belongs or are due to a triplet whose concentration depends on that of the triplet that is ‘tickled’, e.g. because it is populated through triplet energy transfer from the latter triplet state. Note that, because the effects measured are usually very small, adequate care must be taken that interference of the two microwave channels is avoided. One precaution is to insert directional couplers or circulators in the two channels (Fig. 2a). It is important to check before use the isolation provided by these devices, as storage on e.g. a metal-containing shelf may adversely affect the isolation.

A. Triplet-Minus-Singlet (T–S) Absorbance Difference Spectra When a triplet state is present, the absorbance spectrum contains the following contributions: 1. The unperturbed singlet ground state absorbance spectrum transitions) of all molecules that are not in the triplet state

286

Arnold J. Hoff T–S difference spectrum. On the other hand, re­ cording T–S spectra for different ADMR reson­ ance 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 is of much interest. For non-interacting triplet states it provides a very accurate triplet absorbance spec­ trum, since that is given by adding the properly normalized singlet ground state spectrum to the T–S spectrum. For interacting triplet states, for example present in a photosynthetic pigment-protein complex, it records these interactions in a very sensitive way and thus provides a unique means to study pigment configuration.

B. Linear Dichroic T–S Spectroscopy 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 (including 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. 3. The absorbance spectrum of the triplet state transitions. itself, consisting of With square-wave, on-off amplitude-modulated microwaves, the T–S spectrum represents the difference in absorbance of the sample for microwaves on and microwaves off (Fig. 3). It can be shown (den Blanken et al., 1984) that this difference is proportional to the difference in absorbance with and without the triplet state present. In other words, the T–S spectrum repre­ sents the difference of the absorbance of the sam­ ple 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 and and conse­ quently different ODMR resonance frequencies may be present without showing up in the T–S spectrum. Their absorbance is not changed by the microwaves and therefore their contribution to the absorbance cancels in the ADMR-monitored

The microwave transitions between the u and v triplet sublevels are polarized along w = u × 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 perpendic­ ular 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, LD-(T–S) spectroscopy provides a means to ascertain the directions of the magnetic transi­ tion moments. With this knowledge one may then derive from the LD-(T–S) spectra precise struc­ tural information on molecular aggregates. In optical spectroscopy, the transition proba­ bility for a transition with transition dipole p is proportional to where is the angle between p and the electric vector E of the (polar­ ized) incident light. A similar relation holds for the magnetic microwave transitions between the triplet sublevels. Thus, for a microwave transition and an angle between and the moment magnetic vector of the (polarized) microwave field, we have a transition probability It follows that molecules ori­ more or less parallel to have ented with a much higher transition probability than those

ODMR of triplet states oriented about perpendicular to (Of course, the transition probability is exactly for zero.) Hence, for random excitation to the triplet state, molecules oriented in an angular interval close to will experience a much higher change in their relative triplet concentration upon the application of (polarized) resonant micro­ waves than molecules in an interval close to Consequently, the distribution of triplet states, which was isotropic before the application of the microwaves, becomes axially anisotropic when resonant micro­ with the axis parallel to waves are switched on. Microwave-induced selection in the triplet state distribution is very similar to that of photose­ lection. We can therefore partake of the formal­ ism derived for that technique (see e.g. Vermég­ lio et al., 1978) to calculate the functional relationship between and the angle be­ It can be shown that the ratio R tween p and of the intensity of the LD-(T–S) spectrum (which represents the difference and the T–S spectrum is given by (den Blanken et al., 1984; Hoff, 1985)

Eq. (14) is plotted in Fig. 4, together with plots of the LD-(T–S) and T–S intensities versus It is seen that R is quite sensitive to so that with proper calibration of the T–S and LD-(T–S) spectra, can be determined quite accurately. Of course, all this applies rigourously only for single absorbance bands. If bands with different di­ overlap, R will have rections of p vis-à-vis some intermediate value and one will have to simulate the complete LD-(T–S) and T–S spectra to obtain values for the various The angle in Eq. (14) refers to one particular say that corresponding to the x-polarized y transition at frequency Tuning the microwaves to the frequency, or y-polarized transition, allows that is the the recording of LD-(T–S) spectra and the deter­ mination of R for a different viz. the angle between p and y. When p, x and y lie in one since the triplet spin axes x, plane, y and z span a cartesian coordinate frame. If p, x and y are not coplanar, the two measurements

287

uniquely define the orientation of p in the x,y,z coordinate frame. This is a great advantage over the ordinary photoselection experiment, where one determines just one angle between two transi­ tion moments, which leaves one with a conical ambiguity. Note that it suffices to record LD(T–S) spectra for just two of the three possible ADMR transition frequencies. Since the orien­ tations of all values of p are determined in one and the same coordinate frame, their mutual angular dependence immediately follows. 1. Instrumentation for LD-(T–S) Spectroscopy The instrumentation for LD-(T–S) spectroscopy is quite similar to that of isotropic T–S spectros­ copy and we can refer to Fig. 2a for a description. First of all we need polarized microwaves, there­ fore a simple helix is not suitable. We use a splitring or loop-gap cavity as described by Hardy and Whitehead (1981). This design has the advantage that for the rather long wavelength of the micro­ waves (between 50 and 100cm) one still has a cavity of manageable dimensions, which fits easily into a four-window liquid helium cryostat. The field is polarized along the vertical, and there­

288 fore it is perpendicular to the horizontal light beam. The probe light is unpolarized. The field induces an ellipticity in the transmitted light T, which is detected via a photoelastic modulator (PEM) after the sample fol­ lowed by a polarizer. The PEM rotates the ellipse spanned by the unequally-transmitted light vec­ tors and (with respect to by 180° at a frequency of 50 kHz. The analyzer converts this polarization modulation into an amplitude modu­ lation at 100 kHz that is proportional to (T, transmission; A, absorbance) for small differences. The microwaves are as in T–S spectroscopy modulated at low frequency (say 315 Hz), so that the light intensity falling on the photodiode is doubly modulated at 100 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 dif­ Hz gives the ference signal. This procedure is illustrated in Fig. 2c. The signal-to-noise ratio is enhanced by inserting selective amplifiers in both modulation channels. Scanning the monochromator yields si­ multaneously 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, mono­ chromator sensitivity, etc. as a function of wave­ length. To correctly evaluate the ratio R (Eq. (14)) one must mutually calibrate the T–S and LD-(T–S) signals. This calibration includes am­ plification factors, lock-in sensitivities, etc. It is best done by simulating the transmitted modu­ lated light by a modulated light-emitting diode (LED). The amplitude of the 100 kHz modu­ lation at specific instrument settings can then be accurately compared with that of the 315 Hz modulation. Care has to be taken to avoid as much as possible ellipticities induced by ex­ traneous sources, such as the lamp, cryostat win­ dows, sample cell, etc. In our set-up these ex­ traneous ellipticities amounted to less than 1% of the LD-(T–S) signal. V. ODMR in Photosynthesis The use of ODMR in photosynthesis research has proved to be a particularly fruitful field of its application in biology. By far the most important triplet state in photosynthetic membranes is that

Arnold J. Hoff generated on the primary electron donor by rad­ ical recombination under conditions that forward electron transport is blocked by the prereduction of one of the acceptors (or by its physical dele­ tion):

Because is prereduced and cannot normally re­ accept two electrons, the radical pair combines in about 20 to 50 ns to either the singlet excited or ground state of D, or with almost 100% yield at cryogenic temperatures to its triplet state, (Dutton et al., 1972). Applications of ODMR have included fluor­ escence detection (reviewed in Hoff, 1982, 1989) and absorbance detection. Because the latter technique now dominates the field we will mostly discuss applications of ADMR to various photo­ synthetic preparations.

A. ADMR Spectroscopy of Reaction Centers The great advantage of the ADMR variant above 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 gives maximal density can be attained signal). Both conditions hold for the photosyn­ thetic triplets, and in the first application of ADMR to isolated bacterial reaction centers (den Blanken et al., 1982; den Blanken and Hoff, 1982) 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 op­ ened the way to studies of numerous isolated reaction centers, pigment solutions, etc., both of bacterial and plant origin. Accurate values of and the were determined (den Blanken et al., 1982,1983a,b; den Blanken and Hoff, 1982,1983a,b,c; Hoff and Cornelissen, 1982; Vasmel et al., 1984; Ulrich et al. 1987), as well as approximate values of the populating probabilit­ ies (Angerhofer et al., 1991) (earlier determined with FDMR by Hoff and De Vries, 1978). The values of for the primary donor triplet in RCs

ODMR of triplet states of purple bacteria are lower by 20–30% com­ pared to that of the isolated bacteriochlorophyll (BChl) pigment (den Blanken and Hoff, 1983a). From recent EPR data on the reaction center triplet state in single crystals (Norris et al., 1987) and from ADMR spectroscopy (Lous and Hoff, 1987) it was concluded that, at least in Rhodo­ pseudomonas (Rps.) viridis, the triplet state is largely localized on one of the dimer BChls, namely The difference between the in vivo and in vitro value of was ascribed to admixture of charge transfer (CT) states of the form state to the monomeric (Norris et al., 1987). The temperature depen­ dence of the zero-field splitting parameters was investigated with ADMR by Ullrich et al. (1987) and Aust et al. (1990), and yielded further proof that it cannot be explained as a thermally acti­ vated population of one of the accessory BChls, in accordance with temperature-dependent EPR measurements (Hoff and Proskuryakov, 1985). Hole burning double-resonance ODMR spec­ troscopy has provided additional resolution. The first such experiment on photosynthetic RCs was carried out with FDMR (Hoff, 1976). A narrow hole of about 1.2 MHz FWHH was observed, indicating that the FDMR line itself is inhomo­ geneously broadened because of of site-heterogeneity. This result was confirmed with ADMR hole burning on protonated and deuterated RCs of Rhodobacter (Rb.) sphaeroides R26 and proton­ ated RCs of Rps. viridis (Greis et al., 1994), with of < 0.25, 1.0, and 2.0 observed hole width MHz, respectively. The observed hole width in principle yield the transversal relaxation time giving for protonated RCs of Rb. sphaeroides R26. This value differs observed with spin-echo pulsed from the (Lous and Hoff, ADMR of 1.16 ±0.05 1987b). Careful analysis of the physical cause of the hole width resolved this discrepancy (Greis et al., 1994). It was shown that the hole width results from unresolved second-order hyperflne interac­ tions. In a hole burning experiment, many such interactions are time-averaged because of fast nu­ clear spin flip-flops. In time-resolved spin-echo ADMR spectroscopy, however, a limited distri­ bution of flip-flop processes is sampled, leading to an apparently longer than calculated from the hole width. Measured hole widths were well-

289 reproduced when it was assumed that the triplet state of Rb, sphaeroides R26 is fully delocalized, that of Rps. viridis localized on one of the dimer BChls. A further interesting result was the obser­ vation of satellite holes when the ADMR transi­ tion was driven with high microwave powers. Then, normally forbidden quadrupole transitions are induced because of simultaneous electron and nucleus con­ nuclear spin flips, and every nected to the unpaired electrons of the triplet state gives rise to three lines at both sides of the hole. Experimentally, instead of the expected 8 × 6 = 48 lines, only four satellites are observed, suggesting that all eight nitrogens of the primary donor dimer experience about the same electric field gradient, with two quadrupole transitions overlapping. ADMR of triplets of carotenoids present in reaction center and antenna complexes of various photosynthetic bacteria yield accurate and values (Ulrich et al., 1989; Aust et al., 1991). It is even possible to distinguish different naturally ocurring carotenoids in the same antenna pre­ paration (Aust et al., 1991). In plant RCs the values of and the are practically the same as those of monomeric chlorophyll in solution (den Blanken and Hoff, 1983b; den Blanken et al., 1983a). This can be explained by either one of three possibilities: i) the primary donor of both plant photosystems I and II is a monomeric Chl a molecule, ii) the primary donor is a plane-parallel sandwich (Chl dimer with a fully delocalized triplet state (strong exciton coupling), iii) the primary donor is a dimer on which the triplet state is fully lo­ calized 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. More on this in Section V.C.

B. T–S Spectroscopy of Reaction Centers ADMR was first used to record T–S spectra by den Blanken et al. (1982). 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 tech­

290 niques. T–S spectra related to the primary donor triplet have now been recorded for many bacterial photosystems and for PS I and PS II of plants (reviewed by Hoff, 1989,1990,1993). The inter­ pretation of some of these spectra wil be dis­ cussed in the next section. T–S spectra of carot­ enoids in various bacterial reaction centers and antenna complexes were reported by Lous and Hoff (1989), Ulrich et al. (1989) and Aust et al. (1991), of plant antenna complexes by van der Vos et al. (1991) and Carbonera et al. (1992).

C. Linear-Dichroic T–S Spectroscopy As explained in Section IV.B LD-(T–S) spectros­ copy is similar to that of photoselection, in which an oriented distribution of e.g. photo-oxidized primary donors is produced by exciting the immo­ bilized unoriented sample with a beam of linearlypolarized light of a wavelength corresponding to a particular absorption band, e.g. that of D. It has the advantage that, in contrast to photoselec­ tion, the dichroic spectrum can be recorded with respect to two axes of reference that are perpend­ icular to each other. This reduces considerably the ambiguity of unraveling the orientation of the various transition moments in the T–S spectrum. Complex T–S spectra such as those of the photo­ synthetic RC, where many overlapping features are present such as appearing bands, bleachings, band shifts to the red and to the blue, can only be interpreted with certainty if the corresponding LD-(T–S) spectra are available (Hoff et al., 1985). The experimental T–S and LD-(T–S) spectra can be compared with spectra simulated with ex­ citon theory, which describes the interaction be­ tween pigment molecules brought about by the electrostatic dipole–dipole coupling between their electronic transition moments. For two iden­ tical molecules this interaction causes a shift and a splitting of the excited state, which is now com­ posed of two levels with (apart from the shift) energies 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 T–S pigments (Davi­ dov, 1981; Kasha et al., 1965; Pearlstein, 1982). For a known pigment configuration the absorp­ tion spectrum of the excitonically coupled spec-

Arnold J. Hoff trum is then readily obtained. Generally the re­ sulting bands are mixtures of the original uncoupled bands, with transition moments that are vectorial combinations of the original transi­ tion moments. Thus, in general, introducing a triplet state in a coupled pigment system, with corresponding changes in the dipole–dipole coup­ lings, will lead to changes in the position, inten­ sity and the polarization of individual absorption bands. Especially the change in polarization is useful for drawing conclusions on the extent of dipolar coupling between the cofactors in photo­ synthetic reaction centers. 1. Bacterial Reaction Centers Exciton calculations based on the crystal structure of the RC of Rps. viridis (Deisenhofer et al., 1985) yield satisfactory simulations of its ADMRmonitored T–S and LD-(T–S) spectra (Lous and Hoff, 1987a; Knapp et al., 1986; Scherer and Fischer, 1987a) (Fig. 5). The spectral fits allowed to conclude that the triplet is localized on one of The bleaching the BChl components of D, at 990 nm is due to the disappearance of the longwavelength band of D that is shifted to the red because of excitonic coupling between the two BChls of D. Exciton coupling is much weaker in the triplet state of so that in a localized state part of the BChl absorption is bleached and part shifts back to the ‘normal’ wavelength of BChl absorption in a protein matrix. The large positive band at about 830 nm and the two smaller features at the long- and short-wavelength side of the positive band were attributed to band shifts of the two accessory BChls induced by the change (den Blanken and Hoff, 1982). The small positive band at 872 nm was attributed to a triplet–triplet absorption of whose transition axis in moment is oriented along the the BChl macrocycle. The absence of significant bands in the 790 nm region indicates that the two and are only weakly bacteriopheophytins, coupled to D. With appropriate modifications, a similar picture holds for chemically-modified RC of Rb. sphaeroides R-26 (Scherer and Fischer, 1987b; Beese et al., 1988; Angerhofer et al., 1989). The above interpretation has stood the test of time rather well, with the exception of the

ODMR of triplet states

assignment of the band at 872 nm. Similar work was recently carried out on RCs of Rb. sphaero­ ides R26 and its LH(L131), LH(M160), HL(L173) and HL(M202) mutants, and on BChl a in vitro (Vrieze and Hoff, 1995a,b; Vrieze et al., 1995a,b). The orientation of the moment of the primary donor relative to the tri­ plet x- and y-axes was found to be 85 ± 5° and 18 ± 3°, respectively. The absorption region of the accessory BChls was deconvoluted in two bleached bands, corresponding to the two bands of the low-temperature absorption spectrum, and three appearing bands, which included the transi­ tion at 818 nm previously attributed to a triplet– triplet transition by analogy to the interpretation of the 872 nm band for Rps. viridis. This previous

291

assignment could now be excluded on the basis of the measured polarization and the polarization of the in vitro triplet–triplet absorption band (Vrieze and Hoff, 1995a). With the aid of the results for two mutants that were changed in the composition of the primary donor and its environ­ ment, the two bleachings and two of the appear­ ing bands were assigned to band shifts resulting from the introduction of the triplet state. The third band was attributed to mostly a primary donor contribution, likely the “monomer” transi­ tion, although it should be realized that all bands reflect mixed transitions (Fig. 6). Surprisingly, the orientations of the bleached bands and of the corresponding appearing bands were practically the same. Because all reasonable exciton models

292

Arnold J. Hoff

major difference being that one of the accessory BChls is replaced by a BPh (Vasmel, 1986; Vasmel et al., 1987). The T–S spectra of green sulfur bacteria are more complex and have only been tentatively interpreted (den Blanken et al., 1983b; Hoff et al., 1988). ADMR and T–S spec­ tra of membranes of Heliobacterium chlorum, a representative of the Heliobacteriaceae contain­ ing BChl g, were recently recorded by Vrieze et al. (Vrieze et al., 1995c). A variety of triplet states were detected, which with the aid of their corresponding T–S spectrum were assigned to the primary donor and several distinct antenna pig­ ments. 2. Plant Reaction Centers predict considerable changes in the orientation, it was concluded that exciton interaction was not the single determining coupling between the pig­ ments in the reaction center. With minor differences the qualitative aspects of the above picture hold for all purple bacteria investigated (Dijkman et al., 1988). The same applies to the green filamentous bacterium Chlo­ roflexus aurantiacus (Vasmel et al., 1984), whose RC is very similar to that of purple bacteria, the

The first highly-resolved ADMR-recorded T–S spectra of the plant photosystems were obtained already a decade ago (den Blanken and Hoff, 1983b; den Blanken et al., 1983a). They are characterized by a strong bleaching of donor bands at 703 and 682 nm for PS I and II, respec­ tively, and a positive band at the blue side of this bleaching, at ~ 674 nm, close to the wavelength of the absorption of Chl a in vitro (Fig. 7). This

ODMR of triplet states

band, which is more pronounced in intact mem­ brane fragments than in core or RC particles (den Blanken et al., 1983a; van der Vos et al., 1992; Angerhofer et al., 1993; Carbonera et al., 1994a), was tentatively attributed to the appearing ab­ sorption of a monomeric Chl a molecule belong­ ing to a primary donor dimer on which the triplet state was localized on the second Chl a (den Blanken and Hoff, 1983b), supporting the notion that the primary donor of the two photosystems is a dimeric Chl a complex. Recent work employing LD-(T–S) spectroscopy, however, could not con­ firm this assignment (see below). The similarity of the T–S spectra of intact PS I and PS II suggests that the composition and pigment environment of the primary donor of PS I and PS II are similar.

a. Photosystem I The orientations of the of P700 and of the triplet–triplet transition of in the x,yplane of the latter as determined with LD-(T– S) spectroscopy are shown in Fig. 8a. The two orientations are practically identical; both lie ap­ proximately in the triplet x,y-plane and are ro­ tated by about 13° with respect to the correspond­ ing transition moments of Chl a in vitro (Fig. 8b,c). This co-rotation strongly suggests that is localized, as for a delocalized triplet the of P700 is necessarily a delocalized exciton transition, and must either coincide with one of the in-plane triplet axes, or for a parallel

293

dimer, be aligned along the monomer tion, in order to produce a geometry for which the ZFS parameters are identical to those of mon­ omeric Chl a. Neither of these cases obtains for Note that a similar argument holds for of P680 (see the orientation of the below). The orientation of the 674 nm band deduced from LD-(T–S) spectroscopy precludes its assignment to a ‘monomer’ transition of it is likely due to a band shift of an accessory Chl a located close to P700. Small bands at 663 and 670 nm were assigned to the ‘active’ and ‘inactive’ acceptor Chl a. Lastly, the band at 687 nm was attributed to a Chl a chromophore adjacent to P700, and coupled to the primary acceptor (Vrieze et al., 1995d). No evidence was found for a higher-energy exciton component of an excitonically-coupled P700 dimer.

b. Photosystem II The question of the configuration of the primary donor in PS II (Chl a monomer or dimer) was addressed by T–S and LD-(T–S) spectroscopy (van der Vos et al., 1992; van der Vos, 1994; Vrieze and Hoff, 1995b) of the so-called D1/D2– cyt b-559 RC particle (Nanba and Satoh, 1987) and of Chl a in methanol. It was found that the ADMR signals and corresponding primary donor bleaching in the T–S spectrum spectrum are quite heterogeneous, being composed of five distin­

294

guishable bands, all related to active P680. These bands are also present in FDMR signals of the D1/D2 particle (Carbonera et al., 1994b), but largely coalesce for intact PS II membranes (Car­ bonera et al., 1994a), suggesting that the optical and ODMR heterogeneity of the Dl/D2–cyt b­ 559 RC is introduced by the isolation procedure. Note that a similar heterogeneity of the ADMR and optical properties of the triplet state of the primary donor was previously found for bacterial RCs and, to lesser extent, chromatophores (den Blanken and Hoff, 1983c). The angles between the transition moments of the major components of the P680 bleaching and the triplet x,y-axes were determined with LD-(T– S) spectroscopy and proved to be practically the same, ruling out the possibility that one would represent a higher exciton component (van der moment of Vos et al., 1992). The P680 lies approximately in the plane spanned by – x,y angles with the triplet x,y-axes. The their margin of error are displayed in Fig. 9, which also shows the corresponding angles for Chl a in methanol. Two possibilities are con­ is localized on one Chl a of an sidered: (i) excitonically coupled dimer (Fig. 9b), (ii) is localized on one non-excitonically coupled, monomeric Chl a (Fig. 9c). The small rotation (10–20°) observed in Fig. 9c of the

Arnold J. Hoff

moment of P680 compared to the of monomeric Chl a in methanol can easily result from differences in the environment of the triplet state (Vrieze and Hoff, 1995b), and therefore does not necessarily falsify assumption (ii) that P680 is a non-coupled Chl a monomer. Thus, the ADMR data is consistent with, but does not definitely prove, that P680 is a (weakly) excitonically-coupled Chl a dimer. The directional information obtained by LD­ ADMR can be used to extract the orientation of moment of P680 relative to the the membrane with the aid of the orientation of the triplet axes with respect to the membrane plane obtained from EPR on oriented membranes by van Mieghem et al. (1991). For the primary donor pigments angles result of 15.5° and 12.5°, for the accesssory BChls, 21° and 23° (van der Vos et al., 1992). None of these angles are close to the experimental angle, so that the orientation of P680 in the membrane must differ from that of the primary donor of the purple bacteria. The high resolving power of ADMR is nicely demonstrated by the observation of a pheophytin (Pheo) triplet absorbing at 681 nm (Fig. 10) (van der Vos et al., 1992). Because has quite characteristic ZFS parameters, the microwaveoptical double-resonance experiment of Fig. 10

ODMR of triplet states unequivocally demonstrates that at least one Pheo in the D1D2–cyt b-559 complex absorbs at 681 nm and overlaps with the P680 nm absorp­ tion. A microwave double-resonance experiment suggested that is populated indirectly through energy transfer from or enhanced intersystem crossing in RCs ‘closed’ by the forma­ tion of (van der Vos et al., 1992). It was found in later work, however, that the doubleresonance experiment had suffered from interfer­ ence effects (van der Vos and Hoff, 1995), and that is formed in a one-photon process not correlated with the presence of in ‘de­ graded’ RCs, possibly representing a confor­ mationally changed state in which for example the distance between P680 and Pheo is increased (Angerhofer et al., 1994). VI. Concluding Remarks The triplet state is a versatile probe of structure and function in photosynthesis on a molecular level. ODMR, and especially ADMR, in zeromagnetic field of triplet states is a powerful tool for determining triplet properties that give direct information on the structure and interactions of pigments in photosynthetic pigment-protein com­ plexes. Accurate values have been determined of the zero-field splitting parameters D and E and the sublevel decay rates, of the triplet state of the primary donor, and of chlorophyll and carotenoid antenna chromophores, for a variety of photosyn­ thetic preparations, including the two plant RCs, a number of bacterial RCs, and several antenna pigment-protein complexes. ADMR has further allowed the recording of accurate, high-resolution T–S and LD-(T–S) spectra, which have offered insight in the relative orientation of, and the in­ teractions between, reaction center pigments, and in the “multiplicity” of the primary donor. An important finding is that isolated reaction centers generally exhibit a heterogeneity of the optical and magnetic resonance parameters of the pri­ mary donor, which is attributed to somewhat dif­ ferent conformations of the RC pigments. This observation relates to the many experiments car­ ried out on such RCs, for example by fast laser flash photolysis and ENDOR and (high-field) EPR spectroscopy, which normally lack the resol­ ution to discriminate between the various confor­

295 mations of the RC. Consequently, the results of these experiments represent an average over the conformations, static at low temperatures, likely dynamic at higher temperatures, and it may be difficult to compare the results meaningfully with properties calculated from the crystal structure, which presumably is much better defined than the “glassy” matrix of the RC protein in solid and liquid solution. A key result of ADMR-recorded LD-(T–S) spectroscopy is the determination of the orien­ tation of several transition moments. This has recently allowed much better insight in the com­ position of the absorbance spectrum, which is of crucial importance in the interpretation of ul­ trafast pump-probe laser flash spectroscopy, and therefore for our understanding of the details of primary electron transfer. Future applications of ODMR will focus on comparative studies of mutant and chemicallymodified reaction centers, and on RC heteroge­ neity. Pulsed ADMR will become increasingly important, allowing studies of the temperature dependence of the various triplet parameters, and of dynamical aspects of line-broadening, triplet– triplet transfer, etc. Results on the orientation of the various absorption bands in the T–S ab­ sorbance difference spectrum call for renewed ef­ fort in theoretically interpreting the optical pro­ perties of the RC pigment complex.

References Angerhofer A, Beese D, Hoff AJ, Lous EJ and Scheer H (1989) Linear dichroic triplet-minus-singlet absorbance dif­ ference spectra of borohydride treated reaction centres of Rhodobacter sphaeroides R26. In: Singhal G (ed) Appli­ cations of Molecular Biology in Bioenergetics of Photo­ synthesis, pp 197–203. Narosa Publishing House, New Delhi. Angerhofer A, Speer R, Ullrich J, von Schütz JU and Wolf HC (1991) Time resolved ADMR applied to the triplet state of the primary donor of bacterial photosynthetic reac­ tion centers. Appl Magn Reson 2: 203–216. Angerhofer A, Bernlocher D and Robert B (1993) Absorption detected magnetic resonance of D-1/D-2 complexes from Pisum sativum. In: Steiner U (ed) Magnetic Field and Spin Effects in Chemistry, pp 167–180. Oldenburg Verlag, München. Angerhofer A, Friso F, Giacometti GM, Carbonera D and Giacometti G (1994) Optically detected magnetic reson­ ance study on the origin of the pheophytin triplet state in

296 D-1/D-2 cytochrome b-559 complexes. Biochim Biophys Acta 1188: 35–45. Aust V, Angerhofer A, Parot PH, Violette CA and Frank HA (1990) Temperature-dependent ADMR on borohydridetreated reaction centers of Rhodobacter sphaeroides R26. Chem Phys Lett 173: 439–442. Aust V, Angerhofer A, Ullrich J, von Schlitz JU, Wolf HC and Cogdell RJ (1991) ADMR of carotenoid triplet states in bacterial photosynthetic antenna and reaction center complexes. Chem Phys Lett 181: 213–221. Beese D, Steiner R, Scheer H, Robert B, Lutz M and Anger­ hofer A (1988) Chemically modified photosynthetic bac­ terial reaction centers: Circular dichroism, Raman reson­ ance, low temperature absorption, fluorescence and ODMR spectra and polypeptide composition of borohyd­ ride treated reaction centers from Rb. sphaeroides R26. Photochem Photobiol 47: 293–304. Carbonera D, di Valentin M, Giacometti G and Agostini G (1992) FDMR of chlorophyll triplets in integrated particles and isolated reaction centers of Photosystem II. Identifica­ tion of P680 triplet. Biochim Biophys Acta 1185: 167–176. Carbonera D, Giacometti G, Agostini C, Angerhofer A and Aust V (1994a) ODMR of carotenoid and chlorophyll tri­ plets in CP 47 complexes of spinach. Chem Phys Lett 194: 273–281. Carbonera D, Giacometti G and Agostini G (1994b) A well resolved ODMR triplet minus singlet spectrum of P680 from PS II particles. FEBS Lett 343: 200–204. Chan IY (1982) Zero-field ODMR techniques - phosphores­ cence detection. In: Clarke RH (ed) Triplet State ODMR Spectroscopy, pp 1–24. Wiley-Interscience, New York. Davidov AS (1981) Theory of Molecular Excitons, Plenum Press, New York. Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1985) Structure of the protein subunits in the photosynthetic reac­ tion centre of Rhodopseudomonas viridis at 3Å resolution. Nature 318: 618–624. den Blanken HJ and Hoff AJ (1982) High-resolution optical absorption-difference spectra of the triplet state of the pri­ mary donor in isolated reaction centers of the photosyn­ thetic bacteria Rhodopseudomonas sphaeroides R26 and Rhodopseudomonas viridis measured with optically de­ tected magnetic resonance at 1.2 K. Biochim Biophys Acta 681: 365–374. den Blanken HJ and Hoff AJ (1983a) Sublevel decay kinetics of the triplet state of bacteriochlorophyll a and b in methyltetrahydrofuran at 1.2 K. Chem Phys Lett 96: 343–347. den Blanken HJ and Hoff AJ (1983b) High-resolution absorbance-difference spectra of the triplet state of the pri­ mary donor P700 in Photosystem I subchloroplast particles measured with absorbance-detected magnetic resonance at 1.2 K. Evidence that P700 is a dimeric chlorophyll complex. Biochim Biophys Acta 724: 52-61. den Blanken HJ and Hoff AJ (1983c) Resolution enhance­ ment of the triplet–singlet absorbance-difference spectrum and the triplet-ESR spectrum in zero field by the selection of sites. An application to photosynthetic reaction centers. Chem Phys Lett 98: 255–262. den Blanken HJ, van der Zwet GP and Hoff AJ (1982) ESR in zero field of the photoinduced triplet state in isolated

Arnold J. Hoff reaction centers of Rhodopseudomonas sphaeroides R26 detected by the singlet ground state absorbance. Chem Phys Lett 85: 335–338. den Blanken HJ, Hoff AJ, Jongenelis APJM and Diner BA (1983a) High-resolution triplet-minus-singlet absorbance difference spectrum of photosystem II particles. FEBS Lett 157: 21–27. den Blanken HJ, Vasmel H, Jongenelis APJM, Hoff AJ and Amesz J (1983b) The triplet state of the primary donor of the green photosynthetic bacterium Chloroflexus aur­ antiacus. FEBS Lett 161: 185–189. den Blanken HJ, Meiburg RF and Hoff AJ (1984) Polarized triplet-minus-singlet absorbance difference spectra mea­ sured by absorbance detected magnetic resonance. An ap­ plication to photosynthetic reaction centres. Chem Phys Lett 105: 336–342. Dijkman JA, den Blanken HJ and Hoff AJ (1988) Towards a new taxonomy of photosynthetic bacteria: ADMR-monitored triplet difference spectroscopy of reaction center pigment-protein complexes. Isr J Chem 28: 141–148. Dutton PL, Leigh JS and Seibert M (1972) Primary processes in photosynthesis: in situ ESR studies on the light induced oxidized and triplet state of reaction center bacteriochloro­ phyll. Biochem Biophys Res Commun 46: 406–413. Greis JW, Angerhofer A, Norris JR, Scheer H, Struck A and von Schütz JU (1994) Spectral diffusion and quadru­ pole splittings in absorption detected magnetic resonance hole burning spectra of photosynthetic reaction centers. J Chem Phys 100: 4820–4827. Hardy WN and Whitehead LA (1981) Split-ring resonator for use in magnetic resonance from 200–2000 MHz. Rev Sci Instrum 57: 213–216. Hoff AJ (1976) Kinetics of populating and depopulating of the components of the photoinduced triplet state of the photosynthetic bacteria Rhodospirillum rubrum, Rhodop­ seudomonas spheroides (wild type), and its mutant R26. Biochim Biophys Acta 440: 765–771. Hoff AJ (1982) ODMR spectroscopy in photosynthesis II. The reaction center triplet in bacterial photosynthesis. In: Clarke RH (ed) Triplet State ODMR Spectroscopy, pp 367–425. John Wiley and Sons, New York. Hoff AJ (1985) Triplet-minus-singlet absorbance difference spectroscopy of photosynthetic reaction centers by absorbance-detected magnetic resonance. In: Michel-Beyerle ME (ed) Antennas and Reaction Centers of Photosynthetic Bacteria. Structure Interaction and Dynamics, pp 150–163. Springer-Verlag, Berlin. Hoff AJ (1989) Optically-detected magnetic resonance of tri­ plet states. In: Hoff AJ (Ed) Applications in Biology and Biochemistry, pp 633–684. Elsevier, Amsterdam. Hoff AJ (1990) Triplet states in photosynthesis: linear dichroic optical difference spectra via magnetic resonance. In: Lin­ skins HF and Jackson JF (eds) Physical Methods in Plant Sciences Vol II, Modern Methods of Plant Analysis, pp 23–57. Springer-Verlag, Berlin. Hoff AJ (1993) Optically detected magnetic resonance of tri­ plet states in proteins. In: Riordan JF and Vallee BL (eds) Metallobiochemistry Part D Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metallop­

ODMR of triplet states roteins. Methods in Enzymology Vol 227, pp 290–330. Academic Press, San Diego. Hoff AJ and Cornelissen B (1982) Microwave power depen­ dence of triplet state kinetics as measured with fluorescence detected magnetic resonance in zero field. An application to the reaction centre bacteriochlorophyll triplet in bac­ terial photosynthesis. Mol Phys 45: 413–425, Hoff AJ and de Vries HG (1978) Electron spin resonance in zero magnetic field of the reaction center triplet of photo­ synthetic bacteria. Biochim Biophys Acta 503: 94–106. Hoff AJ and Proskuryakov II (1985) Triplet EPR spectra of the primary electron donor in bacterial photosynthesis at temperatures between 15 and 296 K. Chem Phys Lett 115: 303–310. Hoff AJ and van der Waals JH (1976) Zero field resonance and spin alignment of the triplet state of chloroplasts at 2 K. Biochim Biophys Acta 423: 615–620. Hoff AJ, Govindjee and Romijn JC (1977) Electron spin resonance in zero magnetic field of triplet states of chloro­ plasts and subchloroplast particles. FEBS Lett 73: 191– 196. Hoff AJ, den Blanken HJ, Vasmel H and Meiburg RF (1985) Linear-dichroic triplet-minus-singlet absorbance difference spectra of reaction centers of the photosynthetic bacteria Chromatium vinosum, Rhodopseudomonas sphaeroides R­ 26 and Rhodospirillum rubrum S1. Biochim Biophys Acta 806: 389–397. Hoff AJ, Vasmel H, Lous EJ and Amesz J (1988) Tripletminus-singlet optical difference spectroscopy of some green photosynthetic bacteria. In: Olson JM, Ormerod JG, Amesz J, Stackebrandt, E and Trüper HG (eds) Green Photosynthetic Bacteria, pp 119–126. Plenum Press, New York. Kasha M, Rawls HS and El-Bayoumi MA (1965) The exciton model in molecular spectroscopy. Pure Appl Chem 11: 371–392. Knapp EW, Scherer POJ and Fischer SF (1986) Model studies of low-temperature optical transitions of photosynthetic reaction centers. A-, LD-, CD-, ADMR and LD-ADMRspectra for Rhodopseudomonas viridis. Biochim Biophys Acta 852: 295–305. Lous EJ (1988) Interactions between Pigments in Photosyn­ thetic Protein Complexes. An Optically-detected Magnetic Resonance and Magnetic Field Effect Study. Doctoral Thesis, Leiden. Lous EJ and Hoff AJ (1987a) Exciton interactions in reaction centers of the photosynthetic bacterium Rhodopseudo­ monas viridis probed by optical triplet-minus-singlet polar­ ization spectroscopy at 1.2 K monitored through absorbance-detected magnetic resonance. Proc Natl Acad Sci USA 84: 6147–6151. Lous EJ and Hoff AJ (1987b) Absorbance detected electron spin echo spectroscopy of non-radiative states in zero field. An application to the primary donor of the photosynthetic bacterium Rhodobacter sphaeroides R26. Chem Phys Lett 140: 620–625. Lous EJ and Hoff AJ (1989) Isotropic and linear dichroic triplet-minus-singlet absorbance difference spectra of two carotenoid containing bacterial photosynthetic reaction centers in the temperature range 10–288 K. A model for

297 bacteriochlorophyll-carotenoid triplet transfer. Biochim Biophys Acta 974: 88–103. Maki AH (1984) Techniques, theory, and biological appli­ cations of optically-detected magnetic resonance (ODMR). In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance Vol 6, pp 187–294. Plenum Press, New York. Nanba O and Satoh K (1987) Isolation of a Photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome b 559. Proc Natl Acad Sci USA 84: 109–112. Norris JR, Lin CP and Budil DE (1987) Magnetic resonance of ultrafast chemical reactions. Examples from photosynth­ esis. J Chem Soc Faraday Trans 1 83: 13–27. Pearlstein RM (1982) Chlorophyll singlet excitons. In: Gov­ indjee (ed) Energy Conversion in Plants and Bacteria. Pho­ tosynthesis Vol I, pp 293–329. Academic Press, New York. Scherer POJ and Fischer SF (1987a) Model studies to lowtemperature optical transitions of photosynthetic reaction centers of Rhodobacter sphaeroides and Chloroflexus aur­ antiacus. Biochim Biophys Acta 891: 157–164. Scherer POJ and Fischer SF (1987b) Application of exciton theory to optical spectra of sodium borohydride treated reaction centers from Rhodobacter sphaeroides R26. Chem Phys Lett 137: 32–36. Ullrich J, Angerhofer A, von Schütz JU and Wolf HC (1987) Zero-field absorption ODMR of reaction centers of Rhodo­ bacter sphaeroides at temperatures between 4.2 and 75 K. Chem Phys Lett 140: 416–420. Ullrich J, Speer R, Greis J, von Schütz JU, Wolf HC and Cogdell RJ (1989) Carotenoid triplet states in pigmentprotein complexes from photosynthetic bacteria: Absorption-detected magnetic resonance from 4.2–225 K. Chem Phys Lett 155: 363–370. van der Bent SJ, de Jager PA and Schaafsma TJ (1976) Op­ tical detection and electronic simulation of magnetic reson­ ance in zero magnetic field of dihydroporphin free base. Rev Sci Instrum 47: 117–212. van der Vos R (1994) Antenna and Reaction Center Com­ plexes in Photosynthesis. An Absorbance-detected Mag­ netic Resonance and Magnetic Field Effect Study. Doctoral Thesis, Leiden. van der Vos R and Hoff AJ (1995) Optically–detected mag­ netic field effects on the D1–D2 cyt b-559 complex of Photosystem II. Temperature dependence of kinetics and structure. Biochim Biophys Acta 1228: 73–85. van der Vos R, Carbonera D and Hoff AJ (1991) Microwave and optical spectroscopy of carotenoid triplets in lightharvesting complex LHCII of spinach by absorbance-detected magnetic resonance. Appl Magn Reson 2: 179–202. van der Vos R, van Leeuwen PJ, Braun P and Hoff AJ (1992) Analysis of the optical absorbance spectra of D1–D2– cytochrome b-559 complexes by absorbance-detected mag­ netic resonance. Structural properties of P680. Biochim Biophys Acta 1140: 184–196. van Mieghem FJE, Satoh K and Rutherford AW (1991) A chlorophyll tilted 30° relative to the membrane in the Photosystem-II reaction centre. Biochim Biophys Acta 1058: 379–385. Vasmel H (1986) The Photosynthetic Membrane of Green Bacteria. Doctoral Thesis, Leiden. Vasmel H, den Blanken HJ, Dijkman JA, Hoff AJ and Amesz

298 J (1984) Triplet-minus-singlet absorbance difference spec­ tra of reaction centers and antenna pigments of the green photosynthetic bacterium Prosthecochloris aestuarii. Bio­ chim Biophys Acta 767: 200–208. Vasmel H, Meiburg RF, Amesz J and Hoff AJ (1987) Optical properties of the reaction center of Chloroflexus aur­ antiacus at low temperature. Analysis by exciton theory. In: Biggins J (ed) Progress in Photosynthesis Research Vol 1, pp 403–406. Martinus Nijhoff, Dordrecht. Verméglio A, Breton J, Paillotin G and Cogdell R (1978) Orientation of chromophores in reaction centers of Rho­ dopseudomonas sphaeroides: A photoselection study. Biochim Biophys Acta 501: 514–530. Vrieze J, Gast P and Hoff AJ (1992) The structure of the reaction center of Photosystem I investigated with linear ­ dichroic absorbance-detected magnetic resonance at 1.2 K. In: Murata N (ed) Research in Photosynthesis Vol I, pp 553–556. Kluwer Academic Publishers, Dordrecht. Vrieze J and Hoff AJ (1995a) Interactions between chromo­ phores in reaction centers of purple bacteria. A reinterpre­ tation of the triplet-minus-singlet spectra of Rb. sphaero-

Arnold J. Hoff ides R26 and Rps. viridis. Biochim Biophys Acta, submit­ ted. Vrieze J and Hoff AJ (1995b) The orientation of the triplet axes with respect to the optical transition moments in (bacterio)chlorophylls. Chem Phys Lett 237: 493–501. Vrieze J, Williams JC, Allen J and Hoff AJ (1995a) A LD­ ADMR study on reaction centers of the LH(L131) and LH(M160) mutants of Rb. sphaeroides. Biochim Biophys Acta, submitted. Vrieze J, Schenck CC and Hoff AJ (1995b) The triplet state of the primary donor in reaction centers of the HL(L173) and HL(M202) heterodimer mutants of Rb. sphaeroides. Biochim Biophys Acta, submitted. Vrieze J, van de Meent EJ and Hoff AJ (1995c) Bacteriochlor­ ophyll g triplet states of the primary donor and antenna in membranes of Heliobacterium chlorum. Biochemistry, submitted. Vrieze J, Gast P and Hoff AJ (1995d) The structure of the reaction center of Photosystem I of plants. An investigation with linear-dichroic absorbance-detected magnetic reson­ ance. J Phys Chem, submitted.

Chapter 18

Magic Angle Spinning Nuclear Magnetic Resonance of

Photosynthetic Components

Huub J.M. de Groot Leiden Institute of Chemistry, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands

Summary I. Introduction II. Magic Angle Spinning NMR Spectroscopy III. Probing the Local Environment of M(Y)210 in Rb. sphaeroides R26 RC with MAS IV. The Configuration of the Spheroidene in the Rb. sphaeroides RC V. The Asymmetric Binding in Rb. sphaeroides R26 VI. New Developments. CIDNP and Correlation Spectroscopy VII. Concluding Remarks Acknowledgements References

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Summary This chapter is the first extensive review of magic angle spinning (MAS) NMR studies of photosynthetic Rhodobacter components. We describe how the chemical environment of the (M)Y210 in sphaeroides R26 reaction centers was investigated and how labeled Rb. sphaeroides 2.4.1 (M)Y210W mutant reaction centers were used to assign the response of (M)Y210 in R26 to a narrow signal at 152.2 ppm. According to the MAS NMR, the Y(M)210 is in a homogeneously ordered region of the complex, and probably contributes to the fine-tuning of the energy levels of prosthetic groups involved in electron transfer. In another MAS NMR investigation, analysis of the chemical shifts of labels in the configuration for the spheroidene in the Rb. sphaeroides RC. This is carotenoid supports a a detail of the structure that can not yet be resolved unambiguously with modern X-ray techniques. A since the CP/MAS signal of carbonyl 4 is not temperature-dependent asymmetry was reported for K, contrary to the signals from the opposite carbonyl 1. Together, observable at temperatures these studies represent the first systematic MAS investigation of a membrane protein complex. Finally, some new technical developments are discussed. A novel example of MAS photo-CIDNP was discovered recently, and yields strong emissive signals for depleted, or pre-reduced, labeled RC. Using MAS dipolar correlation Spectroscopy on uniformly enriched chlorophyll a/water aggretates, a complete assignment for resonances was obtained and intermolecular transfer of coherence was observed. This development is an important step forward towards structure determination in multispin labeled proteins and complexes that are not accessible to diffraction methods.

Correspondence: Fax: 31-71-5274537; E-mail: [email protected]

299 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 299–313. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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Abbreviations: BChl – bacteriochlorophyll; Chl – chlorophyll; CIDNP – chemically induced dynamic nuclear polarization; CP – cross polarization; FID – free induction decay; LCAO – linear combination – primary of atomic orbitals; MAS – magic angle spinning; MO – molecular orbital; P – special pair; quinone acceptor; – accessory bacteriochlorophyll; RC – reaction center; – ubiquinone-10; – bacteriopheophytin

I. Introduction In this chapter the recent application of the CP/MAS NMR technique for the investigation of photosynthetic pigments, in particular the bac­ terial photosynthetic reaction center of Rb. sphaeroides, is reviewed. MAS NMR, in conjunc­ tion with selective isotope enrichment, is the me­ thod of choice for NMR investigations of mem­ brane protein complexes and other large macromolecular entities. Some years ago, we started, in an intensive collaboration with several other groups, a multidisciplinary effort to investi­ gate the photosynthetic reaction center of Rb. sphaeroides with MAS NMR and site- specific isotope enrichment (de Groot et al., 1990; 1992). Very recently, the group of A.E. McDermott has reported on a first observation of MAS photoCIDNP data of reaction centers (Zysmilich and McDermott, 1994). Altogether, these studies now represent the first systematic investigation of a membrane protein complex with MAS NMR techniques, and they have already provided infor­ mation that is not otherwise accessible. MAS NMR is a technique for obtaining highresolution NMR data from solids. A brief intro­ duction to the technique is presented in section II. Subsequently the results obtained thus far for bacterial photosynthetic reaction centers will be discussed. Section III deals with the MAS NMR characterization of the side chain of the (M)Y210 amino acid, which is highly conserved between different species and has attracted considerable attention over the past few years, since it is thought to be of importance for the fast rate of primary electron transfer. In section IV we de­ scribe how MAS NMR was used to address one specific structural aspect to atomic resolution, the configuration of the spheroidene carotenoid around the central 15 = 15' bond, a detail that could not be unambiguously resolved with X-ray

crystallography and is crucial for understanding the protective triplet quenching mechanism. The investigation of protein-cofactor interactions of importance for the specific one- electron redox properties of the primary quinone are dis­ cussed in section V. Finally, we discuss in section VI new developments concerning photo-CIDNP of RC and MAS dipolar correlation spectroscopy of chlorophyll a model systems. II. Magic Angle Spinning NMR Spectroscopy The standard CP/MAS experiment and a resulting FID are shown schematically in Fig. 1. Following a pulse on the ensemble of abundant nuclear spins, symbolized by I, the net proton magnetization is rotated to the plane perpendicu­ Sub­ lar to the direction of the magnetic field sequently the phase of the proton transmitter is resulting in a spin-lock pulse, that changed by can be interpreted as a small rf field along the magnetization vector, while simultaneously another rf field in the same coil is applied at the frequency of the less-abundant nucleus S, typically with

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the Hartmann–Hahn condition. Since the splitting of the and levels are now the same in the rotating frame, cross polarization, i.e. transfer of coherence between the two spin spe­ cies, can take place via the heteronuclear dipolar interactions. In proteins cross polarization times are typically 1–5 ms, and to a reasonable vary with approximation signal intensities according to the double exponential function (see e.g. Mehring, 1983)

In this expression the sets the amplitude, is the characteristic polarization transfer time as­ sociated with the dipolar coupling and is the longitudinal proton relaxation time in the rotating frame, i.e. under spin-lock con­ ditions. The theoretical maximum for the transfer of the protons into S-spin pola­ of polarization rization is

with the ratio of the number of lessabundant nuclei and abundant protons As the normally and for this represents an enhancement of with respect to thermal equilibrium. For membrane protein studies this enhance­ ment factor is convenient, but not crucial. Since the NMR experiment involves the acquisition of 20,000–50,000 scans, the total time required for obtaining a spectrum is in practice determined by the repetition time between individual scans, which is of the order of the longitudinal relaxation time in membrane proteins can be very long for nuclei located inside a membrane protein complex and direct acquisition of signals is practically impossible. In contrast, longitudinal relaxation proceeds much faster, as the excess energy can rapidly transfer into the lattice via the homonuclear dipolar interactions through e.g. rotating methyl groups with an in­

trinsically short Since in the CP/MAS experiment the is polarized from the spin system, the repetition time between scans is limited by the short This represents a significant gain in overall efficiency, which is in fact crucial. Since strong continuous wave decoupling is applied during acquisition in the CP/MAS experi­ ment, the main linebroadening mechanism for randomly oriented molecules in a powder is due to the anisotropy of the chemical shift,

The effect is illustrated in Fig. 2A, which shows the CP response of a powder sample of [1– The chemical shift is represented by a real symmetric second rank tensor In its is diagonal. In the principal axis frame the discussion of MAS results obtained on membrane proteins, two representations of the are com­ monly used. Most often

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with and the principal components, arranged in such a way that is the most upfield is the most component in the spectrum and downfield component. Alternatively, the iso­ tropic and anisotropic part may be separated ac­ cording to

with the isotropic shift, and 0. By convention and the dif­ ference between the two representations is a reor­ dering of the tensor principal components. Since the anisotropic part is traceless, it can be charac­ terized with two parameters, the anisotropy and the asymmetry The chemical shift broadening is effectively suppressed by macroscopic sample rotation A around an axis at the magic angle detailed treatment of the MAS averaging is be­ yond the scope of this chapter, and the reader is referred to the existing literature (see e.g. Mehr­ ing, 1983). Briefly, during MAS individual mol­ ecules are subject to physical sample rotation and the chemical shift of every molecule varies peri­ the rotation rate. Since many odically with different chemical shift trajectories will be pos­ sible, the macroscopic nuclear spin polarization collapses in a very short time, but refocuses at After every full rotor cycle the signal is recovered, yielding a rotational echo train, modu­ lated by the overall precession frequency corre­ sponding to the resonance offset (Fig. 1). Fourier transformation then gives the the effect of the sample spinning illustrated in Fig. 2B–D. In the frequency domain, MAS generates an infinite number of sidebands at integral multiples of the spinning speed with respect to the average shift experienced by every crystallite during the sample rotation. When the sideband intensities the can be extracted are determined by the from a MAS pattern by the comparison of the experimental data with theoretical simulations (see e.g. de Groot et al., 1991). This can provide invaluable structural information, with respect to conformation and configuration, and provides in-

sight into electrostatic polarization effects at the molecular and atomic level. III. Probing the Local Environment of M(Y)210 in Rb. Sphaeroides R26 RC with MAS The initial electron transfer time in the photosyn­ thetic reaction center of Rb. sphaeroides is sensi­ tive to the replacement of one particular tyrosine, M210, which is highly conserved between differ­ and the ent species, and is located close to P, (Fig. 3; Allen et al., 1987; Chang et al., 1991; Ermler et al., 1994). It has been suggested from electrostatic calculations that the presence of (M)Y210 lowers the energy of which affects the mechanism of electron transfer through the A branch (Parson et al., 1990). In­ deed it was found that the electron transfer kin­ and the redox potential etics from P to of P are altered when this tyrosine, close to P, is substituted by other amino and acids (Dimagno et al., 1992). CP/MAS NMR was used to study structural and electrostatic heterogeneities of the protein matrix in the immediate environment of (M)Y210 (de Groot et al., 1990; Fischer et al., 1992; Sho­ chat et al., 1995). Fig. 4a shows the response

Magic angle spinning NMR

of the Rb, sphaeroides R26 sample, recorded with a spinning speed at T = 230K from frozen RC solutions. The strong signals around 205, 155, and 105 ppm are from the isotope labels. A natural abundance spectrum was collected at the same spinning speed for an unlabeled R26 RC sample (Fig. 4b).

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The difference spectrum, containing essentially only the response from the labels, is shown in Fig.4c. The label signals in Fig. 4 are from 28 different tyrosines. An assignment of the (M)Y210 signal was obtained by comparing with data for labeled (M)Y210W mutant RC, with

304

M210 changed into a tryptophan (Fig. 5). In order to emphasize signals from individual tyrosines in the protein interior the second derivatives of the data are shown. Taking the second derivative is not a commonly accepted procedure in the NMR field. However, in this case it yields surprisingly good results that reproduce extremely well. For instance, in both spectra, which are from different species, a small doublet remains in the residuals after deconvolution, around 154.5 ppm, as indi­ cated by the circles in Fig. 5. It is now obvious that the signal at 152.2 ppm for R26 is associated with (M)Y210. The obser­ vation that only one narrow MAS NMR signal is

Huub J.M. de Groot suppressed by the mutation implies that the direct environment of M210 label is unique. Mattioli et al. (1991) reported that (M)Y210 is probably not hydrogen bonded. This is expected to produce a resonance frequency several ppm upfield from the hydrogen bonded side chains, in agree­ ment with the NMR results. Normally, resonances of proteins, including membrane proteins, are subject to some ob­ servable inhomogeneous broadening under MAS conditions when the temperature is lowered. The signal at 152.2 ppm from (M)Y210 is very narrow and its line width is independent of temperature. Therefore this tyrosine must be located in a well defined and rigid protein environment. Its linewidth of only 34 Hz reveals that the phenolic side chain can be considered static with respect to rotational diffusion on time scales as long as (Fischer et al., 1992). Since the 152.2 ppm reson­ ance is the only signal that is suppressed by the mutation, the MAS data provide strong evidence against structural or electrostatic (functional) het­ erogeneity in the chemical environment of (M)Y210 in the wild type, a possibility that has been suggested to explain the nonexponentiality of the primary charge transfer (Dimagno et al., 1991). On the scale of the MAS NMR, the protein environment of the M210 label is structurally and electrostatically (and in this sense functionally) homogeneous. For the Rb. sphaeroides RC the changes in electron transfer kinetics upon M210 mutation are quite spectacular when the tyrosine is re­ placed by a tryptophan, since the primary charge separation rate is slowed down by more than a factor of 10 (Shochat et al. 1994). This could be related to unanticipated structural changes or increased heterogeneity associated with the mu­ tation. From the decon volution of the data in Fig. 5 it appears that the remaining part of the signals in the carotenoid-less R26 is al­ most identical in the (M)Y210W species, which contains a spheroidenone carotenoid. Notably, the differences in chemical shift for the individual components vary less than 0.4 ppm between R26 and (M)Y210W mutant (Shochat et al., 1995). Since 160 ppm corresponds to 1 unit of charge, this translates into variations of electrostatic pola­ rization on individual residues in the order of electronic equivalents. This is very small,

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considering that the 4'-position in the phenolic side chain of the tyrosine is probing widely differ­ ent aspects of the microscopic chemical environ­ ment, for instance, differences in hydrogen bond­ ing environment, polarization by nearby polar residues or differences in conformation. Using the label signals as internal markers, we conclude that there is very little effect of both the mutation and the presence of the carotenoid on the overall structure and electrostatic properties of the frozen protein complex. In addition, the NMR data effectively exclude that a > 25% fast elec­ tron transfer component could be due to partial back-mutation (Shochat et al. 1994). Since the signal at 152.2 ppm in Figs. 5 and 6 is completely suppressed, it can be concluded that back-mutation in the (M)Y210 is less than 5%. IV. The Configuration of the Spheroidene in the Rb. Sphaeroides RC The photosynthetic reaction center of Rb. sphaeroides 2.4.1 contains one carotenoid, spheroidene, which protects the protein complex against photodestruction, probably by quenching chlorophyll triplet states and preventing the chlorophyll-sensitized formation of singlet state oxygen, a major oxidizing agent. The carotenoid structure deduced from the X-ray diffraction studies suffers from many ambiguities and uncer­ tainties (Ermler et al., 1994). Based on a com­ parison of the Raman spectra of model com­ pounds with the Raman spectrum of the RC, Koyama et al. (1983) proposed a central cis dou­ ble bond for the spheroidene in the RC. The same conclusion was reached by Lutz et al. (1987), who extracted the spheroidene from the RC under low-light conditions followed by NMR analy­ sis of the solution. The structure around the central (15–15' ) dou­ ble bond of the bound spheroidene carotenoid was also investigated in situ with low-temperature MAS NMR and site- specific isotope-labeling (Fig. 6). CP/MAS NMR spectra were ob­ tained of R26 RCs reconstituted with spheroidene specifically enriched at the C-14' or C-15' position (de Groot et al., 1992). The analysis of the configuration around the 15–15' double bond is built upon a characterization of the shifts of the resonances in spheroidene upon isomerization.

Since the 15–15'- cis spheroidene is highly un­ stable and isomerizes to the all-trans form, alltrans and 15–15'- cis were used as models. It appears that both in solution and in iso­ the solid state, the shifts upon merization are less than one ppm, except for the two carbons situated in the 15–15' double bond, and the C-14 and C-14' on either side of it, which shift by 4–6 ppm. This phenomenon may be used to analyze the configuration of the 15-15' double bond of spheroidene in situ by using specific labelling at C-14' and C- 15' in conjunction with CP/MAS NMR. In Fig. 7 the spectrum of spheroidene R26 RCs in frozen solution at T = 220 K and is shown. In this sample the signal of the C-15' is increased by a factor 90 because enrichment. A tiny sharp reson­ of the 99% ance from the label at 126.5 ppm is indicated by an arrow. In Table 1 the protein data for a C-14' and a C-15' label are compared with solution data isomers. for spheroidene and the two It appears that the signals from the labels of the spheroidene incorporated in the RC are both shifted upfield with respect to the all-trans mol­ ecule in solution, as is to be expected for the 15– 15' cis form. In addition, the magnitudes of the shifts compare well with the values observed for isomerization of the It is unlikely that other mechanisms than iso­ merization could be responsible for the upfield shifts upon incorporation of the molecule in the RC. The Raman studies by Lutz et al. (1987)

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Huub J.M. de Groot capabilities of modern X-ray techniques (Ermler et al., 1994).

V. The Asymmetric Sphaeroides R26

have provided evidence for a planar center of the RC-bound spheroidene and the close correspon­ dence between solution and solid state chemical shifts argues against an explanation in terms of small conformational distortions. According to the X-ray data, the labels are probably more than 0.6 nm away from the plane of the accessory bac­ teriochlorophyll monomer (B). This is the nearest macroaromatic cycle, and any ring current shift should be less than 0.5 ppm. Although shifts in the range of 4–6 ppm may in principle be caused by the presence of a polar group in the protein, such an explanation would be in contradiction with the X-ray structure, which reveals an apolar binding site. Hence the NMR data of the specifi­ cally enriched, reconstituted RC complexes provide convincing evidence for a 15–15'-cis con­ formation of the carotenoid. Determining this de­ tail of the structure is at present still beyond the

Binding in Rb.

A first set of CP/MAS NMR results on Rb. site sphaeroides R26 RCs reconstituted at the was obtained by with selectively labeled van Liemt et al. (1995). Using site-specific labe­ ling of positions 1, 2, 3, and 4 forming a path in the quinone ring between the two carbonyls, the protein-cofactor interactions affecting the quin­ one ring system can be investigated. The MAS results confirm asymmetric binding of by the protein. The most remarkable difference in NMR properties between the two carbonyl positions in is the temperature dependence of the signal at position 4, which is not detected at temperatures (Fig. 8). This temperature effect is now observed routinely in our laboratories and its origin is currently being investigated in detail. The differences in isotropic shifts between crystalline and are only 0.2–1.1 ppm for the labelled positions. This contrasts with the ground-state shift of for a mode vibration detected by dominated by the FTIR (Brudler et al., 1994, Breton et al., 1994). However, even without substantial changes of the isotropic shift, variations of principal components of the chemical shift tensor can be quite large. A deconvolution analysis using computer-generated carbonyl data in Fig. 9 MAS patterns of the indicates that the chemical shift asymmetry for the is somewhat smaller than for the 4– position in crystalline (Table 2). In a highly simplified formalism the chemical shielding of atom A can be expressed as the sum of a paramagnetic and a diamagnetic term

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The diamagnetic shift depends on the relative placements of the atoms and the associated elec­ tron densities, while in this description the para­ magnetic term takes into account the hybridiz­ ation effects. In an average energy approximation, using a simple LCAO molecular orbital scheme (Pople, 1962),

308

with e and m the charge and mass of the electron, Planck’s constant divided by c the velocity the average excitation energy of of light and the excited electronic states in the absence of a magnetic field. The summation runs over neigh­ bours B of A, the atom with the nucleus of inter­ est and is proportional to the average value for the 2p orbitals. According to Pople (1962),

and Similar expressions exist for and The is expressed in terms of the gen­ first part eralized charge densities while the nearest neighbour bond effect is expressed in terms Because of of the generalized bond orders the ordering of the MO coefficients in the and the transitions are not contribut­ ing to the in this model. However, bond order changes may be expected to affect the principal components of the chemical shift tensor consider­ ably through the terms that depend on From experimental data for the relation be­ tween carbonyl frequencies and bond orders (Josien et al., 1953), it can be inferred that the shift of the mode dominated by the stretch represents a variation of the bond order of approximately 0.1. A crude estimate based on trends put forward in a recent summary of characteristic shielding anisotropies and approxi­ mate calculation of shielding tensors using Eqs. contributions to the ten­ (8–10), suggests that

Huub J.M. de Groot

sor elements could give rise to changes in the 10 ppm range for a bond order change of ~0.1 (Zilm can and Duchamp, 1992). In addition, the vary depending on the surrounding structure. However, with experimental errors in the range of 10–20 ppm, for the principal components of carbonyls the chemical shift tensor for the listed in Table 2, it was concluded that there is at present no conflict between the NMR shift anisotropy analysis and the quite dramatic 60 shift of the stretch mode observed with infrared spectroscopy (van Liemt et al., 1995). Apparently the two spectroscopic tech­ niques are complementary, since the NMR is most suitable to detect charge densities, while the FTIR is much more sensitive to rehybridization effects. In line with the FTIR results, the esti­ mates of the shift anisotropy indicate an effect at C-4 and not at C-1. The fact that a strong and narrow response for can be detected at ambient tempera­ the ture provides evidence that this carbonyl is static on the scale of the NMR and in a well-defined part of the binding pocket. On the other hand, the loss of signal for should be related to dynamic destructive interference of motion with the NMR signal. The CP/MAS ex­ periment is sensitive to dynamic perturbations with correlation times in the 0.01–1 ms range, and in particular CP and decoupling are very sen­ sitive to local dynamics. Recent additional experi­ ments support these inferences and suggest that sig­ the temperature dependence of the at temper­ nal is related to a shortening of the atures approaching 255 K (B.J. van Rossum et al., 1996).

Magic angle spinning NMR

309

Several possible mechanisms for the tempera­ ture effect in the NMR response have now been put forward in the literature (Brudler et al., 1994; Breton et al., 1994). It could be related to dy­ namic hydrogen bonding interactions on the scale of the NMR, 0.01–1 ms, for instance switching between the two possible H-bond donors. In such a scheme the dynamics would freeze at lower K the temperatures, explaining why for 4 carbonyl signal can be observed. Kleinfeld et al. (1984) inferred that under physiological con­ ditions transitions between different structural affecting states occur on a time scale the distance by However, a definite explanation in terms of a precise molecu­ lar mechanism cannot be given as yet, and further studies are necessary. VI. New Developments. CIDNP and Correlation Spectroscopy Zysmilich and McDermott (1994) reported the observation of MAS photo-CIDNP under con­ enriched RC with fortinuous illumination of ward electron transfer blocked by depletion or by pre-reduction of the This is the first CIDNP in the solid state with observation of the chromophores in the protein driving the nu­ clear spin selection mechanism. According to the discoverers of the effect, it relies on a partial interconversion between and followed by charge recombination, nu­ clear relaxation and ground state decay in this channel. An example of the phenomenon is shown in Fig. 10, for depleted RC in the dark and under continuous illumination. The data under signals illumination reveal strong emissive that are characteristic for the CIDNP effect, with an estimated enhancement of ~300 over the natu­ ral abundance response. The CIDNP response is probably associated with the nuclei of P and the signal strengths are essentially independent of proton decoupling power level. When the precise mechanism of the enhancement is fully under­ stood, this new CIDNP approach could provide information about the electron density in the as opposed to the transient species extensively stable oxidized radical cation

studied by EPR. Moreover, the chemical shifts of the nuclei in the special pair can be characterized. Another recent technical development con­ cerns MAS dipolar correlation Spectroscopy of uniformly enriched chlorophyll a (Chl a), the ubiquitous chromophore involved in light-harvesting and photochemistry in the photosynthetic energy conversion processes of green plants and related organisms (Fig. 11). When exposed to Chl a can aggregate and it is thought that water molecules form intermolecular hydrogenbonded bridges from the Mg to the carbonyls and (Katz et al., 1991). The CP/MAS NMR spectrum of the enriched Chl-a/water aggregate is shown in Fig. 11 (Boender et al., 1995). The Chl- a/water micelle and similar aggre­ gates, for instance the BChl c entities in chloro­ some antennae, provide typical examples of sys­ NMR tems where high-resolution solid state spectroscopy will be indispensable to provide in­

310

formation at the atomic level about the precise structure. Recently, important progress towards a general approach for MAS NMR structure re­ finement using 2-D dipolar correlation spectros­ copy in uniformly enriched moderately sized molecules was reported by Boender et al. (1995). In their study, pure-phase MAS NMR dipolar enriched Chlcorrelation spectra of the a/water aggregates were obtained with the pulse sequence of Fig. 12. An integral multiple n of XY-8 phase alternated rotor-synchronized trains pulses was used to promote exchange of of coherence through dipolar interactions during the (Bennet et al., 1992). mixing period In the average Hamiltonian approximation for a nuclear spin I = 1/2 coupled to another spin S = 1/2 the density matrix is:

Huub J.M. de Groot

Magic angle spinning NMR

at the start of detection time Therefore polar­ ization transfer between and can take place during mixing. In Fig. 13 an example of a 2-D correlation spectrum of the enriched Chl

311

aggregate, collected with an 10,000 ± 5 Hz and ± is shown, with strong 2–D cross-peaks revealing transfer of co­ herence. The horizontal and vertical lines illus­ trate how the molecular framework gives rise to a correlation network, similar to the assignment procedures in solution NMR. A complete assignment of the resonances obtained from the analysis of 2-D correlation spectra is compared with data for the molecule in solution. It appears that the two methyl groups and are shifted upfield by more than 3 ppm in the aggregate with respect to their chemical shifts in solution. According to Boender et al. (1995), this is probably induced by the ring-curof the macroaromatic cycle rent of the

312 of a neighbouring molecule, and would be in agreement with current models for the aggregate structure, in which the edges of the macrocycles of neighbouring molecules are above one an­ are shifted by the

other, and the atoms macroarornatic cycle of a neighbouring molecule.

Moreover, carbon 2 appears to correlate with

carbon 14, carbon has a correlation with car­ bon 20 and carbon 4 as a correlation with carbon 12. The carbon atoms 12, and 14 are on the opposite side of the Chl a ring with respect to carbons 2, 4 and 20. Such cross-peaks are there­ fore most likely associated with short intermolecu­ lar distances. This indicates that the stacking of the Chl a rings proceeds roughly in agreement with the model of Chow et al. (1975), which was based on the X-ray structure of ethyl chloro­ phyllide a, although the presence of the corre­ lation between and 20 suggests that the rings are rotated slightly along an axis perpendicular to the plane of the molecule, generating a curva­ ture of the array of stacked rings which could explain the formation of tubular micelles in the Chl-a/water aggregates. Dipolar correlation spectroscopy of multispin systems as a generally applicable research tool is thus forthcoming, but it should be mentioned that it still requires the development of a new gen­ eration of high-field wide-bore solid NMR instru­ ments (750 MHz) with high-speed (>20 kHz) MAS probes and excellent performance with re­ spect to long-term stability. In addition, further development of NMR-methods and advanced partial labeling schemes of larger systems will be required. Nevertheless, the experiment of Fig. 13 represents an important step in the development of MAS NMR as a tool for comprehensive struc­ ture determination at the atomic level in solids that are neither accessible to X-ray nor to solution NMR methods. Many biological subcellular lifesustaining systems fall into this category. VII. Concluding Remarks In this chapter, we have summarized how MAS NMR was used to investigate structure-function properties of Rb. sphaeroides photosynthetic re­ action centers and their significance to fundamen­ tal molecular mechanisms of photochemical en­ ergy conversion. MAS NMR in conjunction with

Huub J.M. de Groot site-directed specific isotope labeling is at present a technological growth area in the field of mem­ brane protein research. It is rapidly developing into a versatile technique suitable for obtaining information about microstructure and structurefunction relations in essentially unperturbed bio­ logically ordered solid-type systems, even when there is no translation symmetry and X-ray or solution NMR are not applicable. Since in prin­ ciple atomic selectivity can be obtained in systems with molecular weights counting several megadal­ ton, many of the components of the photosyn­ thetic apparatus are already within reach of the technique, and more MAS NMR applications in photosynthetic systems are forthcoming. Acknowledgements The contributions of Ir. G. Boender, Dr. M. Fischer, Prof. H. Frank, Dr. P. Gast, Dr. R. Gebhard, Drs. K. v.d. Hoef, Dr. W. van Liemt, Mrs. S. Shochat, Dr. C. Violette and Dr. C. Winkel to the MAS explorations of the bacterial RC are gratefully acknowledged. Special thanks are accorded to Prof. A.J. Hoff, Dr. J. Raap and Prof. J. Lugtenburg, for continuing collabor­ ations and for making the initial MAS studies of photosynthetic reaction centers possible. This work was supported by the Royal Netherlands Academy of Arts and Sciences (KNAW), the Foundations for Biophysical and Chemical Re­ search (financed by The Netherlands Foundation for Scientific Research), and the Commission of the European Communities. References Allen JP, Feher G, Yeates TO, Komiya H and Rees DC (1987) Structure of the reaction center from Rhodobacter sphaeroides R26: The cofactors. Proc Natl Acad Sci USA 84: 5730-5734. Bennet AE, Ok JH, Griffin RG and Vega S (1992) Chemical shift correlation spectroscopy in rotational solids: Radio frequency- driven dipolar recoupling and longitudinal ex­ change. J Chem Phys 96: 8624–8627. Boender GJ, Raap J, Prytulla S, Oschkinat H and de Groot HJM (1995) MAS NMR structure refinement of uniformly enriched chlorophyll-a/water aggregates with 2-D di­ polar correlation spectroscopy. Chem Phys Lett 237: 502– 508. Breton J, Boullais C, Burie J-R, Nabedryk E and Mioskowski C (1994) Binding sites of quinones in photosynthetic bac­

Magic angle spinning NMR terial reaction centers investigated by light-induced FTIR difference spectroscopy: Assignment of the interactions of each carbonyl of in Rhodobacter sphaeroides using sitespecific ubiquinone. Biochemistry 33: 14378– 14386. Brudler R, de Groot HJM, van Liemt WBS, Steggerda WF, Esmeijer R, Gast P, Hoff AJ, Lugtenburg J, and Gerwert groups K (1994) Asymmetric binding of the 1– and of in Rhodobacter sphaeroides R26 reaction centres monitored by Fourier transform infra-red spectroscopy using site-specific isotopically labelled ubiquinone-10. EMBO J 13: 5523–5530. Chang CH, E-Kabbani O, Tiede D, Norris J and Schiffer M (1991) Structure of the membrane-bound protein photosyn­ thetic reaction center from Rhodobacter sphaeroides. Bio­ chemistry 30: 5352–5360. Chow HC, R. Serlin R and Strouse CE (1975) The crystal and molecular structure and absolute configuration of ethyl chlorophyllide a dihydrate. A model for the different spec­ tral forms of chlorophyll a. J Am Chem Soc 97: 7230–7242. de Groot HJM, Raap J., Winkel C., Hoff AJ and Lugtenburg J (1990) Magic-angle-spinning NMR with atomic resol­ ution of a photosynthetic reaction center enriched in Chem Phys Lett 169: 307–310. de Groot HJM, Smith SO, Kolbert AC, Courtin JML, Winkel C, Lugtenburg J, Herzfeld J and Griffin RG (1991) Iterat­ ive fitting of magic-angle-spinning NMR spectra. J Magn Res 77: 251–257. de Groot HJM, Gebhard G, vd Hoef I, Hoff AJ, Lugtenburg J, Violette CA and Frank HA (1992) magic angle spinning NMR evidence for a configuration of the spheroidene in the Rhodobacter sphaeroides photosynthetic reaction center. Biochemistry 31: 12446–12450. DiMagno TJ, Rosenthal SJ, Xie X, Du CK, Chan CK, Han­ son D, Schiffer M, Norris J and Fleming GR (1992) Recent experimental results for the initial step of bacterial photo­ synthesis. In: Breton J and Verméglio A (ed) The Photo­ synthetic Bacterial Reaction Center II, pp 209–217. Ple­ num Press, New York. Ermler U, Fritzsch G, Buchanan SK and Michel HM (1994) Structure of the photosynthetic reaction centre from Rho­ dobacter sphaeroides at 2.65 Å resolution: cofactors and protein-cofactor interactions. Structure 2: 925–935. Fischer MR, de Groot HJM, Raap J., Winkel C, Hoff AJ magic angle spinning study of and Lugtenburg J (1992) the light-induced and temperature-dependent changes in Rhodobacter sphaeroides R26 reaction centers enriched in tyrosine. Biochemistry 31: 11038–11049. Josien ML, Fuson N, Lebas JM and Gregory TM (1953) An infrared spectroscopic study of the carbonyl stretching frequency in a group of ortho and para quinones. J Chem Phys 21: 331–340. Katz JJ, Bowman MK, Michalski TJ and Worcester DL (1991) Chlorophyll aggregation: Chlorophyll/water micelles as models for in vivo long-wavelength chlorophyll. In: Scheer H (ed) Chlorophylls, pp 211–235. CRC Press, Boca Raton. Kleinfeld D, Okamura MY, and Feher G (1984) Electrontransfer kinetics in photosynthetic reaction centers cooled to cryogenic temperatures in the charge-separated state:

313 Evidence for light- induced structural changes. Biochemis­ try 23: 5780–5786. Koyama Y, Kito M, Takii T, Saiki K, Tsukida K and Yamash­ ita J (1983) Configurations of neurosporene isomers iso­ lated from the reaction center and the light-harvesting com­ plex of Rhodobacter sphaeroides G1C. A resonance Raman, electronic absorption, and study. Photochem Photobiol 48: 107–114. Lutz M, Szaponarski W, Berger G, Robert B and Neumann J (1987) The stereoisomerism of bacterial, reaction centerbound carotenoids revisited: An electronic absorption, res­ onance Raman and study. Biochim Biophys Acta 894: 423–433. Mattioli TA, Gray KA, Lutz M, Oesterhelt D and Robert B (1991) Resonance Raman characterization of Rhodobacter sphaeroides reaction centers bearing site-directed mu­ tations at tyrosine M210. Biochemistry 30: 1715–1722. Mehring, M (1983) High Resolution NMR in Solids. SpringerVerlag, Berlin. Parson WW, Chu ZT and Warshel A (1990) Electrostatic control of charge separation in bacterial photosynthesis. Biochim Biophys Acta 1017: 251–272. Pople JA (1962) Molecular-orbital theory of diamagnetism. I. An approximate LCAO scheme. J Chem Phys 37: 53–59. Shochat S, Arlt T, Francke C, Gast P, van Noort PI, Otte SCM, Schelvis HPM, Schmidt S, Vijgenboom E, Vrieze J, Zinth W and Hoff AJ (1994) Spectroscopic characterization of reaction centers of the (M)Y210W mutant of the photo­ synthetic bacterium Rhodobacter sphaeroides. Photosynth Res 40: 55–66. Shochat S, Gast P, Hoff AJ, Boender GJ, van Leeuwen S, van Liemt WBS, Vijgenboom E, Raap J, Lugtenburg J MAS NMR evidence for a and de Groot HJM (1995) homogeneously ordered environment of tyrosine M210 in reaction centers of Rhodobacter sphaeroides, Spectrochim Acta 51A: 135–144. van Liemt WBS, Boender GJ, Gast P, Hoff AJ, Lugtenburg J and de Groot HJM (1995) magic angle spinning NMR characterization of the functionally asymmetric binding in Rhodobacter sphaeroides R26 photosynthetic reaction centers using site-specific ubiquinone-10, Bio­ chemistry 34: 10229–10236. van Rossum B-J, van Liemt WBS, Boender GJ, Gast P, Hoff AJ, Lugtenburg J and de Groot HJM (1996) MAS NMR relaxation study of the binding in Rhodobacter sphaeroides-R26 reaction centers. In P. Mathis (ed) Photo­ synthesis: From Light to Biosphere Vol. I, pp. 899–902. Kluwer Academic Publisher, Dordrecht. 1995. Zilm KW and Duchamp JC (1992) Comparisons of shielding anisotropies for different nuclei and other insights into shielding from an experimentalist’s viewpoint. In: Tossel JA (ed) Nuclear Magnetic Shieldings and Molecular Struc­ ture, NATO ASI Series, Series C: Mathematical and Physi­ cal Sciences, pp. 315–334. Kluwer Academic Publishers, Dordrecht. Zysmilich M and McDermott AE (1994) Photo-chemically induced dynamic nuclear polarization in the solid state spectra of reaction centers from photosynthetic bacteria Rhodobacter sphaeroides R-26. J Am Chem Soc 116: 8362– 8366.

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PART THREE

Structure and oxygen

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Chapter 19

Structure Determination of Proteins by X-Ray Diffraction† Marianne Schiffer*

Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory,

9700 South Cass Avenue, Argonne, IL 60439–4833, USA

Summary I. Introduction II. Theory, Equations, and Some of the Terms Used in X-Ray Structure Determination III. Determination of Protein Structure A. Characterization of the Crystals B. Data Collection C. Determination of Preliminary Structure (or Phases) D. Refinement of Structure IV. Quality of the Structure V. Comparison with Structural Information Obtained with Other Techniques Acknowledgements References

317 317 318 318 318 319 319 321 323 323 323 324

Summary The arrangement of atoms within protein molecules, as determined by X-ray diffraction of single crystals, forms some of the basic data for many spectroscopic, protein engineering, and computation studies. In this chapter, the terminology used in protein crystallography is explained and a brief description is given of how a protein structure is determined. Further, some ideas are discussed on how to judge X-ray data and the resulting protein structure and how to compare the data derived by X-ray diffraction with data obtained by other methods. I.

Introduction

from the Protein Data Bank at Brookhaven National Laboratory.‡ The number of protein structures is increasing at a very high rate; in 1989, there were 400 structures while the January 1994 release of the Data Bank contained 2143. In the last ten years, protein crystallography has become a significant component of photosynthesis research. It is the aim of this chapter to make the protein crystallography literature more accessible and comprehensible to the typical spec­ troscopist and biologist. Since there are several very good textbooks (e.g., Blundell and Johnson, 1976; McPherson, 1982; McRee, 1993), two volumes in the Methods of Enzymology (Wyckoff et al., 1985a,b), and an excellent mini-review (Eisenberg and Hill, 1989) that describe the determi­

The arrangement of atoms within protein molecules, as determined by X-ray diffraction of single crystals, forms some of the basic data for many spectroscopic, protein engineering, and computational studies. Depending on the specific application, it is important to know how to evaluate the atomic coordinates that can be obtained † The U.S. Government’s right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged. *Correspondence: Fax: 1-708-2525517; E-mail: [email protected]. ‡ Coordinates can be obtained by FTP from pdb.pdb.bnl.gov (Internet address 13.199.144.1).

317 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 317–324. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

318 nation of protein structures by X-ray diffraction, I will concentrate on defining the terminology used in protein crystallography, provide a brief description of how a protein structure is deter­ mined, and give some ideas and personal views on how to judge X-ray data and the resulting protein structure. The emphasis in this chapter is on the methods of structure determination when a suitable protein crystal is already in hand.

II. Theory, Equations, and Some of the Terms Used in X-Ray Structure Determination Electrons within a crystal scatter X-rays in di­ rections that are determined by the geometry of the unit cell of the crystal and its orientation relative to the X-ray beam. The X-rays can be considered as “reflected” from a plane or family of planes of the crystal. Each plane is defined by three indices: h, k, and l. The perpendicular distance between these planes is The Bragg equation relates d with the scat­ and the X-ray wavelength in tering angle angstroms.

The quantity (in angstroms) is quoted in general as the resolution of the dataset. It defines the largest angle of scattering the crystal is cap­ able of, or, in other words, the crystalline order within the crystal. The more ordered the crystal is, the more higher-resolution observations can be made and the more accurately the structure can be determined. The higher the resolution of Data at 2.5 Å resol­ the data, the lower the ution is considered “high” resolution and good for a protein crystal; 1.8 Å resolution is excellent, and 5–6 Å is low resolution. The intensity (I) of the reflected beam is the experimental quantity that is measured for all possible reflections of the crystal. The intensity (I) is proportional to the square of the structure fac­ tor (F). If the crystal structure is known, the structure factor can be calculated according to the follow­ ing formula:

Marianne Schiffer

where is the atomic scattering factor or form factor that depends on the electron distribution in the nth atom (carbon, oxygen, etc.); are the coordinates of the atom and B is the atomic temperature factor. where is the square of the average displacement of the atom. The structure factor of each reflection (hkl) is a vector that consists of an amplitude F(hkl) and a phase angle or phase Only the amplitude of the reflection can be measured directly. The phases of the re­ flections have to be determined using other me­ thods. When both the amplitudes and phases are known, the three-dimensional electron density distribution of the crystal can be calculated. Elec­ tron density at point xyz in the unit cell is defined by a Fourier series in three dimensions. An elec­ tron density map or Fourier map is calculated according to:

The summation is over the three indices, hkl; V is the volume of the unit cell. III. Determination of Protein Structure The determination of a crystal structure has sev­ eral steps: characterization of the crystal, collec­ tion of diffraction data, determination of prelimi­ nary phases (and structure), refinement of phases (and structure), and description of the structure. A review paper by Brünger and Nilges (1993) describes the computational aspects of structure determination and refinement. A review by Fin­ zel (1993) summarizes the most frequently used computer program packages.

A.

Characterization of the Crystals

Unit cell dimensions, three axes (a, b, and c), and and define the basic repeat three angles

X-ray diffraction unit of the crystal. The cell dimensions are deter­ mined from the geometry of the diffraction pat­ tern, obtained from precession photographs or area detector data. The space group is determined from the sym­ metry of the diffraction pattern and from reflec­ tions that are systematically absent. The space group defines the symmetry (symmetry oper­ ators) within the crystal. Possible space groups and their properties are listed in the International Tables for Crystallography, Volume A (Hahn, 1983). The space group symmetry operates on the asymmetric unit of the crystal. The asymmetric unit usually contains one molecule, but it can include more than one molecule or part of the molecule. If it contains more than one molecule (e.g., two molecules), the structure that must be determined is two times larger than the single molecule and, therefore, the unique set of reflec­ tions defining the structure is also two times larger. If a partial molecule is the asymmetric unit, the symmetry element of the unit cell relates two halves of the molecule; e.g., a twofold axis and the number of unique observations defining the molecule is also halved. In other words, the amount of diffraction data required to “solve” a structure is determined not only by the size of the protein itself, but also by the size of the asym­ metric unit. The unit cell volume (calculated from the unit cell dimensions) and the space group determine the numbers of molecules in the unit cell and in values the asymmetric unit. Volume/dalton for soluble proteins range from 1.7 to with an average value of (Matthews, 1968). For membrane proteins, because of the presence of detergent in the crystal, is larger and can vary from 3.5 to The number of molecules in the asymmetric unit can be deter­ mined experimentally by measuring the density of the crystal, but more usually it is done by values and choosing a number calculating the that will result in a value that falls in the expected range.

B. Data Collection There are several different methods for data col­ lection. The method of choice may be determined by crystal cell dimensions and how well the crystal diffracts X-rays. Diffractometers measure one re­

319 flection at a time, and each reflection can be very accurately determined; diffractometers are now mostly used for small molecules. Area detectors can simultaneously measure a plane of reflections (possibly several hundred of them) at one orien­ tation of the crystal; such detectors include elec­ tronic area detectors, X-ray film, and imaging plate detectors (for comparison of these detec­ tors, see Eikenberry et al., 1992). Protein crystals deteriorate (the intensity of the reflections “decays”) in the X-ray beam as a function of time. Although time-dependent decay correction can be applied to the diffraction data, after a certain point a fresh crystal is used. Cor­ rections for the asymmetric absorption of X-rays by the crystal and surrounding mounting material are made using a predetermined function for dif­ fractometer data. For area detector data, the same reflection is measured several times at dif­ ferent crystal settings. Scale factors applied to the data collected at different crystal settings usually correct for absorption and crystal decay. To assess the quality of the data, R-sym, the agreement between equivalent reflections mea­ sured at different geometries, have to be exam­ ined. R-sym is generally better for reflections with higher intensities and, therefore, it gets worse with increasing resolution where the reflections get weaker. Weak, low-intensity reflections are also strongly affected by the background correc­ tion. Of course, R-sym also depends on whether the absorption and decay corrections were effec­ tive. A good dataset will have an overall R-sym of less than 0.1. As in other measurements, maximizing the peak signal-to-background ratio is important. This is usually achieved by using crystals with as large a volume as possible or with intense Xray beams such as those provided by synchrotron radiation sources.

C. Determination of Preliminary Structure (or Phases) The most generally used method for determining the structure of large protein molecules is multi­ ple isomorphous replacement or MIR (Dickerson et al., 1961). If a structure of a homologous pro­ tein molecule is known, then molecular replace­ ment (Rossmann, 1972,1990) can be applied. For example, the MIR method was used to determine

320 the structure of the reaction center from Rps. viridis, (Deisenhofer et al., 1984) while molecular replacement was used to determine the Rb. sphaeroides reaction center structure; this deter­ mination exploited the availability of the coordin­ ates from Rps. viridis as the search structure (Chang et al,. 1986; Allen et al., 1986). These molecules have similar structures but crystallized in different unit cells and had different space groups. In multiple isomorphous replacement, heavy atoms (in the form of salts of heavy atoms; e.g., or organic derivatives, e.g., para-chloroHg-phenol) are introduced into a crystal generally by soaking the “native” crystal in a solution con­ taining the heavy atom (HA). In special cases, the HA can be covalently bound to the protein or involve the replacement of a metal atom in the structure with a heavier one. For this method to work, the HA contribution (1) has to be signifi­ cant, (2) its introduction does not change the unit cell of the protein (the two crystals are isomorph­ ous), and (3) only few atoms or molecules are introduced per molecule of protein, so their posi­ tion can be determined. As a first step, the HA is located in the unit cell (by Patterson or some­ times by direct methods, that are generally used to determine small molecule structures). The Pat­ terson function or map is a Fourier series calcu­ lated with squared structure factors. The peaks in a Patterson map correspond to vectors between atoms. The Patterson map calculated with the difference between the derivative and native squared will give the structure factors vectors between the heavy atoms. When there are relatively few HAs per protein molecule, their locations can be determined from the Patterson map. Direct methods (Karle, 1989; Hauptman, 1989), based on the relationships between struc­ ture factors, are used when Patterson methods fail to locate the HA positions. The positions of the HAs are then refined using the quantity Several HA “de­ rivatives” are prepared and analyzed, and to­ gether they are used to determine the phases for the protein (“native” crystals). The quality of the resultant phases depends on the quality of the HA and native datasets, how isomorphous the derivative is, the interpretation of the HA atom data (where all HA sites are found), and the size

Marianne Schiffer of the contribution of the HA to the native data. If the quality of the phases (and therefore the resulting Fourier or electron-density map) is good, a crystal structure can be interpreted even at 4 Å resolution. Certainly at 3.5 Å resolution, the polypeptide chains can be traced and side chains can be identified. In molecular replacement, a known homolo­ gous structure, the search molecule, is located in the new unit cell by first finding its orientation (the rotation problem) and then its position (the translation problem). In this method, vectors be­ tween objects in the unit cell are analyzed. Sev­ eral methods and program packages are available for these searches (e.g., Fitzgerald, 1988; Brünger, 1990). In general, the signal-to-noise ratio for the rotation problem is better than that for the translation problem. Most often, low res­ olution data (between 20 Å and 6–4 Å resolution) are used for the searches. After the molecule location is found, its position and orientation are refined with rigid body refinement. It is very important to check that the resulting molecular positions in the unit cell make sense; that is to say, two molecules don’t interfere with each other and don’t occupy the same space. An­ other way to check the molecular replacement solution is to see if there is density for parts of the molecule that were not part of the search molecule. For example, if the search molecule had an Ala in a certain position while the mol­ ecule had a Trp, then if the solution is correct, electron density should be present for Trp (see Chang et al., 1986, and Fig. 1). This should also work for longer segments, but very often loops on the surface of the molecule only in contact with the solvent don’t have density. The lack of density could signal physical disorder (more than one conformation). It could also result from a problem with molecular replacement method, be­ cause for the same structure, electron density for the fragment can be found when using MIR me­ thods and not with molecular replacement me­ thod. After preliminary phases are obtained by any method, the molecule is built “into” the electron density map using the amino acid sequence of the protein. The features of the electron density are interpreted in terms of molecular structure. At low resolution, observable features include the

X-ray diffraction

molecular envelope and At medium resolution, the protein backbone and side chains become apparent. Protein crystals don’t diffract highly enough to see individual atoms. “Mini maps”, or small-scale maps, are helpful for trac­ ing the polypeptide chain and locating the alpha carbon positions. Further building of the protein structure is carried out with computer graphics programs like FRODO (Jones, 1978) or O (Jones et al., 1991). The construction of the molecular model takes into account the geometry and con­ tacts that are expected from small molecule and other protein structures. Of course, if the struc­ ture was solved by molecular replacement, the coordinates of the search molecule can be used as the starting point of rebuilding.

D.

Refinement of Structure

The refinement of the “structure” consists of im­ proving the atomic positions in the model and improving the temperature factors so that the or using structure factors calculated the improved structure will more closely approxi­

321

mate the observed structure factors or The quality of the result is expressed by the residual, or R-factor summed over all reflec­ tions, where

Once a preliminary structure is built according to the electron density map, refinement programs can be used. These programs first “regularize” the structure so it will have the expected bond angles, bond lengths, and nonbonded contacts. Then they refine the atomic positions against the “observed” structure factors. One of the methods uses least squares refinement; e.g., programs PROLSQ (Hendrickson, 1985) and TNT (Tron­ rud et al., 1987). In least squares refinement, because the ratio of observations to atomic par­ ameters is relatively low, restraints on the struc­ ture are used as additional observations. The re­ straints are based on the known rules of the stereo chemistry of the molecule, and they keep the molecule close to the ideal geometry. (For small

322 molecular structures that diffract to atomic resol­ there are about ten times as many ution, observations as parameters being refined; there­ fore, the least squares refinement is well be­ haved.) During the refinement, different weights can be given to the observations and the re­ straints. Depending on the relative weights, good geometry can be maintained, or a better fit to the structure factors, as reflected by the R-factor, can be attained. Ideally, the aim is to get a low Rfactor with good geometry. Besides the three co­ of each atom, overall scale ordinates factors and temperature factors are optimized in the beginning of the refinement process. As the refinement progresses, individual atomic temper­ ature factors are introduced. The refinement technique known as simulated annealing (SA) has a larger radius of convergence than least squares refinement. With SA refine­ ment, atoms can move over 2 Å to their correct positions. This method is based on molecular dy­ namics and was introduced by Brünger et al. (1987). In X-PLOR, the total energy of the sys­ tem (the crystal) is refined. In addition to bonded and nonbonded interactions, the experimental diffraction data is also considered as an energy term. After the relative weights are established for the structural and diffraction data, the energy of the system is minimized. This is followed by molecular dynamics calculations that allow the protein to move over energy barriers. After mol­ ecular dynamics calculations at different tempera­ tures, using different protocols, the energy of the system is further minimized. The method is very computer-intensive, but because it can refine structures that are farther from the correct one, less manual rebuilding is needed, and generally the overall refinement process is faster. The SA refinement generates an ensemble of structures that agree with the X-ray data; small outside loops can take different conformations (Xu et al., 1990) and have to be examined carefully. Cycles of automated refinement are inter­ spersed with “manual” refinement that involves examination of the electron density map and making required adjustments by rebuilding the structure using a computer graphics program (e.g., FRODO or O). During the refinement of also im­ the structure, the calculated phases prove and get closer to the correct phases; there-

Marianne Schiffer fore, electron density maps calculated with the improved and will show an improved struc­ ture. The refinement process alternates between improving the coordinates and examining the fit of the coordinates to the calculated electron den­ sity map. (A map calculated with and coef­ ficients reproduces the structure at that point of the refinement. A map calculated with and has the correct measured amplitude for the structure factor; therefore, it will be closer to the real structure.) Difference maps calculated with and areused to coefficients identify errors because they give more infor­ In the­ mation than the maps calculated with ory, when an atom is incorrectly positioned, it map, should be observed at half height in a while another half-height peak should show up in the correct position. In an map, there should be a half- height positive peak in the cor­ rect position and a half-height negative peak in the position where it was introduced by mistake. Therefore, the combination of these two maps should have a posi­ tive peak in the correct position and a negative one in the incorrect one. Difference maps calculated with both and coefficients are used later in the refinement process to locate cofactors, ions, detergent, and water molecules. To identify these additional small molecules, the shape of the “extra” electron density and the hydrogen bonding potential have to be taken into account. The newly rebuilt protein molecule and the additional atoms then are refined further to see if the fit improved in the refinement and whether the new features improve in the subsequent maps. In general, when an atom is introduced, it will appear in the maps, even when its position is incorrect; therefore, it is very hard sometimes to eliminate incorrect features of the molecule that were introduced at some point in the modelbuilding process. If that atom or segment has been part of the molecule for many refinement cycles, removing it from the phase calculation is not sufficient. The structure has to be refined without the segment in question, and it is also helpful to remove some of the surrounding resi­ dues as well before new refinement cycles are carried out and new maps are built. Such con­ siderations led to the recent introduction of the

X-ray diffraction free R-factor (Brünger, 1993), which is a good monitor of the progress of the refinement. To determine the free R-factor, a fraction of the re­ flections are not refined. If the structure improved during the refinement of the majority of the re­ flections, this should improve the R-factor also for the ones that were left out. IV. Quality of the Structure How can you tell how good a structure is? This question has been the topic of discussions (see Bränden and Jones, 1990). The R-factor is one indication. A well refined high resolution 2–2.5 Å structure that includes solvent (water) mol­ ecules will have an R-factor of 0.15–0.20. Me­ dium resolution structure of about 3 Å that does not include solvent molecules will have an Rfactor of 0.20–0.25. But even with such good Rfactors, segments of the structure can be incor­ rect. The accuracy of the structure depends on the goodness of the diffraction data, correctness of phase angles, the resolution of the data, and how carefully the structure was determined. For a de novo structure, the ø, angles (Ramachand­ ran plot) reflecting the nonbonded contacts, dis­ tances between neighboring protein molecules, and in general parameters that were not explicitly restrained can be good indicators of structure quality; they should have values expected from studies of small molecules. The “correctness” of the structure is also a good indicator; i.e., (most) hydrophobic residues are on the inside while hyd­ rophilic residues are on the outside (Lüthy et al., 1992). Judging a solution obtained by molecular replacement is more difficult because this “cor­ rectness” was already met in the search molecule. Here it is important to examine intermolecular contacts; they are expected to make chemical sense. The course of the refinement is also an important indicator. Did the refinement proceed smoothly and did the free R-factor improve upon introduction of new features? For example, lower R-factors can be achieved by introducing water molecules, but they do not necessarily improve the structure.

323 V. Comparison with Structural Information Obtained with other Techniques The refinement statistics give overall values of bond angles, bond lengths, etc. Luzzatti plots (Luzzatti, 1952) give average positional errors in the atomic coordinates. The positional error can vary from 0.2 Å to 0.5 Å, for a refined 2 Å structure to a refined 3 Å structure. Because these are average values, the errors in the specific resi­ dues or chain segments can be much larger; as with all statistical values, some can lie outside the limit. Protein crystal structures are time-averaged structures; examination of the atomic tempera­ ture (B) factors (part of the data deposited in the Protein Data Bank) gives an indication of how well the positions of the chain fragments or resi­ dues are known. The B values are generally low in the middle of the molecule and increase for outside loops or sidechains on the surface of the molecule. High tem­ perature factors can be caused by movement of the residue or chain segment, or by static dis­ order. High B-factor also could mean that part of the molecule is incorrect. Contacts of protein molecules within the crystal (lattice contacts) can influence both the structure and the B-factor at or near the contact point. To compare data obtained by X-ray diffraction with data obtained by other spectroscopic tech­ niques, several facts have to be remembered. Pro­ teins in the crystals are at very “high” concentra­ tions and (sometimes) in the presence of high salt or other precipitants, whereas spectroscopy is usually carried out in solutions which are more diluted. The pH and the composition of the buffer used to obtain the crystal and to carry out the spectroscopic measurement are also likely to be different. Acknowledgements I thank Dr. Fred J. Stevens for helpful dis­ cussions, Dr. Rosemarie Raffen and George Johnson for reading the initial manuscript, Dr. Phani Pokkuluri for making the figure, and David E. Nadziejka for technical editing assistance. Sup­ ported by the U.S. Department of Energy, Office of Health and Environmental Research, under

324 Contract No. W-31–109–ENG-38; also supported by Public Health Service Grant GM36598.

References Allen JP, Feher G, Yeates TO, Rees DC, Deisenhofer J, Michel H and Huber R (1986) Structural homology of reaction centers from Rhodopseudomonas sphaeroides and Rhodopseudomonas viridis as determined by X-ray diffrac­ tion. Proc Natl Acad Sci USA 83: 8589–8593 Blundell TL and Johnson LN (1976) Protein Crystallography. Academic Press, London Bränden CI and Jones A (1990) Between objectivity and sub­ jectivity. Nature 343: 687–689 Brünger AT (1990) Extension of molecular replacement: a new search strategy based on Patterson correlation refine­ ment. Acta Cryst A46: 46–57 Brünger AT (1993) Assessment of phase accuracy by cross validation: the free R value. Methods and applications. Acta Crystallogr D49: 24–36 Brünger, AT and Nilges M (1993) Computational challenges for macromolecular structure determination by X-ray crys­ tallography and solution NMR-spectroscopy. Quart Rev Biophys 26: 49–125 Brünger AT, Kuriyan J. and Karplus M (1987) Crystallo­ graphic R factor refinement by molecular dynamics. Sci­ ence 235: 458–460 Chang CH, Tiede D, Tang J, Smith U, Norris J and Schiffer M (1986) Structure of Rhodopseudomonas sphaeroides R­ 26 reaction center. FEBS Lett 205: 82–86 Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1984) X-ray structure analysis of a membrane protein complex. J Mol Biol 180: 385–398 Dickerson RE, Kendrew JC and Strandberg BE (1961) The crystal structure of myoglobin: phase determination to a resolution of 2 Å by the method of isomorphous replace­ ment. Acta Cryst 14: 1188–1195 Eikenberry EF, Tate MW, Bilderback DH and Gruner SM (1992) X-ray detectors: comparison of film, storage phos­ phors and CCD detectors. In: Photoelectronic Image De­ vices 1991, Bristol: Institute of Physics 2: 273–280 Eisenberg D and Hill CP (1989) Protein crystallography: more surprises ahead. Trends Biochem Sci 14: 260–264 Finzel BC (1993) Software for macromolecular crystallogra­ phy: a user’s overview. Current Opinion in Structural Bi­ ology 3: 741–747 Fitzgerald PMD (1988) MERLOT, an integrated package of

Marianne Schiffer computer programs for determination of crystal structures by molecular replacement. J Appl Crystallogr 21: 273–278 Hahn T (1983) International Tables for Crystallography, Vol­ ume A, Space-group symmetry. D. Reidel Publishing, Dor­ drecht Hauptman HA (1989) The phase problem of X-ray crystal­ lography. Physics Today November 24–29 Hendrickson WA (1985) Sterochemically restrained refine­ ment of macromolecular structures. Methods Enzymol 115: 252–270 Jones TA (1978) A graphics model building and refinement system for macromolecules. J Appl Cryst 11: 268–272 Jones TA, Zou J-Y, Cowan SW and Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Cryst 47: 110–119 Karle J (1989) Direct methods in protein crystallography. Acta Cryst A45: 765–781 Lüthy R, Bowie JU and Eisenberg D (1992) Assessment of protein models with three-dimensional profiles. Nature 356: 83–85 Luzzatti V (1952) Traitement statistique des erreurs dans la détérmination des structures cristallines. Acta Cryst 5: 802– 810 Matthews BW (1968) Solvent content of protein crystals. J Mol Biol 33: 491–497 McPherson A (1982) Preparation and Analysis of Protein Crystals. John Wiley and Sons, New York McRee DE (1993) Practical Protein Crystallography. Aca­ demic Press, New York Rossmann MG (1972) The Molecular Replacement Method: A Collection of Papers on the Use of Non-Crystallographic Symmetry. Gordon and Breach Science Publishers, New York Rossmann MG (1990) The molecular replacement method. Acta Cryst A46: 73–82 Tronrud DE, Ten Eyck LF and Matthews BW (1987) An efficient general-purpose least-squares refinement program for macromolecular structures. Acta Cryst A43: 489–501 Wyckoff HW, Hirs CHW and Timasheff SN (1985a) Diffrac­ tion Methods for Biological Macromolecules, Part A, Me­ thods in Enzymology. Volume 114, Academic Press, New York Wyckoff HW, Hirs CHW and Timasheff SN (1985b) Diffrac­ tion Methods for Biological Macromolecules, Part B, Me­ thods in Enzymology. Volume 115, Academic Press, New York Xu Z-B, Chang C-H and Schiffer M (1990) Testing the proce­ dure of simulated annealing by refining homologous immu­ noglobulin light-chain dimers. Protein Engineering 3: 583–589

Chapter 20

Electron Microscopy

Egbert J. Boekema* and Matthias Rögner1 Bioson Research Institute, Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands 1 Institute of Botany, University of Münster, Schlossgarten 3, D-48149 Münster, Germany Summary I. Principles A. Introduction B. The Electron Microscope C. Specimen Preparation D. Averaging Methods E. Possibilities of EM in Terms of Resolution and in Relation to Object Size and Specimen Preparation II. Periodic Averaging A. Two-Dimensional Crystallization B. Periodic Averaging by Fourier Methods C. Averaging of Photosystem I Crystals D. High-Resolution EM E. Light-Harvesting Complex II III. Single Particle Averaging A. Method 1. Alignment by Correction Methods 2. Multivariate Statistical Analysis 3. Classification Step B. Analysis of Photosystem I Trimers from Cyanobacteria C. Analysis of Photosystem II Dinners from Cyanobacteria IV. Concluding Remarks Acknowledgements References

325 326 326 326 327 328 329 330 330 330 330 331 331 332 332 332 332 333 333 333 335 335 335

Summary Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging of individual projections. Averaging procedures can be divided into crystallographic and non-crystallographic methods and both will be described. Crystallographic averaging, based on two-dimensional crystals of rather small proteins, has the potential of solving a structure to atomic resolution just as the more common techniques of Xray diffraction and NMR. Single particle analysis is an alternative method for large proteins, viruses and all non-crystallizable proteins. It is a fast method to reveal the low-resolution structure with details in the range of maximally 10–15 Å. Results of EM on Light-harvesting complex II (LHC-II) and Photosystem I will be presented as examples for the crystallographic averaging. Trimeric Photosystem I complexes and dimeric Photosystem II complexes will be discussed as examples for the potential of single-particle averaging. *Correspondence: Fax: 31-50-3634800; E-mail: [email protected]

325 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 325–336. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

326

Egbert J. Boekema and Matthias Rögner

I. Principles

A.

Introduction

Direct information about the three-dimensional (3D) structure of a protein is essential for under­ standing its functional organization. At present electron microscopy (EM) is a widely applied technique for studying the structure of proteins and membranes; however, it is still less common than X-ray diffraction, where solving the 3D structure of proteins becomes almost routine, once suitable crystals have been obtained. On the other hand, X-ray diffraction has two disadvan­ tages in comparison to EM. First, X-rays cannot be focussed and only diffraction patterns are ob­ tained, whereas EM results in direct information in the form of images. Second, the interaction of X-rays with material is a factor of about 10,000 weaker than that of electrons. This makes EM a useful technique as images of single protein molecules or one-layer thick crystals can be ob­ tained, whereas X-ray diffraction needs much thicker specimens. In this chapter on EM, the first sections will briefly introduce: the electron microscope with some instrumental aspects (I.B); specimen pre­ paration (I.C); image analysis averaging tech­ niques (I.D) and finally the possible resolution of EM with respect to object size and specimen preparation (I.E). Sections II.A and III.A will focus on image analysis of periodic and non-periodic objects including some examples. For more details concerning theory of the elec­ tron microscope, techniques for recording the sig­ nal and image analysis, we refer to the book by Hawkes and Valdrè (1990), which was written for the field of protein structure determination.

B.

The Electron Microscope

The resolution of a light microscope, which is about 2000 Å, is mainly limited by the wavelength of the light. Improving the resolution of a micro­ scope is only possible by exploiting waves with a much shorter wavelength. According to the well known formula of de Broglie, acceler­ ated particles such as electrons also have a wave character. At an acceleration voltage of 100,000

V the wavelength of the electron beam is 0.037 Å. This is certainly sufficient to enable microscopy at atomic resolution. Shortly after de Broglie had described the wave character of particles, it was discovered, and put into practice, that electrons can be focussed by electric and magnetic fields with axial symmetry. Based on these principles, Ernst Ruska and Max Knoll constructed the first simple electron microscope in 1931, a tube under vacuum with an electron source and several len­ ses. Improvements in the following years en­ hanced the resolution to 100 Å, already much better than the resolution of a light microscope. Since the early days the electron microscope has been gradually but substantially improved to an instrument which can now routinely achieve a resolution of about 2 Å. This resolution is limited by lens geometries and reflects compromises be­ tween several optical parameters, such as minim­ izing the spherical aberration, which is a kind of lens error. Overall, much further improvement in resolution cannot be expected, but 2 Å resolution is sufficient to solve a protein structure at the atomic level. To minimize the lens aberrations, the lenses in the electron microscope have a small opening: the holes in lens apertures are less than 0.1 mm in diameter. This results in a large “depth of field” and “depth of focus” at the object plane and image plane, respectively. As a consequence, EM gives two-dimensional (2D) projections in which the upper- and lower side of a thin object (up to a few 1000 Å) are seen superimposed with the same “sharpness”. As a result, a simple fo­ cussing on selected levels in an object, as is pos­ sible with a light microscope, cannot be done with the electron microscope. To get information about the 3D shape of a protein, the specimen must be tilted in the microscope and the various projections must be compared or combined into a 3D reconstruction. Imaging by EM of, for example, a thin metal foil or a gold cluster will easily provide projec­ tions with atomic resolution, but obtaining struc­ tures of proteins at high resolution is much harder work. Why? The contrast in the electron micro­ scope is caused by scattering. Electrons are de­ flected at atomic nuclei through large angles and by other electrons through small angles. Since the

Electron microscopy scattering by nuclei is proportional to the atomic number, biological material, containing only the lighter elements, will give images with very low contrast. Besides, radiation damage by an elec­ tron beam easily destroys biological samples. Ra­ diation damage cannot be avoided, but only mini­ mized by cooling the specimen to liquid nitrogen or liquid helium temperature and by minimizing the electron dose. As a result, electron microg­ raphs are noisy and objects are hardly visible. Therefore, image analysis techniques have been developed to improve the signal recorded in the noisy EM pictures.

C. Specimen Preparation Since modern electron microscopes have enough resolving power for structural studies of macrom­ olecules, other factors than instrumental ones are of importance. The specimen preparation method is one of these factors and it strongly determines the ultimate resolution that can be achieved. In the negative staining technique the contrast is enhanced by embedding proteins or protein crys­ tals in a heavy metal salt solution. Upon drying, the metal salts fill spacings and cavities around the molecules, but do not penetrate the protein interior. As a result, negatively stained specimens show protein envelopes with good contrast, but with a resolution that usually does not exceed 15 Å, due to the graininess of the contrasting agent. Because of its simplicity, negative staining has been widely applied. Single particles as well as crystals of photosynthetic membrane proteins, such as Photosystem I and II, have been success­ fully prepared by negative staining (Boekema, 1991; Boekema et al., 1994). As an alternative for negative staining, Unwin and Henderson (1975) pioneered the embedding of proteins in other media, such as glucose. It was demonstrated for crystals that images with a resolution of better than 10 Å could be achieved. This opened the way to high-resolution EM for biological macromolecules. A second important discovery in this context was the reduction of radiation damage, leading to a better signal. Specimen holders were developed that could be cooled down to liquid nitrogen-or liquid helium temperature. It was found for organic- and pro­

327 tein crystals that at the temperature of liquid ni­ trogen radiation damage is roughly reduced by a factor of 3–5 and by a factor of 10–20 at liquid helium temperature (Zemlin, 1992). Presently, the instrumental difficulties of practical cryo-EM have been mostly solved and the performance of the newest microscopes at low temperature is almost as good as at room temperature. Cryo-EM was further stimulated by the discov­ ery of vitrification of protein solutions (Adrian et al., 1984). By rapidly cooling a protein solution, the formation of ice crystals can be avoided and proteins embedded in a thin layer of amorphous ice are obtained. Contrast is caused by the differ­ ence in density between amorphous ice (0.93 and protein and is rather low in comparison to negative staining. However, there are several advantages of cryo-EM of vitr­ ified specimens: Specimen flattening and other drying artifacts are circumvented. Moreover, cryo-images better reflect the true density of a protein, because the contrast directly originates from scattering by the protein rather than from the surrounding stain (Fig. 1). Also, negative stain interaction with the protein is often quite complex. In thinner stain layers, the upper part of the protein could easily be less well embedded in the stain layer, as pointed out in Fig. 1. This means that the contribution of the upper- and lower half of a protein in the final recorded image do not have the same weighting. Other techniques are less important for high resolution structural work. Freeze-fracture tech­ niques have been widely applied in research on photosynthesis. Cell or membranes are rapidly frozen, cleaved and replicated. The replicas give useful information about the overall size and dis­ tribution of the complexes embedded in these membranes (Staehelin, 1988). But the resolution is rather limited. Only particle diameters and the overall shape of membrane protein can be ob­ tained. The main value of these techniques lies in the imaging of the in vivo situation of the membranes. It can reveal crystalline packing of photosystems and sometimes the multimeric state of large protein complexes. Evidence for the exis­ tence of a dimeric organization of PS II in vivo was obtained by freeze-fracturing (Mörschel and Schatz, 1987).

328

D. Averaging Methods In EM image analysis, improving the signal of an object recorded in a noisy electron micrograph is performed by averaging. By adding hundreds or, if possible, thousands of projections the signal improves substantially and trustworthy electron density maps are obtained. There are two general methods for averaging of 2D projections, depending on the object. One method is based on

Egbert J. Boekema and Matthias Rögner filtering images of periodic objects, which are usu­ ally 2D crystals. The other one deals with singleparticle projections. Periodic averaging takes advantage of the fact that in crystals, protein molecules are arranged in a regular packing. This means that neighborto-neighbor distances have a fixed value. In other words: the precise position of the molecules can be easily determined, even if the crystal is re­ corded with a low electron dose to prevent radi­ ation damage and the molecules can be barely seen. As a result, higher resolution can be ob­ tained. If 2D crystals with a diameter of at least can be grown, EM can be performed several under cryo-EM conditions at high resolution. For some small membrane proteins with a mass of 20–40 kDa, averaging over very large areas re­ sulted in projection structures with a resolution better than 5 Å. For bacteriorhodopsin, the three-dimensional structure could be determined entirely from EM data by fitting the amino acid chain into the electron density map (Henderson et al., 1990). Another example is in the field of photosynthesis, where a second high resolution structure determination, that of the light-harvesting complex II (LHC-II) from pea was re­ cently completed (Kühlbrandt et al., 1994). The crystallographic method, which is based on Four­ ier methods, is further described in section II, where results on LHC-II will be discussed in more detail. Projections of single particles can be averaged after they have been brought into equivalent posi­ tions by shifting them rotationally and transla­ tionally. This a-periodic averaging technique or single particle analysis is able to reveal the pre­ dominant projections of protein molecules (Frank et al., 1988). The fact that crystallization of the protein is not required is an advantage of this method. A disadvantage is that the maximal possible resolution by single-particle analysis is re­ stricted to about 10–25 Å. This limit is set by the signal-to-noise ratio, which is related to the size of the object and has a relative low value for small objects. The resolution is also limited by the fact that the particles are not fixed in a definite position, as in a crystal. Small tilts from a com­ mon predominant position cause slight differences between similar-looking projections, resulting in an ensemble of projections that are all

Electron microscopy

slightly different. Averaging all of them would give a sum in which the finest details would be blurred out.

E. Possibilities of EM in Terms of Resolution and in Relation to Object Size and Specimen Preparation With the present state of the art in EM, as de­ scribed in the previous sections, we can give an overview of the possibilities of EM in the field of proteins. Fig. 2 describes the potential of 5 types of EM approaches in the field of proteins. The thickness of the lines in Fig. 2 indicates the suit­ ability and the attainable resolution in relation to the molecular mass of a protein. Approach 1. Single-particle averaging of nega­ tively stained specimens is able to resolve the structure up to 15 Å in favorable cases. This resol­ ution is sufficient for the localization of subunit positions in projections. Examples will be given for Photosystem I and II. Approach 2. If the negative staining method is replaced by cryo-EM of vitrified solutions, single particle averaging can be applied to objects with a mass of at least a few hundred kDa. From smaller proteins, especially those with a mass of 100 kDa or lower, the projections from single molecules as recorded by EM are too noisy for accurate averaging. Approach 2 works better than ap­

329

proach 1 for larger objects, for reasons mentioned before, such as removing the flattening upon dry­ ing. Another reason is the lower contrast of cryoEM, which becomes relatively disadvantageous for small molecules, as we will briefly explain. The contrast in biological material is largely caused by the scattering of the electron cloud of the C, N and O elements. This contrast is called phase contrast. The contrast originating from in­ teraction with atomic nuclei, the scattering con­ trast, is relatively unimportant. Enhancement of phase contrast is possible by a stronger defo­ cussing of the objective lens. But this coincides with a degradation and loss of fine details in the image. The smaller the object, the larger the de­ focussing needed to see the object at all. Approach 3. Periodic averaging of stained ob­ jects. The advantage of a periodic object is its regular packing. This enables an accurate deter­ mination of the positions of the molecules and thus an accurate averaging of projections. For crystals of about in size, a resolution of about 15–25 Å is usual. Small proteins (10– 50 kDa) are advantageous, because they allow averaging over a larger number of molecules per area. Approach 4. Cryo-EM of unstained, periodic objects. As for approach 2, the contrast comes largely from phase contrast. But crystals can be considered as a “big object” and can be recorded with small defocus values. Although the signal of one individual molecule is almost buried in the noise, it will still be interpretable, because it is averaged periodically. Small crystals of organic in size molecules and small proteins of a few can be analyzed to high resolution. For such small macromolecules, EM provides the same quality as X-ray diffraction, although the image analysis is not as straightforward. Approach 5. Based on perfect, large 2D crys­ tals atomic resolution is possible for proteins up to at least 50 kDa. For larger proteins, the resol­ ution will gradually decrease. For proteins with a mass between 300 and 600 kDa, like Photosystem I and II and ATP synthase, solving the structure at high-resolution by EM would be difficult. The preferential size of 2D crystals of such objects is Such large crystals are difficult at least to grow and have not yet been obtained.

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Egbert J. Boekema and Matthias Rögner

II. Periodic Averaging

A. Two-Dimensional Crystallization As indicated in Fig. 2, small proteins should be crystallized into 2D crystals, to obtain the best structural information. The best 2D crystals pro­ duced in vitro have been grown from detergentsolubilized, purified material. Starting from highly purified protein, crystallization conditions can be controlled more easily and results are more reproducible (Kühlbrandt, 1992; Jap et al., 1992; Engel et al., 1992). Reconstitution of mem­ brane proteins into lipid bilayers by detergent removal is currently the most universal method of 2D crystallization. A suspension of a lipiddetergent mixture is usually added to a detergentsolubilized protein preparation. Crystallization of the protein into sheets or vesicle crystals is then induced by removal of detergent by means of dialysis or absorption.

B.

Periodic Averaging by Fourier Methods

Usually, images of 2D crystals are recorded on electron micrographs. Fourier analysis has been proven to be very valuable in the processing of micrographs. The Fourier transform is a fre­ quency decomposition in reciprocal space. A 2D Fourier transform calculated from an image gives a 2D diffraction pattern. If the image shows a good 2D crystal, its transform will show a pattern with many spots laying on a regular pattern, as in X-ray diffraction patterns. These spots repre­ sent the structure factor amplitudes and have an amplitude (peak height) and a phase. But the noise in the images will also show up in the dif­ fraction pattern. An illustration of the Fourier techniques will be given in the next section; it shows an effective way to get rid of the noise.

C. Averaging of Photosystem I Crystals At medium resolution no complicated strategy is necessary since correcting for image aberrations is only necessary for high-resolution EM (see II.D). We will show by a simple example the basics of periodic averaging. Monomeric Photosystem I (PS I) has been crystallized into two-dimensional arrays from the cyanobacterium Synechococcus

elongatus (Böttcher et al., 1992) by removal of detergent (Fig. 3A). In a 2D Fourier transform of the PS I crystal (Fig. 3B), we see spots at regular distances. They tell us about the lattice parameters and about resolution. In this case, the lattice of the crystal is rectangular. Based on the Fourier transform of the image, a filtering is performed. Computationally, a mask is constructed that is superimposed on the Fourier transform (Fig. 3C). The mask has holes that neatly surround the peaks, which contain the crystalline information. The mask shields the space between the peaks, which represents noise in the crystal image. With a reverse Fourier-transformation (Fig. 3D) a real image is generated again, in which much of the noise is removed. This procedure is called Fourier-peak filtering and the comparison between Figs. 3E and F illus­

Electron microscopy trates the considerable improvement in signal-tonoise ratio. Further improvement in filtering is also pos­ sible. As crystals never have a perfect lattice, a small bending in the plane results in molecules being slightly displaced from their ideal position. Corrections can be made by “cutting” the crystal into pieces containing one or several molecular projections and shifting them into their ideal posi­ tion. This is done by correlation methods, which have also been used in single particle averaging methods (Frank, 1982). An application of this method is the analysis of PS I crystals (Böttcher et al., 1992).

D. High-Resolution EM While a low-resolution structure by single particle averaging can be obtained within weeks, obtain­ ing a high-resolution structure from EM data may take years. One of the limits of a structure deter­ mination at high resolution is merely the produc­ tion of well-ordered, large crystals. Once this pre­ requisite is fulfilled, a resolution better than 10 Å is possible. However, recording the highest quality images and extensive processing are also time consuming. The signal recorded by EM suf­ fers from aberrations, which increase in severity as the required resolution becomes higher (Hend­ erson et al., 1986). Images providing structural information beyond a resolution of about 5 Å need extensive processing, which is carried out in Fourier space on the raw image amplitudes and phases. The phases are corrected for the effects of the contrast transfer function, beam tilt and phase origin (Henderson et al., 1986). A further treatment and discussion of these image distor­ tions and their analysis and correction is beyond the scope of this contribution. For the interested reader we refer again to the book of Hawkes and Valdrè (1990), as a useful source of further information, and to the paper of Henderson et al. (1990). The microscope can be configured in two dif­ ferent ways: a) image mode and b) diffraction mode, i.e. recording the image formed in the back-focal plane of the objective lens. Electron diffraction (ED) patterns of crystals contain many spots, as in X-ray diffraction patterns and also represent the structure factors without the phase

331

information that is present in calculated Fourier transforms. ED patterns usually have much stronger spots than those from calculated trans­ forms. The reason possibly lies in the specimen movement during image recording. Fourier trans­ formation and diffraction in an optical system have the property that they are “translation in­ variant”. This means that movements during the recording of an ED pattern are less dramatic than during the recording of a real image, because they do not result in blurring the ED. Therefore spots in ED are much stronger and this makes re­ cording of only ED for high resolution work tempting. But then a “phase problem”, as in Xray diffraction, is created and phases need to be generated. Isomorphous replacement, as used in X-ray diffraction, is not a useful method for phas­ ing the ED data from 2D protein crystals because the scattering contrast of heavy atoms is low for electrons and the noise level in the patterns is relatively high. To compute a protein map at high-resolution by Fourier methods, images, are recorded as well. They are necessary to extract the phase from each of the spots. The phases (from the images) and amplitudes (from ED) are finally combined and corrected for some image errors, briefly mentioned in section I.D. This is called a “Fourier synthesis”. An example of the Fourier techniques for high-resolution structure determination will be given in the next section.

E. Light-Harvesting Complex II The light-harvesting chlorophyll a/b protein com­ plex associated with photosystem II (LHC-II) harvests light energy and is capable of passing it to photosystem II. Detergent-solubilized, purified LHC-II forms large, highly ordered 2D crystals, which are ideal objects for recording high-resolution EM. Based on the averaging methods de­ veloped by Henderson et al. (1990), Kühlbrandt et al. (1994) were able to calculate a 3D structure at 3.4 Å resolution. Images were recorded with an electron microscope capable of resolving 2 Å object features at a specimen temperature of 4.2 K. Tilted and untilted images were collected for a 3D reconstruction. Since it is difficult to record images at tilt angles above 60°, there was a region in the Fourier space where amplitudes and phases could not be measured. This caused a reduction

332

Egbert J. Boekema and Matthias Rögner

III. Single-Particle Averaging

A.

Method

Isolated proteins prepared on a carbon support film exhibit a full range of rotational and transla­ tional orientations in the plane of the support film. As a consequence, the projections of the proteins have a random orientation within this plane and computer averaging of such projections can only be achieved after an alignment proce­ dure. Also, proteins often are attached in various ways to the support film (Fig. 1) and this results in a further variety in the obtained projections. To separate the various predominant projections, multivariate statistical analysis together with automated classification was introduced (Van Heel and Frank, 1981). The alignment procedure, multivariate statistical analysis and classification form the main steps in the single particle averag­ ing procedure (Frank et al, 1988). These steps will be briefly discussed. 1. Alignment by Correlation Methods

in the resolution in the direction perpendicular to the membrane plane to between 4.6 and 4.7 Å. But due to the high quality of the phases it was possible to trace the polypeptide chain in the 3D electron density map. About 80% of the amino acids could be fitted, as well as the tetrapyrrole rings of 12 chlorophylls and two carotenoids (Fig. 4). LHC-II is the second protein solved by EM to atomic resolution. No doubt, the work on LHC­ II is a major step forward both for EM and for photosynthesis. To stress the similarities with Xray crystallography, the term electron crystal­ lography has been introduced for high-resolution protein determination by EM.

The first step in comparing images of projections of biomolecules is to bring them into register in the plane: the alignment process. A reference image is taken and each image in the data set is compared with this reference; rotational and translational cross-correlation functions are com­ puted to determine the best angular and transla­ tional shifts to bring the image into a position most similar to the reference image. The align­ ment procedure is an iterative process because sums of projections align the data set better than does a noisy projection of just one molecule. In practice, several references are used and results are combined to circumvent a possibly wrong choice of the first reference. 2. Multivariate Statistical Analysis Once a data set of typically hundreds of projec­ tions has been aligned, they can be compared in some numerical way. In particular, correspon­ dence analysis, a special form of principal compo­ nent analysis, is used to extract relevant feature information from the data set (Van Heel and Harauz, 1988). Each image of × pixels can be

Electron microscopy

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represented as a point in an × dimensional “hyper”space, and the entire set of images forms a cloud in this space. Correspondence analysis de­ termines a new, rotated-coordinate system in which the first axis represents the direction of the greatest inter-image variance, the second axis represents the direction of the largest remaining inter-image variance, and so on. The cloud of images can now be described with respect to this new coordinate system. By describing each image with respect to only the first few components, the images can be considered as points in a much smaller (than n × n) dimensional space. In this way a very large reduction in the amount of data to be analyzed is achieved. 3. Classification Step After the data compression by correspondence analysis, the grouping together of those images that are most similar is achieved by automatic classification schemes. Output of the classification are “classes” of groups of projections that are most similar. In the classification process, projec­ tions are shifted between the classes to optimize the variance between the classes and to minimize the variance of the members belonging to the classes. The number of classes to be chosen is somewhat arbitrary, but usually a number of 6– 12 are chosen. The differences between classes may represent real structural features (as will be illustrated by two examples) or are merely noiserelated when there is not more than one type of projection present in the data set.

B. Analysis of Photosystem I Trimers from Cyanobacteria A first practical example of single particle analysis concerns Photosystem I. In cyanobacteria, such as the thermophilic Synechococcus elongatus and the mesophilic Synechocystis PCC 6803, PS I is arranged as a trimeric complex with a diameter of about 200 Å in the membrane (Boekema et al., 1989; Kruip et al., 1993). From electron mic­ rographs of PS I from Synechocystis PCC 6803 (Kruip et al., 1993), top view projections (Fig. 5) were extracted, aligned, treated by correspon­ dence analysis and classified. The analysis re­ sulted in the separation of the top view projec­

tions into two types, which are mirror-related (Fig. 6). These two types are generated because particles are asymmetric and can be attached to the carbon support film in two ways (upside-up and upside-down or “flip” and “flop”). It should be noted that it is not easy to judge by eye whether the PS I trimer projections (Fig. 5) be­ long to the flip or flop-type, but after a 3–fold averaging most of them (but not all) can be classi­ fied by eye.

C. Analysis of Photosystem II Dimers from Cyanobacteria Photosystem II is another membrane protein with a size large enough to enable single particle analy­ sis. Dimeric particles were purified from the Syne­ chococcus elongatus (Rögner et al., 1987; Dekker

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et al., 1988). We visualized these particles in the presence of the detergent dodecyl maltoside by negative staining with uranyl acetate (Boekema et al., 1994). Averaged top views of dimeric PS II show that the dimers are built up from two monomers which are arranged in an anti-parallel way (Fig. 7A). In contrast to the example of PS I top view projections, there was only one predominant top view present in the data set. The dimer has dimensions of 120 × 155 Å in the membrane (corrected for detergent). The side views, however, show more variations. Original images clearly show protrusions in the side-view position (see Fig. 9 in Boekema et al., 1994). Often two protrusions can be seen on one dimer, but sometimes only one is visible. The protrusions

Egbert J. Boekema and Matthias Rögner

are thought to represent the 33 kDa oxygenevolving subunit resulting in a maximal height of 90 Å for the particles. Image analysis of side views gives another example of the usefulness of single particle analysis. By correspondence analy­ sis and classification, a discrimination between different views was possible, which we interpret as overlap- and non-overlap views. In the non­ overlap views two protrusions can be seen (Fig. 7D, E), whereas the overlap view shows one pro­ trusion (Fig. 7F). Note that the non-overlap view shows a longer projection than the overlap views, indicating a different position of the respective dimers on the carbon support.

Electron microscopy IV. Concluding Remarks In conclusion, EM techniques are available to study isolated (membrane) protein complexes from very small to very large size. In combination with techniques used in cell biology for studying whole cells or cell fragments, EM has the possibil­ ity and the potential to supply the field of photo­ synthesis with a detailed picture of the photosyn­ thetic membrane and its interacting proteins. For structure determination, crystals are more useful than single particles. But not in all cases will it be possible to grow good crystals in a short term for such complicated structures as PS I, PS II and synthase complex including their the interacting donor-, acceptor- and regulatory mol­ ecules and proteins. In the meantime a combi­ nation of low-resolution EM reconstructions of these photosynthetic membrane complexes with atomic structures of their individual components, determined by EM, NMR or X-ray diffraction, will be useful for understanding the structure and function of these proteins. Acknowledgements We are grateful to Dr. W. Keegstra for his help with computer image analysis, Dr. G. Perkins for discussion and Mr. K. Gilissen for photography. Work has been supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO), by a grant from the European Union BIO2CT-930078 (EJB), the Deutsche Forschungsgemeinschaft (MR) and a grant from NEDO/RITE, Japan (MR). References Adrian M, Dubochet J, Lepault J, McDowall AW (1984) Cryoelectron microscopy of viruses. Nature 308: 32–36 Boekema EJ (1991) Negative staining of integral membrane proteins. Micron and Microsc Acta 22: 361–369 Boekema EJ, Dekker JP, Rögner M, Witt I, Witt HT and van Heel, MG (1989) Refined analysis of the trimeric struc­ ture of the isolated Photosystem I complex from the therm­ ophilic cyanobacterium Synechococcus sp. Biochim Bi­ ophys Acta 974: 81–87 Boekema EJ, Boonstra AF, Dekker JP and Rögner (1994) Electron microscopic structural analysis of Photosystem I, Photosystem II, and the cytochrome b6/f complex from

335 green plants and cyanobacteria. J Bioenerg Biomembr 26: 17–29 Böttcher B, Gräber P and Boekema EJ (1992) The structure of Photosystem I from the thermophilic cyanobacterium Synechococcus sp. determined by electron microscopy of two-dimensional crystals. Biochim Biophys Acta 1100: 125–136 Dekker JP, Boekema EJ, Witt HT Rögner M (1988) Refined purification and further characterization of oxygen-evolving and Tris-treated Photosystem II particles from the ther­ mophilic cyanobacterium Synechococcus sp. Biochim Bi­ ophys Acta 936: 307–318 Engel A, Hoenger A, Hefti A, Henn C, Ford RC, Kistler J and Zulauf M (1992) Assembly of 2–D membrane protein crystals: dynamics, crystal order, and fidelity of structure analysis by electron microscopy. J Struct Biol 109: 219–234 Frank J (1982) New methods for averaging non-periodic ob­ jects and distorted crystals in biologic electric microscopy. Optik 63: 67–89 Frank J, Radermacher M, Wagenknecht T and Verschoor A (1988) Studying ribosome structure by electron microscopy and computer-image processing. Methods in Enzymology 164: 3–35 Hawkes PW and Valdrè U (1990) Biophysical Electron Micro­ scopy. Basic concepts and modern techniques. Academic Press, London Henderson R, Baldwin JM, Downing KH, Lepault J and Zemlin F (1986) Structure of purple membrane from Halobacterium halobium: recording, measurement and evalu­ ation of electron micrographs at 3.5 Å resolution. Ultram­ icroscopy 19: 147–178 Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E and Downing KH (1990) Model for the structure of bacteriorhodopsin based on high-resolution electron cryo­ microscopy. J Mol Biol 213: 899–920. Jap BK, Zulauf M, Scheybani T, Hefti A, Baumeister W, Aebi U and Engel A (1992) 2D crystallization: from art to science. Ultramicroscopy 46: 45–84 Kruip J, Boekema EJ, Bald D, Boonstra AF and Rögner M (1993) Isolation and structural characterization of monom­ eric and trimeric Photosystem I complexes and from the cyanobacterium Synechocystis PCC 6803. J Biol Chem 268: 23353–23360 Kühlbrandt W (1992) Two-dimensional crystallization of membrane proteins. Quaterly Rev of Biophys 25: 1–49 Kühlbrandt W, Wang DN and Fujiyoshi Y (1994) Atomic model of plant light-harvesting complex by electron crystal­ lography. Nature 367: 614–621 Mörschel E and Schatz GH (1987) Correlation of photosystem-II complexes with exoplasmic freeze-fracture particles of thylakoids of the cyanobacterium Synechococcus sp. Planta 172: 145–154 Rögner M, Dekker JP, Boekema EJ and Witt HT (1987) Size, shape and mass of the oxygen-evolving photosystem II complex from the thermophilic cyanobacterium Synechoc­ occus sp. FEBS Lett 219: 207–211 Staehelin LA (1988) Chloroplast structure and supramolecular organization of photosynthetic membranes. In: Staehelin LA and Arntzen CJ (eds.) Photosynthesis III, pp. 1–84, Springer-Verlag, Berlin

336 Unwin PNT and Henderson R (1975) Molecular structure determination by electron microscopy of unstained crystalline specimens. J Mol Biol 94: 425–440 Van Heel M and Frank J (1981) Use of multivariate statistics in analysing the images of biological macromolecules. Ultramicroscopy 6: 187–194

Egbert J. Boekema and Matthias Rögner Van Heel H and Harauz G (1988) Biological macromolecules explored by pattern recognition. Scanning Microscopy, Supplement 2: 295–301 Zemlin F (1992) Desired features of a cryoelectron microscope for the electron crystallography of biological ma­ terial. Ultramicroscopy 46: 25–32

Chapter 21 X-Ray Absorption Spectroscopy: Determination of Transition Metal Site Structures in Photosynthesis

Vittal K. Yachandra* and Melvin P. Klein

Structural Biology Division, Lawrence Berkeley National Laboratory, University of California,

Berkeley, CA 94720, USA

Summary I. Introduction II. X-ray Absorption Spectroscopy (XAS) A. X-ray Absorption Near Edge Structure (XANES) B. Extended X-ray Absorption Fine Structure (EXAFS) 1. Basic Theory of EXAFS a. Energy of the Photoelectron b. Definition of EXAFS c. The EXAFS Equation C. Experimental Methodology D. Advantages and Limitations of XAS 1. Advantages 2. Limitations III. Applications of XANES and EXAFS in Photosynthesis A. Fe–S Proteins 1. Soluble Plant Ferredoxin and in Photosystem I 2. Fe–S Acceptors

3. Rieske Fe–S Clusters B. Manganese Oxygen Evolving Complex in Photosystem II IV. Future Directions Acknowledgements References

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Summary This chapter gives a brief description of the theory and experimental methodology involved in X-ray absorption Spectroscopy, both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). The advantages and limitations of the methods are discussed. Several examples of applications of X-ray absorption Spectroscopy in the field of photosynthesis are presented. Abbreviations: EPR – Electron Paramagnetic Resonance; EXAFS – Extended X-ray Absorption Fine Structure; cyt – cytochrome; OEC – Oxygen Evolving Complex; PS I – Photosystem I; PS II – Photosystem II; XANES – X-ray Absorption Near Edge Structure; XAS – X-ray Absorption Spectro­ scopy *Correspondence: Fax: 1-510-4866059; E-mail: [email protected]

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J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 337–354. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

338 I. Introduction X-ray absorption spectroscopy (XAS) is the mea­ surement of transitions from core electronic states of the element to the excited electronic states or continuum states, which is known as X-ray absorption near edge structure (XANES), and the study of the fine structure in the absorption cross section at energies greater than the thresh­ old for electron release, which is known as ex­ tended X-ray absorption fine structure (EXAFS). These two methods give complementary struc­ tural information, the edge-spectra reporting ox­ idation state and symmetry, and the EXAFS re­ porting numbers, types and distances to ligands and neighboring atoms from the absorbing atom. The major application of XAS in biology and in photosynthesis has been in the study of the structure of the metal sites in metallo-proteins and metallo-enzymes (reviewed in Yachandra, 1995; Cramer, 1988; Scott, 1984; Powers, 1982). This was primarily due to the availability of Xray beam lines at synchrotron sources optimized in the X-ray energy region of metal K-edges (from Ca to Mo) and also the ease of making measurements at such X-ray energies, without interference from the protein matrix, water or air. Secondly, advances being made in the field of bioinorganic chemistry were raising important questions of correlation between structure and function of the metal sites in metallo-proteins which were amenable to XAS studies. II. X-Ray Absorption Spectroscopy (XAS)

A. X-Ray Absorption Near Edge Structure (XANES) X-ray absorption spectra of any material, whether it is atomic or molecular in nature, is charac­ terized by sharp increases in absorption at specific X-ray photon energies, which are characteristic of the absorbing element. These sudden increases in absorption are called absorption edges, and correspond to the energy required to eject a core electron into the continuum thus producing a photoelectron. The absorption discontinuity is known as the K-edge, when the photoelectron originates from a 1s core level, and an L-edge

V.K. Yachandra and M.P. Klein when the ionization is from a 2s or 2p level. Fig. 1 shows a typical energy level diagram. The physics of the processes involved as it per­ tains to the K-edge XANES of transition metals has not been satisfactorily explained, despite ad­ vances in theory (Stöhr, 1992; Durham, 1988; Bianconi, 1988). There are two main approaches to XANES analysis. The first method developed by Shulman et al. (1976) in their work with the highly ionic metal fluorides involved transitions to atomic states localized on the metal, and the spectra were rationalized on the basis of ligand field perturbations of the localized atomic or­ bitals. For complexes that contain significant metal-ligand interactions, such as is commonly found in biological materials, this simple approach is not valid; and quantitative treatment probably requires a knowledge of the low lying unoccupied molecular orbitals of the complex. The effect of core holes on the outer lying states is also impor­ tant in the consideration of the L-edges. The se­ cond method has used multiple scattering of the photoelectron from the neighboring atoms to ex­ plain the structure seen on the edges (Kutzler et al., 1980; Pendry 1983; Rehr et al., 1992). The most fruitful use of K-edge spectra in bi­ ology has been in determining the oxidation state of the metal in the active site of metallo-proteins. The oxidation state and the coordination sphere of the metal largely determines the position and shape of the X-ray absorption K-edge. In com­ plexes with similar coordination, as the oxidation state of the metal increases the K-edge shifts to higher energy, because as the effective positive charge increases, it becomes correspondingly more difficult to remove an electron from the metal. However, it is not only the oxidation state of the metal which controls the effective positive charge of the metal, but also the nature and number of the ligands of the metal (Kirby et al., 1981, Conradson et al., 1985).

B. Extended X-Ray Absorption Fine Structure (EXAFS) At higher energies above the edge, the absorption decreases in accordance with the theory of the photoelectric effect, where the excess energy is transferred to the photoelectron as kinetic en­

X-ray absorption spectroscopy

ergy. However, there is a crucial difference in this falloff of absorption between atomic or isolated atoms, and molecular or condensed systems. For the latter, superimposed on the gradual decrease in absorption is a periodic modulation in absorp­ tion, starting immediately past an absorption edge and extending to about 1 keV above the edge. These oscillations in the X-ray absorption spectra are known as Extended X-ray Absorption Fine Structure or EXAFS. A typical X-ray ab­ sorption spectrum at the Mn K-edge is shown in Fig. 2. The EXAFS region starts about 50 eV above the edge, and the region before that is the XANES region. EXAFS oscillations occur only in molecular or condensed systems. For all systems there is no requirement for long range order as exists in crys­ talline materials. EXAFS contains information about the local environment around the absorb­ ing atom (reviewed in Eisenberger and Kincaid, 1978). In fact, the EXAFS oscillations result from the interference between the outgoing photoelec­ tron wave and components of this wave backscatt­ ered from neighboring atoms in the molecule. The importance of the EXAFS technique de­ pends directly on the fact that the EXAFS modu­ lations contain information about the distance be­ tween the absorbing and backscattering atoms within a distance of about 5–6 Å, as well as the identity and number of the backscattering atoms.

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Essentially, EXAFS analysis is used to determine the radial distribution of atoms around a parti­ cular absorbing atom, thus providing a probe for the local structure in the vicinity of the absorbing atom. 1.

Basic Theory of EXAFS

a.

Energy of the Photoelectron

The EXAFS modulations shown in Fig. 2 are a direct consequence of the wave-nature of the photoelectron whose wavelength is given by the de Broglie relation

where h is Planck’s constant, the electron mass, and v is the velocity of the photoelectron, which is the velocity imparted to the photoelec­ tron by the energy of the absorbed X-ray photon which is in excess of the binding or threshold energy for the electron. The kinetic energy of the photoelectron is given by the following relation:

where E is the X-ray photon energy, and is the

ionization or threshold energy for the electron.

The EXAFS modulations are better expressed

V.K. Yachandra and M.P. Klein

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as a function of the photoelectron wave-vector k, which is related to the de Broglie wavelength described above as follows:

which now can be expressed by the substitution of Eqs. (1) and (2) as follows:

or

where E and are expressed in electron volts (eV) and k has the units of inverse angstroms

b.

Definition of EXAFS

The general definition for the EXAFS phenome­ which is the oscillatory portion of the non absorption coefficient, is the difference between and the the observed absorption coefficient nor­ free atom absorption coefficient malized by the free atom contribution:

The absorption coefficient is proportional to the square of the electric dipole transition mo­ which is given quantum mechanically ment by a matrix element between the initial and final states of the photoelectron: In the case of K-edge absorption the initial state wave function is that of the 1s state in the

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atomic core and the final state wave function is that of the photoelectron. In an isolated atom the final state wave function is the wave func­ tion of the outgoing photoelectron In a molecular or condensed system the final state wave function of the photoelectron consists of both the outgoingwave and the backscatt­ ered wave from the neighboring j atoms and the total final wave function is given by the sum of the outgoing and the scattered wave func­ tions from each of the j backscattering atoms. The transition dipole moment can now be ex­ pressed as follows:

and it is the interference between and that lead to the EXAFS modulations, and substitution in Eq. (6) gives,

The electric-dipole moment is non-zero only in the region where the initial state is non­ zero, which is near the center of the absorbing atom. So one needs to know only how the sur­ rounding atoms perturb the outgoing wave and what effect this has at the center of the absorbing atom. One can envision this process more clearly by the help of a schematic of the outgoing and backscattered waves as shown in Fig. 3. As the energy of the photoelectron changes so does the of the photoelectron. At a wavelength, the outgoing and the backparticular energy scattered waves are in phase and constructively interfere, thus increasing the probability of Xray absorption or in other words increase the absorption coefficient. At a different energy the outgoing and backscattered wave are out-of phase and destructively interfere, decreasing the absorption coefficient. This modulation of the ab­ sorption coefficient by the backscattered wave from neighboring atoms is essentially the basic phenomenon of EXAFS.

c.

The EXAFS Equation

A quantitative description of the EXAFS modu­

lation,

depends on an evaluation of the final state wave function, Many excellent

sources exist for a rigorous derivation of the EXAFS equation (Sayers et al., 1971; reviewed can be in Stern, 1988). For our purposes expressed as follows:

where is the number of equivalent backscatter­ from the absorbing ing atoms j at a distance atom, is the backscattering amplitude which is a function of the atomic number of the includes the backscattering element j, and phase shift from the central atom absorber as well as the backscattering element j. The phase shift occurs due to the presence of atomic potentials that the photoelectron experiences as it traverses the potential of the absorber atom, the potential

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of the backscattering atom, and then back through the potential of the absorber atom. There is inherent static disorder due to a distri­ and dynamic disorder due bution of distances to thermal vibrations of the absorbing and scat­ tering atoms. Eq. (10) is modified to include this disorder term or the Debye–Waller factor where is the root-mean-square devi­ ation to give the following equation

The loss of photoelectrons to inelastic scattering processes can be accounted for by including a term, where is the mean free path of the photoelectron. The EXAFS contribution from each backscatt­ ering atom j is a damped sine wave in k-space, with an amplitude, and a phase, which are both dependent on k. EXAFS spectra contain a sig­ nificant amount of information about atomic sur­ roundings of the central absorbing atom in the system being studied and have been used in nu­ merous applications (see for example, Bertagnolli and Ertel, 1994). The contribution and impor­ tance of each of the terms in the EXAFS equation to the EXAFS spectrum, and the analysis proce­ dures used to extract the information contained in the EXAFS equation are described in detail by Teo (1986), and Sayers and Bunker (1988). From the phase of each sine wave the absorber-backscatterer distance can be determined if the phase shift is known. The phase shift is obtained either from theoretical calculations (Rehr et al., 1991; McKale et al., 1988) or empirically from com­ pounds characterized by crystallography with the specific absorber-backscatterer pair of atoms. The depends on both the absorber phase shift, and scatterer atoms. Because one knows the ab­ sorbing atom in an EXAFS experiment, an esti­ mation of the phase shift can be used in identify­ ing the scattering atom. The amplitude function contains the Debye– the number of backscatter­ Waller factor and These two parameters are highly corre­ ers at lated and make the determination of difficult at best. The backscattering amplitude function,

V.K. Yachandra and M.P. Klein depends on the atomic number of the scattering atom and, in principle, can be used to identify the scattering atoms. In practice, how­ ever, the phase shift and backscattering ampli­ tude function, both of which are dependent on the identity of the backscattering atom, can be used only to identify scattering atoms that are well separated by atomic number. The sinusoidal nature of the backscattering contributions makes Fourier analysis particularly appropriate for analyzing EXAFS spectra. A Fourier transform of the EXAFS data in k-space can be employed to separate the different scat­ tering distances into unique peaks in the conju­ gate R-space. The Fourier transform amplitude peaks at the characteristic distances is directly related to the phase shift where in Eq. (11), and it is a powerful visual tool in providing a simple physical picture of the local structure of the metal site. The Fourier transform provides a spectrum which is similar to a radial distribution function with peaks at R', which are but which correspond to shifted from R by the shells of backscatterers around the central absorbing atom. Quantitative information is obtained from however, fits are rarely per­ curve fitting to obtained from raw data, espe­ formed on cially for biological systems because of the signalto-noise limitations. It is desirable to separate the EXAFS contributions into their component shells and to filter out the high frequency noise. The technique employed to achieve this is called Four­ ier filtering and consists of Fourier transforming the data into R-space, next selecting the Fourier peak of interest by applying an appropri­ ate window function, and then back-transforming into k-space. Thus one can isolate the contri­ bution from one shell of backscatterers which can then be fit to the EXAFS equation to obtain the structural parameters for just that shell. This isolation technique works well if the backscatter­ ing atoms are well separated from each other in radial distance from the absorbing atom (~ 1 Å). In its most general form curve fitting consists of (1) proposing a chemically feasible model based on information from other studies and by inspection of the properties of the Fourier trans­ for such a form, and then simulating the model using the EXAFS equation; (2) comparing

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the calculated EXAFS with that from experiment N, and and (3) varying the parameters the Debye–Waller parameter, to minimize the difference between the calculated and observed spectrum using a non-linear least squares minim­ ization procedure. This protocol is followed for each of the Fourier filtered shells individually, hence simplifying the fitting procedure. However, that does not mean that each Fourier peak corre­ sponds to only one kind of atom at one distance. In fact such a case would be an exception in most biological systems. In most cases, curve fitting is a trial and error enterprise consisting of several iterations in the choice of the backscattering atoms and distances and in the number of sub-shells that constitute each Fourier peak. Physical reasonableness and chemical feasibility of the fit parameters always help direct the search for the right fit. There are N, four variables for each shell of atoms, and hence, including another shell doubles the number of variables and most often leads to a better fit. However, it is important to consider the statistical criteria for including another shell, by the degree of improvement in the goodnessof-fit parameter. The statistical rule-of-thumb for the number of independently variable parameters where is the width of the is given by is the window used for Fourier filtering, and For a data length of the data set, set that extends from 3.5–12 and a Fourier window width of 1.5 Å, the maximum number of parameters that can be varied in a multi-shell fit is 8. Data analysis is usually performed from about 3.5 because of problems associated with multiple scattering effects below that energy.

or fluorescent X-rays. Tunable monochromatic X-rays are usually obtained by the use of Bragg diffraction of X-rays by crystals. A detailed ac­ count of the design and use of synchrotron radi­ ation for an XAS experiment is presented by Heald (1988a, b). The sensitivity of the technique is extended several orders of magnitude by fluorescence de­ (Jak­ tection of the absorption spectrum levic et al., 1977). This enhanced sensitivity is essential to the measurement of X-ray absorption spectra of biological systems. In biological sys­ tems one is usually studying a dilute metal embed­ ded in a matrix of protein, lipids and/or nucleic acids, which all scatter X-rays. So ideally one needs to discriminate the fluorescence photons (mostly, 2p to 1s for K-edge measurements) of the element of interest from the large scattering observed from the matrix. At present the detec­ tors best suited for biological spectroscopy are the Si or Ge solid state detector which is placed at right angles to the incident beam (Cramer at al., 1988). In addition a third X-ray detector is used downstream from which is used to mea­ sure simultaneously the spectrum of a reference compound, which provides an energy reference. The essential components described above are shown in Fig. 4. The source used for most biological XAS ex­ periments is the synchrotron radiation produced at electron storage rings. The intense flux gen­ erated by this means is significantly higher than that from any other X-ray source, and this tech­ nology makes X-ray measurements of dilute sys­ tems, which is typical of most biological ma­ terials, feasible.

C. Experimental Methodology

D. Advantages and Limitations of XAS

The X-ray absorption measurements are anal­ ogous to optical absorption and fluorescence exci­ tation measurements. The spectrometer consists of a source of X-rays, a monochromator, an Xray detector which monitors the incident flux, a sample compartment, and a second detector which monitors the flux of X-rays transmitted The most common form through the sample, of collecting X-ray spectra consists of scanning through a range of wavelengths and measuring the flux of the incident and the flux of transmitted

1. Advantages 1) XAS is element specific, so one can focus on one element without interference from other ele­ ments present in the sample. In a system which has more than one metal, like the photosynthetic apparatus, it is possible to study selectively the structural environment of each metal atom. The specificity of the technique also makes it suitable for studying biological systems which contain sev­ eral polypeptides, lipid molecules, pigment mol­

344

ecules which is typical of the photosynthetic sys­ tems (bacterial reaction center, PS I and PS II). The specificity and the fact that it is always possible to obtain an X-ray spectrum of an ele­ ment, provided it is present in sufficient concen­ trations, also means that one ‘sees’ all of the metal of interest that is present in the sample. This makes it imperative that one is sure of the biochemical homogeneity of the sample and, if there is more than one site for the same metal, to resolve the structural parameters of the different sites. 2) Another important advantage of XAS is that the metal of interest is never ‘silent’ with respect to X-ray absorption spectra. The system could be ‘silent’ with respect to EPR, optical or other spectroscopic methods, but one can always probe the metal site structure by XAS. 3) XAS is not limited by the state of the sam­ ple, because it is sensitive only to the local metal site structure. The sample can be prepared as a

V.K. Yachandra and M.P. Klein

powder, a solution or, as is done most often, as a frozen solution for biological samples. The advantages of this are many-fold. It is not neces­ sary to obtain single crystals of the material to examine the local structure of the metal. Obtain­ ing single crystals can often be difficult and some samples do not crystallize. The more important aspect is that one can either trap intermediates in the enzymatic cycle or modify the site by the addition of inhibitors or substrate or generate other chemical modifica­ tions. Such samples can be made as frozen solu­ tions, avoiding the problems of trying to obtain single crystals. 2. Limitations 1) Damage to biological samples by X-rays is cause for serious concern for XAS experiments. It is absolutely essential that the samples are checked for their biochemical integrity before and

X-ray absorption spectroscopy

after exposure to X-rays by an independent me­ thod, whenever possible. The use of a liquid He flow cryostat, where the samples are at ambient pressure in a He atmosphere has greatly reduced the risk of sample damage by X-rays. 2) It is also important to realize the intrinsic limitations of EXAFS, beyond those of a purely experimental nature (Lee et al., 1981). A fre­ quent problem is the inability to distinguish be­ tween scattering atoms with little difference in Z, the atomic number (C, N, O or S, Cl, or Mn, Fe). Care must also be exercised when deciding between atoms that are well separated in Z, be­ cause frequently, it is possible to obtain equally good fits using backscattering atoms which are very different in Z (for example, Mn, or Cl) but which are at different distances from the absorb­ ing atom. This is more acute when dealing with Fourier peaks at greater distances. In bridged multinuclear centers, it is not always possible to unequivocally assign the Fourier peaks at > 3 Å (Scott and Eidsness, 1988), The peaks at > 3 Å could arise from scattering from second or third shell scattering from ligands like the imidazole ring of histidine or the pyrrole ring of hemes, or from backscattering from a bridged metal atom. 3) Distances are usually the most reliably de­ termined structural parameters from EXAFS. But the range of data that can be collected, often­ times due to practical reasons like the presence of the K-edge of another metal, limits the resol­ ution of distance determinations to between 0.1 to 0.2 Å. 4) Determination of coordination numbers or number of backscatterers is fraught with difficult­ ies. The Debye-Waller factor is strongly corre­ lated with the coordination number and one must have recourse to other information, like compari­ son to inorganic model complexes, to narrow the range that is possible from curve fitting analysis alone. The most important point in the analysis is to differentiate between fit parameters which are required and others which are merely consistent with the data. The EXAFS method is most useful when delineating all the structural alternatives based on required fit parameters, or addressing the question of subtle structural changes in sys­ tems well characterized by other techniques like X-ray crystallography.

345 III. Applications of XANES and EXAFS in Photosynthesis

In the two decades since the XAS technique has become practical, it has become a standard me­ thod for probing the metal site structure in metallo-proteins. The technique has been applied to virtually every metallo-protein that has been iso­ lated containing Fe, Mo, V, Cu, Co, Mn, Zn, Ni, Ca and other metals. In this section applications of XAS to Fe-S centers and the Mn oxygen-evolving complex in photosynthesis are described. Other components of the photosynthetic appar­ atus, plastocyanin (reviewed in Blackburn, 1990), the Fe-quinone acceptor complex in bacterial re­ action centers (Bunker et al., 1982; Eisenberger complex et al., 1982), and the of ATPase (Carmelli et al., 1986) have also been studied using XAS.

A. Fe–S Proteins 1. Soluble Plant Ferredoxin Among the first metallo-proteins studied by XAS are the class of non-heme iron sulfur proteins which are found in most redox-mediated path­ ways in biology. Initially, three classes of Fe–S structures containing 1Fe, 2Fe and 4Fe metal sites were recognized. This list has expanded to in­ clude 3Fe sites and higher nuclearity structures containing 7 and 8 Fe atoms in the M and P clusters of nitrogenase. The most common struc­ in 1Fe containing Fe– tural units are in 2Fe– S protein like rubredoxin, 2S proteins like soluble plant ferredoxins, and in 4Fe–4S proteins like ‘high po­ tential iron proteins’ (HIPIP) and other ferredox­ ins generally referred to as bacterial ferredoxins. The Fe EXAFS of these iron-sulfur proteins is dominated by backscattering from sulfur and iron atoms in the active site. In rubredoxin one Fe atom is ligated to four thiolate derived S atoms in near tetrahedral symmetry and it is a good example of a single-backscattering environment. The EXAFS of rubredoxin (see Fig. 5) exhibits a single wave and the Fourier transform shows one distinct peak, indicative of a single-shell sys­ tem with one type of Fe–S distance of 2.26 Å (Teo and Shulman, 1982).

346

V.K. Yachandra and M.P. Klein

In contrast to the EXAFS from rubredoxin, the EXAFS of the 2Fe–2S soluble plant ferre­ doxin and the 4Fe–4S bacterial ferredoxin exhib­ its (see Fig. 5) a beat pattern indicating the pres­ ence of at least two sine waves. The respective Fourier transforms show two peaks. The first peak is due to backscattering from S ligand atoms at ~ 2.23–2.25 Å, and the second peak is due to backscattering from the Fe atoms at 2.73 Å. These distances compare well with distances de­ rived from X-ray crystallography of the 2Fe–2S proteins from Spirulina platensis (Tsukihara et al., 1978), Anabaena 7120 (Rypniewski et al., 1991), 4Fe–4S proteins as well as synthetic anal­ ogs (reviewed in Spiro, 1982; Teo and Shulman, 1982). 2. Fe–S Acceptors Photosystem I

and

in

The electron transfer reactions from the primary donor of PS I to the substrates are initially

mediated by a number of PS I bound electron acceptors, labeled and which are Fe– S clusters with redox potentials of –705, –590, and –530 mV, respectively. XANES and EXAFS studies at the Fe K-edge have been used to study these Fe-S clusters. In the Fe K-edge XANES study of PS I the transition to bound 3d states was used to derive information about the symmetry of the Fe–S ac­ ceptor complexes. The 1s to 3d pre-edge transi­ tion is electric-dipole forbidden, but it is often observed in the edge spectra and is commonly attributed to d-p mixing. In centrosymmetric complexes d–p mixing is symmetry unallowed and the 1s to 3d transition is weak. In non-centrosymmetric complexes d–p mixing is allowed and the 1s to 3d transition is more intense. The inten­ sity of the 1s to 3d pre-edge transition can be used as a marker to differentiate between oc­ tahedral and tetrahedral complexes (Shulman et al., 1976; Roe et al., 1984). Fig. 6 shows the Fe K-edge spectrum of Fe–S

X-ray absorption spectroscopy

centers and The centers are compared to a 4Fe–4S model compound and to a centro­ symmetric hexacoordinate Fe complex. There is a decrease in the intensity of the 1s to 3d transi­ tion, and there is a change in the shape of the spectrum between tetrahedral Fe–S centers and a centrosymmetric hexacoordinate system. The intensity of the pre-edge 1s to 3d transition shows that the Fe–S acceptors and are in tetrahedral Fe–S complexes (McDermott et al., 1988a, 1989). Fe K-edge EXAFS studies of PS I preparations from spinach and the thermophilic cyanobacter­ ium Synechococcus sp. showed that the spectra were similar to those from Fe–S clusters, with a Fourier peak corresponding to S backscattering and another peak corresponding to backscatter­ ing from Fe. The results from PS I preparations containing and showed that the data could be simulated with either three 4Fe–4S clus­ ters or two 4Fe–4S and one 2Fe–2S cluster. This was due to the complexity of dealing with PS

347

I preparations which contained about 11–14 Fe atoms (McDermott et al., 1988a). A subsequent EXAFS study using PS I preparations which con­ tained only clearly showed that it was also a 4Fe–4S cluster (McDermott et al., 1989). The recently determined crystal structure of PS I has confirmed that all three acceptors are 4Fe–4S clusters (Krauss et al., 1993). 3. Rieske Fe–S Clusters

A Rieske Fe–S cluster is part of the cyt com­ plex in higher plants which mediates electron transfer between PS II and PS I. A Rieske Fe–S complex of center is also present in the cyt photosynthetic bacteria which is involved in cyclic electron transport. The Rieske Fe–S centers are characterized by an EPR signal and redox proper­ ties (150–350 mV) which are very different from the other more ubiquitous 2Fe–2S and 4Fe–4S proteins. Analogous Rieske centers are part of

348 the mitochondrial electron transport scheme. Al­ though there are no studies from plant sources, EXAFS studies of Rieske Fe–S protein from the complex from mitochondria (Powers et cyt al., 1989) and phthalate dioxygenase from Pseudomonas cepacia (Tsang et al., 1989) have been reported. Curve fitting results from both studies showed that better fits were obtained by including a mixed S and N coordination to Fe, but the quantitation of the number of nitrogens per Fe was not unequivocal. These studies are in accord with the ENDOR (Gurbiel et al., 1991) and ESEEM (Britt et al., 1991) results which showed there may be two terminal histidine li­ gands per 2Fe–2S cluster. The EXAFS studies found that: the Fe–S binding and terminal dis­ tances and the Fe–Fe distance were similar to the regular 2Fe–2S and 4Fe–4S clusters providing evidence that the unique properties of the Rieske centers are due to the N ligation or due to factors other than differences in Fe–S and Fe–Fe dis­ tances.

B. Manganese Oxygen Evolving Complex in Photosystem II Most of the oxygen in the atmosphere which sup­ ports life on earth is generated by plants by the photo-induced oxidation of water to dioxygen. The reaction shown in Eq. (12) is catalyzed by a tetranuclear Mn complex, which sequentially stores four oxidizing equivalents that are used to oxidize two molecules of water to molecular oxygen. The Mn complex is part of a multiprotein assembly called Photosystem II, which contains the reaction center involved in photosynthetic charge separation and an antenna complex of chlorophyll molecules. The complex also contains cyt and a Fe-quinone electron acceptor com­ plex (reviewed in Debus 1992; Rutherford et al., 1992). Owing to the complexity of the system and the presence of so many pigment and other components, study of the Mn complex by optical and other spectroscopic methods can be difficult. EXAFS is ideally suited for the study of the struc­ ture of the Mn complex because the specificity of the technique allows us to look at the Mn without interference from the pigment molecules, or the

V.K. Yachandra and M.P. Klein protein and membrane matrix, or other metals like Ca, Mg, Cu and Fe which are also present in active preparations. An active oxygen-evolving complex has not yet been crystallized, but EXAFS does not need single crystals; the struc­ tural studies can be performed on frozen solu­ tions. Also, several of the intermediate states mentioned above have been stabilized as frozen solutions and studied by EXAFS. We and others have studied the structure of Mn in the OEC using XAS. An earlier series of papers from our group reported Mn–Mn interac­ tions at 2.7 Å and Mn–O interactions at 1.75 Å, and an additional highly disordered shell of light atom (O, N) scatterers at ~ 2 Å. Similar results were found for both PS II-enriched membrane preparations from spinach chloroplasts and for detergent-solubilized OEC preparations from the thermophilic cyanobacterium Synechococcus sp. (McDermott et al., 1988b). From these results on samples poised in both the and states, it was predicted that the OEC contains binuclear dibridged Mn units whose structures remain largely unchanged upon the advance to (reviewed in Sauer et al., 1992; Klein et al., 1993). More recent EXAFS studies of Mn in the OEC, at substantially lower sample temperatures and with improved signal to noise ratio, have provided evidence for scatterers at > 3 Å in ad­ dition to the interaction at 2.7 Å. These experi­ ments include various combinations of oxygenevolving preparations and EXAFS analysis tech­ niques. George et al. (1989) report Mn scatterers at distances of 2.7 and 3.3 Å in angle-dependent EXAFS studies of whole oriented spinach chloro­ plasts. It was found that the scatterers at these distances exhibited significant dichroism. PennerHahn et al. (1990) also reported the 2.7 Å shell and at least one and possibly two shells of scatter­ ers at > 3 Å in EXAFS experiments using core preparations from spinach. Both these studies re­ ported difficulties in detecting the 1.8 Å shell; this was probably due to Mn(II) contamination in their experiments. MacLachlan et al. (1992) reproduced the 1.8 Å shell along with the 2.7 Å scatterers, but differed from the above experi­ ments by predicting a shell of Ca at a significantly longer distance of 3.7 Å. These results all have different implications for a structural model of the OEC.

X-ray absorption spectroscopy

Fig. 7 shows a typical Fourier transform of Mn in PS II from our data. The first Fourier peak requires two different distances to be fit ad­ equately, one at ~ 1.8 Å and the other 1.95–2.15 Å to light elements like C, N, or O. The second peak on the other hand is best fit by a heavier atom and, as noted above, probably a Mn atom at 2.7 Å; the amplitude is best fit to an average of one such interaction per Mn atom in the complex. Fitting the third peak, at a longer distance is more difficult. It can be fit to C, or Mn or Ca, or to a combination of these entities at 3 Å. The quality of fits is always better when Mn is included, but it is not clear if there is only Mn or also Ca atoms (Latimer et al., 1995). The presence of both the Mn–O (C,N) distances at ~ 1.8 Å and the Mn– Mn distances at 2.7 Å in all known bridged multinuclear Mn complexes, and the presence of a 3.3 Å Mn–Mn separation in monobridged Mn complexes leads to the con­ clusion that both of these structural motifs are present in the Mn complex of PS II (Wieghardt 1989; Pecoraro 1992). The number of such scat­ tering interactions leads to the conclusion (re­ membering that there are four Mn atoms) that bridged there are at least two Mn–Mn

349

units and at least one Mn–Mn bridged unit (DeRose et al., 1994). Figure 8 shows the Fourier transform of the same system in oriented samples (Mukerji et al., 1994). It is evident that the 2.7 and 3.3 Å Mn– Mn vectors (the second and third peak in the Fourier transform) are oriented differently. The 2.7 Å vector is more parallel to the membrane normal and the 3.3 Å is more perpendicular. De­ tailed analysis of the dichroism can be used to determine the angles more accurately. The dichroism requires that the complex be asymmet­ ric. One of the many possible structures (DeRose et al., 1994) consistent with data shown in Fig. 7 and the dichroism measurements is shown in Fig. 9 and provides us with a working model for the Mn cluster (Yachandra et al., 1993) Figure 10 shows the Mn K-edge spectrum from complexes in oxidation states (II), (III) and (IV). The inflection point shifts to higher energy as the oxidation state increases. There is also a dramatic change in the general shape of the edge as shown by the changes in the second derivatives. The shape of the edges is also an important indicator of oxidation state, as seen in Fig. 10 (Yachandra et al., 1993). A combination of the position and the shape were used to assign the oxidation states

350

V.K. Yachandra and M.P. Klein in the and states to and respectively. Fig. 11 shows the Mn K-edge from and states of the preparations in the photosystem II complex, and it shows that the Mn K-edge shifts to higher energy on advancing to and from to indicating oxid­ from ation of Mn (Goodin et al., 1984; McDermott et al., 1988b; Guiles et al., 1990a). However no such shift was evidenced in samples prepared in the state by a cryogenic double turnover protocol, which leads to the conclusion that Mn is not oxid­ to transition. It was proposed ized in the that the oxidation equivalent is stored on a ligand or amino acid residue (Guiles et al., 1990b). Ono et al. (1992) have presented Mn XANES of PS II samples that advanced progressively from by one through five laser flashes through the Kok cycle. Their data were interpreted to provide evidence for Mn oxidation from to and to and then reduction on going from to It is important to point out that Ono et al. have assumed that their samples were advanced on each flash but have presented no substantiating data, such as the pattern of multiline EPR signal intensity vs. flash. We have recently completed such a study (Roelofs et al., 1995, Andrews et al., 1994), and our data support our earlier results to that showed Mn is oxidized during the to transition but that it may not be and oxidized on the to advance.

IV. Future Directions The future holds much promise for the use of XAS in photosynthesis, and bio-inorganic chem­ istry in general. The availability of new storage rings and beamlines at these storage rings dedi­ cated to research in biology is increasing. There has been considerable improvement in the bright­ ness of the X-ray sources and also in detector technology. This makes the measurement of Xray absorption spectra of dilute species easier. The high brightness and the increased sensitivity of the technique are being used to couple micro­ scopy with spectroscopy. XAS in the soft X-ray region, which has been mostly used in materials and surface science stud­ ies, is increasingly being applied to biological problems. Some of the edges of interest in photo­ synthesis include the K-edges of P, S and Cl and also C, N, and O, and the L-edges of transition

X-ray absorption spectroscopy

metals. L-edges, as shown in Fig. 1, are 2p to 3d transitions, and in contrast to the 1s–3d trans­ itions in K-edge spectra are electric dipole al­ lowed and hence are intense. The natural linewidths of K and L-edge transitions, for example, in Mn are 1.12 and 0.32 eV, respectively, making L-edges considerably more sensitive to factors such as symmetry that influence the d orbital splittings and population. Differential absorption of circularly polarized X-rays in the presence of a magnetic field (XMCD, X-ray magnetic circular dichroism) is another method that is being applied to biological systems. X-ray magnetic circular dichroism with its selectivity for paramagnetic species and the relative magnetic orientation of different species in a mulicenter system promises to be a powerful tool for studying metal centers in the photosyn­ thetic apparatus.

351

Acknowledgements We thank Dr. Matthew Latimer and Dr. Annette Rompel for a critical reading of the manuscript. We are grateful to Prof. Kenneth Sauer for his suggestions. The work from our laboratory pre­ sented in this article was supported by the National Science Foundation grant DMB91– 0414, and by the Director, Division of Energy Biosciences, Office of Basic Energy Sciences, De­ partment of Energy (DOE) under contract DEAC03–76SF00098. Synchrotron radiation facili­ ties were provided by the Stanford Synchrotron Radiation Laboratory (SSRL) and the National Synchrotron Light Source (NSLS), both sup­ ported by DOE. The Biotechnology Laboratory at SSRL and Beam Line X9–A at NSLS are sup­ ported by the National Center for Research Re­ sources of the National Institutes of Health.

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V.K. Yachandra and M.P. Klein and Prins R (eds) X-ray Absorption: Principles, Appli­ cations, Techniques of EXAFS, SEXAFS and XANES, pp 53–84. John Wiley and Sons, New York. Eisenberger P and Kincaid BM (1978) EXAFS: New horizons in structure determinations. Science 200: 1441–1447. Eisenberger P, Okamura MY and Feher G (1982) The elec­ tronic structure of in reaction centers from Rhodop­ seudomonas sphaeroides II. Extended X-ray absorption fine structure. Biophys J 37: 523–538. George GN, Prince RC and Cramer SP (1989) The manganese site of the photosynthetic water-splitting enzyme. Science 243: 789–791. Goodin DB (1983) The local structure of manganese in the photosynthetic apparatus and superoxide dismutase: An Xray absorption study. Ph D Dissertation. Universtity of California, Berkeley, CA, USA Lawrence Berkeley Lab­ oratory Report, LBL–16901. Goodin DB, Yachandra VK, Britt RD, Sauer K and Klein MP (1984) State of manganese in the photosynthetic appar­ atus. 3 Light-induced changes in X-ray absorption (K-edge) energies of manganese in photosynthetic membranes. Bi­ ochim Biophys Acta 767: 209–216. Guiles RD (1988) Structure and Function of the Manganese Complex Involved in Photosynthetic Oxygen Evolution Determined by X-ray Absorption Spectroscopy and Elec­ tron Paramagnetic Resonance Spectroscopy. Ph D Disser­ tation University of California, Berkeley, CA, USA, Law­ rence Berkeley Laboratory Report, LBL-25186. Guiles RD, Yachandra VK, McDermott AE, Cole JL, Dexheimer SL, Britt RD, Sauer K and Klein MP (1990a) The state of photosystem II induced by hydroxylamine: differences between the structure of the manganese com­ plex in the and states determined by X-ray absorption spectroscopy. Biochemistry 29: 486–496. Guiles RD, Zimmermann J-L, McDermott AE, Yachandra VK, Cole J, Dexheimer SL, Britt RD, Wieghardt K, Bos­ sek U, Sauer K and Klein MP (1990b) The state of photosystem II: differences between the structure of the manganese complex in the and states determined by X-ray absorption spectroscopy. Biochemistry 29: 471–485. Gurbiel RJ, Ohnishi T, Robertson DE, Daldal F and Hoffman BM (1991) Q-band ENDOR spectra of the Rieske protein from Rhodobactor capsulatus ubiquinol-cytochrome c oxi­ doreductase show two histidines coordinated to the [2Fe– 2S] cluster. Biochemistry 30: 11579–11584. Heald SM (1988a) Design of an EXAFS experiment. In: Kon­ ingsberger DC and Prins R (eds) X-ray Absorption: Prin­ ciples, Applications, Techniques of EXAFS, SEXAFS and XANES, pp 87– 118. John Wiley and Sons, New York. Heald SM (1988b) EXAFS with synchrotron radiation. In: Koningsberger DC and Prins R (eds) X-ray Absorption: Principles, Applications, Techniques of EXAFS, SEXAFS and XANES, pp 119– 161. John Wiley and Sons, New York. Jaklevic J, Kirby JA, Klein MP, Robertson AS, Brown GS and Eisenberger P (1977) Fluorescence detection of EXAFS-sensitivity enhancement for dilute species and thin films. Solid State Commun 23: 679–682. Kirby JA, Goodin DB, Wydrzynski T, Robertson AC and Klein MP (1981) State of manganese in the photosynthetic

X-ray absorption spectroscopy apparatus. 2 X-ray absorption edge studies on manganese in photosynthetic membranes. J Am Chem Soc 103: 5537– 5542. Klein MP, Sauer K and Yachandra VK (1993) Perspectives on the structure of the photosynthetic oxygen evolving manganese complex and its relation to the Kok cycle. Pho­ tosynth Res 38: 265–277. Krauss N, Hinrichs W, Witt I, Fromme P, Pritzkow W, Dauter Z, Betzel C, Wilson KS, Witt H and Saenger W (1993) 3–Dimensional structure of system-I of photosynth­ esis at 6 angstrom resolution. Nature (London) 361: 326– 331. Kutzler FW, Natoli CR, Misemer DK, Doniach S and Hodg­ son KO (1980) Use of one-electron theory for the interpre­ tation of near edge structure in K-shell X-ray absorption spectra of transition metal complexes. J Chem Phys 73: 3274–3288. Latimer MJ, DeRose VJ, Mukerji I, Yachandra VK, Sauer K and Klein MP (1995) Evidence for the proximity of calcium to the manganese cluster of photosystem II: Deter­ mination by X-ray absorption spectroscopy. Biochemistry 34: 10898–10909. Lee PA, Citrin PH, Eisenberger P and Kincaid BM (1981) Extended X-ray absorption fine structure - Its strengths and limitations as a structural tool. Rev Mod Phys 53: 769– 806. MacLachlan DJ, Hallahan BJ, Ruffle SV, Nugent JHA, Evans MCW, Strange RW and Hasnain SS (1992) A EXAFS study of the manganese complex in purified photosystem II membrane fractions. Biochem J 285: 569– 576. McDermott AE, Yachandra VK, Guiles RD, Britt RD, Dexheimer SL, Sauer K and Klein MP (1988a) Low-potential iron–sulfur centers in photosystem I: An X-ray absorp­ tion spectroscopy study. Biochemistry 27: 4013–4020. McDermott AE, Yachandra VK, Guiles RD, Cole JL, Britt RD, Dexheimer SL, Sauer K and Klein MP (1988b) Characterization of the manganese complex and the iron-quinone acceptor complex in photosystem II from a thermophilic cyanobacterium by electron paramag­ netic resonance and X-ray absorption spectroscopy. Bio­ chemistry 27: 4021–4031. McDermott AE, Yachandra VK, Guiles RD, Sauer K, MP Klein, Parrett KG and Golbeck J (1989) EXAFS structural study of the low potential Fe–S center in photosystem I Biochemistry 28: 8056–8059. McKale AG, Veal BW, Paulikas AP, Chan SK and Knapp GS (1988) Improved ab initio calculations of amplitude and phase functions for extended X-ray absorption fine structure spectroscopy. J Am Chem Soc 110: 3763–3768. Mukerji I, Andrews JC, DeRose VJ, Latimer MJ, Yachandra VK, Sauer K and Klein MP (1994) Orientation of the oxygen-evolving manganese complex in a photosystem II membrane preparation: An X-ray absorption spectroscopy study. Biochemistry 33: 9712–9721. Ono T-A, Noguchi T, Inoue Y, Kusunoki M, Matsushita T and Oyanagi H (1992) X-ray detection of the period-four cycling of the manganese cluster in the photosynthetic water oxidizing enzyme. Science 258:1335–1337. Pecoraro VL (1992) Structurally diverse manganese coordi­

353 nation compounds. In: Pecoraro VL (ed) Manganese Redox Enzymes, pp 197–231, VCH Publishers, New York. Pendry JB (1983) The transition between XANES and EXAFS. In: Bianconi A, Incoccia L and Stipcich S (eds) EXAFS and Near Edge Structure, p 4. Springer-Verlag, Berlin. Penner-Hahn JE, Fronko RM, Pecoraro VL, Yocum CF, Betts SD and Bowlby NR (1990) Structural characteriz­ ation of the manganese sites in the photosynthetic oxygenevolving complex using X-ray absorption spectroscopy. J Am Chem Soc 112: 2549–2557. Powers L (1982) X-ray absorption spectroscopy: applications to biological molecules. Biochim Biophys Acta 683: 1– 38. Powers L, Schägger H, von Jagow G, Smith J, Chance B and Ohnishi T (1989) EXAFS studies of the isolated bovine heart Rieske cluster. Biochim Biophys Acta 975: 293–298. Rehr JJ, de Leon JM, Zabinsky SI and Albers RC (1991) Theoretical X-ray absorption fine structure standards. J Am Chem Soc 113: 5135–5140. Rehr JJ, Albers RC and Zabinsky SI (1992) High-order multiple-scattering calculations of X-ray absorption fine struc­ ture. Phys Rev Lett 69: 3397–3400. Roe AL, Schneider DJ, Mayer RJ, Pyrz JW, Widom J and Que L (1984) X-ray absorption spectroscopy of iron-tyrosinate proteins. J Am Chem Soc 106: 1676–1681. Roelofs TA, Liang W, Latimer MJ, Cinco R, Rompel A, Andrews JC, Yachandra VK, Sauer K and Klein MP (1995) Manganese oxidation states of the flash-induced S-states of photosystem II. In: Mathis P (ed) Photosynthesis from Light to Biosphere, Vol. II, pp 459–462. Kluwer Academic Publishers, Dordrecht. Rutherford AW, Zimmermann, J-L and Boussac A (1992) Oxygen evolution. In: Barber J (ed) The Photosystems: Structure, Function and Molecular Biology, pp 179–229. Elsevier, Amsterdam. Rypniewski WR, Breiter DR, Benning MM, Wesenberg G, Oh B-H, Markley JL, Rayment I and Holden HM (1991) Crystallization and structure determination to 2.5 Å resol­ ution of the oxidized [2Fe–2S] ferredoxin isolated from Anabaena 7120 Biochemistry 30: 4126–4131 Sauer K, Yachandra VK, Britt RD and Klein MP (1992) The photosynthetic water oxidation complex studied by EPR and X-ray absorption spectroscopy. In: Pecoraro VL (ed) Manganese Redox Enzymes, pp 141–175. VCH Publishers, New York. Sayers DE and Bunker BA (1988) Data Analysis. In: Kon­ ingsberger DC and Prins R (eds) X-ray Absorption: Prin­ ciples, Applications, Techniques of EXAFS, SEXAFS and XANES, pp 211– 253. John Wiley and Sons, New York. Sayers DE, Stern EA and Lytle FW (1971) New technique for investigating noncrystalline structures: Fourier analysis of the extended X-ray absorption fine structure. Phys Rev Lett 27: 1204–1207. Scott RA (1984) X-ray absorption spectroscopy. In: Rousseau RL (ed) Structural and Resonance Techniques in Biologi­ cal Research, pp 295–362. Academic Press, Orlando, FL. Scott RA and Eidsness MK (1988) The use of X-ray absorp­ tion spectroscopy for detection of metal–metal interac­

354 tions. Applications to copper-containing enzymes. Com­ ments Inorg Chem 7: 235–267. Shulman RG, Yafet Y, Eisenberger P and Blumberg, WE (1976) Observation and interpretation of X-ray absorption edges in iron compounds and proteins. Proc Natl Acad Sci USA, 73: 1384–1388. Spiro TG (1982) Iron–Sulfur Proteins. John Wiley, New York Stern E (1988) Theory of EXAFS. In: Koningsberger DC and Prins R (ed) X-ray Absorption: Principles, Applications, Techniques of EXAFS, SEXAFS and XANES, pp 3– 51. John Wiley and Sons, New York. Stöhr J (1992) NEXAFS Spectroscopy. Springer-Verlag, Berlin. Teo B-K (1986) EXAFS: Basic Principles and Data Analysis. Springer-Verlag, New York. Teo B-K and Shulman RG (1982) X-ray absorption studies of iron–sulfur proteins and related compounds. In: Spiro TG (ed,) pp 343–366. John Wiley and Sons, New York. Tsang H-T, Batie CJ, Ballou DP and Penner-Hahn JE (1989)

V.K. Yachandra and M.P. Klein X-ray absorption spectroscopy of the [2Fe–2S] Rieske clus­ ter in Pseudomonas cepacia phthalate dioxygenase. Deter­ mination of core dimensions and iron ligation. Biochemis­ try 28: 7233–7240. Tsukihara T, Fukuyama K, Tahara H, Katsube Y, Matsuura Y, Tanaka N, Kakudo M, Wada K, and Matsubara H (1978) X-ray analysis of ferredoxin from Spirulina platensis. II Chelate structure of active center. J Biochem (Tokyo) 84: 1645–1647. Wieghardt K (1989) The active site in manganese-containing metalloproteins and inorganic model compounds. Angew Chem Int Ed Engl 28: 1153–1172. Yachandra, VK (1995) X-ray absorption spectroscopy and applications in structural biology. In: Sauer K (ed) Methods in Enzymology. Biochemical Spectroscopy Vol 246. pp 638–675. Academic Press, Orlando, FL. Yachandra VK, DeRose VJ, Latimer MJ, Mukerji I, Sauer K and Klein MP (1993) Where plants make oxygen: A structural model for the photosynthetic oxygen evolving manganese cluster. Science 260: 675–679.

Chapter 22

Mössbauer Spectroscopy

Peter G. Debrunner

Physics Department, University of Illinois at Urbana-Champaign, 1110 W. Green Street,

Urbana, IL 61801–3080, USA

Summary I. Introduction II. Mössbauer Spectroscopy: Physics and Formalism A. Basic Features B. Hyperfine Interactions C. Electronic States of the Iron D. Spin Coupling E. Dynamical Aspects F. Calculation of Mössbauer Spectra G. Experimental Considerations III. Applications A. Iron–quinone Complex B. Iron–sulfur Proteins C. Cytochromes References

355 356 357 357 357 359 361 362 363 364 365 365 368 371 371

Summary spectroscopy (MS) has played an important role in the elucidation of the iron centers in the photosynthetic apparatus. In 1975, G. Feher and collaborators demonstrated that the single iron of bacterial reaction centers (RC) was high-spin ferrous irrespective of the state of Moreover, they showed that reduction of broadened the Mössbauer lines at 4.2K, indicative of spin coupling between the semiquinone and the iron, related to the broadening observed in the EPR signal of the semiquinone (Debrunner et al., 1975). Photosystem I (PSI) of green plants and algae contains three iron–sulfur centers labeled and that have originally been identified by EPR. Evans et al. (1977, 1979, 1981) showed that the Mössbauer spectra of PSI were practically identical with those of the well understood bacterial 4Fe–4S centers. The low-potential center remained controversial, however, as others suggested a 2Fe–2S center instead. The controversy was resolved by Petrouleas et al. (1989), who studied a mutant lacking centers and and found that had indeed all the properties of a 4Fe–4S center. The more difficult task of analyzing Photosystem II (PSII) of green plants was undertaken by Petrouleas and Diner (1982, 1986, 1990), who identified the redox center known as with the iron– quinone complex and showed the iron to be redox active in contrast to the Fe(II) of the bacterial RC. The same group demonstrated, by MS, that formate affected the iron–quinone complex (Diner and Petrouleas, 1987), and finally that the Fe(II) formed an NO derivative.

Correspondence: Fax: 1-217-3339819; E-mail: [email protected]

355 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 355–373. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

356

Peter G. Debrunner

After a review of the methodology, the various iron sites of bacterial RC, of PSI and PSII will be discussed in detail to illustrate the application of the method. Abbreviations: EFG – electric-field gradient; EPR – electron paramagnetic resonance; LDAO – lauryldi­ methylamine N-oxide; MS – Mössbauer spectroscopy; PSI – Photosystem I; PSII – Photosystem II; RC – (bacterial) reaction center

I. Introduction This chapter attempts to acquaint the reader with the basic concepts of spectros­ copy (MS) and its applications in photosynthesis research. is the most important Mössbauer isotope, and since the photosynthetic apparatus contains several prominent iron complexes, MS can be utilized to study the properties of these complexes under various experimental con­ ditions. For more general and in-depth treat­ ments of MS the reader is referred to the litera­ ture (Greenwood and Gibb, 1971; Gonser, 1975; Gibb, 1976; Gütlich et al., 1978; Huynh and Kent, 1984; Cranshaw et al., 1985; Dickson and Berry, 1986; Debrunner, 1993). Since MS involves the resonant absorption of gamma rays by nuclei, it has several features that set it apart from other types of spectroscopy. First of all, the quantum energy of the transition is quite large, in the case of yet the intrinsic linewidth, is suffi­ ciently small to resolve the hyperfine interactions nucleus with its electron shell. The of the applications of MS discussed here indeed use the known electric and magnetic moments of the to probe the internal electric and magnetic fields produced by the electrons and to gather infor­ mation, in the process, about the state of the iron. Secondly, the 14.4 keV radiation to be res­ onantly absorbed by the nuclei is emitted by which decays to a source of radioactive stable via the 14.4 keV excited state. The source thus emits 14.4 keV gamma rays of intrinsic line width (plus other radi­ ations contributing to the non-resonant back­ ground). Given the discrete energy, keV, of the radiation emitted by the source, the spectrum of the absorber can be scanned by mov­

ing the source with velocity v relative to the ab­ sorber, imparting thereby a Doppler shift to the gamma rays,

where c is the speed of light. It is customary in MS to express all energies in terms of the measured velocities v. To convert to more familiar energy units the following conversion factors are useful:

Thirdly, it follows from energy and momentum conservation that Mössbauer transitions of intrin­ can only occur in solids and only sic linewidth in a fraction f < 1 of the cases as will be discussed in Section II.E. A fourth unique feature of MS is its sensitivity to a single isotope, here Since the natural is only 2.2%, biological abundance of samples need to be enriched in to improve the signal-to-noise ratio. As will be discussed in Section II, different oxidation and spin states pro­ duce distinctly different spectral contributions, and with a few empirical rules it is in principle possible to resolve a composite spectrum into its components and to deduce the oxidation and spin state of each iron site. To illustrate this process, Section III discusses the analysis and interpre­ tation of spectra taken on bacterial reaction cen­ ters, PSI and PSII. It will be noted that the appro­ priate level of sophistication in the data analysis depends on the quality of the data. At the very least, MS can assess the purity and composition of a sample as far as its iron content is concerned. Once the quality and reproducibility of the samples are established, the spectral components can be assigned to the various iron species pre­ sent, and the assignments can be verified by titr­

Mössbauer spectroscopy ation, by the expected temperature and field de­ pendences, etc. Finally, it may be possible to characterize the iron environment(s) further by fitting spectra, taken as a function of temperature and field, to a spin Hamiltonian or other theoreti­ cal model.

357 of an nucleus with transition en­ can be written as where depends on the transi­ tion probability and other factors. The intensity transmitted through an absorber with n nuclei per unit area then is

ergy

II. Mössbauer Spectroscopy: Physics and Formalism

A. Basic Features The stable ground state of has nuclear spin I = 1/2 and connects via magnetic dipole transi­ tion to the first excited state at 14.4 keV with spin The Heisen­ I* = 3/2 and mean life berg principle then predicts a Lorentzian energy distribution of the emission or absorption line with intrinsic width where is Planck’s constant. Most experiments are done in a transmission geometry, whereby source the radiation emitted by a single-line impinges on the absorber to be investigated, and the transmitted 14.4 keV gammas are counted in a detector, e.g. a proportional counter. As the Doppler-shifted energy of the incident beam nuclei matches the transition energy of some in the absorber, these nuclei absorb the resonant gammas in proportion to predictable transition probabilities, and the count rate of the detector decreases. Doppler shifts are varied periodically and data are typically accumu­ between lated for many hours. The final spectrum is a plot of the total number of counts, N(v), recorded as a function of Doppler velocity v. To find a quantitative expression for a trans­ mission spectrum let the intensity distribution of v)dE, where is the incident beam be I(E, the center of the Lorentzian line emitted by the source. With the substitutions can be written as which integrates to Here and (below) are the recoilless fractions of the source and ab­ sorber, respectively, and will be discussed further in Section II.E. Similarly, with the absorption cross section

with thin-absorber

The which approximates is valid for most bio­ by of Eq. (4) one logical samples. To has to add an energy-independent background B. The transmission spectrum therefore consists of a (negative) Lorentzian of full width at halfheight and relative height The maximum res­ with no hyperfine onance cross section of splitting is which is much larger than the (non-resonant) absorption cross of iron at 14.4 keV. section of In a more general case several types of iron environments will be found in an absorber, and each is subject to hyperfine interactions, which lift the two- and four-fold degeneracies of the ground and excited states, respectively. The factor n in Eq. (4) then refers to the of a as well as depends on given species, and the nuclear eigenstates involved in the transition as described below. limit,

B. Hyperfine Interactions This section discusses how the electron shell and/or external fields affect the nuclear energy levels and thereby the Mössbauer spectra, and how the electronic properties of the iron can be deduced from the spectra. In addition to the spin angular momenta I and I* of the ground and excited state already mentioned, the Î and nucleus has magnetic moments respectively, with and an electric quadrupole moment

358 where all starred quantities refer to the I* = 3/2 excited state, the ground state of spin I = 1/2 having no measureable quad­ rupole moment. In the expressions for and is the nuclear above magneton. The difference in mean-square charge moreover, gives rise to an radii, electric monopole interaction that shifts the transi­ tion energy proportional to the s-electron density, at the nucleus and manifests itself in a shift of the whole spectrum without affecting its shape. This so-called chemical or isomer shift, is given by the expression

where the subscripts A and S in the last term refer to absorber and source, respectively. In order to define the isomer shift independently of the particular source used, is usually quoted as relative to the centroid of the spectrum of metallic iron taken at 300K. It should be noted that the overall shift has a dynamical contribution as well which will be discussed in Section II.E. The electric quadrupole interaction, an­ other correction to the Coulomb energy of the atom, accounts for the non-spherical charge dis­ tribution of the nucleus in the excited state. It splits the Mössbauer transition into two lines sep­ and oc­ arated by the quadrupole splitting curs whenever an electric-field gradient (EFG) is present at the nucleus. An EFG arises from the valence electrons of the iron and from surround­ ing charges unless excluded by symmetry. In iron proteins high local symmetry with no EFG such as tetrahedral, octahedral, etc., is rare, and a measurable quadrupole splitting is therefore the rule. Since a single 3d-electron can produce a the valence splitting of contribution typically exceeds the lattice contri­ bution which arises from charges outside the elec­ tron shell. The EFG or its negative, x,y,z, is a symmetric, traceless second rank ten­ sor. It is readily calculated from the charge den­ sity of the valence electrons and from any exter­ nal charges. For the covalent, low-symmetry iron sites of interest here a proper treatment requires a molecular orbital approach, but simpler crystal-

Peter G. Debrunner field approximations have been used in the past. Model calculations are complicated by the distor­ tion of the inner electron shells as parametrized by the Sternheimer factors. Several equivalent expressions for the electric quadrupoleinteractions are in use (Abragam and Bleaney (1990))

In the last two expressions are the principal-axes components of and the are the components of the nuclear spin operator, Î, along these axes. The numerical values in Eq. (6) are those for the I* = 3/2 excited state. The last expression uses the condition and the definition of the asymmetry parameter

With the convention, is In the absence limited to the range of magnetic interactions splits the I* = 3/2 excited state into two doublets with a quadrupole splitting of

Since the ground state does not split, the quadru­ pole interaction leads to the characteristic twoline pattern of the Mössbauer spectrum, both lines being Lorentzians of minimum linewidth and equal areas for randomly oriented samples. The magnetic dipole interaction, finally, lifts the degeneracy of the nuclear levels completely, and the magnetic dipole selection rules allow six transitions between the two and the four equidistant levels of the ground and excited state, respectively. In the presence of electric quadrupole interaction, the I* = 3/2 eigenstates are linear combinations states, and up to eight transitions are of the possible. An external magnetic field B leads to the nuclear Zeeman interatction for the ground state,

where the last expression assumes that B defines

Mössbauer spectroscopy the direction of z. An analogous expression holds for the excited state. The magnetic hyperfine interaction couples the nuclear spin operators Î or Î* with the electron spin operators and is given by (Ab­ ragam and Bleaney 1970)

Here, the sum is over all unpaired electrons with position orbital angular momentum and spin and is a numerical factor of roughly 0.35. An analogous expression applies for the excited state. The three contributions in Eq. 10 are the orbital, the traceless spin dipolar and the isotropic Fermi contact term, respectively, where the last one generally dominates. As stated in Eq. 10 the hyperfine tensor à has units of energy and can be compared directly with EPR/ENDOR data. It is convenient, however, to divide à by so that

represents an internal field that can be substituted for, or added to, in Eq. 9. Note that Eq. which is a 11 contains the expection value of classical vector rather than an operator so that the left-hand side can be equated to a field. Whenever a sufficiently large external field is aplied to a sample of spin such that the Zeeman interaction far exceeds the magnetic hyperfine interaction, typically can be calculated from the electron spin then Hamiltonian alone, Eq. 15, as will be discussed later in Section II.C. In field units the Fermi contact term for six-coordinate iron with oxygen/ nitrogen ligands has the value which decreases to coordination for the highly covalent of iron-sulfur proteins. In a general case the nuclear Hamiltonian is given by the sum

If the internal field approach, Eq. 11, is valid, simplifies to

which depends on nuclear spin operators only.

359 Since refers to a molecular frame while is defined in the laboratory, each molecule in a randomly oriented sample will have a different and therefore different nuclear Hamiltonian energy levels and transition probabilities. As a result, the Mössbauer spectrum no longer consists of a set of discrete lines but rather of a continuous distribution, and any quantitative analysis re­ quires computer programs. The information available from such an analysis can be substantial even for the simplest case of a diamagnetic com­ Here, the applied field pound with broadens the quadrupole doublet, and the lineshape allows one to deduce as well as the sign two parameters that characterize the sym­ of metry of the valence electrons in more detail than the quadrupole splitting alone.

C. Electronic States of the Iron Ferric iron has five 3d-electrons with spins ar­ orbital ranged either parallel, resulting in a singlet of total spin S = 5/2, or with four spins paired leaving a single unpaired spin, S = 1/2, in The former, high-spin state an orbital triplet is found in more ionic, weak-field compounds, whereas the latter, low-spin state is found in highly covalent, strong-field compounds, e.g., the ferricytochromes. The spin state of ferric com­ pounds is readily recognizable from MS, and al­ though the same can be said of EPR, which is vastly more sensitive, Mössbauer data can pro­ vide additional information not available other­ wise. The combination of the two methods is clearly most powerful. We begin with a discussion of high-spin Fe(III). The sixfold spin degeneracy of the state is partially lifted by the zero-field splitting which is given to lowest order by the expression

By convention, the rhombic term E in Eq. 13 is

in a ‘proper’ coordinate sys­

limited to

tem. The axial term D is typically small for S =

5/2 iron,

but both signs of D are possible. The eigenstates of are three Kra­

mers doublets; for E = 0 these doublets are the

360 states, while they are lin­ ear combinations of the states for The remaining twofold degeneracy is lifted by the Zeeman interaction where is the Bohr magne­ ton, and is the g-tensor, which is expected to have the spin-only value of since the state allows no orbital contribution. The total electronic spin Hamiltonian is then the sum of Eq. 10 should in principle be added to Eq. 15 since it depends on the electron spin oper­ ator Inclusion of is warranted for van­ requires an expansion ishing only since of the basis set to (2I + 1)(2S + 1), etc., and com­ plicates the solution of Eq. 15 considerably. For is a small perturbation that does not affect appreciably and can therefore be neglected in Eq. 15. Diagonalization of yields the six eigenstates and their energies and allows one to calcu­ late EPR transitions, the spin expectation values of each state, etc. In the pres­ ence of an applied field mT the internal field formalism, Eq. 11, is valid, and the nuclear eigenstates and energies can be found from Eq. Each spin eigenstate produces a different internal field, however, and for each molecular orientation six different component spectra with different Boltzmann factors have to be added. In practice, spectra of high-spin Fe(III) typi­ cally show well resolved bands with overall for splittings of roughly 16 The orbital singlet state, allows no orbital and spin dipolar con­ tribution to Ã, and any deviations reported from isotropy are indeed small. The nominal singlet also predicts zero valence contribution to the qua­ drupole interaction, but does not rule out lattice contributions or effects due to anisotropic elec­ tron delocalization. Typical splittings are of the but exceptional cases with order twice that value are known. orbital triplet, is best Low-spin Fe(III), a understood as a single-hole state in an otherwise filled subshell. The magnetic properties, in particular the g- and A-tensors, are reasonably

Peter G. Debrunner well reproduced by a crystal-field model proposed by Griffith (1957). According to this model the threefold orbital degeneracy obtained in strong octahedral field is lifted by crystal field compo­ nents of lower symmetry, namely an axial and a rhombic component. If the Hamiltonian con­ sisting of the electrostatic potential energy and the known spin-orbit interaction is solved, three Kramers doublets are obtained as linear combi­ orbitals with appropriate spin func­ nations of tions. The splittings are such that only the ground doublet is populated at any accessible tempera­ ture, and once the wavefunction of this ground doublet is known, all observables, in particular the tensors Ã, and can be calculated. In practice, the axial and rhombic crystal field com­ ponents are adjusted to match the measured gvalues, and Taylor (1977) has given a particularly simple algorithm for this purpose. The g-values may deviate substantially from the spin-only value due to orbital contributions, and the Atensors typically are quite anisotropic for the same reason and because of spin dipolar contribu­ tions. The quadrupole splitting reflects the combi­ orbitals that make up the ground nation of state wavefunction: if a single orbital dominates, if all orbitals may be as large as 3 contribute equally, may be close to zero. In low-spin heme compounds the lattice or co­ valency contributions to are substantial (De­ brunner, 1989). Ferrous iron has six 3d-electrons, i.e. one more than Fe(III), and both high- and low-spin states are found in weak- and strong- field compounds, respectively. Low-spin Fe(II) has a filled subshell and is therefore diamagnetic as exem­ plified by ferrocytochromes. The valence contri­ bution to the quadrupole splitting is zero, but the lattice and covalency contributions lead to in the cytochromes. Isomer shifts of the latter are in the range of High-spin Fe(II) is characterized by the largest isomer shifts, 0.9 where the low value applies to the more covalent, 5-coordinate heme proteins, the high value to the most ionic, 6-coordinate compounds. The excep­ coordination of the irontionally covalent sulfur proteins leads to Another characteristic of high-spin Fe(II) is the large quadrupole splitting of

361

Mössbauer spectroscopy

which has a noticeable temperature de­ pendence as illustrated in Fig. 1 and explained below. The extra 3d-electron of high-spin Fe(II), which causes the large leads to a or state in octahedral or tetrahedral symmetry, in respectively. As illustrated for the case of the upper inset of Fig. 1, crystal field components of axial and/or rhombic symmetry lift the orbital degeneracy, and the thermal population of the higher orbital state(s) with different quadrupole and interactions averages out the observable hence makes it temperature dependent. A proper treatment of has to include the spin-orbit coupling as well as the axial and rhombic crystalfield terms (Ingalls, 1964). The magnetic properties of high-spin Fe(II) are usually described by the spin-Hamiltonian, Eq. 15. The zero-field splittings are typically sev­ i.e. larger than in high-spin ferric com­ eral pounds, and the eigenstates of Eq. 13 are singlets rather than Kramers doublets. The last for point is an important, general distinction between integer-spin and half-integer spin systems: For integer-spin, non-Kramers systems the spin ex­ pectation values vanish in zero field, and no spon­ taneous magnetic hyperfine splitting is observed.

The Mössbauer spectra of high-spin Fe(II) there­ fore show unbroadened quadrupole doublets in zero field as illustrated in Fig. 2 (top). The only known exceptions are magnetically ordered ma­ is comparable terials or the unusual case that to the splitting between the eigenstates of Eq. 13. Strong external fields will induce internal fields described by Eq. 11 and thus allow one to deduce the hyperfine tensor à as illustrated in Fig. 2 (bottom), which will be discussed further in Sec­ tion III.A. It should be kept in mind that the parameters of Eq. 15 are constants only as long as the next higher orbital level is far removed in energy from the ground level, a condition that is is strongly tempera­ certainly not satisfied if ture dependent.

D. Spin Coupling The formalism of Section II.C. refers to isolated iron sites. In many biological systems including the photosynthetic apparatus, however, the iron

362 is either found in clusters as in the 4Fe–4S prote­ ins or near a radical as in the ferroquinone com­ plex. In both cases the component spins couple, changing the properties of the coupled system drastically. This subsection therefore attempts to describe the coupled system in terms of the pro­ perties of its parts. The simplest type of coupling between two spin operators and is given by the Heisenberg-Van Vleck expression for isotropic ex­ change, which is the dominant term when the wavehas functions of the two centers overlap. eigenstates of spin and energy For J > 0 the state of lowest energy is thus the one with the smallest spin S, and the opposite is true for J < 0. The situation is simple as long as is much larger than any other term in the spin and This Hamiltonian, Eq. 15, of the spins is the case for the 2Fe–2S proteins, which exist in the oxidation states Fe(III)–Fe(III) and Fe(III)– Fe(II) and have strong antiferromagnetic coup­ ling so their net spins are S = 0 and S = 1/2, resp. The situation is more complicated in the case of the 4Fe – 4S proteins, which exist in an oxidized and a reduced state. The problem is that there are six interacting iron pairs that can not simultaneously align their spin anti­ parallel. The energy of the system can be low­ ered, however, by double exchange (Münck et al., 1988), whereby the extra electron of Fe(II) delocalizes onto Fe(III) so that the average charge per iron is 2.5 as judged by the isomer shift. The spins of the delocalized pair align paral­ has two lel to a spin of 9/2. Oxidized such pairs lining up antiparallel to a cluster spin there is a of S = 0. In reduced delocalized, S' = 9/2 pair and a diferrous pair with aligned spin, S'' = 4 and the cluster spin is S = 1/2. For the system spin is a good quan­ tum number, and using spin algebra the expectation value of any operator involving or can be calculated in any eigenstate of To illus­ trate the process, consider Eq. (10), written here for the intrinsic spin In

Peter G. Debrunner terms of the system spin the Hamiltonian be­ comes where the A-tensor in the S representation is given by If is small or comparable to as is the case in the ferroquinone complex, the full Hamiltonian has to be diagonalized.

E. Dynamical Aspects The vibrational motion of the iron affects the Mössbauer spectra in three significant ways, viz. (i) the recoilless fraction f, (ii) the thermal red­ shift which adds to the isomer shift, and (iii) the spin-lattice coupling, which controls the spin dy­ namics. We will discuss these effects in this order. As stated in the Introduction, the Mössbauer effect is observable in solids only. The reason is that energy and momentum have to be conserved in the emission and absorption processes, and this is possible without recoil energy loss only if the emitting or absorbing atom is part of a quantized vibrational system with discrete energy levels. When a bound iron absorbs a photon, the lattice will on average increase its energy by the recoil energy and it does so by emitting n phonons, n = 0,1,2,..., where the inclusion of n = 0 is crucial as it implies a zero-phonon, recoilless Mössbauer transition. The larger the phonon energy as com­ pared to the recoil energy, the larger the re­ coilless fraction f will be, where f is the fraction of n = 0 events out of the total. For a solid with harmonic forces the recoilless fraction is given by where is the mean-square displacement of the iron in the direction of the gamma ray and pm is the wavelength of the 14.4 keV radiation. Since is a monotonically in­ creasing function of temperature, T, the recoilless fraction decreases rapidly with increasing T. The of iron proteins are zero-point vibrations comparable to those of other iron compounds, and recoilless fractions of are typical at 4.2 K. For iron proteins, the linear increase of for 50K < T < 150K is larger, however, and for T > 150K non-vibrational motions contri­ increasing its rise with T further. bute to

Mössbauer spectroscopy

363 ues in Eq. 11 can be replaced by their thermal where i refers to the (2S + 1) average eigenstates of in Eq. 15. For Fe(III) in weak fields the magnetic hyperfine splitting collapses completely in the fast-fluctuation limit since approaches zero, thus the spectra simplify and the whole intensity concentrates in the emerging quadrupole doublet. While Mössbauer spectra can be simulated for any spin fluctuation rate and the temperature dependence has been modeled successfully (Schulz et al., 1988), it is not always practical to reach the simple limiting cases, and the data analysis then remains difficult.

F. Calculation of Mössbauer Spectra

For T > 230K limited diffusion sets in, which re­ sults in line broadening, motional decrease of etc. (Keller et al., 1980; Aleksandrov et al., 1987). The thermal redshift or second-order Doppler shift is given by

where is the mean-square velocity of the iron, which stays close to the minimum value given by zero-point vibrations for T < 50K and approaches the classical limit of 3kT/M without reaching it at high temperatures. The thermal redshift adds to the isomer shift and is illustrated in Fig. 3. The last dynamical feature to be discussed is the spin–phonon coupling that causes transitions between the eigenstates of the spin Hamiltonian Eq. 15, and is related to the spin relaxation rate in EPR. In the presence of magnetic hyper­ fine interactions spin state fluctuations affect the shape of the spectra profoundly. So far it has been tacitly assumed that the spin states are sta­ tionary on the Mössbauer time scale, a case that typically applies near 4.2K. In the opposite limit of fast spin state fluctuations, which is approached at higher temperatures, the spin expectation val­

A variety of computer programs are available for the quantitative analysis of Mössbauer spectra, and only the general principles will be discussed here. In the simplest case, the spectrum consists of a number of Lorentzians on a constant back­ ground, each characterized by its center, width and height, and a least-squares routine will find an optimum parameter set with standard devia­ tions and correlation coefficients. The results will then have to be interpreted in terms of quadru­ pole doublets and/or single lines attributable to different spectral components. Alternatively, constraints can be incorporated in the fitting rou­ tine, e.g. requiring identical shapes or areas for both lines of a quadrupole doublet. To allow for inhomogeneity in the iron environment a Gaus­ sian distribution of Lorentzians or Voigt line shape can be used with the Gaussian width as a parameter. In an external field each molecule has a differ­ ent nuclear Hamiltonian, Eq. 12 or 12' and the spectra are calculated by adding contributions from an appropriate sample of molecular orien­ tations, typically more than 100. For paramag­ netic centers the spin expectation values or needed in Eq. 12 or 12' will also depend on the molecular orientation, thus both and have to be diagonalized for each orientation. Since and depend on a large number of parameters, a successful analysis is possible only with data of high quality, but the resulting parameter set, in particular Ã, and the relative orientation of these tensors, will characterize the iron environment in great detail.

364

Peter G. Debrunner

G. Experimental Considerations Most Mössbauer measurements are done in trans­ mission, and count rates upward of are obtained with sources of roughly 50 mCi. If N is the total number of counts per data point, the mean square deviation of N equals N, and counts per It therefore takes data point to measure a 1% dip to a standard deviation of 10% and counts to measure a 0.1% dip to the same relative accuracy, etc. Ac­ cording to Section II.A the total area under the absorption spectrum is proportional to the recoilless fraction times the number of atoms per and for a given spectral area the peak absorption is obviously largest if the number of lines and the widths are minimal. Although MS can provide unique information about the environment of the iron in the photo­ synthetic apparatus, its application has been limited by the need for quantities of per sample. Enrichment in is therefore abso­ lutely mandatory, and as long as the iron cannot be exchanged, growth of the photosynthetic or­ ganisms, either bacteria, cyanobacteria or algae, on enriched media has been the only practical approach. As pointed out by Evans et al. (1977), an excess of iron in the growth medium should be avoided. In R. sphaeroides with one iron per RC, samples containing up to of have been obtained, resulting in spectra with a total area of at 4.2K as shown in Fig. 2. Based on figures reproduced here, comparable in PSII preparations are 5.5 and estimates of in Fig. 4b and Fig. 6a,b, respectively. With two irons per PSII, these numbers imply a roughly tenfold lower concentration of RCs than in R. sphaeroides. To achieve equivalent signalto-noise ratios in the two cases, the number of times counts accumulated would have to be larger in the case of PSII. Any increase in the concentration of the iron-bearing proteins as well as any reduction in the number of species present in a sample, e.g. by genetic engineering or chemi­ cal means, will greatly facilitate the Mössbauer experiments. The problems are well illustrated by Fig. 4, which compares the spectra of membranes from cyanobacteria in (a) with oxygen-

evolving core complexes in (b). The total areas of the spectra are 1.8 and respec­ tively, indicating an estimated amount of 15 and of The dominant doublet in Fig. 4a arises mainly from iron–sulfur proteins and cytochromes in PSI and the cytochrome complex; it hides the PSII spectrum of Fig. 4b, im­ which actually contains a cytochrome purity exceeding the amount of cytochrome of PSII (Picorel et al., 1994). PSI contains three 4Fe–4S clusters, and Figs. 10 and 11 indicate that samples of adequate signal-to-noise ratio can be prepared. Temperature is an important variable in any Mössbauer experiment since most samples are measured as frozen solutions, since the recoilless fraction is largest at helium temperatures, and since the spin fluctuation rates are strong func­ tions of T. Accordingly, a variable temperature cryostat is essential. For samples of adequate sig­

Mössbauer spectroscopy nal-to-noise ratio external fields are useful also as illustrated in Figs. 2 and 11. III. Applications This section applies the basic concepts and for­ malism developed so far to the three types of iron sites found in the membrane-bound photosyn­ thetic apparatus of bacteria, cyanobacteria and algae. Among the latter the experiments done on Chlamydomonas reinhardtii stand out. The three types of iron sites are the iron–quinone complex of the bacterial reaction center (RC) and PSII of the higher organisms, the iron–sulfur clusters of PSI, and the cytochrome of PSII. Here, the goal is to illustrate the arguments leading from experi­ mental data to final conclusions and to point out strengths and weaknesses of the method.

A. Iron–quinone Complex Early Mössbauer experiments on bacterial RCs conclusively showed the existence of a ferroquinone complex which is now known in atomic detail from x-ray diffraction (Deisenhofer et al., 1985; Feher et al., 1989), although the function is still elusive (Debus et al., 1985). Equally sig­ nificant was the demonstration of an analogous iron site in PSII, which was not only redox-active in contrast to the bacterial one, but was sensitive to the presence of bicarbonate/formate and able to form an Fe(II)NO adduct of spin S = 3/2. Since the iron quinone complex has no precedent, it will be discussed in some detail starting with the bacterial RC of Rhodobacter sphaeroides, Figs. 1–3 (Debrunner et al., 1975; Boso et al., 1981). A first step in the Mössbauer study of any new sample is to check its iron content, its purity, homogeneity and reproducibility. As shown in Fig. 2 (top), the spectrum of native RC consists of a single quadrupole doublet, i.e. the sample appears to be pure. Based on a calibration with a known absorber, the total area of matches within 10% the of esti­ mated from the optical absorption with the as­ sumption that 90% of the iron comes from the enriched iron in the growth medium. The lineis larger than the mini­

width, mum experimental linewidth,

but is smaller than that observed in ferrous heme

365

proteins. The homogeneity of the iron site is therefore quite high. The combination of finally, leaves and no doubt that the iron is high spin ferrous. The good match of the isomer shift with the values found in compounds of spin S = 2 suggested mixed N- and O-ligands as borne out by x-ray diffraction. Figure 1 illustrates the typical temperature de­ of this high-spin ferrous com­ pendence plex; the lines (A), (B), and (C) represent three attempts to fit using a crystal field model. The upper inset defines the axial and rhombic crystal field terms Dl and D2 and sketches the effect of the spin–orbit coupling which is given in terms of the standard value and an adjustable covalency factor Curves (A) and (B) further assume a lattice or covalency contribution to the EFG, quantified by whereas curve (C) assumes different delocalization for the spin and the charge densities. Obviously, these four-parameter models fit the data reasonably well, yet the strong covalency of the 3d-electrons that all of them require implies that the crystal field ap­ proach is a poor approximation. Figure 2 (bottom) shows the magnetic hyper­ fine broadening brought about by a strong field and a simulation of the spectrum based on eqs. and 15. At a temperature of 156K the limit of fast spin fluctuation rates applies, and the thermal of the spin expectation can there­ average, fore be used in Eq. 11. The spectrum is from a series of measurements taken at different temper­ atures and fields and illustrates the type of infor­ mation that can be extracted, which is summar­ ized by the parameters given in the caption. The main conclusion from the data is that the iron site definitely has low symmetry. The hyperfine tensor is highly anisotropic, and its average value of is well below the Fermi contact term of indicating substantial or­ bital and spin dipolar contributions. Moreover, the quadrupole tensor with asymmetry is rotated relative to the zero-field splitting which was assumed to be coaxial with Ã. As mentioned earlier, the Hamiltonian parameters must be thermal averages rather than constants since Fig. 1 clearly indicates that higher orbital

366

states are being populated over the temperature range of interest. Figure 3, finally, compares the temperature dependences of the isomer shifts of six RC samples with nominally zero, one or two quin­ ones, prepared with different detergents, with and o-phenanthroline, or with chemically reduced as verified by EPR. By and large is identical for all samples, and the temperature de­ pendence is given by the same thermal redshift, Eq. 18. From this result and from the observation (not shown) it clearly of nearly identical follows that the ligands of the iron are the same in all samples, hence neither quinone nor o-phenanthroline bind to the iron. The only discrepancy in Fig. 3 is the 4.2K data point of the semiquinone sample (6). At higher temperatures this sample has 5–10% larger linewidths than all the others, but at 4.2K the quadrupole lines broaden asym­ metrically by roughly a factor 1.7, a clear indi­ cation of unresolved magnetic hyperfine interac­ tion. The explanation is obviously the magnetic coupling of the iron spin, S = 2, to the spin S = 1/2 of the semiquinone, a coupling that can be modeled by Eq. 16. The interaction of the two spins manifests itself not only in spontaneous broadening of the Mössbauer lines in zero field, but also in the large, temperature-dependent broadening of the EPR spectrum. The latter has been modeled quantitatively by Butler et al. (1984) using the spin Hamiltonian, Eq. 15 of the iron. Next, we turn to the work of Petrouleas and Diner (1982,1986,1990) on PSII of Chlamydo­ monas reinhardtii which established the existence of an iron–quinone complex in PSII of green plants. The complex appears to be structurally related to the bacterial one (Michel and Deisen­ hofer, 1988). Fig. 5 compares the Mössbauer spec­ tra of PSII particles taken at 130K (a) and 4.2K (b,c). Obviously, the absorption is much smaller than in Fig. 2, the spectrum is more complex, and one component, an asymmetric doublet labeled (II) in the top trace, apparently vanishes at 4.2K. The major persistent component (I) has par­ ameters characteristic of high-spin Fe(II), and the comparison in Table 1 suggests its analogy with the iron–quinone complex of the bacterial RC. Component (III) may arise from air-oxidized

Peter G. Debrunner

iron–quinone or some unidentified impurity. The properties of doublet (II), which are listed in Table 3, indicate that it is due to ferricytochrome: Magnetic hyperfine splitting broadens it beyond the detection limit at 4.2K, but partial collapse of the magnetic splitting leaves an asymmetric at 130K. This doublet with assignment is confirmed by optical difference

Mössbauer spectroscopy

measurements between reduced and oxidized samples and by EPR. In two subsequent papers, Petrouleas and Diner showed that the Fe(II) of the iron–quinone complex can be reversibly oxidized and identified the Fe(III)/Fe(II) couple with the high-potential of PSII first described by electron acceptor Ikegami and Katoh (1973). Here, only the Mössbauer data will be discussed while all the supporting evidence will be skipped. Figure 6 shows spectra taken at different redox potentials of membranes from a mutant lacking PSI and the cytochrome complex (Diner and Wollman, 1980). Spectrum (a) of the untreated sample is comparable to that of Fig. 5a with the exception of a narrow quadrupole doublet assigned to ferrocytochrome. The spectrum of Fig. 6b was ob­ tained after oxidation of the sample with ferri­ cyanide to a potential of 450 mV. The high-spin Fe(II) doublet of the iron–quinone complex has disappeared completely, and a broad, ill-defined absorption in the range of is seen, comprising contributions from ferricytoch­ rome, ferro-ferricyanide and possibly from highspin Fe(III) with partially collapsed magnetic hy­ perfine splitting. On reduction of this sample with ascorbate to a potential of 300 ± 20 mV, Fig. 6c, the high-spin Fe(II) doublet of the iron–quinone complex reappears, but the spectrum differs from the original one because of the peak near due to ferrocyanide. Similar results were obtained by Picorel et al. (1994) with PSII isolated from the cyanobacter­ ium Phormidium laminosum. Figure 7 shows Mössbauer spectra obtained from oxygen-evolving core complexes at 77K (a) as isolated, (b)

367

after oxidation with ferricyanide, and (c) after removal of the ferricyanide and reduction by di­ thionite. Spectra (a) and (c) are indistinguishable and show two quadrupole doublets, one from the high-spin ferrous iron–quinone complex, the and where other from the cytochromes the latter is an impurity. The rather symmetrical spectrum obtained on oxidation is a superposition of the ferricytochrome doublet and a broad highspin Fe(III) line indicative of relatively fast spin fluctuations. Another important finding of Diner and Pe­ trouleas (1987) was the observation that the quad­ rupole splitting of the iron–quinone complex changes when bicarbonate is replaced by formate and vice versa as illustrated in Fig. 8. Semin et al. (1990) reported analogous results for PSII par­ ticles from Synechococcus elongatus. There must be a reversible change in the iron environment, but it is not clear yet whether either one of the compounds binds directly to the iron. Fig. 9, fin­ ally, shows the effect of NO binding on the Mössbauer spectrum of the iron–quinone com­ plex (Petrouleas and Diner, 1990; Diner and Pe­ trouleas, 1990). The intensity of the S = 2 doublet decreases on treatment with NO, and the lines at and grow instead. The authors do not provide a quantitative analysis, but it is clear that the isomer shift and the quadru­ pole splitting of the adduct are substantially smaller than for the S = 2 state. EPR shows that the Fe(II) NO complex has spin S = 3/2, and Mössbauer as well as EPR data are therefore analogous to those obtained for NO complexes of Fe(II) EDTA and dioxygenases (Arciero et al., 1983).

368

Peter G. Debrunner

B. Iron–sulfur Proteins EPR and MS have long been the major spectro­ scopic probes for the study of iron–sulfur proteins (Cammack et al., 1977), and PSI is no exception. EPR titrations identified three distinct but over­ lapping signals and that were assigned to iron–sulfur centers in PSI (Malkin and Bearden 1971). Evans et al. (1977, 1979, 1981) did the first Mössbauer studies on oxidized and re­ duced PSI samples and concluded that all three centers were 4Fe–4S clusters. Since the low-poremained controversial, having tential center been assigned to a 2Fe–2S cluster as well (Ber­ trand et al., 1988), Petrouleas et al. (1989) exam­ and (Parrett et al., 1989) ined PSI lacking and confirmed it to be a 4Fe–4S cluster. The iron–sulfur clusters of iron–sulfur proteins are built from the same structural unit, namely a high-spin iron coordinated to four sulfurs, where the sulfurs are either cysteine or bridging

Mössbauer spectroscopy

369

depending on how many of these units are joined together. The high covalency of the complexes leads to characteristically small isomer shifts and A-tensors that set them apart from other iron environments. As discussed in Section II.D, the magnetic properties of larger clusters are dominated by the strong exchange interac­ tions, Eq. 16, and for clusters with more than two irons by double exchange leading to delocalized valence pairs. All these properties have been studied extensively in small, well defined iron proteins as well as in model complexes, and the Mössbauer studies of PSI have revealed no new features apart from the unusually low redox po­ tential of the center. Accordingly, the dis­ cussion that follows will be kept brief. To con­ sider the simplest system first, it begins with the

370

mutant containing only and presents the com­ plete PSI later. Figure 10 shows the Mössbauer spectra ob­ tained by Petrouleas et al., (1989) from PSI core protein of Synechococcus 6301, a mutant of this and cyanobacterium that lacks the two centers but still contains The two spectra of the oxidized cluster on the right consist of an asym­ metric, broad doublet with at 77K, indicating that the four iron sites have similar, indistinguishable Mössbauer parameters. On cooling to 4.2K the spectrum just moves slightly to more positive velocities as ex­ pected from the second-order Doppler shift, Eq. 18, but it does not broaden magnetically. As shown in Table 2, the Mössbauer parameters clusters of spin match those of other S = 0. The oxidized protein is known to be EPR inactive, while the reduced protein with S = 1/2 shows the EPR signal that led to its discovery.

Peter G. Debrunner

The Mössbauer spectrum of the reduced pro­ tein on the left is marked ‘D’ after correction for a 15% admixture of the oxidized spectrum. It is still a broad doublet at 77K although with differ­ ent parameters, At 4.2K, on the other hand, the spectrum broad­ ens, extending from to and showing the features typical of magnetic hyp­ erfine splitting in as will be discussed below. Figure 11, finally, shows the 4.2K-Mössbauer spectra of PSI from the cyanobacterium Chlorog­ loea fritschii (Evans et al., 1981). These samples contain all three iron–sulfur centers and were partially reduced to show the EPR spectrum of only in (a), of and in (b), and of

Mössbauer spectroscopy

and in (c). The resolution of the spectra into oxidized and reduced material is shown, based on the spin Hamiltonian parameters de­ duced by Middleton et al. (1978) for the 4Fe­ ferredoxin from Bacillus stearothermophilus. These parameters clearly fit all three spectra quite well, and there is every reason to believe that and are the spectral contributions of indistinguishable, i.e. that all three are generic 4Fe–4S centers. It should be recalled that the has nominal Fe(II)Fe(III) pair in spin S' = 9/2 and delocalized valence while the Fe(II)Fe(II) pair has spin S'' = 4 to give a net spin of S = 1/2. The two pairs have different hyp­ erfine tensors Ã' and Ã'' and therefore contri­ bute distinct Mössbauer spectra as seen in Fig. 11.

C. Cytochromes Cytochrome is an intrinsic part of PSII and therefore appears in the spectra of Figs. 4–9, generally as an undesirable component that over­ laps the low-energy line of the iron-quinone doublet. The few Mössbauer parameters reported are summarized in Table 3. As a low-spin heme is diamagnetic protein, reduced cytochrome and shows a sharp quadrupole doublet in the

371

Mössbauer spectra at all temperatures. Oxidized on the other hand, has spin S = 1/2, is EPRactive and is expected to have a Mössbauer spec­ trum with wide magnetic hyperfine splitting at 4.2K, a spectrum that has collapsed near 80K to a quadrupole doublet with broad, asymmetric lines. Although cytochrome is of consider­ able interest, especially since it can exist in a highand a low-potential form associated, presumably, with a change in orientation of the axial histidine ligands, any meaningful Mössbauer studies would have to be done on isolated cy­ tochrome rather than on PSII preparations to achieve adequate signal-to-noise ratio. Since most of the work has been done by optical techniques and EPR, which are more sensitive (e.g. Babcock et al., 1985), the interested reader is referred to a Mössbauer study of a cytochrome b model (Walker et al., 1986) that deals with the question of the axial ligand orientation. References Abragam A and Bleaney B (1970) Electron Paramagnetic Resonance of Transition Ions. Oxford University Press. Aleksandrov AY, Novakova AA and Semin BK (1987) Mössbauer spectroscopy study of the conformational dy­ namics of native membrane proteins. Phys Lett 123: 151– 154.

372 Arciero DM, Lipscomb JD, Huynh BH, Kent T and Münck E (1983) EPR and Mössbauer studies of protocatechuate 4,5–dioxygenase. Characterization of a new environ­ ment. J Biol Chem 258: 14981–14991. Babcock GT, Widger WR, Cramer WA, Oertling WA and Metz JG (1985) Axial ligands of chloroplast cytochrome Identification and requirement of a heme-cross-linked polypeptide structure. Biochemistry 24: 3638–3645. Bertrand P, Guigliarelli B, Gayda J-P, Sétif P and Mathis P (1988) An interpretation of the peculiar magnetic proper­ ties of center X in Photosystem I in terms of a 2Fe–2S cluster. Biochim Biophys Acta 933: 393–397. Boso B, Debrunner P, Okamura MY, and Feher G (1981) Mössbauer spectroscopy studies of photosynthetic reaction centers from Rhodopseudomonas sphaeroides R-26. Biochim Biophys Acta 638: 173–177. Butler WF, Calvo R, Fredkin DR, Isaacson RA, Okamura MY and Feher G (1984) The electronic structure of in reaction centers from Rhodopseudomonas sphaeroides. III. EPR measurements of the reduced acceptor complex. Biophys J 45: 947–973. Cammack R, Dickson DPE and Johnson CE (1977) Evidence from Mössbauer spectroscopy and magnetic resonance on the active centers of the iron-sulfur proteins. In: Lovenberg W (ed) Iron–Sulfur Proteins Vol. III, pp. 283–330. Aca­ demic Press, New York. Cranshaw TE, Dale BW, Longworth GO and Johnson CE (1985) Mössbauer spectroscopy and its applications. Cam­ bridge University Press, Cambridge. Debrunner PG (1989) Mössbauer Spectroscopy of Iron Por­ phyrins. In: Lever ABP and Gray HB (eds.) Physical Bi­ oinorganic Chemistry Series. Iron Porphyrins Vol 3, pp. 137–234. VCH Publishers. Debrunner PG (1993) Mössbauer spectroscopy of iron prote­ ins. In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance Vol. 13, pp. 59–101. Plenum Press, New York and London. Debrunner PG, Schulz CE, Feher G and Okamura MY (1975) Mössbauer study of reaction centers from R. sphaeroides . Biophys J 15: 226a. Debus RJ, Okamura MY, and Feher G (1985) Reconstitution of iron-depleted reaction centers from Rhodopseudomonas sphaeroides R-26 with Fe, Mn, Cu and Zn. Biophys J 47: 3a. Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1985) Structure of the protein subunits in the photosynthetic reac­ tion centre of Rhodopseudomonas viridis at 3Å resolution. Nature (London) 318: 618–624. Dickson DPE and Berry FJ (1986) Mössbauer spectroscopy. Cambridge University Press, Cambridge. the non-heme iron of Diner BA and Petrouleas V (1987) the Photosystem II iron–quinone complex. A spectroscopic probe of quinone and inhibitor binding to the reaction center. Biochim Biophys Acta 895: 107–125. Diner BA and Petrouleas V (1990) Formation by NO of nitrosyl adducts of redox components of the Photosystem II reaction center. II. Evidence that binds to the acceptor-side non-heme iron. Biochim Biophys Acta 1015: 141–149. Diner BA and Wollman F-A (1980) Isolation of highly active

Peter G. Debrunner Photosystem II particles from a mutant of Chlamydomonas reinhardtii. Eur J Biochem 110: 521–526. Evans EH, Carr NA, Rush JD and Johnson CE (1977) Identi­ fication of a non-magnetic iron centre and an iron-storage or transport material in blue-green algal membranes by Mössbauer spectroscopy. Biochem J 166: 547–551. Evans EH, Rush JD Johnson CE and Evans MCW (1979) Mössbauer spectra of Photosystem I reaction centres from the blue-green alga Chlorogloea fritschii. Biochem J 182: 861–865. Evans EH, Dickson PE, Johnson CE, Rush JD and Evans MCW (1981) Mössbauer spectroscopic studies of the nature of centre X of Photosystem I reaction centres from the cyanobacterium Chlorogloea fritschii. Eur J Biochem 118: 81–84. Feher G, Allen JP, Okamura MY and Rees DC (1989) Struc­ ture and function of bacterial photosynthetic reaction centres. Nature (London) 339: 111–116. Gibb TC (1976) Principles of Mössbauer spectroscopy. Chap­ man and Hall, London. Gonser U (1975) Mössbauer Spectroscopy. Springer-Verlag, New York. Greenwood NN and Gibb TC (1971) Mössbauer Spectros­ copy. Chapman and Hall, London. Griffith JS (1957) Theory of electron resonance in ferrihaemo­ globin azide. Nature (London) 180: 30–31. Gütlich P, Link R and Trautwein A (1978) Mössbauer Spec­ troscopy and Transition Metal Chemistry. Springer-Verlag, New York. Huynh BH and Kent TA (1984) Mössbauer studies of iron proteins. In: Eichhorn GL and Marzili LG (eds.) Advances in Inorganic Biochemistry, Vol. 6, pp. 163–223). Elsevier, Amsterdam. Ikegami I and Katoh S (1973) Studies on chlorophyll fluor­ escence in chloroplasts II. Effect of ferricyanide on the induction of fluorescence in the presence of 3–(3,4–dichlorophenyl)-1,1–dimethylurea. Plant Cell Physiol 14: 829– 836. Ingalls R (1964) Electric-field gradient tensor in ferrous com­ pounds. Phys Rev 133: A781–A795. Keller H and Debrunner PG (1980) Evidence for coforma­ tional and diffusional mean square displacements in frozen aqueous solution of oxymyoglobin. Phys Rev Lett 45: 68– 71. Malkin R and Bearden AJ (1971) Primary reactions of photo­ synthesis. Photoreduction of a bound chloroplast ferre­ doxin at low temperature as detected by EPR spectroscopy. Proc Natl Acad Sci USA 68: 16–19. Michel H and Deisenhofer J (1988) Relevance of the photo­ synthetic reaction center from the purple bacteria to the structure of Photosystem II. Biochemistry 27: 1–7. Middleton P, Dickson DPE, Johnson CE and Rush JD (1978) Interpretation of the Mössbauer spectra of the four-iron ferredoxin from Bacillus stearothermophilus. Eur J Biochem 88: 135–141. Münck E, Papaefthymiou V, Surerus KK and Girerd JJ (1988) Double exchange in reduced clusters and novel clus­ ters with In Metal Clusters in Proteins, Que L (ed.), ACS Symposium Series, Vol. 372, pp. 302–325, Am Chem Soc, Washington, D.C.

Mössbauer spectroscopy Parrett KG, Mehari T, Warren PG and Golbeck JH (1989) Purification and properties of the intact P-700 and taining Photosystem I core protein. Biochim Biophys Acta 973: 324–332. Petrouleas V and Diner BA (1982) Investigation of the iron components in photosystem II by Mössbauer spectroscopy. FEBS Lett 147: 111–114. Petrouleas V and Diner BA (1986) Identification of a high-potential electron acceptor of Photosystem II, with the iron of the quinone–acceptor complex. Biochim Biophys Acta 849: 264–275. Petrouleas V and Diner BA (1990) Formation by NO of nitrosyl adducts of redox components of the Photosystem II reaction center. I NO binds to the acceptor-side nonheme iron. Biochim Biophys Acta 1015: 131–140. Petrouleas V, Brand JJ, Parrett KG, and Golbeck JH (1989) A Mössbauer analysis of the low-potential iron–sulfur cen­ ter in Photosystem I: Spectroscopic evidence that is a [4Fe–4S] cluster. Biochemistry 28: 8980–8983.

373 Picorel R, Williamson DL, Yruela I and Seibert M (1994) The state of iron in the oxygen-evolving core complex of the cyanobacterium Phormidium laminosum: Mössbauer spectroscopy. Biochim Biophys Acta 1184: 171–177. Schulz CE, Nyman P and Debrunner PG (1987) Spin fluctu­ ations of paramagnetic iron centers in proteins and model complexes: Mössbauer and EPR results. J Chem Phys 87: 5077–5091. Semin BK, Loviagina ER, Aleksandrov AY, Kaurov YN and Novakova AA (1990) Effect of formate on Mössbauer par­ ameters of the non-heme iron of PSII particles of cyanobac­ teria. FEBS Lett 270: 184–186. Taylor CPS (1977) The EPR of low spin heme complexes. hole model to the directional properties Relation of the of the g-tensor, and a new method for calculating the ligand field parameters. Biochim Biophys Acta 491: 137–149. Walker FA, Huynh BH, Scheidt WR and Osvath SR (1986) Models of the cytochrome b. Effect of axial ligand plane orientation on the EPR and Mössbauer spectra of low-spin ferrihemes. J Am Chem Soc 108: 5288–5297.

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Chapter 23

Characterization of Photosynthetic Supramolecular Assemblies Using Small Angle Neutron Scattering† David 2M. Tiede1,* and P. Thiyagarajan2

1

Chemistry Division D-200 and Intense Pulsed Neutron Source, Argonne National Laboratory,

Argonne, IL 60439, USA

Summary I. Introduction II. Small Angle Neutron Scattering A. Analysis of Scattering in the Very Small q Domain B. Scattering in the Intermediate q Domain III. SANS Studies of Photosynthetic Complexes A. Crystallization of Photosynthetic Proteins 1. Detergent Micelle Structures in Crystallization Conditions 2. Reaction Center Aggregation States B. Structural Characterization of Photosynthetic Supramolecular Assemblies C. Internal Structure in Supramolecular Assemblies D. New Results IV. Concluding Remarks Acknowledgements References

375 376 377 378 379 379 379 380 382 385 386 388 388 388 389

Summary

Small angle neutron scattering (SANS) offers opportunities for resolution of structure in molecular assemblies that can not be readily accessed by crystallography, such as inherently disordered assemblies, like micelles or vesicles, or multiple protein component complexes that can not be easily crystallized, like RC-cyt complexes or RC-antenna complexes. Scattering measurements on solution samples allow direct correlations to be made between structural features of Supramolecular assemblies and their spectroscopically determined function. Parameters which can be resolved by SANS include the size, shape, molecular weight, volume of macromolecules, internal packing for multiple component protein complexes. This information can be used to discriminate between possible molecular models for supra­ molecular structures. This chapter surveys possibilities for the application of this technique for the characterization of supramolecular assemblies in photosynthesis. SANS has been used to characterize the effect of ionic strength and detergents on reaction center aggregation. These measurements are being used to examine the pathways for reaction center crystallization. Applications are also presented for structural characterization of light-harvesting antenna complexes. Abbreviations: CMC – Critical micelle concentration; cyt – Cytochrome c; HT – Heptane-1,2,3–triol;

LDAO – Lauryldimethylamine-N-oxide; LH – Light-harvesting complex;

PEG – Polyethyleneglycol; RC – Reaction center; SANS – Small angle neutron scattering

†

The US government’s right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.

*Correspondence: Fax: 1-708-2529289, E-mail: [email protected]

375 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 375–390. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

376

I. Introduction Small angle neutron scattering (SANS) offers op­ portunities for the determination of solution structures of macromolecules with sizes in the range of 10Å to 500 Å which complements struc­ tural determination by other techniques. A distin­ guishing feature of neutron scattering compared to x-ray, electron and light scattering is that neu­ trons interact with atomic nuclei instead of elec­ trons. This property allows neutron scattering to be sensitive to both light and heavy atoms in a structure, including protons which are typically not observed in x-ray and electron scattering. Table 1 shows coherent neutron scattering and incoherent scattering cross sec­ lengths, tions, for selected elements compared to their x-ray scattering amplitudes in the forward direction, f(0). The magnitudes of the coherent neutron scattering lengths for H and D are seen to be nearly comparable to those for other atoms found in biological molecules, while large differ­ ences are seen for x-ray scattering amplitudes which increase with increasing atomic number. This implies that H and D will make significant contributions to neutron scattering for biological molecules, while they will make relatively weak contributions for x-ray scattering signals. Another significant feature of neutron scat­ tering that makes it suitable for probing biological structures is the relatively low energy of the neu­ tron beam. At wavelengths compatible for resolv­ ing structure on the 10 Å to 500 Å scale, the

David M. Tiede and P. Thiyagarajan millivolt energy of the neutron beam is a million fold less than the kilovolt energy used in compar­ able x-ray scattering experiments, resulting in several orders of magnitude reduction in radi­ ation damage. Furthermore, the penetration depth of the neutron beam allows neutron scat­ tering analysis to be made on aqueous samples with 1 to 5 millimeter path lengths. This pen­ etration depth allows SANS measurements to be made with samples similar to those encountered for the characterization of photosynthetic prote­ ins by optical spectroscopies. This concurrence in sample constraints will permit the same sample to be analyzed by optical spectroscopy and SANS, enabling direct comparisons between photosyn­ thetic function and structure. SANS is a widely applied scattering technique, and several reviews cover the application to bio­ logical systems (Jacrot, 1976; Stuhrmann and Miller, 1978; Chen, 1986; Feigin and Svergun, 1987). This technique allows extraction of form factors, which are descriptions of macromolecular size and shape, and particle–particle structure factors in solutions. This technique has also been demonstrated to provide structural information on supramolecular assemblies in their native state in aqueous solutions (Ramakrishnan et al., 1984). While form factors are a relatively low resolution image of a macromolecular assembly, this infor­ mation can precisely define the distribution of aggregation states for proteins in solution, charac­ terize protein packing in aggregates, and charac­

Small angle neutron scattering terize the dimensions of multiple component supramolecular assemblies such as protein-detergent complexes, or multiple component pro­ tein complexes. Unique opportunities also exist for the resolution of structure factors for selected components within complex mixtures by combin­ ing SANS measurements with isotopic substitu­ tion. This chapter will examine selected appli­ cations of SANS for resolution of molecular structures in photosynthesis.

377

characterization of the intraparticle structure fac­ tor. The intraparticle structure factor describes the scattering due to the atom position vectors within a particle: F(q) is the particle form factor, defined in terms and the of the atomic scattering lengths, with respect to the atomic position vectors, particle center of mass:

II. Small Angle Neutron Scattering Neutron scattering for proteins or other particles in solution is determined by the atomic composi­ tion and time-averaged position vectors of all nu­ clei in the sample. Neutron scattering is measured as a function of the scattering vector where is the neutron wavelength and is the angle of scattering. Persistent distance correlations between atoms in the sample pro­ duce variations in the scattered intensity due to interference effects of the scattered neutrons. Distance correlations between atoms will arise from the fixed atom positions within a particle, as well as due to the distribution of particles within the sample volume. As a result, neutron scattering, measured as the differential scattering cross section, can be written as the product of the number density of particles, n, the intraparticle structure factor, P(q) and the interparticle struc­ ture factor, S(q): S(q) makes a significant contribution to scattering under conditions in which the location of one particle affects the distribution of other particles around it. This correlation occurs in concentrated samples and in solutions of particles with surface charge (Chen, 1986; Chen et al., 1988). This ef­ fect can be exploited to investigate the nature of particle–particle interactions using SANS. In dilute solutions, or solutions of non-interacting particles, particle locations are not corre­ lated, and S(q) approaches 1. Under these con­ ditions, neutron scattering is determined exclusively by the number density of particles and their structure. Analysis of neutron scattering under these conditions yields the most accurate

The brackets in Eq. (2) indicate the average over all particle orientations. The scattered intensity can be written: where and are the position vectors of the i th and jth atoms within the particle, and and are the atomic scattering lengths. In general the scattering length of an atom, depends on the isotope and its spin states. The examples of scattering lengths listed in Table 1 are averages of scattering lengths taken over all spin states for specific isotopes. The distribution of spin states for a stable nucleus gives rise to two components to neutron scattering of a given atom, which are the coherent and incoherent scat­ tering cross sections. Both contribute to scat­ tering signals. For example H has a large incoher­ ent cross section while D has very little. Hydrogen rich biological molecules produce sig­ nificant amounts of incoherent scattering; how­ ever, if they are deuterated, the incoherent scat­ tering cross sections can be substantially reduced. The differential scattering cross section of a parti­ cle in Eq. (4) thus has two terms, where

The incoherent scattering component does not depend on the position vectors of the atoms. Ex­ perimentally it is observed as a flat background signal uncorrelated to structure. The coherent sig­

378

nal, on the other hand, depends on the position correlations of the atoms in the particle and it is the basis for obtaining structural information from SANS. Equation (4) describes neutron scattering due to intraparticle atom distance correlations in vac­ uum, but does not take into account the effect of placing the particle in a solvent. In the small and intermediate angle scattering domains, atom– atom correlations are measured over distances that are large compared to individual atom bond lengths. In these domains, the solvent acts as a continuum with an average scattering length defined as the sum of the scattering density, lengths for solvent atoms contained within a unit volume. Similarly, the particle can be considered as composed of volume elements whose scattering length densities are determined by the atomic composition within each unit volume: This allows the scattering due to the atom–atom correlations with the particle to be expressed in terms of scattering length density of individual volume elements: The scattering from the particle is detected with respect to the scattering length density of the solvent:

David M. Tiede and P. Thiyagarajan ing sections, analysis of these signals permits determination of size, shape and structural or­ ganization of the molecular assemblies. Table 2 lists mean scattering length densities for molecules relevant to the solubilization and crystallization of isolated photosynthetic proteins. These scattering length densities are seen to fall between the scattering length densities of and This allows scattering from selected components within these mixtures to be minim­ ratio so ized by adjusting the aqueous that the mean scattering length density of the solvent matches that of the selected component. Table 2 also shows the effectiveness of deuter­ ation for the enhancement of scattering length densities of biological molecules. For example the average scattering length density of proteins typi­ cally increases from to upon complete deuteration. The scattering characteristics of a subset of components within a macromolecular assembly can be resolved if they can be selectively deuterated, and by re­ cording the difference in scattering between unla­ belled and labelled material. Elegant demonstra­ tions of this technique include the resolution of phosphatidylcholine and bilesalt organization in rod-like mixed micelles (Hjelm et al., 1992), the resolution of subunit organizations in the 30S (Capel et al., 1987), and 50S (Nowotny et al., 1989) ribosomes and a RNA polymerase (Lederer et al., 1991).

A. Analysis of Scattering in the Very Small q Domain Hence, the difference in the scattering length densities of the particle and the solvent, termed contrast, dictates the intensity of the SANS sig­ nal. Equation 10 indicates that neutron scattering contrast can be varied either by changing the iso­ topic composition of the particle or of the solvent. This allows scattering due to selected volume ele­ ments to be minimized by matching their scat­ tering length densities to that of the solvent, while offering a mechanism for maximizing the contri­ bution of other volume elements by increasing their contrast. Thus, isotopic substitution, in com­ bination with contrast matching, is a powerful approach for the identification of scattering for selected components within complex macromole­ cular assemblies, while minimizing chemical per­ turbation of the system. As outlined in the follow-

At sufficiently low q, Eq. 10 can be shown to reduce to a particularly simple form, termed the Guinier equation (Guinier and Fournet, 1955): The Guinier approximation is valid for the scat-

tering region where the condition is met.

From a plot of ln[I(q)] vs. the size parameter,

and the forward scattering cross section, I(0), can be obtained. is the radius of gyration, which is defined as the root mean squared dis­ tance of all of the atoms to the centroid of the scattering length distribution. While does not directly resolve the macromolecular shape, the determination of by Guinier analysis is a sensi­ tive index for monitoring relative macromolecular

379

Small angle neutron scattering

size and aggregation state. I(0) is the amplitude of the scattering measured at q = 0:

where n is the number of macromolecules per unit volume, and, and V are the average scat­ tering length density and volume of the macromo­ lecule respectively. If SANS data are obtained on an absolute scale, then I(0) can be used for obtaining the molecular weight and volume of the particles, provided the concentration of the particle and the scattering length densities of the solvent and the particles are known.

B. Scattering in the Intermediate q Domain Since the determination of by Guinier analysis does not give information on the particle shape, this information has to be extracted by fitting I(q) data with model form factors. One procedure is to fit experimental scattering data with I(q) profiles calculated using atomic coordinates based upon crystal structures (Feigin and Svergun, 1987; Glatter, 1991). This approach has been used ex­ tensively with x-ray scattering. For example, this procedure has been used to determine the extent to which crystal structures of proteins are compat­ ible with their structure in solution (Fedorov and Denesyuk, 1978; Heidorn and Trewhella, 1988; Hubbard et al., 1988), and for the determination of the structure of protein aggregates in solution (Grossmann et al., 1993). For molecular assemblies for which there are no molecular models, such as with proteins or protein complexes that have not been crys­ tallized, or with inherently disordered structures

like micelles or membrane structures, a second approach has been to fit experimental data with form factors calculated from geometric shapes. Methods have been developed which range from fitting with simple geometric shapes of constant scattering length density, to fitting with multipole expansions of a set of spherically harmonic shape functions (Guinier and Fournet, 1955; Stuhrmann and Miller, 1978; Feigin and Svergun, 1987; Glatter, 1991; Svergun, 1991). These procedures provide tools for analysis of dimensions, shapes, and internal structures of supramolecular assemblies based on SANS mea­ surements. However, without the input of other structural information, SANS cannot generally determine a supramolecular form factor unam­ biguously. Instead, a set of distinct form factors, representing different supramolecular shapes and dimensions, may typically be found to reasonably fit experimental data recorded over a restricted q-range. Often this information can be combined with other physical or chemical data to resolve the most likely molecular structure. The following sections provide examples that illustrate how the SANS technique can contribute towards an understanding of the structural basis for function in photosynthetic supramolecular assemblies.

III. SANS Studies of Photosynthetic Complexes

A. Crystallization of Photosynthetic Proteins The crystallization of photosynthetic proteins is of crucial importance for investigation of photo­

380 synthetic mechanisms. However, there has only been limited success in the production of high quality crystals of photosynthetic membrane pro­ teins. The reasons for success or failure in crys­ tallization are not known, nor are the mechanisms for crystallization. Bacterial photosynthetic reaction centers pro­ vide a useful model for examining mechanisms for membrane protein crystallization. Reaction centers from two different species, Rhodopseudo­ monas viridis and Rhodobacter sphaeroides, have been successfully crystallized from detergent mix­ tures, and their molecular structures have been determined by x-ray diffraction (Miki et al., 1986; Allen et al., 1987; Yeates et al., 1987; Deisen­ hofer and Michel, 1989; Chang et al., 1991). So far, only two detergents have been found to yield crystals suitable for high resolution structural analysis. LDAO has been used successfully to produce high quality crystals of reaction centers, but only in conjunction with the addition of amphiphiles such as HT (Michel, 1982; Allen and Feher, 1990; Buchanan et al., 1993). Alternatively, OG has been used successfully in the absence of ad­ ditional amphiphiles (Allen and Feher, 1984; Chang et al., 1985; Ducruix and Reiss-Husson, 1987). Crystallization in the presence of LDAO was accomplished with a variety of precipitants, including ammonium sulfate (Michel, 1982), pot­ assium phosphate (Buchanan et al., 1993), and polyethylene glycol, PEG/sodium chloride mix­ tures (Allen and Feher, 1984; Allen and Feher, 1990). In contrast, successful crystallization in the presence of OG has only been reported using PEG/sodium chloride mixtures (Allen and Feher, 1984; Chang et al., 1985; Ducruix and Reiss-Husson, 1987; Franck et al., 1987). These crystalliza­ tion studies suggest that LDAO, unlike OG, re­ quires the addition of an amphiphile to permit crystallization, while LDAO is less sensitive to the chemical nature of the precipitant than is OG. The unique suitability of PEG/sodium chloride as a precipitant in the presence of OG is also sug­ gested by a comparison of the crystallization of 21 water-soluble proteins in the presence of OG (McPherson et al., 1986). OG was found to be generally beneficial for crystallization when PEG/sodium chloride mixtures were used as the pre-

David M. Tiede and P. Thiyagarajan cipitant, but not with ammonium sulfate as a pre­ cipitant (McPherson et al., 1986). These crystallization studies indicate that the conditions required for reaction center crystalliza­ tion are significantly regulated by detergent pro­ perties. Causes for the different requirements for crystallization in the presence of OG and LDAO have been suggested from SANS (Thiyagarajan and Tiede, 1994). These studies illustrate some of the capabilities of the SANS technique, and they are summarized below. 1. Detergent Micelle Structures in Crystallization Conditions Detergent micelle size, number density, and nat­ ure of inter-micelle interaction are all likely to be critical in determining micelle compatibility for crystallization. These properties can be directly accessed by SANS. SANS studies have identified clear differences in micelle structure and micelle– micelle interactions for the detergents OG and LDAO under conditions used for crystallization. For example, Fig. 1a and 1b show Guinier plots for 1% solutions of LDAO and OG respec­ tively, as a function of NaCl concentration. For LDAO in the absence of NaCl, scattering inten­ sity is seen to fall-off too quickly at low q. This is a clear indication of repulsive inter-micelle in­ teractions (Chen, 1986; Timmins et al., 1991; Thi­ yagarajan and Tiede, 1994). Much stronger devia­ tions of this type are seen for anionic detergents (Chen, 1986). For LDAO, this presumably re­ flects electrostatic interactions between micelles arising from the zwitterionic character of the mol­ ecule. Fig. 1a also shows that upon addition of 1M NaCl, the deviation from linearity is nearly completely removed, presumably due to dielec­ tric screening. This data shows that in the absence of NaCl, LDAO micelles will experience appreci­ able electrostatic repulsion. This behavior can be contrasted with the non­ ionic OG micelle. In the absence of salt, the mi­ celles were found to be non-interacting, as re­ flected by the linear plot throughout the range. In the case of OG solutions, Fig. 1 b, the addition of 1M NaCl caused a vertical shift of the plot, along with an upward deviation of the Guinier plot at low The upward deviation is a clear indication of a salt-induced aggregation of the

Small angle neutron scattering

OG micelle. This effect is the opposite of that seen with LDAO. The vertical shift is of interest since it reflects an increase in micelle number density. This data shows that the CMC of OG is much more strongly influenced by ionic strength than that of LDAO. The relative insensitivity of the observed slopes to ionic strength demon­ strates that LDAO and OG micelle sizes are not strongly affected by ionic strength. However, these SANS data have shown that these deterg­ ents differ significantly in the nature of inter-micelle interaction and response to salt additions. Fitting of SANS profiles to model form factors has also resolved differences in the size and shape of LDAO and OG micelles. The LDAO micelle was found to be larger, and best fit with an ellip­ soidal shape, while a spherical shape was found for the OG micelle (Thiyagarajan and Tiede, 1994). Using these procedures, a comparative study of OG and LDAO micelles under conditions used for reaction center crystallization has shown that the successful crystallization methods can be ra­ tionalized in terms of an optimization of micelle size, number density, and suppression of intermicelle interactions (Thiyagarajan and Tiede, 1994). LDAO and OG micelle characteristics in different solution conditions are schematically summarized in Figs. 2 and 3 respectively. These studies showed that the requirement for

381

HT for crystallization with LDAO as the solubil­ izing detergent can be understood from the ben­ eficial effects that this reagent had on LDAO micelle size and inter-micelle interactions. As in­ dicated in the top panel in Fig. 2, the LDAO micelle in the presence of NaCl and PEG is noninteracting, but retains the relatively large ellip­ soidal shape that presumably interferes with crys­ tallization. The addition of HT converts these micelles into smaller, spherical mixed-micelles. The bottom panel illustrates the finding that LDAO micelles are strongly associating in the and also retain the ellip­ presence of soidal shape. Under these conditions, the ad­ dition of HT was found to have the remarkable effect of dispersing the LDAO aggregates into smaller, non-interacting, spherical mixed-micelles. The use of HT in crystallization mixtures as with either PEG/NaCl mixtures or protein precipitants result in the formation of HT­ LDAO mixed-micelles having similar character­ istics. This property is consistent with the ob­ served flexibility in the choice of precipitant in crystallization schemes employing HT-LDAO mi­ celles. In the case of OG, both NaCl and additions were found to cause aggregation of mi­ celles, as indicated in the lower panel in Fig. 3. Uniquely, the addition of PEG to OG in NaCl solutions dissociated these aggregates, resulting

382

in the formation of relatively small, spherical, non-interacting micelles that are compatible with crystallization. The effect of PEG appeared to be due to a direct molecular interaction between PEG and OG, as the presence of PEG was found to raise the CMC of OG. These SANS studies have shown that LDAO and OG micelles are fundamentally different in terms of size, shape, and nature of inter-micelle interactions in non-crystallizing conditions. How­ ever, these differences were found to be minim­ ized under conditions used for protein crystalliza­ tion. Under crystallization conditions both LDAO-HT mixed micelles and OG micelles were found to be spherical, possibly reflecting a flexible radius of curvature; they were small relative to the size of the protein (micelle radius 17 Å–23

David M. Tiede and P. Thiyagarajan

Å, reaction center dimensions 74 Å × 70 Å × 40 Å), and non-interacting. These results suggest that these shared micelle characteristics are ne­ cessary features required to permit successful pro­ tein crystallization. Interestingly, these studies also showed that even smaller spherical micelles could be produced by the formation HT-OG mixed micelles in PEG/NaCl mixtures, as indi­ cated in the far-right panel in Fig. 3. These results suggest that conditions may be searched to further minimize possible micelle–micelle con­ tacts in the crystalline lattice. 2. Reaction Center Aggregation States The physical characteristics of isolated detergent micelles can be expected to influence the solubil­ ity and aggregation behavior of corresponding protein-detergent complexes. We have used SANS to study the aggregation states and inter­ particle behavior for the RC solubilized by OG and LDAO as a function of NaCl. Striking depen­ dencies of RC aggregation state on detergent and ionic strength were found. The aggregation be­ havior of the isolated RC can be understood from

Small angle neutron scattering

the ionic strength dependencies for inter-micelle interactions for these detergents. This work de­ monstrates the importance of the detergent mi­ celle characteristics in determining the solubility of the corresponding detergent-protein complex. As an example, Fig. 4 shows neutron scattering intensity, I(q), as a function of q for two fully deuterated RC samples in 0.03% LDAO. The samples are matched in RC and detergent con­ centrations, but differ by the addition or absence ratio was adjusted of 2 M NaCl. The to 5% to contrast match the detergent. Under these conditions, the scattering can unambigu­ ously be assigned as solely due to the RC. The figure shows that the scattering intensity is mark­ edly enhanced for LDAO solubilized RCs in the absence of NaCl compared to that in the presence of 2 M NaCl. The solid lines are fits using the procedure described below. Qualitatively, the marked difference in scattering intensity between the two samples identifies a higher aggregation state for the reaction center in the low-ionicstrength solution. Calculations based upon the reaction center crystal structure showed that the scattering profile for reaction centers in 2 M NaCl, 0.03% LDAO

383

can be fit by a monomeric state of the reaction center. This sample also allowed a test of the fitting of SANS data with low-resolution geomet­ rical form factors. Fig. 5a shows a fit to the scat­ tering profile in 2 M NaCl using a cylindrical form factor that was determined from a maximum entropy search method (Hjelm et al., 1990; Mor­ rison et al., 1992). The algorithm fits the scat­ tering data by searching a predefined dimension space using a distribution of particles of all pos­ sible sizes. This method has proven to be parti­ cularly effective in fitting polydisperse systems. The output shows that the reaction center sample at high ionic strength in LDAO can be fit by a monodispersed distribution of cylindrical part­ icles centered about a diameter of 68 Å and length of 60 Å. These dimensions correlate nicely with the average monomeric dimensions of 70 Å × 74 Å × 40 Å determined from the crystal structure. In contrast to a monomeric state of reaction centers in 2 M NaCl, 0.03% LDAO, fits to the SANS profile at low ionic strength requires a dis­ tribution of particles in two size ranges. A minor component is seen with dimensions consistent with monodispersed RCs. The major portion of the scattering is fit to a distribution of RC aggre­ gates having a length of more than 500 Å, which falls outside the size-domain for the experimental q-range. The increase in the size of the aggregate along a single dimension suggests a linear aggre­ gate. Similar measurements were also done with This RCs in solutions of OG and 17% ratio removes the scattering from OG micelles. Significantly, the aggregation state of the RC was also found to be ionic strength depen­ dent with this detergent, but the ionic-strength effect was opposite to that measured in LDAO. In OG, RCs were found to be predominately monomeric at low ionic strength, but a linear aggregate was found as the predominant form at high ionic strength. By comparison to the ionic strength dependen­ cies for LDAO and OG micelles described above, the SANS results for RCs demonstrate that a monodispersed state for the RC is only found under conditions in which detergent micelle–micelle interactions are removed. This result estab­ lishes the importance of micelle characteristics in

384

determining the solution behavior of the solubil­ ized protein-detergent complex. This approach can be extended to examine the RC aggregation states in crystallization mixtures as the sample proceeds through the pre-saturated, metastable and labile saturated states. This investigation of structural intermediates in crystallization mix­ tures can lead to an identification of crystalliza­ tion mechanism. We have begun such a characterization of the OG/PEG/NaCl crystallization method. As a first step, we have characterized the effect of the ad­ dition of PEG4000 at concentrations below the precipitation threshold for RCs solubilized by OG. Table 2 shows that the scattering length den­ sities for OG and PEG are nearly equivalent, and that both are far from that of a fully deuterated protein. SANS data for the deuterated RCs were which provided a contrast collected in 17% match for OG and PEG4000. Residual scattering due to PEG4000 could be eliminated by subtrac­ tion of scattering profiles collected for appropri­ ate reference samples in the absence of RCs. This method of data acquisition allowed SANS profiles for RCs to be exclusively detected without inter­ ference from either OG or PEG4000. Fig. 6a shows RC SANS profiles in the presence and absence of 10% PEG4000. In the absence of PEG4000 the scattering profile follows that ex­

David M. Tiede and P. Thiyagarajan

pected for monodispersed RCs. The scattering profile in the presence of 10% PEG4000 shows additional scattering at low q, corresponding to RC–RC correlations in aggregated states. A fit to the scattering profile with PEG4000 using the maximum entropy method is shown in Fig. 6b. The fit shows that the RCs are distributed be­ tween the monomeric state and a series of linear aggregated states. The fact that aggregation of OG micelles did not occur in the presence of PEG4000 shows that this effect is a property of the RC protein and not the detergent annulus. RC aggregation is observed with PEG4000 concentrations far from those required for RC precipitation and crystallization. It can be ex­ pected that as crystallization mixtures progress­ ively decrease the solubility of the RC either through increasing the ionic strength with fixed PEG concentration, or by simultaneously increas­ ing PEG and salt concentrations, the observed equilibrium between monomeric and aggregated RC states will be shifted towards the aggregate. The existence of this equilibrium has direct impli­ cations for the mechanism of RC crystallization. A prevalent view of protein crystallization as­ sumes that protein monomers serve as intermedi­ ates in crystallization (Feher and Kam, 1985; Durbin and Feher, 1986; Durbin and Feher, 1991). This view assumes that crystal growth oc­

Small angle neutron scattering

curs by the addition of protein monomers to the crystalline array, and that nucleation arises from an equilibrium between protein monomers and a crystalline aggregate. In this mechanism, the observed RC aggregation will act as a competing pathway, and optimization of crystallization must minimize the participation of non-crystalline ag­ gregation paths. However, an alternative me­ chanism is possible in which the observed aggre­ gates themselves function as intermediates in crystallization. In this mechanism, the key step in crystallization is the conversion of structurally compatible, non-crystalline aggregates into crys­ talline ones for nucleation, and the incorporation of compatible aggregates into a crystalline lattice during crystal growth. The participation of aggregates as intermedi­ ates in crystallization has also been suggested from light scattering (Skouri et al., 1992; Thibault et al., 1992; Forsythe and Pusey, 1994) and SANS (Boue et al., 1993) studies of crystallization mix­ tures for the water-soluble protein lysozyme. The similarity between these results and those from the reaction center SANS studies raises the possi­ bility that non-crystalline aggregates may be a general feautre of protein crystallization path­ ways.

385

B. Structural Characterization of Photosynthetic Supramolecular Assemblies In the SANS studies of RCs in solution, a match­ ing of experimental scattering with that calculated from the RC crystal structure unambiguously identified the scattering profile associated with the monodispersed RC state. Knowledge of the crystallographic dimensions of the RC also per­ mitted identification of the most appropriate fit­ ting of the RC with simple geometric form fac­ tors. The most definitive characterization of a supramolecular structure by SANS is achieved by fitting experimental data with scattering profiles calculated for molecular assemblies built from the crystal structures (Grossmann et al., 1993). Ulti­ mately this technique will be used to accurately characterize the structure of the RC aggregates and complexes of RC with other proteins. In the case of supramolecular assemblies for which there is no data on the structure of the constituents, the strength of SANS analysis lies in providing a test for plausible structural models. This capability can be illustrated by a SANS characterization of the light-harvesting antenna complexes of photosynthetic bacteria. The antenna of the purple bacteria consists of

386 two complexes, LH1 and LH2; for reviews see Hunter et al., (1989) and Zuber and Brunisholz, (1991). Each complex is composed of two short proteins, and containing approximately 50 pair is as­ amino acid residues. In LH1 each sociated with two bacteriochlorophyll molecules, while each pair is associated with three bacteri­ ochlorophylls in LH2. Functional energy delo­ calization occurs throughout an array of these building blocks, involving up to 200 or more chlorophylls (Holzwarth, 1991; Pullerits et al., 1994). There is no definitive characterization of the structure of the antenna assemblies either in the natural membrane or in detergent-isolated states. The fundamental building block of the LH1 and LH2 complexes has been proposed to be either or units, with cylindrically complexes (Hunter et al., 1989; symmetric Zuber and Brunisholz, 1991) or an complex (Kleinekofort et al., 1992) functioning as the min­ imal functional unit. SANS studies can discriminate between these possible models without detailed fitting of scat­ tering profiles to molecular models. For example, the two models for the minimal functional unit for LH2, and differ in molecular mass. Peptide and pigment composition analyses for the LH2 complex from Rb. sphaeroides (Zuber and Brunisholz, 1991) indicates that the molecular masses for these models are 88 kDa and 118 kDa respectively. Figure 7 shows a comparison of scattering pro­ files for deuterated Rb. sphaeroides LH2 and RC. Contributions from the solubilizing detergent, OG, were eliminated by contrast matching through solubilizing both samples in 17% solutions. Scattering profiles for LH2 were indis­ tinguishable with either LDAO solutions or OG as the solubilizing detergent, and using 5% and 17% respectively to contrast match the detergent. Model fitting and Guinier analysis show that both samples are monodispersed. The scattering profiles in Fig. 7 were normalized at low q. This normalization illustrates that the fall­ off in scattering intensity starts at a lower q for LH2 than for RC. This unambiguously establishes that the LH2 assembly has a structure with longer atom-atom distance correlations than does the RC. This is also indicated by a comparison of of 31±1.5 Guinier analysis which yields an

David M. Tiede and P. Thiyagarajan

Å and 38 ± 1.3 Å for the RC and LH2 complex respectively. These observations require that the LH2 complex have slightly larger size than the RC. Maximum entropy fitting procedures found that the scattering profiles for LH2 can be fit using a solid, cylindrical form factor having a particle of 68 Å diameter and length of 64 Å The dimensions and molecular mass of the pro­ posed models (Hunter et al., 1989; Zuber and Brunisholz, 1991) are smaller than those for the RC. Thus, the SANS data rule out the complex as a possible structure for the LH2 in these samples. The molecular mass of the model is 1.17 fold larger than that of the RC. Form factors consistent with this molecular mass can be fit to the LH2 scattering data, and hence the model is more consistent with the SANS results. This example demonstrates how SANS data that resolves only the overall size and mass density of a supramolecular assembly can be used for identification of possible structural models.

C. Internal Structure in Supramolecular Assemblies In addition to a resolution of particle size and mass density, SANS measurements offer oppor­ tunities for resolution of internal structures in

Small angle neutron scattering

supramolecular assemblies. In the q-range up to this can be illustrated with SANS mea­ surements for the LH1 complex. Figure 8 shows SANS profiles for the Rb. sphaeroides LH1 at two different concentrations. Maximum entropy analysis required dispersions of LH1 particle sizes to fit both profiles. Both samples were found to be composed of different mixtures of particles with diameters of approximately 60 Å and 130 Å. A dispersion of particle sizes, which varied as a function of LH1 concentration, was also found by size-exclusion chromatography and electron microscopy of LH1 (Boonstra et al., 1993). This behavior is reflected in the SANS data. The shoulder seen in the scattering profile for the more concentrated sample in Fig. 8 at apis noteworthy, since fea­ proximately tures of this kind have not been seen in any of the monodispered or aggregated RC samples, nor in the LH2 samples. Additionally, this feature was not fit using solid cylindrical form factors in the polydispersity fitting analysis. One intriguing possibility is that this feature arises from the in­ ternal structure, or packing of subunits within the larger LH1 particles, as suggested from the following modelling studies. Figure 9 shows scattering profiles calculated

387

for two model structures. The first was a cylindri­ cally symmetric ring composed of six cylinders, having a 50 Å diameter and 60 Å length. Each cylinder represents a geometrical form factor ap­ proximation to the unit. This model has a hole in the middle with a size sufficient to contain a seventh cylinder. The scattering profile calcu­ lated for this model is shown by the solid line in Fig. 9. One noticeable feature is the shoulder in the q region to that arises from intra-particle interference effects. The shape of this feature is similar to that seen in the experi­ mental data. The sensitivity of the shoulder to the presence of the hole in the model structure is illustrated by the scattering profile marked by the dashed line in Fig. 9, which was calculated for a model consisting of the same 6 cylinder ring, but that additionally contained a seventh cylinder in the center. This filled ring model removed the shoulder seen in the scattering profile in the q region to Fits to the experi­ mental data will require optimization of the model as well as inclusion of effects due to aggre­ gate size dispersity. However, these calculations demonstrate the plausibility of a ring-like as­ sembly to give rise to the observed scattering fine structure for LH1. Hence, the SANS data support structural models for the LH1 at high concentra­ tion that incorporate ring-like structures. The ability to detect internal structure within

388 the LH1 assembly at high concentration arises because of the large size of the aggregated as­ sembly, with corresponding large structural repe­ ats and scattering length density discontinuities. These results offer encouragement for the appli­ cation of SANS measurements at higher q to probe structural repeats of smaller dimension, such as the internal packing of protein in smaller assemblies such as the LH2 and RC complexes.

D. New Results Since the first draft of this chapter, a high resol­ ution, R = 2.5 Å, structure has been determined for the LH2 complex from Rps. acidophila by Xray diffraction on single crystals (McDermott et al., 1995), and a low-resolution, R = 8.5 Å, struc­ ture has been determined for the LH1 from Rho­ dospirillum by electron microscopy on two-dimensional crystals (Karrasch et al., 1995). The crystallography of LH2 showed the com­ plex to be composed of 9 pairs, arranged sym­ metrically in a barrel-shaped bundle, with a 36 Å diameter hole in the center. The overall dimen­ sions of the LH2 complex were 68 Å diameter by 70 Å length (McDermott et al., 1995). These dimensions are in approximate aggreement with the 68 Å by 64 Å dimensions determined by our fitting of the SANS data for the Rb. sphaeroides LH2 complex in solution. It is likely that the differences in measured dimensions arise from the use of cylindrical form factors as approxi­ mations of the protein shape for fitting the SANS data. Å more exact comparison of the crystal and solution structures for LH2 will require compari­ sons of experimental SANS profiles with those calculated from atomic crystal coordinates. As discussed for the LH1 complex, a hollow or ring­ like structure will give rise to characteristic undu­ lations in SANS profiles due to the cylindrical symmetry. Undulations due to a 36 Å diameter hole in the LH2 structure would not be seen in the present SANS profiles recorded in the q-range These features would be expected to appear at higher q-values. The two-dimensional crystals of the R. rubrum LH1 showed the complex to be composed of 16 pairs arranged, on average, in a 116 Å ring with a 68 Å hole in the middle (Karrasch et al.,

David M. Tiede and P. Thiyagarajan 1995). A ring-like structure was also anticipated from SANS data for the Rb. sphaeroides LH1 in solution, (Fig. 8), which has a secondary maxi­ mum at Preliminary modelling sug­ gests that the ring-like structure described by Kar­ rasch et al. can account for the shoulder seen in the scattering profile for the Rb. sphaeroides LH1. However, a detailed modelling of the solu­ tion scattering profiles will require that particle size dispersity be taken into account. The electron microscopy of the two-dimensional LH1 arrays showed that there were small ellipitical distor­ tions in the ring shape in different crystals, and that there were different crystalline forms com­ posed of LH1 particles with different dimensions (Karrasch, S. et al., 1995). A detailed evaluation of the solution SANS data in light of the new structured information is underway. IV. Concluding Remarks SANS studies on photosynthetic complexes de­ monstrate the capability of this technique for re­ solving inter-particle interactions, size, shape and internal order or packing of supramolecular as­ semblies pertinent to photosynthesis. Opportuni­ ties for definitive assignment of supramolecular structures are possible by comparison of experi­ mental scattering data with scattering profiles cal­ culated for molecular models built from crystal structures of the composite proteins. Photosynth­ esis ultimately relies upon a hierarchy of struc­ tures and intermolecular interactions. SANS pro­ vides a complement to crystallographic studies by providing a technique for assessing structure in functional, supramolecular assemblies that can not be examined by crystallography. Acknowledgements The authors thank Dr. Rex Hjelm (Los Alamos National Laboratory) and Dr. D. S. Sivia (ISIS) for use of the maximum entropy fitting program used to characterize the size distributions of pho­ tosynthetic complexes, and Dr. Stephen Hender­ son (Oak Ridge National Laboratory) for the use of his program BIOMOD for the calculation of scattering profiles for model structures in Fig. 9. This work was supported by the U.S Department of Energy, Office of Basic Energy Sciences, Divi­

Small angle neutron scattering sion of Chemical Sciences and the Division of Material Sciences, under Contract W-31–109– Eng-38 and P.T. was additionally supported in part, by a NASA Microgravity Biotechnology Program Grant M951–ES-3–004–2511. References Allen JP and Feher G (1984) Crystallization of reaction center from Rhodopseudomonas sphaeroides: preliminary charac­ terization. Proc Natl Acad Sci USA 81: 4795–4799. Allen JP and Feher G (1990) Crystallization of reaction cen­ ters from Rhodobacter sphaeroides. In: Michel H (ed) Crys­ tallization of Membrane Proteins, pp 137–154. CRC Press, Boca Raton. Allen JP, Feher G, Yeates TO, Komiya H and Rees DC (1987) Structure of the reaction center from Rhodobacter sphaeroides R-26: The protein subunits. Proc Natl Acad Sci. USA 84: 6162–6166. Boonstra AF, Visschers RW, Calkoen F, van Grondelle R, van Bruggen EFJ and Boekema EJ (1993) Structural characterization of the B800–850 and B875 light-harvesting antenna complexes from Rhodobacter sphaeroides by elec­ tron microscopy. Biochim. Biophys Acta 1142: 181–188. Boue F, Lefaucheux F, Robert MC and Rosenman I (1993) Small angle neutron scattering study of lysozyme solutions. J Crystal Growth 133: 246–254. Buchanan SK, Fritzsch G, Ermler U and Michel H (1993) New crystal form of the photosynthetic reaction centre from Rhodobacter sphaeroides of improved diffraction quality. J Mol Biol 230: 1311–1314. Capel MS, Engelman DM, Freeborn BR, Kjeldgaard M, Langer JA, Ramakrishnan V, Schindler DG, Schneider DK, Schoenborn BP, Sillers I-Y, Yabuki S and Moore PB (1987) A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli. Science 238: 1403– 1406. Chang C-H, Schiffer M, Tiede DM, Smith U and Norris JR (1985) Structure of the membrane-bound protein photosyn­ thetic reaction center from Rhodopseudomonas sphaero­ ides R-26 by x-ray diffraction. J. Mol. Biol. 186: 201–203. Chang C-H, El-Kabbani O, Tiede DM, Norris J and Schiffer M (1991) Structure of the membrane-bound protein photo­ synthetic reaction center from Rhodobacter sphaeroides. Biochemistry 30: 5352-5360. Chen SH (1986) Small angle neutron scattering studies of the structure and interaction in micellar and microemulsion systems. Annu Rev Phys Chem 37: 351–399. Chen SH, Sheu EY, Kalus J and Hoffman H (1988) Smallangle neutron scattering investigation of correlations in charged macromolecular and supramolecular solutions. J Appl Cryst 21: 751-769. Deisenhofer J and Michel H (1989) The photosynthetic reac­ tion center from the purple bacterium Rhodopseudomonas viridis (Noble Lecture). Angew Chem Int Ed Engl 28: 829– 968. Ducruix A and Reiss-Husson F (1987) Preliminary charac­ terization by x-ray diffraction of crystals of photochemical

389 reaction centres from wild-type Rhodopseudomonas sphaeroides. J Mol Biol 193: 419–421. Durbin SD and Feher G (1986) Crystal growth studies of lysozyme as a model for protein crystallization. J Crystal Growth 76: 583–592. Durbin SD and Feher G (1991) Simulation of lysozyme crystal growth by the Monte Carlo method. J Crystal Growth 110: 41–51. Fedorov BA and Denesyuk AI (1978) Large-angle x-ray dif­ fuse scattering, a new method for investigating changes in the conformation of globular proteins in solution. J Appl Cryst 11: 473-477. Feher G and Kam Z (1985) Nucleation and growth of protein crystals: general principles and assays. Methods Enzy­ mology 114: 77–112. Feigin LA and Svergun DI (1987) Structure Analysis by Small Angle X-ray and Neutron Scattering 1–335. Forsythe E and Pusey ML (1994) The effects of temperature and NaCl concentration on tetragonal lysozyme face growth rates. J Crystal Growth 139: 89–94. Franck HA, Taremi SS and Knox JR (1987) Crystallization and preliminary x-ray and optical spectroscopic characteriz­ ation of the photochemical reaction center from Rhodo­ pseudomonas sphaeroides strain 2.4.1. J Mol Biol 198:139– 141. Glatter O (1991) Small-angle scattering and light scattering. In: Lindner P and Zemb T (ed) Neutron, X-ray and Light Scattering, pp 33–82. Elsevier, Amsterdam. Grossmann JG, Abraham ZHL, Adman ET, Neu M, Eady RR, Smith BE and Hasnain SS (1993) X-ray scattering using synchrotron radiation shows nitrite reductase from Achromobacter xylosoxidans to be a trimer in solution. Biochemistry 32: 7360–7366. Guinier A and Fournet G (1955) Small Angle Scattering. John Wiley and Sons, New York. Heidorn DB and Trewhella J (1988) Comparison of the crystal and solution structures of calmodulin and troponin c. Bio­ chemistry 27: 909–915. Hjelm RP, Thiyagarajan P, Sivia DS, Lindner P, Alkan HA and Schwahn D (1990) Small-angle neutron scattering from aqueous mixed colloids of lecithin and bile salts. Prog Col­ loid Polym Sci 81: 225–321. Hjelm RP, Thiyagarajan P and Alkan-Onyuksel H (1992) Organization of phosphatidylcholine and bile salt in rodlike mixed micelles. J Phys Chem 96: 8653–8661. Holzwarth AR (1991) Excited state kinetics in chlorophyll systems and its relationship to the functional organization of the photosystems. In: Scheer H (ed) Chlorophylls, pp 1125–152. CRC Press, Boca Raton. Hubbard ST, Hodgson KO and Doniach S (1988) Small-angle x-ray scattering investigation of the solution structure of troponin c. J Biol Chem 263: 4151–4158. Hunter CN, van Grondelle R and Olsen JD (1989) Photosyn­ thetic antenna proteins: 100 ps before photochemistry starts. Trends Bioch Sci 14: 72–76. Jacrot B (1976) The study of biological structures by neutron scattering from solution. Rep Prog Phys 39: 911–953. Karrasch S, Bullough PA and Ghosh R (1995) The 8.5 Å projection map of the light-harvesting complex I from Rho­

390 dospirillum rubrum reveals a ring composed of 16 subunits. EMBO J 14: 631–638. Kleinekofort W, Germeroth L, van der Broek JA, Schubert D and Michel H (1992) The light-harvesting complex II (B800/850) from Rhodospirillum molischianum is an octa­ mer. Biochim Biophys Acta 1140: 102–104. Lederer H, Mortensen K, May RP, Baer G, Crespi H, Dersch D and Heumann H (1991) Spatial arrangement of and core enzyme of Escherichia coli RNA polymerase: A neutron solution scattering study. J Mol Biol 219: 747–755. McDermott G, Prince SM, Freer AA, Hawthornthwaith-Lawless AM, Papiz MZ, Cogdell RJ and Isaacs NW (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374: 517– 521. McPherson A, Koszelak S, Axelrod H, Day J, Williams R, Robinson L, McGrath M and Cascio D (1986) An experi­ ment regarding crystallization of soluble proteins in the J Biol Chem 261: 1969–1975. presence of Michel H (1982) Three-dimensional crystals of a membrane protein complex. The photosynthetic reaction centre from Rhodopseudomonas viridis. J Mol Biol 158: 567–572. Miki K, Saeda M, Masaki K, Kasai N, Miki M and Hayashi K (1986) Crystallization and preliminary x-ray diffraction study of ferrocytochrome from Rhodopseudomonas vir­ idis. J Mol Biol 191: 579–580. Morrison JD, Corcoran JD and Lewis KE (1992) The determi­ nation of particle size distributions in small-angle scattering using the maximum-entropy method. J Appl Cryst 25: 504– 513. Nowotny V, Nowotny P, Voss H, Nierhaus KH and May RP (1989) The quaternary structure of the ribosome from Escherichia coli- A neutron small-angle scattering study. Physica B 156: 499–501. Pullerits T, Visscher KJ, Hess S, Sundstrom V, Freiberg A, Timpmann K and R. vG (1994) Energy transfer in the inhomogeneously broadened core antenna of purple bac­ teria: A simultaneous fit of low-intensity picosecond ab­ sorption and fluorescence kinetics. Biophys J 66: 236–248.

David M. Tiede and P. Thiyagarajan Ramakrishnan VR, Capel M, Kjeldgaard M, Engleman DM and Moore PB (1984) Position of protein S14, protein S18 and protein S20 in the 30S ribosomal subunit of Escherichia coli. J Mol Biol 174: 265–284. Sears VF (1986) Neutron scattering lengths and cross sections. In: Sköld K and Price DL (ed) Neutron Scattering. Me­ thods of experimental physics, Celotta R and Levine J (series eds) 23A, pp 521–549. Academic Press, New York. Skouri M, Munch J-P, Lorber B, Giege R and Candau S (1992) Interactions between lysozyme molecules under precrystallization conditions studied by light scattering. J Crys­ tal Growth 122: 14–20. Stuhrmann HB and Miller A (1978) Small-angle scattering of biological structures. J Appl Cryst 11: 325–345. Svergun DI (1991) General theorems of small-angle scattering by disperse systems. In: Lindner P and Zemb T (ed) Neu­ tron, X-ray and Light Scattering, pp 83–98. North-Holland, Amsterdam. Thibault F, Langowski J and Leberman R (1992) Optimizing protein crystallization by aggregate size distribution analy­ sis using dynamic light scattering. J Crystal Growth 122: 50–59. Thiyagarajan P and Tiede DM (1994) Detergent micelle struc­ ture and micelle-micelle interactions determined by small angle neutron scattering in solution conditions used for membrane protein crystallization. J Phys Chem 98: 10343– 10351. Timmins PA, Hauk J, Wacker T and Welte W (1991) The influence of heptane-1,2,3–triol on the size and shape of LDAO micelles. FEBS Letts 280: 115–120. Yeates TO, Komiya H, Rees DC, Allen JP and Feher G (1987) Structure of the reaction center from Rhodobacter sphaeroides R-26: Membrane-protein interactions. Proc Natl Acad Sci USA 84: 6438–6442. Zuber H and Brunisholz RA (1991) Structure and function of antenna polypeptides and chlorophyll-protein complexes: Principles and variability. In: Scheer H (ed) Chlorophylls, pp 627–703. CRC Press, Boca Raton.

Chapter 24

Measurement of Photosynthetic Oxygen Evolution

Hans J. van Gorkom* and Peter Gast

Department of Biophysics, Huygens Laboratory of the State University, P.O.Box 9504,

2300 RA Leiden, The Netherlands

Summary I. Introduction II. Polarography A. The Clark Electrode B. Unwanted Chemistry at the Bare Cathode C. Rate Electrodes D. Concentration Electrodes E. Polarograms F. Flash-Induced Kinetics III. EPR Oximetry A. EPR Line Broadening by Oxygen B. Oxygen Probes for EPR C. Sensitivity 1. Sensitivity in Direct Linewidth Broadening Measurement 2. Sensitivity in Amplitude Measurement D. Applications in Photosynthesis Research IV. Mass Spectrometry V. Photoacoustic Spectroscopy VI. Galvanic Sensors VII. Prospects Acknowledgements References

391 392 392 392 393 394 395 395 397 398 398 398 399 399 399 401 401 402 402 402 403 403

Summary An overview is presented of methods that have been used to measure photosynthetic oxygen evolution over the past 25 years. Oxygen polarography in its many versions is treated in some detail, the complications caused by a large bare cathode are discussed and an interpretation of the current/voltage characteristic (polarogram) of flash-induced oxygen signals is proposed. The recent controversy on the interpretation of the kinetics of such signals is briefly summarized. The discovery of a new class of spinprobes for EPR oximetry has greatly enhanced its possibilities. The sensitivity of the method is evaluated, consequences of its nonlinearity in time-resolved measurements are indicated, and the first reports on its use in photosynthesis research are summarized. The use of mass spectrometry, photoacoustic spectro­ scopy and galvanic sensors to measure photosynthetic oxygen evolution is briefly reviewed.

*Correspondence: Fax: 31-71-5275819; E-mail: [email protected]

391 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 391–405. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

392

Hans J. van Gorkom and Peter Gast

I. Introduction

II.

Polarography

Oxygen evolution was the first photosynthetic re­ action discovered and two centuries of research have left an impressive track record of methods applied to measure it (Rabinowitch, 1945, 1951, 1956; Burr and Mauzerall, 1968; Joliot, 1993). It is useful to keep old methods in mind. A striking example was the recent application of the classical work of T. Engelmann in the 1880's, who used bacteria with oxotropic motility to locate the source of photosynthetic oxygen at the micros­ copic level: this method allowed V. Zimmermann's group to select the photosynthetically competent cells among hybrids produced by elec­ trofusion (Hampp et al., 1986). Due to its impor­ tant medical and industrial applications, the mea­ surement of oxygen has received much attention. A wide range of methods has been reviewed in a symposium dedicated to the subject (Degn et al., 1976). In biological and medical research, polar­ ography has become the predominant technique, on which extensive documentation is available (Fatt, 1976; Hitchman, 1978; Gnaiger and Forstner, 1983). Also in photosynthesis research polarography has replaced the traditional War­ burg manometry, a variety of electrochemical cells has been designed and other ways of measur­ ing oxygen have been explored. Here we present a survey of such methods as published over the past 25 years or so, after the discovery of the period four oscillation of the oxygen yield with flash number upon illumination of Photosystem II with a series of short saturating flashes (Joliot et al., 1969). This finding marked a shift of scien­ tific interest from rate measurements in continu­ ous light to the measurement of oxygen yields of individual flashes in a series. In recent years also the kinetics of oxygen release after a flash have been at the focus of attention, requiring measure­ ments with even higher time resolution. In this chapter we concentrate on methods meeting these requirements and, due to our own involvement, pay some extra attention to the interpretation of time-resolved polarographic signals and to a promising new class of spin-probes for EPR-oximetry.

A. The Clark Electrode Routine measurements of photosynthetic oxygen evolution are now carried out with commercially available equipment based on the Clark electrode (Clark, 1956). This is a ‘bipolar' electrochemical oxygen sensor: the cathode and anode and the connecting electrolyte solution are separated from the sample by a teflon membrane, which is permeable to oxygen but not to water and ions. The platinum cathode reduces oxygen to hydroxyl ions. At the silver anode initially AgCl is formed and later, as accumulates, also AgOH. Apart from its small-oxygen consumption it is essentially a closed system, isolated from the sam­ ple by the teflon membrane, and can be used to measure oxygen in the gas phase (Delieu and Walker, 1983) as well as in solution. The use of a small cathode and stirring of the sample (if in solution) ensure that the oxygen concentration gradient is restricted to the membrane and the use of a sufficiently low cathode potential, about –0.7 V relative to the anode, ensures that this gradient stays at its maximum value because the cathode surface is kept anaerobic. The measured current is simply proportional to the oxygen ten­ sion (partial pressure) in the sample and is easily calibrated by comparison to the signal in an airsaturated solution at the same temperature. The oxygen concentration then follows from the ox­ ygen solubility in the medium used (which can be much reduced at high salt concentrations). The properties and theoretical background of the Clark electrode are described most comprehen­ sively in the monograph by Hitchman (1978), which also contains oxygen solubility data. In photosynthesis research, situations often arise where the unmodified Clark electrode is too insensitive, too slow, or both. In particular, it does not allow resolution of the individual oxygen yields of successive flashes in a series, which is required to study the 4-flash redox cycle of the oxygen evolving complex. Sufficient sensitivity for this purpose may be reached by a substantial increase of the cathode surface area (Velthuys and Kok, 1978; Lübbers et al., 1993), maintaining the advantages of the physical separation by the teflon membrane between sample and electro­

Oxygen evolution chemistry. However, even with vigorous stirring it will be difficult to prevent the oxygen diffusion gradient from spreading into the the sample and the advantage of easy calibration is probably lost. With a thin membrane, the time-resolution may be good enough for single flash resolution at a usual 1 Hz flash frequency. A further enhance­ ment of sensitivity and time-resolution can only be obtained by removing the membrane, sacrific­ ing all advantages of the Clark electrode concept.

B. Unwanted Chemistry at the Bare Cathode When a bare cathode is used, the sample must contain the electrolyte connecting the cathode and anode and a buffer to avoid excessive pH production at the cathode, increase due to and should preferably not contain any substance that could mediate electrochemical reduction of redox centers in the sample. A polarized bare platinum electrode covered by a thin sample layer is the standard tool for electrochemical titrations and can readily impose its potential on all redox couples in the sample if adequate mediators are present. Fortunately, most redox centers in Pho­ tosystem II are highly inaccessible and the ‘sub­ strate binding sites’, where plastoquinone reduc­ tion and water oxidation take place, seem to allow reaction with a very limited variety of mol­ ecules only. Even the mobile plastoquinone pool is not rapidly reduced by the cathode if no media­ tors are added. However, the cathode itself may produce redox mediators. Hydrogen peroxide, formed at the cathode surface as an intermediate in the re­ duction of to can interact with the ox­ ygen evolving complex, especially in Photosystem II preparations where the protective shield of ex­ trinsic polypeptides has been damaged (Schröder and Åkerlund, 1986). Addition of catalase might help to avoid this complication, but one should keep in mind that there is also evidence for the production of hydrogen peroxide by Photosystem II itself under some circumstances (Wydrzynski et al., 1989). The inhibition observed by Plijter et al. (1988) at cathode potentials lower than –0.5 V vs SHE (Standard Hydrogen Electrode) may be due to hydrogen produced at the cathode surface. As the inhibition appears to be an irreversible

393 all-or-none effect (the shape of the period four oscillation in a flash series remains the same), this might be due to over-reduction and subsequent dissociation from the reaction center of or manganese. This problem can be avoided by weaker polarization, and depends on pH and cathode material, Pt being a much more efficient catalyst for hydrogen production than Au. Last but not least, the cathode removes oxygen and oxygen usually is involved in poising the redox potential in the sample. There have been reports that oxygen evolution requires oxygen (Bader and Schmid, 1988). The oxygen concentration near the surface of a strongly polarized cathode surface can become quite low (Baumgärtl et al., 1974) and it is primarily the oxygen evolution in this region that one measures. The modification of the chemical conditions in the sample by the cathode is normally limited by using a thin sample layer exposed on the other side to a continuous flow of conditioning medium (rate electrodes). It can be avoided altogether by a continuous, very fast sample replacement (Etienne, 1968) or mini­ mized by using a very weak polarization (Plijter et al., 1988) (concentration electrodes). Moreover, the experiment may require the ad­ dition of chemicals, such as an artificial electron acceptor when isolated Photosystem II particles are used. Joliot et al. (1966) introduced modu­ lated illumination and lock-in detection to mea­ sure selectively the electrode current resulting from photosynthetic activity (see also Joliot, 1972). Particularly troublesome, however, are substances which produce spurious signals upon flash illumination due to photochemistry in the sample or at the cathode surface. In some cases such signals can be avoided by applying a thin film of collodion on the cathode. With a flat disk electrode this can be done conveniently by letting a drop of collodion solution fall on the cathode. By choice of solvent, collodion concentration, drop size and speed, one can obtain reproducible films of the right diameter to cover the cathode and thin enough not to affect the kinetics of the flash induced oxygen signals. In one respect the measurement of oxygen evolution should be simplified by the use of a bare cathode. The cathode current is proportional to the oxygen concentration in a boundary layer adjacent to the cathode surface. With a bare cath­

394 ode there is no phase separation between this layer and the sample and one measures concen­ tration rather than partial pressure, independent of the oxygen solubility in the medium used. Un­ fortunately, this potential advantage is more than offset by other difficulties involved in quantitative calibration of signals obtained with a bare cath­ ode. In fact no such calibration seems to have been achieved, except for the turbulent flow – concentration electrode used by Etienne (1968), and one has to resort to comparison of the signal to that obtained with a more easily calibrated method.

C. Rate Electrodes Until about 25 years ago, photosynthesis re­ searchers measuring oxygen evolution were often primarily interested in highly precise measure­ ments of the rate of photosynthesis to study in­ duction phenomena and action spectra and so­ phisticated electrochemical cells had been developed for this purpose, as reviewed by Fork (1972). The design of these cells was based on the pioneering work of L. Blinks and coworkers. Blinks and Skow (1938) already used photosyn­ thetic material appressed to a large surface plati­ num cathode. Haxo and Blinks (1950) introduced the ‘rate electrode’, using a semi-permeable membrane to keep the sample appressed to the cathode and immersing the ensemble in a large volume of stirred or flowing medium to keep the oxygen concentration and other chemical con­ ditions constant. The concentration gradient is now in the thin sample layer and the idea of F. Haxo and L. Blinks was that the rate of oxygen evolution there would simply add up to the back­ ground current due to oxygen flow from the me­ dium, through the sample, towards the cathode. In their conditions most of the photosynthetic oxygen may actually be lost to the medium, but anyway the amplitude of the light-induced in­ crease of the signal is proportional to the rate of oxygen evolution. Later rate electrode designs mostly used a shal­ low sample compartment in which unicellular algae or isolated chloroplasts were allowed to settle in a thin layer on the cathode. Many varia­ tions have been published. A typical system may consist of (from bottom up): Pt cathode, about

Hans J. van Gorkom and Peter Gast sample sediment, about stationary supernatant medium, dialysis membrane, flowing medium of constant composition, and window for illumination. The Ag/AgCl anode, which much be shielded from illumination, may be a ring sur­ rounding the cathode, to reduce electromagnetic interference. The positive spike at the moment of the flash, often seen in reported measurements of flash-induced oxygen evolution, is an artifact caused either by insufficient optical shielding of the Ag/AgCl anode from the flash light, or by insufficient electromagnetical shielding of the electrochemical cell as a whole from the flash tube discharge. The anode can be placed in the sample compartment, in the flowing medium compartment, or in a third compartment sepa­ rated from the conditioning medium by a second dialysis menbrane and itself being part of a se­ cond flow system. The latter arrangement was introduced by Joliot and Joliot (1968) to avoid chemical interference of the conditioning medium with the anode. It should also allow the use of different electrolytes in the sample and anode compartments (e.g. to study chloride depletion), but this facility appears not to have been used. Pickett (1966) placed the anode with the cathode under a teflon membrane, combining the Haxo and Blinks electrode and Clark electrode con­ cepts (used e.g. by Weiss and Sauer, 1970). Den Haan et al. (1976) used a sample compartment of adjustable depth: the stationary supernatant medium was pushed out mechanically through the membrane. Diner and Mauzerall (1973) used a flowing gas instead of a solution as the condition­ ing medium. This may be the most efficient way to supply oxygen and, if necessary, to remove and leaves hydrogen, but it does not remove little room for electrolyte to connect anode and cathode electrically. A too large electrical resis­ tance leads to an electrical potential gradient in the electrolyte, which corresponds to using a weaker polarization, but may also result in a slow response time if the circuit contains a large capac­ itance (Meunier and Popovic, 1988). Rate electrodes can be very sensitive. The modulated rate measurement as described by Joliot (1972) may be the most sensitive way to measure the rate of photosynthesis: an oxygen has been re­ detection limit of ported.

Oxygen evolution

D. Concentration Electrodes Going back to the setup of Blinks and Skow (1938), one might also try to optimize the system for measurement of the oxygen concentration rather than of its rate of change. To avoid oxygen loss, the sample should be bound by an oxygenimpermeable wall, or at least be a thick layer on the cathode. The problem is that the large bare cathode will quickly modify the chemical con­ ditions near its surface. Vigorous stirring (Joliot, 1965) helps to spread the cathode-induced changes in chemical conditions rapidly over the whole sample but does not prevent them. Etienne (1968) used continuous sample re­ placement. A small cathode was placed at the outlet of a capillary tube in which a fast, turbulent sample flow was maintained. By varying the dis­ tance between the cathode and a small, brightly illuminated spot in the capillary, the flash induced kinetics could be determined in steady state mea­ surements, allowing a good signal/noise ratio in spite of the small size of the cathode. The system has limited applicability, but it does prevent all influence of the cathode on the sample. Plijter et al. (1988) used a stationary sample suspension in a closed cuvette and minimized the influence of the cathode by using an extremely weak polarization ( – 0.1 V vs. SHE), compen­ sating the reduction in cathode efficiency by a large cathode surface area Ideally, the cathode should function as a non-disturbing probe and should not create substantial diffusion gradients in the sample. The measured signals were indeed shown to be independent of viscosity (van Gorkom et al., 1989). Electrochemists prob­ ably would not call this method polarography at all. The electrochemical cell described by Plijter et al. (1988) was complicated by the requirement of simultaneous UV absorbance measurements (van Leeuwen et al., 1990) via a high transmitt­ ance grid cathode as used by Marsho and Hom­ mersand (1975), but we have obtained similar results with a very simple system consisting of an 0.5mm thick Pt-plastic-Ag/AgCl sand­ wich inserted in a standard 1 or 2 mm path length spectrophotometer cuvette, the Pt side facing the flash lamp (H.J. van Gorkom and M.A. van Dijk, unpublished). The detection limit of the Plijter electrode, with 10 ms response time and no aver­

395 aging, is an oxygen concentration change of about M. For reasons yet unknown, mutually exclusive results were obtained by Etienne (1968) and Plijter et al. (1988), so these methods should be used with caution.

E. Polarograms A polarogram is a plot of the measured current as a function of the polarization voltage, as in Fig. 1a. As usual, the polarization voltage is indi­ cated by the potential difference applied between the Pt cathode and Ag/AgCl anode, but what happens to oxygen when it hits the cathode de­ pends only on the electrochemical potential of the cathode, which should preferably be indicated relative to the Standard Hydrogen Electrode. If the cathode potential is not measured or fixed relative to a separate reference electrode by a potentiostat circuit, it may be estimated on the basis of the equilibrium potential of the Ag/AgCl couple at the chloride concentration used, but the actual anode potential will be higher. The deviation depends on the current density at the anode and can safely be neglected only if the anode is many times larger than the cathode. The polarogram shows an exponentially rising curve (the cathode potential affects the activation energy of the reaction) until diffusion of oxygen towards the cathode becomes rate limiting: a pla­ teau sets in at the voltage where oxygen in a boundary layer on the cathode surface is reduced more rapidly than it can be replenished from the solution. The steady state concentration in the boundary layer can only approach zero. The max­ imum concentration gradient in the stationary layer between cathode and stirred solution, and hence the rate of oxygen transport towards the cathode and the electrode current, is therefore limited by the concentration in the bulk solution. This is what makes the current in a Clark elec­ trode voltage independent. It does not mean that every oxygen molecule hitting the cathode is re­ duced. Stronger polarization may still allow an exponentially faster oxygen reduction and de­ crease the concentration in the boundary layer manifold, but if that concentration was negligible already the concomitant increase of the diffusion

396

gradient and of the steady state electrode current will be negligible, too. With a rate electrode the background current due to oxygen diffusing from the flowing con­ ditioning medium to the cathode also shows a polarogram with a more or less well-defined pla­ teau; only the current increase due to hydrogen evolution at strong polarization starts earlier due to the lower pH (Fig. 1b, open symbols). Haxo and Blinks (1950) used a polarization voltage in the middle of this plateau. With a rate electrode, however, one measures oxygen evolution in the boundary layer: the sample is on the electrode side of the diffusion gradient and the background current resulting from this gradient is in fact irre­ levant. As just stated, the rate of oxygen reduc­ tion in the boundary layer should increase ex­ ponentially with the voltage, if not limited by the electronics, until every oxygen molecule hitting the cathode is reduced. The polarization voltage at which that happens can be estimated from the

Hans J. van Gorkom and Peter Gast

kinetics of the flash induced signal if the kinetics without disturbance by the measurement are known. The data and calculations of Plijter et al. (1988) indicate that 100% efficiency of the cath­ ode reaction is approached at a cathode potential of about – 0.7 V vs. SHE (or – 1.0 V vs. At this potential hydro­ Ag/AgCl at gen production at a Pt cathode is already causing a considerable background current and all re­ ported oxygen measurements with rate electrodes were carried out at much weaker polarization, mostly around – 0.4 V vs. SHE, where oxygen reduction by the cathode still depends exponen­ tially on the cathode potential and the cathode is not at all the efficient oxygen trap it is usually thought to be. Polarograms of flash-induced oxygen evolution measured with rate electrodes show an exponen­ tial dependence of the current on the polarization voltage only to about – 0.3 V vs. SHE and a less steep increase of the current at stronger polariz­

Oxygen evolution ation (Fig. 1b, solid symbols). This has sometimes been attributed to limitation by O 2 diffusion from – 0.3 V followed by a gradual change from 2­ electron reduction formation) to 4-elecformation) at – 0.7 V, but tron reduction there is little evidence for that. It could be due instead to the convolution of oxygen production and its consumption by the electrode, and satur­ ation of the electrode efficiency could explain the maximum near – 0.7 V vs. SHE. Myers and Gra­ ham (1963) already noted the absence of a flat plateau and emphasized the need to stabilize the cathode potential and minimize the resistance in the circuit. A potentiostat circuit using a separate reference electrode and/or current to voltage con­ version to reduce the resistance are now generally used.

F. Flash-Induced Kinetics The kinetics of the current transient upon flashinduced oxygen evolution in a rate electrode setup is not fully understood. It consists of a sig­ moidal rise in a few ms, followed by a decay in tens of ms. It is agreed that the rise (or its Fourier transform, as measured by Joliot et ah., 1966) is to transition’, the reduction caused by the of the oxygen evolving complex by water, which normally proceeds with a time constant of 2 ms. The generally accepted view is that this coincides with the release of an oxygen molecule, and hence that the rise of the signal shows the release of the oxygen. According to Plijter et ah. (1988), however, a good rate electrode measures the rate of oxygen evolution, so the kinetics of the flashinduced current transient is the first derivative of the oxygen concentration, in agreement with their findings with a concentration electrode. The rise of the signal from a rate electrode in that case means a delay preceding oxygen release, attri­ buted to water oxidation by the oxygen evolving complex, and the time constant of the release process itself is reflected in the decay of the sig­ nal. No valid criticism or feasible alternative expla­ nation of the data or reasoning in the Plijter et ah. paper has been published to date. Also no one has reported an attempt to reproduce the measurements, but the method has been applied ever since in this laboratory, using various elec­

397 trode arrangements and electronics, and we have found nothing wrong with the measurements. The study by Plijter et al. (1988) was prompted by the paradox that after removal of the extrinsic 33 kDa protein the UV absorbance change due to reduc­ tion of the oxygen evolving complex, and hence presumably water oxidation, was much slower than oxygen release as measured by the rise of the signal from a thin sample layer centrifuged onto a strongly polarized electrode (Miyao et al., 1986). Plijter et al. (1988) showed both theoreti­ cally and experimentally that at 100% cathode efficiency a first order oxygen release process in a stationary sample suspension in a closed cuvette produces a transient current increase with a rise time 7 times shorter than the time constant of the release process, and the situation of a very thin sample sediment on a very inefficient cathode under a stationary supernatant is mathematically the same. The combination of a thin layer, strong polarization, and oxygen removal by a flowing medium can only lead to a further acceleration of the signal. It seems inevitable, therefore, that oxygen release is at least 7 times slower than the rise of the signal one would expect to measure with a rate electrode. However, the observed rise kinetics of the sig­ nal is not understood. It is clearly sigmoidal and the initial delay is not accounted for in the model of Plijter et al. It might be due to the chloridedependent artifact described by these authors. A good fit can be obtained by assuming that all oxygen sources are confined to a plane at a dis­ tance close to or even exceeding the thickness of the sample sediment and release oxygen with a time constant several fold less than that of water oxidation (Lupatov, 1979; Lavorel, 1992), but this is clearly unrealistic. More likely values of these parameters do not allow an acceptable fit of the initial delay in the signal and the steep decline after its maximum (M.H. Vos, unpub­ lished). Many authors have claimed inconsistency be­ tween results obtained with the method of Plijter et al. and those obtained with rate electrodes and other methods (Lavergne, 1989; Mauzerall, 1990; Strzalka et al., 1990; Jursinic and Dennenberg, 1990; Schulder et al., 1990, 1992; Meunier and Popovic, 1991; Tang et al., 1991; Lavorel, 1992; Ichimura et al., 1992; Joliot et al., 1992), but in

398 no case this was demonstrated for the same ma­ terial in the same conditions. It is often over­ looked that the release times given in Plijter et al. refer to a temperature of 5°C and decrease with increasing temperature by a factor of 2 per 20°C, and that shorter signal rise times were ob­ served in samples which exhibit flash-induced ox­ ygen uptake as well. It is clear from the previous section, however, that Plijter et al. overemphas­ ized the importance of weak polarization. They actually found similar signal rise times at cathode potentials down to – 0.45 V (– 0.73 V vs. which is well in the range Ag/AgCl at of values commonly used but still implies a low cathode efficiency. The different signal shape ob­ tained with rate electrodes must be primarily due to diffusion of oxygen out of the thin sample layer, away from the cathode. III. EPR Oximetry

A. EPR Line Broadening by Oxygen EPR oximetry has first been applied by the group of Y.N. Molin (Backer et al., 1977). This tech­ nique is based on the fact that molecular oxygen is paramagnetic. As a consequence, collisions be­ tween oxygen and a properly chosen spin probe, will increase spin-spin and spin lattice relaxation of this probe through the so-called Heisenberg exchange mechanism (Windrem and Plachy, 1980; Subczynski and Hyde, 1981; Popp and Hyde, 1981). For homogeneously broadened lines, this will result in broadening of the spec­ trum and changes in its microwave power satur­ ation behavior. Under non-saturating conditions, at low microwave power, the EPR line will broaden and thus the amplitude of the first de­ rivative signal, which is inversely proportional to the square of its linewidth, will decrease. In first approximation, the line broadening is often lin­ early dependent on the oxygen concentration (Windrem and Pachy, 1980; Glockner and Swartz, 1992). Measuring of oxygen levels by li­ newidth broadening can be done in two ways: a) direct measurement of the linewidth of the EPR signal, by recording of the EPR spectrum, and b) indirect measurement of the change in linewidth by recording the change in amplitude of the first derivative EPR spectrum. In the latter method,

Hans J. van Gorkom and Peter Gast the magnetic field sweep is turned off and the magnetic field is set at the maximum of the EPR signal. By comparing the amplitude changes at low and high microwave power distinction can be made between changes due to oxygen concentra­ tion changes and those caused by disappearance of the radical through chemical reaction (Strzalka et al., 1986). Under saturating conditions, at high microwave power, an increase of the signal inten­ sity may be observed under certain conditions, due to de-saturation at elevated oxygen levels, and recently this effect has also been used to measure oxygen concentration changes (Ligeza et al., 1994). EPR oximetry is non-invasive, does not con­ sume oxygen or produce harmful reaction prod­ ucts and does not require stirring of the sample. It can be used to measure the oxygen concentra­ tion in very small systems, like cells. In such ex­ periments a spin probe is added that penetrates the cell and the extracellular nitroxide signal is quenched by adding paramagnetic salts like ferri­ cyanide (Salikhov et al.,1971; Swartz, 1978), which do not enter the cell. The EPR signal of the extracellular nitroxide is broadened to such an extent that it virtually disappears and only the signal from intracellular nitroxides remains. Spinlabels can also be distributed homogeneously throughout an organ or whole body, and 2D and 3D EPR oximetry imaging is possible (Bacic et al.,1988; Demsar et al., 1988; Swartz and Glockner, 1989). For a review on EPR oximetry and its many uses, see Swartz and Glockner (1989).

B.

Oxygen Probes for EPR

A prerequisite for using EPR in oximetry is the presence of a (stable) radical in the system under study. Free radicals are rare in biological systems, and stable radicals are almost absent (an excep­ tion to this is melanin, which can also be used as oxygen probe (Sarna et al., 1980). Therefore these radicals have to be added. For this purpose nitroxide radicals have been widely used and characterized in spin-label experiments as ‘re­ porter’ molecules. A major disadvantage of EPR oximetry has been the fact that many spin labels and especially the nitroxides are rather toxic. Another complica­

Oxygen evolution tion is that there are many parameters other than oxygen that will broaden the EPR line of nitrox­ ide radicals, like the presence of paramagnetic ions (Salikhov et al.,1971), nitroxide concentra­ tion, viscosity, microwave power, pH and tem­ perature. Therefore it is necessary to calibrate the oxygen effect for each system and to keep parameters like temperature, microwave power and nitroxide concentration constant. Further­ more, EPR oximetry is not very sensitive, espe­ cially at high oxygen concentrations (see below). The new oxygen-sensitive probes that have been introduced by the group of H. Swartz in recent years (Swartz et al. 1991; Glockner and Swartz, 1992; Liu et al., 1993) seem to overcome the above mentioned shortcomings of the tra­ ditional, nitroxide-based EPR oximetry. They are fusinite (a certain type of coal), certain chars, soot, lithiumphthalocyanine crystals (PcLi) (Turek et al., 1987) and even Indian Ink. What these new spin-probes have in common is that they are all highly insoluble in most solvents, very sensitive to oxygen, non-toxic, virtually insensi­ tive to the environment and extremely stable (al­ though PcLi in water may be less stable than previously thought, M. Moussavi, personal com­ munication). They are solid particles and measure the oxygen partial pressure, whereas the soluble nitroxides measure concentration (Povitch, 1975) unless enclosed in lipid droplets (Ligeza et al., 1994). The sensitivity and nontoxicity of the new probes has been demonstrated by measuring changes in oxygen concentration in the brain of a living mouse, in living cells, liver, and heart; their stability was convincingly shown by re­ cording oxygen levels in a rat’s leg muscle over a period of more than 150 days (Glockner and Swartz, 1992; Liu et al., 1993).

C. Sensitivity When calculating the sensitivity of an EPR oxygen-probe, distinction should be made between the two ways of measurement: a) direct linewidth broadening measurement and b) amplitude mea­ surement. The amplitude measurement is more sensitive, but cannot always be used since in many systems the nitroxides are rapidly bio-reduced to EPR silent hydroxylamines (Swartz et al., 1986).

399

This means that the spin-concentration is not con­ stant. 1. Sensitivity in Direct Linewidth Broadening Measurement If the broadening is proportional to the oxygen concentration (Windrem and Pachy, 1980; Glockner and Swartz, 1992) the linewidth can be written as: The minimal change in linewidth that can be mea­ sured will depend on the linewidth itself. This is the reason why often deuterated nitroxides are used because of their reduced linewidth. Based on experimental facts, it is not unreasonable to state that for an EPR signal with reasonable signal-to-noise ratio (S/N) a change of 5% in linewidth of an EPR signal can be observed. Therefore:

and If the proportionality constant c is determined from the linewidths at 0 and at the latter being about the concentration in air-saturated water at 20°C (Hitchman, 1978), we get: For the deuterated nitroxide TEMPONE, with (Swartz and and Pals, 1989) the minimally detectable change in is about oxygen concentration at and at this value has increased to 20 The most sensitive probe, PcLi, with a

14 mG and

will have a mini­ mally detectable change at of about 200 nM and at of 15 2. Sensitivity in Amplitude Measurement This method is in principle much more sensitive than the linewidth measurement, since changes in amplitude are more easily measured than changes in linewidth and because the amplitude of the first derivative of an EPR line is pro­ portional to the square of the inverse of the line­

400

Hans J. van Gorkom and Peter Gast

width. The minimally detectable oxygen-change with this method will depend on the S/N of the probe signal. Although there are limitations to the maximum concentration for spin-labels (due to line broadening at high concentration), it is not unreasonable to assume that the S/N at can be 1000 or more for many systems (taking (Glockner the number of spins/g in PcLi as and Swartz, 1992) and the sensitivity of a com­ mercial X-band EPR spectrometer as spins/Gauss, 10 nanogram will be sufficient to achieve this S/N under anaerobic conditions). If we further assume that the minimal detectable amplitude change is a change with S/N = 1 we obtain: which is 100 times less than that obtained in a linewidth measurement. For d-TEMPONE the detection limit becomes at and 75 nM at 0 for PcLi it becomes 150 and 2 nM at 0 The last nM at 300 value is probably comparable to the minimum flash yield detectable with a polarographic rate electrode. It should be noted, however, that these num­ bers apply in rather ideal situations. For instance, when EPR oximetry is used in a whole-body sys­ tem using a low-Q surface probe instead of a standard high-Q cavity, and using low microwave frequency to increase microwave penetration (Nilges et al., 1989; Bacic et al., 1989), or in timeresolved EPR oximetry, the sensitivity may be greatly reduced. If one can exclude or correct for spin-label reduction, both methods will give identical re­ sults, taking the quadratic decrease of the ampli­ tude with increasing linewidth into account. How­ ever, in time-resolved studies where the magnetic field sweep is turned off and the field is set at the maximum of the EPR signal, deviating results may be obtained when the changes in oxygen concentration are large. This is due to the fact that when the oxygen concentration during the measurement is increased or decreased, the linewidth will broaden or narrow, respectively, and as a result, the magnetic field position will ‘slide off the peak of the first derivative spectrum. This is shown in Fig. 2. Here the calculated response is depicted of the EPR amplitude, set at the maxi-

mum of the EPR line at t = 0, to an oxygen con­ centration increase with a time constant of 200 ms. When the change is small, the signal ampli­ tude reflects the oxygen evolution kinetics. How­ ever, when the change in oxygen concentration is large, a more rapid and non-exponential curve is observed. As expected, the deviation becomes significant at much smaller oxygen concentration changes with PcLi than with d-TEMPONE, since PcLi has a much sharper EPR line. In photosyn­ thetic material, such large changes may occur lo­ cally for a short time. For instance, a chloroplast suspension of a chlorophyll concentration of oxygen on a 1 mM may produce more than flash and all that oxygen may initially be confined to a small fraction of the volume: the chloroplasts occupy about 1/40 and the thylakoids only 1/300

Oxygen evolution of the suspension volume (Heldt et al., 1973). On a time scale of some ms, oxygen diffusion processes and the non-linearity of the EPR ampli­ tude may complicate the observed kinetics, de­ pending on the microscopic distribution of the oxygen and that of the spin probes used. On the other hand, this might become a unique tool to obtain time-resolved information on the micros­ copic distribution of oxygen.

D. Applications in Photosynthesis Research Up to now only a few reports have appeared on EPR oximetry using photosynthetic material. Strzalka et al. (1986) used the spin label TEM­ PONE to measure oxygen evolution in thylakoid membranes of spinach. They measured amplitude changes and used the difference in microwave power saturation behavior to distinguish between production of oxygen and photoreduction of the probe, which could be controlled by adding suffi­ cient amounts of an electron acceptor (p-benzoquinone). Belkin et al. (1987) have used EPR oximetry to measure light induced oxygen pro­ duction in whole cells of cyanobacteria under con­ tinuous illumination. Direct linewidth as well as amplitude measurements were used in this study. the By the paramagnetic agent EPR signal from the extracellular oxygen probe was broadened beyond detection and the intra­ cellular oxygen production and photo-inhibition was measured selectively. In a second report from Strzalka et al. (1990) the more sensitive probe perdeuterated TEMPONE was used to mea­ sure the oxygen release time in thylakoids after a short flash by the change in EPR amplitude. This was found to be 0.4–0.5 ms, which is much shorter than the 2 ms time constant of water oxid­ ation and may perhaps have been caused by the non-linearity discussed above. Perhaps contrary to the soluble nitroxides, the new, solid spin pro­ bes should allow unambiguous distinction be­ tween free oxygen and oxygen still associated with the photosynthetic system, if the slow release process postulated by Plijter et al.(1988) exists. The only report so far on the use of PcLi crystals as an oxygen probe in photosynthetic material is from Tang et al. (1991), who found by amplitude measurements an oxygen release time in PSII membranes of 1–2 ms. In a recent article by Dis­

401 mukes et al. (1994) TEMPONE oximetry was used to demonstrate hydrogen peroxide produc­ tion in photosystem II membranes inactivated by depletion: oxygen production was restored not only by readdition of but also by addition of catalase. With oxygen polarography, such ex­ periments are complicated by the production of hydrogen peroxide at the cathode. Ligeza et al. (1994) used the oxygen-dependent desaturation at high microwave power to measure oxygen evol­ ution in leaves, after injection of an emulsion droplets containing a nitroxide enclosed in of paraffin oil covered with serum albumin. This system largely prevents photoreduction of the ni­ oxygen was troxide. A detection limit of reported.

IV. Mass Spectrometry Unlike most processes discussed in this volume, photosynthetic oxygen evolution involves the re­ arrangement of nuclei to form one kind of mol­ ecule from another and can be studied by tra­ ditional biochemical tools using labeling of the substrate by nuclear isotopes and measuring the isotope composition of the product. The useful­ mass spectrometry in photosynthesis ness of research was established by Hoch and Kok (1963), who replaced the sample injection port of a conventional mass spectrometer by a vessel containing a stirred suspension of photosynthetic material separated only by a teflon membrane from the vacuum system. Later authors have mostly used a thin layer of photosynthetic ma­ terial sedimented on the teflon membrane. The selective permeability of the membrane allows direct measurement of the gases dissolved in the sample solution. Sufficient sensitivity and time resolution can be reached to allow individual flash yields in a series to be analysed. The method has been reviewed by Radmer and Ollinger (1980). It has more recently been used e.g. to measure oxygen uptake kinetics during the induction of oxygen evolution upon continuous illumination (Peltier and Ravenel, 1987), to distinguish oxygen evolution from water and that from hydrogen per­ oxide (Mano et al., 1987), and to prove that water to transition is still ex­ oxidized on the changeable in (Radmer and Ollinger, 1986; Bader et al., 1987), the kinetics of which have

402 now been measured with 30 ms time resolution (Messinger et al., 1995). Photosynthetic oxygen evolution is almost al­ ways accompanied by light-dependent oxygen up­ take processes and an unambiguous distinction between the two can normally not be made by other methods, which measure only the net change in oxygen concentration. But also mass spectrometry may not help. It should be kept in mind that these uptake processes take place at short distance and may selectively consume the oxygen just produced photosynthetically. In fact that can be the cause of their light-dependence, as was strikingly illustrated by the measurement of a period four oscillation in the oxidation of mitochondrial cytochrome c upon flash illumi­ nation of a green alga (Lavergne, 1989). Perhaps a quantitative trapping of photosynthetic oxygen before it can leave the cell may explain the appar­ ent oxygen dependence of oxygen evolution re­ ported for a cyanobacterium (Bader and Schmid, 1988).

Hans J. van Gorkom and Peter Gast VI. Galvanic Sensors Galvanic sensors to detect oxygen in the gas phase have been developed for technical appli­ cations mainly (Kleitz and Fouletier, 1976; Heyne, 1976), but there are a few reports of the use of zirconium oxide detectors in photosynth­ esis research (Björkman and Gaul, 1970; Green­ baum and Mauzerall, 1976; Meyer et al., 1989). These devices are based on the phenomenon that conducts at a mixed crystal of high temperatures (around 800°C) and can be used as a solid electrolyte between two Pt elec­ trodes exposed to the gas to be measured and to a reference gas, respectively. Due to the required transport and drying of the gas the time-resolution is poor and the flash number dependence of the oxygen yield can be determined only by the cumulative method (measuring the total yield of a series of 1, 2, 3, etc. flashes). The main advantages of the technique are that it combines single flash sensitivity with easy quantitative cali­ bration, and that there is no chemical interference between sample and detector.

V. Photoacoustic Spectroscopy VII. Prospects The traditional volumetric measurement of pho­ tosynthetic oxygen evolution by the Warburg technique may now be obsolete, but it has a mod­ ern pendant: the pressure increase in the gas phase due to oxygen evolution causes a ‘photoba­ ric’ contribution to photoacoustic signals (Bults et al., 1982). Photoacoustic spectroscopy is re­ viewed elsewhere in this volume by S. Malkin (Chapter 12). The photobaric oxygen signal can be measured with high sensitivity and time resol­ ution. It shows the characteristic period four os­ cillation in a flash series (Canaani et al., 1988) and the flash-induced kinetics have been studied with ms time resolution (Mauzerall, 1990). The concomitant photothermal signal, oxygen uptake phenomena in the leaf discs used, and the use of an open microphone and a.c. amplification led to convoluted signal kinetics, but their analysis according to Mauzerall suggested that oxygen re­ lease was fast. The use of a very thin layer of a PS II preparation which does not show oxygen uptake, and a pressure transducer as detector, might indeed open the road to obtain conclusive evidence on the oxygen release time.

There is no single ‘best’ way to measure photo­ synthetic oxygen evolution. Each of the tech­ niques described above has its own specific pos­ sibilities and drawbacks, and different experimental situations may call for different me­ thods of measurement. Moreover, all these me­ thods still hold potential for further development. Polarography, being relatively cheap and easy to implement, continues to be a rewarding play­ ground for inventive experimenters. The appli­ cation of EPR oximetry in photosynthesis re­ search is in an early, exploratory phase and is just beginning to prove its value; mass spectrometry will remain indispensable to unravel the interplay between oxygen evolution and oxygen consuming processes in photosynthetic organisms. Methods which have so far only been studied for their own interest, but also phenomena now considered as troublesome complications of established me­ thods, may suddenly develop into valuable tools to solve a particular problem. Despite its long history, research on the me­ chanism of photosynthetic oxygen evolution is

Oxygen evolution still in full swing and the oscillatory phenomena caused by the 4–flash redox cycle of the oxygen evolving complex have not lost their charm. It is to be expected that oxygen measurements will continue to play an essential role in their study. Acknowledgements We are grateful to Dr. M.H. Vos for advice and extensive fitting of time-resolved polarographic data kindly supplied by Dr. J. Lavorel, and to Dr. G.H. Schmid for pointing out the benefits of collodion films and how to apply them. Research in this laboratory was supported by the Nether­ lands Foundation for Chemical Research (SON), financed by the Netherlands Organization for Scientific Research (NWO). P.G. is a research fellow of the Royal Netherlands Academy of Arts and Sciences (KNAW). References Bacic G, Demsar F, Zolnai Z and Swartz HM (1988) Contrast enhancement in ESR imaging: role of oxygen. Magn Res Med Biol 1: 54–65. Bacic G, Nilges MJN, Magin RL, Walczak T and Swartz HM (1989) In vivo localized ESR spectroscopy reflecting metabolism. Magn Res Med 10: 266–272. Backer JM, Budker VG, Eremenko SI and Molin YN (1977) Detection of the kinetics of biochemical reactions with oxygen using exchange broadening in the ESR spectra of nitroxide radicals. Biochim Biophys Acta 460: 152–156. Bader KP and Schmid GH (1988) Mass spectrometric analysis of a photosystem II mediated oxygen uptake phenomenon in the filamentous cyanobacterium Oscillatoria chalybea. Biochim Biophys Acta 936: 179–186. Bader KP, Thibault P and Schmid GH (1987) Study on the properties of the by mass spectrometry in the fila­ mentous cyanobacterium Oscillatoria chalybea. Biochim Biophys Acta 893: 564–571. Baumgärtl H, Grunewald W and Lübbers DW (1974) Polaro­ graphic determination of the oxygen partial pressure field field in front of a Pt by Pt microelectrodes using the macroelectrode as a model. Pflügers Arch 347: 49–61. Belkin S, Mehlhorn RJ and Packer L (1987) Determination of dissolved oxygen in photosynthetic systems by nitroxide spin-probe broadening. Arch Biochem Biophys 252: 487– 495. Björkman O and Gaul E (1970) Use of the zirconium oxide ceramic cell for measurement of photosynthetic oxygen evolution by intact leaves. Photosynthetica 4: 123–128. Blinks LR and Skow RK (1938) The time course of photo­ synthesis as shown by a rapid electrode method for oxygen. Proc Natl Acad Sci USA 24: 420–427. Bulls G, Horwitz BA, Malkin S and Cahen D (1982) Photoac­

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Index

Since almost all chapters deal with chlorophyll or bacteriochlorophyll, these words are not used as entry in this index.

A absorbance-detected magnetic resonance, see ADMR, ODMR absorption spectrum 4-6 derivative of 4 dichroic 13, 16, 18, 28, 34 difference, light-induced 4-6 difference, reaction modulated infrared 153 difference, redox-induced 148 difference, electric field-induced 177-188 FTIR 137-157 hole burning 123-134 line width 43, 110, 125 transient 70, 71 X-ray 337-352 accumulated photon echo spectroscopy 109-120 ADMR 277-295 amorphous ice 327 anisotropy of absorbance 14 of emission 26 function 20 of time-resolved fluorescence 57 of transition dipoles 24 antenna, see LH 386 atomic scattering factor 318 temperature factor 318 averaging, a-periodic 328

B bacteriopheophytin 164, 169, 185, 186, 289 bottleneck state 113

C C-phycocyanin 57 windows 155, 156 caged compounds 148 carotenoids 27, 161, 166, 168, 185, 289, 290, 295, 305 charge pairs 97 formation in PSII 95

half lives of 99

recombination of 91

stabilization of 95

chemical shift tensor 306 chemically induced dynamic spin polarization 212 chirality 24, 25, 27, 30 chirally induced differential scattering (CIDS) 30-32 Chlamydomonas reinhardtii 69, 365 Chlorobium limicola 167, 171, 269 tepidum 131 Chloroflexus aurantiacus 29, 148, 292 Chlorogloea fritschii 370, 371 Chromatium vinosum 147, 172 chlorosome 6, 30

circular dichroism 24-34 excitonic 28 fluorescence-detected 13, 26, 34 intrinsic 28 long-range interaction 26 orientation dependence 26 psi-type 27, 31, 33 vibrational 26 Clark electrode 392 concentration electrode 395 Conne’s advantage 143 correlation spectroscopy 309 Cotton effect 25 cross-polarisation 301 cryo-electron microscopy 327, 328 crystal structure, determination of 317-323 cytochrome 359, 365, 375 cytochrome 371

D data fitting 78 dephasing 112, 118 dichroic ratio 18 dichroism, reduced 18 dielectric asymmetry 188 screening 187 difference spectrum, see absorption spectrum differential polarization images 22 microscopy 13 scattering 25 dipolar interactions 301 dipole moment 178 strength 15 dispersion model 119 double exchange 369 double resonance 277, 284, 285, 294, 295

E electric field 51, 177, 178 gradient 358 orientation 24 electroabsorption 177 electrochemical cell 147 oxidation 148 electrochromism, see Stark spectroscopy electrode, Clark 392 concentration 395 electron transfer at 148 gold grid 147 rate 394 electron crystallography 332 density map 320, 322 microscopy 326, 331

Index

408 nuclear double resonance, see ENDOR paramagnetic resonance, see EPR spin echo, see ESE spin echo envelope modulation, see ESEEM electron-phonon coupling 118, 130 Eligeron canadensis 103 emission, see fluorescence, phosphorescenc ENDOR 255-272 bacteriochlorophyll 256 Davies ESE 243 electron acceptors 271 general TRIPLE 261 in frozen solution 263 in liquid solution 258 in single crystals 265 Mims ESE 245 of

269 stochastic 267 TRIPLE 260 triplet states 265, 271 energy storage 195-199 transfer 6, 44, 87, 116, 125, 133, 213, 217, 226 EPR 211-229, 258, 278, 280, 289, 294, 295 direct detection 215 ENDOR-induced 262 Fourier transform 215-217, 226 multiline signal 248, 350 oximetry 398 pulsed 249 time-resolved 211-229 error analysis 85 ESE 235-252 ESEEM 238 EXAFS 338 definition 340 equation 341 Fe 346 Mn 349 theory 339 excitation transfer, see energy transfer excited state, see exciton, energy transfer exciton, annihilation 54 bands 26 circular dichroism 28 coherence 116 coupling 25, 29 dynamics 109, 110 interaction 29, 30, 33 manifold 117 scattering 116 states 116 extended X-ray absorption fine structure, see EXAFS

F FDMR 277, 278, 280-284, 288, 289 Fe centers 348, 355 K-edge spectra 347 quinone complex 365, 366 Fe (II) high-spin 361

Fe (III) high-spin 360 Fe (II) low spin 360 Fe(II) NO complex 367 Fe-S acceptors in photosystem I 346 Fe-S proteins 345, 346, 362, 368 Felgett’s advantage 143 Fenna-Matthews-Olson, see FMO film-stretching 21

flattening effect 4, 26

fluorescence, depolarization of 44, 68

emission 6, 18, 47

spectrum 6, 7, 46

lifetime 44

microwave-induced 278, 285

polarization of 18

relaxation 45

self absorption 48 single photon counting 52 spectrophotometer 45 time-resolved 52, 55 upconversion 53, 64 yield 6, 45, 197, 198 fluorescence-detected magnetic resonance, see FDMR, ODMR FMO complex 7, 116, 117, 131 force field 140 formate 367 Fourier analysis 330 transform infrared, see FTIR transform EPR 215-217, 226 Fourier-peak filtering 330 Franck-Condon principle 43 frequency grating 112 FTIR 137-137, see also infrared amide I mode 144 amide II mode 144 difference spectra 140, 145, 147, 148 protein modes 155 spectrophotometer 141, 145, 153, 154 spectroscopy 137-157, 306 techniques 138 time resolved 148 fusinite 399

G g-factor 258 galvanic sensors 402 gel-squeezing 19, 21 glow curves 94, 98 gold grid electrode 147 group frequencies 140, 141

H Hartmann-Hahn condition 301 Heliobacterium chlorum 292 heterogeneity 278, 289. 294, 295 high pressure hole burning 128 hole burning 117 double resonance ODMR 289

Index high-pressure 128 non-photochemical 124 photochemical 124 spectroscopy 114, 123-134 transient 124 homodyne detection 114 homogeneous linewidth 110 Huang-Rhys factor 125 hydrogen peroxide 393 hyperfine interaction 249, 258, 356, 357

I infrared 137-157 cell 155 detector 137, 141, 143, 144 difference spectroscopy 140, 145, 148 dispersive spectroscopy 153 lasers 153 photometer 153, 154 picosecond spectroscopy 137, 154, 155 spectroscopy 137-157 windows 156 inhomogeneous broadening 43, 125 linewidth 43, 110 interferogram 149 interferometer 141, 142, 149, 152 internal conversion 44 intersystem crossing 44 isomorphous replacement 319 isotope labelling 140, 312

J Jacquinot’s advantage 143

K K-edge spectra 338, 347, see also EXAFS, XANES Krönig-Kramers transforms 25

L Langmuir Blodgett film 16 laser, argon ion 64, 126 diode 127 dye 64, 164 Nd:YAG 165 picosecond infrared 137 regenerative amplifier 65 spectroscopy 63-72, 154, 155 Ti:Sapphire 64-66 tunable infrared 137, 154 LH1 67, 87, 170, 386 LH2 386, 388 LHC II 27, 29-33, 69, 117, 328, 331, 332 light-harvesting, see LH1, LH2, LHCII light scattering 4, 33

409 light-induced difference spectroscopy 147 linear dichroic T-S 286, 278, 290 linear dichroism 13, 16, 18. 34, see also orientation liquid crystal 218, 228 lithium phthalocyanine 399

M macro-organization of chromophores 26, 27, 30, 32 magnetic circular dichroism (MCD) 26, 34 mass spectrometry 401 metallo-proteins 338 microwave-induced absorption (MIA) 278-285 fluorescence (MIF) 278-285 phosphorescence (MIP) 278-285 micelles 380 Mn, see also oxygen evolving complex Mn cluster 246, 249 EXAFS 349 K-edge spectra 350 molecular dynamics calculation 322 molecular replacement 319 Mössbauer spectroscopy 355-372 Mueller images 30 matrix 14, 34 multiplex advantage 143, 151

N negative staining 327, 328, 333 nitroxide radicals 398 NMR 335 chemical shift tensor 306 CIDNP 309 CP/MAS 299, 300 cross polarization 301 Hartmann-Hahn 301 solid state 299-313 two-dimensional 311 non-photochemical hole burning 124 normal mode analysis 140 nuclear magnetic resonance, see NMR Hamiltonian 359

O ODMR 277-295 time-resolved 280 optical rotary dispersion (ORD) 25 optically detected magnetic resonance, see ODMR orientation, angle 17, 21, 22, 28 by electric field 20 by film-stretching 21 by flow 19 by gel-squeezing 19, 21 by magnetic field 20 mechanical 19 selection 263 orientation-dependent circular dichroism 26

410 oxygen diffusion 201 oxygen evolution 391 by photoacoustics 200-202 limiting rate 201-202 S-states 95, 200, 201 oxygen evolving complex 95, 99, 246, 334, see also Mn double hits 100 misses 95 Mn complex 95, 348, 350 S-states 95, 200, 201

P PDMR 278, 280, 282 periodic averaging 328, 330 phase contrast 329 phonon sideband holes 124 Phormidium laminosum 364, 367 phosphorescence 45, 278, 280, 284, 286, 288 microwave-induced 278, 285 phosphorescence-detected magnetic resonance, see PDMR, ODMR photoacoustic spectroscopy 34, 191-204 photobaric oxygen signal 402 photochemical hole burning 124 photon echo 111, see also accumulated photon echo photoselection 18, 20, 287. 290 photosystem I 7, 69, 87, 94, 169, 249, 290, 293, 327, 330, 333, 346-348, 355, 367-371 photosystem II 7, 94-105, 169, 249, 290, 293. 327, 333, 346­ 348, 355, 366, 371 polarizability 178 polarization microscopy 24 polarized IR spectroscopy 19 light 13, 25 polarography 392, 395 Prochlorothrix hollandica 30 Prosthecochloris aestuarii 29, 126 protein crystallography 318 proton release pattern 96 transfer 137 psi-type aggregates 27, 30-33 pulsed microwaves 281 pump-probe techniques 138

Q quadrupole coupling 262 interaction 358 quinone 147, 157, 220, 365, 366 replacement 140

R R-factor 321 radical-pair mechanism 224 Raman spectroscopy 161-173 rate electrode 394 reaction center 118, 129-131, 152, 166-170, 268-271, 288­

Index 296, 302-309, 321, 365, 375, 383 regenerative amplifier 65 resonance Raman spectroscopy 161-173 Rhodobacter capsulatus 68, 71, 135 sphaeroides 68, 69, 71, 118, 125, 129, 133, 147, 155, 166, 169, 170-172, 180, 182, 184, 269, 271, 289, 291, 300, 303-309, 321, 364, 365, 388 Rhodopseudomonas acidophila 388 marina 32 palustris 166 viridis 8, 29, 129, 130, 133, 147, 155, 166, 169, 172, 271, 289, 291, 303-309, 321 Rhodospirillum 388 Rieske Fe-S clusters 348 rotational correlation time 260

S secondary structure 32, 144 sieve effect 4, 26 simulated annealing 322 single-photon counting 52 single-particle averaging 332 small-angle neutron scattering 375-389 space group 319 spectroelectrochemical cell 156 spin coupling 361 Hamiltonian 258, 357, 360, 361

label 398

polarization 212

Stark spectroscopy 177-188 step-scan interferometer 152 Stokes parameters 14, 15 shift 47 Synechococcus elongatus 330, 332, 367 Synechocystis 103, 333 structure factor 318 subtractive mixing techniques 155 supramolecular assemblies 375 synchrotron radiation 343

T T-S spectrum 278, 285-293, 295 TEMPONE 399 thermal deactivation spectrum 198 thermoluminescence 93-104 activation energy 97

application 103

assignments 100

components 101

origin 94

oscillation 99

peak temperature 96

time-averaged structures 323 transient absorption 70, 71 transition dipole moment, electric 15, 16, 278, 279, 286, 287, 290-295

magnetic 25

Index TRIPLE resonance, see ENDOR triplet state 277-298 magnetic field effect on 199 mechanism 213, 218 reaction center 289 spin Hamiltonian 278 triplet-minus-singlet absorbance difference, see T-S two-dimensional crystallization 330 tyrosine 71, 249, 303

U unit cell dimensions 318

V vibrational modes 141 spectroscopy 138 wavepackets 71

W water-oxidation system, see oxygen evolving system

411 X X-ray absorption near edge structure, see XANES absorption spectroscopy 337-352 diffraction 317-323, 326, 335 linear dichroism 16 XANES 338,346 K-edge spectra 338, 346

Z Zeeman frequency 258 zero-quantum beats 228--29 zero-field splitting parameters 277, 278-280, 289, 293-295, 359, 402 zero-phonon hole 124 line 118 ZFS, see zero-field splitting zirconium oxide detector 402