Biophysical Techniques in Photosynthesis

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 multidisciplinary (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 reaction centers to the physiology of the whole plant, and from X-ray crystallography 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

3

3 3 3 4 6 8

Linear and Circular Dichroism Garab

11

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

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

161 161 162 162 163 167 168 169 171 172 173 174 174

177 177 177 178 181 184 188 188

viii

12

Contents 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

235 235 235 238 243 246 249 252 252

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

21

Contents 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

24

Measurement of Photosynthetic Oxygen Evolution Hans J. van Gorkom and Peter Gast Summary I. Introduction II. Polarography III. EPR Oximetry IV. Mass Spectrometry V. Photoacoustic Spectroscopy VI. Galvanic Sensors VII. Prospects Acknowledgements References

Index

337 337 338 338 345 350 351 352

355 355 356 357 365 371

375 375 376 377 379 388 388 389

391 391 392 392 398 401 402 402 402 403 403

407

Preface Progress in photosynthesis research is strongly dependent on instrumentation. It is therefore not surprising 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 analysis, 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 registration. Hand in hand with these developments optical studies of photosynthesis have acquired growing importance for gaining insight in the molecular 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 modern optical methods. This chapter will survey some of the developments during the last 50 years and will discuss some of the “traditional” methods that still play an important role in photosynthesis 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 fluorescence properties of photosynthetic material (Kautsky and Hirsch, 1931; Kautsky and Franck,

In view of the key role of pigments in photosynthesis, 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 photosynthesis. Progress in optical research on photosynthesis, however, was for a long time arrested, mainly because simple and sensitive techniques for measuring and recording light intensities were lacking. About 50 years ago, however, 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 pioneered 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 photosystems 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 transformations in excited pigments (see Chapter 4 by Jimenez 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 conveniently, 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 spectra 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 scattering, if not properly corrected for, not only causes an upward shift and distortion of the absorption 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 latter, however, at the expense of sensitivity. In some cases reliable data can be obtained by fluorescence 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, however, that even a properly measured absorption spectrum is not identical to that of the same pigments 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 interactions the in vivo absorption spectra of photosynthetic 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 resolution of absorption (or other) spectra is by measuring the second or even fourth derivatives (Martin, 1959; Butler and Hopkins, 1970a, 1970b; see Fig. 1). Caution, however, is needed in the interpretation 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 photosynthesis. Although pump-probe measurements with high time resolution are now in the forefront of research (see Chapter 4 by Jimenez and Fleming), 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 measuring 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. photomultiplier 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 modulated signals of intermediates with a sufficiently

5

short lifetime (Kok, 1959; Spruit, 1971; Nishimura et al., 1969). The relatively slow modulation employed in the above mentioned apparatus precludes measurements 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 extensively used in photosynthesis research. Fluorescence artifacts can be corrected for, if necessary, 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 apparatus 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 apparatus for measuring time resolved difference spectra based on an array of pulsed light emitting diodes has been described by Klughammer et al. (1990).

III. Fluorescence Fluorescence from leaves and photosynthetic pigments was first observed by D. Brewster and G.G. Stokes, in the mid-nineteenth century (see Rabinowitch, 1951), but it was only about a century later when fluorescence measurements became an increasingly important tool in photosynthesis research. An interesting quantitative survey of the various topics studied by fluorescence 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 discussion of those measurements the reader is referred 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 excitation 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 processing being standard nowadays. A discussion of some of the pitfalls and possible errors, like those caused by false light and self-absorption of fluorescence can be found in Amesz (1973). An apparatus specially designed for photosynthesis research is now commercially available from Walz, Effeltrich, Germany. Apparatus have also been

Jan Amesz devised for field studies and productivity measurements (Renger and Schreiber, 1986; Öquist and Wass, 1988). The prominent emission bands in photosynthetic 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 usually weaker because thermal equilibrium favors emission from the pigment with the lowest energy. 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 phycobilisomes (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 photosynthetic 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 chlorosomes and FMO protein, whereas emission from the BChls in the core complex is hardly observable (Amesz and Vasmel, 1986; Otte et al., 1991). A quantitative determination, however, of the transfer efficiencies from such measurements requires knowledge of the “intrinsic” fluorescence yield of these antenna components, i.e. the yield in the absence of energy transfer to other pigments. 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 (action) spectrum. Such a spectrum defines the relative efficiencies of absorbed or incident photons of various wavelengths to bring about the phenomenon under study. Unfortunately, published action spectra are often poorly defined. A properly measured action spectrum gives quantitative information about the pigment or pigments that sensitize the reaction, but care must be taken to avoid errors, such as those caused by a nonlinearity of the response with light intensity and self-screening within the sample (Amesz, 1973). Quantitative information on efficiencies of energy 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 information in the range between, say, 90 and 100%

7

transfer efficiency. Fig. 2 shows fluorescene excitation spectra of chloroplasts, measured at low temperature. By choosing the proper emission wavelength, the excitation spectra of Photosystems 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 photosystems are not available, unless one resorts to fractions solubilized by detergents, but the general impression from these and similar experiments 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 obtained in any other way. The anoxygenic photosynthetic bacteria have only one photosystem and do not pose such problems. Fig. 3 shows a fluorescence excitation spectrum 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 chromatophores of the BChl b containing purple bacterium Rhodopseudomonas viridis. The reaction center bands are completely lacking in the excitation 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 fluorescence 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 material. Relative yields can be measured much more easily, and such measurements, as a function of time, of added ions and inhibitors and of light intensity, have been done extensively during the last decades. Starting with the work of Duysens 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 Govindjee (1995). References Allen MB, French CS and Brown JS (1960) Native and extractable forms of chlorophyll in various algal groups. In: Allen MB (ed) Comparative Biochemistry ofPhotoreactive 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 analysis 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 photochemistry and fluorescence emission. J. Photochem Photobiol 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 tunneling. Biophys J 6: 825–847. Duysens LNM (1952) Transfer of Excitation Energy in Photosynthesis. Doctoral Thesis, University of Utrecht. Duysens LNM (1956) The flattening of the absorption spectrum 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) Research in Photosynthesis, pp 59–61. Interscience Publishers, 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 photochemical 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 photosynthetic pigments. In: Johnson FH (ed) The Luminescence of Biological Systems, pp 51–74. Am Ass for the Advancement of Science, Washington DC. French CS and Harper GE (1957) Derivative spectrophotometry. 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 Kohlensäureassimilation. 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 Electron 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 absorbing antenna bacteriochlorophylls to the reaction center. Biochim Biophys Acta 1099: 175–181.

9 Klughammer C, Kolbowski J and Schreiber U (1990) LED array spectrophotometry for time resolved difference spectra in the 530–600 nm wavelength region. Photosynth Res 25: 317–327. Kok B (1959) Light-induced absorption changes in photosynthetic organisms. II: A split-beam difference spectrophotometer. 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 Physiol 34: 193–199. Latimer P and Eubanks CAH (1962) Absorption spectrophotometry 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 principles 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 phosphorescence, 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 bacteriochlorophyll 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 Photosynthetic 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, Orlando. Schreiber U (1986) Detection of rapid induction kinetics with

10 a new type of high-frequency modulated chlorophyll fluorometer. Photosynth Res. 9: 261–272. Schreiber U, Neubauer C and Schlina U (1993) PAM fluorometer based on medium- frequency pulsed Xe- flash measuring light: A highly sensitive new tool in basic and applied photosynthesis research. Photosynth Res. 36: 65–72. Shibata K (1958) Spectrophotometry of intact biological materials. Absolute and relative measurements of their transmission, reflection and absorption spectra. J Biochem (Tokyo) 45: 599–623. Spruit CJP (1971) Sensitive quasi-continuous measurement of photoinduced transmission changes. Meded Landbouwhogeschool 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 absolute quantum yield of fluorescent solutions. Trans Faraday Soc 53: 646–655. Witt HT, Moraw R and Müller A (1959) Blitzlichtphotometrie. 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

11 12 13 15 16 16 17 17 18 18 19 19 20 20 20 21 21 22 23 23 24 24 24 24 25 26 27 27 30 33 33 35 35 35

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]

11 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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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 nevertheless 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 structural 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 structural parameters can be determined only by means of LD and CD investigations. These methods are also indispensable when structural reorganizations 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 qualitative accord with the results of X-ray crystallography. A recent systematic study on bovine gamma-crystallines showed the excellent agreement 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 substitute a systematic review. Throughout the chapter, the emphasis will be placed on the concepts, and the mathematical formalism will be used sparingly. LD and CD spectroscopy have developed significantly 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 combined with novel methods, such as photoacoustic LD et al., 1985), differential polarization microscopy (Finzi et al., 1989) and circular differential polarization scattering (Garab et al., 1988c). Some other techniques, such as vibrational 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),

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and later developments were summarized by Breton (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 reviewed 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 proportional to each other in magnitude, are mutually 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 polarization 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 spectroscopy, 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 superpositions to conceptualize the state of polarization of a light beam. As shown in Fig. 1A and B, circularly polarized light can be represented as the sum of two orthogonal linearly polarized beams in which the amplitudes are equal and the phases are shifted by exactly + or With a phaseretardation of another linearly polarized beam is obtained, the polarization of which is orthogonal 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-

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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 superpositions, circularly or linearly polarized light can easily be constructed. In the following the modulation 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 polarized 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 technique (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, respectively. In most samples, there are correlations between different elements of the matrix. Generally, however, all 16 elements can be independent 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 the absorption LD and CD are determined, mainly because of technical difficulties and the poor understanding of the physical meaning of other elements.

B. Absorbance of Light by Molecules During an optically induced transition, the electron distribution of the molecule oscillates periodically 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 transition dipole moment, which for an optically induced 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

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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 transitions are considered for fixed nuclear positions, i.e. for no vibronic coupling of the transition. The admixture of vibronic components may complicate the interpretation of the polarization measurements. 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 polarized 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 concentration (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 absorbance 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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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 determined 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 transition 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 molecular coordinate system. It should be noted that, for symmetry reasons, not all molecules can exhibit 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 useful 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. Conventionally, the X passes through C7, the position of phytyl substitution, and the Y axis through the N atoms of pyrroles I and III in Fig. 3). (The axes, are selected on the basis of the electronic symmetry of the system.) The main electronic transitions of Chls are labeled with indices X and Y, and and and for the red and the Soret bands, respectively, to indicate that they are polarized along these axes. However, Bauman and Wrobel (1980)

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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 109°, thereby closer to Furthermore, it was concluded that other transitions of Chl a cannot be characterized in simple terms of a transition moment in the molecular frame but must be described in terms of averages of goniometric functions.

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 referred to as and coincide with the preferential alignment of the sample, e.g. the plane of membranes or the long axis of complexes or aggregates. 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 performed:

(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 reduced dichroism and the dichroic ratio (DR):

If in membranes, S > 0 (DR > 1) it can be concluded that the dipoles tend to lie in the membrane plane, with with respect to the normal to the plane is called the magic angle, an orientation which cannot be distinguished from random orientation in an LD experiment. The dichroism of the sample is often characterized 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 symmetry 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 deexcitation processes. (Contributions from the anisotropic 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 emission is polarized because, for the same electronic 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 excited with non-polarized light, and the system must involve perfect energy transfer. For the general case, FP depends not only on the fluorescence, but also on the absorbance of the sample, 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 excitation 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 fluorescence 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. Conventionally, stands for the degree of polarization of fluorescence after polarized excitation of a randomly oriented sample, the indices referring to the polarization directions of the observation with respect to the direction of polarization of the exciting beam (see Chapter 3). In complex systems, e.g. photosynthetic membranes, it is technically difficult to separate FP from P. As recognized by Breton et al. (1973), the intrinsic anisotropy of dipoles, which in oriented samples gives rise to can contribute significantly to the measured value of P even if macroscopically FP = 1. This “residual polarization” 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

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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 depends 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 reflections. This method is useful when the water content 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 spreading (Breton et al., 1973). Some artifacts may be present (Haworth et al., 1982), which are probably 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., 1972) to whole chloroplasts et al., 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 hydrodynamic gradient, is used mainly for long, cylindrical 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 concentration, and there is essentially no restriction as concerns 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 Abdourakhmanov 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 environment 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 temperature measurements. Increasing the concentration of acrylamide and increasing the ratio of bisacrylamide 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, personal 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 polyacrylamide 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 membrane. This can probably account for observations that in heliobacterial membranes BChl g was bleached (van Dorssen et al., 1985) and artifacts appeared in complexes of fucoxanthin-containing algae (Hiller and Breton, 1992). 2. Orientation in a Magnetic Field The magnetically induced orientation of a photosynthetic system was first reported in Chlorella by Geacintov et al. (1972). A major advantage of this method is that practically no restriction applies as concerns the composition of the medium, and the degree of alignment can be nearly 100%. However, as the method is based on the diamagnetic 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 orientation 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 perpendicular 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 electric field orientates so as to minimize the total energy of the system (Charney, 1988). The measurements are usually restricted to low ionic strength. The electrophoretic movement of particles can be prevented by using alternating voltage or pulses. It has been shown that the frequency and the magnitude of the voltage may influence not only the degree, but also the mechanism of orientation through permanent and induced dipole moments (Gagliano et al., 1977; Charney, 1988). Chloroplasts can be oriented in an a.c. (50 Hz) field of about while small particles, e.g. chromatophores or isolated complexes, can be easily oriented by millisecond electric pulses of A quasisteady-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 polarized light, which induces anisotropy in the sample. This occurs alike in intrinsically isotropic samples (e.g. solutions of molecules) and in samples containing randomly oriented intrinsically anisotropic particles (e.g. a membrane suspension). The anisotropy function, r, for absorbance difference is defined as

where the indices refer to the orientation of the polarization direction of the probe light with respect 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 emission of the detected chromophore with polarization directions parallel and perpendicular to the actinic polarization (Breton and Vermeglio, 1982). This technique has become increasingly important in determination of the mutual orientation of the dipoles in the reaction center preparations containing small number of Chl molecules (Kwa et al., 1994). The method of photoselection is also ideally suited to the monitoring 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 membranes cannot be deformed, that there is no fluctuation in the orientation angles and that both LD and absorbance can be measured with high precision. With the exception of the last conditions, in realistic systems these assumptions do not hold and the conclusions from idealized systems remain qualitative. (Although LD and absorbance 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 amplitudes. Most dichrographs measure and use the approximation of which is not true if 1. Degree of Orientation, Distribution Functions In some systems, a large proportion of the particles are found to be perfectly aligned whereas the complementary population remains at random, 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 fragments can not be oriented even at much higher

21

field strengths (Garab et al., 1981). In other systems, e.g. with gel-squeezing or film-stretching techniques, the random suspension is gradually shifted toward the ‘perfect’ alignment, but saturation 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 distribution function and calculations for the unfavorable cases, i.e. rod-shaped particles with unidirectional 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 realistic cases. In our laboratory S values in chloroplasts 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 deformation of the shape. Since, however, distortions may occur it is advisable to carry out the experiments with different known values of the squeezing parameter, with cells of defined dimensions and extrapolate the value of the orientation angle to the non-squeezed case, and/or carry out the calculations 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 homogenous, 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 between two crossed polarizers is bright and uniform. Although the technique of gel-squeezing has been shown to yield reliable data on the orientation 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 chloroplasts, such model calculations led to the conclusion that the long-wavelength emitting dipoles of Chl a lie essentially in the plane of the membranes. It was also shown that dipoles of 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, despite 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 anisotropy (Breton et al., 1973) may contribute significantly to the degree of polarization of fluorescence (see II.B.3) points to the need for microscopic fluorescence polarization investigations, possibly in combination with fluorescence lifetime imaging (Gadella et al., 1993). Such techniques would probably permit estimation of the magnitude of local order in native membranes and/or in lamellar aggregates of purified complexes, provided the local order is longranged enough compared to the resolution. 3. Fluctuations The orientation of a transition dipole may fluctuate in a certain angular interval. The fluctuation of the pigment dipoles can originate from (i) the pigment-protein complexes and/or (ii) the fluctuation 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 orientation 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 protein axis. It may be speculated that major structural rearrangements in the antenna are accompanied by reorientation of some complexes. Such changes may be responsible for the observed LD changes due to state transitions in cyanobacteria (Homer-Dixon et al., 1994). Data obtained on algal mutants and on chloroplasts 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 result in a better alignment for disc- and rod-shaped particles, respectively. Thus, if the shape of the (sufficiently rigid) particle is unknown, measurement 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 macrostructure 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 transitions, however, see Nordén et al., 1992.) Thus, the measurements can be used to resolve overlapping 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 chlorosomes. 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 chloroplasts 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 differential polarization imaging techniques (Kim et al., 1987a) and laser scanning microscopy (Shotton and White, 1989), a more refined use of polarization microscopy seems possible. The fact that chloroplasts can be sliced optically during the recording of LD images (Garab et al., 1991a) opens up the possibility for 3–dimensional reconstruction of the thylakoid membrane system through the use of LD.

CD arises from the intra- or intermolecular asymmetry (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 systems, see below.) Although CD is the most commonly determined chiroptical quantity, it is necessary to recall the correlations between CD and the other manifestations of optical activity: ellipticity, ORD and CB.

LD and CD

25

Absorbance by an optically active sample induces ellipticity in the linearly polarized measuring beam. This can be understood if it is taken into account that linearly polarized light can be decomposed into two oppositely rotating circularly polarized light components of equal amplitudes (see I.A). After the selective absorbance of one of the components, the two intensities do not remain equal and thus the beam will become elliptically polarized. It is obvious that CD and ellipticity are equivalent quantities. Although modern home-built or commercially available dichrographs measure absorbance differences, 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 measurement of time-resolved CD with nanosecond resolution is based on ellipsometry and not (Lewis 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 strength it must be stressed that this does not depend solely on the electric dipole of the transition, but also on the magnetic dipole (m): (19)

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

Im indicates that the rotational strength corresponds to the imaginary part of the scalar product, 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 component parallel to For these to occur, the 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 motion 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 ring, while is in the plane.) This explains the 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 chromophores (Tinoco, 1962; DeVoe, 1965). Excitonic interactions give rise to a conservative band structure (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 differential scattering of left and right circularly polarized light:

Garab

26

Systematic studies have revealed that these signals 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 Bustamante, 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 luminescent, 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, subchloroplast particles and chloroplasts, and demonstrated large differences in both magnitude and sign. Additionally, it was concluded that in chloroplast 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 sensitivity of CPL to scattering and sieve effects (Duysens, 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 (Tinoco 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 photosynthetic 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 technique 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 perturbation that induces CD in chiral or achiral

samples. Since MCD arises via a different mechanism, 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 transitions; 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 chromophores 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 interpretation of data. CD in oriented systems contains additional information due to the fact that chiroptical 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 information on the symmetry of the excitonic aggregate. In photosynthesis, the conclusion reached by Charney in 1979 still applies: “This field remains 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 environment, but the participation of excitonic interactions 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, attributed to the long-range coupling of chromophores 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 corrected 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 significantly 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 distance vector R. If the two molecules are brought close enough to each other to interact electronically, but are still sufficiently apart for the electrons to remain localized on each of the molecules, the absorbance band splits into two bands. The degree of separation of the bands depends on the interaction energy between the two dipoles and

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 molecules, 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 preferentially in-plane orientation of dipoles with 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, respectively, is the band center energy, and the vectors in scalar triple products are unit vectors, It is clear that for the dimer is independent of for the monomers, and i.e. the CD of the couplet is 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 conservative 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 identification of the different bands very difficult. In contrast, CD selectively detects the excitonic interactions on a weak background of the intrinsic CD of the pigment molecules, thereby offering a much better sensitivity than absorbance for identification 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 favorable 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 interactions with known geometry it is possible to calculate these spectra, as in purple bacterial reaction 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 pigment dipoles, respectively. It must also be realized 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 (Breton and Vermeglio, 1982; Garab et al., 1987). The characteristic CD spectra of different particles 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 bacterial 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 interact with the dipole of a nearby molecule (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 excitonic 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 center of a green bacterium, Chloroflexus aurantiacus, was analyzed by using exciton theory and the structure of Rps. viridis (Deisenhofer et al., 1984). The data suggested that the arrangement 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 interactions 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, interpretation of the absorbance and CD of the FMO complex turned out to be more difficult than anticipated, 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 Pearlstein (1993) found that some cryosolvents perturb the structure. Zhou et al. (1994) have reported 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). However, 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 complexes, the lack of high-resolution structure hampered analysis of the CD spectra in terms of exact structural parameters. Analyses of the CD spectra and the amino acid sequences led to the construction of models which satisfactorily described the structure and function of these antenna complexes (Scherz and Parson, 1986; Zuber and Brunischolz, 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 importance 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 analyzed in terms of exciton theory. These CD spectra 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 complex 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 appropriate distances and mutual orientations of the Chl molecules. In chlorosomes, BChl c oligomers appear to be the main building blocks, thus investigations 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 aggregates is commensurate with the wavelength of visible light, and (iii) the pigment molecules appear to be assembled with long-range order. Thus, the CD signals may originate in part from the long-range chiral order of the pigment molecules, 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 macrosystems and determination of the possible contribution 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 (Bustamante et al., 1985). The theory for psi-type aggregates describes the interaction of light with large inhomogenous molecular aggregates containing a high density of intensely interacting chromophores (Keller and Bustamante, 1986a,b). The theory of imaging macrodomains that have different 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 wavelets 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 wavelets maintain a well-defined polarization and phase relationships to each other, the interference phenomenon is greatest when the wavelength of the circularly polarized light closely matches the dimensions of the macrohelix. In the first Born approximation, each dipole is considered independent of the others. The interaction 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 macrostructure. It must be stressed that the angle-depen-

LD and CD

dence of the non-polarized scattering is not related 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 observation, carry useful information on the macrostructural parameters of large objects. The psi-type CD theory (Keller and Bustamante, 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 consider not only these short-range interactions but also long-range effects. In psi-type aggregates the full electrodynamic interaction between the dipoles must be taken into account (Keller and Bustamante, 1986a). In small aggregates, the entire 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 commensurate with the wavelength this is not true and retardation effects can play an important role. At distant points of observation the oscillating dipole can be regarded as a radiating spherical wave. Thus, the chromophores at large distances can be coupled via a radiation coupling mechanism between the dipoles. In large chirally organized aggregates the significance of the radiation 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 aggregates is not the coupling between individual pairs of chromophores, but the coupling between any given chromophore and the rest of the chromophores 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 and dependences, describe the static dipole coupling and the radiation term, respectively. The second term, with is called the intermediate coupling. The final term ensures that the self-interaction is zero. Due to static, intermediate and radiation coupling 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 handedness. In psi-type aggregates, the theoretical prediction is that if the long-range coupling between the dipoles is strong, the excitation generated at one chromophore can delocalize for the entire aggregate. This is called the collective absorbance, which increases or decreases at a given wavelength depending on how well the light is able to produce a collective excitation in the system. When the group polarizabilities are made weaker, the groups are moved farther apart or the density of the chromophores inside the aggregate 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 Bustamante, 1986a,b) reduces to the classical theory of DeVoe (1965), which describes excitonic interactions. Since psi-type aggregates also satisfy the criteria for CIDS, psi-type CD is always accompanied

32

by CIDS. In CIDS alone, the static and intermediate 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 information on the macro-organization of large molecular structures, and resolve microscopic domains 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 physical information on the macro-organization of pigments (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 rearrangements of the pigment-protein complexes, non-polarized scattering yields far less specific information (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 “islands” 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 interpretation 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 excitation energy in the antenna (see e.g., Horton et al., 1991; Istokovics et al., 1992). In granal chloroplasts and LHCII macroaggregates, 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 asymmetry is introduced, e.g. during macroaggregation, these systems satisfy the conditions for psitype aggregates. Barzda et al. (1994) showed that, in accordance with the prediction of psi-type theory, 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. marina (R. Meckenstock, G. Garab, R. A. Brunisholz 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 experimental conditions and on systems permitting realistic model calculations quantitative interpretation of psi-type spectra does not seem possible. It would be equally important to understand the functional significance of the psi-type organization 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 reflects the backbone conformation of the polypeptides. The amide chromophore has plane symmetry and is itself not optically active. In a peptide, a chiral electrostatic field can be provided by the surrounding amides and other polar groups; exciton interactions between chromophores also contribute significantly to the CD signal (Woody, 1985; Johnson, 1990). Some of the interactions can be specifically assigned to certain conformations (Woody, 1985). The conformations are characterized by the 222 nm negative band and an excitonic couplet 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 unordered 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. Semiempirical methods for this analysis are based on different 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 statistical 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 systematic 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%, respectively) . Similar contents were found in other isolated complexes (Breton and Nabedryk, 1987).The orientation of the with respect to the membrane normal was found to be less than 30°, as determined from IR LD measurements (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 secondary 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 orientation and with a mimicked absorbance. In the presence of large particles, which “concentrate” the chromophores and are commensurate 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; corrections can then be difficult and efforts must be concentrated first on correcting for CDS. In a conventional dichrograph, CD of absorbance and CDS are combined into the apparent CD signal. CDS can most easily be recognized by varying the acceptance angle of the photomultiplier, 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 absorbance band, CD is produced by CDS and exhibits a monotonously decreasing signal (“long tail”) on the long wavelength side of the absorbance 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 fluorescing photosynthetic systems, and to my knowledge, 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 conventional 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 anisotropic, 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 heliobacteria. The LD was caused by gravitational orientation of the cells, whereas the LB originated mainly from the strain in the window of the cryostat. The magnitude of the spurious signal increased 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 differential 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 components. In particular, CD image artifacts due to imperfections in circular polarization can be eliminated by using the fact that the CD-related images or are invariant on the rotation whereas contributions from a linear anisotropic signal are sensitive to rotation. CD images of chloroplasts were tested for linear dichroic contributions in both face-aligned (LD = 0) and edge-aligned positions, 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 macroscopic samples with magnetically aligned chloroplasts trapped in gel. CD spectra were recorded in vertical position and at ± 45° and ± 90° with respect to the vertical direction. The contribution of LD to CD (in position) was found to be 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 organelles. For most applications, LD and CD have solid theoretical background and elaborated experimental 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. Conclusions 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 transitions of Chls. The importance of excitonic CD has been demonstrated thoroughly in almost all photosynthetic complexes studied so far. As shown for the case of the FMO complex, CD contains information on virtually all possible pigment–pigment interactions as well as the interactions of the pigments with the protein moieties. This information, 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 absorbance spectra on the basis of the physical correlations 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 appears 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 approach. 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 activities, 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 sphaeroides 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 bacteriochlorophyll 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

41 42 42 42 45 45 46 47 48 49 50 50 51 52 52 52 52 53 54 54 55 57 59 59 60

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 – phycocyanin; TCSPC – time-correlated single-photon counting

I. Introduction Fluorescence emission derives from excited electronic states of molecules. In photosynthesis the molecules of interest are associated with the antenna and the reaction centers. Fluorescence from chlorophylls, bacteriochlorophylls and phycobiliproteins in whole organisms or in preparations 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 energy, 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 involved 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 chromophores in the pigment-protein complexes that are ubiquitous in photosynthetic membranes and their associated components. The resulting delocalization of the excitation is essential to the function 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 excitation 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 fluorescence on a still longer time scale. We see from this scenario that the molecule whose fluorescence 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 measurements; however, steady-state measurements, which average the time-dependent behavior, also provide useful guides to investigating and interpreting 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 characterize this process are given in several monographs (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 thermally 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 mechanical coupling between the ground and excited states.

43 3. Excited electronic states of photosynthetic pigments invariably involve delocalized trons associated with the conjugated or aromatic bonding systems in chlorophylls, openchain tetrapyrroles or carotenoid polyenes. Because the mass of the electron is small compared 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 produced with a nuclear configuration that is initially 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 microstates that is present in the initial thermal distribution of ground state configurations, together with the equally large variety of excited state configurations that can result from the absorption 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 relaxation results from an exchange of energy among internal modes of motion of the chromophore, 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 conversion, ic, from higher energy excited electronic states etc. to the lowest energy excited state having the same spin multiplicity (typically singlet) as the ground state. 6. Thermal equilibration occurs as the excited state configurations attain the distribution corresponding to the temperature of the environment. This occurs typically within a few picoseconds 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 comparison of absorption or excitation spectra with fluorescence emission spectra using relations derived by Stepanov (1957). 7. Several distinct fates are possible for the thermally relaxed excited electronic state. a) Fluorescence – radiative decay from the excited 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 radiation by the ground state. The spectrum of fluorescence is typically red-shifted (Stokes shift) relative to the longest wavelength absorption band, because the excited electronic 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 fluorescence 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 energy 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 quantum mechanically, but for complex molecules 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 photosynthetic materials this occurs by an inductive resonance process described initially by Förster (1948, 1967). The inductive resonance 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 energy overlap between D and A, (2) the inverse 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 involved 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 “isolated” molecule. Trapping of excitation in reaction centers resulting in productive charge separation is an example of photochemical quenching. Other types of quenching 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 quenching. 8. Polarization is a consequence of the vectorial nature of the absorption and emission of radiation. The transition dipole moments associated with these processes have both a magnitude (related to the allowedness or the “oscillator strength” of the transition) and a direction relative to molecular axes. For a molecule 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. Subsequent emission from the excited state is polarized in a manner that depends on the transition moment vector for fluorescence and on the direction of observation relative to that of excitation. Depolarization of the fluorescence can result from: a) Intramolecular relaxation ic from higher excited 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 fluorescence. 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 transition dipole moment for spontaneous emission (Einstein A coefficient). For molecules having similar ground- and excited-state nuclear configurations, 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 decreased 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 competing processes). 11. Fluorescence relaxation for simple molecules in dilute solutions and at low incident light intensities is kinetically first-order in the excited 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 components with different relaxation rates, b) high local concentrations of excited states that result in excitation annihilation (bimolecular), often a consequence of high intensities 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-

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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) electronic components for displaying, recording or storing output information in a form that is accessible 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 arranged 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. Polarizers 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 photomultiplier or a solid state photodiode, must be sensitive 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 electronics, 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 importance 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 exciting 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 sample may result in fluorescence, phosphorescence, 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 illuminated at low intensities, both and are linearly 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 dependence 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 pigments 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, indicating 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 chlorosomes to the membranes is disrupted by cell breakage excitation transfer from the accessory pigments is no longer effective. Thus, wavelengths absorbed by the accessory pigments, mainly Chl b and carotenoids in the case of spinach chloroplast thylakoids (Fig. 3), do not lead to Chl a fluorescence, and the fluorescence excitation 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

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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 incompletely resolved for large chromophores in condensed media), the fluorescence emission spectrum 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 extract, 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). Failure of this relation may indicate situations where there is (1) significant overlap of more than one electronic transition in the absorption spectrum, as is the case for the and transitions of Chl a or Chl b, (2) incomplete relaxation or thermalization of the excited state prior to emission, or (3) excitation transfer among identical molecules in different local environments that produce different spectral shifts, or among chromophores that are chemically distinct but have overlapping absorption 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, submitted). 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 triplet state or may have symmetry elements that cause it not to couple radiatively with the ground state. Nevertheless, molecules such as carotenoids can transfer excitation to “sensitize” the fluorescence of nearby chlorophyll molecules in photosynthetic membranes. The fluorescence of mixtures of chromophores of different chemical types is, in general, an additive 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 molecular 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 selected. Useful assays of mixtures of photosynthetic 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 extract using the facts that (1) absorption (excitation) 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 obviously necessary to avoid higher pigment concentrations where excitation transfer from chlorophyll 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 sample 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 signal 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 different experimental set-ups, a good rule of thumb is to use samples with absorbance (optical density) 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, because 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 directly 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 concentration (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 extent, 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 detailed dependence on angle is different. Scattering 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 coupling 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 scattering to occur. The scattered light is readily detected by sensitive fluorescence spectrophotometers, 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 methods to measure the fluorescence at the same wavelength as the exciting light, even for homogeneous solutions that are “dust-free”. However,

Fluorescence when the scattering is not too intense, the fluorescence excitation and emission wavelengths can be within a few nanometers of one another, depending 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 molecular 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 excitation 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 vectorial 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 predominantly in directions that are not along the transition moment vector. Between the excitation and emission processes, events such as internal conversion, 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 sample. 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. Unpolarized 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 illuminated from one direction is always anisotropic. 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 anisotropy 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 direction). Depending on which orientation of the polarizer is chosen, a different subset of the chromophores will be excited, determined by the projection of the oscillating electric field of the exciting light on the absorption transition dipoles of the individual chromophores. Depending on which subset is excited, the projection of the ensemble of molecular vectors is different in the direction of propagation of the fluorescence emission. 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 sample fluorescence can be readily determined from the relative intensities of emitted light detected when the analyzer is oriented eithe parallel vertical) or perpendicular horizontal) to the 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 polarization effects quite apart from those of the sample, 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 correction is described by Houssier and Sauer (1969) and by Lakowicz (1983).) For purposes of interpretation of polarization measurements for a particular sample, the measured (and corrected) polarized fluorescence intensities are combined to give a value for the polarization anisotropy, A, where

Each of the quantities involved in Eq. (2) is dependent on the wavelengths of excitation and emission. (The reader should be aware that an alternative description of the polarization anisotropy is sometimes seen, especially in the earlier literature, 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 relevant 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 membrane fragments, (2) rotation of the chromophore during the excited state lifetime, and (3) excitation 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 anisotropy value of zero. For an isotropic sample where the individual molecules are (1) effectively fixed in their orientation 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 dipoles are parallel to one another, to – 1/3, when they are mutually perpendicular. Intermediate values of the polarization anisotropy can be interpreted in terms of the angle between the ab-

Kenneth Sauer and Martin Debreczeny sorption and emission transition dipoles. If excitation transfer occurs among the molecules in the sample, the polarization anisotropy can be interpreted in terms of the relative orientation of the absorption transition moment of the chromophore 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 significant 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 directions of the absorption transition moments for transitions to different electronic excited states of pigment molecules like chlorophyll, bacteriochlorophyll or protochlorophyll have been derived from experimental measurements and compared with theoretical deductions (Gouterman and Stryer, 1962). Studies of fluorescence depolarization of pigment molecules in solution as a function 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 preparations, 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 arrangement of the chromophores where such information 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 fluorescence and photochemical trapping in reaction centers. As a consequence of this competition, fluorescence yields are complementary to the yields of productive electron transport initiated by the reaction centers (Latimer et al., 1956; Govindjee et al., 1986; Krause and Weis, 1991; Dau, 1994). In Photosystem II and in purple bacteria, closing the reaction centers by strong illumination, using inhibitors of primary electron transport or adding reducing agents that keep the endogenous 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 preparations. Special instrumentation has been developed to measure variable fluorescence yields and the associated kinetics of fluorescence induction. Depending on the light intensity used to close the traps and on the conditions of inhibition, the process occurs primarily on the nanoseconds to seconds 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 understood, Photosystem 1 makes little or no contribution to the variable fluorescence signals (Briantais et al., 1986). Instrument requirements for these studies differ significantly depending on the time scale involved. Because light plays the dual role of stimulating 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 illumination, one of which provides “exciting” light that is chopped or modulated at a frequency of several hundred and the other provides steady (unmodulated) “actinic” light when it is turned on. The exciting light intensity is set

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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 simultaneously 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 distribution of Photosystem II in thylakoid membranes (Melis, 1991). 2. Electric Fields Influence Fluorescence Associated with Photosynthetic Materials Because the transport of electrons across photosynthetic 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 chemical potential for driving some of the dark energyconserving biochemical processes, but also an interaction with pigment molecules in the membranes that absorb or fluoresce strongly. (See chapter by S. Boxer in this volume.) The direct effect of the field produces carotenoid and chlorophyll 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 of (Lockhart et al., 1988). In such experiments the samples are generally spatially isotropic, 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 between 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 relative salt concentrations of the solutions used in rapid mixing experiments (Dau and Sauer, 1991). Based on both steady-state changes in fluorescence intensity and time-resolved fluorescence relaxation measurements, the effect of the applied 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 detect, indicating that they reflect only a small portion of the pigment molecules present. Because the excited states responsible for the long wavelength fluorescence are at energies close to or even lower than the excited states of the associated reaction centers, the role of these pigments in the collection of light and the funneling of excitation to the reaction centers is of considerable interest. Nevertheless, little is known at present about the mechanism of the very effective quenching of this fluorescence at room temperature. 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 homogeneous dilute solution, the effect of lowering the temperature of the sample is primarily to narrow the spectral widths of the components of the emission spectrum (also of the absorption and excitation spectrum), thus improving the spectral resolution. Apart from this sharpening of the incompletely resolved spectra, there are no dramatic changes in fluorescence yield. Many photosynthetic 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 between room temperature and 77 K. Furthermore, the spectrum of the fluorescence increase is typically 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 continuous 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 arriving 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 fluorescence is widely used and its practical implementation 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 relatively short time. The time resolution of this technique 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 limiting factor for the TCSPC technique is usually the response time of the detector. Micro-channel plate detectors, a type of photomultiplier designed to achieve optimal time resolution, currently 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 response 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 fluorescence is upconverted at a particular crystal orientation, a time-resolved fluorescence spectrum 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; Debreczeny, 1994). The chief disadvantage of the upconversion technique is the low efficiency with which currently employed crystals upconvert fluorescence. 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 fluorescence data. The fluorescence upconversion technique of time-resolving fluorescence is similar to the pump-probe technique of measuring transient absorption 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 interpretation of pump-probe experiments, are avoided in the fluorescence upconversion experiment. (See chapter by G Fleming in this volume.) 3. Deconvolution An experimentally observed time-resolved fluorescence signal consists of the molecular fluorescence signal of interest convoluted with the instrument 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 instantaneous and the convolution integral ignored. However, because interesting events in photosynthesis 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 technique, 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 wavelengths. As a consequence there is some uncertainty in the designation of time zero. Data from individual decays at different fluorescence wavelengths 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 measurement of time zero. The IRF can be measured with the same geometric arrangement as the fluorescence upconversion signal by tuning the nonlinear 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 upconversion 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 average laser power. High pulse energies at low repetition rates have been used to study photostable dye molecules. However, in multi-chromophore systems like photosynthetic proteins, high energy pulses can lead to exciton annihilation

Fluorescence (Geacintov and Breton, 1982). Exciton annihilation can occur when two or more chromophores that are coupled by energy transfer each absorbs a photon. The experimental result is that the fluorescence decay profile shows an excitation intensity 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 context 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 annihilation effects are to be avoided. Using the above equation, the extent of exciton annihilation in the TCSPC technique and the upconversion technique are compared, choosing the trimeric aggregate of C-phycocyanin (PC) excited at 624 nm with a repetition rate of 4 MHz as an example. In a typical TCSPC experiment the laser power at the sample is 0.5 mW, and the beam waist is roughly (unfocused). The average number of photons absorbed per pulse per PC timer in such an experiment is In a fluorescence upconversion experiment a typical power seen by the sample is 1 mW and the laser is focused onto the sample to have a beam waist. The number of photons absorbed per pulse per PC trimer under these conditions is If much larger complexes (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 fluorescence and shown below in Eq. (4), the fluorescence 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 by the relative extinction coefficients, of the 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 homogeneous chromophores, P(t) will decay as a single exponential. The contribution of each chromophore 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 differences 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 applicable), Eq. (4) still holds. However, the population term, P(t), is no longer necessarily described 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 increases 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 included 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 absorption 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 unambiguous measure of excited-state decay without interference from product formation. Unlike dye molecules free in solution, the chromophores in photosynthetic systems are organized 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 energy transfer from a donor to acceptor chromophore in a light-harvesting complex can be described by a narrow range of rate constants and is often well approximated by a single rate constant. Similarly, electron transfer reactions in photosynthetic 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 constants 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 ionstate 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 same rate constants, However, the amplitudes, associated with each exponential term will be different for the different chromophore species. By incorporating Eq. (6) into Eq. (4) we can see that the isotropic time-resolved fluorescence signal is described by a sum of exponentials. It is for this reason that the most common method of analysis of isotropic time-resolved fluorescence 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 constants for energy transfer and electron transfer between chromophores. A common analysis technique that relies on the assumption that the rate constants are independent of the probed emission wavelength is to fit data simultaneously at multiple 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 techniques can increase the number of resolved kinetic components. Unfortunately, without simplifying 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 developed 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. Holzwarth in this volume). In this case the difficulty often lies in establishing the uniqueness of a particular solution or model. When using the summed exponential model to describe time-resolved fluorescence 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 fluorescence 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, referred to as and according to the amino acid residue to which the linear tetrapyrrole chromophore 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 increasing 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 chromophores are excited by the laser pulse, the higher energy chromophore is preferentially excited. 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 lower-energy chromophore with concurrent loss of the excited-state population of the higherenergy chromophore With prior knowledge of the relative extinction coefficients of the two chromophore types, the time-resolved spectra at all times were modeled, and information about the rate constants for energy transfer and the fluorescence spectra of the individual chromophores 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 fluorescence spectra of the and chromophores 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 interactions. At inter-chromophore separation distances of <20 Å, however, strong energetic interactions 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 chromophores and Eq. (4), which treats the net fluorescence 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 arranged 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 observable 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 isotropic fluorescence spectroscopy. If, however, the polarization of the emitted fluorescence is measured 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 (calculated according to Eq. (2)) can be interpreted as being a function of the dot product of the absorption transition dipole, induced by the excitation pulse, with the emission transition dipole, observed at some later time, t.

is the second Legendre polynomial in x, and the brackets around represent a correlation function that includes the rotational averaging necessary 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 rotation 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 absorption transition dipole can be extracted from the residual anisotropy level. If energy transfer between chromophores is being probed, fluorescence anisotropy will be sensitive 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 rotational diffusion). If the emitting chromophore is different from the initially absorbing chromophore, the fluorescence anisotropy value will decrease according to the extent by which the emission transition dipole is rotated away from the orientation of the absorbing transition dipole. In a typical case, the observed fluorescence spectrum 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 different energies, the anisotropy in the long wavelength region of the emission spectrum will normally decrease more rapidly than that in the short wavelength region, because energy transfer occurs preferentially downhill energetically. The reader is referred elsewhere for derivations of the model function used to fit timeresolved anisotropic fluorescence of photosynthetic 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 monomers and trimers isolated from a genetically engineered strain of Synechococcus sp. PCC 7002 (Debreczeny et al., 1995). The mutant strain was engineered to be missing one of the three 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 the trimer with symmetry (axis of symmetry perpendicular to the plane of the diagram). Trimers of PC stack to form the “rods” of the phycobilisome, 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 present in this system (the and chromophores), 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 parallel). Using Förster theory, a decay time of 158 ± 12 ps can be predicted, in reasonable agreement with the measured value (Debreczeny et al., 1995). When the monomer PC is aggregated into trimers, 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 possible to estimate the angle between the transition dipoles of the and chromophores on adjacent monomers (52°) and to restrict the allowed values of the angles formed between the axis of symmetry and the transition dipoles of the and chromophores. Further structural information could be obtained if oriented rather than isotropic samples of PC were used in these experiments. (See chapter by G. Garab in this volume.) IV. Conclusion

anisotropy decay. The PC trimer anisotropy decays to a much lower value and reaches its residual anisotropy level much more quickly than does the monomer. Two distinct regions of the PC trimer anisotropy are evident, showing characteristic 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 between the and chromophores on adjacent 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 photosynthetic energy conversion. Knowledge of the energies and lifetimes of the excited states of pigment molecules provides insights into events associated with excitation transfer among antenna chromophores, trapping and charge separation in reaction centers, charge recombination, quenching processes, etc. Existing quantum mechanical theory 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 experimental values for excitation transfer rates in chromophore 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 questions 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 contribution. We are especially grateful to Mary Talbot 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 Sciences, Energy Biosciences Division, of the U.S. Department of Energy under Contract No. DEAC03–76SF00098.

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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 Spectroscopy, 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 fluorescence 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, fluorescence 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 Kohlensäureassimilation. 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) Bioenergetics 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 emission using frequency-dependent phase angle and modulation 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 bacterial 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.

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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. ConcludingRemarks References

63 63 64 64 64 65 65 66 66 68 70 70 71 72 72

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 experiments. 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 spectroscopy 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]

63 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 developed as have extensions into the infrared spectral region. The reader is referred to recent literature for details of these techniques (Diller et al., 1992; Maiti et al., 1993, Durrant et al. 1994). Solid state lasers, in particular Ti:sapphire lasers have greatly simplified the practice of, and extended the capabilities of ultrafast spectroscopy. We describe apparatuses based only on such lasers in the belief that dye laser-based systems will become obsolete within the next five to seven years. One aspect of ultrafast spectroscopy that contrasts 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 respect to a vibration. In these cases, vibrational quantum beats may be observed in the pump probe or spontaneous emission signals. The beat frequencies allow measurement of vibrational 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) discuss these points in detail. The rate of disappearance 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 observation 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 principles of fluorescence upconversion are described, along with a discussion of the optical arrangements 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 amplifiers 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 generation and pulse compression. The cavity arrangement 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 pulses with 1–6 nJ energy at a repetition rate around 80 MHz, with pulses as short as 11 fs. Cavity

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dumping of these oscillators has also been demonstrated, 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 combined 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. Furthermore, 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 amplifiers are available commercially. The first category is that of Nd:YAG pumped amplifiers. The pump source is the second harmonic 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 operate at 1–10 kHz repetition rates and typically utilize pre-amplification pulse stretching, electrooptic switching, and post-amplification compression (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 injected and ejected acousto-optically (electro-optic devices are limited to 10 kHz or lower). Pulse stretching prior to amplification may be employed, but more commonly the pulse is stretched by dispersive elements within the amplifier cavity. As usual, the pulse is recompressed after amplification. 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 shifting 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 technique is continuum generation. When an amplified 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 modulation which creates a stable white light continuum extending across the entire visible spectrum. 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 enough light to serve as the excitation pulse for a fluorescence upconversion experiment, or the probe pulse for a transient absorption experiment. Selected portions of the continuum may be amplified by optical parametric amplification (Reed et al., 1994). One way of doing this involves temporally and spatially overlapping a portion 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 demonstrated 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 nonlinear 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 wavelength of fluorescence upconverted. The advantage 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 counter. A double monochromator is very helpful because despite the non-collinear geometry, the signal is contaminated with doubled gate beam and doubled excitation beam, both of which are orders of magnitude more intense than the upconverted signal. A prism may also be used for dispersing 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 arrangement, 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 excitation beam. This technique makes it easier to measure fluorescence wavelengths near the excitation wavelength without upconverting the excitation beam. The time resolution of the upconversion experiment is determined by the instrument response function (IRF), which is approximately given by the cross-correlation of the excitation pulse with the gate pulse:

67

where and are the intensity profiles of the excitation and gate pulses, respectively. 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 excitation pulse) are temporally and spatially overlapped. 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 mismatch for their 0.4mm crystal to be 115– 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 measurement (Shah, 1988). Thus, limitations of the upconversion technique make experiments with IRFs of significantly less than 100 fs difficult, even with the availability of shorter pulses. Furthermore, the necessity of using thin crystals with large upconverted bandwidths causes a difficulty in discriminating 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 upconversion 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 measurements resolve the isotropic emission from a sample 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 transfer. Performing these measurements requires the addition of a polarizer to each beam and a waveplate for rotating the polarization of the excitation beam. In addition, it must be verified that the optical arrangement preserves the anisotropy of the fluorescence by ensuring that the anisotropy of a standard system (such as Rb. sphaeroides R26 reaction centers) is 0.4. A further complication in these studies is the presence of singlet–singlet and singlet–triplet annihilation processes whenever high excitation densities and/or high repetition rates are used (van Grondelle, 1985). These processes shorten the fluorescence lifetime of pigment-protein complexes, and may affect depolarization measurements when the systems are highly ordered. Low temperature fluorescence upconversion studies have not yet become common. Signal averaging 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 challenge 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 purple bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides. The timescale for electron transfer from the primary donor, an excitonically coupled dimer of bacteriochlorophyll a (denoted 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 measurements of the decay of P* by stimulated emission, most workers have made measurements at or near the isosbestic point in the spectrum consisting of ground state bleaching and absorption of the radical cation of P and P*. However, such a procedure makes it difficult to observe longer decay components in the stimulated emission. Fluorescence upconversion measurements performed by Du and coworkers clearly showed behavior which had been difficult to observe in pump-probe experiments. After exciting the special pair’s or bands and time resolving the 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 fluctuations on the time scale of the electron transfer (Gehlen et al., 1994; Jia et al., 1994). A second application of fluorescence upconversion to photosynthesis is to the study of energy transfer in light harvesting systems. Energy transfer 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 depolarization of fluorescence from membranes of PSIonly and LHC-II-only strains of Chlamydomonas reinhardtii. The average single-step energy transfer 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 fluorescence studies of photosynthetic systems, we consider the energy transfer processes in bacterial light harvesting complexes. The purple photosynthetic bacteria contain two types of light harvesting 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 complex 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 resolved the single-step hopping of electronic excitation 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 process. As an example of an unusual anisotropy decay, Fig. 7 shows the decay of anisotropy from the bacteriochlorophyll fluorescence upon excitation 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 dipoles

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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 monitoring the intensity of a weak probe beam transmitted through a sample subsequent to the passage of a much stronger pump beam. The differential absorbance of the sample is monitored by a detector, 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 multichannel detection may be used in order to frequency resolve the signal. Lock-in detection may be used with high repetition rate sources, detection being referenced to a chopper placed in the pump beam. Experiments with low repetition rate sources cannot efficiently utilize lock-in detection, so this type of measurement can suffer from

high background levels. Shot-by-shot normalization 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. Multicolor pump probe experiments require care to ensure that the pump and probe beams are focussed 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.

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71

B. Some Examples Extensive measurements of the rates of primary charge separation in reaction centers from Rhodobacter 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 transfer 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 temperature 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 coupled to the electron transfer. A remarkable series of pump-probe measurements 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 Rhodobacter capsulatus Martin and coworkers observed oscillations in the 77 K pump-probe signals (Fig. 10) which they interpret as arising from vibrational wavepackets in the excited state. These oscillations persist for many picoseconds, and indicate 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 reaction centers in their native membrane environment (Vos et al., 1994). Observation of an oscillatory contribution to the signal calls into question the conventional assumption that vibrational dephasing and relaxation occur on much shorter timescales than does the electron-transfer step. A full description of electron transfer incorporating a realistic model of the protein dynamics is still an active area of theoretical development (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 satisfactorily 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 complex 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 scattering 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 bacteriorhodopsin, probed by time resolved infrared spectroscopy. Biochemistry 31: 5567–5572. Du M, Rosenthal SJ, Xie X, DiMagno TJ, Schmidt M, Hanson DK, Schiffer M, Norris JR and Fleming GR (1992) Femtosecond spontaneous emission studies of reaction centers 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 observation of ultrafast energy transfer processes in light harvesting complex II. J Phys Chem 98: 4736–4741. Durrant JR, Knoester J and Wiersma DA (1994) Local energetic 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 affecting 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 condensed 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) Ultrafast Processes in Chemistry and Photobiology Blackwell Scientific Publications, Oxford. Jonas DM, Bradforth SE, Passino SA and Fleming GR (1994) Femtosecond wavepacket spectroscopy: Influence of temperature, 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 studies 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 Rhodopseudomonas 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) Femtosecond 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 a mode-locked laser with reduced third-order 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 components 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 Gallagher 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 repetition rates in Opt Lett 18: 625–627. Stanley R and Boxer SG (1995) Oscillations in the spontaneous 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 Biophys 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 relationship 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) Regenerative 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

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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 limitations are then discussed in detail. Further subsections deal with the mathematical optimization procedures, proper weighting of data points, and the importance of performing an error analysis. As a firsthand 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 photosynthetic 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 techniques can be observed in all areas of science, and photosynthesis research represents no exception. Using advanced time-resolved techniques researchers 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 complex 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 methods 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 sophisticated 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 fluorescence 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 exemplify the principles and the capability of the discussed 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 statements and relationships discussed. Rather the emphasis is on the principles. Numerous examples from other research groups who have applied 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 dependent quantity (fluorescence intensity, absorption difference etc.) vs. time at a certain detection wavelength The signal is usually the response to an optical excitation by a brief exciting light pulse of wavelength If the system under study is a multicomponent one, as almost all biological systems and photosynthetic systems in particular one, the kinetics observed will in general depend both on the detection wavelength and on the excitation wavelength The time-dependent 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 radical 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 homogeneous 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

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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 four parameters, two amplitudes and two lifetimes, for each experiment at a particular excitation/detection wavelength pair. One such experiment will not be sufficient to characterize the system. Carrying out the analysis for a set of M experiments will result in parameters for the whole data set, i.e. 2*M amplitudes 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) experiments. 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 account 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 hold for This is in fact the simplest form of a parameter connection across experiments but is also the most common one in time-resolved spectroscopy. It is important to realize that any parameter connection can in principle be achieved, provided it can be formulated in some mathematical terms, either analytically or numerically. The nature of this connection does not follow from the time-resolved experiment itself but must always 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 The latter is always larger than for M > 1. 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 M + N vs. Without giving any proof, we may expect as a consequence that global 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 components. How large is the improvement resulting from global over single-decay analysis? This is a difficult 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 numerical simulations of experimental data sets should be carried out taking into account the experimental 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 depend on the type of noise (i.e. constant, poissonian, signal-dependent etc. noise) which in turn depends on the type of experiment (transient absorption, fluorescence etc.) and on the difference in the spectra of the components. A similar relationship 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 applications 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 described in the previous paragraph, aims at an

Data analysis of time-resolved measurements

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adequate mathematical description of the measured (kinetic) data. A mathematical model containing parameters is fitted to the data. Consequently 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 fitting procedure” leads to the “mathematical parameters” that describe the kinetics. This is the point where data analysis often ends. Unfortunately, however, these “mathematical parameters” 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 mathematical function to the data and then try to indirectly 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 mathematical 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 modelling (Beechem et al., 1985b; Löfroth, 1986) depending on the application and also on the preferences 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, however: Direct derivation of the physical/chemical parameters of interest from the original (lifetime) data rather than from any intermediate mathematical fitting parameters. This principle is illustrated 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 minimally required number of unknown parameters that are of real interest to understand the problem. The second advantage is even more important, however, and must be stressed strongly: It involves a drastic change in the “philosophy” of the data analysis such that mathematical data fitting 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 parameters 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 examples) 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 immediately as a reasonable description of the system. This possibility to definitely exclude certain models 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 equations describe the relationships between these terms and between mathematical and physical parameters of the model: For the two-component mixture we would measure two DAS, and with corresponding lifetimes and (Fig. 1B). The DAS (amplitudes of Eq. (1) as a function of emission or excitation wavelength) and the lifetimes would result from a global data fitting within a sums of exponentials model. They do not represent the physical parameters of interest, however. Rather the physical parameters of interest are the SAES or SAAS (Fig. 1B), which actually are the real emission or absorption spectra, respectively, of the two species, and the rate constants The following equations show the relationships between these parameters (in case of a two-component system):

It follows from (Eq. (2)) that the DAS are generally 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 excitation wavelength, and the extinction coefficients of the species involved (Eq. (3)). The fluorescence decay is a linear combination of the exponential terms with the DAS as weighting factors (amplitudes). These equations correspondingly hold for transient absorption measurements if the SAES are replaced by the corresponding species-associated absorption difference spectra (SADS). The relationships defined in Eq. (3) for the example of the two-component mixture with energy 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 definitions: X(t) = (n × 1) vector of concentrations of (excited 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 lifetimes (the mathematical parameters) and the physical parameters become clear from these equations. The relationship between the eigenvalues 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 constants, 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 parameters 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 photosynthesis 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 restrictions with respect to the values and range or the mutual relationships of the physical fitting parameters can be implemented easily if such information is available. It could come either from other measurements or could be introduced deliberately 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 formulated. 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 information may be spectroscopic, structural, thermodynamic 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-

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senting antenna energy transfer/charge separation processes. Due to the complexity of an antenna/reaction center complex certain simplifying assumption must be made. The level of approximation 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. phycobiliproteins (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 compartment 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 possible, the proper or “reasonable” compartmentalization of the system tells us a great deal about the physical nature and behavior of the system. Since any compartmentalization of a system represents an approximation, a compartment model can only answer a limited number of questions about a system. Thus as the most stringent restriction, 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 compartment, 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 implicitely that usually a physical model testing approach 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 “compartment” 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 assumed 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 assumed 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 mathematical 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 procedures are the subject of an extended area of numerical mathematics and no attempt will be made here for an extensive in-depth presentation. The

83 principal procedures may be found in monographs on numerical data analysis (see e.g. Bevington, 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 optimization are the same for both approaches. The first question to be answered is what we define as an “optimal” value for a fitting parameter. 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 deviations of the fitting function from the data. Taking into account the non-linear nature of the problems this is called non-linear least squares minimization. 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 nevertheless is a useful and practical definition. We thus define a function (called the reduced which has to be minimized using appropriate methods in order to determine the optimal values for the elements of the parameter set

In this equation the double summation runs over all experiments and all data points of 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 minimization 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

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value and computes in a next step corrections this parameter set such that the decreases. This operation in repeated until the minimal value has been found, i.e. until the method converges. 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 the uncorrelated parameters Thus it has the 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 minimum, this procedure either fails or at least shows very poor convergence. The Gauss–Newton method 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 minimum. 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 (remember that the in general is a hypersurface of dimension p + 1 when p is the number of parameters). If the is smooth, 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 parameter set. One has to realize that in practice one can not have the guarantee to find the local minimum of a complex by using any 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 derivatives of the or other fitting functions 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 algorithms” (Goldberg and Richardson, 1987; Goldberg et al., 1989). To my knowledge so far hardly any applications of these algorithms to data analysis 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 to the The standard deviation is a measure of the noise in the data. Often in nonlinear fitting problems this weighting factor is set to one, resulting in an equal weighting of all data points and thus ignoring the fact that experimental 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 capability to resolve complex fitting problems is impeded. Therefore the use of proper weighting factors in the definition of the (Eq. (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 Poissonian nature and thus the standard deviation is given by the square root of the number of counts in a channel. In a transient absorption experiment measuring small absorption differences, the standard deviation is usually the same for all data points in a single experiment (constant noise independent 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 deviation of the measured signals however.

85 Finally it is noted without proof here that the type of noise present in a signal (poissonian, constant 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 computer resources than the actual parameter optimization 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 confidence 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 parameters is not taken into account properly. Furthermore 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 interval 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 parameter 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 parameter. The estimates to both sides of the optimal value where the has increased by the same pre-defined amount then define the confidence 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 parameters 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 parameters. This may serve as an example that error analysis is indeed important and essential for distinguishing 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 testing vs. mathematical data fitting have been pointed 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 accuracy of the data set (a low accuracy would simply lead to a larger confidence interval). Rather this is an inherent mathematical or information theoretical problem. Let us take our simple example of a two-component mixture with energy transfer. Suppose we have measured the fluorescence kinetics 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 amplitudes are usually not measured as absolute values but only as relative numbers (due to the presence of unknown instrumental proportionality factors), for each detection wavelength there exist only three independent parameters, two lifetimes and one (relative) amplitude. The other amplitude is a dependent one, i.e. it is the difference from 100%. Thus not enough linearly independent equations exist in order to determine four rate constants. If one were to allow for freely adjustable rate constants in the fit, a mathematical solution (good fit) would definitely result. However, the fit would not be unique and three of the rate constants would be parametrically dependent 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 mathematically good description of the data set). This may serve as an example that it is absolutely 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 tedious 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 common furthermore that only target analysis was capable of determining the underlying mechanisms 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 lifetime data was in good agreement with such a model (Müller et al., 1993). Interestingly the global 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 constants 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 target 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, assuming 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 system. Note that in addition the two parts of the parallel scheme II are linked by assuming equation rate constants for transfer (rate 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 essential to exclude the wrong model.

B. Photosystem I Kinetics Fig. 7 shows different compartment models (compartments are antenna pools 1–3) for the core antenna (RC complex of photosystem I from cyanobacteria (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 different 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 reasonable, was already too complex given the details and resolution of the experimental data sets (two lifetime components were found by global lifetime analysis in fluorescence and three in transi-

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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 experimental data one could try to test this model again. Both models 7A and 7C were tested extensively on the fluorescence and the transient absorption data set. Again both models gave a (mathematically) perfect fit to the data. The resulting SAES (from the fluorescence data) and the SADS (from the transient absorption data) are shown in Fig. 8 for each model. The fluorescence 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 correct. Further analysis of the SAES and SADS revealed that, despite the good fit for both models, in fact only model 7A was physically reasonable 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 proportional 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 conditions 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 violate the Boltzmann principle. In further analysis data about the pool sizes could be estimated from the target data which were in very good agreement 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 application 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 components 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 parameters 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 various 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 involved in these procedures, as is occasionally implied 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 described here are applicable as well to data analysis problems outside kinetic spectroscopy. Finally I should like to mention a couple of important additional papers from research areas outside photosynthesis where similar analysis procedures as treated here have been described and/or applied in a highly fruitful fashion (Nagle, 1991; Beechem, 1988; Lakowicz et al. 1984; Beechem et al. 1991; Christian et al. 1981; Halvorson 1981; Hessling 1992). The interested reader is referred

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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 described 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 testing many complex data analysis cases and finding out about their capabilities and limitations.

References Ameloot M and Hendrickx H (1982) Model building in fluorescence relaxation experiments. Arch Int Physiol Biochim 90:46–47. Ameloot M, Beechem JM, and Brand L (1986) Compartmental modeling of excited-state reactions: Identifiability of the rate constants from fluorescence decay surfaces. Chem Phys Lett 129:211–219. Beechem JM (1989) A second generation global analysis program for the recovery of complex inhomogeneous fluorescence decay kinetics. Chem Phys Lipids 50:237–251 . Beechem JM, Knutson JR, and Brand L (1984) Simultaneous analysis of multiple fluorescence emission anisotropy decays. Biophys J 45:A127. Beechem JM, Ameloot M, and Brand L (1985a) Global analysis of fluorescence decay surfaces: Excited-state reactions. Chem Phys Lett 120:466–472. Beechem JM, Ameloot M, and Brand L (1985b) Global and target analysis of complex decay phenomena. Anal Instrum 14:379–402. Beechem JM, Gratton E, Ameloot M, Knutson JR, and Brand L (1991) The Global Analysis of Fluorescence Intensity and Anisotropy Decay Data – 2nd-Generation Theory and Programs. In: Lakowicz JR (ed) Topics in Fluorescence Spectroscopy. 2, pp 241–305, Plenum Press, New York. Bevington PR (1969) Data reduction and error analysis for the physical sciences. McGraw Hill, New York, pp 1–336. Christian GD, Callis JB, and Davidson ER (1981) Array detectors and excitation-emission matrices in multicomponent analysis. In: Wehry EL (ed) Modern Fluorescence Spectroscopy 4, Plenum Press, New York, pp 111–166. Di Cera E (1992) Use of weighting function in data fitting. In: Brand L and Johnson ML (eds) Methods in Enzymology, Vol 210 Numerical Computer Methods, Academic Press, San Diego, pp 68–87. Finkele U, Lauterwasser C, Struck A, Scheer H, and Zinth

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 Genetic 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 bacteriochlorophyll anion radical during the primary charge separation 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 Publishers, 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 fluorescence 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 photosystem 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 Methods. 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 Limkeman M (1984) Analysis of fluorescence decay kinetics from variable frequency phase shift and modulation data. Biophys J 46:463–477. Livesey AK and Brochon J-C (1988) Maximum entropy data analysis of dynamic parameters from pulsed-fluorescent decays. In: Harding SE and Rowe AJ (eds) Dynamic Properties of Biomolecular Assemblies, Royal Society of Chemistry, 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 estimation 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 centers 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 Rhodobacter sphaeroides studied by picosecond fluorescence kinetics. 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 Rhodobacter 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. Biophys 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 kinetics from pea chloroplasts. A new approach to the characterization 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 photosystem 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 Computer 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 rubrum 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. Biochim Biophys Acta 1187:1–65. Weidner R and Georghiou S (1990) Global methods for timeresolved and steady-state fluorescence and steady-state absorption 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 andAnalyses 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

93 94 94 94 94 95 96 98 98 98 99 100 100 101 101 102 102 102 102 103 103 103 103 104 105 105

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

93 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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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 acceptor of PSII; PQ – plastoquinone; PSI –.photosystem I; PSII – photosystem II; – the primary acceptor quinone of PSII; – the secondary acceptor quinone of PSII; – – redox states of the 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 preilluminated 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 generated by photoreactions and trapped or stabilized 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 condition of preillumination. Through extensive studies during 20 years, many of these photosynthetic TL components were separated and characterized in detail. It has now been firmly established 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 between the positive charges accumulated in the intermediates of the water oxidation system on its donor side and the negative charges stabilized on primary or secondary quinone acceptors 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 knowledge about photosynthetic TL and discuss some possible new research areas to which TL technique can be applied.

II. Origins of TL from Photosynthetic Apparatus

A. Processes Involved in Photosynthetic TL 1. Historical Aspects and Basic Phenomenology TL from plant material was first observed by Arnold and Sherwood (1957) subsequent to the discovery 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 different emission temperatures, indicative of participation 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 characterization 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 involved 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 sequential 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 subsequently led to assignment of specific S-state intermediates as the positive charge carriers responsible 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 turnovers 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 temperature in the mutant plants, indicating that a significant alteration in properties occurs when some amino acids are replaced in the D1 protein. Supported by these as basic findings, photosynthetic TL has now become widely used as a simple but efficient probe of PSII functioning not only for biophysical purposes but also for assisting genetical 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 symmetric way like in the reaction center of nonsulfur purple bacteria. However, in order to retain the functional Mn-cluster and thereby the ability of water oxidation, several more membrane proteins and one extrinsic protein are required 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 transfer from to the primary one-electron acceptor quinone of PSII, which is then followed by further transfer of the electron to the secondary two-electron acceptor quinone of PSII in Reduced is stable for tens of seconds or even to hours at room temperature and constitutes the major trap of negative charges in photosynthetic TL phenomena, while does not, because of its short life, unless the electron transport between and is blocked by some means, e.g. by herbicides. Upon the second photochemistry in the same reaction center, is further reduced to but this species is readily replaced by a plastoquinone (PQ) of the pool and does not participate in photosynthetic TL under normal conditions. Stabilization of the charge separated state occurs also on donor side of PSII. The cation radical of P680, is reduced by Yz, the Tyr 161 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 = 0–4) with and as the lowest and highest oxidation states, respectively. Repeated photochemistry in PSII reaction center advances the Sstates by one step each, and abstraction of four electrons from two molecules of water finally results in release of one molecule of Among the five intermediate S-states, and are stable in dark-adapted condition, but they do not participate in TL because they do not carry any positive charge, whereas and carry a positive equivalent (see later) and are stable for tens of

96

seconds at room temperature, so that they constitute the major source of positive charges for photosynthetic 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 photosynthetic TL are believed to be the and Beside Yz, another auxiliary secondary donor to P680 is known; the redox active Tyr 160 residue of D2 protein denoted as Its oxidized form known as EPR Signal IIs is stable in darkness 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 b559 and some of the chlorophyll molecules surrounding 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 photochemistry 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 recombination between stabilized donor and acceptor at a photochemical reaction center. In PSII, the primary product of light-induced charge separation is a singlet radical pair, which rapidly proceeds to stabilization of positive and negative equivalents as forms of oxidized donor and reduced acceptor and regenerates a 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).

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The process of recombination of is not always understood in detail, but it is generally considered to proceed through a series of equilibrations among various intermediate charge separated 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 nonradiatively 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 expressions of the same process: the former is due to spontaneous recombination at a constant (usually 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 heating. In fact, more or less the same period-four oscillation has been observed for both phenomena, 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 flash(es) of a few duration at a constant temperature (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 gradually 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 photomultiplier 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 illuminations 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 treated 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, respectively. 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 overlapping TL bands and also to determine the activation energy for each TL components. An efficient 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 counterpart 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 analyses (Table 1).

Among these parameters derived from TL measurements, the peak temperature gives us the most useful information. However, the peak temperature 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 difference in positioning of the thermocouple around the sample or sample holder. Despite the significant differences, a shift in peak temperature induced by treating the sample can easily and correctly 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 recombination, indicative of induction of a deeper trap by the treatment, while a downshift indicates induction 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 potential of the positively or negatively charged species, 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 usually the most dominant species among the centers. 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 section, the probability of positive charge stabilization on the donor side oscillates in a 1,1,0,0 pattern, 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 gate mechanism of the quinone. Thus the probability of stabilization of both positive and negative charges in one PSII reaction center, which is a prerequisite for emission of TL, oscillates in a 1,0,0,0 pattern. In actual PSII preparations, this pattern is perturbed by several factors: (i) initial distribution of (ii) initial distribution of (iii) the 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%, respectively, and the TL yield ratio of In contrast, the initial ratio of varies depending on the sample and relaxation conditions: 50:50 in thylakoids after brief dark-relaxation or in well dark-adapted intact chloroplasts, whereas it is 25:75 in well darkrelaxed thylakoids or in PSII membrane fragments, 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 typical initial ratios are illuminated with a series of flashes. Distribution of to the centers

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 obtained. These patterns agree well with observed ones, supporting the mechanisms of TL oscillation 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 deviates from these standard patterns, which gives information about the functioning of the S-state cycle and the electron gate in inhibited 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, responsible charge pairs have been identified (Table

Yorinao Inoue

3). In the following we discuss information currently available about the properties of the respective 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 enzyme (Inoue and Shibata, 1977a), in particular with the presence of functionally active Mn (Inoue, 1976; Rozsa and Demeter, 1982), and has been assigned to and charge recombination (Rutherford et al., 1982, 1985). When excited 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 fragments (Rutherford et al., 1984). Analysis of the oscillation pattern indicates that both the and are involved in generation of this TL component (Rutherford et al., 1982; Demeter and Vass, 1984). Participation of as the negative charge trap has been evidenced by modulation of the initial ratio by chemical or

Thermoluminescence, a specific probe of PSII

101 shape, but below pH 6.0, they exhibit two distinguishable components denoted and 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 concomitant 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 negative charge detectable by TL instead of The Q-band is often used to analyze the features of stabilization of the

3. The A-band

light pretreatment or by removal and reconstitution of (Rutherford et al., 1982; Demeter 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 assigned to arise from charge recombination based on the following considerations: illumination with two flashes at room temperature generates an state which is incapable of TL due to the absence of negative charge (see Table 2), but the 77 K illumination oxidizes cytochromeb559 or chlorophyll instead of the Mn-cluster (in and generates which recombines with the previously formed by illumination 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 band is called the to distinguish it from the A-band having the same emission temperature (Koike et al., 1986). The origin of this component 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 modified 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 origin is not yet clear. The participation of as the negative counterpart has recently been confirmed by reconstitution of artificial in purified 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 participation of as the negative counterpart is likely, judging from enhancement of its intensity in the presence of high concentration of DCMU. The C-band is reported to exhibit a period-four oscillation with maxima in the and 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 equivalent in and Demeter et al. (1993), Krieger et al. (1993) and Johnson et al. (1994) reported that this component arises from charge recombination between and in normal and PSII as well.

7. The Z-band A strong and broad TL band appears at around 110K when various plant materials are illuminated at 77 K with continuous light. This component, 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 temperature 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 recently that LHCI and LHCII, that have no photochemical 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 preferentially 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 conformational changes in LHC concomitant with the accumulation of zeaxanthin via the xanthophyll cycle.

Thermoluminescence, a specific probe of PSII

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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 remarkably 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 between and is decreased from 70 to 30 mV due to a structural alteration of the site by the mutation. Analysis of cyanobacterial 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 phenylureas, suggesting a more important role of Ser264 in as compared with Phe255 (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 as and respectively (Noguchi et al., 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 components are essentially the same as those of the Zband: being excited preferentially by blue light and emitted more strongly from LHCs and aggregated 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 difference in emission temperature between the Bband and Q-band, which arise from and 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 Notably, however, the emission temperature of the resulting Q-band differs depending on the chemical structure of the herbicide employed: phenolic type herbicides induce a Q-band emitting between –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 to a lowered redox potential of or to the ADRY (acceleration of deactivation reactions in

B. Donor side of PSII The generation of the B-bands and the A-band involves the and so that the emission temperature and oscillatory behavior of those TL bands provide useful information regarding the properties of the S-states and the role of various cofactors involved in S-state transitions. 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 oscillation 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 studies reveal that and 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 temperature”, a low-temperature sensitive intermediate 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 electrostatically 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 extrinsic proteins, these proteins can be reversibly removed and reconstituted with concomitant inactivation and reactivation of oxygen-evolving activity. Upon removal of the set of proteins, oxygen evolution is almost lost whereas the TL Bband can still be fully induced. Although PSII depleted of the extrinsic proteins exhibits normal oscillation up to the the final 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, suggesting no appreciable change in the stability of the charge pair. In contrast, however, the peak temperature of the Q-band is remarkably upshifted by 20–25 °C indicative of deeper stabilization of the state in the absence of the extrinsic proteins (Vass et al., 1987a). The different stabilization 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 obtained 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 thylakoids. TL data implicating an incomplete association of the 33 kDa protein with PSII membranes have also been collected for cyanobactrial

Yorinao Inoue mutants in which a few amino acids on the lumenal loop of the CP47 protein are deleted (Gleiter et al., 1994). By analogy to these, TL has now been extensively applied in probing redox properties of the donor side of PSII (Ono and Inoue, 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 fundamental concept about this phenomenon were established. 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. Following 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 genetically 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 coupled, also in this case, with genetically engineered mutants. Alterations in properties 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 photoinhibition that affect the functioning of PSII. Taking all these into consideration, the advantages 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 physiological 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 theoretically 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 Technology Agency of Japan (STA) which has been financially supporting the TL studies during the last 15 years under the title of Solar Energy Conversion by Means of Photosynthesis. The author is also grateful to Drs. Mamiko Kimimura-Taguchi and Han Kab-Cho for their technical assistance in preparing the manuscript, and also to Professor J. Amesz for his valuable suggestions.

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Yorinao Inoue dues on the donor side of the photosystem II D1 polypeptide. 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 accompanied by a shift in the midpoint redox potential of the primary quinone acceptor Biochim Biophys Acta 1144: 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) Thermoluminescence emission at liquid helium temperature from photosynthetic 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 thermoluminescence: 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 treatment 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 unknown positive charge accumulator. Biochim Biophys Acta 1020: 269–277. Ono T and Inoue Y (1991a) Biochemical evidence for histidine 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 isolated 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) Thermoluminescence 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 accumulation and photochemistry in leaves studied by thermoluminescence 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 the evolving enzyme. Biochim Biophys Acta 767: 548– 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 photosystem II. A new model for the release of protons ac-

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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

109 109 110 111 111 111 113 114 116 118 119 120 120

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

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

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 compete very effectively with intrinsic molecular relaxation processes such as fluorescence, intersystem crossing and internal conversion. For a full understanding and description of the molecular 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 separation, and for understanding the underlying intermolecular interactions in relation to the molecular 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 photon 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 measurements. In the second part we discuss the results which have been obtained by measurements on isolated photosynthetic pigment–protein complexes. 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 measurements. Data from both types of measurements should be considered to be complementary, and as such will be important for the correct interpretation of the excited state dynamics involved. II. Homogeneous and Inhomogeneous Linewidths Optical transitions of a molecule are characterized by a finite bandwidth, usually referred to as the homogeneous linewidth, which is determined 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, characterized by time constants and respectively. Population relaxation involves transitions between 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 modulation 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 homogeneous line width is related to the inverse of the overall dephasing time

This equation for the homogeneous linewidth is essentially a manifestation of the Heisenberg-uncertainty principle. At sufficiently low temperatures, such that bath fluctuations are completely quenched, the homogeneous linewidth is determined by For an ‘isolated’ molecule in a host matrix, will in general be equal to the fluorescence lifetime. Intermolecular interactions between the guest-molecules may give rise to additional 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 detail. The experimentally observed linewidth, however, is often much broader than the homogeneous linewidth. This is due to the effects of inhomogeneous 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 permanent dipole moment. To uncover the homogeneous linewidth parameters from the inhomogeneously broadened line, special methods are required. They can be divided in two main categories: those that operate in the frequency domain such as transient and permanent hole burning 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 relationship 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 polarization in the medium, which is oscillating at the driving optical frequency. The amplitude of this polarization is proportional to the number of excited molecules. Hence, the intensity of the radiation emitted by a coherent ensemble of dipole oscillators will be proportional to (Dicke, 1954), whereas in an incoherent emission process this intensity is proportional to However, even in the absence of scattering processes, the coherent superposition is lost very rapidly because of the dephasing which arises from the different resonance frequencies in the case of inhomogeneous broadening. A. Two-Pulse Photon Echo In the absence of phase-perturbing scattering processes, the time development of the relative phases of the oscillators in an inhomogeneously broadened system is well-defined despite the variation in resonance frequency. The magic of the two-pulse photon echo arises from the fact that this phase development can be reversed by applying 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 superposition is restored at a time after the second excitation pulse. This state gives rise to an intense burst of coherent radiation, proportional to which is known as the photon

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echo. The photon echo intensity will have a maximum when the pulse areas, defined as are equal to and for the first and second excitation pulse, respectively (Allen and Eberly, 1975). Here, is the electric field amplitude, and the pulse area is a measure 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 irreversible 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 coherent 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 polarization can be manipulated by other pulse sequences (Allen and Eberly, 1975). The most familiar 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 periodically varying amplitude with a fringe-spacing given by over a frequency range deter-

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mined by the optical bandwidth of the light pulses. If this pulse-pair excites an inhomogeneously broadened molecular system, this pattern gives rise to a similarly periodic variation of the population 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 population 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 relaxation, these elements can be written as (Hesselink, 1980):

In this equation, and are the wave vectors and phases of the excitations, is the 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 obtained if this is a pulse. The effect of this third pulse is to transfer the phase information that was ‘stored’ in the population grating in frequency 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 a time after the third pulse the macroscopic

polarization is reestablished, resulting in the emission 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 homogeneous dephasing which occurs during the interval between the first two excitation pulses, while the second exponential factor reflects eradication of the frequency grating through depopulation of the excited state. It can be seen that by measuring the stimulated photon echo intensity as a function of the waiting time for a fixed it is possible to measure the lifetime of the excited state. On the other hand, can be measured by varying while keeping fixed. An excited molecular system will ultimately

Accumulated photon echo measurements decay to the ground state, either directly by fluorescence or radiationless relaxation, or via relaxation through some long-lived intermediate state, such as the lowest triplet state. In the former case, the frequency grating will disappear on a time scale of but in the latter case the frequency 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 which persists on a time scale of The amplitude of this persistent grating, and therefore the intensity of the anomalous photon echo, is determined 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 necessary 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 example by frequency up-conversion (Hesselink and Wiersma, 1978; Meijers and Wiersma, 1992). Therefore, such experiments present a significant

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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 repetition 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 modulation depth of the frequency grating. The contribution 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 transformation upon optical excitation. After the sample – a polymerized porphyrazine styrol solution – was excited with a train of pulse-pairs, the frequency 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 relationship between the phase difference and the frequency grating has been examined experimentally by Jefferson and Meixner (1992). Each of the pulses in the train of pulse pairs incident on the sample, while sustaining the accumulated frequency grating, simultaneously gives rise to a stimulated echo. The intensity of this echo is strongly enhanced by the accumulation 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 determined 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

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pulse (1 and 2 in Fig. 2B) in a pulse pair, which are separated by a time delay We examine the accumulated photon echo generated by the first pulse (3 in Fig. 2B) of a subsequent pulse pair with a wave vector According to the 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 interference occurs between the accumulated photon 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 detection scheme provides a greatly enhanced sensitivity.

Thijs J. Aartsma et al. So far we have assumed that the inhomogeneous 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 systems, 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 consequence 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 experiment (Narasimhan et al., 1990). In accumulated 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 spectral diffusion can be neglected, the information derived from both types of experiments is expected to be the same. Finally we note that it is not absolutely necessary to use transform-limited pulses for the generation 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 correlation function of the excitation pulses. Experimentally, 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 resolution is obtained at the expense of spectral resolution. 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 noncollinear 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 translation stage. In this configuration, the sample is excited by a succession of pulse pairs at a repetition 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 significantly 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 photodiode 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,

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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 photon 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 population of the excited state, but this is typically several orders of magnitude smaller than the intensity of the accumulated photon echo. Note that the diagram in Fig. 3 is actually identical to that of a pump-probe setup for measurements 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 relationship 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 lifetime 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 dephasing 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 (Förster, 1948). In the strong coupling limit, the excited states of the system are more properly described in terms of exciton states. Such states are formally described by diagonalizing the Hamiltonian 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 temperature, exciton scattering processes may be very fast, and under such conditions energy transfer is essentially an incoherent hopping process except, possibly, at very short times (tens of femtoseconds) 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 containing 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 spectroscopic 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 (Matthews 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 approximately 24 Å, yielding dipolar interactions between the BChls with a maximum of and respectively. The absorption and circular dichroism spectra 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 measurements 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 decreases, with time constants varying from subpicoseconds at the shortest to hundreds of picosecond at the longest wavelengths (Louwe and Aartsma, 1994). At all wavelenghts the photon echo decay is strongly multiexponential. However, 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 energy 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 temperature, 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 transitions 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 experimental observation. In addition, the relaxation 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 homogeneous 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 dependence 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 transitions in the manifold. Relatively narrow holes width, limited by spectral resolution)

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were only observed in the longest wavelength region of the absorption band of the FMO complex. At shorter wavelengths broad holes were obtained 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 photon echo. At present, the origin of this discrepancy 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 complex LHCII of green plants, which has a absorption 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 complex 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 reactions that occur in photosynthetic reaction centers (RCs) have been the subject of numerous investigations (reviewed in Kirmaier and Holten, 1993; Zinth and Kaiser, 1993; Shuvalov, 1993). Most of these investigations involved measurements to provide information about the populations dynamics of the states involved. In the accumulated photon echo experiments described 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 concluded 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 experiments, 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 bacterium Rb. sphaeroides R26 was recently reexamined, and extended to measurements on the mutant (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 conditions 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 characterized by a very sharp feature around t = 0, and a longer-lived component with a much smaller amplitude. This decay pattern is in good agreement with the shape of the homogeneous line obtained by hole burning measurements (Jankowiak and Small, 1993). Considering the Fourier-transform relationship, the sharp feature in the photon echo decay can be attributed to a contribution 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 (Johnson et al., 1989). Closer inspection of the longer decay component shows that it is biexponential, with time constants of 1.2 and 8 ps. The biexponential kinetics has also been observed in pump-probe and fluorescence lifetime measurements at room temperature as well as at cryogenic temperatures (Kirmaier and Holten, 1993; Müller et al., 1992; Nagarajan et al., 1990). These low temperature data agree reasonably well with the values obtained from the accumulated photon echo measurements. 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 electronic 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 intrinsic dynamics of the primary step of charge separation. Several explanations have been put forward to account for this nonexponential kinetics which is also observed in pump-probe and fluorescence decay experiments. From our measurements, 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

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between these two cases on the basis of the accumulated 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 includes photosynthetic pigment–protein complexes. A prerequisite for the observation of the accumulated photon echo is the efficient population of a long-lived intermediate state in the relaxation 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 conditions. At sufficiently low temperatures, pure optical dephasing is negligible, and the accumulated photon echo decay is dominated by population relaxation. In photosynthetic reaction centers this decay is determined by primary charge separation. 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 suggested 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 intrinsically 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 appropriate. 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 investigations. 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 amorphous 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 bacteriochlorophyll 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 repopulation 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 Netherlands. Dicke RH (1954) Coherence in spontaneous radiation processes. Phys Rev 93: 99–110. Duppen K and Wiersma DA (1986) Picosecond multiple-pulse experiments involving spatial and frequency gratings: a unifying 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 chlorosomes 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 bacterial 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, heterogeneity, 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 measurements 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 primary 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 Prosthecochloris bacteriochlorophyll a-protein optical spectra improved by parametric computer search. Photochem Photobiol 57: 86–91. Lyle PA and Struve WS (1990) Evidence for ultrafast localization 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 bacteriochlorophyll 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) Photon echo stimulated from optically induced nuclear spin polarization. Chem Phys Lett 64: 1–4. Morsink JBW, Hesselink WH and Wiersma DA (1982) Photon 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 Rhodobacter sphaeroides studied by picosecond fluorescence kinetics. 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 primary photosynthtic electron-transfer process in Rhodobacter 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 Photosynthetic Bacteria, pp 161–178. Plenum Press, New York. Pearlstein RM (1992) Theory of the optical spectra of the bacteriochlorophyll a-antenna protein trimer from Prosthecochloris 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 myoglobin. 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 characterization 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 Photosynthetic 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 micelles. 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 photosynthetic 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 temperatures. For about ten years now, SHB has been fruitfully applied to photosynthetic protein complexes. 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 addressed, be it the role of phonons and structural heterogeneity in energy or electron transfer, exciton level structure in complexes containing several 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 dynamics of photosynthetic protein complexes have been the subject of reviews by Reddy et al. (1992a), Jankowiak and Small (1993) and Jankowiak et al. (1993). These reviews provide broad coverage of the complexes which have been studied 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 limitations in application to photosynthetic complexes. 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 photosynthetic complexes. Those interested in the connection (via of the nonlinear optical susceptibility) between SHB and photon-echo spectroscopies 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 narrow isochromat of chromophores within an inhomogeneously broadened absorption band. SHB occurs when the decay of the excited isochromat to the ground state is either blocked (persistent hole burning) or delayed (transient hole burning). Blocking of the decay to the ground state can be due to a photochemical reaction initiated in the excited state such as the inner proton-tautomerization of the porphyrins or due to a rearrangement of the host environment (typically 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 intermediate state or a shortlived product (that reverts to the initial material) in a photochemical reaction is used to store (temporarily) the depleted 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 pseudovibronic 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 detergent) to stems from the fact that the members of an ensemble of a given complex have slightly different structures (conformations), a natural consequence of the “glass-like “ nature of proteins. By determining the dependence of the hole structure (ZPH and its PSBH) on the burn frequency within the origin band, one can not only determine but also the center frequency of the inhomogeneous distribution of zerophonon line (ZPL) transition frequencies. It has been found that is depending 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., 1991) the mean phonon frequency, is ~20– the width of the one-phonon profile is and that the coupling is weak, S < ~0.6. The quantity can be viewed as the optical reorganization energy; would be the Stokes shift (when the state being probed is fluorescent). 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 uncommon 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 temperatures much below room temperature (~200 K) because of the inhomogeneous broadening. Required for a correct calculation of the temperature dependence of the rate is the single complex spectrum, comprised of the ZPL and phonon sideband, in the low temperature limit. (The spectrum for higher temperature can then be calculated.) Also required is the distribution of values. Fortunately, it has been shown that, if and are the inhomogeneous broadenings, the width of the is Thus, all ingredients are at hand for a calculation of the temperature dependence 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 dispersive kinetics stemming from a static distribution of energy gap values (Lyle et al., 1993). As reviewed by G.S. Small and coworkers (Reddy et al., 1992a; Jankowiak and Small, 1993; Jankowiak et al., 1993), the width of the ZPH burned into the origin band of a can also yield the lifetime of that state in the low temperature 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 thermalization of chromophore and bath modes precedes ultra-fast electron and energy transfer as first discussed by Tang et al. (1989) and Johnson et al. (1990). Finally, we emphasize that the superior resolution afforded by SHB can often lead to resolution of closely spaced This has proven 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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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 exciton levels occurs in ~100 fs at 4.2 K. This result was later confirmed by the femtosecond experiments. 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 inhomogeneously 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 (provided 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 photomultipliers 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 fluorescence 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 is scanned by scanning the intracavity 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 bottleneck state has a sufficiently long lifetime (<~100 Wannemacher et al. (1993) developed the experimental setup shown in Fig. 2. It is based on burn-scan technique (Cone et al., 1984) and employs 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 transmission mode (using a photomultiplier and signal averaging) is accomplished with a digital oscilloscope and a personal computer. The time between 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 usefulness of SHB in studies of energy/electron transfer and excited state structure in photosynthetic complexes. This problem can be overcome by employing a Fourier transform (FT) spectrometer that replaces everything after the light attenuating

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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 spectrometer 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 intensity at the crossing of the two beams forms a spatial grating in the medium. The spatial grating is due to spatially modulated absorption and refractive index coefficients. For readout, one tunable 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 biological samples is however, limited since the diffraction efficiency is a function of read frequency and the stringent requirement that the optical quality of the sample be high.

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For SHB studies in antenna and reaction center complexes at variable pressure and temperature (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 compressor 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 helium 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 pressure cell) is kept constant. The maximum pressure 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 access 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 region 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 transmission 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 several 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) photochemical 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 broadening 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 corresponding 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 dependence 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 intriguing, 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 phenylalanine 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 commensurate with the standard diabatic state model for charge separation is currently under investigation 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 bottleneck state for hole burning is In the region of P960 one observes a weak ZPH coincident with the burn frequency as well as the progression associated with the aforementioned special pair marker mode, The responses of 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 spectrum) which is the upper excitonic dimer component of the special pair. Reddy et al. (1993a) succeeded in obtaining nonphotochemical 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 relative to the origin component of the P960 absorption band, i.e. the photoinduced structural transformation stabilizes P960* The largest 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 the anti-holes can be explained in terms 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 absorption band B is predominantly due to the out-ofphase linear combination of the 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 identified, for the first time, all six of a bacterial 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 pressures has been used with systems that exhibit very sharp ZPH to measure compressibilities, solvent shifts etc. in amorphous (protein) hosts (Zollfrank and Friedrich, 1992a, 1992b). Only modest values of high pressure (< ~10 MPa) were required 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 inhomogeneous line shape itself, has been developed by Laird and Skinner (1989). This theory assumes that a) the transition frequency of a solute molecule can be described by a sum of pairwise interactions with each solvent and b) each solvent molecule occupies a position that is independent 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 Skinner 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 interactions 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 description 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 independence 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) interactions 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 relatively 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 solvent shift is and that for excitonic splitting is However, another 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. Freiberg 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 Rhodopseudomonas 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 differences 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 investigations on both P870 and P960 at high pressures. An important question in the study of the function of transport proteins (e.g. horse radish peroxidase 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. Recently, this aspect was investigated for mesoporphyrin substituted (for heme) horse radish peroxidase (Friedrich et al., 1994) using high pressure hole burning. Low temperature absorption spectrum 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 induced by small changes in the prosthetic groups. Similar correlation was observed for protoporphyrin 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

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Rhodobacter sphaeroides observed persistent narrow 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 width is due to inhomogeneous 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 coincident 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 function 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 excitonic 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) obtained 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 parameters 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 University 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 quadrupole 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 photosynthesis. In:Breton J and Vermeglio A (eds) Photosynthetic Bacterial Reaction Center II: Structure, Spectroscopy, 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 fluorescence emission spectra and lifetimes of the photosynthetic 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 photosynthetic 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 conformational substates in chromoproteins. Proc Natl Acad Sci (USA) 91:1029–1033 Gillie JK, Small GJ and Golbeck JH (1989) Nonphotochemical 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, heterogeneity, 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 organic molecules by laser irradiation. Opt Commun 12:191– 194 Laird B and Skinner JL (1989) Microscopic theory of reversible pressure broadening in hole-burning spectra of impurities in glasses. J Chem Phys. 90:3274–3281 Lu X and Pearlstein RM (1993). Simulations of Prosthecochloris bacteriochlorophyll a protein optical spectra improved by parametric computer search. Photochem Photobiol 57:86 – 91 Lyle PA, Kolaczkowski SV and Small GJ (1993) Photochemical hole-burned spectra of protonated and deuterated reaction centers of Rb. sphaeroldes. J Phys Chem 97:6924–6933 Moerner WE (ed.) (1987) Topics in Current Physics, Persistent Spectral Hole Burning: Science and Applications, Vol. 44, Springer-Verlag, New York Mukamel S (1995) Principles of Nonlinear Spectroscopy, Oxford 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 experiments. Chem Rev 90, 439 – 457 Reddy NRS, Small GJ, Seibert M and Picorel R (1991) Energy 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 reaction 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 photoinduced 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) Holographic 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 temperature 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) Structured hole burned spectra of the primary donor state absorption 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 diffusion in organic glasses. Temperature dependence of permanent 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 pressure: 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

137 138 139 139 141 141 144 145 145 147 148 148 149 151 151 152 153 153 154 155 157 157 157

Summary

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]

137 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; FTIR – Fourier transform infrared; – primary electron accepter (pheophytin); MCT – mercurycadmium-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 transmission 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). Nichols noted that “the diathermancy of chlorophyll solutions depends on the type of solvent” he used, and thus was the first one to discover spectral shifts of chlorophyll absorption due to aggregation states. Although we would nowadays consider 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 advancement of infrared instrumentation, vibrational spectroscopy in the mid-infrared region became interesting for structural studies of the photosynthetic pigments, and a paper by Stair and Coblentz (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 aggregation and hydration (Katz et al., 1966, 1978; Ballschmiter and Katz, 1968, 1969). Although all these studies were on isolated pigments in solvents, it is obvious that they were undertaken in order to explain certain properties of the pigments 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 polypeptides, pointed out the specific IR absorption of polypepide secondary structures, long before crystallography could provide us with atomic information on protein structures. This spectroscopic approach has become well-established with a theoretical background and numerous applications 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 developments had to be awaited. A breakthrough has been the development, the availability, and the “affordability” of Fourier transform IR spectrophotometers, which, for reasons given below, can provide the sensitivity needed to monitor vibrational 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 dedicated to the discussion of FTIR techniques which provide the sensitivity to monitor changes at individual 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 spectroscopy, 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 reaction centers. A section on sample preparation for infrared spectroscopy and one on the sisyphean task of IR band assignment will provide the reader with the information that no photosynthetic 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 properties 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 pigments, the quinones, lipids, and water constituting the entire complex, and thus contains abundant 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 energies 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 properties of the RC. Infrared spectroscopy probes vibrational modes of the pigments, the quinones, the polypeptide 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 complementary to Raman spectroscopy, where reson-

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ance enhancement can yield spectra of individual pigments even in a complex photosynthetic membrane (Lutz and Mäntele, 1991). A problem of the non-selectivity of IR spectroscopy is the background 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 spectroscopies where two different samples are compared, “reaction-induced” IR difference techniques on a single sample have to be used. The “trigger” inducing the reaction should provide a minimum disturbance of the sample, but specifically 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, intrinsic photochemical reactions, electrochemical reactions in a spectroelectrochemical cell, and indirect photochemical reactions releasing an effector molecule which then starts the desired reaction, 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 dynamic 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 phenomena, but kinetically uncoupled from these. Vibrational spectroscopy can thus provide us with a detailed molecular view of the role of the protein 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 capacity, 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 vibrational modes from the backbone, from amino acid side chain groups, from cofactors, lipids, detergent and buffer molecules, and water. Coupling of such modes is possible, but will only result in small shifts depending on the interactions. For each of these group frequencies, an empirical assignment is then attempted. One of the strategies for an empirical assignment consists of isotope labelling of particular bonds and comparison of the observed with the calculated frequency shifts. In the case of a C=O stretching mode, replacement of by 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 suspensions 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 different from those in with the result that a clear assignment is not possible either. The probability 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 assignment. Other mass labels, like or can be introduced biosynthetically by growing microorganisms on isotope-labeled nutrients, but will end unspecifically. Ideal, but still in its infancy, is site-directed isotope labeling, i.e. the placement of a non-natural amino acid in an expression system to a specific position in the polypeptide chain. Although the feasibility has been demonstrated 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 spectroscopy 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 monitors the changes of dipole moments) is extremely sensitive. The balance seems to require generation of mutants with sufficiently small perturbations, 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 analogs 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 labelled 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 reaction centers with the spectra of the isolated cofactors, 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 radical, 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 significantly 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 electronic 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 involved in an between 250 and 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) of around However, since the 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 suspensions or hydrated films have to be used. Table 1 summarizes the vibrational modes which can be expected to contribute to the infrared 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 frequencies (i.e. below very little use 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 diagnostic purposes, but this region is in general too congested for a detailed analysis.

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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 dispersive 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 detectors similar to an optical multichannel detecting system or a diode array, but unfortunately multiple IR detector elements from semiconductors (e.g., HgCdTe, InSb) are extremely costly and can hardly be fabricated with constant specifications as compared to silicon diodes. A Fourier transform spectrophotometer makes use of an interferometer, rather than scanning the individual wavelengths successively. An interferometer (typically of the Michelson type) produces an interference modulated beam which is passed through the sample and then reaches the detector. The detector intensity as a function of the interferometer position is called the interferogram. 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 reviews 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 transmission or reflection properties of all optical components, and of the spectral sensitivity of the detector. The exact position of the moving mirror is

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determined by a He–Ne laser reference interferogram which is generated with the same interferometer and which controls, by detection of constructive and destructive interference pattern of the He–Ne laser, the timing of the data acquisition and digitization. The resolution of an FTIR instrument is determined by the distance d which the moving mirror travels from the point (“