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 3 Å (Scott and Eidsness, 1988), The peaks at > 3 Å could arise from scattering from second or third shell scattering from ligands like the imidazole ring of histidine or the pyrrole ring of hemes, or from backscattering from a bridged metal atom. 3) Distances are usually the most reliably determined structural parameters from EXAFS. But the range of data that can be collected, oftentimes due to practical reasons like the presence of the K-edge of another metal, limits the resolution of distance determinations to between 0.1 to 0.2 Å. 4) Determination of coordination numbers or number of backscatterers is fraught with difficulties. The Debye-Waller factor is strongly correlated with the coordination number and one must have recourse to other information, like comparison to inorganic model complexes, to narrow the range that is possible from curve fitting analysis alone. The most important point in the analysis is to differentiate between fit parameters which are required and others which are merely consistent with the data. The EXAFS method is most useful when delineating all the structural alternatives based on required fit parameters, or addressing the question of subtle structural changes in systems well characterized by other techniques like X-ray crystallography.

345 III. Applications of XANES and EXAFS in Photosynthesis

In the two decades since the XAS technique has become practical, it has become a standard method for probing the metal site structure in metallo-proteins. The technique has been applied to virtually every metallo-protein that has been isolated containing Fe, Mo, V, Cu, Co, Mn, Zn, Ni, Ca and other metals. In this section applications of XAS to Fe-S centers and the Mn oxygen-evolving complex in photosynthesis are described. Other components of the photosynthetic apparatus, plastocyanin (reviewed in Blackburn, 1990), the Fe-quinone acceptor complex in bacterial reaction centers (Bunker et al., 1982; Eisenberger et al., 1982), and the complex of ATPase (Carmelli et al., 1986) have also been studied using XAS.

A. Fe–S Proteins 1. Soluble Plant Ferredoxin Among the first metallo-proteins studied by XAS are the class of non-heme iron sulfur proteins which are found in most redox-mediated pathways in biology. Initially, three classes of Fe–S structures containing 1Fe, 2Fe and 4Fe metal sites were recognized. This list has expanded to include 3Fe sites and higher nuclearity structures containing 7 and 8 Fe atoms in the M and P clusters of nitrogenase. The most common structural units are in 1Fe containing Fe– S protein like rubredoxin, in 2Fe– 2S proteins like soluble plant ferredoxins, and in 4Fe–4S proteins like ‘high potential iron proteins’ (HIPIP) and other ferredoxins generally referred to as bacterial ferredoxins. The Fe EXAFS of these iron-sulfur proteins is dominated by backscattering from sulfur and iron atoms in the active site. In rubredoxin one Fe atom is ligated to four thiolate derived S atoms in near tetrahedral symmetry and it is a good example of a single-backscattering environment. The EXAFS of rubredoxin (see Fig. 5) exhibits a single wave and the Fourier transform shows one distinct peak, indicative of a single-shell system with one type of Fe–S distance of 2.26 Å (Teo and Shulman, 1982).

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V.K. Yachandra and M.P. Klein

In contrast to the EXAFS from rubredoxin, the EXAFS of the 2Fe–2S soluble plant ferredoxin and the 4Fe–4S bacterial ferredoxin exhibits (see Fig. 5) a beat pattern indicating the presence of at least two sine waves. The respective Fourier transforms show two peaks. The first peak is due to backscattering from S ligand atoms at ~ 2.23–2.25 Å, and the second peak is due to backscattering from the Fe atoms at 2.73 Å. These distances compare well with distances derived from X-ray crystallography of the 2Fe–2S proteins from Spirulina platensis (Tsukihara et al., 1978), Anabaena 7120 (Rypniewski et al., 1991), 4Fe–4S proteins as well as synthetic analogs (reviewed in Spiro, 1982; Teo and Shulman, 1982). 2. Fe–S Acceptors Photosystem I

and

in

The electron transfer reactions from the primary donor of PS I to the substrates are initially

mediated by a number of PS I bound electron acceptors, labeled and which are Fe– S clusters with redox potentials of –705, –590, and –530 mV, respectively. XANES and EXAFS studies at the Fe K-edge have been used to study these Fe-S clusters. In the Fe K-edge XANES study of PS I the transition to bound 3d states was used to derive information about the symmetry of the Fe–S acceptor complexes. The 1s to 3d pre-edge transition is electric-dipole forbidden, but it is often observed in the edge spectra and is commonly attributed to d-p mixing. In centrosymmetric complexes d–p mixing is symmetry unallowed and the 1s to 3d transition is weak. In non-centrosymmetric complexes d–p mixing is allowed and the 1s to 3d transition is more intense. The intensity of the 1s to 3d pre-edge transition can be used as a marker to differentiate between octahedral and tetrahedral complexes (Shulman et al., 1976; Roe et al., 1984). Fig. 6 shows the Fe K-edge spectrum of Fe–S

X-ray absorption spectroscopy

centers and The centers are compared to a 4Fe–4S model compound and to a centrosymmetric hexacoordinate Fe complex. There is a decrease in the intensity of the 1s to 3d transition, and there is a change in the shape of the spectrum between tetrahedral Fe–S centers and a centrosymmetric hexacoordinate system. The intensity of the pre-edge 1s to 3d transition shows that the Fe–S acceptors and are in tetrahedral Fe–S complexes (McDermott et al., 1988a, 1989). Fe K-edge EXAFS studies of PS I preparations from spinach and the thermophilic cyanobacterium Synechococcus sp. showed that the spectra were similar to those from Fe–S clusters, with a Fourier peak corresponding to S backscattering and another peak corresponding to backscattering from Fe. The results from PS I preparations containing and showed that the data could be simulated with either three 4Fe–4S clusters or two 4Fe–4S and one 2Fe–2S cluster. This was due to the complexity of dealing with PS

347

I preparations which contained about 11–14 Fe atoms (McDermott et al., 1988a). A subsequent EXAFS study using PS I preparations which contained only clearly showed that it was also a 4Fe–4S cluster (McDermott et al., 1989). The recently determined crystal structure of PS I has confirmed that all three acceptors are 4Fe–4S clusters (Krauss et al., 1993). 3. Rieske Fe–S Clusters

A Rieske Fe–S cluster is part of the cyt complex in higher plants which mediates electron transfer between PS II and PS I. A Rieske Fe–S center is also present in the cyt complex of photosynthetic bacteria which is involved in cyclic electron transport. The Rieske Fe–S centers are characterized by an EPR signal and redox properties (150–350 mV) which are very different from the other more ubiquitous 2Fe–2S and 4Fe–4S proteins. Analogous Rieske centers are part of

348 the mitochondrial electron transport scheme. Although there are no studies from plant sources, EXAFS studies of Rieske Fe–S protein from the cyt complex from mitochondria (Powers et al., 1989) and phthalate dioxygenase from Pseudomonas cepacia (Tsang et al., 1989) have been reported. Curve fitting results from both studies showed that better fits were obtained by including a mixed S and N coordination to Fe, but the quantitation of the number of nitrogens per Fe was not unequivocal. These studies are in accord with the ENDOR (Gurbiel et al., 1991) and ESEEM (Britt et al., 1991) results which showed there may be two terminal histidine ligands per 2Fe–2S cluster. The EXAFS studies found that: the Fe–S binding and terminal distances and the Fe–Fe distance were similar to the regular 2Fe–2S and 4Fe–4S clusters providing evidence that the unique properties of the Rieske centers are due to the N ligation or due to factors other than differences in Fe–S and Fe–Fe distances.

B. Manganese Oxygen Evolving Complex in Photosystem II Most of the oxygen in the atmosphere which supports life on earth is generated by plants by the photo-induced oxidation of water to dioxygen. The reaction shown in Eq. (12) is catalyzed by a tetranuclear Mn complex, which sequentially stores four oxidizing equivalents that are used to oxidize two molecules of water to molecular oxygen. The Mn complex is part of a multiprotein assembly called Photosystem II, which contains the reaction center involved in photosynthetic charge separation and an antenna complex of chlorophyll molecules. The complex also contains cyt and a Fe-quinone electron acceptor complex (reviewed in Debus 1992; Rutherford et al., 1992). Owing to the complexity of the system and the presence of so many pigment and other components, study of the Mn complex by optical and other spectroscopic methods can be difficult. EXAFS is ideally suited for the study of the structure of the Mn complex because the specificity of the technique allows us to look at the Mn without interference from the pigment molecules, or the

V.K. Yachandra and M.P. Klein protein and membrane matrix, or other metals like Ca, Mg, Cu and Fe which are also present in active preparations. An active oxygen-evolving complex has not yet been crystallized, but EXAFS does not need single crystals; the structural studies can be performed on frozen solutions. Also, several of the intermediate states mentioned above have been stabilized as frozen solutions and studied by EXAFS. We and others have studied the structure of Mn in the OEC using XAS. An earlier series of papers from our group reported Mn–Mn interactions at 2.7 Å and Mn–O interactions at 1.75 Å, and an additional highly disordered shell of light atom (O, N) scatterers at ~ 2 Å. Similar results were found for both PS II-enriched membrane preparations from spinach chloroplasts and for detergent-solubilized OEC preparations from the thermophilic cyanobacterium Synechococcus sp. (McDermott et al., 1988b). From these results on samples poised in both the and states, it was predicted that the OEC contains binuclear dibridged Mn units whose structures remain largely unchanged upon the advance to (reviewed in Sauer et al., 1992; Klein et al., 1993). More recent EXAFS studies of Mn in the OEC, at substantially lower sample temperatures and with improved signal to noise ratio, have provided evidence for scatterers at > 3 Å in addition to the interaction at 2.7 Å. These experiments include various combinations of oxygenevolving preparations and EXAFS analysis techniques. George et al. (1989) report Mn scatterers at distances of 2.7 and 3.3 Å in angle-dependent EXAFS studies of whole oriented spinach chloroplasts. It was found that the scatterers at these distances exhibited significant dichroism. PennerHahn et al. (1990) also reported the 2.7 Å shell and at least one and possibly two shells of scatterers at > 3 Å in EXAFS experiments using core preparations from spinach. Both these studies reported difficulties in detecting the 1.8 Å shell; this was probably due to Mn(II) contamination in their experiments. MacLachlan et al. (1992) reproduced the 1.8 Å shell along with the 2.7 Å scatterers, but differed from the above experiments by predicting a shell of Ca at a significantly longer distance of 3.7 Å. These results all have different implications for a structural model of the OEC.

X-ray absorption spectroscopy

Fig. 7 shows a typical Fourier transform of Mn in PS II from our data. The first Fourier peak requires two different distances to be fit adequately, one at ~ 1.8 Å and the other 1.95–2.15 Å to light elements like C, N, or O. The second peak on the other hand is best fit by a heavier atom and, as noted above, probably a Mn atom at 2.7 Å; the amplitude is best fit to an average of one such interaction per Mn atom in the complex. Fitting the third peak, at a longer distance is more difficult. It can be fit to C, or Mn or Ca, or to a combination of these entities at 3 Å. The quality of fits is always better when Mn is included, but it is not clear if there is only Mn or also Ca atoms (Latimer et al., 1995). The presence of both the Mn–O (C,N) distances at ~ 1.8 Å and the Mn– Mn distances at 2.7 Å in all known bridged multinuclear Mn complexes, and the presence of a 3.3 Å Mn–Mn separation in monobridged Mn complexes leads to the conclusion that both of these structural motifs are present in the Mn complex of PS II (Wieghardt 1989; Pecoraro 1992). The number of such scattering interactions leads to the conclusion (remembering that there are four Mn atoms) that there are at least two Mn–Mn bridged

349

units and at least one Mn–Mn bridged unit (DeRose et al., 1994). Figure 8 shows the Fourier transform of the same system in oriented samples (Mukerji et al., 1994). It is evident that the 2.7 and 3.3 Å Mn– Mn vectors (the second and third peak in the Fourier transform) are oriented differently. The 2.7 Å vector is more parallel to the membrane normal and the 3.3 Å is more perpendicular. Detailed analysis of the dichroism can be used to determine the angles more accurately. The dichroism requires that the complex be asymmetric. One of the many possible structures (DeRose et al., 1994) consistent with data shown in Fig. 7 and the dichroism measurements is shown in Fig. 9 and provides us with a working model for the Mn cluster (Yachandra et al., 1993) Figure 10 shows the Mn K-edge spectrum from complexes in oxidation states (II), (III) and (IV). The inflection point shifts to higher energy as the oxidation state increases. There is also a dramatic change in the general shape of the edge as shown by the changes in the second derivatives. The shape of the edges is also an important indicator of oxidation state, as seen in Fig. 10 (Yachandra et al., 1993). A combination of the position and the shape were used to assign the oxidation states

350

V.K. Yachandra and M.P. Klein in the and states to and respectively. Fig. 11 shows the Mn K-edge from preparations in the and states of the photosystem II complex, and it shows that the Mn K-edge shifts to higher energy on advancing from to and from to indicating oxidation of Mn (Goodin et al., 1984; McDermott et al., 1988b; Guiles et al., 1990a). However no such shift was evidenced in samples prepared in the state by a cryogenic double turnover protocol, which leads to the conclusion that Mn is not oxidized in the to transition. It was proposed that the oxidation equivalent is stored on a ligand or amino acid residue (Guiles et al., 1990b). Ono et al. (1992) have presented Mn XANES of PS II samples that advanced progressively from by one through five laser flashes through the Kok cycle. Their data were interpreted to provide evidence for Mn oxidation from to and to and then reduction on going from to It is important to point out that Ono et al. have assumed that their samples were advanced on each flash but have presented no substantiating data, such as the pattern of multiline EPR signal intensity vs. flash. We have recently completed such a study (Roelofs et al., 1995, Andrews et al., 1994), and our data support our earlier results that showed Mn is oxidized during the to and to transition but that it may not be oxidized on the to advance.

IV. Future Directions The future holds much promise for the use of XAS in photosynthesis, and bio-inorganic chemistry in general. The availability of new storage rings and beamlines at these storage rings dedicated to research in biology is increasing. There has been considerable improvement in the brightness of the X-ray sources and also in detector technology. This makes the measurement of Xray absorption spectra of dilute species easier. The high brightness and the increased sensitivity of the technique are being used to couple microscopy with spectroscopy. XAS in the soft X-ray region, which has been mostly used in materials and surface science studies, is increasingly being applied to biological problems. Some of the edges of interest in photosynthesis include the K-edges of P, S and Cl and also C, N, and O, and the L-edges of transition

X-ray absorption spectroscopy

metals. L-edges, as shown in Fig. 1, are 2p to 3d transitions, and in contrast to the 1s–3d transitions in K-edge spectra are electric dipole allowed and hence are intense. The natural linewidths of K and L-edge transitions, for example, in Mn are 1.12 and 0.32 eV, respectively, making L-edges considerably more sensitive to factors such as symmetry that influence the d orbital splittings and population. Differential absorption of circularly polarized X-rays in the presence of a magnetic field (XMCD, X-ray magnetic circular dichroism) is another method that is being applied to biological systems. X-ray magnetic circular dichroism with its selectivity for paramagnetic species and the relative magnetic orientation of different species in a mulicenter system promises to be a powerful tool for studying metal centers in the photosynthetic apparatus.

351

Acknowledgements We thank Dr. Matthew Latimer and Dr. Annette Rompel for a critical reading of the manuscript. We are grateful to Prof. Kenneth Sauer for his suggestions. The work from our laboratory presented in this article was supported by the National Science Foundation grant DMB91– 0414, and by the Director, Division of Energy Biosciences, Office of Basic Energy Sciences, Department of Energy (DOE) under contract DEAC03–76SF00098. Synchrotron radiation facilities were provided by the Stanford Synchrotron Radiation Laboratory (SSRL) and the National Synchrotron Light Source (NSLS), both supported by DOE. The Biotechnology Laboratory at SSRL and Beam Line X9–A at NSLS are supported by the National Center for Research Resources of the National Institutes of Health.

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353 nation compounds. In: Pecoraro VL (ed) Manganese Redox Enzymes, pp 197–231, VCH Publishers, New York. Pendry JB (1983) The transition between XANES and EXAFS. In: Bianconi A, Incoccia L and Stipcich S (eds) EXAFS and Near Edge Structure, p 4. Springer-Verlag, Berlin. Penner-Hahn JE, Fronko RM, Pecoraro VL, Yocum CF, Betts SD and Bowlby NR (1990) Structural characterization of the manganese sites in the photosynthetic oxygenevolving complex using X-ray absorption spectroscopy. J Am Chem Soc 112: 2549–2557. Powers L (1982) X-ray absorption spectroscopy: applications to biological molecules. Biochim Biophys Acta 683: 1– 38. Powers L, Schägger H, von Jagow G, Smith J, Chance B and Ohnishi T (1989) EXAFS studies of the isolated bovine heart Rieske cluster. Biochim Biophys Acta 975: 293–298. Rehr JJ, de Leon JM, Zabinsky SI and Albers RC (1991) Theoretical X-ray absorption fine structure standards. J Am Chem Soc 113: 5135–5140. Rehr JJ, Albers RC and Zabinsky SI (1992) High-order multiple-scattering calculations of X-ray absorption fine structure. Phys Rev Lett 69: 3397–3400. Roe AL, Schneider DJ, Mayer RJ, Pyrz JW, Widom J and Que L (1984) X-ray absorption spectroscopy of iron-tyrosinate proteins. J Am Chem Soc 106: 1676–1681. Roelofs TA, Liang W, Latimer MJ, Cinco R, Rompel A, Andrews JC, Yachandra VK, Sauer K and Klein MP (1995) Manganese oxidation states of the flash-induced S-states of photosystem II. In: Mathis P (ed) Photosynthesis from Light to Biosphere, Vol. II, pp 459–462. Kluwer Academic Publishers, Dordrecht. Rutherford AW, Zimmermann, J-L and Boussac A (1992) Oxygen evolution. In: Barber J (ed) The Photosystems: Structure, Function and Molecular Biology, pp 179–229. Elsevier, Amsterdam. Rypniewski WR, Breiter DR, Benning MM, Wesenberg G, Oh B-H, Markley JL, Rayment I and Holden HM (1991) Crystallization and structure determination to 2.5 Å resolution of the oxidized [2Fe–2S] ferredoxin isolated from Anabaena 7120 Biochemistry 30: 4126–4131 Sauer K, Yachandra VK, Britt RD and Klein MP (1992) The photosynthetic water oxidation complex studied by EPR and X-ray absorption spectroscopy. In: Pecoraro VL (ed) Manganese Redox Enzymes, pp 141–175. VCH Publishers, New York. Sayers DE and Bunker BA (1988) Data Analysis. In: Koningsberger DC and Prins R (eds) X-ray Absorption: Principles, Applications, Techniques of EXAFS, SEXAFS and XANES, pp 211– 253. John Wiley and Sons, New York. Sayers DE, Stern EA and Lytle FW (1971) New technique for investigating noncrystalline structures: Fourier analysis of the extended X-ray absorption fine structure. Phys Rev Lett 27: 1204–1207. Scott RA (1984) X-ray absorption spectroscopy. In: Rousseau RL (ed) Structural and Resonance Techniques in Biological Research, pp 295–362. Academic Press, Orlando, FL. Scott RA and Eidsness MK (1988) The use of X-ray absorption spectroscopy for detection of metal–metal interac-

354 tions. Applications to copper-containing enzymes. Comments Inorg Chem 7: 235–267. Shulman RG, Yafet Y, Eisenberger P and Blumberg, WE (1976) Observation and interpretation of X-ray absorption edges in iron compounds and proteins. Proc Natl Acad Sci USA, 73: 1384–1388. Spiro TG (1982) Iron–Sulfur Proteins. John Wiley, New York Stern E (1988) Theory of EXAFS. In: Koningsberger DC and Prins R (ed) X-ray Absorption: Principles, Applications, Techniques of EXAFS, SEXAFS and XANES, pp 3– 51. John Wiley and Sons, New York. Stöhr J (1992) NEXAFS Spectroscopy. Springer-Verlag, Berlin. Teo B-K (1986) EXAFS: Basic Principles and Data Analysis. Springer-Verlag, New York. Teo B-K and Shulman RG (1982) X-ray absorption studies of iron–sulfur proteins and related compounds. In: Spiro TG (ed,) pp 343–366. John Wiley and Sons, New York. Tsang H-T, Batie CJ, Ballou DP and Penner-Hahn JE (1989)

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Chapter 22 Mössbauer Spectroscopy Peter G. Debrunner Physics Department, University of Illinois at Urbana-Champaign, 1110 W. Green Street, Urbana, IL 61801–3080, USA

Summary I. Introduction II. Mössbauer Spectroscopy: Physics and Formalism A. Basic Features B. Hyperfine Interactions C. Electronic States of the Iron D. Spin Coupling E. Dynamical Aspects F. Calculation of Mössbauer Spectra G. Experimental Considerations III. Applications A. Iron–quinone Complex B. Iron–sulfur Proteins C. Cytochromes References

355 356 357 357 357 359 361 362 363 364 365 365 368 371 371

Summary spectroscopy (MS) has played an important role in the elucidation of the iron centers in the photosynthetic apparatus. In 1975, G. Feher and collaborators demonstrated that the single iron of bacterial reaction centers (RC) was high-spin ferrous irrespective of the state of Moreover, they showed that reduction of broadened the Mössbauer lines at 4.2K, indicative of spin coupling between the semiquinone and the iron, related to the broadening observed in the EPR signal of the semiquinone (Debrunner et al., 1975). Photosystem I (PSI) of green plants and algae contains three iron–sulfur centers labeled and that have originally been identified by EPR. Evans et al. (1977, 1979, 1981) showed that the Mössbauer spectra of PSI were practically identical with those of the well understood bacterial 4Fe–4S centers. The low-potential center remained controversial, however, as others suggested a 2Fe–2S center instead. The controversy was resolved by Petrouleas et al. (1989), who studied a mutant lacking centers and and found that had indeed all the properties of a 4Fe–4S center. The more difficult task of analyzing Photosystem II (PSII) of green plants was undertaken by Petrouleas and Diner (1982, 1986, 1990), who identified the redox center known as with the iron– quinone complex and showed the iron to be redox active in contrast to the Fe(II) of the bacterial RC. The same group demonstrated, by MS, that formate affected the iron–quinone complex (Diner and Petrouleas, 1987), and finally that the Fe(II) formed an NO derivative.

Correspondence: Fax: 1-217-3339819; E-mail: [email protected]

355 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 355–373. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

356

Peter G. Debrunner

After a review of the methodology, the various iron sites of bacterial RC, of PSI and PSII will be discussed in detail to illustrate the application of the method. Abbreviations: EFG – electric-field gradient; EPR – electron paramagnetic resonance; LDAO – lauryldimethylamine N-oxide; MS – Mössbauer spectroscopy; PSI – Photosystem I; PSII – Photosystem II; RC – (bacterial) reaction center

I. Introduction This chapter attempts to acquaint the reader with the basic concepts of spectroscopy (MS) and its applications in photosynthesis research. is the most important Mössbauer isotope, and since the photosynthetic apparatus contains several prominent iron complexes, MS can be utilized to study the properties of these complexes under various experimental conditions. For more general and in-depth treatments of MS the reader is referred to the literature (Greenwood and Gibb, 1971; Gonser, 1975; Gibb, 1976; Gütlich et al., 1978; Huynh and Kent, 1984; Cranshaw et al., 1985; Dickson and Berry, 1986; Debrunner, 1993). Since MS involves the resonant absorption of gamma rays by nuclei, it has several features that set it apart from other types of spectroscopy. First of all, the quantum energy of the transition is quite large, in the case of yet the intrinsic linewidth, is sufficiently small to resolve the hyperfine interactions of the nucleus with its electron shell. The applications of MS discussed here indeed use the known electric and magnetic moments of the to probe the internal electric and magnetic fields produced by the electrons and to gather information, in the process, about the state of the iron. Secondly, the 14.4 keV radiation to be resonantly absorbed by the nuclei is emitted by a source of radioactive which decays to stable via the 14.4 keV excited state. The source thus emits 14.4 keV gamma rays of intrinsic line width (plus other radiations contributing to the non-resonant background). Given the discrete energy, keV, of the radiation emitted by the source, the spectrum of the absorber can be scanned by mov-

ing the source with velocity v relative to the absorber, imparting thereby a Doppler shift to the gamma rays,

where c is the speed of light. It is customary in MS to express all energies in terms of the measured velocities v. To convert to more familiar energy units the following conversion factors are useful:

Thirdly, it follows from energy and momentum conservation that Mössbauer transitions of intrinsic linewidth can only occur in solids and only in a fraction f < 1 of the cases as will be discussed in Section II.E. A fourth unique feature of MS is its sensitivity to a single isotope, here Since the natural abundance of is only 2.2%, biological samples need to be enriched in to improve the signal-to-noise ratio. As will be discussed in Section II, different oxidation and spin states produce distinctly different spectral contributions, and with a few empirical rules it is in principle possible to resolve a composite spectrum into its components and to deduce the oxidation and spin state of each iron site. To illustrate this process, Section III discusses the analysis and interpretation of spectra taken on bacterial reaction centers, PSI and PSII. It will be noted that the appropriate level of sophistication in the data analysis depends on the quality of the data. At the very least, MS can assess the purity and composition of a sample as far as its iron content is concerned. Once the quality and reproducibility of the samples are established, the spectral components can be assigned to the various iron species present, and the assignments can be verified by titr-

Mössbauer spectroscopy ation, by the expected temperature and field dependences, etc. Finally, it may be possible to characterize the iron environment(s) further by fitting spectra, taken as a function of temperature and field, to a spin Hamiltonian or other theoretical model.

357 of an nucleus with transition encan be written as where depends on the transition probability and other factors. The intensity transmitted through an absorber with n nuclei per unit area then is

ergy

II. Mössbauer Spectroscopy: Physics and Formalism

A. Basic Features The stable ground state of has nuclear spin I = 1/2 and connects via magnetic dipole transition to the first excited state at 14.4 keV with spin I* = 3/2 and mean life The Heisenberg principle then predicts a Lorentzian energy distribution of the emission or absorption line with intrinsic width where is Planck’s constant. Most experiments are done in a transmission geometry, whereby the radiation emitted by a single-line source impinges on the absorber to be investigated, and the transmitted 14.4 keV gammas are counted in a detector, e.g. a proportional counter. As the Doppler-shifted energy of the incident beam matches the transition energy of some nuclei in the absorber, these nuclei absorb the resonant gammas in proportion to predictable transition probabilities, and the count rate of the detector decreases. Doppler shifts are varied periodically between and data are typically accumulated for many hours. The final spectrum is a plot of the total number of counts, N(v), recorded as a function of Doppler velocity v. To find a quantitative expression for a transmission spectrum let the intensity distribution of the incident beam be I(E, v)dE, where is the center of the Lorentzian line emitted by the source. With the substitutions can be written as which integrates to Here and (below) are the recoilless fractions of the source and absorber, respectively, and will be discussed further in Section II.E. Similarly, with the absorption cross section

with thin-absorber

The which approximates by is valid for most biological samples. To of Eq. (4) one has to add an energy-independent background B. The transmission spectrum therefore consists of a (negative) Lorentzian of full width at halfheight and relative height The maximum resonance cross section of with no hyperfine splitting is which is much larger than the (non-resonant) absorption cross section of of iron at 14.4 keV. In a more general case several types of iron environments will be found in an absorber, and each is subject to hyperfine interactions, which lift the two- and four-fold degeneracies of the ground and excited states, respectively. The factor n in Eq. (4) then refers to the of a given species, and as well as depends on the nuclear eigenstates involved in the transition as described below. limit,

B. Hyperfine Interactions This section discusses how the electron shell and/or external fields affect the nuclear energy levels and thereby the Mössbauer spectra, and how the electronic properties of the iron can be deduced from the spectra. In addition to the spin angular momenta I and I* of the ground and excited state already mentioned, the nucleus has magnetic moments Î and respectively, with and an electric quadrupole moment

358 where all starred quantities refer to the I* = 3/2 excited state, the ground state of spin I = 1/2 having no measureable quadrupole moment. In the expressions for and above is the nuclear magneton. The difference in mean-square charge radii, moreover, gives rise to an electric monopole interaction that shifts the transition energy proportional to the s-electron density, at the nucleus and manifests itself in a shift of the whole spectrum without affecting its shape. This so-called chemical or isomer shift, is given by the expression

where the subscripts A and S in the last term refer to absorber and source, respectively. In order to define the isomer shift independently of the particular source used, is usually quoted as relative to the centroid of the spectrum of metallic iron taken at 300K. It should be noted that the overall shift has a dynamical contribution as well which will be discussed in Section II.E. The electric quadrupole interaction, another correction to the Coulomb energy of the atom, accounts for the non-spherical charge distribution of the nucleus in the excited state. It splits the Mössbauer transition into two lines separated by the quadrupole splitting and occurs whenever an electric-field gradient (EFG) is present at the nucleus. An EFG arises from the valence electrons of the iron and from surrounding charges unless excluded by symmetry. In iron proteins high local symmetry with no EFG such as tetrahedral, octahedral, etc., is rare, and a measurable quadrupole splitting is therefore the rule. Since a single 3d-electron can produce a splitting of the valence contribution typically exceeds the lattice contribution which arises from charges outside the electron shell. The EFG or its negative, x,y,z, is a symmetric, traceless second rank tensor. It is readily calculated from the charge density of the valence electrons and from any external charges. For the covalent, low-symmetry iron sites of interest here a proper treatment requires a molecular orbital approach, but simpler crystal-

Peter G. Debrunner field approximations have been used in the past. Model calculations are complicated by the distortion of the inner electron shells as parametrized by the Sternheimer factors. Several equivalent expressions for the electric quadrupoleinteractions are in use (Abragam and Bleaney (1990))

In the last two expressions are the principal-axes components of and the are the components of the nuclear spin operator, Î, along these axes. The numerical values in Eq. (6) are those for the I* = 3/2 excited state. The last expression uses the condition and the definition of the asymmetry parameter

With the convention, is limited to the range In the absence of magnetic interactions splits the I* = 3/2 excited state into two doublets with a quadrupole splitting of

Since the ground state does not split, the quadrupole interaction leads to the characteristic twoline pattern of the Mössbauer spectrum, both lines being Lorentzians of minimum linewidth and equal areas for randomly oriented samples. The magnetic dipole interaction, finally, lifts the degeneracy of the nuclear levels completely, and the magnetic dipole selection rules allow six transitions between the two and the four equidistant levels of the ground and excited state, respectively. In the presence of electric quadrupole interaction, the I* = 3/2 eigenstates are linear combinations of the states, and up to eight transitions are possible. An external magnetic field B leads to the nuclear Zeeman interatction for the ground state,

where the last expression assumes that B defines

Mössbauer spectroscopy the direction of z. An analogous expression holds for the excited state. The magnetic hyperfine interaction couples the nuclear spin operators Î or Î* with the electron spin operators and is given by (Abragam and Bleaney 1970)

Here, the sum is over all unpaired electrons with position orbital angular momentum and spin and is a numerical factor of roughly 0.35. An analogous expression applies for the excited state. The three contributions in Eq. 10 are the orbital, the traceless spin dipolar and the isotropic Fermi contact term, respectively, where the last one generally dominates. As stated in Eq. 10 the hyperfine tensor à has units of energy and can be compared directly with EPR/ENDOR data. It is convenient, however, to divide à by so that

represents an internal field that can be substituted for, or added to, in Eq. 9. Note that Eq. 11 contains the expection value of which is a classical vector rather than an operator so that the left-hand side can be equated to a field. Whenever a sufficiently large external field is aplied to a sample of spin such that the Zeeman interaction far exceeds the magnetic hyperfine interaction, typically then can be calculated from the electron spin Hamiltonian alone, Eq. 15, as will be discussed later in Section II.C. In field units the Fermi contact term for six-coordinate iron with oxygen/ nitrogen ligands has the value which decreases to for the highly covalent coordination of iron-sulfur proteins. In a general case the nuclear Hamiltonian is given by the sum

If the internal field approach, Eq. 11, is valid, simplifies to

which depends on nuclear spin operators only.

359 Since refers to a molecular frame while is defined in the laboratory, each molecule in a randomly oriented sample will have a different nuclear Hamiltonian and therefore different energy levels and transition probabilities. As a result, the Mössbauer spectrum no longer consists of a set of discrete lines but rather of a continuous distribution, and any quantitative analysis requires computer programs. The information available from such an analysis can be substantial even for the simplest case of a diamagnetic compound with Here, the applied field broadens the quadrupole doublet, and the lineshape allows one to deduce as well as the sign of two parameters that characterize the symmetry of the valence electrons in more detail than the quadrupole splitting alone.

C. Electronic States of the Iron Ferric iron has five 3d-electrons with spins arranged either parallel, resulting in a orbital singlet of total spin S = 5/2, or with four spins paired leaving a single unpaired spin, S = 1/2, in an orbital triplet The former, high-spin state is found in more ionic, weak-field compounds, whereas the latter, low-spin state is found in highly covalent, strong-field compounds, e.g., the ferricytochromes. The spin state of ferric compounds is readily recognizable from MS, and although the same can be said of EPR, which is vastly more sensitive, Mössbauer data can provide additional information not available otherwise. The combination of the two methods is clearly most powerful. We begin with a discussion of high-spin Fe(III). The sixfold spin degeneracy of the state is partially lifted by the zero-field splitting which is given to lowest order by the expression

By convention, the rhombic term E in Eq. 13 is limited to in a ‘proper’ coordinate system. The axial term D is typically small for S = 5/2 iron, but both signs of D are possible. The eigenstates of are three Kramers doublets; for E = 0 these doublets are the

360 states, while they are linear combinations of the states for The remaining twofold degeneracy is lifted by the Zeeman interaction where is the Bohr magneton, and is the g-tensor, which is expected to have the spin-only value of since the state allows no orbital contribution. The total electronic spin Hamiltonian is then the sum of Eq. 10 should in principle be added to Eq. 15 since it depends on the electron spin operator Inclusion of is warranted for vanishing only since requires an expansion of the basis set to (2I + 1)(2S + 1), etc., and complicates the solution of Eq. 15 considerably. For is a small perturbation that does not affect appreciably and can therefore be neglected in Eq. 15. Diagonalization of yields the six eigenstates and their energies and allows one to calculate EPR transitions, the spin expectation values of each state, etc. In the presence of an applied field mT the internal field formalism, Eq. 11, is valid, and the nuclear eigenstates and energies can be found from Eq. Each spin eigenstate produces a different internal field, however, and for each molecular orientation six different component spectra with different Boltzmann factors have to be added. In practice, spectra of high-spin Fe(III) typically show well resolved bands with overall splittings of roughly 16 for The orbital singlet state, allows no orbital and spin dipolar contribution to Ã, and any deviations reported from isotropy are indeed small. The nominal singlet also predicts zero valence contribution to the quadrupole interaction, but does not rule out lattice contributions or effects due to anisotropic electron delocalization. Typical splittings are of the order but exceptional cases with twice that value are known. Low-spin Fe(III), a orbital triplet, is best understood as a single-hole state in an otherwise filled subshell. The magnetic properties, in particular the g- and A-tensors, are reasonably

Peter G. Debrunner well reproduced by a crystal-field model proposed by Griffith (1957). According to this model the threefold orbital degeneracy obtained in strong octahedral field is lifted by crystal field components of lower symmetry, namely an axial and a rhombic component. If the Hamiltonian consisting of the electrostatic potential energy and the known spin-orbit interaction is solved, three Kramers doublets are obtained as linear combinations of orbitals with appropriate spin functions. The splittings are such that only the ground doublet is populated at any accessible temperature, and once the wavefunction of this ground doublet is known, all observables, in particular the tensors Ã, and can be calculated. In practice, the axial and rhombic crystal field components are adjusted to match the measured gvalues, and Taylor (1977) has given a particularly simple algorithm for this purpose. The g-values may deviate substantially from the spin-only value due to orbital contributions, and the Atensors typically are quite anisotropic for the same reason and because of spin dipolar contributions. The quadrupole splitting reflects the combination of orbitals that make up the ground state wavefunction: if a single orbital dominates, may be as large as 3 if all orbitals contribute equally, may be close to zero. In low-spin heme compounds the lattice or covalency contributions to are substantial (Debrunner, 1989). Ferrous iron has six 3d-electrons, i.e. one more than Fe(III), and both high- and low-spin states are found in weak- and strong- field compounds, respectively. Low-spin Fe(II) has a filled subshell and is therefore diamagnetic as exemplified by ferrocytochromes. The valence contribution to the quadrupole splitting is zero, but the lattice and covalency contributions lead to in the cytochromes. Isomer shifts of the latter are in the range of High-spin Fe(II) is characterized by the largest isomer shifts, 0.9 where the low value applies to the more covalent, 5-coordinate heme proteins, the high value to the most ionic, 6-coordinate compounds. The exceptionally covalent coordination of the ironsulfur proteins leads to Another characteristic of high-spin Fe(II) is the large quadrupole splitting of

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which has a noticeable temperature dependence as illustrated in Fig. 1 and explained below. The extra 3d-electron of high-spin Fe(II), which causes the large leads to a or state in octahedral or tetrahedral symmetry, respectively. As illustrated for the case of in the upper inset of Fig. 1, crystal field components of axial and/or rhombic symmetry lift the orbital degeneracy, and the thermal population of the higher orbital state(s) with different quadrupole interactions averages out the observable and hence makes it temperature dependent. A proper treatment of has to include the spin-orbit coupling as well as the axial and rhombic crystalfield terms (Ingalls, 1964). The magnetic properties of high-spin Fe(II) are usually described by the spin-Hamiltonian, Eq. 15. The zero-field splittings are typically several i.e. larger than in high-spin ferric compounds, and the eigenstates of Eq. 13 are singlets for rather than Kramers doublets. The last point is an important, general distinction between integer-spin and half-integer spin systems: For integer-spin, non-Kramers systems the spin expectation values vanish in zero field, and no spontaneous magnetic hyperfine splitting is observed.

The Mössbauer spectra of high-spin Fe(II) therefore show unbroadened quadrupole doublets in zero field as illustrated in Fig. 2 (top). The only known exceptions are magnetically ordered materials or the unusual case that is comparable to the splitting between the eigenstates of Eq. 13. Strong external fields will induce internal fields described by Eq. 11 and thus allow one to deduce the hyperfine tensor à as illustrated in Fig. 2 (bottom), which will be discussed further in Section III.A. It should be kept in mind that the parameters of Eq. 15 are constants only as long as the next higher orbital level is far removed in energy from the ground level, a condition that is certainly not satisfied if is strongly temperature dependent.

D. Spin Coupling The formalism of Section II.C. refers to isolated iron sites. In many biological systems including the photosynthetic apparatus, however, the iron

362 is either found in clusters as in the 4Fe–4S proteins or near a radical as in the ferroquinone complex. In both cases the component spins couple, changing the properties of the coupled system drastically. This subsection therefore attempts to describe the coupled system in terms of the properties of its parts. The simplest type of coupling between two spin operators and is given by the Heisenberg-Van Vleck expression for isotropic exchange, which is the dominant term when the wavefunctions of the two centers overlap. has eigenstates of spin and energy For J > 0 the state of lowest energy is thus the one with the smallest spin S, and the opposite is true for J < 0. The situation is simple as long as is much larger than any other term in the spin Hamiltonian, Eq. 15, of the spins and This is the case for the 2Fe–2S proteins, which exist in the oxidation states Fe(III)–Fe(III) and Fe(III)– Fe(II) and have strong antiferromagnetic coupling so their net spins are S = 0 and S = 1/2, resp. The situation is more complicated in the case of the 4Fe – 4S proteins, which exist in an oxidized and a reduced state. The problem is that there are six interacting iron pairs that can not simultaneously align their spin antiparallel. The energy of the system can be lowered, however, by double exchange (Münck et al., 1988), whereby the extra electron of Fe(II) delocalizes onto Fe(III) so that the average charge per iron is 2.5 as judged by the isomer shift. The spins of the delocalized pair align parallel to a spin of 9/2. Oxidized has two such pairs lining up antiparallel to a cluster spin of S = 0. In reduced there is a delocalized, S' = 9/2 pair and a diferrous pair with aligned spin, S'' = 4 and the cluster spin is S = 1/2. For the system spin is a good quantum number, and using spin algebra the expectation value of any operator involving or can be calculated in any eigenstate of To illustrate the process, consider Eq. (10), written here for the intrinsic spin In

Peter G. Debrunner terms of the system spin the Hamiltonian becomes where the A-tensor in the S representation is given by If is small or comparable to as is the case in the ferroquinone complex, the full Hamiltonian has to be diagonalized.

E. Dynamical Aspects The vibrational motion of the iron affects the Mössbauer spectra in three significant ways, viz. (i) the recoilless fraction f, (ii) the thermal redshift which adds to the isomer shift, and (iii) the spin-lattice coupling, which controls the spin dynamics. We will discuss these effects in this order. As stated in the Introduction, the Mössbauer effect is observable in solids only. The reason is that energy and momentum have to be conserved in the emission and absorption processes, and this is possible without recoil energy loss only if the emitting or absorbing atom is part of a quantized vibrational system with discrete energy levels. When a bound iron absorbs a photon, the lattice will on average increase its energy by the recoil energy and it does so by emitting n phonons, n = 0,1,2,..., where the inclusion of n = 0 is crucial as it implies a zero-phonon, recoilless Mössbauer transition. The larger the phonon energy as compared to the recoil energy, the larger the recoilless fraction f will be, where f is the fraction of n = 0 events out of the total. For a solid with harmonic forces the recoilless fraction is given by where is the mean-square displacement of the iron in the direction of the gamma ray and pm is the wavelength of the 14.4 keV radiation. Since is a monotonically increasing function of temperature, T, the recoilless fraction decreases rapidly with increasing T. The zero-point vibrations of iron proteins are comparable to those of other iron compounds, and recoilless fractions of are typical at 4.2 K. For iron proteins, the linear increase of for 50K < T < 150K is larger, however, and for T > 150K non-vibrational motions contribute to increasing its rise with T further.

Mössbauer spectroscopy

363 ues in Eq. 11 can be replaced by their thermal average where i refers to the (2S + 1) eigenstates of in Eq. 15. For Fe(III) in weak fields the magnetic hyperfine splitting collapses completely in the fast-fluctuation limit since approaches zero, thus the spectra simplify and the whole intensity concentrates in the emerging quadrupole doublet. While Mössbauer spectra can be simulated for any spin fluctuation rate and the temperature dependence has been modeled successfully (Schulz et al., 1988), it is not always practical to reach the simple limiting cases, and the data analysis then remains difficult.

F. Calculation of Mössbauer Spectra

For T > 230K limited diffusion sets in, which results in line broadening, motional decrease of etc. (Keller et al., 1980; Aleksandrov et al., 1987). The thermal redshift or second-order Doppler shift is given by

where is the mean-square velocity of the iron, which stays close to the minimum value given by zero-point vibrations for T < 50K and approaches the classical limit of 3kT/M without reaching it at high temperatures. The thermal redshift adds to the isomer shift and is illustrated in Fig. 3. The last dynamical feature to be discussed is the spin–phonon coupling that causes transitions between the eigenstates of the spin Hamiltonian Eq. 15, and is related to the spin relaxation rate in EPR. In the presence of magnetic hyperfine interactions spin state fluctuations affect the shape of the spectra profoundly. So far it has been tacitly assumed that the spin states are stationary on the Mössbauer time scale, a case that typically applies near 4.2K. In the opposite limit of fast spin state fluctuations, which is approached at higher temperatures, the spin expectation val-

A variety of computer programs are available for the quantitative analysis of Mössbauer spectra, and only the general principles will be discussed here. In the simplest case, the spectrum consists of a number of Lorentzians on a constant background, each characterized by its center, width and height, and a least-squares routine will find an optimum parameter set with standard deviations and correlation coefficients. The results will then have to be interpreted in terms of quadrupole doublets and/or single lines attributable to different spectral components. Alternatively, constraints can be incorporated in the fitting routine, e.g. requiring identical shapes or areas for both lines of a quadrupole doublet. To allow for inhomogeneity in the iron environment a Gaussian distribution of Lorentzians or Voigt line shape can be used with the Gaussian width as a parameter. In an external field each molecule has a different nuclear Hamiltonian, Eq. 12 or 12' and the spectra are calculated by adding contributions from an appropriate sample of molecular orientations, typically more than 100. For paramagnetic centers the spin expectation values or needed in Eq. 12 or 12' will also depend on the molecular orientation, thus both and have to be diagonalized for each orientation. Since and depend on a large number of parameters, a successful analysis is possible only with data of high quality, but the resulting parameter set, in particular Ã, and the relative orientation of these tensors, will characterize the iron environment in great detail.

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G. Experimental Considerations Most Mössbauer measurements are done in transmission, and count rates upward of are obtained with sources of roughly 50 mCi. If N is the total number of counts per data point, the mean square deviation of N equals N, and It therefore takes counts per data point to measure a 1% dip to a standard deviation of 10% and counts to measure a 0.1% dip to the same relative accuracy, etc. According to Section II.A the total area under the absorption spectrum is proportional to the recoilless fraction times the number of atoms per and for a given spectral area the peak absorption is obviously largest if the number of lines and the widths are minimal. Although MS can provide unique information about the environment of the iron in the photosynthetic apparatus, its application has been limited by the need for quantities of per sample. Enrichment in is therefore absolutely mandatory, and as long as the iron cannot be exchanged, growth of the photosynthetic organisms, either bacteria, cyanobacteria or algae, on enriched media has been the only practical approach. As pointed out by Evans et al. (1977), an excess of iron in the growth medium should be avoided. In R. sphaeroides with one iron per RC, samples containing up to of have been obtained, resulting in spectra with a total area of at 4.2K as shown in Fig. 2. Based on figures reproduced here, comparable estimates of in PSII preparations are 5.5 and in Fig. 4b and Fig. 6a,b, respectively. With two irons per PSII, these numbers imply a roughly tenfold lower concentration of RCs than in R. sphaeroides. To achieve equivalent signalto-noise ratios in the two cases, the number of counts accumulated would have to be times larger in the case of PSII. Any increase in the concentration of the iron-bearing proteins as well as any reduction in the number of species present in a sample, e.g. by genetic engineering or chemical means, will greatly facilitate the Mössbauer experiments. The problems are well illustrated by Fig. 4, which compares the spectra of membranes from cyanobacteria in (a) with oxygen-

evolving core complexes in (b). The total areas of the spectra are 1.8 and respectively, indicating an estimated amount of 15 and of The dominant doublet in Fig. 4a arises mainly from iron–sulfur proteins and cytochromes in PSI and the cytochrome complex; it hides the PSII spectrum of Fig. 4b, which actually contains a cytochrome impurity exceeding the amount of cytochrome of PSII (Picorel et al., 1994). PSI contains three 4Fe–4S clusters, and Figs. 10 and 11 indicate that samples of adequate signal-to-noise ratio can be prepared. Temperature is an important variable in any Mössbauer experiment since most samples are measured as frozen solutions, since the recoilless fraction is largest at helium temperatures, and since the spin fluctuation rates are strong functions of T. Accordingly, a variable temperature cryostat is essential. For samples of adequate sig-

Mössbauer spectroscopy nal-to-noise ratio external fields are useful also as illustrated in Figs. 2 and 11. III. Applications This section applies the basic concepts and formalism developed so far to the three types of iron sites found in the membrane-bound photosynthetic apparatus of bacteria, cyanobacteria and algae. Among the latter the experiments done on Chlamydomonas reinhardtii stand out. The three types of iron sites are the iron–quinone complex of the bacterial reaction center (RC) and PSII of the higher organisms, the iron–sulfur clusters of PSI, and the cytochrome of PSII. Here, the goal is to illustrate the arguments leading from experimental data to final conclusions and to point out strengths and weaknesses of the method.

A. Iron–quinone Complex Early Mössbauer experiments on bacterial RCs conclusively showed the existence of a ferroquinone complex which is now known in atomic detail from x-ray diffraction (Deisenhofer et al., 1985; Feher et al., 1989), although the function is still elusive (Debus et al., 1985). Equally significant was the demonstration of an analogous iron site in PSII, which was not only redox-active in contrast to the bacterial one, but was sensitive to the presence of bicarbonate/formate and able to form an Fe(II)NO adduct of spin S = 3/2. Since the iron quinone complex has no precedent, it will be discussed in some detail starting with the bacterial RC of Rhodobacter sphaeroides, Figs. 1–3 (Debrunner et al., 1975; Boso et al., 1981). A first step in the Mössbauer study of any new sample is to check its iron content, its purity, homogeneity and reproducibility. As shown in Fig. 2 (top), the spectrum of native RC consists of a single quadrupole doublet, i.e. the sample appears to be pure. Based on a calibration with a known absorber, the total area of matches within 10% the of estimated from the optical absorption with the assumption that 90% of the iron comes from the enriched iron in the growth medium. The linewidth, is larger than the minimum experimental linewidth, but is smaller than that observed in ferrous heme

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proteins. The homogeneity of the iron site is therefore quite high. The combination of and finally, leaves no doubt that the iron is high spin ferrous. The good match of the isomer shift with the values found in compounds of spin S = 2 suggested mixed N- and O-ligands as borne out by x-ray diffraction. Figure 1 illustrates the typical temperature dependence of this high-spin ferrous complex; the lines (A), (B), and (C) represent three attempts to fit using a crystal field model. The upper inset defines the axial and rhombic crystal field terms Dl and D2 and sketches the effect of the spin–orbit coupling which is given in terms of the standard value and an adjustable covalency factor Curves (A) and (B) further assume a lattice or covalency contribution to the EFG, quantified by whereas curve (C) assumes different delocalization for the spin and the charge densities. Obviously, these four-parameter models fit the data reasonably well, yet the strong covalency of the 3d-electrons that all of them require implies that the crystal field approach is a poor approximation. Figure 2 (bottom) shows the magnetic hyperfine broadening brought about by a strong field and a simulation of the spectrum based on eqs. and 15. At a temperature of 156K the limit of fast spin fluctuation rates applies, and the thermal average, of the spin expectation can therefore be used in Eq. 11. The spectrum is from a series of measurements taken at different temperatures and fields and illustrates the type of information that can be extracted, which is summarized by the parameters given in the caption. The main conclusion from the data is that the iron site definitely has low symmetry. The hyperfine tensor is highly anisotropic, and its average value of is well below the Fermi contact term of indicating substantial orbital and spin dipolar contributions. Moreover, the quadrupole tensor with asymmetry is rotated relative to the zero-field splitting which was assumed to be coaxial with Ã. As mentioned earlier, the Hamiltonian parameters must be thermal averages rather than constants since Fig. 1 clearly indicates that higher orbital

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states are being populated over the temperature range of interest. Figure 3, finally, compares the temperature dependences of the isomer shifts of six RC samples with nominally zero, one or two quinones, prepared with different detergents, with and o-phenanthroline, or with chemically reduced as verified by EPR. By and large is identical for all samples, and the temperature dependence is given by the same thermal redshift, Eq. 18. From this result and from the observation of nearly identical (not shown) it clearly follows that the ligands of the iron are the same in all samples, hence neither quinone nor o-phenanthroline bind to the iron. The only discrepancy in Fig. 3 is the 4.2K data point of the semiquinone sample (6). At higher temperatures this sample has 5–10% larger linewidths than all the others, but at 4.2K the quadrupole lines broaden asymmetrically by roughly a factor 1.7, a clear indication of unresolved magnetic hyperfine interaction. The explanation is obviously the magnetic coupling of the iron spin, S = 2, to the spin S = 1/2 of the semiquinone, a coupling that can be modeled by Eq. 16. The interaction of the two spins manifests itself not only in spontaneous broadening of the Mössbauer lines in zero field, but also in the large, temperature-dependent broadening of the EPR spectrum. The latter has been modeled quantitatively by Butler et al. (1984) using the spin Hamiltonian, Eq. 15 of the iron. Next, we turn to the work of Petrouleas and Diner (1982,1986,1990) on PSII of Chlamydomonas reinhardtii which established the existence of an iron–quinone complex in PSII of green plants. The complex appears to be structurally related to the bacterial one (Michel and Deisenhofer, 1988). Fig. 5 compares the Mössbauer spectra of PSII particles taken at 130K (a) and 4.2K (b,c). Obviously, the absorption is much smaller than in Fig. 2, the spectrum is more complex, and one component, an asymmetric doublet labeled (II) in the top trace, apparently vanishes at 4.2K. The major persistent component (I) has parameters characteristic of high-spin Fe(II), and the comparison in Table 1 suggests its analogy with the iron–quinone complex of the bacterial RC. Component (III) may arise from air-oxidized

Peter G. Debrunner

iron–quinone or some unidentified impurity. The properties of doublet (II), which are listed in Table 3, indicate that it is due to ferricytochrome: Magnetic hyperfine splitting broadens it beyond the detection limit at 4.2K, but partial collapse of the magnetic splitting leaves an asymmetric doublet with at 130K. This assignment is confirmed by optical difference

Mössbauer spectroscopy

measurements between reduced and oxidized samples and by EPR. In two subsequent papers, Petrouleas and Diner showed that the Fe(II) of the iron–quinone complex can be reversibly oxidized and identified the Fe(III)/Fe(II) couple with the high-potential electron acceptor of PSII first described by Ikegami and Katoh (1973). Here, only the Mössbauer data will be discussed while all the supporting evidence will be skipped. Figure 6 shows spectra taken at different redox potentials of membranes from a mutant lacking PSI and the cytochrome complex (Diner and Wollman, 1980). Spectrum (a) of the untreated sample is comparable to that of Fig. 5a with the exception of a narrow quadrupole doublet assigned to ferrocytochrome. The spectrum of Fig. 6b was obtained after oxidation of the sample with ferricyanide to a potential of 450 mV. The high-spin Fe(II) doublet of the iron–quinone complex has disappeared completely, and a broad, ill-defined absorption in the range of is seen, comprising contributions from ferricytochrome, ferro-ferricyanide and possibly from highspin Fe(III) with partially collapsed magnetic hyperfine splitting. On reduction of this sample with ascorbate to a potential of 300 ± 20 mV, Fig. 6c, the high-spin Fe(II) doublet of the iron–quinone complex reappears, but the spectrum differs from the original one because of the peak near due to ferrocyanide. Similar results were obtained by Picorel et al. (1994) with PSII isolated from the cyanobacterium Phormidium laminosum. Figure 7 shows Mössbauer spectra obtained from oxygen-evolving core complexes at 77K (a) as isolated, (b)

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after oxidation with ferricyanide, and (c) after removal of the ferricyanide and reduction by dithionite. Spectra (a) and (c) are indistinguishable and show two quadrupole doublets, one from the high-spin ferrous iron–quinone complex, the other from the cytochromes and where the latter is an impurity. The rather symmetrical spectrum obtained on oxidation is a superposition of the ferricytochrome doublet and a broad highspin Fe(III) line indicative of relatively fast spin fluctuations. Another important finding of Diner and Petrouleas (1987) was the observation that the quadrupole splitting of the iron–quinone complex changes when bicarbonate is replaced by formate and vice versa as illustrated in Fig. 8. Semin et al. (1990) reported analogous results for PSII particles from Synechococcus elongatus. There must be a reversible change in the iron environment, but it is not clear yet whether either one of the compounds binds directly to the iron. Fig. 9, finally, shows the effect of NO binding on the Mössbauer spectrum of the iron–quinone complex (Petrouleas and Diner, 1990; Diner and Petrouleas, 1990). The intensity of the S = 2 doublet decreases on treatment with NO, and the lines at and grow instead. The authors do not provide a quantitative analysis, but it is clear that the isomer shift and the quadrupole splitting of the adduct are substantially smaller than for the S = 2 state. EPR shows that the Fe(II) NO complex has spin S = 3/2, and Mössbauer as well as EPR data are therefore analogous to those obtained for NO complexes of Fe(II) EDTA and dioxygenases (Arciero et al., 1983).

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B. Iron–sulfur Proteins EPR and MS have long been the major spectroscopic probes for the study of iron–sulfur proteins (Cammack et al., 1977), and PSI is no exception. EPR titrations identified three distinct but overlapping signals and that were assigned to iron–sulfur centers in PSI (Malkin and Bearden 1971). Evans et al. (1977, 1979, 1981) did the first Mössbauer studies on oxidized and reduced PSI samples and concluded that all three centers were 4Fe–4S clusters. Since the low-potential center remained controversial, having been assigned to a 2Fe–2S cluster as well (Bertrand et al., 1988), Petrouleas et al. (1989) examined PSI lacking and (Parrett et al., 1989) and confirmed it to be a 4Fe–4S cluster. The iron–sulfur clusters of iron–sulfur proteins are built from the same structural unit, namely a high-spin iron coordinated to four sulfurs, where the sulfurs are either cysteine or bridging

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depending on how many of these units are joined together. The high covalency of the complexes leads to characteristically small isomer shifts and A-tensors that set them apart from other iron environments. As discussed in Section II.D, the magnetic properties of larger clusters are dominated by the strong exchange interactions, Eq. 16, and for clusters with more than two irons by double exchange leading to delocalized valence pairs. All these properties have been studied extensively in small, well defined iron proteins as well as in model complexes, and the Mössbauer studies of PSI have revealed no new features apart from the unusually low redox potential of the center. Accordingly, the discussion that follows will be kept brief. To consider the simplest system first, it begins with the

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mutant containing only and presents the complete PSI later. Figure 10 shows the Mössbauer spectra obtained by Petrouleas et al., (1989) from PSI core protein of Synechococcus 6301, a mutant of this cyanobacterium that lacks the two centers and but still contains The two spectra of the oxidized cluster on the right consist of an asymmetric, broad doublet with at 77K, indicating that the four iron sites have similar, indistinguishable Mössbauer parameters. On cooling to 4.2K the spectrum just moves slightly to more positive velocities as expected from the second-order Doppler shift, Eq. 18, but it does not broaden magnetically. As shown in Table 2, the Mössbauer parameters match those of other clusters of spin S = 0. The oxidized protein is known to be EPR inactive, while the reduced protein with S = 1/2 shows the EPR signal that led to its discovery.

Peter G. Debrunner

The Mössbauer spectrum of the reduced protein on the left is marked ‘D’ after correction for a 15% admixture of the oxidized spectrum. It is still a broad doublet at 77K although with different parameters, At 4.2K, on the other hand, the spectrum broadens, extending from to and showing the features typical of magnetic hyperfine splitting in as will be discussed below. Figure 11, finally, shows the 4.2K-Mössbauer spectra of PSI from the cyanobacterium Chlorogloea fritschii (Evans et al., 1981). These samples contain all three iron–sulfur centers and were partially reduced to show the EPR spectrum of only in (a), of and in (b), and of

Mössbauer spectroscopy

and in (c). The resolution of the spectra into oxidized and reduced material is shown, based on the spin Hamiltonian parameters deduced by Middleton et al. (1978) for the 4Feferredoxin from Bacillus stearothermophilus. These parameters clearly fit all three spectra quite well, and there is every reason to believe that the spectral contributions of and are indistinguishable, i.e. that all three are generic 4Fe–4S centers. It should be recalled that the nominal Fe(II)Fe(III) pair in has spin S' = 9/2 and delocalized valence while the Fe(II)Fe(II) pair has spin S'' = 4 to give a net spin of S = 1/2. The two pairs have different hyperfine tensors Ã' and Ã'' and therefore contribute distinct Mössbauer spectra as seen in Fig. 11.

C. Cytochromes Cytochrome is an intrinsic part of PSII and therefore appears in the spectra of Figs. 4–9, generally as an undesirable component that overlaps the low-energy line of the iron-quinone doublet. The few Mössbauer parameters reported are summarized in Table 3. As a low-spin heme protein, reduced cytochrome is diamagnetic and shows a sharp quadrupole doublet in the

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Mössbauer spectra at all temperatures. Oxidized on the other hand, has spin S = 1/2, is EPRactive and is expected to have a Mössbauer spectrum with wide magnetic hyperfine splitting at 4.2K, a spectrum that has collapsed near 80K to a quadrupole doublet with broad, asymmetric lines. Although cytochrome is of considerable interest, especially since it can exist in a highand a low-potential form associated, presumably, with a change in orientation of the axial histidine ligands, any meaningful Mössbauer studies would have to be done on isolated cytochrome rather than on PSII preparations to achieve adequate signal-to-noise ratio. Since most of the work has been done by optical techniques and EPR, which are more sensitive (e.g. Babcock et al., 1985), the interested reader is referred to a Mössbauer study of a cytochrome b model (Walker et al., 1986) that deals with the question of the axial ligand orientation. References Abragam A and Bleaney B (1970) Electron Paramagnetic Resonance of Transition Ions. Oxford University Press. Aleksandrov AY, Novakova AA and Semin BK (1987) Mössbauer spectroscopy study of the conformational dynamics of native membrane proteins. Phys Lett 123: 151– 154.

372 Arciero DM, Lipscomb JD, Huynh BH, Kent T and Münck E (1983) EPR and Mössbauer studies of protocatechuate 4,5–dioxygenase. Characterization of a new environment. J Biol Chem 258: 14981–14991. Babcock GT, Widger WR, Cramer WA, Oertling WA and Metz JG (1985) Axial ligands of chloroplast cytochrome Identification and requirement of a heme-cross-linked polypeptide structure. Biochemistry 24: 3638–3645. Bertrand P, Guigliarelli B, Gayda J-P, Sétif P and Mathis P (1988) An interpretation of the peculiar magnetic properties of center X in Photosystem I in terms of a 2Fe–2S cluster. Biochim Biophys Acta 933: 393–397. Boso B, Debrunner P, Okamura MY, and Feher G (1981) Mössbauer spectroscopy studies of photosynthetic reaction centers from Rhodopseudomonas sphaeroides R-26. Biochim Biophys Acta 638: 173–177. Butler WF, Calvo R, Fredkin DR, Isaacson RA, Okamura MY and Feher G (1984) The electronic structure of in reaction centers from Rhodopseudomonas sphaeroides. III. EPR measurements of the reduced acceptor complex. Biophys J 45: 947–973. Cammack R, Dickson DPE and Johnson CE (1977) Evidence from Mössbauer spectroscopy and magnetic resonance on the active centers of the iron-sulfur proteins. In: Lovenberg W (ed) Iron–Sulfur Proteins Vol. III, pp. 283–330. Academic Press, New York. Cranshaw TE, Dale BW, Longworth GO and Johnson CE (1985) Mössbauer spectroscopy and its applications. Cambridge University Press, Cambridge. Debrunner PG (1989) Mössbauer Spectroscopy of Iron Porphyrins. In: Lever ABP and Gray HB (eds.) Physical Bioinorganic Chemistry Series. Iron Porphyrins Vol 3, pp. 137–234. VCH Publishers. Debrunner PG (1993) Mössbauer spectroscopy of iron proteins. In: Berliner LJ and Reuben J (eds) Biological Magnetic Resonance Vol. 13, pp. 59–101. Plenum Press, New York and London. Debrunner PG, Schulz CE, Feher G and Okamura MY (1975) Mössbauer study of reaction centers from R. sphaeroides . Biophys J 15: 226a. Debus RJ, Okamura MY, and Feher G (1985) Reconstitution of iron-depleted reaction centers from Rhodopseudomonas sphaeroides R-26 with Fe, Mn, Cu and Zn. Biophys J 47: 3a. Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1985) Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3Å resolution. Nature (London) 318: 618–624. Dickson DPE and Berry FJ (1986) Mössbauer spectroscopy. Cambridge University Press, Cambridge. Diner BA and Petrouleas V (1987) the non-heme iron of the Photosystem II iron–quinone complex. A spectroscopic probe of quinone and inhibitor binding to the reaction center. Biochim Biophys Acta 895: 107–125. Diner BA and Petrouleas V (1990) Formation by NO of nitrosyl adducts of redox components of the Photosystem II reaction center. II. Evidence that binds to the acceptor-side non-heme iron. Biochim Biophys Acta 1015: 141–149. Diner BA and Wollman F-A (1980) Isolation of highly active

Peter G. Debrunner Photosystem II particles from a mutant of Chlamydomonas reinhardtii. Eur J Biochem 110: 521–526. Evans EH, Carr NA, Rush JD and Johnson CE (1977) Identification of a non-magnetic iron centre and an iron-storage or transport material in blue-green algal membranes by Mössbauer spectroscopy. Biochem J 166: 547–551. Evans EH, Rush JD Johnson CE and Evans MCW (1979) Mössbauer spectra of Photosystem I reaction centres from the blue-green alga Chlorogloea fritschii. Biochem J 182: 861–865. Evans EH, Dickson PE, Johnson CE, Rush JD and Evans MCW (1981) Mössbauer spectroscopic studies of the nature of centre X of Photosystem I reaction centres from the cyanobacterium Chlorogloea fritschii. Eur J Biochem 118: 81–84. Feher G, Allen JP, Okamura MY and Rees DC (1989) Structure and function of bacterial photosynthetic reaction centres. Nature (London) 339: 111–116. Gibb TC (1976) Principles of Mössbauer spectroscopy. Chapman and Hall, London. Gonser U (1975) Mössbauer Spectroscopy. Springer-Verlag, New York. Greenwood NN and Gibb TC (1971) Mössbauer Spectroscopy. Chapman and Hall, London. Griffith JS (1957) Theory of electron resonance in ferrihaemoglobin azide. Nature (London) 180: 30–31. Gütlich P, Link R and Trautwein A (1978) Mössbauer Spectroscopy and Transition Metal Chemistry. Springer-Verlag, New York. Huynh BH and Kent TA (1984) Mössbauer studies of iron proteins. In: Eichhorn GL and Marzili LG (eds.) Advances in Inorganic Biochemistry, Vol. 6, pp. 163–223). Elsevier, Amsterdam. Ikegami I and Katoh S (1973) Studies on chlorophyll fluorescence in chloroplasts II. Effect of ferricyanide on the induction of fluorescence in the presence of 3–(3,4–dichlorophenyl)-1,1–dimethylurea. Plant Cell Physiol 14: 829– 836. Ingalls R (1964) Electric-field gradient tensor in ferrous compounds. Phys Rev 133: A781–A795. Keller H and Debrunner PG (1980) Evidence for coformational and diffusional mean square displacements in frozen aqueous solution of oxymyoglobin. Phys Rev Lett 45: 68– 71. Malkin R and Bearden AJ (1971) Primary reactions of photosynthesis. Photoreduction of a bound chloroplast ferredoxin at low temperature as detected by EPR spectroscopy. Proc Natl Acad Sci USA 68: 16–19. Michel H and Deisenhofer J (1988) Relevance of the photosynthetic reaction center from the purple bacteria to the structure of Photosystem II. Biochemistry 27: 1–7. Middleton P, Dickson DPE, Johnson CE and Rush JD (1978) Interpretation of the Mössbauer spectra of the four-iron ferredoxin from Bacillus stearothermophilus. Eur J Biochem 88: 135–141. Münck E, Papaefthymiou V, Surerus KK and Girerd JJ (1988) Double exchange in reduced clusters and novel clusters with In Metal Clusters in Proteins, Que L (ed.), ACS Symposium Series, Vol. 372, pp. 302–325, Am Chem Soc, Washington, D.C.

Mössbauer spectroscopy Parrett KG, Mehari T, Warren PG and Golbeck JH (1989) Purification and properties of the intact P-700 and taining Photosystem I core protein. Biochim Biophys Acta 973: 324–332. Petrouleas V and Diner BA (1982) Investigation of the iron components in photosystem II by Mössbauer spectroscopy. FEBS Lett 147: 111–114. Petrouleas V and Diner BA (1986) Identification of a high-potential electron acceptor of Photosystem II, with the iron of the quinone–acceptor complex. Biochim Biophys Acta 849: 264–275. Petrouleas V and Diner BA (1990) Formation by NO of nitrosyl adducts of redox components of the Photosystem II reaction center. I NO binds to the acceptor-side nonheme iron. Biochim Biophys Acta 1015: 131–140. Petrouleas V, Brand JJ, Parrett KG, and Golbeck JH (1989) A Mössbauer analysis of the low-potential iron–sulfur center in Photosystem I: Spectroscopic evidence that is a [4Fe–4S] cluster. Biochemistry 28: 8980–8983.

373 Picorel R, Williamson DL, Yruela I and Seibert M (1994) The state of iron in the oxygen-evolving core complex of the cyanobacterium Phormidium laminosum: Mössbauer spectroscopy. Biochim Biophys Acta 1184: 171–177. Schulz CE, Nyman P and Debrunner PG (1987) Spin fluctuations of paramagnetic iron centers in proteins and model complexes: Mössbauer and EPR results. J Chem Phys 87: 5077–5091. Semin BK, Loviagina ER, Aleksandrov AY, Kaurov YN and Novakova AA (1990) Effect of formate on Mössbauer parameters of the non-heme iron of PSII particles of cyanobacteria. FEBS Lett 270: 184–186. Taylor CPS (1977) The EPR of low spin heme complexes. Relation of the hole model to the directional properties of the g-tensor, and a new method for calculating the ligand field parameters. Biochim Biophys Acta 491: 137–149. Walker FA, Huynh BH, Scheidt WR and Osvath SR (1986) Models of the cytochrome b. Effect of axial ligand plane orientation on the EPR and Mössbauer spectra of low-spin ferrihemes. J Am Chem Soc 108: 5288–5297.

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Chapter 23 Characterization of Photosynthetic Supramolecular Assemblies Using Small Angle Neutron Scattering† David 2M. Tiede1,* and P. Thiyagarajan2

1

Chemistry Division D-200 and Intense Pulsed Neutron Source, Argonne National Laboratory, Argonne, IL 60439, USA

Summary I. Introduction II. Small Angle Neutron Scattering A. Analysis of Scattering in the Very Small q Domain B. Scattering in the Intermediate q Domain III. SANS Studies of Photosynthetic Complexes A. Crystallization of Photosynthetic Proteins 1. Detergent Micelle Structures in Crystallization Conditions 2. Reaction Center Aggregation States B. Structural Characterization of Photosynthetic Supramolecular Assemblies C. Internal Structure in Supramolecular Assemblies D. New Results IV. Concluding Remarks Acknowledgements References

375 376 377 378 379 379 379 380 382 385 386 388 388 388 389

Summary

Small angle neutron scattering (SANS) offers opportunities for resolution of structure in molecular assemblies that can not be readily accessed by crystallography, such as inherently disordered assemblies, like micelles or vesicles, or multiple protein component complexes that can not be easily crystallized, like RC-cyt complexes or RC-antenna complexes. Scattering measurements on solution samples allow direct correlations to be made between structural features of Supramolecular assemblies and their spectroscopically determined function. Parameters which can be resolved by SANS include the size, shape, molecular weight, volume of macromolecules, internal packing for multiple component protein complexes. This information can be used to discriminate between possible molecular models for supramolecular structures. This chapter surveys possibilities for the application of this technique for the characterization of supramolecular assemblies in photosynthesis. SANS has been used to characterize the effect of ionic strength and detergents on reaction center aggregation. These measurements are being used to examine the pathways for reaction center crystallization. Applications are also presented for structural characterization of light-harvesting antenna complexes. Abbreviations: CMC – Critical micelle concentration; cyt – Cytochrome c; HT – Heptane-1,2,3–triol; LDAO – Lauryldimethylamine-N-oxide; LH – Light-harvesting complex; PEG – Polyethyleneglycol; RC – Reaction center; SANS – Small angle neutron scattering †

The US government’s right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged. *Correspondence: Fax: 1-708-2529289, E-mail: [email protected]

375 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 375–390. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

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I. Introduction Small angle neutron scattering (SANS) offers opportunities for the determination of solution structures of macromolecules with sizes in the range of 10Å to 500 Å which complements structural determination by other techniques. A distinguishing feature of neutron scattering compared to x-ray, electron and light scattering is that neutrons interact with atomic nuclei instead of electrons. This property allows neutron scattering to be sensitive to both light and heavy atoms in a structure, including protons which are typically not observed in x-ray and electron scattering. Table 1 shows coherent neutron scattering lengths, and incoherent scattering cross sections, for selected elements compared to their x-ray scattering amplitudes in the forward direction, f(0). The magnitudes of the coherent neutron scattering lengths for H and D are seen to be nearly comparable to those for other atoms found in biological molecules, while large differences are seen for x-ray scattering amplitudes which increase with increasing atomic number. This implies that H and D will make significant contributions to neutron scattering for biological molecules, while they will make relatively weak contributions for x-ray scattering signals. Another significant feature of neutron scattering that makes it suitable for probing biological structures is the relatively low energy of the neutron beam. At wavelengths compatible for resolving structure on the 10 Å to 500 Å scale, the

David M. Tiede and P. Thiyagarajan millivolt energy of the neutron beam is a million fold less than the kilovolt energy used in comparable x-ray scattering experiments, resulting in several orders of magnitude reduction in radiation damage. Furthermore, the penetration depth of the neutron beam allows neutron scattering analysis to be made on aqueous samples with 1 to 5 millimeter path lengths. This penetration depth allows SANS measurements to be made with samples similar to those encountered for the characterization of photosynthetic proteins by optical spectroscopies. This concurrence in sample constraints will permit the same sample to be analyzed by optical spectroscopy and SANS, enabling direct comparisons between photosynthetic function and structure. SANS is a widely applied scattering technique, and several reviews cover the application to biological systems (Jacrot, 1976; Stuhrmann and Miller, 1978; Chen, 1986; Feigin and Svergun, 1987). This technique allows extraction of form factors, which are descriptions of macromolecular size and shape, and particle–particle structure factors in solutions. This technique has also been demonstrated to provide structural information on supramolecular assemblies in their native state in aqueous solutions (Ramakrishnan et al., 1984). While form factors are a relatively low resolution image of a macromolecular assembly, this information can precisely define the distribution of aggregation states for proteins in solution, characterize protein packing in aggregates, and charac-

Small angle neutron scattering terize the dimensions of multiple component supramolecular assemblies such as protein-detergent complexes, or multiple component protein complexes. Unique opportunities also exist for the resolution of structure factors for selected components within complex mixtures by combining SANS measurements with isotopic substitution. This chapter will examine selected applications of SANS for resolution of molecular structures in photosynthesis.

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characterization of the intraparticle structure factor. The intraparticle structure factor describes the scattering due to the atom position vectors within a particle: F(q) is the particle form factor, defined in terms of the atomic scattering lengths, and the atomic position vectors, with respect to the particle center of mass:

II. Small Angle Neutron Scattering Neutron scattering for proteins or other particles in solution is determined by the atomic composition and time-averaged position vectors of all nuclei in the sample. Neutron scattering is measured as a function of the scattering vector where is the neutron wavelength and is the angle of scattering. Persistent distance correlations between atoms in the sample produce variations in the scattered intensity due to interference effects of the scattered neutrons. Distance correlations between atoms will arise from the fixed atom positions within a particle, as well as due to the distribution of particles within the sample volume. As a result, neutron scattering, measured as the differential scattering cross section, can be written as the product of the number density of particles, n, the intraparticle structure factor, P(q) and the interparticle structure factor, S(q): S(q) makes a significant contribution to scattering under conditions in which the location of one particle affects the distribution of other particles around it. This correlation occurs in concentrated samples and in solutions of particles with surface charge (Chen, 1986; Chen et al., 1988). This effect can be exploited to investigate the nature of particle–particle interactions using SANS. In dilute solutions, or solutions of non-interacting particles, particle locations are not correlated, and S(q) approaches 1. Under these conditions, neutron scattering is determined exclusively by the number density of particles and their structure. Analysis of neutron scattering under these conditions yields the most accurate

The brackets in Eq. (2) indicate the average over all particle orientations. The scattered intensity can be written: where and are the position vectors of the i th and jth atoms within the particle, and and are the atomic scattering lengths. In general the scattering length of an atom, depends on the isotope and its spin states. The examples of scattering lengths listed in Table 1 are averages of scattering lengths taken over all spin states for specific isotopes. The distribution of spin states for a stable nucleus gives rise to two components to neutron scattering of a given atom, which are the coherent and incoherent scattering cross sections. Both contribute to scattering signals. For example H has a large incoherent cross section while D has very little. Hydrogen rich biological molecules produce significant amounts of incoherent scattering; however, if they are deuterated, the incoherent scattering cross sections can be substantially reduced. The differential scattering cross section of a particle in Eq. (4) thus has two terms, where

The incoherent scattering component does not depend on the position vectors of the atoms. Experimentally it is observed as a flat background signal uncorrelated to structure. The coherent sig-

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nal, on the other hand, depends on the position correlations of the atoms in the particle and it is the basis for obtaining structural information from SANS. Equation (4) describes neutron scattering due to intraparticle atom distance correlations in vacuum, but does not take into account the effect of placing the particle in a solvent. In the small and intermediate angle scattering domains, atom– atom correlations are measured over distances that are large compared to individual atom bond lengths. In these domains, the solvent acts as a continuum with an average scattering length density, defined as the sum of the scattering lengths for solvent atoms contained within a unit volume. Similarly, the particle can be considered as composed of volume elements whose scattering length densities are determined by the atomic composition within each unit volume: This allows the scattering due to the atom–atom correlations with the particle to be expressed in terms of scattering length density of individual volume elements: The scattering from the particle is detected with respect to the scattering length density of the solvent:

David M. Tiede and P. Thiyagarajan ing sections, analysis of these signals permits determination of size, shape and structural organization of the molecular assemblies. Table 2 lists mean scattering length densities for molecules relevant to the solubilization and crystallization of isolated photosynthetic proteins. These scattering length densities are seen to fall between the scattering length densities of and This allows scattering from selected components within these mixtures to be minimized by adjusting the aqueous ratio so that the mean scattering length density of the solvent matches that of the selected component. Table 2 also shows the effectiveness of deuteration for the enhancement of scattering length densities of biological molecules. For example the average scattering length density of proteins typically increases from to upon complete deuteration. The scattering characteristics of a subset of components within a macromolecular assembly can be resolved if they can be selectively deuterated, and by recording the difference in scattering between unlabelled and labelled material. Elegant demonstrations of this technique include the resolution of phosphatidylcholine and bilesalt organization in rod-like mixed micelles (Hjelm et al., 1992), the resolution of subunit organizations in the 30S (Capel et al., 1987), and 50S (Nowotny et al., 1989) ribosomes and a RNA polymerase (Lederer et al., 1991).

A. Analysis of Scattering in the Very Small q Domain Hence, the difference in the scattering length densities of the particle and the solvent, termed contrast, dictates the intensity of the SANS signal. Equation 10 indicates that neutron scattering contrast can be varied either by changing the isotopic composition of the particle or of the solvent. This allows scattering due to selected volume elements to be minimized by matching their scattering length densities to that of the solvent, while offering a mechanism for maximizing the contribution of other volume elements by increasing their contrast. Thus, isotopic substitution, in combination with contrast matching, is a powerful approach for the identification of scattering for selected components within complex macromolecular assemblies, while minimizing chemical perturbation of the system. As outlined in the follow-

At sufficiently low q, Eq. 10 can be shown to reduce to a particularly simple form, termed the Guinier equation (Guinier and Fournet, 1955): The Guinier approximation is valid for the scattering region where the condition is met. From a plot of ln[I(q)] vs. the size parameter, and the forward scattering cross section, I(0), can be obtained. is the radius of gyration, which is defined as the root mean squared distance of all of the atoms to the centroid of the scattering length distribution. While does not directly resolve the macromolecular shape, the determination of by Guinier analysis is a sensitive index for monitoring relative macromolecular

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Small angle neutron scattering

size and aggregation state. I(0) is the amplitude of the scattering measured at q = 0:

where n is the number of macromolecules per unit volume, and, and V are the average scattering length density and volume of the macromolecule respectively. If SANS data are obtained on an absolute scale, then I(0) can be used for obtaining the molecular weight and volume of the particles, provided the concentration of the particle and the scattering length densities of the solvent and the particles are known.

B. Scattering in the Intermediate q Domain Since the determination of by Guinier analysis does not give information on the particle shape, this information has to be extracted by fitting I(q) data with model form factors. One procedure is to fit experimental scattering data with I(q) profiles calculated using atomic coordinates based upon crystal structures (Feigin and Svergun, 1987; Glatter, 1991). This approach has been used extensively with x-ray scattering. For example, this procedure has been used to determine the extent to which crystal structures of proteins are compatible with their structure in solution (Fedorov and Denesyuk, 1978; Heidorn and Trewhella, 1988; Hubbard et al., 1988), and for the determination of the structure of protein aggregates in solution (Grossmann et al., 1993). For molecular assemblies for which there are no molecular models, such as with proteins or protein complexes that have not been crystallized, or with inherently disordered structures

like micelles or membrane structures, a second approach has been to fit experimental data with form factors calculated from geometric shapes. Methods have been developed which range from fitting with simple geometric shapes of constant scattering length density, to fitting with multipole expansions of a set of spherically harmonic shape functions (Guinier and Fournet, 1955; Stuhrmann and Miller, 1978; Feigin and Svergun, 1987; Glatter, 1991; Svergun, 1991). These procedures provide tools for analysis of dimensions, shapes, and internal structures of supramolecular assemblies based on SANS measurements. However, without the input of other structural information, SANS cannot generally determine a supramolecular form factor unambiguously. Instead, a set of distinct form factors, representing different supramolecular shapes and dimensions, may typically be found to reasonably fit experimental data recorded over a restricted q-range. Often this information can be combined with other physical or chemical data to resolve the most likely molecular structure. The following sections provide examples that illustrate how the SANS technique can contribute towards an understanding of the structural basis for function in photosynthetic supramolecular assemblies.

III. SANS Studies of Photosynthetic Complexes

A. Crystallization of Photosynthetic Proteins The crystallization of photosynthetic proteins is of crucial importance for investigation of photo-

380 synthetic mechanisms. However, there has only been limited success in the production of high quality crystals of photosynthetic membrane proteins. The reasons for success or failure in crystallization are not known, nor are the mechanisms for crystallization. Bacterial photosynthetic reaction centers provide a useful model for examining mechanisms for membrane protein crystallization. Reaction centers from two different species, Rhodopseudomonas viridis and Rhodobacter sphaeroides, have been successfully crystallized from detergent mixtures, and their molecular structures have been determined by x-ray diffraction (Miki et al., 1986; Allen et al., 1987; Yeates et al., 1987; Deisenhofer and Michel, 1989; Chang et al., 1991). So far, only two detergents have been found to yield crystals suitable for high resolution structural analysis. LDAO has been used successfully to produce high quality crystals of reaction centers, but only in conjunction with the addition of amphiphiles such as HT (Michel, 1982; Allen and Feher, 1990; Buchanan et al., 1993). Alternatively, OG has been used successfully in the absence of additional amphiphiles (Allen and Feher, 1984; Chang et al., 1985; Ducruix and Reiss-Husson, 1987). Crystallization in the presence of LDAO was accomplished with a variety of precipitants, including ammonium sulfate (Michel, 1982), potassium phosphate (Buchanan et al., 1993), and polyethylene glycol, PEG/sodium chloride mixtures (Allen and Feher, 1984; Allen and Feher, 1990). In contrast, successful crystallization in the presence of OG has only been reported using PEG/sodium chloride mixtures (Allen and Feher, 1984; Chang et al., 1985; Ducruix and Reiss-Husson, 1987; Franck et al., 1987). These crystallization studies suggest that LDAO, unlike OG, requires the addition of an amphiphile to permit crystallization, while LDAO is less sensitive to the chemical nature of the precipitant than is OG. The unique suitability of PEG/sodium chloride as a precipitant in the presence of OG is also suggested by a comparison of the crystallization of 21 water-soluble proteins in the presence of OG (McPherson et al., 1986). OG was found to be generally beneficial for crystallization when PEG/sodium chloride mixtures were used as the pre-

David M. Tiede and P. Thiyagarajan cipitant, but not with ammonium sulfate as a precipitant (McPherson et al., 1986). These crystallization studies indicate that the conditions required for reaction center crystallization are significantly regulated by detergent properties. Causes for the different requirements for crystallization in the presence of OG and LDAO have been suggested from SANS (Thiyagarajan and Tiede, 1994). These studies illustrate some of the capabilities of the SANS technique, and they are summarized below. 1. Detergent Micelle Structures in Crystallization Conditions Detergent micelle size, number density, and nature of inter-micelle interaction are all likely to be critical in determining micelle compatibility for crystallization. These properties can be directly accessed by SANS. SANS studies have identified clear differences in micelle structure and micelle– micelle interactions for the detergents OG and LDAO under conditions used for crystallization. For example, Fig. 1a and 1b show Guinier plots for 1% solutions of LDAO and OG respectively, as a function of NaCl concentration. For LDAO in the absence of NaCl, scattering intensity is seen to fall-off too quickly at low q. This is a clear indication of repulsive inter-micelle interactions (Chen, 1986; Timmins et al., 1991; Thiyagarajan and Tiede, 1994). Much stronger deviations of this type are seen for anionic detergents (Chen, 1986). For LDAO, this presumably reflects electrostatic interactions between micelles arising from the zwitterionic character of the molecule. Fig. 1a also shows that upon addition of 1M NaCl, the deviation from linearity is nearly completely removed, presumably due to dielectric screening. This data shows that in the absence of NaCl, LDAO micelles will experience appreciable electrostatic repulsion. This behavior can be contrasted with the nonionic OG micelle. In the absence of salt, the micelles were found to be non-interacting, as reflected by the linear plot throughout the range. In the case of OG solutions, Fig. 1 b, the addition of 1M NaCl caused a vertical shift of the plot, along with an upward deviation of the Guinier plot at low The upward deviation is a clear indication of a salt-induced aggregation of the

Small angle neutron scattering

OG micelle. This effect is the opposite of that seen with LDAO. The vertical shift is of interest since it reflects an increase in micelle number density. This data shows that the CMC of OG is much more strongly influenced by ionic strength than that of LDAO. The relative insensitivity of the observed slopes to ionic strength demonstrates that LDAO and OG micelle sizes are not strongly affected by ionic strength. However, these SANS data have shown that these detergents differ significantly in the nature of inter-micelle interaction and response to salt additions. Fitting of SANS profiles to model form factors has also resolved differences in the size and shape of LDAO and OG micelles. The LDAO micelle was found to be larger, and best fit with an ellipsoidal shape, while a spherical shape was found for the OG micelle (Thiyagarajan and Tiede, 1994). Using these procedures, a comparative study of OG and LDAO micelles under conditions used for reaction center crystallization has shown that the successful crystallization methods can be rationalized in terms of an optimization of micelle size, number density, and suppression of intermicelle interactions (Thiyagarajan and Tiede, 1994). LDAO and OG micelle characteristics in different solution conditions are schematically summarized in Figs. 2 and 3 respectively. These studies showed that the requirement for

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HT for crystallization with LDAO as the solubilizing detergent can be understood from the beneficial effects that this reagent had on LDAO micelle size and inter-micelle interactions. As indicated in the top panel in Fig. 2, the LDAO micelle in the presence of NaCl and PEG is noninteracting, but retains the relatively large ellipsoidal shape that presumably interferes with crystallization. The addition of HT converts these micelles into smaller, spherical mixed-micelles. The bottom panel illustrates the finding that LDAO micelles are strongly associating in the presence of and also retain the ellipsoidal shape. Under these conditions, the addition of HT was found to have the remarkable effect of dispersing the LDAO aggregates into smaller, non-interacting, spherical mixed-micelles. The use of HT in crystallization mixtures with either PEG/NaCl mixtures or as protein precipitants result in the formation of HTLDAO mixed-micelles having similar characteristics. This property is consistent with the observed flexibility in the choice of precipitant in crystallization schemes employing HT-LDAO micelles. In the case of OG, both NaCl and additions were found to cause aggregation of micelles, as indicated in the lower panel in Fig. 3. Uniquely, the addition of PEG to OG in NaCl solutions dissociated these aggregates, resulting

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in the formation of relatively small, spherical, non-interacting micelles that are compatible with crystallization. The effect of PEG appeared to be due to a direct molecular interaction between PEG and OG, as the presence of PEG was found to raise the CMC of OG. These SANS studies have shown that LDAO and OG micelles are fundamentally different in terms of size, shape, and nature of inter-micelle interactions in non-crystallizing conditions. However, these differences were found to be minimized under conditions used for protein crystallization. Under crystallization conditions both LDAO-HT mixed micelles and OG micelles were found to be spherical, possibly reflecting a flexible radius of curvature; they were small relative to the size of the protein (micelle radius 17 Å–23

David M. Tiede and P. Thiyagarajan

Å, reaction center dimensions 74 Å × 70 Å × 40 Å), and non-interacting. These results suggest that these shared micelle characteristics are necessary features required to permit successful protein crystallization. Interestingly, these studies also showed that even smaller spherical micelles could be produced by the formation HT-OG mixed micelles in PEG/NaCl mixtures, as indicated in the far-right panel in Fig. 3. These results suggest that conditions may be searched to further minimize possible micelle–micelle contacts in the crystalline lattice. 2. Reaction Center Aggregation States The physical characteristics of isolated detergent micelles can be expected to influence the solubility and aggregation behavior of corresponding protein-detergent complexes. We have used SANS to study the aggregation states and interparticle behavior for the RC solubilized by OG and LDAO as a function of NaCl. Striking dependencies of RC aggregation state on detergent and ionic strength were found. The aggregation behavior of the isolated RC can be understood from

Small angle neutron scattering

the ionic strength dependencies for inter-micelle interactions for these detergents. This work demonstrates the importance of the detergent micelle characteristics in determining the solubility of the corresponding detergent-protein complex. As an example, Fig. 4 shows neutron scattering intensity, I(q), as a function of q for two fully deuterated RC samples in 0.03% LDAO. The samples are matched in RC and detergent concentrations, but differ by the addition or absence of 2 M NaCl. The ratio was adjusted to 5% to contrast match the detergent. Under these conditions, the scattering can unambiguously be assigned as solely due to the RC. The figure shows that the scattering intensity is markedly enhanced for LDAO solubilized RCs in the absence of NaCl compared to that in the presence of 2 M NaCl. The solid lines are fits using the procedure described below. Qualitatively, the marked difference in scattering intensity between the two samples identifies a higher aggregation state for the reaction center in the low-ionicstrength solution. Calculations based upon the reaction center crystal structure showed that the scattering profile for reaction centers in 2 M NaCl, 0.03% LDAO

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can be fit by a monomeric state of the reaction center. This sample also allowed a test of the fitting of SANS data with low-resolution geometrical form factors. Fig. 5a shows a fit to the scattering profile in 2 M NaCl using a cylindrical form factor that was determined from a maximum entropy search method (Hjelm et al., 1990; Morrison et al., 1992). The algorithm fits the scattering data by searching a predefined dimension space using a distribution of particles of all possible sizes. This method has proven to be particularly effective in fitting polydisperse systems. The output shows that the reaction center sample at high ionic strength in LDAO can be fit by a monodispersed distribution of cylindrical particles centered about a diameter of 68 Å and length of 60 Å. These dimensions correlate nicely with the average monomeric dimensions of 70 Å × 74 Å × 40 Å determined from the crystal structure. In contrast to a monomeric state of reaction centers in 2 M NaCl, 0.03% LDAO, fits to the SANS profile at low ionic strength requires a distribution of particles in two size ranges. A minor component is seen with dimensions consistent with monodispersed RCs. The major portion of the scattering is fit to a distribution of RC aggregates having a length of more than 500 Å, which falls outside the size-domain for the experimental q-range. The increase in the size of the aggregate along a single dimension suggests a linear aggregate. Similar measurements were also done with RCs in solutions of OG and 17% This ratio removes the scattering from OG micelles. Significantly, the aggregation state of the RC was also found to be ionic strength dependent with this detergent, but the ionic-strength effect was opposite to that measured in LDAO. In OG, RCs were found to be predominately monomeric at low ionic strength, but a linear aggregate was found as the predominant form at high ionic strength. By comparison to the ionic strength dependencies for LDAO and OG micelles described above, the SANS results for RCs demonstrate that a monodispersed state for the RC is only found under conditions in which detergent micelle–micelle interactions are removed. This result establishes the importance of micelle characteristics in

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determining the solution behavior of the solubilized protein-detergent complex. This approach can be extended to examine the RC aggregation states in crystallization mixtures as the sample proceeds through the pre-saturated, metastable and labile saturated states. This investigation of structural intermediates in crystallization mixtures can lead to an identification of crystallization mechanism. We have begun such a characterization of the OG/PEG/NaCl crystallization method. As a first step, we have characterized the effect of the addition of PEG4000 at concentrations below the precipitation threshold for RCs solubilized by OG. Table 2 shows that the scattering length densities for OG and PEG are nearly equivalent, and that both are far from that of a fully deuterated protein. SANS data for the deuterated RCs were collected in 17% which provided a contrast match for OG and PEG4000. Residual scattering due to PEG4000 could be eliminated by subtraction of scattering profiles collected for appropriate reference samples in the absence of RCs. This method of data acquisition allowed SANS profiles for RCs to be exclusively detected without interference from either OG or PEG4000. Fig. 6a shows RC SANS profiles in the presence and absence of 10% PEG4000. In the absence of PEG4000 the scattering profile follows that ex-

David M. Tiede and P. Thiyagarajan

pected for monodispersed RCs. The scattering profile in the presence of 10% PEG4000 shows additional scattering at low q, corresponding to RC–RC correlations in aggregated states. A fit to the scattering profile with PEG4000 using the maximum entropy method is shown in Fig. 6b. The fit shows that the RCs are distributed between the monomeric state and a series of linear aggregated states. The fact that aggregation of OG micelles did not occur in the presence of PEG4000 shows that this effect is a property of the RC protein and not the detergent annulus. RC aggregation is observed with PEG4000 concentrations far from those required for RC precipitation and crystallization. It can be expected that as crystallization mixtures progressively decrease the solubility of the RC either through increasing the ionic strength with fixed PEG concentration, or by simultaneously increasing PEG and salt concentrations, the observed equilibrium between monomeric and aggregated RC states will be shifted towards the aggregate. The existence of this equilibrium has direct implications for the mechanism of RC crystallization. A prevalent view of protein crystallization assumes that protein monomers serve as intermediates in crystallization (Feher and Kam, 1985; Durbin and Feher, 1986; Durbin and Feher, 1991). This view assumes that crystal growth oc-

Small angle neutron scattering

curs by the addition of protein monomers to the crystalline array, and that nucleation arises from an equilibrium between protein monomers and a crystalline aggregate. In this mechanism, the observed RC aggregation will act as a competing pathway, and optimization of crystallization must minimize the participation of non-crystalline aggregation paths. However, an alternative mechanism is possible in which the observed aggregates themselves function as intermediates in crystallization. In this mechanism, the key step in crystallization is the conversion of structurally compatible, non-crystalline aggregates into crystalline ones for nucleation, and the incorporation of compatible aggregates into a crystalline lattice during crystal growth. The participation of aggregates as intermediates in crystallization has also been suggested from light scattering (Skouri et al., 1992; Thibault et al., 1992; Forsythe and Pusey, 1994) and SANS (Boue et al., 1993) studies of crystallization mixtures for the water-soluble protein lysozyme. The similarity between these results and those from the reaction center SANS studies raises the possibility that non-crystalline aggregates may be a general feautre of protein crystallization pathways.

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B. Structural Characterization of Photosynthetic Supramolecular Assemblies In the SANS studies of RCs in solution, a matching of experimental scattering with that calculated from the RC crystal structure unambiguously identified the scattering profile associated with the monodispersed RC state. Knowledge of the crystallographic dimensions of the RC also permitted identification of the most appropriate fitting of the RC with simple geometric form factors. The most definitive characterization of a supramolecular structure by SANS is achieved by fitting experimental data with scattering profiles calculated for molecular assemblies built from the crystal structures (Grossmann et al., 1993). Ultimately this technique will be used to accurately characterize the structure of the RC aggregates and complexes of RC with other proteins. In the case of supramolecular assemblies for which there is no data on the structure of the constituents, the strength of SANS analysis lies in providing a test for plausible structural models. This capability can be illustrated by a SANS characterization of the light-harvesting antenna complexes of photosynthetic bacteria. The antenna of the purple bacteria consists of

386 two complexes, LH1 and LH2; for reviews see Hunter et al., (1989) and Zuber and Brunisholz, (1991). Each complex is composed of two short proteins, and containing approximately 50 amino acid residues. In LH1 each pair is associated with two bacteriochlorophyll molecules, while each pair is associated with three bacteriochlorophylls in LH2. Functional energy delocalization occurs throughout an array of these building blocks, involving up to 200 or more chlorophylls (Holzwarth, 1991; Pullerits et al., 1994). There is no definitive characterization of the structure of the antenna assemblies either in the natural membrane or in detergent-isolated states. The fundamental building block of the LH1 and LH2 complexes has been proposed to be either or units, with cylindrically symmetric complexes (Hunter et al., 1989; Zuber and Brunisholz, 1991) or an complex (Kleinekofort et al., 1992) functioning as the minimal functional unit. SANS studies can discriminate between these possible models without detailed fitting of scattering profiles to molecular models. For example, the two models for the minimal functional unit for LH2, and differ in molecular mass. Peptide and pigment composition analyses for the LH2 complex from Rb. sphaeroides (Zuber and Brunisholz, 1991) indicates that the molecular masses for these models are 88 kDa and 118 kDa respectively. Figure 7 shows a comparison of scattering profiles for deuterated Rb. sphaeroides LH2 and RC. Contributions from the solubilizing detergent, OG, were eliminated by contrast matching through solubilizing both samples in 17% solutions. Scattering profiles for LH2 were indistinguishable with either LDAO solutions or OG as the solubilizing detergent, and using 5% and 17% respectively to contrast match the detergent. Model fitting and Guinier analysis show that both samples are monodispersed. The scattering profiles in Fig. 7 were normalized at low q. This normalization illustrates that the falloff in scattering intensity starts at a lower q for LH2 than for RC. This unambiguously establishes that the LH2 assembly has a structure with longer atom-atom distance correlations than does the RC. This is also indicated by a comparison of Guinier analysis which yields an of 31±1.5

David M. Tiede and P. Thiyagarajan

Å and 38 ± 1.3 Å for the RC and LH2 complex respectively. These observations require that the LH2 complex have slightly larger size than the RC. Maximum entropy fitting procedures found that the scattering profiles for LH2 can be fit using a solid, cylindrical form factor having a particle of 68 Å diameter and length of 64 Å The dimensions and molecular mass of the proposed models (Hunter et al., 1989; Zuber and Brunisholz, 1991) are smaller than those for the RC. Thus, the SANS data rule out the complex as a possible structure for the LH2 in these samples. The molecular mass of the model is 1.17 fold larger than that of the RC. Form factors consistent with this molecular mass can be fit to the LH2 scattering data, and hence the model is more consistent with the SANS results. This example demonstrates how SANS data that resolves only the overall size and mass density of a supramolecular assembly can be used for identification of possible structural models.

C. Internal Structure in Supramolecular Assemblies In addition to a resolution of particle size and mass density, SANS measurements offer opportunities for resolution of internal structures in

Small angle neutron scattering

supramolecular assemblies. In the q-range up to this can be illustrated with SANS measurements for the LH1 complex. Figure 8 shows SANS profiles for the Rb. sphaeroides LH1 at two different concentrations. Maximum entropy analysis required dispersions of LH1 particle sizes to fit both profiles. Both samples were found to be composed of different mixtures of particles with diameters of approximately 60 Å and 130 Å. A dispersion of particle sizes, which varied as a function of LH1 concentration, was also found by size-exclusion chromatography and electron microscopy of LH1 (Boonstra et al., 1993). This behavior is reflected in the SANS data. The shoulder seen in the scattering profile for the more concentrated sample in Fig. 8 at approximately is noteworthy, since features of this kind have not been seen in any of the monodispered or aggregated RC samples, nor in the LH2 samples. Additionally, this feature was not fit using solid cylindrical form factors in the polydispersity fitting analysis. One intriguing possibility is that this feature arises from the internal structure, or packing of subunits within the larger LH1 particles, as suggested from the following modelling studies. Figure 9 shows scattering profiles calculated

387

for two model structures. The first was a cylindrically symmetric ring composed of six cylinders, having a 50 Å diameter and 60 Å length. Each cylinder represents a geometrical form factor approximation to the unit. This model has a hole in the middle with a size sufficient to contain a seventh cylinder. The scattering profile calculated for this model is shown by the solid line in Fig. 9. One noticeable feature is the shoulder in the q region to that arises from intra-particle interference effects. The shape of this feature is similar to that seen in the experimental data. The sensitivity of the shoulder to the presence of the hole in the model structure is illustrated by the scattering profile marked by the dashed line in Fig. 9, which was calculated for a model consisting of the same 6 cylinder ring, but that additionally contained a seventh cylinder in the center. This filled ring model removed the shoulder seen in the scattering profile in the q region to Fits to the experimental data will require optimization of the model as well as inclusion of effects due to aggregate size dispersity. However, these calculations demonstrate the plausibility of a ring-like assembly to give rise to the observed scattering fine structure for LH1. Hence, the SANS data support structural models for the LH1 at high concentration that incorporate ring-like structures. The ability to detect internal structure within

388 the LH1 assembly at high concentration arises because of the large size of the aggregated assembly, with corresponding large structural repeats and scattering length density discontinuities. These results offer encouragement for the application of SANS measurements at higher q to probe structural repeats of smaller dimension, such as the internal packing of protein in smaller assemblies such as the LH2 and RC complexes.

D. New Results Since the first draft of this chapter, a high resolution, R = 2.5 Å, structure has been determined for the LH2 complex from Rps. acidophila by Xray diffraction on single crystals (McDermott et al., 1995), and a low-resolution, R = 8.5 Å, structure has been determined for the LH1 from Rhodospirillum by electron microscopy on two-dimensional crystals (Karrasch et al., 1995). The crystallography of LH2 showed the complex to be composed of 9 pairs, arranged symmetrically in a barrel-shaped bundle, with a 36 Å diameter hole in the center. The overall dimensions of the LH2 complex were 68 Å diameter by 70 Å length (McDermott et al., 1995). These dimensions are in approximate aggreement with the 68 Å by 64 Å dimensions determined by our fitting of the SANS data for the Rb. sphaeroides LH2 complex in solution. It is likely that the differences in measured dimensions arise from the use of cylindrical form factors as approximations of the protein shape for fitting the SANS data. Å more exact comparison of the crystal and solution structures for LH2 will require comparisons of experimental SANS profiles with those calculated from atomic crystal coordinates. As discussed for the LH1 complex, a hollow or ringlike structure will give rise to characteristic undulations in SANS profiles due to the cylindrical symmetry. Undulations due to a 36 Å diameter hole in the LH2 structure would not be seen in the present SANS profiles recorded in the q-range These features would be expected to appear at higher q-values. The two-dimensional crystals of the R. rubrum LH1 showed the complex to be composed of 16 pairs arranged, on average, in a 116 Å ring with a 68 Å hole in the middle (Karrasch et al.,

David M. Tiede and P. Thiyagarajan 1995). A ring-like structure was also anticipated from SANS data for the Rb. sphaeroides LH1 in solution, (Fig. 8), which has a secondary maximum at Preliminary modelling suggests that the ring-like structure described by Karrasch et al. can account for the shoulder seen in the scattering profile for the Rb. sphaeroides LH1. However, a detailed modelling of the solution scattering profiles will require that particle size dispersity be taken into account. The electron microscopy of the two-dimensional LH1 arrays showed that there were small ellipitical distortions in the ring shape in different crystals, and that there were different crystalline forms composed of LH1 particles with different dimensions (Karrasch, S. et al., 1995). A detailed evaluation of the solution SANS data in light of the new structured information is underway. IV. Concluding Remarks SANS studies on photosynthetic complexes demonstrate the capability of this technique for resolving inter-particle interactions, size, shape and internal order or packing of supramolecular assemblies pertinent to photosynthesis. Opportunities for definitive assignment of supramolecular structures are possible by comparison of experimental scattering data with scattering profiles calculated for molecular models built from crystal structures of the composite proteins. Photosynthesis ultimately relies upon a hierarchy of structures and intermolecular interactions. SANS provides a complement to crystallographic studies by providing a technique for assessing structure in functional, supramolecular assemblies that can not be examined by crystallography. Acknowledgements The authors thank Dr. Rex Hjelm (Los Alamos National Laboratory) and Dr. D. S. Sivia (ISIS) for use of the maximum entropy fitting program used to characterize the size distributions of photosynthetic complexes, and Dr. Stephen Henderson (Oak Ridge National Laboratory) for the use of his program BIOMOD for the calculation of scattering profiles for model structures in Fig. 9. This work was supported by the U.S Department of Energy, Office of Basic Energy Sciences, Divi-

Small angle neutron scattering sion of Chemical Sciences and the Division of Material Sciences, under Contract W-31–109– Eng-38 and P.T. was additionally supported in part, by a NASA Microgravity Biotechnology Program Grant M951–ES-3–004–2511. References Allen JP and Feher G (1984) Crystallization of reaction center from Rhodopseudomonas sphaeroides: preliminary characterization. Proc Natl Acad Sci USA 81: 4795–4799. Allen JP and Feher G (1990) Crystallization of reaction centers from Rhodobacter sphaeroides. In: Michel H (ed) Crystallization of Membrane Proteins, pp 137–154. CRC Press, Boca Raton. Allen JP, Feher G, Yeates TO, Komiya H and Rees DC (1987) Structure of the reaction center from Rhodobacter sphaeroides R-26: The protein subunits. Proc Natl Acad Sci. USA 84: 6162–6166. Boonstra AF, Visschers RW, Calkoen F, van Grondelle R, van Bruggen EFJ and Boekema EJ (1993) Structural characterization of the B800–850 and B875 light-harvesting antenna complexes from Rhodobacter sphaeroides by electron microscopy. Biochim. Biophys Acta 1142: 181–188. Boue F, Lefaucheux F, Robert MC and Rosenman I (1993) Small angle neutron scattering study of lysozyme solutions. J Crystal Growth 133: 246–254. Buchanan SK, Fritzsch G, Ermler U and Michel H (1993) New crystal form of the photosynthetic reaction centre from Rhodobacter sphaeroides of improved diffraction quality. J Mol Biol 230: 1311–1314. Capel MS, Engelman DM, Freeborn BR, Kjeldgaard M, Langer JA, Ramakrishnan V, Schindler DG, Schneider DK, Schoenborn BP, Sillers I-Y, Yabuki S and Moore PB (1987) A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli. Science 238: 1403– 1406. Chang C-H, Schiffer M, Tiede DM, Smith U and Norris JR (1985) Structure of the membrane-bound protein photosynthetic reaction center from Rhodopseudomonas sphaeroides R-26 by x-ray diffraction. J. Mol. Biol. 186: 201–203. Chang C-H, El-Kabbani O, Tiede DM, Norris J and Schiffer M (1991) Structure of the membrane-bound protein photosynthetic reaction center from Rhodobacter sphaeroides. Biochemistry 30: 5352-5360. Chen SH (1986) Small angle neutron scattering studies of the structure and interaction in micellar and microemulsion systems. Annu Rev Phys Chem 37: 351–399. Chen SH, Sheu EY, Kalus J and Hoffman H (1988) Smallangle neutron scattering investigation of correlations in charged macromolecular and supramolecular solutions. J Appl Cryst 21: 751-769. Deisenhofer J and Michel H (1989) The photosynthetic reaction center from the purple bacterium Rhodopseudomonas viridis (Noble Lecture). Angew Chem Int Ed Engl 28: 829– 968. Ducruix A and Reiss-Husson F (1987) Preliminary characterization by x-ray diffraction of crystals of photochemical

389 reaction centres from wild-type Rhodopseudomonas sphaeroides. J Mol Biol 193: 419–421. Durbin SD and Feher G (1986) Crystal growth studies of lysozyme as a model for protein crystallization. J Crystal Growth 76: 583–592. Durbin SD and Feher G (1991) Simulation of lysozyme crystal growth by the Monte Carlo method. J Crystal Growth 110: 41–51. Fedorov BA and Denesyuk AI (1978) Large-angle x-ray diffuse scattering, a new method for investigating changes in the conformation of globular proteins in solution. J Appl Cryst 11: 473-477. Feher G and Kam Z (1985) Nucleation and growth of protein crystals: general principles and assays. Methods Enzymology 114: 77–112. Feigin LA and Svergun DI (1987) Structure Analysis by Small Angle X-ray and Neutron Scattering 1–335. Forsythe E and Pusey ML (1994) The effects of temperature and NaCl concentration on tetragonal lysozyme face growth rates. J Crystal Growth 139: 89–94. Franck HA, Taremi SS and Knox JR (1987) Crystallization and preliminary x-ray and optical spectroscopic characterization of the photochemical reaction center from Rhodopseudomonas sphaeroides strain 2.4.1. J Mol Biol 198:139– 141. Glatter O (1991) Small-angle scattering and light scattering. In: Lindner P and Zemb T (ed) Neutron, X-ray and Light Scattering, pp 33–82. Elsevier, Amsterdam. Grossmann JG, Abraham ZHL, Adman ET, Neu M, Eady RR, Smith BE and Hasnain SS (1993) X-ray scattering using synchrotron radiation shows nitrite reductase from Achromobacter xylosoxidans to be a trimer in solution. Biochemistry 32: 7360–7366. Guinier A and Fournet G (1955) Small Angle Scattering. John Wiley and Sons, New York. Heidorn DB and Trewhella J (1988) Comparison of the crystal and solution structures of calmodulin and troponin c. Biochemistry 27: 909–915. Hjelm RP, Thiyagarajan P, Sivia DS, Lindner P, Alkan HA and Schwahn D (1990) Small-angle neutron scattering from aqueous mixed colloids of lecithin and bile salts. Prog Colloid Polym Sci 81: 225–321. Hjelm RP, Thiyagarajan P and Alkan-Onyuksel H (1992) Organization of phosphatidylcholine and bile salt in rodlike mixed micelles. J Phys Chem 96: 8653–8661. Holzwarth AR (1991) Excited state kinetics in chlorophyll systems and its relationship to the functional organization of the photosystems. In: Scheer H (ed) Chlorophylls, pp 1125–152. CRC Press, Boca Raton. Hubbard ST, Hodgson KO and Doniach S (1988) Small-angle x-ray scattering investigation of the solution structure of troponin c. J Biol Chem 263: 4151–4158. Hunter CN, van Grondelle R and Olsen JD (1989) Photosynthetic antenna proteins: 100 ps before photochemistry starts. Trends Bioch Sci 14: 72–76. Jacrot B (1976) The study of biological structures by neutron scattering from solution. Rep Prog Phys 39: 911–953. Karrasch S, Bullough PA and Ghosh R (1995) The 8.5 Å projection map of the light-harvesting complex I from Rho-

390 dospirillum rubrum reveals a ring composed of 16 subunits. EMBO J 14: 631–638. Kleinekofort W, Germeroth L, van der Broek JA, Schubert D and Michel H (1992) The light-harvesting complex II (B800/850) from Rhodospirillum molischianum is an octamer. Biochim Biophys Acta 1140: 102–104. Lederer H, Mortensen K, May RP, Baer G, Crespi H, Dersch D and Heumann H (1991) Spatial arrangement of and core enzyme of Escherichia coli RNA polymerase: A neutron solution scattering study. J Mol Biol 219: 747–755. McDermott G, Prince SM, Freer AA, Hawthornthwaith-Lawless AM, Papiz MZ, Cogdell RJ and Isaacs NW (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374: 517– 521. McPherson A, Koszelak S, Axelrod H, Day J, Williams R, Robinson L, McGrath M and Cascio D (1986) An experiment regarding crystallization of soluble proteins in the presence of J Biol Chem 261: 1969–1975. Michel H (1982) Three-dimensional crystals of a membrane protein complex. The photosynthetic reaction centre from Rhodopseudomonas viridis. J Mol Biol 158: 567–572. Miki K, Saeda M, Masaki K, Kasai N, Miki M and Hayashi K (1986) Crystallization and preliminary x-ray diffraction study of ferrocytochrome from Rhodopseudomonas viridis. J Mol Biol 191: 579–580. Morrison JD, Corcoran JD and Lewis KE (1992) The determination of particle size distributions in small-angle scattering using the maximum-entropy method. J Appl Cryst 25: 504– 513. Nowotny V, Nowotny P, Voss H, Nierhaus KH and May RP (1989) The quaternary structure of the ribosome from Escherichia coli- A neutron small-angle scattering study. Physica B 156: 499–501. Pullerits T, Visscher KJ, Hess S, Sundstrom V, Freiberg A, Timpmann K and R. vG (1994) Energy transfer in the inhomogeneously broadened core antenna of purple bacteria: A simultaneous fit of low-intensity picosecond absorption and fluorescence kinetics. Biophys J 66: 236–248.

David M. Tiede and P. Thiyagarajan Ramakrishnan VR, Capel M, Kjeldgaard M, Engleman DM and Moore PB (1984) Position of protein S14, protein S18 and protein S20 in the 30S ribosomal subunit of Escherichia coli. J Mol Biol 174: 265–284. Sears VF (1986) Neutron scattering lengths and cross sections. In: Sköld K and Price DL (ed) Neutron Scattering. Methods of experimental physics, Celotta R and Levine J (series eds) 23A, pp 521–549. Academic Press, New York. Skouri M, Munch J-P, Lorber B, Giege R and Candau S (1992) Interactions between lysozyme molecules under precrystallization conditions studied by light scattering. J Crystal Growth 122: 14–20. Stuhrmann HB and Miller A (1978) Small-angle scattering of biological structures. J Appl Cryst 11: 325–345. Svergun DI (1991) General theorems of small-angle scattering by disperse systems. In: Lindner P and Zemb T (ed) Neutron, X-ray and Light Scattering, pp 83–98. North-Holland, Amsterdam. Thibault F, Langowski J and Leberman R (1992) Optimizing protein crystallization by aggregate size distribution analysis using dynamic light scattering. J Crystal Growth 122: 50–59. Thiyagarajan P and Tiede DM (1994) Detergent micelle structure and micelle-micelle interactions determined by small angle neutron scattering in solution conditions used for membrane protein crystallization. J Phys Chem 98: 10343– 10351. Timmins PA, Hauk J, Wacker T and Welte W (1991) The influence of heptane-1,2,3–triol on the size and shape of LDAO micelles. FEBS Letts 280: 115–120. Yeates TO, Komiya H, Rees DC, Allen JP and Feher G (1987) Structure of the reaction center from Rhodobacter sphaeroides R-26: Membrane-protein interactions. Proc Natl Acad Sci USA 84: 6438–6442. Zuber H and Brunisholz RA (1991) Structure and function of antenna polypeptides and chlorophyll-protein complexes: Principles and variability. In: Scheer H (ed) Chlorophylls, pp 627–703. CRC Press, Boca Raton.

Chapter 24 Measurement of Photosynthetic Oxygen Evolution Hans J. van Gorkom* and Peter Gast Department of Biophysics, Huygens Laboratory of the State University, P.O.Box 9504, 2300 RA Leiden, The Netherlands

Summary I. Introduction II. Polarography A. The Clark Electrode B. Unwanted Chemistry at the Bare Cathode C. Rate Electrodes D. Concentration Electrodes E. Polarograms F. Flash-Induced Kinetics III. EPR Oximetry A. EPR Line Broadening by Oxygen B. Oxygen Probes for EPR C. Sensitivity 1. Sensitivity in Direct Linewidth Broadening Measurement 2. Sensitivity in Amplitude Measurement D. Applications in Photosynthesis Research IV. Mass Spectrometry V. Photoacoustic Spectroscopy VI. Galvanic Sensors VII. Prospects Acknowledgements References

391 392 392 392 393 394 395 395 397 398 398 398 399 399 399 401 401 402 402 402 403 403

Summary An overview is presented of methods that have been used to measure photosynthetic oxygen evolution over the past 25 years. Oxygen polarography in its many versions is treated in some detail, the complications caused by a large bare cathode are discussed and an interpretation of the current/voltage characteristic (polarogram) of flash-induced oxygen signals is proposed. The recent controversy on the interpretation of the kinetics of such signals is briefly summarized. The discovery of a new class of spinprobes for EPR oximetry has greatly enhanced its possibilities. The sensitivity of the method is evaluated, consequences of its nonlinearity in time-resolved measurements are indicated, and the first reports on its use in photosynthesis research are summarized. The use of mass spectrometry, photoacoustic spectroscopy and galvanic sensors to measure photosynthetic oxygen evolution is briefly reviewed.

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

391 J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 391–405. © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

392

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I. Introduction

II.

Polarography

Oxygen evolution was the first photosynthetic reaction discovered and two centuries of research have left an impressive track record of methods applied to measure it (Rabinowitch, 1945, 1951, 1956; Burr and Mauzerall, 1968; Joliot, 1993). It is useful to keep old methods in mind. A striking example was the recent application of the classical work of T. Engelmann in the 1880's, who used bacteria with oxotropic motility to locate the source of photosynthetic oxygen at the microscopic level: this method allowed V. Zimmermann's group to select the photosynthetically competent cells among hybrids produced by electrofusion (Hampp et al., 1986). Due to its important medical and industrial applications, the measurement of oxygen has received much attention. A wide range of methods has been reviewed in a symposium dedicated to the subject (Degn et al., 1976). In biological and medical research, polarography has become the predominant technique, on which extensive documentation is available (Fatt, 1976; Hitchman, 1978; Gnaiger and Forstner, 1983). Also in photosynthesis research polarography has replaced the traditional Warburg manometry, a variety of electrochemical cells has been designed and other ways of measuring oxygen have been explored. Here we present a survey of such methods as published over the past 25 years or so, after the discovery of the period four oscillation of the oxygen yield with flash number upon illumination of Photosystem II with a series of short saturating flashes (Joliot et al., 1969). This finding marked a shift of scientific interest from rate measurements in continuous light to the measurement of oxygen yields of individual flashes in a series. In recent years also the kinetics of oxygen release after a flash have been at the focus of attention, requiring measurements with even higher time resolution. In this chapter we concentrate on methods meeting these requirements and, due to our own involvement, pay some extra attention to the interpretation of time-resolved polarographic signals and to a promising new class of spin-probes for EPR-oximetry.

A. The Clark Electrode Routine measurements of photosynthetic oxygen evolution are now carried out with commercially available equipment based on the Clark electrode (Clark, 1956). This is a ‘bipolar' electrochemical oxygen sensor: the cathode and anode and the connecting electrolyte solution are separated from the sample by a teflon membrane, which is permeable to oxygen but not to water and ions. The platinum cathode reduces oxygen to hydroxyl ions. At the silver anode initially AgCl is formed and later, as accumulates, also AgOH. Apart from its small-oxygen consumption it is essentially a closed system, isolated from the sample by the teflon membrane, and can be used to measure oxygen in the gas phase (Delieu and Walker, 1983) as well as in solution. The use of a small cathode and stirring of the sample (if in solution) ensure that the oxygen concentration gradient is restricted to the membrane and the use of a sufficiently low cathode potential, about –0.7 V relative to the anode, ensures that this gradient stays at its maximum value because the cathode surface is kept anaerobic. The measured current is simply proportional to the oxygen tension (partial pressure) in the sample and is easily calibrated by comparison to the signal in an airsaturated solution at the same temperature. The oxygen concentration then follows from the oxygen solubility in the medium used (which can be much reduced at high salt concentrations). The properties and theoretical background of the Clark electrode are described most comprehensively in the monograph by Hitchman (1978), which also contains oxygen solubility data. In photosynthesis research, situations often arise where the unmodified Clark electrode is too insensitive, too slow, or both. In particular, it does not allow resolution of the individual oxygen yields of successive flashes in a series, which is required to study the 4-flash redox cycle of the oxygen evolving complex. Sufficient sensitivity for this purpose may be reached by a substantial increase of the cathode surface area (Velthuys and Kok, 1978; Lübbers et al., 1993), maintaining the advantages of the physical separation by the teflon membrane between sample and electro-

Oxygen evolution chemistry. However, even with vigorous stirring it will be difficult to prevent the oxygen diffusion gradient from spreading into the the sample and the advantage of easy calibration is probably lost. With a thin membrane, the time-resolution may be good enough for single flash resolution at a usual 1 Hz flash frequency. A further enhancement of sensitivity and time-resolution can only be obtained by removing the membrane, sacrificing all advantages of the Clark electrode concept.

B. Unwanted Chemistry at the Bare Cathode When a bare cathode is used, the sample must contain the electrolyte connecting the cathode and anode and a buffer to avoid excessive pH increase due to production at the cathode, and should preferably not contain any substance that could mediate electrochemical reduction of redox centers in the sample. A polarized bare platinum electrode covered by a thin sample layer is the standard tool for electrochemical titrations and can readily impose its potential on all redox couples in the sample if adequate mediators are present. Fortunately, most redox centers in Photosystem II are highly inaccessible and the ‘substrate binding sites’, where plastoquinone reduction and water oxidation take place, seem to allow reaction with a very limited variety of molecules only. Even the mobile plastoquinone pool is not rapidly reduced by the cathode if no mediators are added. However, the cathode itself may produce redox mediators. Hydrogen peroxide, formed at the cathode surface as an intermediate in the reduction of to can interact with the oxygen evolving complex, especially in Photosystem II preparations where the protective shield of extrinsic polypeptides has been damaged (Schröder and Åkerlund, 1986). Addition of catalase might help to avoid this complication, but one should keep in mind that there is also evidence for the production of hydrogen peroxide by Photosystem II itself under some circumstances (Wydrzynski et al., 1989). The inhibition observed by Plijter et al. (1988) at cathode potentials lower than –0.5 V vs SHE (Standard Hydrogen Electrode) may be due to hydrogen produced at the cathode surface. As the inhibition appears to be an irreversible

393 all-or-none effect (the shape of the period four oscillation in a flash series remains the same), this might be due to over-reduction and subsequent dissociation from the reaction center of or manganese. This problem can be avoided by weaker polarization, and depends on pH and cathode material, Pt being a much more efficient catalyst for hydrogen production than Au. Last but not least, the cathode removes oxygen and oxygen usually is involved in poising the redox potential in the sample. There have been reports that oxygen evolution requires oxygen (Bader and Schmid, 1988). The oxygen concentration near the surface of a strongly polarized cathode surface can become quite low (Baumgärtl et al., 1974) and it is primarily the oxygen evolution in this region that one measures. The modification of the chemical conditions in the sample by the cathode is normally limited by using a thin sample layer exposed on the other side to a continuous flow of conditioning medium (rate electrodes). It can be avoided altogether by a continuous, very fast sample replacement (Etienne, 1968) or minimized by using a very weak polarization (Plijter et al., 1988) (concentration electrodes). Moreover, the experiment may require the addition of chemicals, such as an artificial electron acceptor when isolated Photosystem II particles are used. Joliot et al. (1966) introduced modulated illumination and lock-in detection to measure selectively the electrode current resulting from photosynthetic activity (see also Joliot, 1972). Particularly troublesome, however, are substances which produce spurious signals upon flash illumination due to photochemistry in the sample or at the cathode surface. In some cases such signals can be avoided by applying a thin film of collodion on the cathode. With a flat disk electrode this can be done conveniently by letting a drop of collodion solution fall on the cathode. By choice of solvent, collodion concentration, drop size and speed, one can obtain reproducible films of the right diameter to cover the cathode and thin enough not to affect the kinetics of the flash induced oxygen signals. In one respect the measurement of oxygen evolution should be simplified by the use of a bare cathode. The cathode current is proportional to the oxygen concentration in a boundary layer adjacent to the cathode surface. With a bare cath-

394 ode there is no phase separation between this layer and the sample and one measures concentration rather than partial pressure, independent of the oxygen solubility in the medium used. Unfortunately, this potential advantage is more than offset by other difficulties involved in quantitative calibration of signals obtained with a bare cathode. In fact no such calibration seems to have been achieved, except for the turbulent flow – concentration electrode used by Etienne (1968), and one has to resort to comparison of the signal to that obtained with a more easily calibrated method.

C. Rate Electrodes Until about 25 years ago, photosynthesis researchers measuring oxygen evolution were often primarily interested in highly precise measurements of the rate of photosynthesis to study induction phenomena and action spectra and sophisticated electrochemical cells had been developed for this purpose, as reviewed by Fork (1972). The design of these cells was based on the pioneering work of L. Blinks and coworkers. Blinks and Skow (1938) already used photosynthetic material appressed to a large surface platinum cathode. Haxo and Blinks (1950) introduced the ‘rate electrode’, using a semi-permeable membrane to keep the sample appressed to the cathode and immersing the ensemble in a large volume of stirred or flowing medium to keep the oxygen concentration and other chemical conditions constant. The concentration gradient is now in the thin sample layer and the idea of F. Haxo and L. Blinks was that the rate of oxygen evolution there would simply add up to the background current due to oxygen flow from the medium, through the sample, towards the cathode. In their conditions most of the photosynthetic oxygen may actually be lost to the medium, but anyway the amplitude of the light-induced increase of the signal is proportional to the rate of oxygen evolution. Later rate electrode designs mostly used a shallow sample compartment in which unicellular algae or isolated chloroplasts were allowed to settle in a thin layer on the cathode. Many variations have been published. A typical system may consist of (from bottom up): Pt cathode, about

Hans J. van Gorkom and Peter Gast sample sediment, about stationary supernatant medium, dialysis membrane, flowing medium of constant composition, and window for illumination. The Ag/AgCl anode, which much be shielded from illumination, may be a ring surrounding the cathode, to reduce electromagnetic interference. The positive spike at the moment of the flash, often seen in reported measurements of flash-induced oxygen evolution, is an artifact caused either by insufficient optical shielding of the Ag/AgCl anode from the flash light, or by insufficient electromagnetical shielding of the electrochemical cell as a whole from the flash tube discharge. The anode can be placed in the sample compartment, in the flowing medium compartment, or in a third compartment separated from the conditioning medium by a second dialysis menbrane and itself being part of a second flow system. The latter arrangement was introduced by Joliot and Joliot (1968) to avoid chemical interference of the conditioning medium with the anode. It should also allow the use of different electrolytes in the sample and anode compartments (e.g. to study chloride depletion), but this facility appears not to have been used. Pickett (1966) placed the anode with the cathode under a teflon membrane, combining the Haxo and Blinks electrode and Clark electrode concepts (used e.g. by Weiss and Sauer, 1970). Den Haan et al. (1976) used a sample compartment of adjustable depth: the stationary supernatant medium was pushed out mechanically through the membrane. Diner and Mauzerall (1973) used a flowing gas instead of a solution as the conditioning medium. This may be the most efficient way to supply oxygen and, if necessary, to remove hydrogen, but it does not remove and leaves little room for electrolyte to connect anode and cathode electrically. A too large electrical resistance leads to an electrical potential gradient in the electrolyte, which corresponds to using a weaker polarization, but may also result in a slow response time if the circuit contains a large capacitance (Meunier and Popovic, 1988). Rate electrodes can be very sensitive. The modulated rate measurement as described by Joliot (1972) may be the most sensitive way to measure the rate of photosynthesis: an oxygen detection limit of has been reported.

Oxygen evolution

D. Concentration Electrodes Going back to the setup of Blinks and Skow (1938), one might also try to optimize the system for measurement of the oxygen concentration rather than of its rate of change. To avoid oxygen loss, the sample should be bound by an oxygenimpermeable wall, or at least be a thick layer on the cathode. The problem is that the large bare cathode will quickly modify the chemical conditions near its surface. Vigorous stirring (Joliot, 1965) helps to spread the cathode-induced changes in chemical conditions rapidly over the whole sample but does not prevent them. Etienne (1968) used continuous sample replacement. A small cathode was placed at the outlet of a capillary tube in which a fast, turbulent sample flow was maintained. By varying the distance between the cathode and a small, brightly illuminated spot in the capillary, the flash induced kinetics could be determined in steady state measurements, allowing a good signal/noise ratio in spite of the small size of the cathode. The system has limited applicability, but it does prevent all influence of the cathode on the sample. Plijter et al. (1988) used a stationary sample suspension in a closed cuvette and minimized the influence of the cathode by using an extremely weak polarization ( – 0.1 V vs. SHE), compensating the reduction in cathode efficiency by a large cathode surface area Ideally, the cathode should function as a non-disturbing probe and should not create substantial diffusion gradients in the sample. The measured signals were indeed shown to be independent of viscosity (van Gorkom et al., 1989). Electrochemists probably would not call this method polarography at all. The electrochemical cell described by Plijter et al. (1988) was complicated by the requirement of simultaneous UV absorbance measurements (van Leeuwen et al., 1990) via a high transmittance grid cathode as used by Marsho and Hommersand (1975), but we have obtained similar results with a very simple system consisting of an 0.5mm thick Pt-plastic-Ag/AgCl sandwich inserted in a standard 1 or 2 mm path length spectrophotometer cuvette, the Pt side facing the flash lamp (H.J. van Gorkom and M.A. van Dijk, unpublished). The detection limit of the Plijter electrode, with 10 ms response time and no aver-

395 aging, is an oxygen concentration change of about M. For reasons yet unknown, mutually exclusive results were obtained by Etienne (1968) and Plijter et al. (1988), so these methods should be used with caution.

E. Polarograms A polarogram is a plot of the measured current as a function of the polarization voltage, as in Fig. 1a. As usual, the polarization voltage is indicated by the potential difference applied between the Pt cathode and Ag/AgCl anode, but what happens to oxygen when it hits the cathode depends only on the electrochemical potential of the cathode, which should preferably be indicated relative to the Standard Hydrogen Electrode. If the cathode potential is not measured or fixed relative to a separate reference electrode by a potentiostat circuit, it may be estimated on the basis of the equilibrium potential of the Ag/AgCl couple at the chloride concentration used, but the actual anode potential will be higher. The deviation depends on the current density at the anode and can safely be neglected only if the anode is many times larger than the cathode. The polarogram shows an exponentially rising curve (the cathode potential affects the activation energy of the reaction) until diffusion of oxygen towards the cathode becomes rate limiting: a plateau sets in at the voltage where oxygen in a boundary layer on the cathode surface is reduced more rapidly than it can be replenished from the solution. The steady state concentration in the boundary layer can only approach zero. The maximum concentration gradient in the stationary layer between cathode and stirred solution, and hence the rate of oxygen transport towards the cathode and the electrode current, is therefore limited by the concentration in the bulk solution. This is what makes the current in a Clark electrode voltage independent. It does not mean that every oxygen molecule hitting the cathode is reduced. Stronger polarization may still allow an exponentially faster oxygen reduction and decrease the concentration in the boundary layer manifold, but if that concentration was negligible already the concomitant increase of the diffusion

396

gradient and of the steady state electrode current will be negligible, too. With a rate electrode the background current due to oxygen diffusing from the flowing conditioning medium to the cathode also shows a polarogram with a more or less well-defined plateau; only the current increase due to hydrogen evolution at strong polarization starts earlier due to the lower pH (Fig. 1b, open symbols). Haxo and Blinks (1950) used a polarization voltage in the middle of this plateau. With a rate electrode, however, one measures oxygen evolution in the boundary layer: the sample is on the electrode side of the diffusion gradient and the background current resulting from this gradient is in fact irrelevant. As just stated, the rate of oxygen reduction in the boundary layer should increase exponentially with the voltage, if not limited by the electronics, until every oxygen molecule hitting the cathode is reduced. The polarization voltage at which that happens can be estimated from the

Hans J. van Gorkom and Peter Gast

kinetics of the flash induced signal if the kinetics without disturbance by the measurement are known. The data and calculations of Plijter et al. (1988) indicate that 100% efficiency of the cathode reaction is approached at a cathode potential of about – 0.7 V vs. SHE (or – 1.0 V vs. Ag/AgCl at At this potential hydrogen production at a Pt cathode is already causing a considerable background current and all reported oxygen measurements with rate electrodes were carried out at much weaker polarization, mostly around – 0.4 V vs. SHE, where oxygen reduction by the cathode still depends exponentially on the cathode potential and the cathode is not at all the efficient oxygen trap it is usually thought to be. Polarograms of flash-induced oxygen evolution measured with rate electrodes show an exponential dependence of the current on the polarization voltage only to about – 0.3 V vs. SHE and a less steep increase of the current at stronger polariz-

Oxygen evolution ation (Fig. 1b, solid symbols). This has sometimes been attributed to limitation by O 2 diffusion from – 0.3 V followed by a gradual change from 2electron reduction formation) to 4-electron reduction formation) at – 0.7 V, but there is little evidence for that. It could be due instead to the convolution of oxygen production and its consumption by the electrode, and saturation of the electrode efficiency could explain the maximum near – 0.7 V vs. SHE. Myers and Graham (1963) already noted the absence of a flat plateau and emphasized the need to stabilize the cathode potential and minimize the resistance in the circuit. A potentiostat circuit using a separate reference electrode and/or current to voltage conversion to reduce the resistance are now generally used.

F. Flash-Induced Kinetics The kinetics of the current transient upon flashinduced oxygen evolution in a rate electrode setup is not fully understood. It consists of a sigmoidal rise in a few ms, followed by a decay in tens of ms. It is agreed that the rise (or its Fourier transform, as measured by Joliot et ah., 1966) is caused by the to transition’, the reduction of the oxygen evolving complex by water, which normally proceeds with a time constant of 2 ms. The generally accepted view is that this coincides with the release of an oxygen molecule, and hence that the rise of the signal shows the release of the oxygen. According to Plijter et ah. (1988), however, a good rate electrode measures the rate of oxygen evolution, so the kinetics of the flashinduced current transient is the first derivative of the oxygen concentration, in agreement with their findings with a concentration electrode. The rise of the signal from a rate electrode in that case means a delay preceding oxygen release, attributed to water oxidation by the oxygen evolving complex, and the time constant of the release process itself is reflected in the decay of the signal. No valid criticism or feasible alternative explanation of the data or reasoning in the Plijter et ah. paper has been published to date. Also no one has reported an attempt to reproduce the measurements, but the method has been applied ever since in this laboratory, using various elec-

397 trode arrangements and electronics, and we have found nothing wrong with the measurements. The study by Plijter et al. (1988) was prompted by the paradox that after removal of the extrinsic 33 kDa protein the UV absorbance change due to reduction of the oxygen evolving complex, and hence presumably water oxidation, was much slower than oxygen release as measured by the rise of the signal from a thin sample layer centrifuged onto a strongly polarized electrode (Miyao et al., 1986). Plijter et al. (1988) showed both theoretically and experimentally that at 100% cathode efficiency a first order oxygen release process in a stationary sample suspension in a closed cuvette produces a transient current increase with a rise time 7 times shorter than the time constant of the release process, and the situation of a very thin sample sediment on a very inefficient cathode under a stationary supernatant is mathematically the same. The combination of a thin layer, strong polarization, and oxygen removal by a flowing medium can only lead to a further acceleration of the signal. It seems inevitable, therefore, that oxygen release is at least 7 times slower than the rise of the signal one would expect to measure with a rate electrode. However, the observed rise kinetics of the signal is not understood. It is clearly sigmoidal and the initial delay is not accounted for in the model of Plijter et al. It might be due to the chloridedependent artifact described by these authors. A good fit can be obtained by assuming that all oxygen sources are confined to a plane at a distance close to or even exceeding the thickness of the sample sediment and release oxygen with a time constant several fold less than that of water oxidation (Lupatov, 1979; Lavorel, 1992), but this is clearly unrealistic. More likely values of these parameters do not allow an acceptable fit of the initial delay in the signal and the steep decline after its maximum (M.H. Vos, unpublished). Many authors have claimed inconsistency between results obtained with the method of Plijter et al. and those obtained with rate electrodes and other methods (Lavergne, 1989; Mauzerall, 1990; Strzalka et al., 1990; Jursinic and Dennenberg, 1990; Schulder et al., 1990, 1992; Meunier and Popovic, 1991; Tang et al., 1991; Lavorel, 1992; Ichimura et al., 1992; Joliot et al., 1992), but in

398 no case this was demonstrated for the same material in the same conditions. It is often overlooked that the release times given in Plijter et al. refer to a temperature of 5°C and decrease with increasing temperature by a factor of 2 per 20°C, and that shorter signal rise times were observed in samples which exhibit flash-induced oxygen uptake as well. It is clear from the previous section, however, that Plijter et al. overemphasized the importance of weak polarization. They actually found similar signal rise times at cathode potentials down to – 0.45 V (– 0.73 V vs. Ag/AgCl at which is well in the range of values commonly used but still implies a low cathode efficiency. The different signal shape obtained with rate electrodes must be primarily due to diffusion of oxygen out of the thin sample layer, away from the cathode. III. EPR Oximetry

A. EPR Line Broadening by Oxygen EPR oximetry has first been applied by the group of Y.N. Molin (Backer et al., 1977). This technique is based on the fact that molecular oxygen is paramagnetic. As a consequence, collisions between oxygen and a properly chosen spin probe, will increase spin-spin and spin lattice relaxation of this probe through the so-called Heisenberg exchange mechanism (Windrem and Plachy, 1980; Subczynski and Hyde, 1981; Popp and Hyde, 1981). For homogeneously broadened lines, this will result in broadening of the spectrum and changes in its microwave power saturation behavior. Under non-saturating conditions, at low microwave power, the EPR line will broaden and thus the amplitude of the first derivative signal, which is inversely proportional to the square of its linewidth, will decrease. In first approximation, the line broadening is often linearly dependent on the oxygen concentration (Windrem and Pachy, 1980; Glockner and Swartz, 1992). Measuring of oxygen levels by linewidth broadening can be done in two ways: a) direct measurement of the linewidth of the EPR signal, by recording of the EPR spectrum, and b) indirect measurement of the change in linewidth by recording the change in amplitude of the first derivative EPR spectrum. In the latter method,

Hans J. van Gorkom and Peter Gast the magnetic field sweep is turned off and the magnetic field is set at the maximum of the EPR signal. By comparing the amplitude changes at low and high microwave power distinction can be made between changes due to oxygen concentration changes and those caused by disappearance of the radical through chemical reaction (Strzalka et al., 1986). Under saturating conditions, at high microwave power, an increase of the signal intensity may be observed under certain conditions, due to de-saturation at elevated oxygen levels, and recently this effect has also been used to measure oxygen concentration changes (Ligeza et al., 1994). EPR oximetry is non-invasive, does not consume oxygen or produce harmful reaction products and does not require stirring of the sample. It can be used to measure the oxygen concentration in very small systems, like cells. In such experiments a spin probe is added that penetrates the cell and the extracellular nitroxide signal is quenched by adding paramagnetic salts like ferricyanide (Salikhov et al.,1971; Swartz, 1978), which do not enter the cell. The EPR signal of the extracellular nitroxide is broadened to such an extent that it virtually disappears and only the signal from intracellular nitroxides remains. Spinlabels can also be distributed homogeneously throughout an organ or whole body, and 2D and 3D EPR oximetry imaging is possible (Bacic et al.,1988; Demsar et al., 1988; Swartz and Glockner, 1989). For a review on EPR oximetry and its many uses, see Swartz and Glockner (1989).

B.

Oxygen Probes for EPR

A prerequisite for using EPR in oximetry is the presence of a (stable) radical in the system under study. Free radicals are rare in biological systems, and stable radicals are almost absent (an exception to this is melanin, which can also be used as oxygen probe (Sarna et al., 1980). Therefore these radicals have to be added. For this purpose nitroxide radicals have been widely used and characterized in spin-label experiments as ‘reporter’ molecules. A major disadvantage of EPR oximetry has been the fact that many spin labels and especially the nitroxides are rather toxic. Another complica-

Oxygen evolution tion is that there are many parameters other than oxygen that will broaden the EPR line of nitroxide radicals, like the presence of paramagnetic ions (Salikhov et al.,1971), nitroxide concentration, viscosity, microwave power, pH and temperature. Therefore it is necessary to calibrate the oxygen effect for each system and to keep parameters like temperature, microwave power and nitroxide concentration constant. Furthermore, EPR oximetry is not very sensitive, especially at high oxygen concentrations (see below). The new oxygen-sensitive probes that have been introduced by the group of H. Swartz in recent years (Swartz et al. 1991; Glockner and Swartz, 1992; Liu et al., 1993) seem to overcome the above mentioned shortcomings of the traditional, nitroxide-based EPR oximetry. They are fusinite (a certain type of coal), certain chars, soot, lithiumphthalocyanine crystals (PcLi) (Turek et al., 1987) and even Indian Ink. What these new spin-probes have in common is that they are all highly insoluble in most solvents, very sensitive to oxygen, non-toxic, virtually insensitive to the environment and extremely stable (although PcLi in water may be less stable than previously thought, M. Moussavi, personal communication). They are solid particles and measure the oxygen partial pressure, whereas the soluble nitroxides measure concentration (Povitch, 1975) unless enclosed in lipid droplets (Ligeza et al., 1994). The sensitivity and nontoxicity of the new probes has been demonstrated by measuring changes in oxygen concentration in the brain of a living mouse, in living cells, liver, and heart; their stability was convincingly shown by recording oxygen levels in a rat’s leg muscle over a period of more than 150 days (Glockner and Swartz, 1992; Liu et al., 1993).

C. Sensitivity When calculating the sensitivity of an EPR oxygen-probe, distinction should be made between the two ways of measurement: a) direct linewidth broadening measurement and b) amplitude measurement. The amplitude measurement is more sensitive, but cannot always be used since in many systems the nitroxides are rapidly bio-reduced to EPR silent hydroxylamines (Swartz et al., 1986).

399

This means that the spin-concentration is not constant. 1. Sensitivity in Direct Linewidth Broadening Measurement If the broadening is proportional to the oxygen concentration (Windrem and Pachy, 1980; Glockner and Swartz, 1992) the linewidth can be written as: The minimal change in linewidth that can be measured will depend on the linewidth itself. This is the reason why often deuterated nitroxides are used because of their reduced linewidth. Based on experimental facts, it is not unreasonable to state that for an EPR signal with reasonable signal-to-noise ratio (S/N) a change of 5% in linewidth of an EPR signal can be observed. Therefore:

and If the proportionality constant c is determined from the linewidths at 0 and at the latter being about the concentration in air-saturated water at 20°C (Hitchman, 1978), we get: For the deuterated nitroxide TEMPONE, with and (Swartz and Pals, 1989) the minimally detectable change in oxygen concentration at is about and at this value has increased to 20 The most sensitive probe, PcLi, with a 14 mG and will have a minimally detectable change at of about 200 nM and at of 15 2. Sensitivity in Amplitude Measurement This method is in principle much more sensitive than the linewidth measurement, since changes in amplitude are more easily measured than changes in linewidth and because the amplitude of the first derivative of an EPR line is proportional to the square of the inverse of the line-

400

Hans J. van Gorkom and Peter Gast

width. The minimally detectable oxygen-change with this method will depend on the S/N of the probe signal. Although there are limitations to the maximum concentration for spin-labels (due to line broadening at high concentration), it is not unreasonable to assume that the S/N at can be 1000 or more for many systems (taking the number of spins/g in PcLi as (Glockner and Swartz, 1992) and the sensitivity of a commercial X-band EPR spectrometer as spins/Gauss, 10 nanogram will be sufficient to achieve this S/N under anaerobic conditions). If we further assume that the minimal detectable amplitude change is a change with S/N = 1 we obtain: which is 100 times less than that obtained in a linewidth measurement. For d-TEMPONE the detection limit becomes at and 75 nM at 0 for PcLi it becomes 150 nM at 300 and 2 nM at 0 The last value is probably comparable to the minimum flash yield detectable with a polarographic rate electrode. It should be noted, however, that these numbers apply in rather ideal situations. For instance, when EPR oximetry is used in a whole-body system using a low-Q surface probe instead of a standard high-Q cavity, and using low microwave frequency to increase microwave penetration (Nilges et al., 1989; Bacic et al., 1989), or in timeresolved EPR oximetry, the sensitivity may be greatly reduced. If one can exclude or correct for spin-label reduction, both methods will give identical results, taking the quadratic decrease of the amplitude with increasing linewidth into account. However, in time-resolved studies where the magnetic field sweep is turned off and the field is set at the maximum of the EPR signal, deviating results may be obtained when the changes in oxygen concentration are large. This is due to the fact that when the oxygen concentration during the measurement is increased or decreased, the linewidth will broaden or narrow, respectively, and as a result, the magnetic field position will ‘slide off the peak of the first derivative spectrum. This is shown in Fig. 2. Here the calculated response is depicted of the EPR amplitude, set at the maxi-

mum of the EPR line at t = 0, to an oxygen concentration increase with a time constant of 200 ms. When the change is small, the signal amplitude reflects the oxygen evolution kinetics. However, when the change in oxygen concentration is large, a more rapid and non-exponential curve is observed. As expected, the deviation becomes significant at much smaller oxygen concentration changes with PcLi than with d-TEMPONE, since PcLi has a much sharper EPR line. In photosynthetic material, such large changes may occur locally for a short time. For instance, a chloroplast suspension of a chlorophyll concentration of 1 mM may produce more than oxygen on a flash and all that oxygen may initially be confined to a small fraction of the volume: the chloroplasts occupy about 1/40 and the thylakoids only 1/300

Oxygen evolution of the suspension volume (Heldt et al., 1973). On a time scale of some ms, oxygen diffusion processes and the non-linearity of the EPR amplitude may complicate the observed kinetics, depending on the microscopic distribution of the oxygen and that of the spin probes used. On the other hand, this might become a unique tool to obtain time-resolved information on the microscopic distribution of oxygen.

D. Applications in Photosynthesis Research Up to now only a few reports have appeared on EPR oximetry using photosynthetic material. Strzalka et al. (1986) used the spin label TEMPONE to measure oxygen evolution in thylakoid membranes of spinach. They measured amplitude changes and used the difference in microwave power saturation behavior to distinguish between production of oxygen and photoreduction of the probe, which could be controlled by adding sufficient amounts of an electron acceptor (p-benzoquinone). Belkin et al. (1987) have used EPR oximetry to measure light induced oxygen production in whole cells of cyanobacteria under continuous illumination. Direct linewidth as well as amplitude measurements were used in this study. By the paramagnetic agent the EPR signal from the extracellular oxygen probe was broadened beyond detection and the intracellular oxygen production and photo-inhibition was measured selectively. In a second report from Strzalka et al. (1990) the more sensitive probe perdeuterated TEMPONE was used to measure the oxygen release time in thylakoids after a short flash by the change in EPR amplitude. This was found to be 0.4–0.5 ms, which is much shorter than the 2 ms time constant of water oxidation and may perhaps have been caused by the non-linearity discussed above. Perhaps contrary to the soluble nitroxides, the new, solid spin probes should allow unambiguous distinction between free oxygen and oxygen still associated with the photosynthetic system, if the slow release process postulated by Plijter et al.(1988) exists. The only report so far on the use of PcLi crystals as an oxygen probe in photosynthetic material is from Tang et al. (1991), who found by amplitude measurements an oxygen release time in PSII membranes of 1–2 ms. In a recent article by Dis-

401 mukes et al. (1994) TEMPONE oximetry was used to demonstrate hydrogen peroxide production in photosystem II membranes inactivated by depletion: oxygen production was restored not only by readdition of but also by addition of catalase. With oxygen polarography, such experiments are complicated by the production of hydrogen peroxide at the cathode. Ligeza et al. (1994) used the oxygen-dependent desaturation at high microwave power to measure oxygen evolution in leaves, after injection of an emulsion containing a nitroxide enclosed in droplets of paraffin oil covered with serum albumin. This system largely prevents photoreduction of the nitroxide. A detection limit of oxygen was reported.

IV. Mass Spectrometry Unlike most processes discussed in this volume, photosynthetic oxygen evolution involves the rearrangement of nuclei to form one kind of molecule from another and can be studied by traditional biochemical tools using labeling of the substrate by nuclear isotopes and measuring the isotope composition of the product. The usefulness of mass spectrometry in photosynthesis research was established by Hoch and Kok (1963), who replaced the sample injection port of a conventional mass spectrometer by a vessel containing a stirred suspension of photosynthetic material separated only by a teflon membrane from the vacuum system. Later authors have mostly used a thin layer of photosynthetic material sedimented on the teflon membrane. The selective permeability of the membrane allows direct measurement of the gases dissolved in the sample solution. Sufficient sensitivity and time resolution can be reached to allow individual flash yields in a series to be analysed. The method has been reviewed by Radmer and Ollinger (1980). It has more recently been used e.g. to measure oxygen uptake kinetics during the induction of oxygen evolution upon continuous illumination (Peltier and Ravenel, 1987), to distinguish oxygen evolution from water and that from hydrogen peroxide (Mano et al., 1987), and to prove that water oxidized on the to transition is still exchangeable in (Radmer and Ollinger, 1986; Bader et al., 1987), the kinetics of which have

402 now been measured with 30 ms time resolution (Messinger et al., 1995). Photosynthetic oxygen evolution is almost always accompanied by light-dependent oxygen uptake processes and an unambiguous distinction between the two can normally not be made by other methods, which measure only the net change in oxygen concentration. But also mass spectrometry may not help. It should be kept in mind that these uptake processes take place at short distance and may selectively consume the oxygen just produced photosynthetically. In fact that can be the cause of their light-dependence, as was strikingly illustrated by the measurement of a period four oscillation in the oxidation of mitochondrial cytochrome c upon flash illumination of a green alga (Lavergne, 1989). Perhaps a quantitative trapping of photosynthetic oxygen before it can leave the cell may explain the apparent oxygen dependence of oxygen evolution reported for a cyanobacterium (Bader and Schmid, 1988).

Hans J. van Gorkom and Peter Gast VI. Galvanic Sensors Galvanic sensors to detect oxygen in the gas phase have been developed for technical applications mainly (Kleitz and Fouletier, 1976; Heyne, 1976), but there are a few reports of the use of zirconium oxide detectors in photosynthesis research (Björkman and Gaul, 1970; Greenbaum and Mauzerall, 1976; Meyer et al., 1989). These devices are based on the phenomenon that a mixed crystal of conducts at high temperatures (around 800°C) and can be used as a solid electrolyte between two Pt electrodes exposed to the gas to be measured and to a reference gas, respectively. Due to the required transport and drying of the gas the time-resolution is poor and the flash number dependence of the oxygen yield can be determined only by the cumulative method (measuring the total yield of a series of 1, 2, 3, etc. flashes). The main advantages of the technique are that it combines single flash sensitivity with easy quantitative calibration, and that there is no chemical interference between sample and detector.

V. Photoacoustic Spectroscopy VII. Prospects The traditional volumetric measurement of photosynthetic oxygen evolution by the Warburg technique may now be obsolete, but it has a modern pendant: the pressure increase in the gas phase due to oxygen evolution causes a ‘photobaric’ contribution to photoacoustic signals (Bults et al., 1982). Photoacoustic spectroscopy is reviewed elsewhere in this volume by S. Malkin (Chapter 12). The photobaric oxygen signal can be measured with high sensitivity and time resolution. It shows the characteristic period four oscillation in a flash series (Canaani et al., 1988) and the flash-induced kinetics have been studied with ms time resolution (Mauzerall, 1990). The concomitant photothermal signal, oxygen uptake phenomena in the leaf discs used, and the use of an open microphone and a.c. amplification led to convoluted signal kinetics, but their analysis according to Mauzerall suggested that oxygen release was fast. The use of a very thin layer of a PS II preparation which does not show oxygen uptake, and a pressure transducer as detector, might indeed open the road to obtain conclusive evidence on the oxygen release time.

There is no single ‘best’ way to measure photosynthetic oxygen evolution. Each of the techniques described above has its own specific possibilities and drawbacks, and different experimental situations may call for different methods of measurement. Moreover, all these methods still hold potential for further development. Polarography, being relatively cheap and easy to implement, continues to be a rewarding playground for inventive experimenters. The application of EPR oximetry in photosynthesis research is in an early, exploratory phase and is just beginning to prove its value; mass spectrometry will remain indispensable to unravel the interplay between oxygen evolution and oxygen consuming processes in photosynthetic organisms. Methods which have so far only been studied for their own interest, but also phenomena now considered as troublesome complications of established methods, may suddenly develop into valuable tools to solve a particular problem. Despite its long history, research on the mechanism of photosynthetic oxygen evolution is

Oxygen evolution still in full swing and the oscillatory phenomena caused by the 4–flash redox cycle of the oxygen evolving complex have not lost their charm. It is to be expected that oxygen measurements will continue to play an essential role in their study. Acknowledgements We are grateful to Dr. M.H. Vos for advice and extensive fitting of time-resolved polarographic data kindly supplied by Dr. J. Lavorel, and to Dr. G.H. Schmid for pointing out the benefits of collodion films and how to apply them. Research in this laboratory was supported by the Netherlands Foundation for Chemical Research (SON), financed by the Netherlands Organization for Scientific Research (NWO). P.G. is a research fellow of the Royal Netherlands Academy of Arts and Sciences (KNAW). References Bacic G, Demsar F, Zolnai Z and Swartz HM (1988) Contrast enhancement in ESR imaging: role of oxygen. Magn Res Med Biol 1: 54–65. Bacic G, Nilges MJN, Magin RL, Walczak T and Swartz HM (1989) In vivo localized ESR spectroscopy reflecting metabolism. Magn Res Med 10: 266–272. Backer JM, Budker VG, Eremenko SI and Molin YN (1977) Detection of the kinetics of biochemical reactions with oxygen using exchange broadening in the ESR spectra of nitroxide radicals. Biochim Biophys Acta 460: 152–156. Bader KP and Schmid GH (1988) Mass spectrometric analysis of a photosystem II mediated oxygen uptake phenomenon in the filamentous cyanobacterium Oscillatoria chalybea. Biochim Biophys Acta 936: 179–186. Bader KP, Thibault P and Schmid GH (1987) Study on the properties of the by mass spectrometry in the filamentous cyanobacterium Oscillatoria chalybea. Biochim Biophys Acta 893: 564–571. Baumgärtl H, Grunewald W and Lübbers DW (1974) Polarographic determination of the oxygen partial pressure field by Pt microelectrodes using the field in front of a Pt macroelectrode as a model. Pflügers Arch 347: 49–61. Belkin S, Mehlhorn RJ and Packer L (1987) Determination of dissolved oxygen in photosynthetic systems by nitroxide spin-probe broadening. Arch Biochem Biophys 252: 487– 495. Björkman O and Gaul E (1970) Use of the zirconium oxide ceramic cell for measurement of photosynthetic oxygen evolution by intact leaves. Photosynthetica 4: 123–128. Blinks LR and Skow RK (1938) The time course of photosynthesis as shown by a rapid electrode method for oxygen. Proc Natl Acad Sci USA 24: 420–427. Bulls G, Horwitz BA, Malkin S and Cahen D (1982) Photoac-

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Index Since almost all chapters deal with chlorophyll or bacteriochlorophyll, these words are not used as entry in this index.

A absorbance-detected magnetic resonance, see ADMR, ODMR absorption spectrum 4-6 derivative of 4 dichroic 13, 16, 18, 28, 34 difference, light-induced 4-6 difference, reaction modulated infrared 153 difference, redox-induced 148 difference, electric field-induced 177-188 FTIR 137-157 hole burning 123-134 line width 43, 110, 125 transient 70, 71 X-ray 337-352 accumulated photon echo spectroscopy 109-120 ADMR 277-295 amorphous ice 327 anisotropy of absorbance 14 of emission 26 function 20 of time-resolved fluorescence 57 of transition dipoles 24 antenna, see LH 386 atomic scattering factor 318 temperature factor 318 averaging, a-periodic 328

B bacteriopheophytin 164, 169, 185, 186, 289 bottleneck state 113

C C-phycocyanin 57 windows 155, 156 caged compounds 148 carotenoids 27, 161, 166, 168, 185, 289, 290, 295, 305 charge pairs 97 formation in PSII 95 half lives of 99 recombination of 91 stabilization of 95 chemical shift tensor 306 chemically induced dynamic spin polarization 212 chirality 24, 25, 27, 30 chirally induced differential scattering (CIDS) 30-32 Chlamydomonas reinhardtii 69, 365 Chlorobium limicola 167, 171, 269 tepidum 131 Chloroflexus aurantiacus 29, 148, 292 Chlorogloea fritschii 370, 371 Chromatium vinosum 147, 172 chlorosome 6, 30

circular dichroism 24-34 excitonic 28 fluorescence-detected 13, 26, 34 intrinsic 28 long-range interaction 26 orientation dependence 26 psi-type 27, 31, 33 vibrational 26 Clark electrode 392 concentration electrode 395 Conne’s advantage 143 correlation spectroscopy 309 Cotton effect 25 cross-polarisation 301 cryo-electron microscopy 327, 328 crystal structure, determination of 317-323 cytochrome 359, 365, 375 cytochrome 371

D data fitting 78 dephasing 112, 118 dichroic ratio 18 dichroism, reduced 18 dielectric asymmetry 188 screening 187 difference spectrum, see absorption spectrum differential polarization images 22 microscopy 13 scattering 25 dipolar interactions 301 dipole moment 178 strength 15 dispersion model 119 double exchange 369 double resonance 277, 284, 285, 294, 295

E electric field 51, 177, 178 gradient 358 orientation 24 electroabsorption 177 electrochemical cell 147 oxidation 148 electrochromism, see Stark spectroscopy electrode, Clark 392 concentration 395 electron transfer at 148 gold grid 147 rate 394 electron crystallography 332 density map 320, 322 microscopy 326, 331

Index

408 nuclear double resonance, see ENDOR paramagnetic resonance, see EPR spin echo, see ESE spin echo envelope modulation, see ESEEM electron-phonon coupling 118, 130 Eligeron canadensis 103 emission, see fluorescence, phosphorescenc ENDOR 255-272 bacteriochlorophyll 256 Davies ESE 243 electron acceptors 271 general TRIPLE 261 in frozen solution 263 in liquid solution 258 in single crystals 265 Mims ESE 245 of 269 stochastic 267 TRIPLE 260 triplet states 265, 271 energy storage 195-199 transfer 6, 44, 87, 116, 125, 133, 213, 217, 226 EPR 211-229, 258, 278, 280, 289, 294, 295 direct detection 215 ENDOR-induced 262 Fourier transform 215-217, 226 multiline signal 248, 350 oximetry 398 pulsed 249 time-resolved 211-229 error analysis 85 ESE 235-252 ESEEM 238 EXAFS 338 definition 340 equation 341 Fe 346 Mn 349 theory 339 excitation transfer, see energy transfer excited state, see exciton, energy transfer exciton, annihilation 54 bands 26 circular dichroism 28 coherence 116 coupling 25, 29 dynamics 109, 110 interaction 29, 30, 33 manifold 117 scattering 116 states 116 extended X-ray absorption fine structure, see EXAFS

F FDMR 277, 278, 280-284, 288, 289 Fe centers 348, 355 K-edge spectra 347 quinone complex 365, 366 Fe (II) high-spin 361

Fe (III) high-spin 360 Fe (II) low spin 360 Fe(II) NO complex 367 Fe-S acceptors in photosystem I 346 Fe-S proteins 345, 346, 362, 368 Felgett’s advantage 143 Fenna-Matthews-Olson, see FMO film-stretching 21 flattening effect 4, 26 fluorescence, depolarization of 44, 68 emission 6, 18, 47 spectrum 6, 7, 46 lifetime 44 microwave-induced 278, 285 polarization of 18 relaxation 45 self absorption 48 single photon counting 52 spectrophotometer 45 time-resolved 52, 55 upconversion 53, 64 yield 6, 45, 197, 198 fluorescence-detected magnetic resonance, see FDMR, ODMR FMO complex 7, 116, 117, 131 force field 140 formate 367 Fourier analysis 330 transform infrared, see FTIR transform EPR 215-217, 226 Fourier-peak filtering 330 Franck-Condon principle 43 frequency grating 112 FTIR 137-137, see also infrared amide I mode 144 amide II mode 144 difference spectra 140, 145, 147, 148 protein modes 155 spectrophotometer 141, 145, 153, 154 spectroscopy 137-157, 306 techniques 138 time resolved 148 fusinite 399

G g-factor 258 galvanic sensors 402 gel-squeezing 19, 21 glow curves 94, 98 gold grid electrode 147 group frequencies 140, 141

H Hartmann-Hahn condition 301 Heliobacterium chlorum 292 heterogeneity 278, 289. 294, 295 high pressure hole burning 128 hole burning 117 double resonance ODMR 289

Index high-pressure 128 non-photochemical 124 photochemical 124 spectroscopy 114, 123-134 transient 124 homodyne detection 114 homogeneous linewidth 110 Huang-Rhys factor 125 hydrogen peroxide 393 hyperfine interaction 249, 258, 356, 357

I infrared 137-157 cell 155 detector 137, 141, 143, 144 difference spectroscopy 140, 145, 148 dispersive spectroscopy 153 lasers 153 photometer 153, 154 picosecond spectroscopy 137, 154, 155 spectroscopy 137-157 windows 156 inhomogeneous broadening 43, 125 linewidth 43, 110 interferogram 149 interferometer 141, 142, 149, 152 internal conversion 44 intersystem crossing 44 isomorphous replacement 319 isotope labelling 140, 312

J Jacquinot’s advantage 143

K K-edge spectra 338, 347, see also EXAFS, XANES Krönig-Kramers transforms 25

L Langmuir Blodgett film 16 laser, argon ion 64, 126 diode 127 dye 64, 164 Nd:YAG 165 picosecond infrared 137 regenerative amplifier 65 spectroscopy 63-72, 154, 155 Ti:Sapphire 64-66 tunable infrared 137, 154 LH1 67, 87, 170, 386 LH2 386, 388 LHC II 27, 29-33, 69, 117, 328, 331, 332 light-harvesting, see LH1, LH2, LHCII light scattering 4, 33

409 light-induced difference spectroscopy 147 linear dichroic T-S 286, 278, 290 linear dichroism 13, 16, 18. 34, see also orientation liquid crystal 218, 228 lithium phthalocyanine 399

M macro-organization of chromophores 26, 27, 30, 32 magnetic circular dichroism (MCD) 26, 34 mass spectrometry 401 metallo-proteins 338 microwave-induced absorption (MIA) 278-285 fluorescence (MIF) 278-285 phosphorescence (MIP) 278-285 micelles 380 Mn, see also oxygen evolving complex Mn cluster 246, 249 EXAFS 349 K-edge spectra 350 molecular dynamics calculation 322 molecular replacement 319 Mössbauer spectroscopy 355-372 Mueller images 30 matrix 14, 34 multiplex advantage 143, 151

N negative staining 327, 328, 333 nitroxide radicals 398 NMR 335 chemical shift tensor 306 CIDNP 309 CP/MAS 299, 300 cross polarization 301 Hartmann-Hahn 301 solid state 299-313 two-dimensional 311 non-photochemical hole burning 124 normal mode analysis 140 nuclear magnetic resonance, see NMR Hamiltonian 359

O ODMR 277-295 time-resolved 280 optical rotary dispersion (ORD) 25 optically detected magnetic resonance, see ODMR orientation, angle 17, 21, 22, 28 by electric field 20 by film-stretching 21 by flow 19 by gel-squeezing 19, 21 by magnetic field 20 mechanical 19 selection 263 orientation-dependent circular dichroism 26

410 oxygen diffusion 201 oxygen evolution 391 by photoacoustics 200-202 limiting rate 201-202 S-states 95, 200, 201 oxygen evolving complex 95, 99, 246, 334, see also Mn double hits 100 misses 95 Mn complex 95, 348, 350 S-states 95, 200, 201

P PDMR 278, 280, 282 periodic averaging 328, 330 phase contrast 329 phonon sideband holes 124 Phormidium laminosum 364, 367 phosphorescence 45, 278, 280, 284, 286, 288 microwave-induced 278, 285 phosphorescence-detected magnetic resonance, see PDMR, ODMR photoacoustic spectroscopy 34, 191-204 photobaric oxygen signal 402 photochemical hole burning 124 photon echo 111, see also accumulated photon echo photoselection 18, 20, 287. 290 photosystem I 7, 69, 87, 94, 169, 249, 290, 293, 327, 330, 333, 346-348, 355, 367-371 photosystem II 7, 94-105, 169, 249, 290, 293. 327, 333, 346348, 355, 366, 371 polarizability 178 polarization microscopy 24 polarized IR spectroscopy 19 light 13, 25 polarography 392, 395 Prochlorothrix hollandica 30 Prosthecochloris aestuarii 29, 126 protein crystallography 318 proton release pattern 96 transfer 137 psi-type aggregates 27, 30-33 pulsed microwaves 281 pump-probe techniques 138

Q quadrupole coupling 262 interaction 358 quinone 147, 157, 220, 365, 366 replacement 140

R R-factor 321 radical-pair mechanism 224 Raman spectroscopy 161-173 rate electrode 394 reaction center 118, 129-131, 152, 166-170, 268-271, 288-

Index 296, 302-309, 321, 365, 375, 383 regenerative amplifier 65 resonance Raman spectroscopy 161-173 Rhodobacter capsulatus 68, 71, 135 sphaeroides 68, 69, 71, 118, 125, 129, 133, 147, 155, 166, 169, 170-172, 180, 182, 184, 269, 271, 289, 291, 300, 303-309, 321, 364, 365, 388 Rhodopseudomonas acidophila 388 marina 32 palustris 166 viridis 8, 29, 129, 130, 133, 147, 155, 166, 169, 172, 271, 289, 291, 303-309, 321 Rhodospirillum 388 Rieske Fe-S clusters 348 rotational correlation time 260

S secondary structure 32, 144 sieve effect 4, 26 simulated annealing 322 single-photon counting 52 single-particle averaging 332 small-angle neutron scattering 375-389 space group 319 spectroelectrochemical cell 156 spin coupling 361 Hamiltonian 258, 357, 360, 361 label 398 polarization 212 Stark spectroscopy 177-188 step-scan interferometer 152 Stokes parameters 14, 15 shift 47 Synechococcus elongatus 330, 332, 367 Synechocystis 103, 333 structure factor 318 subtractive mixing techniques 155 supramolecular assemblies 375 synchrotron radiation 343

T T-S spectrum 278, 285-293, 295 TEMPONE 399 thermal deactivation spectrum 198 thermoluminescence 93-104 activation energy 97 application 103 assignments 100 components 101 origin 94 oscillation 99 peak temperature 96 time-averaged structures 323 transient absorption 70, 71 transition dipole moment, electric 15, 16, 278, 279, 286, 287, 290-295 magnetic 25

Index TRIPLE resonance, see ENDOR triplet state 277-298 magnetic field effect on 199 mechanism 213, 218 reaction center 289 spin Hamiltonian 278 triplet-minus-singlet absorbance difference, see T-S two-dimensional crystallization 330 tyrosine 71, 249, 303

U unit cell dimensions 318

V vibrational modes 141 spectroscopy 138 wavepackets 71

W water-oxidation system, see oxygen evolving system

411 X X-ray absorption near edge structure, see XANES absorption spectroscopy 337-352 diffraction 317-323, 326, 335 linear dichroism 16 XANES 338,346 K-edge spectra 338, 346

Z Zeeman frequency 258 zero-quantum beats 228--29 zero-field splitting parameters 277, 278-280, 289, 293-295, 359, 402 zero-phonon hole 124 line 118 ZFS, see zero-field splitting zirconium oxide detector 402