Advances in Photosynthesis Research Proceedings of the Vlth International Congress on Photosynthesis, Brussels, Belgium, August 1-6, 1983

Volume I

edited by

C . S Y B E S M A

Biophysics Laboratory Vrije Universiteit Brüssel Β-1050 Brussels Belgium

1984 M A R T I N U S N I J H O F F / D R W. J U N K P U B L I S H E R S i ^ f l a member of the KLUWER ACADEMIC PUBLISHERS GROUP |SBj THE HAGUE / BOSTON / LANCASTER

CORE Metadata, citation and similar papers at core.ac.uk

Provided by Open Access LMU

ν G E N E R A L C O N T E N T S

Volume I

1. Excitation Energy Transfer 1

2. Primary Reactions 89

3. Oxygen Evolution 227

4. Photosynthetic Electron Transport I : Plants and Algae 399

5. Photosynthetic Electron Transport I I : Photosynthetic Bacteria 621

6. Physical Parameters; Special Methods; Model Systems 677

Volume I I

1. Pigment and Pigment-Protein Complexes 1

2. Reaction Center and Light-Harvesting Complexes I : Plants, Algae and Cyanobacteria 73

3. Reaction Center and Light-Harvesting Complexes I I : Photosynthetic Bacteria 153

4. Membrane Pontentials and Ion Gradients 233

5. Membrane Bioenergetics; Membrane Transport 371

6. Coupling Factors and ATPase 477

7. Cyanobacteria 625

8. Photosynthesis and Solar Energy Technology 727

Volume I I I

1. The Architecture of Photosynthetic Membranes I : General; Envelope Membranes; Membrane Fluidity; Stacking 1

2. The Architecture of Photosynthetic Membranes I I : Membrane Proteins and Lipids 91

3. The Architecture of Photosynthetic Membranes I I I : Photosystem Particles; Electron Transport Components 195

4. The Architecture of Photosynthetic Membranes IV: Dynamic Aspects 263

V I

5. The Architecture of Photosynthetic Membranes V: Prokaryotic Membranes 335

6. Carbon Metabolism I : Metabolic Pathways; Metabolites; Enzymes 381

7. Carbon Metabolism I I : Regulation, Induction and Activation; Transport 557

8. Ribulose Bisphosphate Carboxylase/Oxygenase 717

9. Photorespiration; Amino Acid Synthesis; Nitrogen Metabolism 811

Volume IV

1. Herbicides 1

2. The Productivity of Photosynthesis 85

3. Environmental Influences on Photosynthesis 181

4. Photosynthesis under Stress Conditions 345

5. Molecular Genetics of the Photosynthetic Apparatus 483

6. Chloroplast Development 595

7. Light-Controlled Development of the Photosynthetic Apparatus 783

V I I

C O N T E N T S T O V O L U M E I

Preface XXI

In Memory of Kazuo Shibata XXII I

Sponsors XXV

International Photosynthesis Committee 1980-1983 XXVII

Local Organizing Committee XXIX

1. Excitation Energy Transfer

Excitation Energy Transfer 1 D. Frackowiak

Energy Transfer: New Concepts 5 G. Paillotin

Photosynthetic Exciton Migration and Trapping 13 R. Pearlstein

Excitation Energy Transfer in Phycobilin-chlorophyll a System in Blue-green and Red Algae 21 M. Mimuro, I . Yamazaki, T. Murao, T. Yamazaki, K. Yoshihara, Y. Fujita

Optimization Factors of the Light-harvesting Antenna Structure Affecting the Rate of Excitation Energy Trapping by Reaction Centers 29 Z. Fetisova, M. Fok, A. Borisov

Energy Transfer and Geometry of Covalently Linked Porphyrin-dimers 33 L. Benthem, R. Koehorst, L. de Graaff, T. Schaafsma

Picosecond Pulse Energy Dependence of Singlet-singlet Exciton Annihilation and Fluorescence Induction in Chloroplasts 37 N. Geacintov, G. Paillotin, J. Deprez, A. Dobek, J. Breton

Theoretical Investigation of Energy Transfer in Bacterial Photosynthesis. Influence of Structural Heterogeneity 41 L. Valkunas, S. Kudzmauskas, V. Liuolia

V I I I

Excitation Energy Transfer in Bacterial Photosynthesis Studied by Picosecond Laser Spectrochronography 45 A. Freiberg, V. Godik, K. Timpmann

Charge Transfer State in Photosynthetic Bacteria Antenna Complexes 49 E. Gaizauskas, G. Trinkunas, L. Valkunas

Magnetic Field Effects on the Fluorescence of Rhodopseudomonas Capsulata 53 Η. Frank, W. McGann

The Formation of the Antenna Carotenoid Triplet in the Isolated B800-B850 Light-harvesting Complex of Rhodopseudomonas sphaeroides G1C 57 H. Kingma, R. van Grondelle, L. Duysens, F. van der Kooy, M. Vos

Fluorescence Lifetime Spectra of the c71 Reaction Center-less Mutant and of the B800-850 Pigment-protein Complex from Rhodopseudomonas Sphaeroides 61 P. Sebban, G. Jolchine, I . Moya

Picosecond Carotenoid Triplet Formation in the Antenna of Rhodospirillum rubrum as Measured by Absorbance Difference Spectroscopy 65 A. Nuijs, A. van Bochove, H. Joppe, L. Duysens

Excitation Energy Transfer in Fucoxanthin Containing Algae 69 L. Caron, H. Jupin, C. Berkaloff

Picosecond Fluorescence Kinetics and Energy Transfer in Antennae Chlorophylls of Green Algae and Membrane Fractions of Thylakoids 73 A. Holzwarth, W. Haehnel, J. Wendler, G. Suter, R. Ratajczak

Picosecond Kinetics of Energy Transfer in Isolated Phycobilisomes from Porphyridium cruentum 77 A. Holzwarth, J. Wendler, W. Wehrmeyer

Polarized Time-resolved Fluorescence of c-phycocyanin from Mastigocladus laminosus 81 W. John, H. Scheer, P. Hefferle, S. Schneider

Leaf Fluorescence Emission Characteristics as a Function of Excitation Wavelength 85 M. Percival, N. Baker

2. Primary Reactions

Electron Transport Chains of the Plant and Bacterial Photosystems Α. Hoff

89

I X

Main Features of the Primary Charge Separation in Photosynthetic Reaction Centers 93 V. Shuvalov

Probing Fluorescence Induction in Chloroplasts on a Nanosecond Scale Utilizing Picosecond Pulse Pairs 101 A. Dobek, J. Deprez, N. Geacintov, J. Breton

A Comparative Study of the Split pH - EPR Signal and of the 77 Κ Fluorescence Yield in Spinach Chloroplasts 105 J. Breton, A. Rutherford

Function of Chlorophylls and Carotenoids in Thylakoid Membranes. Is Pheophytin a Substantial Part of Photosystem I Reaction Centre in Spinach Thylakoids? 109 I . Damm, Τ. Gropper, Τ. Braumann, L. Grimme

Potentiometrie Titrations of the Fluorescence Induction Curves From Pea Chloroplasts 113 D. Bell, M. Hipkins

Fluorescence Redox Titrations in Isolated Chloroplasts 117 A. Schulze, G. Renger

Photosystem 2 and Water Oxidation in Higher Plants 121 K. Sauer, Μ. Boska, J. Casey, K. Karukstis

Primary Electron Transfer in the Photosystem I I Reaction Centre - Characterisation of the Electron Acceptor Complex 127 M. Evans, Y. Atkinson, R. Ford

Charge Separation in Photosystem-II Reaction Centers. The Role of Pheophytin and Manganese 131 V. Klimov

Electron Carriers in Photosystem 2 from Phormidium laminosum 139 Y. Atkinson, M. Evans

Time-resolved Optical Spectroscopy in Photosystem 2: Ki Modifies P680+

Reduction Kinetics 143 J. Warden, P. Lyford

Kinetics of P680+ Reduction in Oxygen-evolving Photosystem 2 Preparations from Lettuce and Phormidium laminosum 147 R. Ford, M. Evans, Y. Atkinson

Heterogeneity of Photosystem I I Acceptors 151 R. Meiburg, H. van Gorkum, R. van Dorssen

Influence of Temperature on the Variable Fluorescence of Purified Photosystem-II and Light-harvesting Particles 155 P. Mathis

χ

Short-lived Fluorescence Quenchers in PS I I of Green Plants 159 G. Laczko, P. Maroti, L. Szalay

Effects of Continuous Background Irradiance on Xenon-flash-induced Fluorescence Yields in Marine Microalgae 163 P. Falkowski, K. Wyman, D. Mauzerall

Analysis of the System I I Reaction Pattern by UV-absorption Changes in Tris-washed Chloroplasts 167 W. Weiss, G. Renger

Absorbance Changes of Z + , the Component Responsible for EPR Signal I I Fast in Tris-treated Photosystem I I Particles 171 J. Dekker, M. Brök, Η. van Gorkum

Comparative Measurements of the Chl-a* Reduction Kinetics in Oxygen Evolving PS I I Particles at 680 nm and 874 nm 175 E. Schlodder, K. Brettel, G. Schatz, Η. Witt

Structure-function Studies of Chloroplast Photosystem I 179 R. Malkin, E. Lam, W. Ortiz

Primary Reactions in Bacterial Reaction Centers 187 W. Parson, D. Holten, C. Kirmaier, A. Scherz, Ν. Woodbury

A Temperature Dependence Study of the Fe^ -Q Acceptor in PS I I Particles by Mössbauer Spectroscopy 195 V. Petrouleas, B. Diner

Evaluation of the Plastoquinone Pool Size in Oxygen-evolving Photosystem I I (OES II) Preparations 199 J. Lavorel, M. Seibert, Β. Maison-Peteri, J. Briantais

Primary Photochemistry and Pigment Composition of Reaction Centers Isolated from the Green Photosynthetic Bacterium Chloroflexus aurantiacus 203 R. Blankenship, L. Mancino, R. Feick, R. Fuller, J. Machnicki, Η. Frank, C. Kirmaier, D. Holten

Inner Structure and Primary Reactions of Bacterial Reaction Centers, Studied by Electrical Techniques of High Time Resolution 207 H. Trissl

Nanosecond Time Resolved Magnetic Resonance Spectra of Pf in R. sphaeroides and in R. viridis Reaction Centers. Orientation of the Primary Donor in Single Crystals of R. viridis Reaction Centers 211 M. Wasielewski, P. Gast, J. Norris

Time-resolved EPR of Bacterial Photosynthetic Reaction Centers 215 A. de Groot, P. Gast, Α. Hoff

X I

Radical Pair Decay Characteristics in Chromatophores of Purple Bacteria 219 A. van Bochove, R. van Grondelle, R. Hof, L. Duysens

Magnetic Field Effects and nmr of Bacterial Reaction Centers 223 S. Boxer, C. Chidsey, J. Fisher, L. Takiff, C. Kirmaier, D. Holten

3. Oxygen Evolution

Photosystem I I : the Oxygen Evolving System of Photosynthesis 227 Govindjee

Photosynthetic Oxygen Evolution: an Overview 239 C. Yocum

Structures and Electron Transfer Reactions on the Oxidizing Side of Photosystem I I 243 G. Babcock, W. Buttner, D. Ghanotakis, P. O'Malley, C. Yerkes, C. Yocum

Studies on the Functional Mechanism and Structural Organization of Water Cleavage by System I I 253 G. Renger, W. Weiss

Thermoluminescence as a Probe of PS I I in Leaves 261 A. Rutherford, Govindjee, Y. Inoue

Oscillations of the Thermoluminescence Bands of Chloroplasts Induced by Flash Excitation 265 S. Demeter, I . Vass, Z. Rozsa

Studies of Oxygen Evolution Using Substituted Hydroxylamine and Hydrazine Compounds as Water Analogs 269 R. Radmer, O. Ollinger

The Effect of Chloride Deprivation on Oxygen Evolution from Spinach Chloroplasts 273 J. Sinclair

A Probable Model of Catalytic Binuclear Manganese Complex for Photosynthetic Water Splitting and Oxygen Evolving Reaction 275 M. Kusunoki

Inhibitory Treatments of Oxygen Evolution and their Effects on Manganese Content, Z + Behavior and Polypeptide Composition 279 D. Ghanotakis, G. Babcock, C. Yocum

The Effect of Chloride and Other Anions on the Thermal Inactivation of Oxygen Evolution in Spinach Thylakoids 283 W. Coleman, I . Baianu, H. Gutowsky, Govindjee

X I I

S-states and Oxygen Evolution in a Filamentous Cyanobacterium 287 K. Bader

Relations Between the Auxiliary Donor D, State S2 and Unequal Misses Obtained from Oxygen and Fluorescence Measurements 291 M. Delrieu

Dependence of the Chl-a^ Reduction Kinetics on the S-state of the Oxygen Evolving System 295 K. Brettel, Ε. Schlodder, Η. Witt

Salt Treatment of Oxygen-evolving Photosystem I I Particles Affects the Higher S-state Transitions 299 J. Wensink, H. van Gorkum

Characterization of the Glow Peaks of Chloroplast Membranes: Relationship with S-states and Redox Components 303 P. Sane, T. Desai, S. Rane, V. Tatake

Studies on the Multiline EPR Signal Associated with State S2 of the Oxygen-Evolving System 307 0. Hansson, L. Andreasson, T. Vängard

Identification of Polypeptides Essential for Oxygen Evolution by Extraction and Mutational Analyses 311 K. Cammarata, N. Tamura, R. Sayre, G. Cheniae

Further Assessment through Mutational Analysis of Chloroplast Membrane Polypeptides Required in Water Photolysis 321 N. Bishop

The Oxygen Evolution System of Photosystem II Particles from Spinach Chloroplasts 329 N. Murata, M. Miyao, T. Omata, T. Kuwabara

Structure of the 0 2-evolution Enzyme Complex in a Highly Active 02-evolving Photosystem-II Preparation 333 Y. Yamamoto, M. Nishimura

Reconstitutability of 0 2 Evolution in Thylakoid Vesicles from Sonicated Chloroplasts 337 N. Ramaswamy, S. Izawa

Factors Affecting Inactivation and Reconstitution of 0 2 Evolution in the Isolated PS I I Complex 341 C. Yocum, P. Sandusky, D. Ghanotakis, G. Babcock

Binding of Polypeptides Involved in the Oxygen Evolution System to Photosystem II Particles 345 M. Miyao, N. Murata

Manganese Proteins from Spinach Thylakoids and 0 2 — Evolving Photosystem-II Enriched Membranes (OEPS II ) : Their Role in Oxygen Evolution D. Abramowicz, T. Raab, C. Dismukes

Manganese Release Following Inhibition of Photosynthetic Oxygen Evolution by Heat Treatment: Topology and Stoichiometry M. Miller, R. Cox

Preparation of Oxygen-evolving PS I I Particles from Soybean and the Identification of Polypeptide Involved in Photosynthetic Oxygen Evolution P. Tang, Y. Dai, C. Xu, F. Zhao, K. Wang, C. Li

Immunological Analyses of the Oxygen Evolving Complex with Special Emphasis on the 23 and 16 kDa Proteins C. Larsson, C. Jansson, U. Ljungberg, H. Äkerlund, Β. Andersson

Studies on the Polypeptide Composition of the Photosynthetic Oxygen Evolving Complex Using Photosystem 2 Particles U. Ljungberg, C. Jansson, C. Larsson, H. Äkerlund, Β. Andersson

Chemical and Physicochemical Characterization of the Proteins Involved in the Oxygen Evolution System T. Kuwabara, N. Murata

Proteins Involved in Photosynthetic Oxygen Evolution - Isolation and Characterization C. Jansson

Solubilization of Thylakoid Polypeptides and Inhibition of Photosynthet Oxygen Evolution L. Franzen, L. Andreasson

Comparative Biochemical Properties of Oxygen-evolving Photosystem I I Particles and of Chloroplasts Isolated from Intermittently-flashed Wheat Leaves Τ. Ono, H. Nakatani, E. Johnson, C. Arntzen, Y. Inoue

Polypeptide Composition Related to Function of Oxygen-evolving Photosystem 2 Particles from Anacystis nidulans R. England, E. Evans

Oxygen Flash-yield Sequence of Inside-out Thylakoids — Effects of Salt-Washing and Readdition of a 23 kDa Protein H. Äkerlund

Characterization of an Oxygen-evolving Membrane Fraction from the Cyanobacterium, Anacystis nidulans R2 H. Pakrasi, L. Sherman

X I V

4. Photosynthetic Electron Transport I : Plants and Algae

Mechanisms of the Electron Transfer between the Two Photosystems 399 P. Joliot, A. Joliot

Optical Spectrum and Kinetics of the Secondary Electron Donor, Z, of Photosystem I I 407 Β. Diner, C. de Vitry

Fluorescence Induction in Isolated Barley Protoplasts 413 P. Quick, P. Horton

The Effect of Cations and LHCP Phosphorylation on the Fo and Slow Phase of Fluorescence Induction Curves in DCMU-treated Pea Chloroplasts 417 M. Hodges, J. Barber

Effects of Linolenic Acid on Fluorescence Kinetics, Delayed Luminescence and P700 of Chloroplast Membrane 421 5. Lin, C. Lou, Q. Zhang, C. Tang, T. Kuang, P. Tang

How K + or Mg2 Affects Variable Fluorescence of Chlorophyll in situ 425 Y. L i , S. Ueng

Alpha and Beta Centres Revisited 429 G. Dohnt, G. Renger

Properties of Photosystem II-alpha and photosystem II-beta in Spinach Chloroplasts 433 T. Brearley, P. Horton

Elective Inhibition of Photosystem I I Reactions from Water Splitting to Q Reduction in Thylakoid Samples by Lauroyl Choline Chloride: Evidence from Fluorescence Transient Measurements 437 T. Wydrzynski, B. Huggins, P. Jursinic

A Photosystem Il-specific Calcium Site in Spinach Chloroplasts 441 R. Barr, F. Crane

EPR of PS II-interactions, Herbicide Effects and a New Signal 445 A. Rutherford, J. Zimmermann, P. Mathis

Active PS I I Particles from a Brown Alga: Spectral and Electrophoretic Characterization 449 C. Berkaloff, J. Duval, B. Rousseau

Active 02-evolving Photosystem I I Phycobilisome Particles from Porphyridium cruentum 453 J. Clement-Metrai, E. Gantt

Purification of a Functional PS I I Complex from the Blue-green Alga Phormidium Laminosum 457 J. Bowes, A. Stewart, D. Bendall

X V

Reactions of Quinones at Catalytic Sites; a Diffusional Role in H-transfer 461 A. Crofts, H. Robinson, M. Snozzi

Plastoquinone Binding Proteins in Photosynthetic Electron Transport 469 W. Oettmeier, K. Masson, H. Soll, E. Olschewski

Plastoquinone and Inhibitor Binding to Photosystem I I Particles from Chlamydomonas Reinhardtii 473 E. Neumann, B. Depka, W. Oettmeier

Kinetics of Proton Uptake and the Oxidation-reduction Reactions of the Quinone Acceptor Complex of PSII from Pea Chloroplasts 477 H. Robinson, A. Crofts

The Effects of Ca 2 + on Electron Transport Rates in Chloroplasts 481 E. Evans, S. Keskin

Photosystem I I / I Stoichiometry by the Methods of Absorbance Change at 325 and 705 nm, 14C-atrazine Binding, and Oxygen Flash Yields 485 P. Jursinic

A Mechanism for the ADRY Induced Photo-oxidation of Cytochrome b-559 489 C. Yerkes, A. Crofts

Evidence for a Heterogeneous Population of High Potential Cytochrome b-559 in the Thylakoid Membrane 493 M. Selak, B. Koch-Whitmarsh, J. Whitmarsh

Studies on Cytochrome b-559 in Higher Plant Thylakoid Membranes 497 D. Whitford, K. Gounaris, J. Barber

Purification and Partial Sequence of the Chloroplast mr= 10,000 Cytochrome b-559 Polypeptide 501 W. Widger, W. Cramer, M. Hermodson, M. Gullifor, D. Meyer

Asymmetric Position of the two Hemes of Cytochrome b6 in the Chloroplast Membrane 505 W. Widger, W. Cramer, R. Herrmann, A. Trebst

Role of Copper in Photosynthesis: new Sites of Action in the Electron Transport Chain 509 G. Horvath, M. Droppa, N. Terry

Photosynthetic Redox Proteins and Electron Transport in Iron- and Copper-depleted Cells 513 G. Sandmann

Low Potential Cytochrome c550 from Cyanobacteria and Algae 517 D. Krogmann, J. Alam, J. Sprinkle

The Primary Structure of Cytochrome f from Spinach and Cyanobacteria 521 J. Alam, D. Krogmann

X V I

The Spin State of Cytochrome b-563 in the Cytochrome bf Complex from Spinach Chloroplasts 525 J. Bergstrom, L. Andreasson, T. Vänngard

Kinetic and Spectroscopic Studies of Cytochrome b-563 in Isolated Cytochrome b/f Complex and in Thylakoid Membranes 529 G. Hind, R. Clark, J. Houchins

Conformational Changes in Spinach Plastocyanin 537 E. Gross, J. Draheim, R. Pan, B. Tripathy, G. Anderson, L. Rellick, J. Duane

On the Mechanism of Ferrodoxin-NADP+ Reductase: Involvement of Essential Histidines 541 N. Carrillo, R. Vallejos

On the Lateral Electron Transport Between the two Light Reactions in Spinach Chloroplasts 545 W. Haehnel

Quantitation of the Inter-system Electron Transport Carriers in Chloroplasts 549 T. Graan, D. Ort

Role of Carbonic Anhydrase in Electron Transport of Photosynthesis 553 S. Vaklinova, G. Lazova, L. Goushtina

Mechanism and Physiological Role of Bicarbonate Action on Electron Flow; a Possible Link Between Photorespiration and Photosynthetic Electron Flow? 557 J. Snel, A. Groote-Schaarsberg, J. van Rensen

Is the Rate of Oxygen Uptake by Reduced Ferredoxin Sufficient to Account for Photosystem I-mediated 0 2 Reduction? 561 J. Golbeck, R. Radmer

Evidence for two Electron Acceptors in the Intermediary Electron Carrier Complex of Photosystem I 565 J. Bonnerjea, M. Evans

Plastocyanin as Direct Electron Donor to P700 569 H. Bottin, P. Mathis

Influence of the Interaction of Ferredoxin-NADP Reductase with Thylakoids on the Catalysis of NADP Reduction 573 G. Forti, M. Bracale

Cyclic Electron Transport Around Photosystem I with Native and Artificial Cofactors 577 D. Moss, D. Bendall

X V I I

NADPH-dependent Cyclic Electron Transport of P-700 (ms) in the Presence of DCMU 581 Η. Rurainski, U. Stauder

Electron Paramagnetic Resonance Studies of the Primary Electron Acceptors of Photosystem I 585 J. McCracken,K. Sauer

Electron Acceptors in PS I : Comparison of A2 and X 589 P. Setif, P. Mathis, B. Lagoutte, J. Duranton

Effects of pH and Mg 2 on the Electron Transfer Reactions between p700-chlorophyll a Protein Complex and Plastocyanin 593 T. Takabe, H. Ishikawa, S. Niwa, S. Itoh

Carbodiimide-promoted Formation of a Covalent Complex between Ferredoxin and Ferredoxin-NADP+ Reductase from Spinach 597 G. Zanetti, A. Aliverti, B. Curti

The Production of Hydrogen Peroxide in Relation to Photosynthesis in Intact Chloroplasts from Spinach 601 M. Räntfors, Η. Egneus

The Dependence on pH of the Trypsination Pattern in Inside-out Thylakoids 605 M. Völker, G. Renger

Electron Transfer Patterns During C 0 2 Fixation 609 D. Crowther, P. Horton

Reactivation of Electron Transport in C02-depleted Chloroplasts by Bicarbonate is Influenced by Thylakoid Surface Potential 613 J. van Rensen, J. Hobe, R. Werner, J. Snel

The Effects of Loosely Bound Inhibitors as a Test of the Pool Function of Q in Plant Mitochondria 617 M. Fernandes Moreira

5. Photosynthetic Electron Transport I I : Photosynthetic Bacteria

Electron Transport in Green Bacteria 621 J. Amesz

The Acceptor Quinone Complexes of Purple Photosynthetic Bacteria 629 C. Wraight, R. Stein, R. Shopes, J. McComb

The Functionally Distinct Quinones of the Cyclic Electron Transfer Pathway of Rhodopseudomonas sphaeroides 637 R. Prince, D. Robertson, P. Dutton

X V I I I

Photosynthetic and Respiratory Electron Transfer Systems in an Aerobic Photosynthetic Bacterium Erythrobacter sp. Strain OCH 114 641 K. Okamura, K. Takamiya, M. Nishimura

A. Single Pool of Cytochrome c2 is Shared by Cytochrome Oxidase and Photoreaction Center in Rhodospirillum rubrum 645 C. Fenoll, S. Gomez-Amores, G. Gimenez-Gallego, J. Ramirez

A new Effect of Antimycin on the b Cytochromes of Rhodopseudomonas sphaeroides 649 S. Meinhardt, A. Crofts

Temperature Dependence of High-potential Cytochrome Photooxidation in C. vinosum 653 A. Sarai, D. Devault

Flash-induced Electron Transport by b- and c-type Cytochromes in Chromatophores of Rhodospirillum rubrum in the Presence of Various Inhibitors. The Oxidation of the High-potential b-type Cytochrome b-428 in the Presence of Antimycin 657 R. van Grondelle, H. van der Wal

Studies on the Mechanism of NAD + Photoreduction by Chromatophores of Rhodopseudomonas capsulata 661 D. Zannoni, R. Borghese, B. Melandri

Base-induced Luminescence From Rhodopseudomonas viridis 665 D. Fleischman, S. Perkins, M. Carlson

Opportunity for the Kinetic Resolution of Functional Cytochrome Orientations in Chromatophores of Rps sphaeroides 669 D. Tiede, Y. Choquet, J. Breton

Isolation and Characterization of Heme C-containing Proteins from Chromatium vinosum 673 D. Gaul, G. Gray,D. Knaff

6. Physical Parameters; Special Methods; Model Systems

EPR Studies of Signal I I at 9.1 and 35.2 GHz 677 M. Brök, Α. de Groot, A. Hoff

Action Spectra for Thermal Deactivation and Photosynthesis in Anacystis nidulans 681 R. Leblanc, R. Carpentier, B. LaRue

Further Characterization of Electrophotoluminescence in Chloroplasts 685 N. Symons, R. Korenstein, S. Malkin

Proton Spin-echo nmr Spectra of the Thylakoid Membrane 689 R. Sharp

X I X

Orientation of Absorbing and Fluorescing Dipoles in PS I and PS I I Particles 693 P. Tapie, S. Acker, C. Arntzen, Y. Choquet, P. Delepelaire, B. Diner, F. Wollman, J. Breton

A Proposed Monomeric Nature for Antenna and Reaction Center Chlorophylls 697 P. O'Malley, G. Babcock

Variation in the Orientation of Chlorophyll Forms in Scenedesmus as Shown by Dichroism of Fluorescence and Absorption 701 T. Szito , J. Kiss, L. Mustardy, A. Faludi-Daniel

Orientation of Phycobilin pigments in the red Algae Porphyridium: Electric Linear Dichroism of the Intact Membrane, Phicobilisome and its Dissociated Components 705 J. Hoarau, A. Gagliano, N. Geacintov, J. Breton

Laser-spectroscopic Results on the Homogeneous Broadening of the Red Chl-a Absorption Band in Primary Leaves of Triticum aestivum L. 709 P. Hoffmann, D. Leupold

Photoacoustic Spectra of Chlorophyll a and Chlorophyll b in Liquid Crystal Matrix 713 D. Frackowiak, S. Hotchandani, R. Leblanc, J. Szurkowski

Zeaxanthin in a Fraction with an Excess of Lutein 717 E. Meek, R. Strasser

Intramolecular Electron Transfer in Substituted Porphyrins 721 H. Krüger, R. Koehorst, A. van Hoek, T. Schaafsma, M. Michel-Beyerle

One Electron Oxidation of Carotene and Chlorophylls in Micelles. Electron Transfers Involving Carotene Cations and Chlorophyll Pigments 725 J. Chauvet, L. Viovy, E. Land, R. Santus, T. Truscott

Energy Transfer and Photoinduced Charge Separation in a Caroteno-porphyrinquinone Triad Molecule 729 T. Moore, P. Mathis, D. Gust, A. Moore, P. Liddell, G. Nemeth, W. Lehman, R. Bensasson, E. Land, C. Chachaty

Spectral Properties of Acridine Orange-detergent Solutions 733 J. Hevesi, E. Bälint

Energetic Characterization of the Thermoluminescence in Isolated Chloroplasts 737 I . Vass, S. Demeter

Energy Transfer in Chl-protein - Galactolipid Particles 741 D. Siefermann-Harms, H. Ninnemann, J. Ross, H. Yamamoto

Surfactant Manganese Complexes as Models for the Oxidation of Water 745 R. Wohlgemuth, J. Otvos, M. Calvin

X X

Reconstitution of Photosynthetic Oxygen Evolution in Liposomes 749 W. Butler, H. Matsuda

Diffusion Controlled Rates in Bacterial Photosynthetic Electron Transport as Studied by Fusion of Chromatophores of Rps sphaeroides with Liposomes 755 M. Snozzi, A. Crofts

The Effect of External pH on Photophosphorylation in Liposomes with Incorporated Granal and Stromal Photosystems 759 Ε. Kulig, J. Kieleczawa, W. Hendrich

Index of Names 763

1.1. 81

POLARIZED TIME-RESOLVED FLUORESCENCE OF C-PHYCOCYANIN FROM

MASTIGPCLADUS LAMINOSUS.

W. J o h n0

, I I . S c h e e r° , Ρ· H e f f e r l e+

, S. Schneider"*" Botanisches I n s t i t u t der Universität M ü n c h e n , FRG

+

I n s t i t u t für P h y s i k a l i s c h e und Theoretische Chemie, Technische Universität M ü n c h e n , Garching, FRG.

I n t r o d u c t i o n : P h y c o b i l i p r o t e i n s are photosynthetic l i g h t - h a r v e s ­t i n g pigments i n blue-green and red a l g a e , where they are organized - together with uncolored peptides - into complex s t r u c t u r e s , the phycobilisomes, attached to the outer t h y l a k o i d s u r f a c e . Within the phycobilisome the energy i s probably t r a n s ­f e r r e d by the Förster mechanism, but the d e t a i l s are yet i n ­s u f f i c i e n t l y understood. I n an approach complementary to the i n ­v e s t i g a t i o n of phycobilisomes and large f r a c t i o n s t h e r e o f , we are c u r r e n t l y studying i s o l a t e d p h y c o b i l i p r o t e i n s and t h e i r aggregates by time-resolved fluorescence spectroscopy. The expected under­standing of t h e i r chromophore-protein and chromophore-chromophore i n t e r a c t i o n s should give u s e f u l information on the energy t r a n s ­f e r i n the more complex systems. Since fluorescence i s one of the most s e n s i t i v e i n d i c a t o r s for any conformational changes of the chromophore, i t can also be used to study the ( p a r t i a l ) denatu-r a t i o n of b i 1 i p r o t e i n s . Here we wish to report on the fluorescence of C-Phycocyanin and i t s subunits from Mastigocladus laminosus.

B i l i p r o t e i n s : M. laminosus was grown on Castenholz medium and harvested a f t e r 3 days. C-Phycocyanin was i s o l a t e d with minor modifications as described e a r l i e r ( l ) . The subunits were se­parated by p r e p a r a t i v e e l e c t r o f o c u s i n g on Sephadex G 75 i n 8M urea on a S e r v a l y t g r a d i e n t , pH 4-9. The <x und ß subunits were eluted and renatured on a Biogel P6 column without delay. Phyco-cyanin aggregates ( t r i m e r s = (c*,ß)_) were d i s s o c i a t e d with NaSCN(lM) to monomers ( c * , ß ) . The Sedimentation c o e f f i c i e n t s of the samples (extrapolated to t = 0 ) , are shown i n t a b l e 1. The molecular weights and sedimentation c o e f f i c i e n t s of the d i s s o ­c i a t e d pigments correspond to the (cx,ß) monomer, those of the aggregates are between d i - and trimers (shape, p a r t i a l d i s s o c i ­a t i o n ? ) and have been i n t e r p r e t e d as trimers (εχ,β)^ from com­par i s o n with e a r l i e r r e s u l t s of other l a b o r a t o r i e s . A l l measure­ments were done i n potassium phosphate b u f f e r (80 mM, pH 6.0).

Measurement and Data A n a l y s i s : The fluorescence decay k i n e t i c s were measured with a synchronously pumped mode-locked dye l a s e r (rhodamine 6G, 80 MHz r e p e t i t i o n r a t e , pulse w i d t h ^ 2 p s e c ) and a r e p e t i t i v e l y working s t r e a k camera (nadland P h o t o n i c s ) . The time r e s o l u t i o n of t h i s system i s appx. 25 psec without deconvolution procedure. The apparatus allows measurements with small e x c i t a t i o n i n t e n s i t i e s (10*3 photons per pulse and cm^) with good s i g n a l to noise r a t i o due to the high r e p e t i t i o n r a t e . P o l a r i z e r s were placed i n the e x c i t a t i o n and emission path for p o l a r i z e d measure­ments .

Sybesma, C. (ed.), Advances in Photosynthesis Research, Vol. I. ISBN 90-247-2942-4. © 1984 Martinus Nijhoff/Dr W. Junk Publishers, The Hague/Boston/Lancaster. Printed in The Netherlands.

1.1. 8 2

From the s e p a r a t e l y recorded p o l a r i z e d fluorescence decay curves I _ l ( t ) and I// ( t ) , the functions I

0 ( t ) - I

lf (t)+2« I ^ ( t ) and

= 1

/ / [ ( t ) - I j L ( t ) are c a l c u l a t e d (dotted curves i n the fig u r e s ) . The b e s t f i t s for both functions ( l

0 ( t ) and D ( t ) ) are deter­

mined under the assumption of a biexponential response function by means of a Marquardt-Algorithm. The f i t curves as w e l l as t h e i r decay times and r e l a t i v e amplitudes are shown i n the f i g u r e s .

R e s u l t s and D i s c u s s i o n :

Rather complex decay laws can be expected to d e s c r i b e the f l u o r e s c e n c e decay curves i n view of the composite nature of the systems under study. The p r e s e n t l y a v a i l a b l e S/N r a t i o and dynamic range does not allow a d i s t i n c t i o n between the p r e d i c t i o n s of the various models. Although i t i s found th a t a l l decay curves can be f i t t :d w e l l as convolutions of a b i e x p o n e n t i a l , the r a t e con­s t a n t s presented i n t h i s paper must s t i l l be considered as purely d e s c r i p t i v e , ( t a b l e l )

The c a l c u l a t e d i s o t r o p i c fluorescence decay ( l0 ( t ) ) agrees w e l l

with the experimentally observed decay when the p o l a r i z e r i s s e t at the magic angle. The a n i s o t r o p i c decay components ( D ( t ) ) are p r i n c i p a l l y s h o r t e r l i v e d , and t h e i r f a s t decaying c o n t r i b u t i o n i s more pronounced than i n the i s o t r o p i c decay. D e p o l a r i z a t i o n by r o t a t i o n of the e n t i r e p r o t e i n can be excluded with the molecular weight being i n the range between 18 (o<-subunit)'and 120 kDalton ( t r i m e r (^,β)^). Local motions of the chromophore region are a l s o u n l i k e l y , because the chromophores are b e l i e v e d to be held r i g i d l y by the p r o t e i n , and large angular motions would be r e q u i r e d for the observed d e p o l a r i z a t i o n . The a l t e r n a t i v e d e p o l a r i z a t i o n mechanism i s then energy t r a n s f e r among neighbouring, n o n p a r a l l e l chromophores, which has a l r e a d y been studied by s e v e r a l groups b y

2\ s t a t i c p o l a r i z e d fluorescence

measurements of b i l i p r o t e i n s · We r e l a t e t h e r e f o r e the f a s t de­caying component of the fluorescence to energy t r a n s f e r between s e n s i t i z i n g ( = " s

M

) and f l u o r e s c i n g (="f") chromophores as w e l l as to t r a n s f e r among the "f"-chromophores, which would both lead to d e p o l a r i z a t i o n . I t i s , however, not the absolute value of the short components, but r a t h e r t h e i r d i f f e r e n c e ( i s o t r o p i c v s . a n i s o t r o p i c ) from which t h i s time constant can u l t i m a t e l y be d e r i v e d . I n the d e s c r i p t i v e two-exponential formalism, energy t r a n s f e r time constants between 20 and 100 psec are estimated.

A monoexponential decay would be excepted on t h i s b a s i s for the c<-subunit, which c a r r i e s only a s i n g l e chromophore. A b i ­exponential was recorded i n s t e a d , s i m i l a r to the r e s u l t s found for the -subunit of phycocyanin i n S p i r u l i n a p l a t e n s i s (these authors, i n the p r e s s ) . Aggregation and energy t r a n s f e r among the aggregates i s u n l i k e l y i n view of the sedimentation data. We r a t h e r suggest that two d i f f e r e n t subsets of the pigment e x i s t i n t h i s s u b u n i t , e i t h e r a lready i n the n a t i v e pigments, or as a r e s u l t of the subunit s e p a r a t i o n . The l a t t e r r e q u i r e s denaturation w i t h urea over long times (about l 6 hours) and subsequent r e n a t u r a t i o n , which may lead to incomplete r e s t o r a t i o n of the o r i g i n a l s t a t u s of the chromophore. To our expierence i s the s e p a r a t i o n by e l e c t r o f o c u s i n g most gentle and complete, i f judged from SDS-PAGE and absorption spectroscopy, but p o s s i b l y

Pigment T(°C) I s o t r o p i c decay Anisotrogi Lc deca£

<x -Sub u n i t 18

36

52

1 0 6 l(74jt)

9 7 5 ( 7 1 $ )

639(80$)

2 6 8 1 ( 2 6 $ )

2 1 3 4 ( 2 9 $ )

2564 (20$)

634(84$)

567 (61$)

313 -

2147(16$)

1297 (39$)

P> -Subunit 18

36

52

363(51$)

2 5 9 ( 4 9 $ )

2 7 7 ( 6 8 $ )

1644(49$)

1075(51$)

1191(32$).

191(47$)

101(53$)

9 5 ( 3 7 $ !

1041(53$)

7 6 9 ( 4 7 $ )

469(63^1

Monomer 18

36

52

428 (52$)

3 0 4 ( 6 5 $ )

1520(48$)

1650(35$)

2 7 2 ( 5 9 $ )

228 (52$)

2 0 2 ( 5 8 $ )

2200(41$)

1600(48$)

1200(42$)

Trimer

<*03

18

36

52

4 6 2 ( 5 5 $ )

3 0 7 ( 5 3 $ )

280 -

2 1 0 0 ( 4 5 $ )

1 6 0 0 ( 4 7 $ )

2060 —

2 0 6 ( 2 3 $ )

1 3 2 ( 7 0 $ )

9 5 ( 7 3 $ !

1375(77$)

1800(30$)

1 1 0 0 ( 2 7 $ !

Table 1: Fluorescence decay c h a r a c t e r i s t i c s of C-Phycocyanin from Mastigocladus

laminosus i n d i f f e r e n t aggretation s t a t e s , and of i t s s u b u n i t s . The decay

curves were f i t with a b i e x p o n e n t i a l , for which the time constants T ( i n psec)

and the r e l a t i v e amplitudes ( $ j i n b r a c k e t s ) are given.

1.1. 8 4

not yet gentle anough. The i n t e r p r e t a t i o n of two subsets of the o<-subunit with each i n d i v i d u a l polypeptide being disaggregated and bearing only one i s o l a t e d chromophore i s supported by the unusually long time constant of the " f a s t " component (as compared to a l l other pigments i n which energy t r a n s f e r i s p o s s i b l e ) and by the comparably slow d e p o l a r i z a t i o n .

The fluorescence decay of a l l samples becomes f a s t e r at higher temperatures. This i s i n c o n t r a s t to e a r l i e r r e s u l t s with PC from S p i r u l i n a p l a t e n s i s (these authors, i n the p r e s s ) , which showed a decreased decay ra t e for the e x c i t e d s t a t e population at higher temperature. This may r e f l e c t an adaption of the thermophilic Mastigocladus laminosus.

The r e s u l t s are summarized i n the f i g s . 2 and 3 · The data for i n t a c t phycobilisomes and for e n t i r e c e l l s are shown for compari­son i n f i g . 4 . The fluorescence i s r a p i d l y d e polarized i n both cases due to the large number of p o s s i b l e energy acceptors i n these u n i t s . The l o n g - l i v e d component i n phycobilisomes corresponds to the r a d i a t i v e l i f e t i m e of the terminal a c c e p t o r , allophyco-cyanin ( λ

e m) / 650 nm); the comparable short l i f e t i m e observed i n

whole c e l l s r e f l e c t s the quenching by the c h l o r o p h y l l s i n the photosynthetic membrane, ( f i g s , not shown i n t h i s summary).

Acknowledgements: This work was supported by the Deutsche Forschungsgemeinschaft, Bonn (SFB 143).

References; ( 1 ) Kufer, W., Scheer, H. 1979, Hoppe-Seyler

!

s Z. P h y s i o l . Chem. 360 pp 9 3 5 - 9 5 6 . ( 2 ) Grabowski, J . Gantt, E. 1978a, Photochem. P h o t o b i o l . 28 3 9 - 4 5 ; T e a l e , F.W.J. , Dale R.E. ( 1970) Biochem. J . J J J ) , l * S l - l 6 9 ; Zickendraht-Wendelstadt. Β., F r i e d r i c h , J . , R ü d i g e r , W. 1980, Photochem.Photobiol. 2L, 367-376 .