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THE PHOTOSYNTHETIC REACTION CENTER

F r o n t cover

p h o t o : Top view of the R h o c i o h a c t e r s p h a e r o u i c s

reaction center protein

showing the surface exposed to the cell periplasmic aqueous phase. The three protein subunits, L , M , and H , are shown in red, green. and blue, respectively.

This book is printed on acid-free p a p e r . ©

Copyright © 1993 by A C A D E M I C PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording. or any information storage and retrieval System, without permission in writing from the publisher.

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Academic Press Limited 24-28 Oval Road, London NW1 7 D X

Library of Congress Cataloging-in-Publication Data The

Photosynthetic reaction center /edited by Johann Deisenhofcr and James R. Norris. p.

cm.

Includes bibliographical references and index. I S B N 0-12-208661-9 (v. 1). - I S B N 0-12-208662-7 (v. 2) 1. Photosynthetic reaction centers. II. Norris, James R., QP517.P45P46

I. Deisenhofen Johann.

Date.

1993

581.1 '3342-dc20

92-43633 CIP

PRINTED IN THE UNITED STATES OF AMERICA 93 94 95 96 97 98

BC

9 8 7 6 5 4 3 2 1

Contents

Contents of T h e P h o t o s y n t h e t i c R e a c t i o n Center, Contributors

Volume I I

x

xi

Preface xiü

1

Structure and Function of the Photosynthetic Reaction Center of R h o d o b a c t e r sphaeroides Marianne Schiffer>(James R. Norris

I. Reaction center complex 1 II. Interactions within the complex References 10

2

6

Refinement of the Structure of a Water-Soluble Antenna Complex from Green Photosynthetic Bacteria by Incorporation of the Chemically Determined Amino Acid Sequence Dale E. Tronrud/Brian W. Matthews

I. II. III. IV. V.

Introduction 13 X-ray sequence analysis 14 Refinement using the chemical sequence Deductions from the new model 18 Conclusions 20 References 21

16

v

Preparation, Purification, and Crystallization of Purple Bacteria Antenna Complexes Richard J. C o g d e l l / A n n a M . Hawthornthwaitc

I. Introduction 23 II. Purification of bacterial antenna complexes 26 III. Crystallization of purple bacteria antenna complexes 28 IV. Current Status of crystallographic studies on the BB800-850 complex of R h o d o p s e n d o m o n a s a c i d o p h i l a strain 10050 36 References 41 Structural Features of Photosynthetic LightHarvesting Systems H. Zuber

I. II. III. IV. V. VI.

Introduction 43 Antenna System of purple bacteria 46 Antenna System of green photosynthetic bacteria 57 Antenna Systems of cyanobacteria and red algae 60 Antenna Systems of algae and higher plants 68 Concluding remarks 76 References 78

Genetic Analysis of Photosynthetic Membrane Biogenesis in R h o d o b a c t e r s p h a e r o i d e s Samuel Kaplan / T i m o t h y J. Donohue

I. Introduction 101 II. Critical membrane components and their structural genes 102 III. Exploiting genetics for the study of bacterial photosynthesis 105 IV. Genetic analyses of critical membrane components V. Summary 125 References 125 Digital Imaging Spectroscopy Adam P. Arkin/Douglas C . Youvan

I. Introduction 133 II. Construction of a spectral "tester" strain

134

107

vii

Ccntenis

III. Digital imaging spectrophotometer hardware specifications 137 IV. Digital imaging spectrophotometer Software specifications 140 V. System development 153 VI. Conclusions 154 References 154

7

Bacterial Reaction Centers with Modified Tetrapyrrole Chromophores Hugo Scheer/Andreas Struck

I. II. III. IV.

Introduction 157 Methods 161 Selectivity of the exchange 170 Static spectroscopy of reaction centers with modified pigments 177 V. Time-resolved spectroscopy 183 VI. Prospective 184 References 185

8

Electron and Proton Transfer in the Acceptor Quinone Complex of Reaction Centers of Phototrophic Bacteria Vladimir P. Shinkarev/Colin A. Wraight

I. Introduction 194 II. Acceptor quinone complex of photosynthetic reaction centers 195 III. Kinetics of the dark reduction of P870 in the absence of secondary electron donors 201 IV. Kinetics of the dark cytochrome reduction 205 V. Exchange of quinone 206 VI. Mechanism of inhibitor action 215 VII. Kinetic analysis of inhibitor action 217 VIII. Dependence of the one-electron equilibrium o n p H 219 IX. p H dependence of the rate of electron transfer between Q and Q 226 X. Charge recombination of the second electron 230 XI. Binary oscillations of the Q semiquinone 231 XII. Electron and proton transfer in the production of quinol 237 +

A

B

B

viii

Contents

XIII. Conclusion 243 Appendix I: General Solution for constant quinone concentration 245 Appendix II: General System of differential equations for P dark relaxation 246 References 247 +

9

Electron Transfer between Bacterial Reaction Centers and Mobile c-Type Cytochromes David M. Tiede/P. Leslie Dutton

I. Introduction 258 II. Kinetics of cytochrome oxidation with R h o d o b a c t e r sphaeroides reaction centers 260 III. Other photosynthetic bacteria 269 IV. Physical characterization of reaction center-cytochrome complexes 271 V. Chemical modification of c-cytochromes as a probe of interaction domains 274 VI. Models of the R h o d o b a c t e r sphaeroides reaction center - cytochrome complex 276 VII. Electrostatic mechanisms for assembly of the R h o d o b a c t e r sphaeroides reaction center-cytochrome complex 278 VIII. Relation to nonphotosynthetic Systems 283 EX. Future directions 283 References 284

10

Isolation and Properties of the Photosystem II Reaction Center Kimiyiiki Satoh

I. Introduction 289 II. Isolation 291 III. Properties of the isolated photosystem II reaction center 293 IV. Prospects 312 References 313

ix

Conicnts

11

Biochemical, Biophysical, and Structural Characterization of the Isolated Photosystem II Reaction Center Complex Michael Seibert

I. Introduction 319 II. Stabilization of the isolated photosystem II reaction center 322 III. Biochemical characterization of the isolated photosystem II reaction center 325 IV. Functional properties of the isolated photosystem II reaction center 328 V. Structural aspects 399 VI. Summary 345 References 347

12

Photosytem II Reaction Center and Bicarbonate Govindjee/JackJ. S. van Rensen

I. Introduction 357 II. Photosystem II electron acceptor side and the bicarbonate effect 364 III. D l protein and the bicarbonate effect 369 IV. D2 protein and the bicarbonate effect 373 V. Mechanism of the bicarbonate effect in photosystem II 377 VI. Summary and conclusions 381 References 382

X3

Structure and Function of the Reaction Center Cofactors in Oxygenic Organisms M. C. W. Evans/J. H. A. Nugent

I. Photosystem II 391 II. Photosystem I 402 References 411 Index

417

H u g o Scheer B o t a n i s c h e s I n s t i t u t der Universität München, Germany

Andreas Struck

1

Kraft General Foods Research a n d Development München, G e r t n a n y

Bacterial Reaction Centers with Modified Tetrapyrrole Chromophores I. Introduction II. Methods A. Pigment synthesis B. Pigment exchange C. Analysis III. Selectivity of the exchange A. Influence of central metals B. Peripheral substituents in bacteriochlorophylls C. D.

Peripheral substituents in bacteriopheophytins Species specificity

IV. Static spectroscopy of reaction centers with modified pigments A.

Structural integrity of modiiied reaction centers

B. Environmental effects on pigments at sites B C. Interactions of pigments at sites B with the primary donor (P) A B

AiB

D.

Interactions of pigments at sites B

A11

and H

A B

V. Time-resolved spectroscopy VI. Prospective References

I.

Introduction

Several different methods are available for preparing reaction centers with nonnative chromophores: (1) (re)assembly from the Polypeptides, pigments, and other components such as quinones, iron, and possibly others; (2) exchange of chromophores into native or partly denatured reaction centers; (3) modification of chromophores in native or partly denatured reaction centers by chemical or enzymatic means; (4) modification of binding sites to incorporate different chromophores, for example, by mutagenesis, and (5) mutagenic modifications of the pigments biosynthetic pathways. With the exception of the last one, all these approaches have been used on different TUE PHOTOSYNTHETIC REACTION CENTER, Volume I Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

157

158

Hugo Scheer/Andreas Struck

(bacterio)chlorophyll proteins, but only Methods 2 and 4 have been successful with reaction centers. Complete reconstitution of the complexes (Method 1) from the isolated constituent Polypeptides and suitably modified pigments is likely to have the greatest potential, but to date has been impossible with reaction centers. This technique has been applied successfully to the core light-harvesting complexes (LHCI) of purple bacteria (Miller et a l , 1987; Ghosh et a l , 1988; Parkes-Loach et a l , 1988,1990; Chang et a l , 1990a,b; Heller and Loach, 1990) and to the peripheral light-harvesting chromoprotein LHCII-ß of higher plants (Plumley and Schmidt, 1987). The method has been extended by Paulsen e t a t . (1990,1991) to the full-length and modified LHCII-ß precursor Polypeptides expressed in and isolated from Escherichia c o l i and by Schmidt et al. (1991) to other green plant peripheral light-harvesting complexes. Site-directed mutagenesis of the binding pockets (Method 4) has been used extensively to exchange bacteriochlorophylls (Behls) with their metalfree derivatives, for example, bacteriopheophytins (Bphes) and vice versa in reaction centers (RCs) from R h o d o b a c t e r ( R b . ) capsulatus and Rb. sphaeroides (Table I)- With only a Single exception, whenever the histidine(s) ligating the central magnesium atom(s) of the Bchl(s) were replaced by hydrophobic amino acids, stably assembled RCs were obtained in which the Behls at the respective site(s) were replaced by Bphe to yield stably assembled and isolatable RCs (Bylina and Youvan, 1988; Coleman and Youvan, 1990; Robles et a l , 1990; Schenck e t a l , 1990; Woodbury et a l , 1990). In reversing this reaction, replacement of leucine or isoleucine next to the central N-4 cavity of Bphe with histidine resulted in binding of Behl at the respective site. Some results are available with amino acid residues of intermediate ligation strength. Serine or threonine selected binding of Behl at B and B (Bylina et a l , 1990); glutamine (mutants H-L173Q and H-M200Q ) at the sites of the special pair resulted in binding of Behl also (Bylina and Youvan, 1988). Omission of Bphe H was found in a mutant of Rb. capsulatus in which part of the D helix of subunit L was replaced by the symmetrically equivalent Stretch of helix D . The resulting RC mutant, called D , lacked Bphe H , presumably because a tryptophan residue was now occupying (part of) the Space usually occupied by Bphe (Robles et al., 1990). The reader interested in this approach is referred to an excellent review (Coleman and Youvan, 1990) and to a series of papers in the book edited by Michel-Beyerle (1990). A natural example of this type of pigment exchange exists in Chloroßexus a u r a n t i a cus: RCs are of the purple bacteria type (type II) but a significant difference is the exchange of His M l 8 0 , which is normally the ligand of Behl B , by A

B

2

L

M

u

A

B

Residue numbering aecording to "best alignment" with R p s . v i r i d i s RCs and the Single letter amino acid code are used throughout this chapter when naming mutants. 2

TABLE I

BChl/BPhe Modüications Induced by Site-Directed Mutagenesis o f Potential Ligands to the Central Magnesium

Site

Residue number"

PA

L173

Amino acid"

Species'

Remarks

Pigment b o u n d

Reference

H

Type II

BChla or b

Native Situation

L

Rb.

caps.

BPhea

Heterodimer, PS~

Bylina and Youvan (1988)

Q" I.

Rb.

caps.

BChla

Heterodimer, PS"

Bylina and Youvan (1988)

Rb.

spbe.

BPhea

Heterodimer

H u b e * e t a l . (1990); Schenck e t a l . (1990)

PB

BA

BB

M200

LI 53

M180

H

Type II

BChla o r b

Native Situation

L

Rb.

caps

BPhea

Heterodimer, PS

Bylina and Youvan (1988)

F

Rb.

caps.

BPhea

Heterodimer, PS

Bylina and Youvan (1988)

Q L

Rb.

caps.

BPhea

Heterodimer, PS'

Bylina and Youvan (1988)

Rb.

spbe.

BPhea

Heterodimer

Huber e t a l . (1990); Schenck e t a l . (1990)

H

Type II Rb.

caps.

BChla or b BChla

Native Situation

S' L

Rb.

caps.

7

PS*

Bylina e t a l . (1990)

Native Situation Native Situation

Ovchinnikov e t a l . (1988)

H L

Type II Cf. a u r .

BChla or b BPhea

Bylina e t a l . (1990)

Shiozawa e t a l . (1989) S T

HB

M212

LI 87

caps.

BChla

Rb.

caps.

BChla

Bylina e t a l . (1990) Bylina e t a l . (1990)

R b . caps. R b . caps. Type II

>

BPhea or b

PS' PS Native Situation

W

Rb.

caps.

None

D

L

Rb.

caps.

BPhea

Revcruiu of D

Type II

BPhea or b

Mutant Native Situation

Robles e t a t . (1990)

L F

Rb.

BPhea

D

Robles e t a l . (1990)

L HA

Rb.

R L

?

caps

u

MM

+ mutant

mutant

Bylina e t a l . (1990) Bylina e t a l . (1990) R o b l e s e / « / . (1990)

LL

Breton e t a l . (1990)

"Residue numbering aecording to "best alignment" with R p . v i r i d i s RC. "One-letter coding of amino acids. 'Type II refers to purple bacterial PSII and related RC. R b . caps.,

R h o d o b a c t e r c a p s u l a t u s . R b . sphe.,

aurantiacus. " Double mutant H-L173-Q/H-M200-Q binds two BChl a at P „ and is PS *. A

"Double mutant H-L153-S/H-M180-S binds two BChl a at B

A1I

.

R h o d o b a c t e r s p h a e r o i d e s . Cf a u r ,

Chloroßexus

160

Hugo Scheer/Andreas Struck

He M178 (Ovchinnikov et a l , 1988; Shiozawa et a l , 1989). In these RCs, for one of the four Bchlas is exchanged for Bphea at the same time, and it is likely to be the one located at B . A series of histidine mutations in the core antennas of photosystem II (PSII) resulted in distinet reduetions of photosynthetic efficiency, but the pigment contents of these mutant complexes are not yet established (Kuhn and Vermaas, 1991; Shen et a l , 1991). Due to heterotrophic growth problems, mutagenesis has proven difficult with Rps. v i r i d i s , but progress in two laboratories shows promise that mutated RC with Bchlb/Bpheb-exchanges will soon be available from this organism (Bylina, 1992; Laußermair and Oesterhelt, 1992). Genetic modification of Behl biosynthesis (Method 5) has, to our knowledge, not yet been applied to obtain modified Behl proteins. Little was known about the last steps of this process until the Publishing of several reports (Burke et a l , 1991; Leeper, 1991; Richards and Fidai, 1991; Suzuki and Bauer, 1991). One example of the apparent selective modification of one pigment in RCs (Method 3) is presented in the literature. Treatment of isolated RCs from Rb. sphaeroides R26 with borohydride (BH ~ ) resulted in the loss of about 40% of the absorption at 800 nm normally ascribed to the two Behls at sites B (Ditson et a l , 1984). Therefore, the researchers concluded that one of these pigments (most likely B ) was attacked selectively by the reagent and subsequently lost from the RC (Ditson e t a l , 1984; Maroti e t a l , 1985; Beese e t a l , 1987; Chadwick e t a l , 1987; Frank, 1990; Frank and Violette, 1989). However, this Interpretation has been challenged by the finding of (1) an unchanged pigment composition in B H -treated RCs (Struck et a l , 1992a), (2) a tetrapyrrolic pigment at the B„ site in the poorly resolved X-ray structure (J. Allen, personal communication, 1991), and (3) at least partial proteolysis of the M subunit (Beese et a l , 1987). The B H ~ reduction also leads to distinet changes in Chlorophyll proteins of higher plants, but these changes have not yet been investigated at a molecular level (Scheer et a l , 1989). A n enzymatic modification of several Chlorophyll proteins with the degradative enzyme chlorophyllase has been reported by Schoch and Brown (1986). Modifications on other complexes involve the demetalation of the FennaMatthews-Olson (FMO) antenna protein of C l o r o b i u m (Ghosh et a l , 1968) and the selective loss of the 800-nm absorption band in the B 8 0 0 - 8 5 0 antenna complex of Rb. sphaeroides (Clayton and Clayton, 1981). In the latter case, the decrease again has been shown to be due not to loss of any pigment but to a detergent effect (Chadwick et a l , 1987). The first attempt to exchange pigments into native or partially denatured reaction centers (Method 4) is likely to be that published by Loach et a l (1975), who worked on the so-called alcaline urea-triton X I 0 0 (AUT) particles of R h o d o s p i r i l l u m (Rs.) r u b r u m . From line narrowing of the light-induced B

4

A B

B

4

4

161

7 Modified Tetrapyrrole Chromophores

Signal after treatment with deuterated Bchla, an exchange of the primary donor Behl r\ was concluded. This Interpretation was challenged (Norris, see discussion in Loach et a l , 1975) and the authors concluded subsequently that the line narrowing was artefactual and no exchange had taken place (P. Loach, personal communication, 1980). The method was modified, and an exchange of the monomeric Bchla at sites B or of Bphea at sites H with chemically modified pigments became possible in RCs of Rb. sphaeroides R26 and some other purple bacteria (Scheer et a l , 1988; Beese, 1989; Struck et a l , 1990a,b,1992; Struck, 1991). This chapter details the procedures involved and summarizes the results obtained with this method. B

A B

II.

A B

Methods

Successful exchange experiments rely on three combined methods: (1) synthesis and structure analysis of the pigments to be introduced, (2) Optimum conditions for the exchange itself, and (3) a reliable, sensitive, and selective pigment analysis for optimization and verification. All three conditions must be met, but in every special case the emphasis may be on a different parameter(s) which shall, therefore, be discussed separately.

A. Pigment synthesis A brief description of partial pigment synthesis, especially of selective modifications at positions C-3 and C-13 , and some of the physical and spectroscopic properties of these pigments are described in Struck et al. (1992b). A selection of the modified pigments is presented in Figure 1. The starting material for all syntheses was Bchla extracted from Rb. sphaeroides or Chla extracted from S p i r u l i n a g e i t l e r i (Scheer, 1988; Struck, 1991; Svec, 1991). Purification was done on DEAE-cellulose (Omata and Murata, 1983). Following the rather lengthy procedure of washing and equilibrating the material and increasing the methanol concentration in small increments is critical to preventing poor adsorption of the pigments to the material and, hence, poor separations. Light must be dim throughout, and the extraction and chromatography should be done without interruptions. The purified pigments are dried with a stream of Ar or N , then in a vaeuum exsiccator, and stored under A r in the deep freezer. The purity of the material should be checked by visible/near-infrared (Vis/NIR) absorption spectroscopy a n d chromatography, because some common alteration produets have absorption spectra very similar to those of the respective parent pigments (see, for example, Scheer, 1988). 2

2

Substituent Pigment

R

Bacteriochlorophylls (M = Mg) BChla BChla' (3-Vinyl) BChla (3-Vinyl> 13 -hydroxy BChla (3-«-Hydroxyethyl) BChla (3-ot-Hydroxyethyl>l 3 -hydroxy BChla 13 Hydroxy BChla 13 -Demethoxycarbonyl BChla (17MMethyl) BChla 13 -Hydroxy