Biochimica et Biophysica, Acta 416 (1975) 105-149 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 86022
THE
PRIMARY
PHOTOCHEMICAL
REACTION
OF BACTERIAL
PHOTOSYNTHESIS WILLIAM W. PARSON and R I C H A R D J. COGDELL
Department of Biochemistry, University of Washington, Seattle, Wash. 98195 (U.S.A.) (Received October 21st, 1974)
CONTENTS I. II.
Introduction
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pa7o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Early observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Bacteriochlorophyll and bacteriopheophytin in the reaction center . . . . . . . . . C. Protein composition of reaction center . . . . . . . . . . . . . . . . . . . .
III. Photochemistry of bacteriochlorophyll in vitro . . . . . . . . . . . . . . . . . . A. Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Triplet states and electron transfer from them
106 106 106 107
115 115 115
. . . . . . . . . . . . . . . . .
116
C. Electron transfer from excited singlet states . . . . . . . . . . . . . . . . . . . D. Photoreduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
119 12~
IV. Quantum yield for P87o photooxidation . . . . . . . . . . . . . . . . . . . . . .
121
V.
121 121 125
Kinetics and mechanism of PaTo photooxidation . . . . . . . . . . . . . . . . . . A. Kinetics and fluorescence lifetimes . . . . . . . . . . . . . . . . . . . . . . B. Transient states at low redox potentials . . . . . . . . . . . . . . . . . . . .
VI. The primary electron acceptor . . . . . . . . . . . . A. Overview . . . . . . . . . . . . . . . . . . . B. Acceptor pool size and reoxidation kinetics . . . . . C. Redox titrations of the primary acceptor . . . . . . D. Evidence implicating Fe . . . . . . . . . . . . . E. Evidence implicating Q . . . . . . . . . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
132 132 133 134 136 139
VII. Free energy capture in the primary reaction and the reversal of the primary reaction . VIII. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
142 145
Acknowledgements
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
146
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
146
Abbreviations: bacteriochlorophyll+, the cationic free radical produced by one-electron oxidation of bacteriochlorophyll; bacteriochlorophyli-, the anionic free radical produced by one-eleciron reduction of bacteriochlorophyll; bacteriopheophytin+, the cationic radical produced by oxidation of bateriopheophytin; P87o, the pigment complex which acts as the primary electron donor in bacterial photosynthesis; PaTo+, the cationic radical produced by oxidation of PaTO; Q, ubiquinone.
106 I. INTRODUCTION The primary electron-transfer reaction in bacterial photosynthesis occurs within a membrane-bound complex of pigments and proteins which has come to be known as "the reaction center". Antenna bacteriochlorophyll and carotenoids serve to funnel the radiant energy, which they absorb, to the reaction center, where useful photochemistry can occur. The migration of energy to the reaction center and its subsequent capture in an electron-transfer reaction both are extremely efficient. In many species, at least 90 ~ of the photons that are absorbed result in electron transfer, with less than 5 ~ being wasted as fluorescence. What is the nature of the photochemical reaction center? How does the photochemical apparatus operate with such high quantum efficiency? What stabilizes the products of the electron transfer reaction, preventing the squandering of energy in a back-reaction? The answers to these questions are still largely unknown, but the last few years have seen considerable progress toward their solutions. In this review, we shall survey recent work on the components and structure of the reaction center. As a basis for discussing the mechanism of the photochemistry which occurs in the reaction center, we shall describe some of the transient states, excited singlets, triplets, radical cations, and radical anions, which can appear when one illuminates bacteriochlorophyll in vitro. For different perspectives on these topics and on other aspects of bacterial photosynthesis, the reader may turn to several other recent reviews [1-6].
lI. P87o IIA. Early observations Our knowledge of the electron donor in the primary electron transfer reaction stems from the work of Duysens in the 1950's. On illuminating suspensions of Chromatium vinosum, Duysens [7,8] detected small absorbance changes in the near infrared. These included a bleaching in an absorption band near 870 nm and a blue shift in another band near 800 nm. Similar absorbance changes were found to be generated by mild chemical oxidation [9,10]. These observations were taken up by Clayton, who developed the idea that the absorbance changes reflected the oxidation of a special, reactive bacteriochlorophyll component, which comprised 2-5 ~ of the total [11,12]. The photooxidation occurs reversibly with relatively high quantum yield even at I ° K [13,14]. Significantly, a mutant strain of Rhodopseudomonas spheroides which was incompetent at photosynthesis was found to lack the reactive bacteriochlorophyll [15]. When C. vinosum chromatophores were exposed to a short flash of light, the photooxidation of the reactive bacteriochlorophyll was found to occur in less than 0.5/zs with a quantum yield near 1.0 [16]. Following this step, the oxidized bacteriochlorophyll extracted an electron from a c-type cytochrome. The second reaction, which had a half-time of 2/~s, also proceeded with a quantum yield near 1.0.
107 We shall call the reactive bacteriochlorophyll complex "P87o", with "P" for pigment, and "870" indicating approximately the kmax of its major infrared-absorption band. The actual position of the absorption band varies by ± 15 nm among different species of bacteria which contain bacteriochlorophyll a, and by even more if one turns to species with bacteroichlorophyll b. Many authors therefore use the designation "P" with different subscripts to indicate the actual ~'max depending on the species. The use of "P87o" as a general term is simpler, however, and seems adequate because the basic properties of the complex probably do not vary fundamentally. (Many authors also use P with different subscripts to indicate the wavelengths of other maxima in the absorbance spectrum of what has proven to be the same bacteriochlorophyll complex. Others have used subscripts to indicate midpoint redox potentials. The proliferation of subscripts has placed a heavy burden on the uninitiated reader.) To indicate the oxidized form of the reactive bacteriochlorophyll complex, we shall use "P87o +'' It has been suggested frequently that some species of photosynthetic bacteria (particularly C. vinosum and Rhodospirillum rubrum) contain more than one type of reaction center. In C. vinosum, this seems to have been disproved, and the reviewers feel that the present experimental evidence supports the view that there is only one type of reaction center present in each species so far studied. For a fuller discussion of this point, see ref. 4.
liB. Bacteriochlorophyll and bacteriopheophytin in the reaction center Our understanding of the nature of PaTo has enjoyed considerable expansion in the last few years. Much of this expansion stems from Reed and Clayton's success [17], in 1968, in purifying a reaction center preparation free of the bulk antenna bacteriochlorophyll. Earlier attempts at this goal had involved selective photooxidation or chemical oxidation of the antenna bacteriochlorophyll. Reed and Clayton turned to detergents. Using Triton X-100, they obtained from a carotenoidless mutant of Rps. spheroides (strain R-26) a particle with a molecular weight of approx. 6.5 x 105, containing P87o, ubiquinone (Q), b- and c-type cytochromes, non-heme Fe and Cu. Further efforts by Clayton, Reed, Feher and their coworkers [18-22] have led to the isolation of simpler and purer preparations from the same strain. These have particle weights near 7 × 104. The most refined preparations contain approximately one equivalent of Q and one of non-heme Fe per mole of P87o, but they are free of cytochromes, Cu, and antenna bacteriochlorophyll. A current procedure [19] for isolating such a preparation calls for the disruption of chromatophores with the nonionic detergent dodecyldimethylamine oxide, followed by sucrose density gradient centrifugation, ammonium sulfate fractionation, and Agarose gel chromatography. Success with other strains and species has demanded variations on this theme, ranging from subtle adjustments of the fractionation conditions to the use of completely different types of detergent. Ref. 6 includes a survey of the various preparations that have been obtained to date. In what follows, we describe numerous experiments that have been performed
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THE
PRIMARY
PHOTOCHEMICAL
REACTION
OF BACTERIAL
PHOTOSYNTHESIS WILLIAM W. PARSON and R I C H A R D J. COGDELL
Department of Biochemistry, University of Washington, Seattle, Wash. 98195 (U.S.A.) (Received October 21st, 1974)
CONTENTS I. II.
Introduction
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pa7o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Early observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Bacteriochlorophyll and bacteriopheophytin in the reaction center . . . . . . . . . C. Protein composition of reaction center . . . . . . . . . . . . . . . . . . . .
III. Photochemistry of bacteriochlorophyll in vitro . . . . . . . . . . . . . . . . . . A. Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Triplet states and electron transfer from them
106 106 106 107
115 115 115
. . . . . . . . . . . . . . . . .
116
C. Electron transfer from excited singlet states . . . . . . . . . . . . . . . . . . . D. Photoreduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
119 12~
IV. Quantum yield for P87o photooxidation . . . . . . . . . . . . . . . . . . . . . .
121
V.
121 121 125
Kinetics and mechanism of PaTo photooxidation . . . . . . . . . . . . . . . . . . A. Kinetics and fluorescence lifetimes . . . . . . . . . . . . . . . . . . . . . . B. Transient states at low redox potentials . . . . . . . . . . . . . . . . . . . .
VI. The primary electron acceptor . . . . . . . . . . . . A. Overview . . . . . . . . . . . . . . . . . . . B. Acceptor pool size and reoxidation kinetics . . . . . C. Redox titrations of the primary acceptor . . . . . . D. Evidence implicating Fe . . . . . . . . . . . . . E. Evidence implicating Q . . . . . . . . . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
132 132 133 134 136 139
VII. Free energy capture in the primary reaction and the reversal of the primary reaction . VIII. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
142 145
Acknowledgements
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
146
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
146
Abbreviations: bacteriochlorophyll+, the cationic free radical produced by one-electron oxidation of bacteriochlorophyll; bacteriochlorophyli-, the anionic free radical produced by one-eleciron reduction of bacteriochlorophyll; bacteriopheophytin+, the cationic radical produced by oxidation of bateriopheophytin; P87o, the pigment complex which acts as the primary electron donor in bacterial photosynthesis; PaTo+, the cationic radical produced by oxidation of PaTO; Q, ubiquinone.
106 I. INTRODUCTION The primary electron-transfer reaction in bacterial photosynthesis occurs within a membrane-bound complex of pigments and proteins which has come to be known as "the reaction center". Antenna bacteriochlorophyll and carotenoids serve to funnel the radiant energy, which they absorb, to the reaction center, where useful photochemistry can occur. The migration of energy to the reaction center and its subsequent capture in an electron-transfer reaction both are extremely efficient. In many species, at least 90 ~ of the photons that are absorbed result in electron transfer, with less than 5 ~ being wasted as fluorescence. What is the nature of the photochemical reaction center? How does the photochemical apparatus operate with such high quantum efficiency? What stabilizes the products of the electron transfer reaction, preventing the squandering of energy in a back-reaction? The answers to these questions are still largely unknown, but the last few years have seen considerable progress toward their solutions. In this review, we shall survey recent work on the components and structure of the reaction center. As a basis for discussing the mechanism of the photochemistry which occurs in the reaction center, we shall describe some of the transient states, excited singlets, triplets, radical cations, and radical anions, which can appear when one illuminates bacteriochlorophyll in vitro. For different perspectives on these topics and on other aspects of bacterial photosynthesis, the reader may turn to several other recent reviews [1-6].
lI. P87o IIA. Early observations Our knowledge of the electron donor in the primary electron transfer reaction stems from the work of Duysens in the 1950's. On illuminating suspensions of Chromatium vinosum, Duysens [7,8] detected small absorbance changes in the near infrared. These included a bleaching in an absorption band near 870 nm and a blue shift in another band near 800 nm. Similar absorbance changes were found to be generated by mild chemical oxidation [9,10]. These observations were taken up by Clayton, who developed the idea that the absorbance changes reflected the oxidation of a special, reactive bacteriochlorophyll component, which comprised 2-5 ~ of the total [11,12]. The photooxidation occurs reversibly with relatively high quantum yield even at I ° K [13,14]. Significantly, a mutant strain of Rhodopseudomonas spheroides which was incompetent at photosynthesis was found to lack the reactive bacteriochlorophyll [15]. When C. vinosum chromatophores were exposed to a short flash of light, the photooxidation of the reactive bacteriochlorophyll was found to occur in less than 0.5/zs with a quantum yield near 1.0 [16]. Following this step, the oxidized bacteriochlorophyll extracted an electron from a c-type cytochrome. The second reaction, which had a half-time of 2/~s, also proceeded with a quantum yield near 1.0.
107 We shall call the reactive bacteriochlorophyll complex "P87o", with "P" for pigment, and "870" indicating approximately the kmax of its major infrared-absorption band. The actual position of the absorption band varies by ± 15 nm among different species of bacteria which contain bacteriochlorophyll a, and by even more if one turns to species with bacteroichlorophyll b. Many authors therefore use the designation "P" with different subscripts to indicate the actual ~'max depending on the species. The use of "P87o" as a general term is simpler, however, and seems adequate because the basic properties of the complex probably do not vary fundamentally. (Many authors also use P with different subscripts to indicate the wavelengths of other maxima in the absorbance spectrum of what has proven to be the same bacteriochlorophyll complex. Others have used subscripts to indicate midpoint redox potentials. The proliferation of subscripts has placed a heavy burden on the uninitiated reader.) To indicate the oxidized form of the reactive bacteriochlorophyll complex, we shall use "P87o +'' It has been suggested frequently that some species of photosynthetic bacteria (particularly C. vinosum and Rhodospirillum rubrum) contain more than one type of reaction center. In C. vinosum, this seems to have been disproved, and the reviewers feel that the present experimental evidence supports the view that there is only one type of reaction center present in each species so far studied. For a fuller discussion of this point, see ref. 4.
liB. Bacteriochlorophyll and bacteriopheophytin in the reaction center Our understanding of the nature of PaTo has enjoyed considerable expansion in the last few years. Much of this expansion stems from Reed and Clayton's success [17], in 1968, in purifying a reaction center preparation free of the bulk antenna bacteriochlorophyll. Earlier attempts at this goal had involved selective photooxidation or chemical oxidation of the antenna bacteriochlorophyll. Reed and Clayton turned to detergents. Using Triton X-100, they obtained from a carotenoidless mutant of Rps. spheroides (strain R-26) a particle with a molecular weight of approx. 6.5 x 105, containing P87o, ubiquinone (Q), b- and c-type cytochromes, non-heme Fe and Cu. Further efforts by Clayton, Reed, Feher and their coworkers [18-22] have led to the isolation of simpler and purer preparations from the same strain. These have particle weights near 7 × 104. The most refined preparations contain approximately one equivalent of Q and one of non-heme Fe per mole of P87o, but they are free of cytochromes, Cu, and antenna bacteriochlorophyll. A current procedure [19] for isolating such a preparation calls for the disruption of chromatophores with the nonionic detergent dodecyldimethylamine oxide, followed by sucrose density gradient centrifugation, ammonium sulfate fractionation, and Agarose gel chromatography. Success with other strains and species has demanded variations on this theme, ranging from subtle adjustments of the fractionation conditions to the use of completely different types of detergent. Ref. 6 includes a survey of the various preparations that have been obtained to date. In what follows, we describe numerous experiments that have been performed
108 I
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I
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900
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"lL1300
X, n m
r
r
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