Photosynthesis Research 17:189-216 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands Minireview
Structure and function of the chloroplast cytochrome bf complex D A N I E L P. O ' K E E F E Central Research and Development Department, E.L duPont deNemours and Company, Inc., Experimental Station, Bldg. 402, Wilmington, DE 19898, USA Received: 1 December 1987; accepted in revised form 2 February 1988
Key words: cytochrome, electrogenic, oxidoreductase, plastoquinone Abstract. The chloroplast cytochrome bfcomplex is an intrinsic multisubunit protein from the
thylakoid membrane consisting of four polypeptides: cytochrome f, a two heine containing cytochrome b6, the Rieske iron-sulfur protein, and a 17 kD polypeptide of undefined function. The complex functions in electron transfer between PSII and PSI, where most mechanisms suggest that the transfer of a single reducing equivalent from plastoquinol to plastocyanin results in the translocation of two protons across the membrane. Primary sequence analyses, dichroism studies, and functional considerations allow the construction of an approximate structural model of a monomeric complex, although some evidence exists for a dimeric structure. Resolution of the properties of the two cytochrome b6 heroes has relied upon the availability of purified solubilized complex, while evidence in the thylakoid suggests the difference between the two hemes are not as great in situ. Such variability in the spectroscopic and electrochemical properties of the cytochrome b6 is a major concern during the experimental use of the purified complex. There is a general consensus that the complex contains a plastoquinol oxidizing (Qz) site, although the evidence for a plastoquinone reduction (Qc) site, called for in most mechanistic hypotheses, is less substantive. Probably the most severe challenge to the so called Q-cycle mechanism comes from experimental observations made with cytochrome b6 initially reduced, where proposed interpretations more closely resemble a b-cycle than a Q-cycle. Although functional during cyclic electron transfer, the role of the complex and its possible interaction with other proteins, has not been completely resolved. Abbreviations: Cytochrome bH-high potential cytochrome b6, Cytochrome bL-low potential
cytochrome b6, DBMIB-2,~-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DNP-INT-2iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether, FNR -ferredoxin:NADP oxidoreductase, HQNO-2-n-heptyl-4-hydroxyquinoline-N-oxide, NQNO-2-n-nonyl-4hydroxyquinoline-N-oxide, Qc-quinone binding site on the cytochrome bfcomplex near the outside of the thylakoid membrane, alternatively designated "centre i" or "centre r", Qz-quinone binding site on the cytochrome bfcomplex near the inside of the thylakoid membrane, alternatively designated "centre o"
190
Introduction The cytochrome bfcomplex of the chloroplast thylakoid membrane plays a central role in the transfer of electrons between the two photosystems, serving as the site of energy conserving proton translocation associated with photosystem I. Together with the two photochemical reaction centers, water oxidizing complex, light harvesting proteins, and the ATP synthetase complex, the cytochrome bf complex is a major functional component of the thylakoid membrane photosynthesis machinery. Analogous in function to the cytochrome bcl complex found in mitochondria and photosynthetic bacteria, the cytochrome bf complex has one of the simplest polypeptide compositions, but exhibits a different substrate:product specificity (plastoquinol:plastocyanin oxidoreductase as compared to ubiquinol:cytochrome c oxidoreductase), and is inhibited by a unique spectrum of compounds. While the implicit assumption of many researchers is that the mechanistic details of the cytochrome bf and bc~ complexes are probably very similar, attempts to incorporate reactions associated with cyclic electron transfer around PSI have also led to proposals utilizing electron transfer pathways unique to the cytochrome bf complex. Many excellent detailed reviews of the literature on various aspects of the cytochrome bfand bcl complexes are available (Bendall 1982, Prince et al. 1982a, Crofts and Wraight 1983, Hauska et al. 1983, Rich 1984, Dutton 1986, Hauska 1986b, Joliot and Joliot 1986b, 1986c). Nonetheless, the past several years have produced new information on the cytochrome bf complex, related to its overall composition and structure, physical properties, and its role in the energy conserving reactions of the chloroplast. The purpose of the current exposition is to provide a brief overview of the approaches recently used to obtain a general idea of the structure of this protein, and how this relates to its function in electron and proton transport in the thylakoid membrane.
Purified preparations That cytochromesfand b6 (also known as b-563) occur in thylakoid membranes as members of a distinct stable protein complex was first demonstrated by Nelson and Neumann (Nelson and Neumann 1972). The polypeptide composition of this preparation was not reported, and while it exhibited some plastoquinol:plastocyanin reductase activity, it was also contaminated with low potential cytochrome b-559 (Rich et al. 1980). Subsequently, Hurt and Hauska reported a procedure for the isolation of a
191 five polypeptide complex, exhibiting plastoquinol:plastocyanin reductase activity (Hurt and Hauska 1981, 1982a), as well as proton translocating activity when reconstituted into phospholipid vescicles (Hurt et al. 1982). Several other procedures for isolating the complex from spinach have also appeared (Clark and Hind 1983a, Doyle and Yu 1985, Black et al. 1987), and with the exception of the procedure of Clark and Hind, all follow essentially the method of Hurt and Hauska. Briefly, this involves washing thylakoid membranes with NaBr to remove extrinsic proteins, solubilization in octyl glucoside or MEGA-9, ammonium sulfate fractionation, and final purification of the cytochrome bfcomplex by either a sucrose gradient (Hurt and Hauska 1981, 1982a, Clark and Hind 1983a), or cellulose phosphate chromatography step (Doyle and Yu 1985, Black et al. 1987). The cellulose phosphate chromatography reportedly allows larger amounts of material to be purified in one preparation (Black et al. 1987), although it is the earlier steps, and not the use of a sucrose gradient that is the most limiting aspect of other procedures. There is some variability in the polypeptide profile of the purified complex. Five polyacrylamide gel bands at 34, 33, 23, 20 and 17 kD reportedly correspond to two cytochrome f polypeptides, cytochrome b6, the Rieske iron sulfur protein, and a 17kD protein (with no known redox function), respectively (Hurt and Hauska 1981, 1982a, Hurt et al. 1981). The two cytochrome f polypeptides both stain positive for heme, are not distinct either by immunological techniques or peptide mapping, and it is necessary to add their contributions together in densitometric scans of electrophoresis gels in order to obtain stoichiometric amounts with the other members of the complex (Hurt and Hauska 1982a). These results suggest that a single cytochrome f polypeptide is partially modified in some way which slightly alters its electrophoretic mobility. Some investigators (Clark and Hind 1983a, Black et al. 1987, DP O'Keefe and KJ Leto, unpublished) have been unable to observe the closely spaced cytochrome f doublet, and some confusion about the nature of the observed doublet is possible when the flavoprotein, FNR (ferredoxin:NADP oxidoreductase, Mr ~ 37kD) copurifies in substantial quantities (Clark et al. 1984). This protein is easily separated from the complex by gel filtration over Sephacryl S-200 (DP O'Keefe and KJ Leto unpublished). The presence of ferredoxin:NADP reductase in any particular cytochrome bfpreparation is readily indicated by rapid reduction of cytochrome f in the presence of NADPH. This ferredoxin:NADP reductase (Forti 1971), is not inhibited in the isolated complex by DBMIB, DNP-INT, antimycin A (O'Keefe 1983), or HQNO (DP O'Keefe unpublished) and occurs in the absence of the complex using purified cytochrome f (Forti 1971). This suggests that the reaction is the
192 result of direct reduction of cytochromefby the flavoprotein, made possible by the accessibility of the cytochrome f heme in the solubilized complex. Reduction of the lower potential components of the complex by NADPH requires ferredoxin in addition to FNR (Lam and Malkin 1982, O'Keefe 1983), further suggesting that there is no direct electron transfer between the flavoprotein and components of the cytochrome bfcomplex. The possibility that the 17 kD polypeptide of the cytochrome bfcomplex is responsible for binding ferredoxin NADP reductase to the thylakoid membrane has been briefly entertained (Vallejos et al. 1984) and rejected (Coughlin et al. 1985). The cytochrome bfpreparation of Black and coworkers resolves many of the above issues in that it only exhibits one cytochromefband, contains no flavoprotein, and has high plastoquinol:plastocyanin oxidoreductase activity (Black et al. 1987). This permits a more categorical statement that a functional cytochrome bfcomplex has a minimal subunit composition of 4 polypeptides:cytochrome f ( ~ 33 kD), cytochrome b 6 (,'~ 23 kD), Rieske iron-sulfur protein (~ 20 kD), and a 17 kD subunit. These polypeptides are present in a single copy per complex (Hurt and Hauska 1982a). The most consistent results in more than one laboratory would suggest that these 4 polypeptides (bearing in mind that the possibility for observing an 'extra' cytochromefpolypeptide exists), are all that is required for the observation in the isolated complex of plastoquinol:plastocyanin oxidoreductase activity at rates (Hauska 1986a, Black et al. 1987) approaching the maximum possible rate of intersystem electron transfer (Whitmarsh and Cramer 1980). However, it should be pointed out that these measurements must be made in the absence of triton X-100, and have all used plastoquinols (PQH2-1 or PQH:-2) of lower molecular weight than the physiological donor, plastoquinol-A(PQH:-9).
Depletion of individual cytochrome bf components The Rieske iron-sulfur protein of the isolated complex can be removed by chromatography on hydroxyapatite in 0.5% Triton X-100 (Hurt et al. 1981). Although attempts at reconstitution of plastoquinol :plastocyanin oxidoreductase with depleted cytochrome bfcomplex, and purified iron sulfur protein were unsuccessful, ferredoxin reduction of cytochrome b6 was still possible in the iron-sulfur depleted complex (Lam and Malkin 1982). A remarkably stoichiometric quantity of nearly 1 plastoquinone per cytochromefco-purifies with the cytochrome bfcomplex (Hurt and Hauska 1982b, O'Keefe 1987). This has been removed by a variety of techniques including organic solvent extraction (Hurt and Hauska 1982b), chromatographic techniques (Doyle and Yu 1985, O'Keefe 1987), a second sucrose gradient in the presence of detergent (Chain 1985), or a second ammonium
193 sulfate precipitation (Nitschke et al. 1987). Most evidence suggests that the copurified plastoquinone is not a specific tightly bound plastoquinone, and procedures which remove other lipophilic pigments are also effective at removing this plastoquinone. It has been reported that the 'bound' quinone could be plastoquinone-C (Hurt and Hauska 1982b), however, high resolution chromatographic techniques demonstrate that the copurified plastoquinone is nearly entirely plastoquinone-A (O'Keefe 1987).
Sources of the cytochrome bf complex Active cytochrome bfcomplex have been most successfully prepared, in the highest number of laboratories, almost exclusively from spinach chloroplasts. However, published procedures do exist for the preparation of cytochrome bf complexes from Chlamydomonas reinhardt (Lemaire et al. 1986) and Anabaena variabilis (Krinner et al. 1982), as well as reported success of preparations from a number of higher plant species (Hauska 1986a). The cytochrome bc~ complex has been purified from the photosynthetic bacterium, Rhodopseudomonas ( Rhodobacter) sphaeroides (Gabellini et al. 1982); however, the electron transfer partners, inhibitor sensitivity, and properties of the Qc semiquinone (see section on mechanism, to follow) suggest that the complex for the photosynthetic bacteria is more closely related to the mitochondrial cytochrome bc~ complex (Prince et al. 1982, Robertson et al. 1984b, Ljungdahl et al. 1987).
Spectroscopic and electrochemical properties These properties of the thylakoid election transfer components have been reviewed at length elsewhere (Cramer and Whitmarsh 1977, Bendall 1982, Malkin 1982b, Rich and Bendall 1980). Further detailed discussion is compelling for only the most recent developments, with a brief summary for others. In this latter regard, cytochromefis a high potential c-type cytochrome with a characteristic absorption maximum of the ferrocytochrome f ~-band at 554 nm, and an EPR signal associated with the ferricytochrome at g ~ 3.5 (Nugent and Evans 1980, Rich et al. 1980, Bergstrom and Vanngard 1982, Crowder et al. 1982). The Rieske iron-sulfur protein contains a 2Fe-2S cluster, and exhibits a broad nearly featureless absorption in the visible spectrum, but the protein does display distinct characteristic EPR signals at g values of 2.02 and 1.89 (Malkin and Aparicio 1975, Hurt et al. 1981). Values for the redox midpoint potential at pH 7 for cytochrome frange from 330 to 390 mV (Cramer and Whitmarsh 1977, Malkin 1982b), with a pKa on the oxidized form at 9.0 (Rich and Bendall 1980). The Rieske
194 - -
('ylochrome I) H
--
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--
563
(-80 vs 235 mV) Cyh)chr0me bL (-260 vs -80 mY)
0.02 AA
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540
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I
1
560
580
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I I I I I I [ I I I I I I I I I [ I I
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Era7 =-70mV, n=l
0.06 ~'Em7 = -25 mV, n=l LEm7 =-120 mV, n=l
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Structure and function of the chloroplast cytochrome bf complex D A N I E L P. O ' K E E F E Central Research and Development Department, E.L duPont deNemours and Company, Inc., Experimental Station, Bldg. 402, Wilmington, DE 19898, USA Received: 1 December 1987; accepted in revised form 2 February 1988
Key words: cytochrome, electrogenic, oxidoreductase, plastoquinone Abstract. The chloroplast cytochrome bfcomplex is an intrinsic multisubunit protein from the
thylakoid membrane consisting of four polypeptides: cytochrome f, a two heine containing cytochrome b6, the Rieske iron-sulfur protein, and a 17 kD polypeptide of undefined function. The complex functions in electron transfer between PSII and PSI, where most mechanisms suggest that the transfer of a single reducing equivalent from plastoquinol to plastocyanin results in the translocation of two protons across the membrane. Primary sequence analyses, dichroism studies, and functional considerations allow the construction of an approximate structural model of a monomeric complex, although some evidence exists for a dimeric structure. Resolution of the properties of the two cytochrome b6 heroes has relied upon the availability of purified solubilized complex, while evidence in the thylakoid suggests the difference between the two hemes are not as great in situ. Such variability in the spectroscopic and electrochemical properties of the cytochrome b6 is a major concern during the experimental use of the purified complex. There is a general consensus that the complex contains a plastoquinol oxidizing (Qz) site, although the evidence for a plastoquinone reduction (Qc) site, called for in most mechanistic hypotheses, is less substantive. Probably the most severe challenge to the so called Q-cycle mechanism comes from experimental observations made with cytochrome b6 initially reduced, where proposed interpretations more closely resemble a b-cycle than a Q-cycle. Although functional during cyclic electron transfer, the role of the complex and its possible interaction with other proteins, has not been completely resolved. Abbreviations: Cytochrome bH-high potential cytochrome b6, Cytochrome bL-low potential
cytochrome b6, DBMIB-2,~-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DNP-INT-2iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether, FNR -ferredoxin:NADP oxidoreductase, HQNO-2-n-heptyl-4-hydroxyquinoline-N-oxide, NQNO-2-n-nonyl-4hydroxyquinoline-N-oxide, Qc-quinone binding site on the cytochrome bfcomplex near the outside of the thylakoid membrane, alternatively designated "centre i" or "centre r", Qz-quinone binding site on the cytochrome bfcomplex near the inside of the thylakoid membrane, alternatively designated "centre o"
190
Introduction The cytochrome bfcomplex of the chloroplast thylakoid membrane plays a central role in the transfer of electrons between the two photosystems, serving as the site of energy conserving proton translocation associated with photosystem I. Together with the two photochemical reaction centers, water oxidizing complex, light harvesting proteins, and the ATP synthetase complex, the cytochrome bf complex is a major functional component of the thylakoid membrane photosynthesis machinery. Analogous in function to the cytochrome bcl complex found in mitochondria and photosynthetic bacteria, the cytochrome bf complex has one of the simplest polypeptide compositions, but exhibits a different substrate:product specificity (plastoquinol:plastocyanin oxidoreductase as compared to ubiquinol:cytochrome c oxidoreductase), and is inhibited by a unique spectrum of compounds. While the implicit assumption of many researchers is that the mechanistic details of the cytochrome bf and bc~ complexes are probably very similar, attempts to incorporate reactions associated with cyclic electron transfer around PSI have also led to proposals utilizing electron transfer pathways unique to the cytochrome bf complex. Many excellent detailed reviews of the literature on various aspects of the cytochrome bfand bcl complexes are available (Bendall 1982, Prince et al. 1982a, Crofts and Wraight 1983, Hauska et al. 1983, Rich 1984, Dutton 1986, Hauska 1986b, Joliot and Joliot 1986b, 1986c). Nonetheless, the past several years have produced new information on the cytochrome bf complex, related to its overall composition and structure, physical properties, and its role in the energy conserving reactions of the chloroplast. The purpose of the current exposition is to provide a brief overview of the approaches recently used to obtain a general idea of the structure of this protein, and how this relates to its function in electron and proton transport in the thylakoid membrane.
Purified preparations That cytochromesfand b6 (also known as b-563) occur in thylakoid membranes as members of a distinct stable protein complex was first demonstrated by Nelson and Neumann (Nelson and Neumann 1972). The polypeptide composition of this preparation was not reported, and while it exhibited some plastoquinol:plastocyanin reductase activity, it was also contaminated with low potential cytochrome b-559 (Rich et al. 1980). Subsequently, Hurt and Hauska reported a procedure for the isolation of a
191 five polypeptide complex, exhibiting plastoquinol:plastocyanin reductase activity (Hurt and Hauska 1981, 1982a), as well as proton translocating activity when reconstituted into phospholipid vescicles (Hurt et al. 1982). Several other procedures for isolating the complex from spinach have also appeared (Clark and Hind 1983a, Doyle and Yu 1985, Black et al. 1987), and with the exception of the procedure of Clark and Hind, all follow essentially the method of Hurt and Hauska. Briefly, this involves washing thylakoid membranes with NaBr to remove extrinsic proteins, solubilization in octyl glucoside or MEGA-9, ammonium sulfate fractionation, and final purification of the cytochrome bfcomplex by either a sucrose gradient (Hurt and Hauska 1981, 1982a, Clark and Hind 1983a), or cellulose phosphate chromatography step (Doyle and Yu 1985, Black et al. 1987). The cellulose phosphate chromatography reportedly allows larger amounts of material to be purified in one preparation (Black et al. 1987), although it is the earlier steps, and not the use of a sucrose gradient that is the most limiting aspect of other procedures. There is some variability in the polypeptide profile of the purified complex. Five polyacrylamide gel bands at 34, 33, 23, 20 and 17 kD reportedly correspond to two cytochrome f polypeptides, cytochrome b6, the Rieske iron sulfur protein, and a 17kD protein (with no known redox function), respectively (Hurt and Hauska 1981, 1982a, Hurt et al. 1981). The two cytochrome f polypeptides both stain positive for heme, are not distinct either by immunological techniques or peptide mapping, and it is necessary to add their contributions together in densitometric scans of electrophoresis gels in order to obtain stoichiometric amounts with the other members of the complex (Hurt and Hauska 1982a). These results suggest that a single cytochrome f polypeptide is partially modified in some way which slightly alters its electrophoretic mobility. Some investigators (Clark and Hind 1983a, Black et al. 1987, DP O'Keefe and KJ Leto, unpublished) have been unable to observe the closely spaced cytochrome f doublet, and some confusion about the nature of the observed doublet is possible when the flavoprotein, FNR (ferredoxin:NADP oxidoreductase, Mr ~ 37kD) copurifies in substantial quantities (Clark et al. 1984). This protein is easily separated from the complex by gel filtration over Sephacryl S-200 (DP O'Keefe and KJ Leto unpublished). The presence of ferredoxin:NADP reductase in any particular cytochrome bfpreparation is readily indicated by rapid reduction of cytochrome f in the presence of NADPH. This ferredoxin:NADP reductase (Forti 1971), is not inhibited in the isolated complex by DBMIB, DNP-INT, antimycin A (O'Keefe 1983), or HQNO (DP O'Keefe unpublished) and occurs in the absence of the complex using purified cytochrome f (Forti 1971). This suggests that the reaction is the
192 result of direct reduction of cytochromefby the flavoprotein, made possible by the accessibility of the cytochrome f heme in the solubilized complex. Reduction of the lower potential components of the complex by NADPH requires ferredoxin in addition to FNR (Lam and Malkin 1982, O'Keefe 1983), further suggesting that there is no direct electron transfer between the flavoprotein and components of the cytochrome bfcomplex. The possibility that the 17 kD polypeptide of the cytochrome bfcomplex is responsible for binding ferredoxin NADP reductase to the thylakoid membrane has been briefly entertained (Vallejos et al. 1984) and rejected (Coughlin et al. 1985). The cytochrome bfpreparation of Black and coworkers resolves many of the above issues in that it only exhibits one cytochromefband, contains no flavoprotein, and has high plastoquinol:plastocyanin oxidoreductase activity (Black et al. 1987). This permits a more categorical statement that a functional cytochrome bfcomplex has a minimal subunit composition of 4 polypeptides:cytochrome f ( ~ 33 kD), cytochrome b 6 (,'~ 23 kD), Rieske iron-sulfur protein (~ 20 kD), and a 17 kD subunit. These polypeptides are present in a single copy per complex (Hurt and Hauska 1982a). The most consistent results in more than one laboratory would suggest that these 4 polypeptides (bearing in mind that the possibility for observing an 'extra' cytochromefpolypeptide exists), are all that is required for the observation in the isolated complex of plastoquinol:plastocyanin oxidoreductase activity at rates (Hauska 1986a, Black et al. 1987) approaching the maximum possible rate of intersystem electron transfer (Whitmarsh and Cramer 1980). However, it should be pointed out that these measurements must be made in the absence of triton X-100, and have all used plastoquinols (PQH2-1 or PQH:-2) of lower molecular weight than the physiological donor, plastoquinol-A(PQH:-9).
Depletion of individual cytochrome bf components The Rieske iron-sulfur protein of the isolated complex can be removed by chromatography on hydroxyapatite in 0.5% Triton X-100 (Hurt et al. 1981). Although attempts at reconstitution of plastoquinol :plastocyanin oxidoreductase with depleted cytochrome bfcomplex, and purified iron sulfur protein were unsuccessful, ferredoxin reduction of cytochrome b6 was still possible in the iron-sulfur depleted complex (Lam and Malkin 1982). A remarkably stoichiometric quantity of nearly 1 plastoquinone per cytochromefco-purifies with the cytochrome bfcomplex (Hurt and Hauska 1982b, O'Keefe 1987). This has been removed by a variety of techniques including organic solvent extraction (Hurt and Hauska 1982b), chromatographic techniques (Doyle and Yu 1985, O'Keefe 1987), a second sucrose gradient in the presence of detergent (Chain 1985), or a second ammonium
193 sulfate precipitation (Nitschke et al. 1987). Most evidence suggests that the copurified plastoquinone is not a specific tightly bound plastoquinone, and procedures which remove other lipophilic pigments are also effective at removing this plastoquinone. It has been reported that the 'bound' quinone could be plastoquinone-C (Hurt and Hauska 1982b), however, high resolution chromatographic techniques demonstrate that the copurified plastoquinone is nearly entirely plastoquinone-A (O'Keefe 1987).
Sources of the cytochrome bf complex Active cytochrome bfcomplex have been most successfully prepared, in the highest number of laboratories, almost exclusively from spinach chloroplasts. However, published procedures do exist for the preparation of cytochrome bf complexes from Chlamydomonas reinhardt (Lemaire et al. 1986) and Anabaena variabilis (Krinner et al. 1982), as well as reported success of preparations from a number of higher plant species (Hauska 1986a). The cytochrome bc~ complex has been purified from the photosynthetic bacterium, Rhodopseudomonas ( Rhodobacter) sphaeroides (Gabellini et al. 1982); however, the electron transfer partners, inhibitor sensitivity, and properties of the Qc semiquinone (see section on mechanism, to follow) suggest that the complex for the photosynthetic bacteria is more closely related to the mitochondrial cytochrome bc~ complex (Prince et al. 1982, Robertson et al. 1984b, Ljungdahl et al. 1987).
Spectroscopic and electrochemical properties These properties of the thylakoid election transfer components have been reviewed at length elsewhere (Cramer and Whitmarsh 1977, Bendall 1982, Malkin 1982b, Rich and Bendall 1980). Further detailed discussion is compelling for only the most recent developments, with a brief summary for others. In this latter regard, cytochromefis a high potential c-type cytochrome with a characteristic absorption maximum of the ferrocytochrome f ~-band at 554 nm, and an EPR signal associated with the ferricytochrome at g ~ 3.5 (Nugent and Evans 1980, Rich et al. 1980, Bergstrom and Vanngard 1982, Crowder et al. 1982). The Rieske iron-sulfur protein contains a 2Fe-2S cluster, and exhibits a broad nearly featureless absorption in the visible spectrum, but the protein does display distinct characteristic EPR signals at g values of 2.02 and 1.89 (Malkin and Aparicio 1975, Hurt et al. 1981). Values for the redox midpoint potential at pH 7 for cytochrome frange from 330 to 390 mV (Cramer and Whitmarsh 1977, Malkin 1982b), with a pKa on the oxidized form at 9.0 (Rich and Bendall 1980). The Rieske
194 - -
('ylochrome I) H
--
t
--
563
(-80 vs 235 mV) Cyh)chr0me bL (-260 vs -80 mY)
0.02 AA
//",,,. / /" I
I
540
I
I
1
560
580
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I I I I I I [ I I I I I I I I I [ I I
560
570
Wavelength (nm) 0.10 /
Era7 =-70mV, n=l
0.06 ~'Em7 = -25 mV, n=l LEm7 =-120 mV, n=l
tt~
.<
