Electron transfer reactions of chemically modified plastocyanin with P700 and cytochrome f. Importance of local charges

Chemically modified spinach plastocyanin, in which negatively charged carboxyl residues are replaced with positively charged amino residues, has been prepared. Four distinct species of chemically modified plastocyanin, having 1 to 4 mol of modified carboxyl residue per mol of plastocyanin, could be separated by ion-exchange chromatography on DEAE-Sephacel. The rate of electron transfer from reduced cytochrome f to oxidized singly substituted plastocyanin was 30% of that of the native unmodified plastocyanin, and the reaction rate decreased further with increasing number of modified carboxyl residues. These results indicate the importance of electrostatic interactions between the negative charges on plastocyanin and the positive charges on cytochrome f in this reaction. Since the overall net charge of cytochrome f is negative at neutral pH, the positive charges on cytochrome f involved in the reaction should be localized ones. On the other hand, the rates of electron transfer from reduced singly and doubly substituted plastocyanin to photooxidized P700 in the P700-chlorophyll alpha protein complex were similar to that of native plastocyanin, which suggests that these carboxyl residues have only a minor role in the electron transfer to P700. Although divalent cation is essential for the electron transfer from native plastocyanin to P700 at neutral pH, the triply substituted plastocyanin could donate electrons to P700 even without MgCl2, and the rate of this reaction reached the maximum at a low concentration of MgCl2 (less than 2.5 mM). The modification of four carboxyl residues per plastocyanin molecule activated this reaction to the maximum level without MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro.

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.     

Quantitation of the rapid electron donors to P700, the functional plastoquinone pool, and the ratio of the photosystems in spinach chloroplasts

Recent studies of chloroplast architecture have emphasized the segregation of photosystem I and photosystem II in different regions of the lamellar membrane. The apparent localization of photosystem II reaction centers in regions of membrane appression and of photosystem I reaction centers in regions exposed to the chloroplast stroma has focused attention on the intervening electron carriers, carriers which must be present to catalyze electron transfer between such spatially separated reaction sites. Information regarding the stoichiometries of these intermediate carriers is essential to an understanding of the processes that work together to establish the mechanism and to determine the rate of the overall process. We have reinvestigated the numbers of photosystem I and photosystem II reaction centers, the numbers of intervening cytochrome b6/f complexes, and the numbers of molecules of the relatively mobile electron carriers plastoquinone and plastocyanin that are actively involved in electron transfer. Our investigations were based on a new experimental technique made possible by the use of a modified indophenol dye, methyl purple, the reduction of which provides a particularly sensitive and accurate measure of electron transfer. Using this dye, which accepts electrons exclusively from photosystem I, it was possible to drain electrons from each of the carriers. Thus, by manipulation of the redox condition of the various carriers and through the use of specific inhibitors we could measure the electron storage capacity of each carrier in turn. We conclude that the ratio of photosystem I reaction centers to cytochrome b6/f complexes to photosystem II reaction centers is very nearly 1:1:1. The pool of rapid donors of electrons to P700 includes not only the 2 reducing equivalents stored in the cytochrome b6/f complex but also those stored in slightly more than 2 molecules of plastocyanin per P700. More slowly available are the electrons from about 6 plastoquinol molecules per P700.     

Photoelectric studies of the transmembrane charge transfer reactions in photosystem I pigment-protein complexes

The results of studies of charge transfer in cyanobacterial photosystem I (PS I) using the photoelectric method are reviewed. The electrogenicity in the PS I complex and its interaction with natural donors (plastocyanin, cytochrome c(6)), natural acceptors (ferredoxin, flavodoxin), or artificial acceptors and donors (methyl viologen and other redox dyes) were studied. The operating dielectric constant values in the vicinity of the charge transfer carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment-protein complex (from plastocyanin or cytochrome c(6) through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and electron transfer rate constant were discussed.     

Interaction of plastocyanin with photosystem I: a chemical cross-linking study of the polypeptide that binds plastocyanin

Plastocyanin has been covalently cross-linked to photosystem I (PSI) by using a water-soluble cross-linker, N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linking reaction is light stimulated and results in the disappearance of a single 19-kDa subunit of PSI with the formation of a new protein-staining component of 31 kDa. The new product at 31 kDa reacts with both plastocyanin and 19-kDa subunit antibodies. Carboxyl group modified plastocyanin does not form a cross-linked product with PSI, implying that the negatively charged surface-exposed groups on plastocyanin are necessary to stabilize binding. These results demonstrate a specific interaction of plastocyanin with PSI and further implicate a specific protein to which plastocyanin binds to facilitate electron transfer to the P700 reaction center.     

The plastocyanin binding domain of photosystem I.

The molecular recognition between plastocyanin and photosystem I was studied. Photosystem I and plastocyanin can be cross-linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant plastocyanins are cross-linked with lysine residues of a domain near the N-terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of plastocyanin by site-directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha-helices. The interacting N-terminal helix lines up six lysines on one side which may guide a fast one-dimensional diffusion of plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two-step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.     

Reactivity of cytochromes c and f with mutant forms of spinach plastocyanin.

The reduction of plastocyanin by cytochromes c and f has been investigated with mutants of spinach plastocyanin in which individual, highly conserved surface residues have been modified. These include Leu-12 and Phe-35 in the 'northern' hydrophobic patch and Tyr-83 and Asp-42 in the 'eastern' acidic patch. The differences observed all involved binding rather than the intrinsic rates of electron transfer. The Glu-12 and Ala-12 mutants showed small but significant decreases in binding constant with cytochrome c, even though the cytochrome is not expected to make contact with the northern face of plastocyanin. These results, and small changes in the EPR parameters, suggested that these mutations cause small conformational changes in surface residues on the eastern face of plastocyanin, transmitted through the copper centre. In the case of cytochrome f, the Glu-12 and Ala-12 mutants also bound less strongly, but Leu12Asn showed a marked increase in binding constant, suggesting that cytochrome f can hydrogen bond directly to Asn-12 in the reaction complex. A surprising result was that the kinetics of reduction of Asp42Asn were not significantly different from wild type, despite the loss of a negative charge.     

Electron transfer between plastocyanin and P700 in highly-purified photosystem I reaction center complex. Effects of pH, cations, and subunit peptide composition

Treatment of isolated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel chromatographies yielded a highly purified P700-chlorophyll a protein complex complex which consists of five polypeptides. The protein complex is virtually free of chlorophyll b (Ch1 alpha/Ch1 b greater than 10) with approximately 30 chlorophylls per P700, and contains iron-sulfur centers A, B, and X. At pH values higher than 6, divalent cations, but not monovalent or trivalent cations, efficiently accelerated the electron transfer from reduced spinach plastocyanin to the photooxidized P700 in the P700-chlorophyll alpha protein complex. At pH values lower than 6, the reaction rate drastically increased with decreasing pH with a maximum at about pH 4.3 without cations. Divalent salts as well as monovalent or trivalent salts decreased the P700 reduction rate at low pH, indicating the involvement of electrostatic interaction in those pH regions. The rate of electron transfer from plastocyanin to the photooxidized P700 in the reaction center protein, which consists of only the largest peptide subunit and no iron-sulfur centers, was reduced only 50% at pH 7.0 in the presence of MgCl2 as compared to the case of P700-chlorophyll alpha protein complex. Essentially similar effects of pH and metal ions on this electron transfer reaction were observed as in the case of P700-chlorophyll alpha protein complex. These results strongly suggest that plastocyanin donates electrons directly to the largest peptide of P700-chlorophyll alpha protein complex and the observed effects of pH and cations are mainly due to the interaction between the largest peptide of P700-chlorophyll alpha protein complex and plastocyanin. The four small subunits in the protein complex seemed to have only a minor role in the reaction with plastocyanin.     

Identification of the plastocyanin binding subunit of photosystem I

Plastocyanin is specifically cross-linked by incubation with N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to a subunit of photosystem I in stroma lamellae and in isolated photosystem I complex. SDS-PAGE shows the disappearance of a 18.5 kDa subunit and the appearance of a new 31.5 kDa protein which was recognized by anti-plastocyanin antibodies. The isolated subunit was identified by its N-terminal amino acid sequence as the mature peptide coded by the nuclear gene psaF [Steppuhn et al. (1988) FEBS Lett. 237, 218–224]. P700⁺ was reduced by cross-linked plastocyanin with the same halftime of 13 μs as found in the native complex. This is evidence that cross-linking conserved the orientation of the complex and that the 18.5 kDa subunit provides the conformation of photosystem I necessary for the extremely rapid electron transfer from plastocyanin to P700⁺.     

Electrogenic reactions in the photosystem I

The processes of electron transfer in cyanobacterial photosystem I (PS I) and photoelectric methods of the studies were reviewed. Particular emphasis was placed on structural and kinetic characteristics of the electron transport chain. The electrogenicity in PS I complex and its interaction with natural donors (plastocyanin, cytochrome c6), natural acceptors (ferredoxin, flavodoxin), and artificial acceptors and donors (methyl viologen and other redox dyes) were studied. On the basis of photoelectric measurements and the X-ray structural data, the operating dielectric constants in the vicinity of charge carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment-protein complex (from plastocyanin or cytochrome c6 through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and the electron transfer rate constant in the corresponding segment of the chain were discussed.     

Interaction of cytochrome c with reaction centers of Rhodopseudomonas sphaeroides R-26: localization of the binding site by chemical cross-linking and immunochemical studies

The location of the cytochrome binding site on the reaction center of Rhodopseudomonas sphaeroides was studied by two different approaches. In one, cross-linking agents, principally dithiobis(propionimidate) and dimethyl suberimidate, were used to link cytochrome c and cytochrome c2 to reaction centers; in the other, the inhibition of electron transfer by antibodies against the subunits was investigated. Cytochrome c (horse) cross-linked to the L and M subunits, whereas cytochrome c2 (R. sphaeroides) cross-linked only to the L subunit. The cross-linked reaction center-cytochrome complexes were isolated by affinity chromatography. The rate of electron transfer in the cross-linked cytochrome c2 complex was the same as that in the un-cross-linked complex. However, when cytochrome c was used, the rate in the cross-linked complex was about 15 times slower than that in the un-cross-linked complex. Fab fragments of antibodies specific against the L and M subunits blocked electron transfer from both cytochrome c (horse) and cytochrome c2 (R. sphaeroides). Antibodies specific for the H subunit did not block either reaction. We conclude that the cytochrome binding site on the reaction center is close (approximately 10 A) to both the L and M subunits, possibly in a cleft between them.     

The interaction of nitrotyrosine-83 plastocyanin with cytochromes f and c: pH dependence and the effect of an additional negative charge on plastocyanin.

Spinach plastocyanin was selectively modified using tetranitromethane which incorporates a nitro group ortho to the hydroxyl group of tyrosine 83 (Anderson, G.P., Draheim, J.E. and Gross, E.L. (1985) Biochim. Biophys. Acta 810, 123-131). This tyrosine residue has been postulated to be part of the cytochrome f binding site on plastocyanin. Since the hydroxyl moiety of nitrotyrosine 83 is deprotonated above its pK of 8.3, it provides a useful modification for studying the effect of an extra negative charge on the interaction of plastocyanin with cytochrome f. No effect on cytochrome f oxidation was observed at pH 7 under conditions in which the hydroxyl moiety is protonated. However, the rate of cytochrome f oxidation increased at pH values greater than 8, reaching a maximum at pH 8.6 and decreasing at still higher pH values. The increase was half-maximal at pH 8.3 which is the pK for the hydroxyl moiety on nitrotyrosine 83. In contrast, the rate of cytochrome f oxidation for control plastocyanin was independent of pH from pH 7 to 8.6. These results show that increasing the negative charge on plastocyanin at Tyr-83 increases the ability to react with cytochrome f, supporting the hypothesis that cytochrome f interacts with plastocyanin at this location. In contrast, the reaction of Ntyr-83 plastocyanin with mammalian cytochrome c was independent of pH, suggesting that its mode of interaction with plastocyanin is different from that of cytochrome f. A comparison of the effects of Ntyr-83 modification of plastocyanin with the carboxyl- and amino-group modifications reported previously suggests that plastocyanin binds to cytochrome f in such a way that electrons could be donated to plastocyanin at either of its two binding sites.     

Electron transfer from plastocyanin to photosystem I.

Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site-specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C-terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid-targeting domain that acts as a bacterial export signal. The identity of the purified plastocyanins was verified by matrix-assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin. We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.     

Inhibition of Plastocyanin to P700+ Electron Transfer in Chlamydomonas reinhardtii by Hyperosmotic Stress

Oxygen electrode and fluorescence studies demonstrate that linear electron transport in the freshwater alga Chlamydomonas reinhardtii can be completely abolished by abrupt hyperosmotic shock. We show that the most likely primary site of inhibition of electron transfer by hyperosmotic shock is a blockage of electron transfer between plastocyanin (PC) or cytochrome c(6) and P(700). The effects on this reaction were reversible upon dilution of the osmolytes and the stability of plastocyanin or photosystem (PS) I was unaffected. Electron micrographs of osmotically shocked cells showed a significant decrease in the thylakoid lumen volume. Comparison of estimated lumenal width with the x-ray structures of plastocyanin and PS I suggest that lumenal space contracts during HOS so as to hinder the movement of docking to PS I of plastocyanin or cytochrome c(6).     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Dynamic interaction of plastocyanin with the cytochrome bf complex

The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of plastocyanin, which has been proposed to be the site of electron transfer from cytochrome f. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of plastocyanin to the intact cytochrome bf complex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.     

The surface-exposed tyrosine residue Tyr83 of pea plastocyanin is involved in both binding and electron transfer reactions with cytochrome f.

Site-directed mutants of the pea plastocyanin gene in which the codon for the surface-exposed Tyr83 has been changed to codons for Phe83 and Leu83 have been expressed in transgenic tobacco plants. The mutant proteins have been purified to homogeneity and their conformations shown not to differ significantly from the wild-type plastocyanin by 1H-NMR and CD. Overall rate constants for electron transfer (k2) from cytochrome f to plastocyanin have been measured by stopped-flow spectrophotometry and rate constants for binding (ka) and association constants (KA) have been measured from the enhanced Soret absorption of cytochrome f on binding plastocyanin. These measurements allow the calculation of the intrinsic rate of electron transfer in the binary complex. An 8-fold decrease in the overall rate of electron transfer to the Phe83 mutant is due entirely to a decreased association constant for cytochrome f, whereas the 40-fold decrease in the overall rate of electron transfer to the Leu83 mutant is due to weaker binding and a lower intrinsic rate of electron transfer. This indicates that Tyr83 is involved in binding to cytochrome f and forms part of the main route of electron transfer.     

Photosynthetic electron transfer through the cytochrome b6f complex can bypass cytochrome f

The cytochrome b(6)f complex is an obligatory electron transfer and proton-translocating enzyme in all oxygenic photosynthesis. Its operation has been described by the "Q-cycle." This model proposes that electrons are transferred from plastoquinol to plastocyanin (the reductant of P700 in Photosystem I) through, obligatorily in series, the iron-sulfur and the cytochrome f redox centers in the cytochrome b(6)f complex. However, here we demonstrate that (a) the iron-sulfur center-dependent reductions of plastocyanin and P700 are much faster than cytochrome f reduction, both in Chlamydomonas reinhardtii cytochrome f mutants and in the wild type, and (b) the steady-state photosynthetic electron transport does not correlate with strongly inhibited cytochrome f reduction kinetics in the mutants. Thus, cytochrome f is not an obligatory intermediate for electrons flowing through the cytochrome b(6)f complex. The oxidation equivalents from Photosystem I are delivered to the high potential chain of the cytochrome b(6)f complex both at the cytochrome f level and, independently, at another site connected to the quinol-oxidizing site, possibly the iron-sulfur center.     

Conformational changes in plastocyanin

The visible and near-uv absorption and circular dichroic spectra were determined for spinach and poplar plastocyanin under a variety of conditions. The visible spectra showed that the copper center was invariant to changes in species, chemical modification with ethylenediamine, and addition of high concentrations of salt [2.7 M (NH4)2SO4]. In contrast, the near-uv spectra were sensitive to these conditions. Reduction of plastocyanin also altered its near-uv absorption and circular dichroic spectra. It is unlikely that these spectral changes were due to charge transfer bands since the near-uv CD spectrum of apo-plastocyanin was almost identical to that of reduced plastocyanin. There were no corresponding changes in the far-uv spectra which monitor protein secondary structure. The most likely explanation is that the protein has a flexible tertiary conformation. Conformational changes may be important in regulating electron transport. If plastocyanin is a mobile electron carrier, differential binding of the oxidized and reduced forms of plastocyanin to its reaction partners cytochrome f and P700 could facilitate electron transport.     

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa. II. Reduction kinetics and electric field increase in the 10 ms range

On dark-adapted Chlorella, after one flash, plastocyanin (PC) undergoes reduction with a half-time of 7 ms. After 4 or 5 flashes, the reduction of PC+ in the 10 ms range is suppressed, and the level of oxidized plastocyanin increases during the next few flashes before reaching a stationary value. Cytochrome f exhibits approximately the same pattern. The reduction of PC+ and cytochrome f+ in the 10 ms range is correlated with an increase of the electrice field named phase b (Joliot, P. and Delosme, R., Biochim. Biophys. Acta 357 (1974) 267-284). Both need the presence of a compound R' in the reduced state. A dark electron transfer involving a carrier of electrons across the membrane, a proton carrier, R' as terminal reducant, PC+ and cytochrome f+ as terminal oxidants, would account for this field generation. Cooperation between the electron transfer chains is implied at the level of plastocyanin oxidation. An equilibrium constant of about 2 is observed between cytochrome f and plastocyanin before 1 ms and after 500 ms after the photochemical reactions. We observe that cytochrome f and plastocyanin are not connected from 1 to 100 ms after a photochemical reaction. The equilibrium constant between plastocyanin and P-700 remains large [20] under these conditions.