Structure of the intermolecular complex between plastocyanin and cytochrome f from spinach

In oxygenic photosynthesis, plastocyanin shuttles electrons between the membrane-bound complexes cytochrome b6f and photosystem I. The homologous complex between cytochrome f and plastocyanin, both from spinach, is the object of this study. The solution structure of the reduced spinach plastocyanin was determined using high field NMR spectroscopy, whereas the model structure of oxidized cytochrome f was obtained by homology modeling calculations and molecular dynamics. The model structure of the intermolecular complex was calculated using the program AUTODOCK, taking into account biological information obtained from mutagenesis experiments. The best electron transfer pathway from the heme group of cytochrome f to the copper ion of plastocyanin was calculated using the program HARLEM, obtaining a coupling decay value of 1.8 x 10(-4). Possible mechanisms of interaction and electron transfer between plastocyanin and cytochrome f were discussed considering the possible formation of a supercomplex that associates one cytochrome b6f, one photosystem I, and one plastocyanin.     

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.     

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.     

Transient binding of plastocyanin to its physiological redox partners modifies the copper site geometry

The transient complexes of plastocyanin with cytochrome f and photosystem I are herein used as excellent model systems to investigate how the metal sites adapt to the changes in the protein matrix in transient complexes that are involved in redox reactions. Thus, both complexes from the cyanobacterium Nostoc sp. PCC 7119 (former Anabaena sp. PCC 7119) have been analysed by X-ray absorption spectroscopy. Our data are consistent with a significant distortion of the trigonal pyramidal geometry of the Cu coordination sphere when plastocyanin binds to cytochrome f, no matter their redox states are. The resulting tetrahedral geometry shows a shortening of the distance between Cu and the S(delta) atom of its ligand Met-97, with respect to the crystallographic structure of free plastocyanin. On the other hand, when plastocyanin binds to photosystem I instead of cytochrome f, the geometric changes are not significant but a displacement in charge distribution around the metal centre can be observed. Noteworthy, the electronic density around the Cu atom increases or decreases when oxidised plastocyanin binds to cytochrome f or photosystem I, respectively, thus indicating that the protein matrix affects the electron transfer between the two partners during their transient interaction.     

In vivo photosystem I reduction in thermophilic and mesophilic cyanobacteria: the thermal resistance of the process is limited by factors other than the unfolding of the partners

Photosystem I reduction by plastocyanin and cytochrome c(6) in cyanobacteria has been extensively studied in vitro, but much less information is provided on this process inside the cell. Here, we report an analysis of the electron transfer from both plastocyanin and cytochrome c(6) to photosystem I in intact cells of several cyanobacterial species, including a comparative study of the temperature effect in mesophilic and thermophilic organisms. Our data show that cytochrome c(6) reduces photosystem I by following a reaction mechanism involving complex formation, whereas the copper-protein follows a simpler collisional mechanism. These results contrast with previous kinetic studies in vitro. The effect of temperature on photosystem I reduction leads us to conclude that the thermal resistance of this process is determined by factors other than the proper stability of the protein partners.     

Laser flash-induced kinetic analysis of cytochrome f oxidation by wild-type and mutant plastocyanin from the cyanobacterium Nostoc sp. PCC 7119

Oxidation of the soluble, truncated form of cytochrome f by wild-type and mutant species of plastocyanin has been analyzed by laser flash absorption spectroscopy in the cyanobacterium Nostoc (formerly, Anabaena) sp. PCC 7119. At low ionic strengths, the apparent electron transfer rate constant of cytochrome f oxidation by wild-type plastocyanin is 1.34 x 10(4) s(-)(1), a value much larger than those determined for the same proteins from other organisms. Upon site-directed mutagenesis of specific residues at the plastocyanin interaction area, the rate constant decreases in all cases yet to varying extents. The only exception is the D54K variant, which exhibits a higher reactivity toward cytochrome f. In most cases, the reaction rate constant decreases monotonically with an increase in ionic strength. The observed changes in the reaction mechanism and rate constants are in agreement with the location of the mutated residues at the interface area, as well as with the peculiar orientation of the two partners within the Nostoc plastocyanin-cytochrome f transient complex, whose NMR structure has been determined recently. Furthermore, the experimental data herein reported match well the kinetic behavior exhibited by the same set of plastocyanin mutants when acting as donors of electrons to photosystem I [Molina-Heredia, F. P., et al. (2001) J. Biol. Chem. 276, 601-605], thus indicating that the copper protein uses the same surface areas-one hydrophobic and the other electrostatic-to interact with both cytochrome f and photosystem I.     

Interaction between immobilized plastocyanin and cytochrome f and the photosystem I reaction center in a reconstituted system

The effects of redox conversions of plastocyanin copper chromophore on the formation of plastocyanin complexes with cytochrome f and the reaction center of photosystem I from pea chloroplasts were studied. In order to investigate the complex formation plastocyanin and cytochrome f were immobilized on Sephadex G-200. The cytochrome f and reaction center assembly takes place on the immobilized plastocyanin, which is necessary for cytochrome f photooxidation. It was found that in a reconstituted system the reduced plastocyanin forms more stable complexes with the proteins than the oxidized one, which is due to its lower pI value.     

Kinetic studies on a cross-linked complex between plastocyanin cytochrome f

A cross-linked complex between plastocyanin and cytochrome f was prepared by incubation in the presence of a water soluble carbodiimide and its kinetic properties were studied. The optical spectra, oxidation-reduction potentials and isoelectric pH of plastocyanin and cytochrome f did not change upon the formation of the cross-linked complex. Studies on the ionic strength effect on the electron transfer rate from cross-linked plastocyanin to ferricyanide indicated that the negative charge on the reaction site of plastocyanin was masked upon the cross-linking. It was also suggested that the sign of the net charge near the cytochrome f heme edge changed from positive to negative upon the cross-linking. On the other hand, electrostatic interactions between cross-linked plastocyanin and P700 seemed to be essentially the same as those in the case of native plastocyanin, although the rate of electron transfer from cross-linked plastocyanin to P700 was severely reduced. We also measured the intra-complex electron transfer from cytochrome f to plastocyanin. This suggested that the covalently cross-linked complex is a valid model of the electron transfer encounter complex. Based on these results, the reaction sites of plastocyanin with P700 and cytochrome f were discussed.     

The cytochrome f-plastocyanin complex as a model to study transient interactions between redox proteins

Transient complexes, with a lifetime ranging between microseconds and seconds, are essential for biochemical reactions requiring a fast turnover. That is the case of the interactions between proteins engaged in electron transfer reactions, which are involved in relevant physiological processes such as respiration and photosynthesis. In the latter, the copper protein plastocyanin acts as a soluble carrier transferring electrons between the two membrane-embedded complexes cytochrome b(6)f and photosystem I. Here we review the combination of experimental efforts in the literature to unveil the functional and structural features of the complex between cytochrome f and plastocyanin, which have widely been used as a suitable model for analyzing transient redox interactions.     

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.     

Respiratory cytochrome c oxidase can be efficiently reduced by the photosynthetic redox proteins cytochrome c6 and plastocyanin in cyanobacteria

Plastocyanin and cytochrome c6 are two small soluble electron carriers located in the intrathylacoidal space of cyanobacteria. Although their role as electron shuttle between the cytochrome b6f and photosystem I complexes in the photosynthetic pathway is well established, their participation in the respiratory electron transport chain as donors to the terminal oxidase is still under debate. Here, we present the first time-resolved analysis showing that both cytochrome c6 and plastocyanin can be efficiently oxidized by the aa3 type cytochrome c oxidase in Nostoc sp. PCC 7119. The apparent electron transfer rate constants are ca. 250 and 300 s(-1) for cytochrome c6 and plastocyanin, respectively. These constants are 10 times higher than those obtained for the oxidation of horse cytochrome c by the oxidase, in spite of being a reaction thermodynamically more favourable.     

Direct simulation of plastocyanin and cytochrome f interactions in solution

Most biological functions, including photosynthetic activity, are mediated by protein interactions. The proteins plastocyanin and cytochrome f are reaction partners in a photosynthetic electron transport chain. We designed a 3D computer simulation model of diffusion and interaction of spinach plastocyanin and turnip cytochrome f in solution. It is the first step in simulating the electron transfer from cytochrome f to photosystem 1 in the lumen of thylakoid. The model is multiparticle and it can describe the interaction of several hundreds of proteins. In our model the interacting proteins are represented as rigid bodies with spatial fixed charges. Translational and rotational motion of proteins is the result of the effect of stochastic Brownian force and electrostatic force. The Poisson-Boltzmann formalism is used to determine the electrostatic potential field generated around the proteins. Using this model we studied the kinetic characteristics of plastocyanin-cytochrome f complex formation for plastocyanin mutants at pH 7 and a variety of ionic strength values.     

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.     

Photo-dependent conversions of cytochrome f in bean chloroplasts and leaves treated with polyene antibiotics

The photo-dependent absorption changes of cytochrome f in bean chloroplasts and native leaves treated with the polyene antibiotics surgumycin and filipin were studied. Upon incubation of the chloroplasts or leaves with the antibiotics the value of the photo-induced signal of cytochrome f decreased considerably; however, the kinetics of the cytochrome oxidation under the effect of the exciting light and dark reduction remained unchanged. An addition of plastocyanin to the suspension of the antibiotic-treated chloroplasts, which contained no artificial donors and acceptors, only slightly increased the absolute value of the photo-induced signal of cytochrome f. An addition of plastocyanin to the chloroplasts containing the dichlorophenolindophenol-ascorbate-methylviologen system, sharply changed the kinetics of the cytochrome f photoconversions. A simultaneous registration of the photo-induced signal of cytochrome f and the photochemical activity of photosystem I of the antibiotic-treated chloroplasts revealed differences in the degree of inhibition of the photosystem I activity and decrease of the absolute value of the cytochrome f signal. The data obtained are discussed in terms of possible alternative pathways of electron transfer in the part of the electron transporting chain under study.     

A laser flash-induced kinetic analysis of in vivo photosystem I reduction by site-directed mutants of plastocyanin and cytochrome c6 in Synechocystis sp. PCC 6803

In cyanobacteria, plastocyanin and cytochrome c6 are two soluble metalloproteins which can alternately serve as electron donors to photosystem I. From site-directed mutagenesis studies in vitro, it is well-established that both hydrophobic and electrostatic forces are involved in the interaction between the donor proteins and photosystem I. Hence, two isofunctional areas, a hydrophobic one in the north and an acidic one in the east, have been described on the surface of both electron donors. In this work, we have tested the relevance of such kinds of interactions in the photosystem I reduction inside the cell. Several plastocyanin and cytochrome c6 site-directed mutant strains affecting both the acidic and hydrophobic regions of the two metalloproteins, which were previously characterized in vitro, have been constructed. The photosystem I reduction kinetics of the different mutants have been analyzed by laser flash absorption spectroscopy. Relevant differences have been found between the in vitro and in vivo results, mainly regarding the role played by the electrostatic interactions. Adding positive electrostatic charges to the acidic patch of plastocyanin and cytochrome c6 promotes an enhanced interaction with photosystem I in vitro but yields the opposite effect in vivo. These discrepancies are discussed in view of the different environmental conditions, in vitro and in vivo, for the reaction mechanism of photosystem I reduction, namely, differential interaction of the electron donors with the thylakoidal membrane and kinetics of donor exchange.     

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)     

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).     

Multiparticle computer simulation of protein interactions in the photosynthetic membrane

The basic principles of the design of direct multiparticle models and the results of multiparticle computer simulation of electron transfer by mobile protein carriers in the photosynthetic membrane of a chloroplast thylakoid are presented. The reactions of complex formation of the plastocyanin with cytochrome f and the pigment-protein complex of photosystem I, as well as of ferredoxin with FNR and photosystem I are considered. The regulatory role of diffusion and electrostatic interactions as well as the effect of the shape of the reaction volume and ionic strength on the rate of electron transport are discussed.     

The reduction of plastocyanin by plastoquinol-1 in the presence of chloroplasts. A dark electron transfer reaction involving components between the two photosystems.

The reduction of plastocyanin by plastoquinol-1 was efficiently catalysed by disrupted chloroplasts or etioplasts in the dark. The reaction was inhibited by 2,5-dibromomethylisopropyl-p-benzo-quinone which inhibits photosynthetic electron transport between plastoquinone and cytochrome f. Evidence is presented that the reduction took place via cytochrome f, and that plastoquinone-9 was not involved. Triton X-100 and organic solvents were inhibitory, but partial fractionation was achieved without loss of activity by density gradient centrifugation in the presence of high digitonin concentrations. All active material contained cytochromes b-559LP and b-563 in addition to cytochrome f, but these b-type cytochromes were not directly involved. Other 1-electron acceptors could be used in place of plastocyanin, for instance ferricyanide and Pseudomonas cytochrome c-551. The reaction can be applied to give a sensitive dark assay for active cytochrome f. It is suggested that cytochrome f possesses two sites for interaction with redox reagents: a hydrophilic site with which plastocyanin reacts by electron transfer and a hydrophobic site with which plastoquinol reacts by hydrogen atom transfer.     

Study of electrostatic potential surface distribution of wild-type plastocyanin Synechocystis solution structure determined by homonuclear NMR

Plastocyanin is a small (approximately 10 kDa), type I blue copper protein that works as an electron donor to photosystem I from cytochrome f in both chloroplast systems and in some strains of cyanobacteria. Comparative studies of the kinetic mechanisms of plastocyanins in different organisms show that the electron transfer from photosystem I happens by simple collision in cyanobacteria but through a intermediate transition complex in green algae and superior plants. Previous work has proved that this effect cannot be explained by structural variations across the different plastocyanins but it can be explained by differences in the electrostatic potential distribution at the protein surface. In that case, minor conformational errors at the amino acid side chain level may imply an important effect in the electrostatic potential distribution calculation. In this work we present a high resolution study of side chain conformation by homonuclear NMR for the reduced wild-type plastocyanin Synechocystis using intensity ratios for 2D-NOESY and 2D-H,H-TOCSY cross peaks at different mixing times. We also present the corresponding comparison with different plastocyanin structures and the effect in the electrostatic potential distribution at the protein surface. We discuss the importance of indirect J-coupling information from TOCSY-type experiments as complement for intraresidue distances derived from NOESY experiments in the determination of side chain orientation and stereo-specific assignments.