The efficient functioning of photosynthesis and respiration in Synechocystis sp. PCC 6803 strictly requires the presence of either cytochrome c6 or plastocyanin

In cyanobacteria, cytochrome c6 and plastocyanin are able to replace each other as redox carriers in the photosynthetic and respiratory electron transport chains with the synthesis of one or another protein being regulated by the copper concentration in the culture medium. However, the presence of a third unidentified electron carrier has been suggested. To address this point, we have constructed two deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803, each variant lacking either the petE or petJ gene, which respectively codes for the copper or heme protein. The photoautotrophic and heterotrophic growth rate of the two mutants in copper-free and copper-supplemented medium as well as their photosystem I reduction kinetics in vivo were compared with those of wild-type cells. The two mutant strains grow at equivalent rates and show similar in vivo photosystem I reduction kinetics as wild-type cells when cultured in media that allow the expression of just one of the two electron donor proteins, but their ability to grow and reduce photosystem I is much lower when neither cytochrome c6 nor plastocyanin is expressed. These findings indicate that the normal functioning of the cyanobacterial photosynthetic and respiratory chains obligatorily depends on the presence of either cytochrome c6 or plastocyanin.     

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.     

Site-directed mutagenesis of cytochrome c6 from Synechocystis sp. PCC 6803. The heme protein possesses a negatively charged area that may be isofunctional with the acidic patch of plastocyanin

This paper reports the first site-directed mutagenesis analysis of any cytochrome c6, a heme protein that performs the same function as the copper-protein plastocyanin in the electron transport chain of photosynthetic organisms. Photosystem I reduction by the mutants of cytochrome c6 from the cyanobacterium Synechocystis sp. PCC 6803 has been studied by laser flash absorption spectroscopy. Their kinetic efficiency and thermodynamic properties have been compared with those of plastocyanin mutants from the same organism. Such a comparative study reveals that aspartates at positions 70 and 72 in cytochrome c6 are located in an acidic patch that may be isofunctional with the well known "south-east" patch of plastocyanin. Calculations of surface electrostatic potential distribution in the mutants of cytochrome c6 and plastocyanin indicate that the changes in protein reactivity depend on the surface electrostatic potential pattern rather than on the net charge modification induced by mutagenesis. Phe-64, which is close to the heme group and may be the counterpart of Tyr-83 in plastocyanin, does not appear to be involved in the electron transfer to photosystem I. In contrast, Arg-67, which is at the edge of the cytochrome c6 acidic area, seems to be crucial for the interaction with the reaction center.     

Balanced regulation of expression of the gene for cytochrome cM and that of genes for plastocyanin and cytochrome c6 in Synechocystis

The cytM gene for cytochrome cM was previously found in Synechocystis sp. PCC 6803. Northern blotting analysis revealed that the cytM gene was scarcely expressed under normal growth conditions but its expression was enhanced when cells were exposed to low temperature or high-intensity light. By contrast, the expression of the genes for cytochrome c6 and plastocyanin was suppressed at low temperature or under high-intensity light. These observations suggest that plastocyanin and/or cytochrome c6, which are dominant under non-stressed conditions, are replaced by cytochrome cM under the stress conditions.     

Copper-mediated regulation of cytochrome c553 and plastocyanin in the cyanobacterium Synechocystis 6803.

In certain cyanobacteria and algae, cytochrome c553 or plastocyanin can serve to carry electrons from the cytochrome bf complex to photosystem I. The availability of copper in the growth medium regulates which protein is present. To investigate copper induced control of gene expression we isolated these proteins from the cyanobacterium Synechocystis 6803. Using immunodetection and optical spectroscopy, the steady state levels of cytochrome c553 and plastocyanin were measured in cells grown at different copper concentrations. The results show that in cells grown in 20-30 nM copper, cytochrome c553 was present, whereas plastocyanin was not detected. The opposite behavior was observed in cells grown in the presence of 1 microM copper; plastocyanin was present, whereas cytochrome c553 could not be detected. Both proteins were present in cells grown in 0.3 microM copper. Northern analysis of total RNA, probed with a gene fragment for cytochrome c553 or the plastocyanin gene, showed that cells grown in the presence of 20-30 nM copper have message for cytochrome c553, but not for plastocyanin, whereas cells grown in 1 microM copper have message for plastocyanin, but not for cytochrome c553. These results demonstrate that copper regulates expression of both of the genes encoding cytochrome c553 and plastocyanin prior to translation in Synechocystis 6803.     

Plastocyanin cytochrome f interaction

Spinach plastocyanin and turnip cytochrome f have been covalently linked by using a water-soluble carbodiimide to yield an adduct of the two proteins. The redox potential of cytochrome f in the adduct was shifted by -20 mV relative to that of free cytochrome f, while the redox potential of plastocyanin in the adduct was the same as that of free plastocyanin. Solvent perturbation studies showed the degree of heme exposure in the adduct to be less than in free cytochrome f, indicating that plastocyanin was linked in such a way as to bury the exposed heme edge. Small changes were also observed when the resonance Raman spectrum of the adduct was compared to that of free cytochrome f. The adduct was incapable of interacting with or donating electrons to photosystem I. Peptide mapping and sequencing studies revealed two sites of linkage between the two proteins. In one site of linkage, Asp-44 of plastocyanin is covalently linked to Lys-187 of cytochrome f. This represents the first identification of a group on cytochrome f that is involved in the interaction with plastocyanin. The other site of linkage involves Glu-59 and/or Glu-60 of plastocyanin to as yet unidentified amino groups on cytochrome f. Euglena cytochrome c-552 could also be covalently linked to turnip cytochrome f, although with a lower efficiency than spinach plastocyanin. In contrast, a variety of cyanobacterial cytochrome c-553's and a cyanobacterial plastocyanin could not be covalently linked to turnip cytochrome f.     

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.     

Site-directed mutagenesis of cytochrome c(6) from Anabaena species PCC 7119. Identification of surface residues of the hemeprotein involved in photosystem I reduction

A number of surface residues of cytochrome c(6) from the cyanobacterium Anabaena sp. PCC 7119 have been modified by site-directed mutagenesis. Changes were made in six amino acids, two near the heme group (Val-25 and Lys-29) and four in the positively charged patch (Lys-62, Arg-64, Lys-66, and Asp-72). The reactivity of mutants toward the membrane-anchored complex photosystem I was analyzed by laser flash absorption spectroscopy. The experimental results indicate that cytochrome c(6) possesses two areas involved in the redox interaction with photosystem I: 1) a positively charged patch that may drive its electrostatic attractive movement toward photosystem I to form a transient complex and 2) a hydrophobic region at the edge of the heme pocket that may provide the contact surface for the transfer of electrons to P(700). The isofunctionality of these two areas with those found in plastocyanin (which acts as an alternative electron carrier playing the same role as cytochrome c(6)) are evident.     

Tracking the function of the cytochrome c6-like protein in higher plants

The contention that plastocyanin is the only mobile electron donor to photosystem I in higher plants was recently shaken by the discovery of a cytochrome c(6)-like protein in Arabidopsis and other flowering plants. However, the genetic and biochemical data presented in support of the idea that the cytochrome c(6) homologue can replace plastocyanin have now been challenged by two complementary studies. This re-opens the debate on the real function(s) of cytochrome c in the chloroplasts of higher plants.     

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.     

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.     

Unexpected changes in photosystem I function in a cytochrome c6-deficient mutant of the cyanobacterium Synechocystis PCC 6803

Cytochrome c6, the product of the petJ gene, is a photosynthetic electron carrier in cyanobacteria, which transfers electrons to photosystem I and which is synthesised under conditions of copper deficiency to functionally replace plastocyanin. The photosystem I photochemical activity (energy storage, photoinduced P700 redox changes) was examined in a petJ-null mutant of Synechocystis PCC 6803. Surprisingly, photosystem I activity in the petJ-null mutant grown in the absence of copper was not much affected. However, in a medium with a low inorganic carbon concentration and with NH4+ ion as nitrogen source, the mutant displayed growth inhibition. Analysis showed that, especially in the latter, the isiAB operon, encoding flavodoxin and CP43', an additional chlorophyll a antenna, was strongly expressed in the mutant. These proteins are involved in photosystem I function and organisation and are proposed to assist in prevention of overoxidation of photosystem I at its lumenal side and overreduction at its stromal side.     

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.     

A single arginyl residue in plastocyanin and in cytochrome c(6) from the cyanobacterium Anabaena sp. PCC 7119 is required for efficient reduction of photosystem I

Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c(6) from Anabaena (Molina-Heredia, F. P., Diaz-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 33565-33570) and Synechocystis (De la Cerda, B., Diaz-Quintana, A., Navarro, J. A. , Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292-13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin.     

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.     

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c ₆ are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b ₆ f and Photosystem I. Despite plastocyanin and cytochrome c ₆ differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c ₆ reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c ₆ does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electron transfers amongst cytochrome f, plastocyanin and photosystem I: kinetics and mechanisms

The review covers the theory and practice of the determination of kinetic constants for the electron transfer reactions in chloroplast thylakoid membranes between plastocyanin and cytochrome f in cytochrome bf complexes, and between plastocyanin and the reaction centre of photosystem I. Effects of ionic strength and pH are featured. The contribution of mutant studies is included. It is concluded that nearly all data from in vitro experiments can be interpreted with a reaction scheme in which an encounter complex between donor and acceptor is formed by long-range electrostatic attraction, followed by rearrangement during which metal centres become close enough for rapid intra-complex electron transfer. In vivo experiments so far cast doubt on this particular sequence, but their interpretation is not straightforward. Means of modelling the bimolecular complex between cytochrome f and plastocyanin are outlined, and two likely structures are illustrated. The complex formed by plastocyanin and photosystem I in higher plants involves the PsaF subunit, but its structure has not been fully determined.     

Kinetics of electron transfer between plastocyanin and the soluble CuA domain of cyanobacterial cytochrome c oxidase

It has been shown that efficient functioning of photosynthesis and respiration in the cyanobacterium Synechocystis PCC 6803 requires the presence of either cytochrome c6 or plastocyanin. In order to check whether the blue copper protein plastocyanin can act as electron donor to cytochrome c oxidase, we investigated the intermolecular electron transfer kinetics between plastocyanin and the soluble CuA domain (i.e. the donor binding and electron entry site) of subunit II of the aa3-type cytochrome c oxidase from Synechocystis. Both copper proteins were expressed heterologously in Escherichia coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (5.1+/-0.2) x 10(4) M(-1) s(-1) and (8.5+/-0.4) x 10(5) M(-1) s(-1), respectively (20 mM phosphate buffer, pH 7). This corresponds to an apparent equilibrium constant of 0.06 in the physiological direction (reduction of CuA), which is similar to Keq values calculated for the reaction between c-type cytochromes and the soluble fragments of other CuA domains. The potential physiological role of plastocyanin in cyanobacterial respiration is discussed.     

Structure of cytochrome c6A, a novel dithio-cytochrome of Arabidopsis thaliana, and its reactivity with plastocyanin: implications for function

Cytochrome c6A is a unique dithio-cytochrome present in land plants and some green algae. Its sequence and occurrence in the thylakoid lumen suggest that it is derived from cytochrome c6, which functions in photosynthetic electron transfer between the cytochrome b6f complex and photosystem I. Its known properties, however, and a strong indication that the disulfide group is not purely structural, indicate that it has a different, unidentified function. To help in the elucidation of this function the crystal structure of cytochrome c6A from Arabidopsis thaliana has been determined in the two redox states of the heme group, at resolutions of 1.2 A (ferric) and 1.4 A (ferrous). These two structures were virtually identical, leading to the functionally important conclusion that the heme and disulfide groups do not communicate by conformational change. They also show, however, that electron transfer between the reduced disulfide and the heme is feasible. We therefore suggest that the role of cytochrome c6A is to use its disulfide group to oxidize dithiol/disulfide groups of other proteins of the thylakoid lumen, followed by internal electron transfer from the dithiol to the heme, and re-oxidation of the heme by another thylakoid oxidant. Consistent with this model, we found a rapid electron transfer between ferro-cytochrome c6A and plastocyanin, with a second-order rate constant, k2=1.2 x 10(7) M(-1) s(-1).