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1.
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.  相似文献   

2.
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.  相似文献   

3.
Cytochrome cM is a new c-class photosynthetic haem protein whose physiological role is still unknown. It has been proposed previously that cytochrome cM can replace cytochrome c6 and plastocyanin in transferring electrons between the two membrane complexes cytochrome b6-f and photosystem I in organisms growing under stress conditions. The experimental evidence herein provided allows us to discard such a hypothesis. We report a procedure to overexpress cytochrome cM from the cyanobacterium Synechocystis sp. PCC 6803 in Escherichia coli cells in mg quantities. This has allowed us to perform a comparative laser flash-induced kinetic analysis of photosystem I reduction by the three metalloproteins from Synechocystis. The bimolecular rate constant for the overall reaction is up to 100 times lower with cytochrome cM than with cytochrome c6 or plastocyanin. In addition, the redox potential value and surface electrostatic potential distribution of cytochrome cM are quite different from those of cytochrome c6 and plastocyanin. These findings strongly indicate that cytochrome cM cannot be recognised by and interact with the same redox partners as the other two metalloproteins.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
Plastocyanin and cytochrome c 6, the alternate donor proteins to photosystem I, can be acidic, neutral or basic; the role of electrostatics in their interaction with photosystem I vary accordingly for cyanobacteria, algae and plants. The effect of different crowding agents on the kinetics of the reaction between plastocyanin or cytochrome c 6 and photosystem I from three different cyanobacteria, Synechocystis PCC 6803, Nostoc PCC 7119 and Arthrospira maxima, and a green alga, Monoraphidium braunii, has been investigated by laser flash photolysis, in order to elucidate how molecular crowding affects the interaction between the two donor proteins and photosystem I. The negative effect of viscosity on the interaction of the two donors with photosystem I for the three cyanobacterial systems is very similar, as studied by increasing sucrose concentration. Bovine serum albumin seems to alter the different systems in a specific way, probably by means of electrostatic interactions with the donor proteins. Ficoll and dextran behave in a parallel manner, favouring the interaction by an average factor of 2, although this effect is somewhat less pronounced in Nostoc. With regards to the eukaryotic system, a strong negative effect of viscosity is able to overcome the favourable effect of any crowding agent, maybe due to stronger donor/photosystem I electrostatic interactions or the structural nature of the eukaryotic photosystem I-enriched membrane particles.  相似文献   

8.
The plastocyanin binding domain of photosystem I.   总被引:2,自引:0,他引:2       下载免费PDF全文
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.  相似文献   

9.
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.  相似文献   

10.
In cyanobacteria, plastocyanin and cytochrome c 6, the alternate donor proteins to Photosystem I, can be acidic, neutral or basic; the role of electrostatics in their interaction with photosystem I varies accordingly. In order to elucidate whether these changes in the electron donors’ properties correlate with complementary changes in the docking site of the corresponding photosystem, we have investigated the kinetics of reactions between three cytochrome c 6 with isoelectric points of 5.6, 7.0 and 9.0, with Photosystem I particles from the same three genera of cyanobacteria which provided the cytochromes. The model systems compared here thus sample the full range of charge properties observed in cytochromes c 6: acidic, basic and neutral. The rate constants and dependence on ionic strength for photosystem I reduction were distinctive for each cytochrome c 6, but independent of Photosystem I. We conclude that the specific structural features of each cytochrome c 6 dictate their different kinetic behaviours, whereas the three photosystems are relatively indiscriminate in docking with the electron donors.  相似文献   

11.
The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
The dependence of the P(700)(+)/P(700) midpoint potential on kinetics of reduction of P(700)(+) in vivo has been examined in a series of site-directed mutants of Chlamydomonas reinhardtii in which the histidyl axial ligand to the Mg(2+) of the P(700) chlorophyll a has been changed to several different amino acids. In wild-type photosystem I, the potential of P(700)(+)/P(700) is 447 mV and the in vivo half-time of P(700)(+) reduction by its natural donor, plastocyanin, is 4 micros. Substitution of the axial histidine ligand with cysteine increases the potential of P(700)(+)/P(700) to 583 mV and changes the rate of P(700)(+) reduction to 0.8 micros. Mutants with a range of potentials between 447 and 583 mV show a strong correlation of the P(700)(+)/P(700) potential to the rate of reduction of P(700)(+) by plastocyanin. There is also an increase in the rate of photosystem I-mediated electron transfer from the artificial electron donor DCPIP to methyl viologen in thylakoid membranes. The results indicate that the overall rate constant of P(700)(+) reduction is determined by the rate of electron transfer between the copper and P(700)(+) and confirmed that in vivo there is a preformed complex between plastocyanin and photosystem I. Using approximations of the Marcus electron transfer theory, it is possible to estimate that the distance between the copper of plastocyanin and P(700)(+) is approximately 15 A. On the basis of this distance, the plastocyanin docking site should lie in a 10 A hollow formed by the lumenal exposed loops between transmembrane helices i and j of PsaA and PsaB.  相似文献   

16.
On the lumenal side of photosystem I (PSI), each of the two large core subunits, PsaA and PsaB, expose a conserved tryptophan residue to the surface. PsaB-Trp(627) is part of the hydrophobic recognition site that is essential for tight binding of the two electron donors plastocyanin and cytochrome c(6) to the donor side of PSI (Sommer, F., Drepper, F., and Hippler, M. (2002) J. Biol. Chem. 277, 6573-6581). To examine the function of PsaA-Trp(651) in binding and electron transfer of both donors to PSI, we generated the mutants PsaA-W651F and PsaA-W651S by site-directed mutagenesis and biolistic transformation of Chlamydomonas reinhardtii. The protein-protein interaction and the electron transfer between the donors and PSI isolated from the mutants were analyzed by flash absorption spectroscopy. The mutation PsaA-W651F completely abolished the formation of a first order electron transfer complex between plastocyanin (pc) and the altered PSI and increased the dissociation constant for binding of cytochrome (cyt) c(6) by more than a factor of 10 as compared with wild type. Mutation of PsaA-Trp(651) to Ser had an even larger impact on the dissociation constant. The K(D) value increased another 2-fold when the values obtained for the interaction and electron transfer between cyt c(6) and PSI from PsaA-W651S and PsaA-W651F are compared. In contrast, binding and electron transfer of pc to PSI from PsaA-W651S improved as compared with PSI from PsaA-W651F and admitted the formation of an inter-molecular electron transfer complex, resulting in a K(D) value of about 554 microm that is still five times higher than observed for wild type. These results demonstrate that PsaA-Trp(651) is, such as PsaB-Trp(627), crucial for high affinity binding of pc and cyt c(6) to PSI. Our results also indicate that the highly conserved structural recognition motif that is formed by PsaA-Trp(651) and PsaB-Trp(627) confers a differential selectivity in binding of both donors to PSI.  相似文献   

17.
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.  相似文献   

18.
The role played by the residues Leu12 and Lys33 – which are both located at the north hydrophobic patch of plastocyanin – in the interaction of the copper protein with Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 has been investigated by site-directed mutagenesis. A thermodynamic analysis of PS I reduction by wild-type and mutant plastocyanins has been performed by laser-flash absorption spectroscopy. In all cases, the electron transfer is impaired by mutations, which induce drastic changes in the apparent activation entropy of the overall reaction. Substitution of Leu12 by alanine specifically affects the hydrophobic interactions with PS I, whereas replacement of Lys33 by glutamate not only induces local electrostatic changes, but also alters the hydrophobic interactions with the photosystem. The thermodynamic analysis of the reactivity of K33E mutant towards PS I reveals that the effect of the mutation can be reversed by addition of magnesium cations, which probably bind at a place close to Glu33. The electrostatic surface potential does thus modulate the hydrophobic interactions with PS I by altering the solvent accessibility of some surface residues. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

19.
Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.  相似文献   

20.
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.  相似文献   

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