Mutagenesis of prochlorothrix plastocyanin reveals additional features in photosystem I interactions

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.     

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.     

Plastocyanin-cytochrome f interactions: the influence of hydrophobic patch mutations studied by NMR spectroscopy

Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 (+/-2) x 10(3) M(-1) for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemical-shift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed.     

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.     

Mutations in both leucine 12 and lysine 33 in plastocyanin from Synechocystis sp. PCC 6803 induce drastic changes in the hydrophobic interactions with Photosystem I

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.     

Role of electrostatics in the interaction between plastocyanin and photosystem I of the cyanobacterium Phormidium laminosum.

The interactions between photosystem I and five charge mutants of plastocyanin from the cyanobacterium Phormidium laminosum were investigated in vitro. The dependence of the overall rate constant of reaction, k2, on ionic strength was investigated using laser flash photolysis. The rate constant of the wild-type reaction increased with ionic strength, indicating repulsion between the reaction partners. Removing a negative charge on plastocyanin (D44A) accelerated the reaction and made it independent of ionic strength; removing a positive charge adjacent to D44 (K53A) had little effect. Neutralizing and inverting the charge on R93 slowed the reaction down and increased the repulsion. Specific effects of MgCl2 were observed for mutants K53A, R93Q and R93E. Thermodynamic analysis of the transition state revealed positive activation entropies, suggesting partial desolvation of the interface in the transition state. In comparison with plants, plastocyanin and photosystem I of Phormidium laminosum react slowly at low ionic strength, whereas the two systems have similar rates in the range of physiological salt concentrations. We conclude that in P. laminosum, in contrast with plants in vitro, hydrophobic interactions are more important than electrostatics for the reactions of plastocyanin, both with photosystem I (this paper) and with cytochrome f[Schlarb-Ridley, B.G., Bendall, D.S. & Howe, C.J. (2002) Biochemistry41, 3279-3285]. We discuss the implications of this conclusion for the divergent evolution of cyanobacterial and plant plastocyanins.     

A Brownian dynamics study of the interaction of Phormidium cytochrome f with various cyanobacterial plastocyanins

Brownian dynamics simulations were used to study the role of electrostatic forces in the interactions of cytochrome f from the cyanobacterium Phormidium laminosum with various cyanobacterial plastocyanins. Both the net charge on the plastocyanin molecule and the charge configuration around H92 (H87 in higher plants) are important in determining the interactions. Those plastocyanins (PCs) with a net charge more negative than -2.0, including those from Synechococcus sp. PCC7942, Synechocystis sp. 6803, and P. laminosum showed very little complex formation. On the other hand, complex formation for those with a net charge more positive than -2.0 (including Nostoc sp. PCC7119 and Prochlorothrix hollandica) as well as Nostoc plastocyanin mutants showed a linear dependence of complex formation upon the net charge on the plastocyanin molecule. Mutation of charged residues on the surface of the PC molecules also affected complex formation. Simulations involving plastocyanin mutants K35A, R93A, and K11A (when present) showed inhibition of complex formation. In contrast, D10A and E17A mutants showed an increase in complex formation. All of these residues surround the H92 (H87 in higher plant plastocyanins) ligand to the copper. An examination of the closest electrostatic contacts shows that these residues interact with D63, E123, R157, D188, and the heme on Phormidium cytochrome f. In the complexes formed, the long axis of the PC molecule lies perpendicular to the long axis of cytochrome f. There is considerable heterogeneity in the orientation of plastocyanin in the complexes formed.     

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.     

Electron donor-specificity observed in photosystem I reactions of membrane fragments of the blue-green alga Anabaena variabilis and the higher plant Spinacea oleracea

Electron donating activities of plastocyanins and c-type cytochromesof various organisms for photosystem I reactions were studiedwith membrane fragments of the blue-green alga Anabaena variabilisand the higher plant Spinacea oleracea. In the Anabaena photosystem I reaction, basic but not acidicplastocyanin and c-type cytochromes acted as efficient electrondonors, while only acidic redox proteins were active in thespinach photosystem I reaction. The selective reactivity ofredox proteins in the two photosystem I reactions was observedwith both plastocyanin (or cytochrome) limited and saturatedconditions. These data support our previous observation that photosystemI of blue-green algae differs from those of other green plantswith respect to specificity to the proteinous electron donor(1). (Received August 17, 1971; )     

Crystal structures of wild-type and mutant plastocyanins from a higher plant, Silene

Plastocyanin functions as an electron carrier between the cytochrome b6f complex and photosystem I. The crystal structures of the wild-type and E43K/D44K double mutant from the higher plant, Silene, have been determined at 2.0 and 1.75 A resolution, respectively. The wild-type plastocyanin comprises two monomers per asymmetric unit, one of which shows the unusually great distance between the copper ion and the Ndelta1 atom of H87 because of the hydrogen bond network formation between H87 and symmetry-related G10. The root mean square deviation for Ca atoms between the wild-type and mutant plastocyanins is 0.44 A, however, the electrostatic potential maps of their molecular surfaces are remarkably different. The low electron-transfer rate in the E43K/D44K mutant results from the hindrance of electrostatic interactions, not from the structural change due to the mutation.     

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.     

Computational simulation of the docking of Prochlorothrix hollandica plastocyanin to potosystem I: modeling the electron transfer complex

We have used several docking algorithms (GRAMM, FTDOCK, DOT, AUTODOCK) to examine protein-protein interactions between plastocyanin (Pc)/photosystem I (PSI) in the electron transfer reaction. Because of the large size and complexity of this system, it is faster and easier to use computer simulations than conduct x-ray crystallography or nuclear magnetic resonance experiments. The main criterion for complex selection was the distance between the copper ion of Pc and the P700 chlorophyll special pair. Additionally, the unique tyrosine residue (Tyr(12)) of the hydrophobic docking surface of Prochlorothrix hollandica Pc yields a specific interaction with the lumenal surface of PSI, thus providing the second constraint for the complex. The structure that corresponded best to our criteria was obtained by the GRAMM algorithm. In this structure, the solvent-exposed histidine that coordinates copper in Pc is at the van der Waals distance from the pair of stacked tryptophans that separate the chlorophylls from the solvent, yielding the shortest possible metal-to-metal distance. The unique tyrosine on the surface of the Prochlorothrix Pc hydrophobic patch also participates in a hydrogen bond with the conserved Asn(633) of the PSI PsaB polypeptide (numbering from the Synechococcus elongatus crystal structure). Free energy calculations for complex formation with wild-type Pc, as well as the hydrophobic patch Tyr(12)Gly and Pro(14)Leu Pc mutants, were carried out using a molecular mechanics Poisson-Boltzman, surface area approach (MM/PBSA). The results are in reasonable agreement with our experimental studies, suggesting that the obtained structure can serve as an adequate model for P. hollandica Pc-PSI complex that can be extended for the study of other cyanobacterial Pc/PSI reaction pairs.     

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.     

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.     

The structural homology of amicyanin from Thiobacillus versutus to plant plastocyanins

The complete amino acid sequence of the blue copper protein amicyanin of Thiobacillus versutus, induced when the bacterium is grown on methylamine, has been determined as follows: QDKITVTSEKPVAAADVPADAVVVGIEKMKYLTPEVTIKAGETVYWVNGEVMPHNVA FKKGIVGEDAFRGEMMTKDQAYAITFNEAGSYDYFCTPHPFMRGKVIVE. The four copper ligand residues in this 106-residue-containing polypeptide chain are His54, Cys93, His96, and Met99. The Thiobacillus amicyanin is 52% similar to the amicyanin of Pseudomonas AM1, the only other copper protein known with the same spacing between the second histidine ligand and the methionine ligand. T. versutus amicyanin contains no cysteine bridge and is more closely related to the plant copper protein plastocyanin than to the bacterial copper protein azurin. Alignment of the two known amicyanin sequences with the consensus sequence of the plastocyanins and comparison with the known three-dimensional structure of poplar leaves plastocyanin reveals that the bacterial proteins have the same overall structure with two beta-sheets packed face to face. The major structural differences between the amicyanins and the plastocyanins appear to be located in two of the five loops that connect the six identified beta-strands of the amicyanins. The first of these two loops, connecting strands F and G, contains a ligand histidine and must have a different conformation from the same loop in the plastocyanins because it is shorter by two amino acids. Further differences occur in the loop connecting the strands D and E. This loop contains only 17 residues in amicyanin whereas the corresponding loop of plastocyanin contains 25 residues. Despite these differences the amicyanins appear much closer related to the plastocyanins than to the azurins. The present findings demonstrate that the occurrence of blue copper proteins with clearly plastocyanin-like features is not restricted to photosynthetic redox chains.     

Mutational analysis of photosystem I of Synechocystis sp. PCC 6803: the role of four conserved aromatic residues in the j-helix of PsaB

Photosystem I is the light-driven plastocyanin-ferredoxin oxidoreductase in the photosynthetic electron transfer of cyanobacteria and plants. Two histidyl residues in the symmetric transmembrane helices A-j and B-j provide ligands for the P700 chlorophyll molecules of the reaction center of photosystem I. To determine the role of conserved aromatic residues adjacent to the histidyl molecule in the helix of B-j, we generated six site-directed mutants of the psaB gene in Synechocystis sp. PCC 6803. Three mutant strains with W645C, W643C/A644I and S641C/V642I substitutions could grow photoautotrophically and showed no obvious reduction in the photosystem I activity. Kinetics of P700 re-reduction by plastocyanin remained unaltered in these mutants. In contrast, the strains with H651C/L652M, F649C/G650I and F647C substitutions could not grow under photoautotrophic conditions because those mutants had low photosystem I activity, possibly due to low levels of proteins. A procedure to select spontaneous revertants from the mutants that are incapable to photoautotrophic growth resulted in three revertants that were used in this study. The molecular analysis of the spontaneous revertants suggested that an aromatic residue at F647 and a small residue at G650 may be necessary for maintaining the structural integrity of photosystem I. The (P700⁺ - P700) steady-state absorption difference spectrum of the revertant F647Y has a ∼5 nm narrower peak than the recovered wild-type, suggesting that additional hydroxyl group of this revertant may participate in the interaction with the special pair while the photosystem I complexes of the F649C/G650T and H651Q mutants closely resemble the wild-type spectrum. The results presented here demonstrate that the highly conserved residues W645, W643 and F649 are not critical for maintaining the integrity and in mediating electron transport from plastocyanin to photosystem I. Our data suggest that an aromatic residue is required at position of 647 for structural integrity and/or function of photosystem I.     

Spectroscopic characterization of plastocyanin from hornwort

Plastocyanin isolated from an aquatic higher plant, Ceratophyllum demersum L. (hornwort), has been characterized by electronic absorption, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopies. The visible absorption, CD, and EPR spectra of hornwort plastocyanin indicated a complete similarity of blue copper center to those of terrestrial higher plants and algae. However, the ultraviolet absorption spectrum of hornwort plastocyanin exhibited a lower tyrosine (Tyr) and a higher phenylalanine (Phe) content of the protein comparing with other plastocyanins. The ratio of Phe/Tyr residues was estimated to be 9 by amino acid analysis. The hornwort plastocyanin resembles in amino acid composition terrestrial higher plant plastocyanins rather than alga plastocyanins but is peculiar in the content of Phe and Tyr residues.     

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.     

Modulation of copper site properties by remote residues determines the stability of plastocyanins

The metal cofactor determines the thermal stability in cupredoxins, but how the redox state of copper modulates their melting points remains unknown. The metal coordination environment is highly conserved in cyanobacterial plastocyanins. However, the oxidised form is more stable than the reduced one in thermophilic Phormidium, but the opposite occurs in mesophilic Synechocystis. We have performed neutral amino-acid substitutions at loops of Phormidium plastocyanin far from the copper site. Notably, mutation P49G/G50P confers a redox-dependent thermal stability similar to that of the mesophilic plastocyanin. Moreover, X-ray absorption spectroscopy reveals that P49G/G50P mutation makes the electron density distribution at the oxidised copper site shift towards that of Synechocystis plastocyanin.     

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.