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)     

Electrostatic Interaction between Plastocyanin and P700 in the Electron Transfer Reaction of Photosystem I-Enriched Particles

The nature of the electron transfer reaction between reducedplastocyanin and P700 oxidized by flash illumination was studiedin P700-enriched Triton subchloroplast fraction 1 particles.An addition of monovalent salts to the suspension at neutralpH increased the reaction rate at low concentrations (>20mM). Salts of divalent cations showed a similar effect at muchlower concentrations (>2 mM), This effect was not dependenton the concentration and the valence of anions. The increaseof rate at low salt concentrations was observed at pH's above5, but below pH 5 the rate was decreased by adding salts. Atabout pH 5, the rate was not affected by salts. Apart from thesesalt effects, the optimum pH for the reaction rate was observedbetween 5.5 and 6.5. The reduction rate depended sigmoidally on the added plastocyaninconcentration at pH 6.8 and 4. A Michaelis-Menten type relationshipwas observed at about pH 5. The half-saturation concentrationof plastocyanin became lower as the salt concentration increasedat pH 6.8, while it became higher by adding salt at pH 4. The effects of salts on the rate of electron donation from othermetalloproteins and artificial electron donors to P700 werealso studied. It is concluded, from the analysis with the Gouy-Chapmantheory, that the net charges on the electron donors and themembrane surfaces mainly determine the response of the P700reduction rate to salt addition. The salt addition changes mainlythe local concentration or accessibility of electron donorsto P700. (Received January 12, 1981; Accepted March 6, 1981)     

Polylysine-enhanced effectiveness of plastocyanin in photosystem I

1. Chloroplast fragments from either Chlamydomonas reinhardi or spinach, which lack plastocyanin, or from Euglena gracilis depleted of cytochrome c552, require a large excess of exogenously added plastocyanin or cytochrome c552 to restore Photosystem I activity.2. In the presence of a small amount of polylysine, Photosystem I activity of chloroplast fragments is stimulated greatly by plastocyanin or cytochrome c552, and the reaction is saturated at a lower concentration of these proteins. Higher concentrations of polylysine inhibit Photosystem I activity; the inhibition is not reversed by plastocyanin or cytochrome c552.3. Salt protects chloroplast fragments from stimulation by polylysine plus plastocyanin or cytochrome c552, and also reverses this stimulation.4. The data suggest that polylysine, at low concentration, enhances binding of plastocyanin or cytochrome c552 to chloroplast membranes, thereby increasing the effective concentration at their site of function. The total inhibition of Photosystem I activity, independent of the presence of plastocyanin or cytochrome c552, at higher polylysine concentrations is similar probably to that observed previously in chloroplasts which retain their plastocyanin.     

Regulation by copper of the expression of plastocyanin and cytochrome c552 in Chlamydomonas reinhardi.

Plastocyanin and cytochrome c552 are interchangeable electron carriers in the photosynthetic electron transfer chains of some cyanobacteria and green algae (P. M. Wood, Eur. J. Biochem. 87:9-19, 1978; G. Sandmann et al., Arch. Microbiol. 134:23-27, 1983). Chlamydomonas reinhardi cells respond to the availability of copper in the medium and accordingly accumulate either plastocyanin (if copper is available) or cytochrome c552 (if copper is not available). The response occurs in both heterotrophically and phototrophically grown cells. We have studied the molecular level at which this response occurs. No immunoreactive polypeptide is detectable under conditions where the mature protein is not spectroscopically detectable. Both plastocyanin and cytochrome c552 appear to be translated (in vitro) from polyadenylated mRNA as precursors of higher molecular weight. RNA was isolated from cells grown either under conditions favorable for the accumulation of plastocyanin (medium with Cu2+) or for the accumulation of cytochrome c552 (without Cu2+ added to the medium). Translatable mRNA for preapoplastocyanin was detected in both RNA preparations, although mature plastocyanin was detected in C. reinhardi cells only when copper was added to the culture. Translatable mRNA for preapocytochrome, on the other hand, was detected only in cells grown under conditions where cytochrome c552 accumulates (i.e., in the absence of copper). We conclude that copper-mediated regulation of plastocyanin and cytochrome c552 accumulation is effected at different levels, the former at the level of stable protein and the latter at the level of stable mRNA.     

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.     

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

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

Ion binding to cytochrome c studied by nuclear magnetic quadrupole relaxation.

The enhancement of the 35Cl- transverse relaxation rate on binding of chloride ions to oxidized and reduced cytochrome c has been studied under conditions of variable sodium chloride concentration, temperature, pH, sodium phosphate, iron hexacyanide, and sodium cyanide concentration. The results revealed the presence of a strong binding site(s) for chloride in both oxidized and reduced cyt c, with a higher affinity in ferrocytochrome c. Competition experiments suggest that these sites also bind iron hexacyanide and phosphate. Cyanide binding to the iron in ferricytochrome c at alkaline and neutral pH was shown to decrease the binding of chloride. The pH dependence of the 35Cl- relaxation rate has been fitted by using literature pK values for ionizable groups. No indications of Na+ binding to oxidized and reduced cytochrome c have been observed by using 23Na+ NMR. Our results suggest that chloride is bound near the exposed heme edge and that the surface structure or dynamics in this region are different in the two oxidation states.     

The steady-state rate equation for cytochrome c oxidase based on a minimal kinetic scheme

A minimal catalytic cycle for cytochrome c oxidase has been suggested, and the steady-state kinetic equation for this mechanism has been derived. This equation has been used to simulate experimental data for the pH dependence of the steady-state kinetic parameters, kcat and Km. In the simulations the rate constants for binding and dissociation of cytochrome c and for two internal electron-transfer steps have been allowed to vary, whereas fixed experimental values (for pH 7.4) have been used for the other rate constants. The results show that the dissociation of the product, ferricytochrome c, cannot be rate-limiting under all conditions, but that intramolecular electron-transfer steps also limit the rate. They also demonstrate that Km can differ considerably from the dissociation constant for the cytochrome c-oxidase complex. Published values for the rate constant for the dissociation of ferricytochrome c are too small to account for the steady-state rates. It is suggested that, at high concentrations, ferryocytochrome c transfers an electron to a cytochrome c molecule which remains bound to the oxidase. This can also explain the nonhyperbolic kinetics, which is observed at low substrate concentrations.     

Proton transfer during the reaction between fully reduced cytochrome c oxidase and dioxygen: pH and deuterium isotope effects.

The pH dependence of proton uptake and electron transfers during the reaction between fully reduced cytochrome c oxidase and oxygen has been studied using the flow-flash method. Proton uptake was monitored using different pH indicators. We have also investigated the effect of D2O on the electron-transfer reactions. Proton uptake was biphasic throughout the pH range studied (6.3-9.3), and the decrease of the observed rate constants at increasing pH could be described by titration curves with pKa values of 8-8.5. Of the four phases resolved in the redox reaction, the rate constants for the first two were independent of pH, whereas that of the third decreased at increasing pH with a pKa of 7.9. All phases except the first were slower in D2O than in H2O. The values obtained for kH/kD were 1.0 for the first phase, 1.4 for the second and third phases, and 2.5 for the fourth phase. We suggest from these results that the fast phase of proton uptake is initiated by the second phase of the redox reaction and that this step includes a partially rate-limiting internal proton transfer. The third and fourth phases of the redox reaction are suggested to be rate limited by proton uptake from the medium. The pH dependencies of the proton uptake reactions are consistent with the participation of a titrable group in the protein in proton transfer from the medium to the oxygen-binding site.     

Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris

The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.     

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

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

Photoinduced electron transfer within complexes between plastocyanin and ruthenium bisbipyridine dicarboxybipyridine cytochrome c derivatives

A new technique has been developed to measure intracomplex electron transfer between cytochrome c and its redox partners. Cytochrome c derivatives labeled at single lysine amino groups with ruthenium bisbipyridine dicarboxybipyridine were prepared as previously described [Pan, L.P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Excitation of RuII with a short light pulse resulted in the formation of the excited-state RuII*, which rapidly transferred an electron to the ferric heme group to form FeII and RuIII. Aniline was included in the buffer to reduce RuIII to RuII, leaving the heme group in the ferrous state. This process was complete within the lifetime of the light pulse. When plastocyanin was present in the solution, electron transfer from the ferrous heme of cytochrome c to CuII in plastocyanin was observed. All of the ruthenium cytochrome c derivatives formed electrostatic complexes with plastocyanin at low ionic strength, allowing intracomplex electron-transfer rate constants to be measured. The rate constants for derivatives modified at the indicated lysines were as follows: Lys 13, 1920 s-1; Lys 8, 1480 s-1; Lys 7, 1340 s-1; Lys 86, 1020 s-1; Lys 25, 820 s-1; Lys 72, 800 s-1; Lys 27, 530 s-1. It is interesting that the derivative modified at lysine 13 at the top of the heme crevice had the largest rate constant, while lysine 27 at the right side of the heme crevice had the smallest.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.     

Preferred sites on cytochrome c for electron transfer with two positively charged blue copper proteins, Anabaena variabilis plastocyanin and stellacyanin

Rate constants for the reactions of horse cytochrome c (E'0 of +260 mV) with the copper proteins Anabaena variabilis plastocyanin (E'0 of +360 mV) used as oxidant and stellacyanin (E'0 of +187 mV) used as reductant have been determined at 25 degrees C, pH 7.5 and 7.0, respectively, and an ionic strength of 0.10 M (NaCl). These rate constants were also measured with eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse cytochrome c derivatives, modified at lysine-7, -13, -25, -27, -60, -72, -86, or -87 and with the trinitrophenyl (TNP) derivative modified at lysine-13. The influence of the modifications on the bimolecular rate constants for these reactions defines the region on the protein that is involved in the electron-exchange reactions and demonstrates that the preferred site is at or near the solvent-accessible edge of the heme prosthetic group on the "front" surface of the molecule. Both reactions are strongly influenced by the lysine-72 modification to the left of the exposed heme edge and, to this extent, behave similar to the earlier studied reaction with azurin. These effects span only an order of magnitude in rate constants and are thus many times smaller than those for the physiological protein redox partners of cytochrome c. While the preferred sites of reaction on the surface of cytochrome c for small inorganic complexes appear to be dependent only on the net charge of the reactants, with the copper proteins additional factors intervene. These influences are discussed in terms of hydrophobic patches and the distribution of charges on the surface of the four copper proteins so far examined.     

Complex formation between the copper protein, azurin and the cytochrome c peroxidase of Pseudomonas aeruginosa.

Reduced azurin reacts with the resting, oxidized cytochrome c peroxidase of Pseudomonas aeruginosa to yield time courses observed at 420 nm, which consist of the sum of two exponential processes. Each process exhibits a hyperbolic dependence of the observed rate constant on the reduced azurin concentration. The fraction of the total optical density change which each process contributes is found to be dependent on the reduced azurin concentration. This pattern of reactivity is maintained at pH values between 5.5 and 8.0. The data has been analyzed in terms of a complex formation between the two proteins followed by an intramolecular electron exchange reaction. This analysis yields values for the binding constants at each pH value. The intramolecular exchange reaction is independent of pH, whilst the pH dependence of the binding reaction suggests the involvement of a histidine residue in this process.     

Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids

The lateral distribution of plastocyanin in the thylakoid lumen of spinach and pea chloroplasts was studied by combining immunocytochemical localization and kinetic measurements of P700+ reduction at high time resolution. In dark-adapted chloroplasts, the concentration of plastocyanin in the photosystem I containing stroma membranes exceeds that in photosystem II containing grana membranes by a factor of about two. Under these conditions, the reduction of P700+ with a halftime of 12 microseconds after a laser flash of saturating intensity indicates that to greater than 95% of total photosystem I a plastocyanin molecule is bound. An analysis of the labeling densities, the length of the different lumenal regions, and the total amounts of plastocyanin and P700 shows that most of the remaining presumable mobile plastocyanin is found in the granal lumen. This distribution of plastocyanin is consistent with a more negative surface charge density in the stromal than in the granal lumen. During illumination the concentration of plastocyanin in grana increases at the expense of that in stroma lamellae, indicating a light-driven diffusion from stroma to grana regions. Our observations provide evidence that a high concentration of plastocyanin in grana in the light favors the lateral electron transport from cytochrome b6/f complexes in appressed grana across the long distance to photosystem I in nonappressed stroma membranes.     

Cytochrome c-induced acceleration of the reaction of muscle actin polymerization

The reaction of rabbit skeletal muscle actin polymerization initiated by the addition of neutral salts is accelerated in the presence of mitochondrial cytochrome c. The observed effect is specific, since the addition of serum albumin does not change the initial velocity of this process. The dependence of the rate of actin polymerization and exogenous cytochrome c concentration correlates within the molar protein ratios from 43:1 to 9:1, respectively. The increase in the polymerization rate occurs immediately after addition of cytochrome c to the reaction mixture; however, the maximal effect is observed only after 5 min coincubation of the proteins. The ability of cytochrome c to stimulate this process is abolished at alkaline values of pH (8.5), which points to the significant role of the molecule positive charge which, in all probability, serves as a primer of the muscle actin polymerization reaction.     

Kinetics of electron transfer between cytochrome c and laccase.

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting Rhus vernicifera laccase as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 125 M-1 s-1 at 25 degrees C in 0.2 M phosphate buffer at pH 6.0 with the activation parameters delta H++ = 16.2 kJ mol-1 and delta S++ = 28.9 J mol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that electron transfer from cytochrome c to laccase is favored by the local electrostatic interaction (ZAZB = -0.9 at pH 6 and -1.3 at pH 4.8) between the basic proteins with positive net charges. From the increase of the rate of electron transfer with decreasing pH, one of the driving forces of the reaction was suggested to be the difference in the redox potentials between the type 1 copper in laccase and the central iron in cytochrome c. Further, on addition of one hexametaphosphate anion per cytochrome c molecule, the rate of the electron transfer was increased, probably because the association of both proteins became more favorable.     

The effect of ethylenediamine chemical modification of plastocyanin on the rate of cytochrome f oxidation and P-700+ reduction

Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6-8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42-45 and Nos. 59-61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42-45 and Nos. 59-61. Modification of plastocyanin at residues Nos. 42-45 showed no effect on the rate of P-700+ reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700+ reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59-61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 +/- 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42-45 or Nos. 59-61.