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.

The protein-protein electron transfer reactions between cytochrome f and plastocyanin, both purified from Brassica komatsuna (Brassica rapa L. var. pervirdis Bailey), have been studied as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of ferrocytochrome f by plastocyanin was found to be k = 4.5 X 10(7) M-1 x S-1 at pH 7.0 mu 0.2 M, and 20 degrees C, with activation parameters delta H not equal to = 8.4 kcal/mol and delta S not equal to = 4.9 cal/mol x deg. Respective rate constant and activation parameters obtained for the reduction of ferricytochrome f by plastocyanin were k = 1.9 X 10(7) M-1 x S-1, delta H not equal to = 8.6 kcal/mol, and delta S not equal to = 3.9 cal/mol x deg. The high rate constants for these reactions and delta S not equal to = 4.9 cal/mol x deg. Respective rate constant and activation parameters obtained for the reduction of ferricytochrome f by plastocyanin were k = 1.9 X 10(7) M-1 x S-1, delta H not equal to = 8.6 kcal/mol, and delta S not equal to = 3.9 cal/mol x deg. The high rate constants for these reactions are attributable not to a low activation enthalpy but to a positive activation entropy term. The rate constants both for the oxidation and the reduction of cytochrome f by plastocyanin drastically decreased with increasing ionic strength, indicating the importance of electrostatic interactions. Divalent cations are more effective than monovalent cations in reducing the rates of these reactions. The rate constants for the oxidation of cytochrome f by plastocyanin are constant between pH 6.0 and 9.0 but decrease markedly above pH 9.0 and below pH 6.0. In the case of the reduction of cytochrome f by plastocyanin, an optimum pH around 7.0 was obtained and a biphasic feature was observed at alkaline pH. The results are discussed in relation to photosynthetic electron transport systems.

The autoreduction of spinach plastocyanin has been studied by fluorescence, absorption, and paramagnetic resonance spectroscopy. Decolorization of oxidized plastocyanin, due to the autoreduction of plastocyanin, occurred rapidly at alkaline pH (half-life 20 min at pH 10.2). Its rate also increased with increasing ionic strength. In the presence of a hydrophobic fluorescent probe such as 2-p-toluidino-naphthalene-6-sulfonate (TNS), the fluorescence intensity of TNS due to binding to oxidized plastocyanin gradually increased following the autoreduction of plastocyanin, which suggests that copper is autoreduced in parallel with the exposure of a hydrophobic site (probably near Cys 84) to the solvent. The EPR spectra also supported the change of coordination geometry of copper. The reduction rate of plastocyanin by ferrocyanide at alkaline pH was almost the same as that at neutral pH, and TNS did not alter the exogenous reduction rate even at alkaline pH. These results suggest that exogenous and endogenous reduction take place independently at different reduction sites in spinach plastocyanin.

The electron transfer reactions of membrane-bound monomeric cytochrome f from Brassica komatosuna (Brassica rapa L. var. perviridis Bailey) with hexacyanoferrate (II)-(III) have been studied as a function of pH, ionic strength and temperature. The second-order rate constant for the oxidation of cytochrome f by Fe(CN)6(3-) at pH 7.0, mu 0.1 M, and 20 degrees C is 1.7 X 10(5) M-1 X S-1, which is similar to the value of oligomeric cytochrome f from parsley. The activation parameters obtained were delta H not equal to = -0.87 kcal/mol and delta S not equal to - -38 cal/mol x deg. Respective rat constant and activation parameters obtained for the reduction of cytochrome f by Fe(CN)6(4-) were k = 1.7 X 10(4) M-1 x S-1, delta H not equal to = +6.7 kcal/mol, and delta S not equal to -16 cal/mol x deg. Both the rate constants for the oxidation and the reduction of cytochrome f markedly decreased with increasing ionic strength. The results indicate that the oxidation and the reduction take place at a positively charged site on the cytochrome f surface, and electrostatic interactions are important for these reactions. The participation of protons and specific amino acid residues in electron transfer reactions of cytochrome f is implied from the pH results. Alkaline isomerization of ferricytochrome f was not observed. The midpoint potential of cytochrome f has a constant value of 360 mV between pH 5.0-8.9, and decreases by about 55 mV per pH unit above 8.9. The results are compared with the data for horse heart cytochrome c and Euglena gracilis cytochrome c-552. These data are discussed in relation to the theories of electrostatic corrected outer-sphere electron transfer of Marcus and multiphonon nonadiabatic electron tunneling of Jortner and Hopfield.

Kinetic studies of the reduction of plastocyanin from spinach chloroplasts by ascorbate were carried out using a stopped flow apparatus. The reduction of spinach plastocyanin by ascorbate followed first-order kinetics in the reductant with a rate constant k = 415 M-1.S-1 [20 degrees C, 0.1 M NaCl, pH 8.0 (10 mM Tris-HCl)]. The second-order rate constants for the reduction of plastocyanin by ascorbate increased dramatically with increasing pH and ionic strength. The cationic micelles of cetyltrimethylammonium bromide also accelerated this electron-transfer reaction. The importance of electrostatic interactions between plastocyanin and ascorbate is discussed in relation to the photosynthetic electron-transport reactions.