Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction

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Abstract
Horse heart cytochrome c and French bean plastocyanin are cross-linked one-to-one by a carbodiimide [Geren, L. M., Stonehuerner, J., Davis, D. H., and Millett, F. (1983) Biochim. Biophys. Acta 724, 62] in the same general orientation in which they associate electrostatically [King, G. C., Binstead, R. A., and Wright, P. E. (1985) Biochim. Biophys. Acta 806, 262]. The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV vs NHE; the EPR spectra of the two metals are not perturbed by cross-linking. Four isomers of the covalent diprotein complex, which probably differ slightly from one another in the manner of cross-linking, are separated efficiently by cation-exchange chromatography. Stopped-flow spectrophotometric experiments with the covalent diprotein complex show that the presence of plastocyanin somewhat inhibits oxidation of ferrocytochrome c by [Fe(CN)6]3- and somewhat promotes oxidation of this protein by [Fe(C5H5)2]+. These changes in reactivity are explained in terms of electrostatic and steric effects. Pulse-radiolysis experiments with the electrosatic diprotein complexy yield association constants of .gtoreq.5 .times. 106 and 1 7x 105 M-1 at ionic strengths of 1 and 40 mM, rspectively, and the rate constant of 1.05 .times. 103 s-1, regardless of the ionic strength, for the intracomplex electron-transfer reaction. Analogous pulse-radiolysis experiments with each of the four isomers of the covalent diprotein complex, at ionic strengths of both 2 and 200 mM, show an absence of the intracomplex electron-transfer reaction. A rearrangement of the proteins for this reaction seems to be possible (or unnecessary) in the electrostatic complex but impossible in the covalent complex.

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