UNIMOLECULAR AND BIOMOLECULAR OXIDOREDUCTION REACTIONS INVOLVING DIPROTEIN COMPLEXES OF CYTOCHROME-C AND PLASTOCYANIN - DEPENDENCE OF ELECTRON-TRANSFER REACTIVITY ON CHARGE AND ORIENTATION OF THE DOCKED METALLOPROTEINS

Cytochrome c and plastocyanin form an electrostatic complex, which can be reinforced by amide bonds in the presence of a carbodiimide. Besides this cross-linking, carbodiimide also converts carboxylate side chains into neutral N-acylurea groups. Four derivatives of the covalent diprotein complex, which differ in the degree of this charge neutralization, are separated by cation-exchange chromatography. Electron-transfer reactions at different ionic strengths involving the electrostatic complex and the four derivatives of the covalent complex are studied by laser flash photolysis with flavin semiquinones as reducing agents. The reactivity of the associated proteins toward external reductants cannot be predicted simply on the basis of this reactivity of the separate proteins. Qualitative analysis of the dependence on ionic strength of the reactions between FMN semiquinone and the covalent derivatives indicates sites at which this reductant interacts with the cross-linked proteins. The surprisingly small steric shielding of the protein redox sites in the covalent complex, as deduced from the reactions at high ionic strength, may indicate that the proteins have multiple reaction domains on their surfaces or that the complex is dynamical or both. The intracomplex (unimolecular) electron-transfer reaction is fast in the electrostatic complex (k(et) = 1300 +/- 200 s-1) but undetectably slow in each of the four derivatives of the covalent complex (k(et) < 0.2 s-1). This reaction apparently requires a migration of cytochrome c from the acidic domain in plastocyanin, which is far from the copper redox site, to the hydrophobic domain, which is near this site. This migration is possible in the electrostatic complex, which is flexible, but impossible in the covalent complex, which contains rigid cross-links.