Cross-linked metalloproteins: Novel systems for the study of intraprotein electron-transfer reactions.

摘要

My initial research shows, for the first time, that proteins can be cross-linked selectively via transition-metal compounds to form stable protein complexes. Incubation of horse heat cytochrome c (designated cyt) with reagents PtCl(sub 4)(sup 2(minus)) and trans-(Pt(2-Fpy)(sub 2)Cl(sub 2)) under mild conditions yields stable diprotein complexes trans-(PtCl(sub 2)(cyt)(sub 2)) and trans-(Pt(2-Fpy)(sub 2)(cyt)(sub 2)), respectively (2-Fpy is 2-fluoropyridine). The complexes are purified and characterized chromatographically. Spectroscopic and electrochemical measurements indicate that the structural and redox properties of the cytochrome c molecules remain virtually unaltered upon cross-linking. The diprotein complexes are stable indefinitely under ordinary conditions and yet they can be cleaved, and the native protein restored, in a mild reaction. Subsequent research focused on oxidoreduction reactions involving the electrostatic and the covalent complex of horse heart cytochrome c and Phaseolus vulgaris plastocyanin, and exploring the importance of the protein rearrangement for the intracomplex electron-transfer reaction. Cytochrome c and plastocyanin are cross-linked one-to-one by a carbodiimide, in the same general orientation in which they associate electrostatically. 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. Electron transfer kinetics of the covalent and electrostatic diprotein complexes are studied using stopped-flow, pulse radiolysis, and flash photolysis experiments. 135 refs., 5 figs., 15 tabs. (ERA citation 15:035958)