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

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 heart cytochrome c (designated cyt) with reagents PtCl[subscript]4[superscript]2- and trans- (Pt(2-Fpy)[subscript]2Cl[subscript]2) under mild conditions yields stable diprotein complexes trans- (PtCl[subscript]2(cyt)[subscript]2) and trans- (Pt(2-Fpy)[subscript]2(cyt)[subscript]2), respectively (2-Fpy is 2-fluoropyridine). The complexes are purified and characterized chromatographically. The protein molecules are coordinated to the Pt(II) atom through the thioether side chains of their respective Met 65 residues. 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. Platinum compounds hold promise as selective and versatile reagents for cross-linking proteins;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. The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV versus 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. Electron transfer kinetics of the covalent and electrostatic diprotein complexes are studied using stopped-flow, pulse radiolysis, and flash photolysis experiments. The electrostatic complex undergoes intracomplex electron transfer with a rate constant of 1.05 x 10[superscript]3s[superscript]-1. However, there appears to be a complete absence of intracomplex electron transfer within all isomers of the covalent complex. A rearrangement of the proteins for this reaction seems to be possible (or unnecessary) in the electrostatic complex but impossible in the covalent complex

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

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 heart cytochrome c (designated cyt) with reagents PtCl[subscript]4[superscript]2- and trans- (Pt(2-Fpy)[subscript]2Cl[subscript]2) under mild conditions yields stable diprotein complexes trans- (PtCl[subscript]2(cyt)[subscript]2) and trans- (Pt(2-Fpy)[subscript]2(cyt)[subscript]2), respectively (2-Fpy is 2-fluoropyridine). The complexes are purified and characterized chromatographically. The protein molecules are coordinated to the Pt(II) atom through the thioether side chains of their respective Met 65 residues. 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. Platinum compounds hold promise as selective and versatile reagents for cross-linking proteins;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. The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV versus 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. Electron transfer kinetics of the covalent and electrostatic diprotein complexes are studied using stopped-flow, pulse radiolysis, and flash photolysis experiments. The electrostatic complex undergoes intracomplex electron transfer with a rate constant of 1.05 x 10[superscript]3s[superscript]-1. However, there appears to be a complete absence of intracomplex electron transfer within all isomers of the covalent complex. A rearrangement of the proteins for this reaction seems to be possible (or unnecessary) in the electrostatic complex but impossible in the covalent complex.</p