Intracomplex Electron-Transfer between Ruthenium-Cytochrome-C Derivatives and Cytochrome-C(1)

by Heacock, Donald H., II; Liu, Rui Qin; Yu, Chang An; Yu, Linda; Durham, Bill; Millett, Francis

The reactions of a beef heart cytochrome c1 preparation containing the hinge protein with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine) ruthenium(II) (Ru(II)) were studied by flash photolysis. All of the ruthenium-cytochrome c derivatives formed complexes with cytochrome c1 in low ionic strength buffer (5 mm sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II) with a 0.4-mus laser flash resulted in rapid electron transfer to the ferric heme group in cytochrome c, followed by electron transfer from the ferrous heme group of cytochrome c to the ferric heme group of cytochrome c1. The kinetic difference spectra displayed maxima at 546 nm and minima at 554 nm characteristic of electron transfer between the two cytochromes. The rate constants were independent of concentration at low ionic strength, indicating intracomplex electron transfer. The rate constants were 4,800, 6,800, 22,000, and 22,000 s-1 for cytochrome c derivatives modified at lysines 13, 27, 25, and 72, respectively. The observed rate constants were independent of ionic strength up to about 50 mm and then decreased progressively with further increases in ionic strength indicating dissociation of the complex. Second-order kinetics were observed at 310 mm ionic strength, with rate constants of 1.0 x 10(6), 1.6 x 10(7), 1.2 x 10(8), and 3.0 x 10(7) M-1 s-1 for the derivatives modified at lysines 13, 27, 25, and 72, respectively. The ionic strength dependence of the second-order rate constants is comparable to that involving native horse cytochrome c and is consistent with electron transfer reactions between oppositely charged proteins.

Journal of Biological Chemistry
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1083-351X; 0021-9258