Genetic-Engineering of Redox Donor Sites - Measurement of Intracomplex Electron-Transfer between Ruthenium-65 Cytochrome-B5 and Cytochrome-C

by Willie, Anne; Stayton, Patrick S.; Sligar, Stephen G.; Durham, Bill; Millett, Francis

The de novo design and synthesis of ruthenium-labeled cytochrome b5 that is optimized for the measurement of intracomplex electron transfer to cytochrome c are described. A single cysteine was substituted for Thr-65 of rat liver cytochrome b5 by recombinant DNA techniques [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome b5 was then labeled with [4-(bromomethyl)-4'-methylbipyridine](bisbipyridine)ruthenium2+ to form Ru-65-cyt b5. The ruthenium group at Cys-65 is only 12 angstrom from the heme group of cytochrome b5 but is not located at the binding site for cytochrome c. Laser excitation of the complex between Ru-65-cyt b5 and cytochrome c results in electron transfer from the excited state Ru(II*) to the heme group of Ru-65-cyt b5 with a rate constant greater than 10(6) s-1. Subsequent electron transfer from the heme group of Ru-65-cyt b5 to the heme group of cytochrome c is biphasic, with a fast-phase rate constant of (4 +/- 1) X 10(5) s-1 and a slow-phase rate constant of (3 +/- 1) x 10(4) s-1. This suggests that the complex can assume two different conformations with different electron-transfer properties. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased, indicating dissociation of the complex. The ionic strength dependence of the second-order rate constant is nearly the same as for the reaction between native cytochrome b5 and cytochrome c [Eltis, L. D., Herbert, R. G., Barker, P. D., Mauk, A. G., & Northrup, S. H. (1991) Biochemistry 30, 3663-3674], indicating that the same electrostatic interactions are involved in both reactions.

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