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 bs by recombinant DNA techniques [Stayton, P, S., Fisher, M.T., and Sligar, S.G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome bs 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 Å 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 bs 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 106 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 105 s-1 and a slow-phase rate constant of (3 ± 1) X 104 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., and Northrup, S.H. (1991) Biochemistry 30, 3663-3674], indicating that the same electrostatic interactions are involved in both reactions.
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