A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase. Site-directed mutagenesis was used to replace His on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4`methylbipyridine) (bisbipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 ± 2.0) 10 s, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 ± 0.2) 10 s. Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH 7) resulted in electron transfer from photoreduced heme c to Cu with a rate constant of (6 ± 2) 10 s, followed by electron transfer from Cu to heme a with a rate constant of (1.8 ± 0.3) 10 s. Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated. The second-order rate constant is (2.5 ± 0.4) 10M s at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.

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