Intracomplex Electron-Transfer between Ruthenium-65-Cytochrome-B5 and Position-82 Variants of Yeast Iso-1-Cytochrome-C

by Willie, Anne; McLean, Mark; Liu, Rui Qin; Hilgen-Willis, Sharon; Saunders, Aleister J.; Pielak, Gary J.; Sligar, Stephen G.; Durham, Bill; Millett, Francis

We tested the idea that the aromatic ring on the invariant residue Phe-82 in cytochrome c acts as an electron-transfer bridge between cytochrome c and cytochrome b5. Ru-65-cyt b5 was prepared by labeling the single sulfhydryl group on T65C cytochrome b5 with [4-(bromomethyl)-4-methylbipyridine] [bis(bipyridine)] ruthenium2+ as previously described [Willie, A., Stayton, P. S., Sligar, S. G., Durham, B., & Millett, F. (1992) Biochemistry 31, 7237-7242]. Laser excitation of the complex formed between Ru-65-cyt b5 and Saccharomyces cerevisiae iso-1-cytochrome c at low ionic strength results in rapid electron transfer from the excited-state Ru(II*) to the heme group of Ru-65-cyt b5 followed by biphasic electron transfer to the heme group of cytochrome c with rate constants of (1.0 +/- 0.2) x 10(5) s-1 and (2.0 +/- 0.4) x 10(4) s-1. Variants of iso-1-cytochrome c substituted at Phe-82 with Tyr, Gly, Leu, and Ile have fast-phase rate constants of 0.4, 1.9, 2.1, and 2.0 x 10(5) s-1 and slow-phase rate constants of 5.3, 3.5, 2.4, and 2.0 x 10(3) s-1, respectively. Increasing the ionic strength to 50 mM results in single-phase intracomplex electron transfer with rate constants of 3.8, 3.1, 3.0, 5.0, and 4.5 x 10(4) s-1 for the wild-type, Tyr, Gly, Leu, and Ile variants, respectively. These results demonstrate that an aromatic side chain at residue 82 is not needed for rapid electron transfer with cytochrome b5. Furthermore, two conformational forms of the complex are present at low ionic strength with fast and slow electron-transfer rates. When the ionic strength is increased to 50 mM, interconversion between the two forms of the complex becomes rapid, resulting in a single phase for electron transfer with a rate constant that is intermediate between the slow and fast rates. At still higher ionic strength the complex dissociates, and second-order kinetics are observed.

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