Design of a Ruthenium Cytochrome-C Derivative to Measure Electron-Transfer to the Initial Acceptor in Cytochrome-C-Oxidase
by Geren, Lois M.; Beasley, James R.; Fine, Bryan R.; Saunders, Aleister J.; Hibdon, Sharon; Pielak, Gary J.; Durham, Bill; Millett, Francis
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(39) 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) (bis-bipyridine)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) x 10(5) s(-1), followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 +/- 0.2) x 10(6) s(-1), 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-A with a rate constant of (6 +/- 2) x 10(4) s(-1), followed by electron transfer from Cu-A to heme a with a rate constant of (1.8 +/- 0.3) x 10(4) s(-1), 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) x 10(7) M(-1) s(-1) at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.
- Journal
- Journal of Biological Chemistry
- Volume
- 270
- Issue
- 6
- Year
- 1995
- Start Page
- 2466-2472
- URL
- https://dx.doi.org/10.1074/jbc.270.6.2466
- ISBN/ISSN
- 1083-351X; 0021-9258
- DOI
- 10.1074/jbc.270.6.2466