Design of a ruthenium-cytochrome c derivative to measure electron transfer to the initial acceptor in cytochrome c oxidase.
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 His39 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, pH7) resulted in electron transfer from photoreduced heme c to CuA with a rate constant of (6 +/- 2) x 10(4) s-1, followed by electron transfer from CuA 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-1s-1 at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
L M
LM
Geren
J R
JR
Beasley
B R
BR
Fine
A J
AJ
Saunders
S
S
Hibdon
G J
GJ
Pielak
B
B
Durham
F
F
Millett
D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012428:Ruthenium / Q000737:chemistry*
D003574:Cytochrome c Group; D012428:Ruthenium; D003576:Electron Transport Complex IV