Use of Ruthenium Photoreduction Techniques to Study Electron Transfer in Cytochrome Oxidase

by Geren, Lois M.; Durham, Bill; Millett, Francis Spencer

Ruthenium photoreduction methods are described to study electron transfer from cytochrome c to cytochrome c oxidase and within cytochrome oxidase. Methods are described to prepare a ruthenium cytochrome c derivative Ru-39-Cc, by labeling the single sulfhydryl group on horse K39C with (4-bromomethyl-4'-methylbipyridine) (bis-bipyridine)ruthenium(II). The ruthenium complex attached to Cys-39 on the opposite side of the heme crevice does not interfere with the interaction with cytochrome oxidase. Laser flash photolysis of a 1:1 complex between Ru-39-Cc and bovine cytochrome oxidase results in photoreduction of heme c within 1 mu sec, followed by electron transfer from heme c to Cu-A in cytochrome oxidase with a rate constant of 60,000 s(-1) and from Cu-A to heme a with a rate constant of 20,000 s-1. A new ruthenium dimer, Ru(2)Z, has been developed to reduce Cu-A within 1 mu sec with a yield of 60%, followed by electron transfer from Cu-A to heme a and then to the heme a(3)/Cu-B binuclear center. Methods are described to measure the single-electron reduction of each of the intermediates involved in reduction of oxygen to water by cytochrome oxidase, including P-m, F, O-H, and E.

Methods in Enzymology
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