Use of ruthenium photooxidation techniques to study electron transfer in the cytochrome bc(1) complex

by Millett, Francis Spencer; Durham, Bill

Ruthenium photooxidation methods are presented to study electron transfer between the cytochrome bc(1) complex and cytochrome c and within the cytochrome bc(1) complex. Methods are described to prepare a ruthenium cytochrome c derivative, Ru-z-39-Cc, by labeling the single sulfhydryl on yeast H39C;C102T iso-1-Cc with the reagent Ru(bPZ)(2)(4-bromomethyl-4'-methylbipyridine). The ruthenium complex attached to wCys-39 on the opposite side of Cc from the heme crevice does not affect the interaction with cyt bc(1). Laser excitation of reduced Ru-z-39-Cc results in photooxidation of heme c within 1 mu sec with a yield of 20%. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc(1) and Ru-z-39-Cc leads to electron transfer from heme cl to heme c with a rate constant of 1.4 x 10(4) S-1. Methods are described for the use of the ruthenium dimer, Ru2D, to photooxidize cyt c(1) in the cytochrome bc(1) complex within 1 mu sec with a yield of 20%. Electron transfer from the Rieske iron-sulfur center [2Fe2S] to cyt c(1) was detected with a rate constant of 6 x 10(4) S-1 in R. sphaeroides cyt bc(1) with this method. This electron transfer step is rate-limited by the rotation of the Rieske iron-sulfur protein in a conformational gating mechanism. This method provides critical information on the dynamics of rotation of the iron-sulfur protein (ISP) as it transfers electrons from QH(2) in the Q(o) site to cyt c(1). These ruthenium photooxidation methods can be used to measure many of the electron transfer reactions in cytochrome bc(1) complexes from any source.

Methods in Enzymology
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