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Millett F, Durham B: Chapter 5 Use of ruthenium photooxidation techniques to study electron transfer in the cytochrome bc1 complex. Methods Enzymol. 2009;456:95-109.
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 Cys-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 microsec 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 c (1) 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, Ru (2) D, to photooxidize cyt c (1) in the cytochrome bc (1) complex within 1 microsec 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. |
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