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• W2158736835 abstract "The reductive part of the catalytic cycle of cytochrome c oxidase from Paracoccus denitrificans was examined by using time-resolved potential measurements on black lipid membranes. Proteoliposomes were adsorbed to the black lipid membranes and Ru II (2,2′-bipyridyl) 3 2+ was used as photoreductant to measure flash-induced membrane potential generation. Single-electron reduction of the oxidized wild-type cytochrome c oxidase reveals two phases of membrane potential generation (τ 1 ≈ 20 μs and τ 2 ≈ 175 μs) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from Cu A to heme a . The slower phase is inhibited completely by cyanide and shows a kinetic deuterium isotope effect by a factor of 2–3. Although two enzyme variants mutated in the so-called D pathway of proton transfer (D124N and E278Q) show the same time constants and relative amplitudes as the wild-type enzyme, in the K pathway variant K354M, τ 2 is increased to 900 μs. This result suggests uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. After the second laser flash under anaerobic conditions, a third electrogenic phase with a time constant of ≈1 ms appears. The amplitude of this phase grows with increasing flash number. We explain this growth by injection of a second electron into the single-electron reduced enzyme. On multiple flashes, both D pathway mutants behave differently compared with the wild type and two additional slow phases of τ 3 ≈ 2 ms and τ 4 ≈ 15 ms are observed. These results suggest that the D pathway is involved in proton transfer coupled to the uptake of the second electron." @default.
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• W2158736835 date "2000-04-18" @default.
• W2158736835 modified "2023-10-03" @default.
• W2158736835 title "Single-electron reduction of the oxidized state is coupled to proton uptake via the K pathway in <i>Paracoccus denitrificans</i> cytochrome <i>c</i> oxidase" @default.
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• W2158736835 doi "https://doi.org/10.1073/pnas.080079097" @default.
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