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 RuII(2,2′-bipyridyl)32+ 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 (τ1 ≈ 20 μs and τ2 ≈ 175 μs) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from CuA 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, τ2 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 τ3 ≈ 2 ms and τ4 ≈ 15 ms are observed. These results suggest that the D pathway is involved in proton transfer coupled to the uptake of the second electron
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