Single-Electron Reduction of the Oxidized State Is Coupled to Proton Uptake via the K Pathway in Paracoccus denitrificans Cytochrome C Oxidase

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)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 (τ1≈ 20 μ s and τ2≈ 175 μ s) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from CuAto 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, τ2is increased to 900 μ s. 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.