Time-resolved generation of a membrane potential by ba₃ cytochrome c oxidase from Thermus thermophilus: Evidence for reduction-induced opening of the binuclear center

ba₃-type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash-induced generation of a membrane potential by the enzyme has been studied in a μs/ms time scale with Ru(II)-tris-bipyridyl complex (RuBpy) as a photoreductant. Flash-induced single electron reduction of the aerobically oxidized ba₃ by RuBpy results in two phases of membrane potential generation by the enzyme with τ values of about 20 and 300 μs at pH 8 and 23°C. Spectrophotometric experiments show that oxidized ba₃ reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide. At the same time, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H₂O₂ with a second order rate constant of ~2x10³ M^-1 s^-1. The data indicate that single electron reduction of ba₃ oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba₃ is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba₃ oxidase as compared to the oxidases of the aa₃-type. Copyright (C) 1999 Federation of European Biochemical Societies.