Time-resolved generation of a membrane potential by ba(3) cytochrome c oxidase from Thermus thermophilus - Evidence for reduction-induced opening of the binuclear center

ba(3)-type cytochrome c oxidase purified from the thermophilic bacterium Th ermus thermophilus has been reconstituted in phospholipid vesicles and lase r flash-induced generation of a membrane potential by the enzyme has been s tudied in a mu s/ms time scale with Ru(II)-tris-bipyridyl complex (RuBpy) a s a photoreductant. Flash-induced single electron reduction of the aerobica lly oxidized ba(3) by RuBpy results in two phases of membrane potential gen eration by the enzyme with tau values of about 20 and 300 mu s at pH 8 and 23 degrees 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, photored uction of the enzyme by RuBpy triggers the electrogenic reaction with H2O2 with a second order rate constant of similar to 2X10(3) M-1 s(-1). The data indicate that single electron reduction of ba(3) oxidase opens the binucle ar center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba(3) is much less than in bovine oxidase, pointing to a possibility of a differ ent electrogenic mechanism of the ba(3) oxidase as compared to the oxidases of the aa(3)-type, (C) 1999 Federation of European Biochemical Societies.