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 microsecond/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 tau values of about 20 and 300 microseconds 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, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H2O2 with a second order rate constant of approximately 2 x 10(3) 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.
Siletskiy
S
S
A.N. Belozerskiy Institute of Physico-Chemical Biology, Moscow State University, Russia.
Soulimane
T
T
Azarkina
N
N
Vygodina
T V
TV
Buse
G
G
Kaulen
A
A
Konstantinov
A
A
D002384:Catalysis; D003573:Cytochrome b Group / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials; D017494:Proton Pumps; D013053:Spectrophotometry; D016963:Thermus thermophilus / Q000201:enzymology*; D013997:Time Factors
D003573:Cytochrome b Group; D017494:Proton Pumps; D006861:Hydrogen Peroxide; C058557:cytochrome ba3; D003576:Electron Transport Complex IV