The rate-limiting step in O2 reduction by cytochrome ba3 from Thermus thermophilus

Cytochrome ba(3) (ba(3)) of Therm us thermophilus (T. thermophilus) is a member of the heme-copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a(3)) and a copper (Cu-B). The heme-copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba(3) with O-2 using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (similar to 1 ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k = 400 s(-1)) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1 ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O-2 binds rapidly to heme a(3) in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O-2 binds transiently to a trap, likely Cu-B, prior to its migration to heme a(3) for the oxidative reaction, highlighting the critical role of Cu-B in regulating the oxygen reaction kinetics in the oxidase super-family. This article is part of a Special Issue entitled: Respiratory Oxidases. (C) 2011 Elsevier B.V. All rights reserved.