Mechanism of Cytochrome c Oxidase-Catalyzed Reduction of Dioxygen to Water: Evidence for Peroxy and Ferryl Intermediates at Room Temperature

Bruce C. Hill
Biochemistry 35: 6136-6143, 1996

SUMMARY:The reaction of CO and O₂ with fully reduced cytochrome caa3 from Bacillus subtilis has been studied by rapid reaction spectrophotometry. The fully reduced caa₃ complex reacts with CO to give a spectrum that is characteristic of formation of ferrocytochrome a₃ÐCO. This adduct is photosensitive, and its recombination rate is proportional to CO concentration with a bimolecular value of 1.2 x 10⁵M_-1 s^-1. When the CO compound of the reduced complex is exposed to O₂, the rate of oxidation proceeds at 0.1 s^-1, which is assigned as the CO off rate. These kinetic constants give an equilibrium dissociation constant for the CO complex of 0.83 mM. Photolysis of the CO adduct in the presence of O₂ reveals three reaction phases over the first 3 ms and an additional phase on the second time scale. A kinetic model is proposed in which fully reduced oxidase first combines with O₂ and then electron transfer commences from both cytochrome a and a₃, followed rapidly by electron input from Cu_A and the cytochrome c domain. An equivalent kinetic model has been used to account for the reactivity of mammalian cytochrome c oxidase in its electrostatic complex with soluble cytochrome c [Hill, B. C., (1994) J. Biol. Chem. 269, 2419-2425]. However, unlike the mitochondrial complex, the reactivity of cytochrome c in the B. subtilis caa₃ complex is unaffected by ionic strength. Thus the cytochrome c moiety in the B. subtilis caa₃ complex seems to be fixed in a reactive orientation by its covalent association with the rest of the oxidase complex. The pathway of electron transfer from cytochrome c to O₂ appears very well conserved from B. subtilis to the mammalian respiratory chain, making the B. subtilis protein a good model to prove intersite electron transfer within the cytochrome cÐcytochrome oxidase complex.