文摘
Cytochrome bo3 ubiquinol oxidase from Escherichia coli catalyzes the reduction of O2 to water by ubiquinol. The reaction mechanism and the role of ubiquinol continue to be a subject of discussion. In this study, we report a detailed kinetic scheme of the reaction of cytochrome bo3 with O2 with steps specific to ubiquinol. The reaction was investigated using the CO flow-flash method, and time-resolved optical absorption difference spectra were collected from 1 渭s to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 渭s, 30 渭s, 42 渭s, 470 渭s, and 2.0 ms. The reaction mechanism was derived by an algebraic kinetic analysis method using frequency-shifted spectra of known bovine states to identify the bo3 intermediates. It shows 42 渭s O2 binding (3.8 脳 107 M鈥? s鈥?), producing compound A, followed by faster (22 渭s) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 渭s), producing the F intermediate and semiquinone. In the 470 渭s step, the o3 F state is converted into the o33+ oxidized state, presumably by semiquinone/ubiquinol, without the concomitant oxidation of heme b. The final 2 ms step shows heme b reoxidation and the partial rereduction of the binuclear center and, following O2 binding, the formation of a mixture of P and F during a second turnover cycle. The results show that ubiquinol/semiquinone plays a complex role in the mechanism of O2 reduction by bo3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.