Kinetics and intermediates of the reaction of fully reduced escherichia coli bo ₃ ubiquinol oxidase with O₂

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 bo ₃ 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 μs to 20 ms after photolysis. Singular value decomposition-based global exponential fitting resolved five apparent lifetimes, 22 μs, 30 μs, 42 μs, 470 μs, 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 μs O₂ binding (3.8 × 10⁷ M^-1 s^-1), producing compound A, followed by faster (22 μs) heme b oxidation, yielding a mixture of P _R and F, and rapid heme b rereduction by ubiquinol (30 μs), producing the F intermediate and semiquinone. In the 470 μs step, the o ₃ 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 O ₂ reduction by bo₃, displaying kinetic steps that have no analogy in the Cu_A-containing heme-copper oxidases.