Kinetics and Intermediates of the Reaction of Fully Reduced Escherichia coli bo 3 Ubiquinol Oxidase with O2

• Published in: Biochemistry (Easton) Vol. 53; no. 33; pp. 5393 - 5404
• Main Authors: Szundi, Istvan, Kittredge, Clive, Choi, Sylvia K, McDonald, William, Ray, Jayashree, Gennis, Robert B, Einarsdóttir, Ólöf
• Format: Journal Article
• Language: English
• Published: American Chemical Society 26-08-2014
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi500567m

Cytochrome bo 3 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 bo 3 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 o 3 3+ 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 bo 3, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.