title: Kinetics and intermediates of the reaction of fully reduced Escherichia coli bo₃ ubiquinol oxidase with O₂.
abstract: 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 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 × 10(7) M(-1) s(-1)), producing compound A, followed by faster (22 μs) heme b oxidation, yielding a mixture of PR and F, and rapid heme b rereduction by ubiquinol (30 μs), producing the F intermediate and semiquinone. In the 470 μs step, the o₃ F state is converted into the o₃(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 bo₃, displaying kinetic steps that have no analogy in the CuA-containing heme-copper oxidases.
authors: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.
authors: Istvan
authors: I
authors: Szundi
authors: Clive
authors: C
authors: Kittredge
authors: Sylvia K
authors: SK
authors: Choi
authors: William
authors: W
authors: McDonald
authors: Jayashree
authors: J
authors: Ray
authors: Robert B
authors: RB
authors: Gennis
authors: Ólöf
authors: Ó
authors: Einarsdóttir
mesh_terms: D001671:Biochemistry / Q000379:methods; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D009569:Nitric Oxide / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C083850:cytochrome bo3, E coli; D009569:Nitric Oxide; D006418:Heme; D010088:Oxidoreductases; C432586:ubiquinol oxidase; D010100:Oxygen