Reaction of dioxygen with cytochrome c oxidase reduced to different degrees: indications of a transient dioxygen complex with copper-B.

The reactions of the fully reduced, three-electron-reduced, and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, starting from the CO complexes, at 445 and 830 nm at pH 7.4 and 25 degrees C. With the fully reduced and the three-electron-reduced enzyme, four kinetic phases with rate constants in the range from 1 x 10(5) to 10(3) s-1 can be observed. The initial fast phase is associated with an absorbance increase at 830 nm. This is followed by an absorbance decrease (2.8 x 10(4) s-1), the amplitude of which increases with the degree of reduction of the oxidase. The third phase (6 x 10(3) s-1) displays the largest absorbance change at both wavelengths in the fully reduced enzyme and is not seen in the mixed-valence oxidase at 830 nm; a change with opposite sign but with a similar rate constant is found at 445 nm in this enzyme form. The slowest phase (10(3) s-1) is also largest in the fully reduced oxidase and not seen in the mixed-valence enzyme. It is suggested that O2 initially binds to reduced CuB and is then transferred to cytochrome a3 before electron transfer from cytochrome a/CuA takes place. The fast oxidation of cytochrome a seen with the fully reduced enzyme is suggested not to occur during natural turnover. A reaction cycle for the complete turnover of the enzyme is presented. In this cycle, the oxidase oscillates between electron input and output states of the proton pump, characterized by cytochrome a having a high and a low reduction potential, respectively.