Mixed-valence Oxidase Research Articles

Dioxygen activation in enzymatic systems and in inorganic models

Cytochrome oxidase reduces O 2 to water quickly and with low overpotential. In addition, it uses the exergonicity of this reaction to pump protons against their thermodynamic gradient, thus contributing directly to the chemiosmotic potential that is used to synthesize ATP. In recent work, we have developed means by which to use time-resolved resonance Raman to study transient heme iron-bound oxygen intermediates that occur during the reduction of O 2 by cytochrome oxidase. Thus far, five different oxygen isotope sensitive modes have been observed; the temporal behavior of each during the reaction sequence has been characterized roughly. By combining the structure-specific vibrational results with optical data from other labs on the same reaction, we have constructed an overall working model for the dioxygen reduction reaction and have calculated concentration/time profiles for key intermediates. As opposed to most O 2 metabolizing enzymes, these calculations indicate that the oxidase/O 2 reaction is under proton control, which allows transient intermediates to build to detectable concentrations. We have linked this behavior to the proton-pump function of the enzyme and have postulated that proton control allows tight coupling between the oxygen chemistry and the proton translocations it drives. Recent findings from several laboratories have shown that a direct connection can be made between the intermediates that occur during oxygen reduction by fully reduced and mixed-valence oxidase and in its reaction with peroxide. This requires a branching reaction at the peroxide level in the reaction sequence. This modification to the mechanism is presented. In this article, these findings are reviewed and the mechanism by which O 2 reduction is catalyzed by cytochrome oxidase is compared and contrasted with other O 2-metabolizing heme enzymes. The continuing importance of vibrational spectroscopic approaches that rely on stable isotope substitution for mode identification is highlighted by reviewing recent developments in other laboratories on O 2 activation in the non-heme iron class of oxygen-metabolizing enzymes. This group of catalysts includes ribonucleotide reductase, methane monooxygenase, and fatty acid Δ 9-desaturase and has been recognized as a distinct, oxygen-metabolizing enzyme class only recently. Finally, recent inorganic model compound work on O 2 activation is briefly summarized.

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.

Interconversion between states in cytochrome oxidase: Interpretation of kinetic data on mixed-valence oxidase

We have proposed [l] a two-state model to account for the functional properties of cytochrome c oxidase. The basis of the model was the demonstration that the enzyme may exist either in a ‘resting’ (R) or in a ‘pulsed’ (P) state, the two states being interconvertible and characterized by different kinetic and spectral properties. Pulsed oxidase as an activated state of the enzyme was originally discovered in [2] on the basis of rapid mixing experiments in which the kinetic properties of the enzyme were tested immediately (milliseconds) after exposure of reduced oxidase to oxygen. Its properties have since been investigated [3-61, and a pulsed state has been identified for oxidases from different organisms. Pulsed oxidase may also be obtained by oxidation of the reduced enzyme by ferricyanide [7] and thus its formation is not absolutely dependent on the presence of dioxygen. Simulation of the time course of activation using a complete reaction scheme [I], has led to an estimate of the rate constant for the transition R, P, (kl = 10’ lo3 s-l). Here, we report the interpretation of flow-flash experiments carried out some years ago [9] starting from the mixedvalence CO derivative of cytochrome c oxidase isolated from beef heart, and showing that the observations are quantitatively consistent with the predictions of the two-state model. Moreover, it appears that interconversions between oxidised