On the mechanism of the cytochrome c oxidase reaction. Bound Fe(III) cytochrome c as an intermediate in the reductive half reaction.

Abstract

The reaction between cytochrome c and Cytochrome aa3 in the presence of reductant was studied using transient and steady‐state kinetic methods. To investigate the effect of complex formation the reaction was studied at both high and low ionic strength in potassium phosphate buffer (pH 7.4). Steady‐state kinetic experiments were performed in which the oxidation level of cytochrome c was measured during the anaerobic‐aerobic transition of a system containing cytochrome aa3, cytochrome c and ascorbate. Such measurements confirm previous suggestions [Mochan and Nicholls (1972) Biochim, Biophys. Acta. 172, 37–51] that cytochrome c forms a complex with cytochrome oxidase which is not easily accessible to ascorbate reduction. The results suggest that one oxidised cytochrome c per oxidase is bound at low ionic strength. The reduction of ‘oxygen‐pulsed’ oxidase in the presence of dithionite and various concentrations of cytochrome c was investigated by stopped flow spectrophotometry. Exposure of buffer containing cytochrome aa3, cytochrome c and dithionite to a brief pulse of oxygen caused an immediate oxidation of the oxidase and of up to one equivalent of cytochrome c per oxidase. Under conditions where complex formation is favoured the subsequent reduction of cytochrome c is many times slower than can be accounted for by the reaction between dithionite and free cytochrome c. This suggests that the bound oxidised cytochrome c is not readily accessible to dithionite reduction. It is proposed that the complex between oxidised cytochrome c and the oxidase is an intermediate of the reductive half‐reaction and it is shown that the kinetic responses of the complex are rapid enough to be in agreement with this suggestion.