EFFECTS OF CYTOCHROME-C ON THE OXIDATION OF REDUCED CYTOCHROME-C-OXIDASE BY HYDROGEN-PEROXIDE

The oxidation of the redox centres in reduced cytochrome c oxidase by hydrogen peroxide was studied by stopped-flow spectrophotometry in the absence and presence of reduced cytochrome c. The oxidation rate of cytochrome a decreased in the presence of cytochrome c. This effect was more pronounced at low than at high ionic strength. Cytochrome c did not influence the time-course of the oxidation of Cu-A or cytochrome a(3). The oxidation of cytochrome c itself was faster at low ionic strength. The results suggest that the effect of cytochrome c is caused by re-reduction of cytochrome a by cytochrome c, the rate of which is dependent upon the ionic strength. We conclude that cytochrome a and cytochrome c are in equilibrium and that the equilibrium constant depends on the ionic strength. At low ionic strength, as a complex is formed between cytochrome c and cytochrome c oxidase, cytochrome a is more reduced than at high ionic strength conditions, when no such complex exists. Since Cu-A is oxidized at the same rate whether cytochrome c is present or not, we conclude that electron transfer from cytochrome a or cytochrome c to Cu-A is slower than electron transfer from Cu-A to cytochrome a or/and to the cytochrome a(3)-Cu-B couple.