Electron transfer to the binuclear center in cytochrome oxidase: catalytic significance and evidence for an additional intermediate.

We have followed, by transient kinetics, the reduction of cytochrome a3 in the presence of carbon monoxide under different experimental conditions. We have observed that the internal electron transfer rate accounts for the turnover number, and both display the same pH and temperature dependence [pKa = 7.4 and activation energy (Ea) = 14.7 +/- 0.1 kcal/mol]. Moreover, comparison of the time course of cytochrome c oxidation and cytochrome a3 reduction indicates that two electrons are transferred internally and with different rates to the oxygen-binding site. A kinetic model based on sequential internal electron transfer pathways, describing quantitatively the experimental data, is presented and discussed.