Proton Translocation by a Native and Subunit 111 - depleted Cytochrome c Oxidase Recon ~ tituted into P ~

The existence of a proton pump associated with bovine cytochrome c oxidase (EC 1.9.3.1) has over the last few years been a matter of considerable dispute. In an attempt to resolve some of the problems with the measuring system we have synthesized ffuoresceinphosphatidylethanolamine which when reconstituted with cytochrome c oxidase into phospholipid vesicles provided a reliable indicator of the intravesicular pH. It was observed that cytochrome c oxidase catalyzed the abstraction of almost 2 protons from the intravesicular medium/molecule of ferroeytochrome c oxidized. In parallel experiments whereby the xtravesicular pH was measured with an electrode it was found that the enzyme appeared to be responsible for the appearance of almost 1.0 proton/molecule of ferrocytochrome c oxidized. Taken together these data unequivocally demonstrate that cytochrome c oxidase behaves as a proton pump. Furthermore, the other proton which was abstracted is believed to be used for the process of the reduction of oxygen. Similar experiments were performed with a cytochrome c oxidase preparation which was devoid of subunit 111. Under these circumstances the enzyme appeared to be unable to translocate protons across the vesicular membrane but was competent to abstract protons from the intravesicular medium for the reduction of oxygen.