Proton transfer reactions associated with the reaction of the fully reduced, purifierd cytochome c oxidase with molecular oxygen and ferrycianide

A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from ≈3.0 H+/COX at pH 6.2−6.3 to ≈1.2 H+/COX at pH 8.0−8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from ≈1.0 H+/COX at pH 6.2−6.3 to ≈2.8 H+/COX at pH 8.0−8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3−1.6 H+/COX in the pH range 6.2−8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O2 to 2H2O. The results show that the expected stoichiometric proton consumption of 4H+ in the reduction of O2 to 2H2O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H+/COX are initially taken up in the reduction of O2 to two OH- groups bound to the binuclear Fe a3−CuB center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H2O. At alkaline pHs the third and fourth protons are taken up with formation of 2H2O only upon rereduction of COX