The mechanism of second H2O formation in fully reduced cytochrome c oxidase is examined by sequential additions of one electron and two protons to a [Fe(IV)=O, Cu(II)] compound II, which is the final intermediate of first H2O formation. It is found that the addition of one electron induces the concerted proton-electron transfer from the Cu-B to Fe=O moieties with no energy barriers. The H2O molecule coordinating to the Cu atom is a key molecule for the proton transfer from the Cu-B moiety to the Fe=O and/or Fe-OH moieties. It is also found from the results in previous and the present works that one can realize the reduction process of dioxygen by fully reduced cytochrome c oxidase. (C) 2007 Elsevier B.V. All rights reserved.