A LIGAND-EXCHANGE MECHANISM OF PROTON-PUMPING INVOLVING TYROSINE-422 OF SUBUNIT-I OF CYTOCHROME-OXIDASE IS RULED OUT

The molecular mechanism by which proton pumping is coupled to electron transfer in cytochrome c oxidase has not yet been determined. However , several models of this process have been proposed which are based on changes occurring in the vicinity of the redox centers of the enzyme. Recently, a model was described in which a well-conserved tyrosine re sidue in subunit I (Y422) was proposed to undergo ligand exchange with the histidine ligand (H419) of the high-spin heme a(3) during the cat alytic cycle, allowing both residues to serve as part of a proton tran sporting system. Site-directed mutants of Y422 have been constructed i n the aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides to te st this hypothesis (Y422A, Y422F). The results demonstrate that Y422 i s not an essential residue in the electron transfer and proton pumping mechanisms of cytochrome c oxidase. However, the results support the predicted proximity of Y422 to heme a(3), as now confirmed by crystal structure. In addition, it is shown that the pH-dependent reversed ele ctron transfer between heme a(3) and heme a(3) is normal in the Y422F mutant. Hence, these data also demonstrate that Y422 is not the residu e previously postulated to interact electrostatically with heme a(3), nor is it responsible for the unique EPR characteristics of heme a in this bacterial oxidase.