Multiple proton-conducting pathways in cytochrome oxidase and a proposed role for the active-site tyrosine

AbstractDuring each turnover of cytochrome oxidase (dioxygen reduced to water), eight protons are taken up from the inside of the membrane, four protons being used for chemistry (water formation) and four protons being pumped across the membrane. The two available X-ray structures of cytochrome c oxidases indicate three possible pathways for proton conduction within the enzyme: the D-, K- and H-channels. The putative H-channel is most clearly defined in the structure of the bovine oxidase. Mutants designed to examine the role of the H-channel in bacterial oxidases have failed to provide convincing evidence, so far, concerning the functional importance of this putative channel. In contrast, studies utilizing site-directed mutants provide strong evidence that the D-channel and K-channel are functionally important. Mutants in the K-channel are clearly defective in one or more steps associated with the first part of the catalytic cycle, i.e., the reduction of the heme/copper binuclear center. Mutants in the D-channel are defective in steps following the interaction of dioxygen with the reduced heme/copper center. A possible explanation of these observations is offered that proposes a direct hydrogen atom transfer from a modified tyrosine at the active site to the distal oxygen atom of dioxygen bound to heme a3