Electron transfer coupled to proton translocation is the basic mechanism of energy generation in most living organisms, but the molecular mechanism is not understood. A key enzyme in all eukaryotic and most prokaryotic electron transfer systems is cytochrome c oxidase,which accepts electrons derived from food and donates them to oxygen, generating a pH and electrical gradient to drive ATP synthesis.
We are studying mammalian, plant and bacterial cytochrome c oxidases which differ in peptide composition but carry out the same reactions using the same metal centers to catalyze the process. Each of these enzymes offers different advantages for investigating the molecular mechanism of energy transduction by a variety of approaches, including kinetic analysis, chemical modification, physical/spectral techniques, genetic engineering and crystallography. To understand the molecular basis of electron transfer and coupled proton translocation, mutants have been prepared in highly conserved residues predicted to be metal ligands or proton ligands. Extensive spectral and biochemical analysis has led to a model of the active site and proposed pathways for proton translocation, which are confirmed by recent high resolution crystal structures for both prokaryotic and eukaryotic enzymes. On-going work involves the design of site-directed mutants to further test these models and efforts to crystallize the oxidase from Rhodobacter sphaeroides and its mutant forms. The goal is to elucidate how the oxygen chemistry drives a proton pump, how the process is regulated to balance fat storage and heat generation, and how aging and disease are associated with loss of efficiency of energy production.