STRUCTURE AND FUNCTION OF CUA IN CYTOCHROME C OXIDASE

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Cytochrome c oxidase catalyzes the 4-electron reduction of O2 to water in the final step of cellular respiration in all higher forms of life. In the 50 years since its discovery, it has been found that the enzyme contains 4 redox-active metal centers. More recently, it has become clear that coupled to this redox chemistry, cytochrome c oxidase also pumps protons across the mitochondrial membrane to add to the electrochemical energy gradient generated by respiration. The mechanism of this redox-linked pumping remains elusive and it is still not known which metal center, if any, is involved. It was proposed some time ago that the spectroscopically unusual Cu(A) center has an unprecedented dithiolate coordination and that this coordination is central to its role in proton pumping. Previous studies have conclusively shown that Cu(A) has at least one cysteine ligand, but the proposal of an unusual second cysteine ligand remains unproven. Evolutionary information additionally requires that Cu(A) be coordinated by one or both of two specific cysteines found close to each other in a highly conserved region of the protein sequence for subunit II. Until recently, site-directed mutagenesis of eukaryotic cytochrome c oxidase was not technically possible. The transmembranous enzyme contains 2 heme irons and is composed of 12 or more different subunits, 3 encoded by mitochondrial DNA (one or more of these contains all 4 metal centers), and the remaining encoded by nuclear genes. However, it has recently become possible to transform yeast mitochondria with exogenous DNA and, fortuitously, the procedure was developed using the gene (COX2) for subunit II of yeast cytochrome c oxidase. The goals of this proposal are to develop a new approach to probing metal center structure and function, to test and further refine detailed models for the coordination of Cu(A) and to assess the involvement of this center in the mechanism of electron transfer and proton pumping. Individual Cys, His and Tyr residues within subunit II which have been implicated in the above models will be altered by site-directed mutagenesis in yeast. The effects of these mutations on the structure and function of the Cu(A) site in yeast in vivo and in detergent-solubilized enzyme will be probed by functional assays and by optical and EPR spectroscopies. If the enzyme fails to assemble correctly for a given mutation, second-site revertants will be selected for, and the resultant mutant enzymes will be similarly analyzed. These results will compared with predictions afforded by the structural models. Finally, the proton pumping activities of these mutants will be compared in order to provide strong evidence for or against a model of redox-linked proton pumping by this unusual copper center.