The proposed research is focused on the catalytic mechanisms and structures of respiratory oxidases. These membrane enzymes catalyze the reduction of 02 to H20, and conserve the considerable free energy liberated by this reaction as a proton motive force. All respiratory oxidases belong to either the proton pumping """"""""heme-copper oxidase"""""""" superfamily or the non- pumping """"""""tri-heme oxidase"""""""" superfamily. The heine-copper oxidases include all the mitochondrial cytochrome oxidases. As a crucial enzyme in cellular bioenergetics, human cytochrome oxidase is of medical significance, and has been studied as the locus of a set of genetic diseases. The prokaryotic enzyme we are studying from Rhodobacter sphaeroides is an excellent model system for understanding how this enzyme functions. The cbb3-type oxidase from Vibrio cholerae is a second member of the home-copper oxidase superfamily that will be studied. Learning how the same functions are accomplished in these two distantly related enzymes should be revealing. Interest in cytochrome oxidase research is primarily directed towards understanding the proton pump mechanism. For each O2 reduced to H2O, eight charges are driven across the membrane, generating a substantial transmembrane voltage. Research on the aa3-type oxidase from R. sphaeroides utilizes site-directed mutagenesis in combination with a several spectroscopic techniques, including solid state NMR and FTIR difference spectroscopy. The X-ray structure of this enzyme is known and serves as a guide to planning and interpreting experiments. One mutant of particular interest is N139D. This mutation results in increasing the cytochrome oxidase activity of the enzyme by at least 50% over the wild type, but completely eliminates proton pumping. Proton uptake, proton release and intra-protein proton movements will be examined. Understanding how the proton pump is decoupled from the catalytic function will provide fundamental information about how the proton pump works. The tri-heme oxidases are unique to prokaryotes, but are also of medical significance as potential drug targets because of their apparent importance for the virulence of some pathogenic bacteria. Research on the tri-heme oxidases will focus on the cytochrome bd from E. coli and the cytochrome bb"""""""" from V. cholerae. The bb'-type enzyme has just recently been discovered and part of the research plan is to purify and characterize this enzyme for the first time. The tri-heme oxidases have two closely interacting heroes at the active site, and spectroscopic methods will be used to elucidate the details of the home-home interaction. Identifying residues involved in intra-protein proton movement from the bulk solution to the active site will also be a goal in the near future. Finally, it is essential to obtain a structure of this enzyme by X-ray crystallography, and efforts will be made to accomplish this.
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