ORGANIZATION AND CONTROL OF ELECTRON TRANSFER CHAINS

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The coupling of electron transfer to proton translocation is the basic mechanism of energy conservation in aerobic organisms. This proposal is aimed at elucidating the molecular basis of this process. Three systems will be used to address different aspects of the problem. The bacterium, Rhodobacter sphaeroides, is considered one of the closest evolutionary relatives of eukaryotic mitochondria. It produces a cytochrome aa3-type oxidase that is structurally and functionally homologous to the mammalian enzyme. We have isolated and characterized the oxidase and cloned and sequenced its genes. In a collaborative effort using site-directed mutagenesis techniques, we have defined the ligands of three of the four metal centers and produced a model of the three dimensional structure of the catalytic site. The first aim of this proposal is to elucidate the mechanism of proton translocation by mutational analysis, selecting residues to be altered on the basis of a high degree of conservation, their predicted relationship to the metal centers, and the model provided by bacteriorhodopsin. Other aspects of the structural basis of oxidase function that will be addressed include: the ligation of CuA, the binding domain for cytochrome c, the interaction between subunits I and II, and the protein environment of the binuclear metal center. Eukaryotic cytochrome c oxidases are more complex in structure than bacterial enzymes, containing from four to ten nuclear encoded peptides. The nuclear peptides differ not only among various organisms but also in different tissues. Their functional significance is a major unsolved question. The plant cytochrome c oxidase has a unique subunit composition and we have evidence of a development form. We propose to explore this possibility by developing antibodies to a putative developmental peptide observed in wheat germ oxidase and to screen for its presence at different stages of growth. Sequence information on the nuclear subunits will also be obtained to relate these peptides to those of other organisms. Our recent results and those of other investigators indicate that conversion of mitochondrial genes into their universal codon equivalents may be an effective approach to allowing the application of genetic tools to the complex mammalian enzymes. We propose to further explore this direction by investigating heterologous expression systems for the mammalian gene for COX II which we have already expressed in oocytes and in vitro. Rhodobacter sphaeroides and Saccharomyces cereviseae will be tested initially. The results of these studies are expected to yield substantial new information regarding the mechanism and regulation of electron and energy transfer in cytochrome c oxidase.