Our objective is to continue elucidation of the molecular reaction mechanisms of the ubiquinone (Q)-mediated mitochondrial electron transfer and energy conservation through study of the two Q-binding protein systems, QPs and QPc, in succinate-Q and ubiquinol-cytochrome c reductase, respectively. The involvement of specific Q-binding proteins (sites) in the mitochondrial electron transfer system has been established and isolated or identified during past support periods. The next step will be the isolation and characterization of active Q-binding proteins, and elucidation of the structure of the Q-binding sites. Multiple approaches will be taken to study the Q-binding sites and Q:protein interaction in both ubiquinol-cytochrome c reductase and succinate-Q reductase. These include: (1) isolation and sequencing of the Q-binding peptides from the isolated Q-binding proteins, (2) organic synthesis of Q derivatives with different functional (electron donating or accepting) properties, and Q derivatives with detectable groups for studying the Q:protein interaction, (3) chemical modification of the Q-binding site, and (4) immunological characterization of the Q-binding sites and proton translocation sites (domains) of Q-binding proteins. The interaction between succinate-Q reductase and ubiquinol cytochrome reductase will be studied by chemical and photoaffinity double labeling technique and by differential scanning calorimetry. The possible involvement of free Q in electron transfer and proton translocation will also be examined. The acceptance of the principle of the chemiosmotic energy coupling hypothesis has given Q a central role in bioenergetics. Success in the proposed research will increase our knowledge of electron transfer and proton translocation reactions. In addition, elucidation of the Q-binding site and Q:protein interaction will provide information crucial to understanding the mechanism of the generation of superoxide, and thus be useful in future investigations of cytotoxicity and aging.
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