The overall objectives of this program are: (a) to study the molecular mechanisms of electron transport, energy conservation and transfer, and ATP synthesis and hydrolysis in beef-heart mitochondria; (b) to purify and determine the composition, activities and lipid requirements of the protein components involved in electron transfer, energy transduction, and ATP synthesis; and (c) to identify structure-function relationships by stepwise resolution and reconstitution of the electron transport/oxidative phosphorylation system. Past studies have resulted in stepwise resolution of this system in to 5 enzyme complexes; characterization of each complex with respect to activities, inhibitor response properties, nature of electron carriers, and polypeptide composition; reconstitution of the respiratory chain; resolution and reconsitution of Complexes II and V; purification and study of composition and activities of NADH dehydrogenase, succinate dehydrogenase (SDH), cyt. b560, coupling factor B; discovery of mitochonrial uncoupler-binding site by photoaffinity labeling; study of mechanism of action of energy-linked nicotinamide nucleotide transhydrogenase (TH), Beta-hydroxybutyrate dehydrogenase, and SDH. Proposed in this application are: (a) Study of mechanism of H+ utilization for ATP synthesis and """"""""uphill"""""""" electron transfer. The hypothesis developed for the mechanism of TH, and to be tested for ATP synthesis, NTP-Pi exchange, and reverse electron transfer, involves H+-induced enzyme conformation change resulting in increased enzyme-substrate affinity and consequent facilitation of the reaction rate under study. Preliminary results have been highly encouraging. (b) Study of mechanism of proton translocation, and of what determines its directionality. (c) Continuation of ongoing work on SDH; cyt. b560; ATPase inhibitor protein; FeS centers of Complex I, NADH dehydrogenase and SDH; role and binding site of oligomycin sensitivity-conferring protein; resolution of membrane sector of Complex V (ATP synthetase complex), isolation of polypeptides and study of their role through reconstitution.
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