This project proposes new approaches to an old problem, namely how adenosine triphosphate (ATP) is made using energy from oxidations and light. ATP serves to drive many biological processes, and an active human turns over about a body weight of ATP each day. How this is accomplished is basic to an understanding of how optimum health can be maintained. One principal objective will be to make critical tests of a proposed mechanism for ATP synthesis. We will use heart and liver mitochondria, chloropast thylakoids, and E. coli membranes to explore the following suggested features of this binding change mechanism: conformational changes driven by the electrochemical potential of the coupling membrane serve to promote competent binding of Pi and ADP and the release of ATP from catalytic sites of the ATP synthase; three catalytic sites on the multisubunit synthase participate in coordinated alternation; and during ATP synthesis there is a rotational change in position of catalytic subunits relative to noncatalytic core subunits occurs. A second major objective will be to explore how the energy-requiring conformational changes are driven: indirectly by the electrochemical potential or directly by oxidation-reduction enzymes. A new concept for energy transmission to the synthase from oxidation-reduction enzymes is presented for experimental testing. Procedures proposed include selected kinetic evaluations; measurements of amounts and properties of bound intermediates; measurement of phosphate-oxygen exchanges and of the 18O-species formed; application of present and the development of new specific cross-linking agents; chemical derivatization with various agents and inhibitors; effects of catalysis on the chemical reactivity and on the physical properties of bound agents; energy transfer measurements between intrinsic and added probes; the effects of selected mutants of the E. coli enzyme on catalytic parameters; the role of subunits as probed by deletion and recombination experiments; and the possible crystallization of the catalytic Beta-subunit for X-ray studies. Plans also call for assembly and publication of the methodology for 18O analysis in phosphates that we have accummulated over the past several years.
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