ATP synthesis by the proton-translocating F1F0 ATPase can be energized by an imposed, bulk, electrochemical gradient of protons (DM-H+) or even an external electric field. Nonetheless, a variety of experimental findings have led some investigators to propose that a more localized form of energy is actually the direct intermediate between natural proton pumps and the F1F0 ATPase that catalyzes ATP synthesis. This hypothesis, which underlies the current proposal, is consistent with data collected thus far on ATP synthesis by uncoupler-resistant mutant strains of Bacillus megaterium and Bacillus subtilis and by obligately alkalophilic bacilli. These organisms synthesize ATP via proton-translocating ATPases at apparently low Dm-H+ values, and show a """"""""preference"""""""" for a Dm-H+ generated by natural pumps to an artificially generated DmH+ of the same magnitude. In the proposed studies, the mechanisms of energy-coupling to ATP synthesis in these two systems will be further explored. In uncoupler-resistant strains of B. subtilis studies will include: the relationship between the phosphorylation potential DGp and the magnitude of the Dm-H+ as a function of the mode of energization; relative sensitivity of ATP synthesis by the uncoupler-resistant and wild type strains to uncouplers and various energy transfer inhibitors; structural studies of the F1F0 ATPase designed to elucidate the structural changes that affect energy-coupling; and cloning of the gene for uncoupler-resistance so that, among other things, the gene product can be characterized in a minicell system. In the alkalophiles studies will include: comparative determinations of Dm-H+, DGp, and Dm solutes (Na+ vs H+ coupled) as a function of the mode of energization; isolation of mutants which have lost the special properties of energy-coupling and characterization of the gene products; and mixed reconstitution experiments in which gene products that may be related to energy-coupling can be examined. The results of these experiments may or may not support the hypothesis of a localized gradient, but they should clarify the actual pathway of protons that naturally couples bioenergetic work to proton pumping events. The genetic and structural analyses should further identify protein(s) and structural features that are involved in these bioenergetic processes.
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