Oxidative phosphorylation in protonophore-resistant mutants of Bacillus subtilis and in extremely alkalophilic Bacillus species yields higher concentrations of ATP than can be accounted for, given the magnitude of the bulk electrochemical proton gradient, by Mitchell's chemosmotic hypothesis that posits that oxidative phosphorylation is directly, completely, and obligatorily coupled to electrochemical proton gradient. Our working hypothesis for these two model systems, in which oxidative phosphorylation depends upon a proton-translocating ATPase, is that synthesis of ATP at submaximal (in protonophore-resistant mutants) or very low (in the alkalophiles) values of electrochemical proton gradient is facilitated by direct contact between the proton pumping respiratory chain components and the ATPase so that the protons can be """"""""handed off"""""""" directly between residues thereof. We hypothesize that chance collision brings about this kind of """"""""localized"""""""" proton movement and that increased frequency of those collisions occurs in our systems by evolutionary adaptations that increase the concentrations of pumps and their diffusion rates (alkalophiles) or mutational changes that allow domains of liquid crystalline membrane to form with increased concentrations of pumps and ATPase (protonophore- resistant mutants). Another necessary feature in both systems is a high resistance of the bulk proton pathway so that protons do not leak out instead of moving inward through the ATPase at low electrochemical proton gradient. We will directly test this hypothesis by: fusion experiments between ATP-synthesizing vesicles and liposomes in which the spaces between pumps and ATPase are increased; modeling the systems in proteoliposomes in which localized coupling effects can be correlated with enzyme concentration and proximity, and with the physical state of the membrane; and direct measurements of the resistance of the bulk proton pathway and the basis for such resistance. In the alkalophile, a critical pK may lead to a rather abrupt diminution of proton movement to and from the bulk at around pH 9.0-9.2; in the protonophore-resistant strains, the membrane lipid changes that lead to the phenotype may affect the proton movement from the bulk through the F o. These possibilities will be directly examined. In both systems, we will also study: the concentration of F1Fo- ATPase and its possible regulation, decoupling and uncoupling agents, and energy-coupling mutants, in order to further define the elements involved in oxidative phosphorylation at low electrochemical proton gradient.
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