This project will clarify the mechanisms used by alkaliphilic Bacillus for the two processes that move protons inward at high external pH: how extremely robust proton-coupled oxidative phosphorylation (OXPHOS) occurs under conditions in which the chemiosmotic driving force (negative and alkaline in) is very low; and how the novel properties of the Mrp sodium/proton antiporter underpin its capacity to support remarkable alkaline pH homeostasis, resulting in a """"""""reversed pH gradient"""""""" at external pH values such that the cytoplasmic pH is more than 2 units below an optimal external pH of 10.5.
Three specific aims will focus on the following questions, (i) How do specific features of the membrane-embedded a- and c-subunits of ATP synthase make distinct critical contributions to proton capture and movement through the ATP synthase? The approaches will include analyses of site-directed mutants that alter properties of critical ATP synthase residues in the proton path and studies of the c-subunit stoichiometry of the synthase. (ii) What is the special involvement of a particular respiratory chain complex, the Cta cytochrome oxidase, in partnering with the ATP synthase at high pH for sequestered proton transfers to the synthase? Protein-protein interactions between Cta and ATP synthase as well as specific conditions and features of the two complexes involved in such interactions will be the focus, (iii) What are the roles of the seven Mrp proteins and the properties of the Mrp antiporter that account for its unusual complexity and efficacy? A multi-pronged biochemical, genetic and physiological approach will test the hypotheses that: (a) this secondary sodium/proton antiporter functions as a novel, hetero-oligomeric complex; and (b) some Mrp proteins have distinct catalytic activities that have positive synergy with Mrp-dependent antiport. Two areas are studied bacteria that thrive in alkali. They are a model system for unraveling details of chemical energy production (as ATP) by oxygen-dependent cells. These bacteria are hypothesized to share strategies with mitochondria so the findings on ATP synthesis have pharmacalogical implications for normal and pathological states of cellular respiration. The studies of the Mrp system characterize a novel protein carrier found in many bacterial pathogens. Mrp is a potential target for development of new antibiotics.
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