Abstract
Oxidative phosphorylation by extremely alkaliphilic Bacillus species violates two major predictions of the chemiosmotic hypothesis: the magnitude of the chemiosmotic driving force, the delta p (electrochemical proton gradient), is too low to account for the phosphorylation potentials observed during growth at pH 10.5 without using a much higher H+/ATP stoichiometry than used during growth at pH 7.5, and artificially imposed diffusion potentials fail to energize ATP synthesis above about pH 9.5 (Guffanti, A. A., and Krulwich, T. A. (1989) Annu. Rev. Microbiol. 43, 435-463). To further examine the latter observation, large valinomycin-mediated potassium diffusion potentials were imposed across starved cells of Bacillus firmus OF4 at various pH values from pH 7.5 to 10.5. As the external pH increased above pH 8, there was a sharp decrease in the rate of ATP synthesis in response to an imposed diffusion potential. The rate of ATP synthesis fell to zero by pH 9.2 and 9.4, respectively, in the presence and absence of a small inwardly directed Na+ gradient. Electrogenic Na+/H+ antiport and Na+/alpha-aminoisobutyric acid symport proceeded at substantial rates throughout. When synthesis was energized by an electron donor, cells under comparable conditions synthesized ATP at rapid rates up to pH 10.5. The proton transfers that occur during respiration-dependent oxidative phosphorylation at pH 10.5 may depend upon specific complexes. Cells grown at pH 7.5, which have one-third the levels of the caa3-type terminal oxidase, and slightly lower levels of certain other respiratory chain complexes than pH 10.5-grown cells, support only low rates of ATP synthesis at pH 10.5, although energy-dependent symport and antiport rates are comparable with those in pH 10.5-grown cells. A model is presented for oxidative phosphorylation by the alkaliphilic Bacillus that involves a nonchemiosmotic direct intramembrane transfer of protons from specific respiratory chain complexes to the F0 sector of the ATPase, whereas remaining respiratory chain complexes extrude protons into the bulk to generate the bulk potential required both for ATP synthesis and other bioenergetic work. A pK-regulated gate or a delocalized proton pathway that fails to work above pH 9.5 are suggested as possible features that account for the loss of efficacy of a bulk-imposed diffusion potential in energizing ATP synthesis above pH 9.4.
Highlights
From the Department of Biochemistry, Mount Sinai School of Medicine, City University of New York, New York, New York 10029
Cells grown at pH 7.5, which have one-third the levels of the caas-type terminaol xidase, and slightly lower levels of certain other respiratory chain complexes than pH 10.5-grown cells, support only low rates of ATP synthesis a t pH 10.5, should vary directly with the magnitude of the bulk Ap and that an artificially imposed Ap and respiration-generated Ap should function identically if the forces are of the same magnitude
Energization of Several Energy-dependent Iontr-anslocating Processes in B. firmus OF4 by a Potassium Diffusion Potential at Various p H Values-A valinomycin-mediated potassium diffusion potential of -176 mV was imposed across starved respiration-inhibited cells of B. firmus OF4 at a range of pH values, either in the absence (Fig. l a ) or presence (Fig. l b ) of a small inwardly directed Na+ gradient
Summary
Energization of Several Energy-dependent Iontr-anslocating Processes in B. firmus OF4 by a Potassium Diffusion Potential at Various p H Values-A valinomycin-mediated potassium diffusion potential of -176 mV was imposed across starved respiration-inhibited cells of B. firmus OF4 at a range of pH values, either in the absence (Fig. l a ) or presence (Fig. l b ) of a small inwardly directed Na+ gradient. Cells in which a diffusion potential was generated were concentrated in either 100 mM potassium phosphate, pH 7.5, or 100 mM potassium carbonate, pH 10.5 plus 10 PM valinomycin and 10 mM KCN. At pH 7.5 or 10.5cells were diluted 1:lOOO or 1:5000 into 100 mM sodium phosphate, pH 7.5, or 100 mM sodium carbonate, pH 10.5, respectively. Controls were dilution buffers containing 100 mM potassium phosphate or potassium carbonate. The A$ of growing cells was measured by washing and suspending cells (0.05 mgof protein/ml) growing at pH 7.5 or 10.5 in 100 mM sodium phosphate 7.5 or 100 mM sodium carbonate 10.5, respectively, plus 10 mM sodium malate. Uptake of 100 p M "Rb+ plus 1p~ valinomycin was assayed by filtration but binding controls were cells without valinomycin. S.D..and reuresent at least five indeDendent exueriments. each one done in dudicate
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