Oxidative phosphorylation by membrane vesicles from Bacillus alcalophilus

ADP and P_i-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with ascorbate N,N,N′,N′-tetramethyl-p-phenylenediamine. ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force Δ \ ̄gm_H⁺ was as low as -30 mV. The phosphate potentials (ΔG_p) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the Δ \ ̄gm_H⁺ values in vesicles at these two pH values were quite different (-40 ± 20 mV at pH 10.5 and -125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na⁺ or K⁺ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low Δ \ ̄gm_H⁺.