Failure of an alkalophilic bacterium to synthesize ATP in response to a valinomycin-induced potassium diffusion potential at high pH

Starved whole cells of the obligately alkalophilic Bacillus firmus RAB synthesize ATP upon addition of l-malate at pH 9.0, as expected of an aerobic organism that grows oxidatively on nonfermentable carbon sources at pH values as high as 11.0. The current study was a detailed examination of the perplexing inability of such cells to exhibit ATP synthesis in response to a valinomycin-mediated potassium diffusion potential at pH 9.0. While there were minor differences in the patterns of generation of the potential and the proton influx that accompanies its generation in the three different buffering systems employed, the magnitude of the transmembrane electrochemical potential of protons was at least as high at pH 9.0 as at pH 7.0. Nevertheless, a diffusion potential consistently energized ATP synthesis at pH 7.0 but not at 9.0; these findings were independent of the presence or absence of Tris or of Na⁺. By contrast, the artificial electron donor ascorbate, in the presence of phenazine metho-sulfate, energized ATP synthesis by the starved whole cells at both pH values.The same phenomenon, i.e., efficacy of a respiration-derived potential but not of a diffusion potential at pH 9.0, was demonstrated in ADP + P_i-loaded membrane vesicles. On the other hand, electrogenic Na⁺-coupled solute transport could be energized by both ascorbate/phenazine methosulfate and a diffusion potential in the vesicles at pH 9.0. The results are discussed in connection with models of a localized path of proton flow between proton pumps and the ATP synthase.