Generation of an electrochemical proton gradient by nitrate respiration in membrane vesicles from anaerobically grown Escherichia coli.

Abstract

In membrane vesicles, isolated from Escherichia coli ML 308-225, grown anaerobically on glucose in the presence of nitrate, nitrate respiration results in the generation of a membrane potential, ΔΨ, as indicated by the accumulation of the lipophilic cation triphenylmethylphosphonium, and a transmembrane pH gradient, ΔpH, as indicated by the accumulation of the weak acid acetate in flow dialysis experiments. Under anaerobic conditions, and low concentrations of formate, the electrochemical proton gradient, ΔμH+, generated by nitrate respiration at pH 6.6 is at least–160 mV, consisting of a ΔΨ of about –90 mV and a ΔpH of about –75 mV. Under aerobic conditions, with ascorbatephenazine methosulphate as electron donor the ΔμH+ is almost –180 mV, consisting of a ΔΨ and a ΔpH both of –90 mV. The undissociated form of formate is membrane-permeable and external formate concentrations of about 10 mM dissipate the ΔpH. The ΔμH+ consists then almost solely of a ΔΨ of about –90 mV. Active transport of l-glutamate in these membrane vesicles is energized by both the membrane potential and the pH gradient as is demonstrated by the effects of the ionophores valinomycin and nigericin under aerobic and anaerobic conditions. The electrochemical proton gradient thus functions as the driving force for solute transport under anaerobic conditions in a similar way as was demonstrated for aerobic conditions in Escherichia coli [Ramos and Kaback (1977) Biochemistry, 16, 854–859].