Hydrogenase activity and proton-motive force generation by Escherichia coli during glycerol fermentation

Proton motive force (Delta p) generation by Escherichia coli wild type cells during glycerol fermentation was first studied. Its two components, electrical-the membrane potential (a dagger phi) and chemical-the pH transmembrane gradient (Delta pH), were established and the effects of external pH (pH(ex)) were determined. Intracellular pH was 7.0 and 6.0 and lower than pH(ex) at pH 7.5 and 6.5, respectively; and it was higher than pH(ex) at pH 5.5. At high pH(ex), the increase of a dagger phi (-130 mV) was only partially compensated by a reversed Delta pH, resulting in a low Delta p. At low pH(ex) a dagger phi and consequently Delta p were decreased. The generation of Delta p during glycerol fermentation was compared with glucose fermentation, and the difference in Delta p might be due to distinguished mechanisms for H+ transport through the membrane, especially to hydrogenase (Hyd) enzymes besides the F0F1-ATPase. H+ efflux was determined to depend on pH(ex); overall and N,N'-dicyclohexylcarbodiimide (DCCD)-inhibitory H+ efflux was maximal at pH 6.5. Moreover, Delta pH was changed at pH 6.5 and Delta p was different at pH 6.5 and 5.5 with the hypF mutant lacking all Hyd enzymes. DCCD-inhibited ATPase activity of membrane vesicles was maximal at pH 7.5 and decreased with the hypF mutant. Thus, Delta p generation by E. coli during glycerol fermentation is different than that during glucose fermentation. Delta p is dependent on pH(ex), and a role of Hyd enzymes in its generation is suggested.