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

Abstract

Proton motive force (Δp) generation by Escherichia coli wild type cells during glycerol fermentation was first studied. Its two components, electrical-the membrane potential (∆φ) and chemical-the pH transmembrane gradient (ΔpH), were established and the effects of external pH (pHex) were determined. Intracellular pH was 7.0 and 6.0 and lower than pHex at pH 7.5 and 6.5, respectively; and it was higher than pHex at pH 5.5. At high pHex, the increase of ∆φ (-130 mV) was only partially compensated by a reversed ΔpH, resulting in a low Δp. At low pHex ∆φ and consequently Δp were decreased. The generation of Δp during glycerol fermentation was compared with glucose fermentation, and the difference in Δp 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 pHex; overall and N,N'-dicyclohexylcarbodiimide (DCCD)-inhibitory H(+) efflux was maximal at pH 6.5. Moreover, ΔpH was changed at pH 6.5 and Δp 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, Δp generation by E. coli during glycerol fermentation is different than that during glucose fermentation. Δp is dependent on pHex, and a role of Hyd enzymes in its generation is suggested.

Impact of membrane-associated hydrogenases on the FOF1-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: ATPase activity and its inhibition by N,N′-dicyclohexylcarbodiimide in the mutants lacking hydrogenases

“…indirectly due to some relation of Hyd-3 to Hyd-1, as suggested [36]. This seems to be likely to that Fdh-H is required to stabilize Hyd-3 but it is not essential for activity [38].…”

Hydrogen production from glycerol by Escherichia coli and other bacteria: An overview and perspectives

“…The intracellular pH and, hence, proton-motive force were lower at pH 7.5 compared with those during glucose fermentation [50]. Proton motive force generation is changed in E. coli hypF mutant lacking all Hyd enzymes [50] due to the effects on the F O F 1 -ATPase too [52].…”

“…It should be noted that DCCD inhibition was reversed during glucose fermentation [34,49]. Moreover, under glycerol fermentation Hyd-2 activity could disappear by protonophore action indicating the requirement of a proton-motive force [50,51].…”

“…H 2 production is inhibited completely by 0.5 mM N-ethylmaleimide (NEM) [50,51], an inhibitor of glycerol kinase [53], which catalyzes the formation of GP and then DHAP (see Fig. 2).…”

Hydrogen production by Escherichia coli during glucose fermentation: Effects of oxidative and reductive routes used by the strain lacking hydrogen oxidizing hydrogenases 1 (hya) and 2 (hyb)

“…The E. coliBW25113 (lacl q rrnB T14 DlacZ W116 hsdR514 DaraBAD AH33 Drha BAD LD78 ) wild type and MW1000 (BW25113DhyaB DhybC) mutant with defective Hyd-1 and Hyd-2 [29] were used. The mutant was provided by Prof. T. Wood (Pennsylvania State University, University Park, PA, USA).…”