Role of E. coli hydrogenases in proton motive force generation during glycerol fermentation at pH 7.5

Abstract

E. coli is known well to process four membrane-bound [Ni-Fe] hydrogenases (Hyd), responsible enzymes of hydrogen metabolism [1]. Hyd-1 and Hyd-2 are reversible Hyd enzymes depending on carbon source (glucose and glycerol) and pH [2]. Hyd-3 and Hyd-4 are mainly H2 producing Hyd enzymes but they can work in reverse H2 uptake mode during glycerol fermentation at different pH [3]. Interestingly, Hyd-3 and Hyd-4 yd-4Hare suggested to have Htranslocating activity playing a role in generation of proton-motive force (PMF) [1]. In this study, it was shown that during glycerol fermentation at pH 7.5 E. coli wild type cell's membrane potential was -130 mV (determined by the distribution of tetraphenylphosphonium ion) and the cytoplasmic pH (pHin) (determined by the fluorescence quenching of 9-aminoacridine) was 7.0. This was in the contrast to glucose fermentation at the same pH when pHin was 7.5. The PMF value calculated during glycerol fermentation was, therefore, 99.4 mV which was lower than during bacterial cell growth on glucose. Interestingly, in E. coli hypFmutant (unable to synthesize all four Hyd enzymes) membrane potential was higher than that of the wild type and equal to -153 mV. But pHin was the same as in the wild type. The PMF generated in this mutant was 123 mV which was higher compared to the wild type. Moreover, N'N-dicyclohexylcarbodiimide (DCCD), which is non specific inhibitor of the F0F1-ATPase but becomes specific one under anaerobic conditions [2,3], does not affect on membrane potential (even in the higher concentration, 0.5 mM). Taken together these results suggest that E. coli Hyd enzymes have a vital role in forming the overall PMF besides the F0F1-ATPase. The different value of PMF in hypF mutant during glycerol fermentation might point out that the reversibility of Hyd enzymes and their different operation mode can be due to the changing of PMF. In addition, no effect of DCCD on membrane potential might be due to the interaction between Hyd enzymes and the F0F1-ATPase suggested.