Hydrogenase 2 is most and hydrogenase 1 is less responsible for H2 production by Escherichia coli under glycerol fermentation at neutral and slightly alkaline pH

Escherichia coli possesses different membrane-bound hydrogenases under certain conditions: the two enzymes are hydrogenase 3 (Hyd-3) and hydrogenase 4 (Hyd-4) responsible for hydrogen gas (H2) production and the other two enzymes - hydrogenase 1 (Hyd-1) and hydrogenase 2 (Hyd-2) responsible for H2 uptake. H2 production by these bacteria grown under anaerobic conditions upon glycerol fermentation at neutral and slightly alkaline pH was studied under glycerol fermentation at pH 7.5 and was compared with that under glucose fermentation. The rate of H2 production under glycerol fermentation was determined in the wild type strain to be ∼2.5-times lower than that under glucose fermentation. This was ∼2-fold increase in the mutant with deletion of the fhlA gene, coding transcriptional activator for Hyd-3 and Hyd-4, upon glycerol fermentation. H2 production by these strains was inhibited by N,N′-dicyclohexylcarbodiimide and sodium azide, inhibitors of the F0F1-ATPase, suggesting a role of this ATPase. Moreover, H2 production by the fhlA mutant was blocked by osmotic downshock. Under glycerol fermentation H2 production rate was lowered significantly in the hyaB and hybC mutants with deleted large subunits of Hyd-1 and Hyd-2, respectively; however, it was ∼4-fold less in hyaB than in hybC mutants. In contrast to glucose fermentation, H2 production upon glycerol fermentation in the conditions used was, therefore, found to be mostly Hyd-2-dependent. Hyd-1 but not fhlA-determined Hyd-3 and/or Hyd-4 might be also involved in H2 production under the conditions above. Neither Hyd-2 nor Hyd-1 is responsible for H2 production under glucose fermentation. Hyd-2 is suggested to be a reversible enzyme working in a different manner under glycerol fermentation.