Hydrogenase 2 is most and hydrogenase 1 is less responsible for H_2 production by Escherichia coli under glycerol fermentation at neutral and slightly alkaline pH

摘要

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 (H_2) production and the other two enzymes - hydrogenase 1 (Hyd-1) and hydrogenase 2 (Hyd-2) responsible for H_2 uptake. H_2 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 H_2 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 tran-scriptional activator for Hyd-3 and Hyd-4, upon glycerol fermentation. H_2 production by these strains was inhibited by N,N'-dicyclohexylcarbodiimide and sodium azide, inhibitors of the F_0F_1-ATPase, suggesting a role of this ATPase. Moreover, H_2 production by the fhlA mutant was blocked by osmotic downshock. Under glycerol fermentation H_2 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, H_2 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 H_2 production under the conditions above. Neither Hyd-2 nor Hyd-1 is responsible for H_2 production under glucose fermentation. Hyd-2 is suggested to be a reversible enzyme working in a different manner under glycerol fermentation.