The roles of hydrogenases 3 and 4, and the F₀F₁-ATPase, in H₂ production by Escherichia coli at alkaline and acidic pH

The hyc operon of Escherichia coli encodes the H₂-evolving hydrogenase 3 (Hyd-3) complex that, in conjunction with formate dehydrogenase H (Fdh-H), constitutes a membrane-associated formate hydrogenlyase (FHL) catalyzing the disproportionation of formate to CO₂ and H₂ during fermentative growth at low pH. Recently, an operon (hyf) encoding a potential second H₂-evolving hydrogenase (Hyd-4) was identified in E. coli. In this study the roles of the hyc- and hyf-encoded systems in formate-dependent H₂ production and Fdh-H activity have been investigated. In cells grown on glucose under fermentative conditions at slightly acidic pH the production of H₂ was mostly Hyd-3- and Fdh-H-dependent, and Fdh-H activity was also mainly Hyd-3-dependent. However, at slightly alkaline pH, H₂ production was found to be largely Hyd-4, Fdh-H and F₀F₁-ATPase-dependent, and Fdh-H activity was partially dependent on Hyd-4 and F₀F₁-ATPase. These results suggest that, at slightly alkaline pH, H₂ production and Fdh-H activity are dependent on both the F₀F₁-ATPase and a novel FHL, designated FHL-2, which is composed of Hyd-4 and Fdh-H, and is driven by a proton gradient established by the F₀F₁-ATPase.