HyfF subunit of hydrogenase 4 is crucial for regulating FOF1 dependent proton/potassium fluxes during fermentation of various concentrations of glucose.

Escherichia coli anaerobically ferment glucose and perform proton/potassium exchange at pH 7.5. The role of hyf (hydrogenase 4) subunits (HyfBDF) in sensing different concentrations of glucose (2 g L^-1 or 8 g L^-1) via regulating H⁺/K⁺ exchange was studied. HyfB, HyfD and HyfF part of a protein family of NADH-ubiquinone oxidoreductase ND2, ND4 and ND5 subunits is predicted to operate as proton pump. Specific growth rate was optimal in wild type and mutants grown on 2 g L^-1 glucose reaching ~ 0.8 h^-1. It was shown that in wild type cells proton but not potassium fluxes were stimulated ~ 1.7 fold reaching up to 1.95 mmol/min when cells were grown in the presence of 8 g L^-1 glucose. Interestingly, cells grown on peptone only had similar proton/potassium fluxes as grown on 2 g L^-1glucose. H⁺/K⁺ fluxes of the cells grown on 2 g L^-1 but not 8 g L^-1 glucose depend on externally added glucose concentration in the assays. DCCD-sensitive H⁺ fluxes were tripled and K⁺ fluxes doubled in wild type cells grown on 8 g L^-1 glucose compared to 2 g L^-1 when in the assays 2 g L^-1glucose was added. Interestingly, in hyfF mutant when cells were grown on 2 g L^-1glucose and in 2 g L^-1 assays DCCD-sensitive fluxes were not determined compared to wild type while in hyfD mutant it was doubled reaching up to 0.657 mmol/min. In hyf mutants DCCD-sensitive K⁺ fluxes were stimulated in hyfD and hyfF mutants compared to wild type but depend on external glucose concentration. DCCD-sensitive H⁺/K⁺ ratio was equal to ~ 2 except hyfF mutant grown and assayed on 2 g L^-1glucose while in 8 g L^-1 conditions role of hyfB and hyfD is considered. Taken together it can be concluded that Hyd-4 subunits (HyfBDF) play key role in sensing glucose concentration for regulation of DCCD-sensitive H⁺/K⁺ fluxes for maintaining proton motive force generation.