Glucose concentration is determinant for the functioning of hydrogenase 1 and hydrogenase 2 in regulating the proton and potassium fluxes in Escherichia coli at pH 7.5.

This study examines how F_OF₁-ATPase, hydrogenases (Hyd-1 and Hyd-2), and potassium transport systems (TrkA) interact to maintain the proton motive force (pmf) in E. coli during fermentation of different glucose concentrations (2 g L^-1 and 8 g L^-1). Our findings indicate that mutants lacking the hyaA-hyaC genes exhibited a 30 % increase in total proton flux compared to the wild type when grown with 2 g L^-1 glucose. This has been observed during assays where similar glucose levels were supplemented. Disruptions in proton pumping, particularly in hyaB and hyaC single mutants, led to increased potassium uptake. The hyaB mutant showed a threefold increase in the contribution of F_OF₁-ATPase to proton flux, suggesting a significant role for Hyd-1 in proton translocation. In the hybC mutant grown in 2 g L^-1 glucose conditions, DCCD-sensitive fluxes decreased by 70 %, indicating critical role of Hyd-2 in proton transport and F_OF₁ function. When cells were grown with 8 g L^-1 glucose, the 2H⁺/1K⁺ ratio was significantly disturbed in both wild type and mutants. Despite these perturbances, mutants with disruptions in Hyd-1 and Hyd-2 maintained constant F_OF₁ function, suggesting that this enzyme remains stable in glucose-rich environments. These results provide valuable insights into how Hyd-1 and Hyd-2 contribute to the regulation of ion transport, particularly proton translocation, in response to glucose concentration. Our study uncovered potential complementary mechanisms between Hyd-1 and Hyd-2 subunits, suggesting a complex interplay between these enzymes via metabolic cross talk with F_OF₁ in response to glucose concentrations to maintain pmf.