This study investigates the roles of specific subunits in Hyd-1 and Hyd-2 and their impact on proton and potassium fluxes during fermentation of glucose in Escherichia coli (E. coli). By examining conditions of varying glucose availability, we aimed to uncover how these subunits influence the metabolic adaptability of E. coli under fermentative conditions by altering ion fluxes, indicative of their roles in hydrogenase activity and cellular energy metabolism. Notably, hyaD and hyaF mutants exhibited a 50% increase in DCCD (N, N'-dicyclohexylcarbodiimide)-sensitive proton flux, underlining their critical roles in Hyd-1 activity. Exclusively, the hybF mutant demonstrated an increase in DCCD-sensitive flux, suggesting cross-regulation between hydrogenases, particularly underscoring the role of HybF in Hyd-1 activity, resembling the observations with hyaB data. Thus, in the presence of low glucose, HybF is presumably less involved in the maturation of Hyd-2. The data for hybE and hybF strongly imply that under conditions of low glucose growth supplemented with high glucose are crucial for Hyd-2 activity. HyaD and hyaF mutants exhibited a 50% increase in DCCD-sensitive proton flux, indicating their essential roles in Hyd-1 activity. Taken together, our results suggest that depending on glucose concentration specific subunits may have altered and cross-regulated roles in maturation of Hyd enzymes.
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