Escherichia coli uptake potassium ions with the coupling of proton efflux and energy utilization via proton FOF1-ATPase. In this study contribution of formate hydrogen lyase (FHL) complexes in the proton/potassium fluxes and the formation of proton conductance (CMH+) were investigated using fhlA mutant strain. The proton flux rate (JH+) decreased in fhlA by ∼ 25% and ∼ 70% during the utilization of glucose and glycerol, respectively, at 20 h suggesting H+ transport via or through FHL complexes. The decrease in JK+ in fhlA by ∼ 40% proposed the interaction between FHL and Trk secondary transport system during mixed carbon fermentation. Moreover, the usage of N,N’-dicyclohexylcarbodiimide (DCCD) demonstrated the mediation of FOF1-ATPase in this interaction. CMH+ was 13.4 nmol min−1 mV-1 in WT at 20 h, which decreased by 20% in fhlA. Taken together, FHL complexes have a significant contribution to the modulation of H+/ K+ fluxes and the CMH+ for efficient energy transduction and regulation of the proton motive force during mixed carbon sources fermentation.