Relationship between formate hydrogen lyase and proton-potassium pump under heterolactic fermentation in Escherichia coli: functional multienzyme associations in the cell membrane.

Anaerobically grown glucose-fermenting E. coli cells produce molecular hydrogen, acidify the medium and uptake potassium ions. It was shown that the H2 release and the proton-potassium exchange with the fixed (2H+/K+) stoichiometry of the initial DCC-sensitive fluxes were lost in mutants with the deleted fdhF gene or the hycA-H operon responsible for the biosynthesis of formate dehydrogenase H (FDH,H) or hydrogenase 3 (H3), respectively, which are the main components of the formate hydrogen lyase FHL(H). However, both processes occurred in mutants with the deleted hycE, hycF or hycG genes encoding the major and minor components of H3, respectively. The K+ uptake was sensitive to the osmotic shock resulting from glucose addition to the medium and decreased significantly in the presence of valinomycin. The H2 release and the 2H+/K+ exchange were absent in the mutant with the deleted hycB gene encoding the corresponding minor component of H3. This mutant acidified the medium and uptook K+ with Km typical for TrkA, but the stoichiometry of the DCC-inhibited fluxes was variable, and the K+ gradient between the cytoplasm and the medium in this mutant was lower than in the mutants lacking other minor components of H3. The results obtained suggest that the hycB gene product, FdhF and HycE, form probably the FHL(H) complex that directly interacts with the H+-ATPase complex F0F1 and the TrkA(H) system of K+ uptake. Such a multienzyme association is responsible for the H2 production and 2H+/K+ exchange. The major and other minor components of H3 have probably no direct role in the H2 production and 2H+/K+ exchange. H2 production by precursor's or hycE mutant's protoplasts treated with toluene was shown to occur upon addition of the thiol reagent dithiothreitol to the medium containing ATP, potassium ions, NAD+, and NADH. H2 production was inhibited by DCC. The quantity of available thiol groups in membrane vesicles of the precursor or the hycE, hycF or hycG mutants, in which the H2 production and 2H+/K+ exchange were observed, was larger than in other mutants. The number of SH groups decreased in the presence of DCC. These results indicate a significance of the thiol groups for the function of the proposed association.