Formate hydrogenlyase is essential for pH homeostasis, maintenance of ATP levels, and CO 2 provision in stationary-phase Escherichia coli

ABSTRACT Formate hydrogenlyase (FHL) enzymes are evolutionarily related to mitochondrial complex I. Formate-inducible FHL-1 of Escherichia coli catalyzes disproportionation of formic acid into H 2 and CO 2 during mixed-acid fermentation. In contrast to FHL-2 enzymes found in many microorganisms, including E. coli , FHL-1 shares only two of the usual five membrane-domain subunits, perhaps explaining its apparent inability to translocate protons. This raises the question, however, as to the physiological function of the FHL-1 enzyme. Here, we demonstrate that FHL-1 serves to regulate intracellular pH and CO 2 levels in stationary-phase fermenting cells. Determination of intracellular and extracellular pH values in E. coli strains with and without an FHL-1 enzyme revealed an alkalinization (0.5 pH units) of the cytoplasm in cells lacking FHL-1. This increase in pH i correlated with a concomitant threefold decrease in the ATP concentration in these cells, while the parental strain retained both high ATP levels and a pH i of 6.7. The reduction in ATP levels in the mutant correlated with an increase in F 1 F o -ATPase activity. Remarkably, ATP levels and pH i in the FHL-1-negative mutant could be restored to near wild-type levels by growth in the presence of bicarbonate. Together, these data indicate that FHL-1 functions to provide CO 2 /bicarbonate for key carboxylation reactions, concomitantly maintaining intracellular pH homeostasis and thereby indirectly optimizing ATP levels by preventing excessive ATP hydrolysis via F 1 F o -ATPase in its attempt to neutralize pH i . CO 2 production by cytoplasmically oriented FHL-1 substitutes for a pyruvate decarboxylation reaction, and explains why maintaining formate homeostasis is central to mixed-acid fermentation. IMPORTANCE The signature molecule of enterobacterial mixed-acid fermentation, formate, is disproportionated into H 2 and CO 2 by formate hydrogenlyase (FHL), an evolutionarily ancient enzyme phylogenetically related to proton-translocating complex I. However, as FHL-1 of Escherichia coli lacks the capacity to translocate protons, its physiological role remains unclear. We show that FHL-1 functions to maintain pH homeostasis and intracellular CO 2 /bicarbonate levels in stationary-phase cells. Mutants lacking a functional FHL-1 fail to generate H 2 and CO 2 , causing activation of F 1 F o -ATPase, which hydrolyzes ATP to maintain neutral cytoplasmic pH. The balance between formate synthesis and its intracellular disproportionation is necessary to maintain the supply of bicarbonate for carboxylation reactions, thus ensuring pH homeostasis and ultimately optimal ATP levels, which together aid stationary-phase survival.