A FocA variant incapable of formate import but retaining formic acid efflux highlights the distinct mechanisms governing bidirectional formate translocation.

Abstract

FocA belongs to the formate-nitrite transporter (FNT) superfamily of pentameric membrane proteins, which translocate small, monovalent anions across the cytoplasmic membrane of bacteria, archaea and certain protists. FocA translocates formate anions or formic acid bidirectionally through a hydrophobic pore present in each protomer. This pore has two highly conserved amino acid residues, threonine 91 and histidine 209 that are proposed to protonate the anion during the translocation process. Current evidence suggests that different mechanisms control efflux and influx of formate. Determination of changes in extracellular and intracellular formate levels were used to characterize new amino acid variants of FocA in which H209 was exchanged for cysteine or serine. While the FocAH209S mutant excreted formic acid very efficiently, the mutant synthesizing FocAH209C translocated formic acid out of the cell poorly. These different efflux efficiencies of formic acid through FocA clearly suggest that the reactivity of the sulfur atom in cysteine accounts for the inefficient translocation of formic acid by the FocAH209C variant. Mutants synthesizing the FocAH209S or FocAH209C variants were incapable to importing formate, or its toxic chemical analogue hypophosphite, a phenotype similar to previously identified H209-exchange variants. Notably, a mutant lacking a functional formate hydrogenlyase (FHL-1) complex, which under physiological conditions disproportionates formate to H2 and CO2, retained sensitivity to hypophosphite, but accumulated formate externally. Our findings indicate that, while coupling between FocA and FHL-1 controls formate import, the import of hypophosphite is not dependent on FHL-1. Further, our data support a model in which two mechanisms for formate import exist, depending on the external formate concentration: at low concentration, protonation of formate or hypophosphite by H209 facilitates anion translocation; at high concentration, formic acid is directed to FHL-1 where it is disproportionated to H2 and CO2.