A single amino acid exchange converts FocA into a unidirectional efflux channel for formate.

During mixed-acid fermentation, initially translocates formate out of the cell, but re-imports it at lower pH. This is performed by FocA, the archetype of the formate-nitrite transporter (FNT) family of pentameric anion channels. Each protomer of FocA has a hydrophobic pore through which formate/formic acid is bidirectionally translocated. It is not understood how the direction of formate/formic acid passage through FocA is controlled by pH. A conserved histidine residue (H209) is located within the translocation pore, suggesting that protonation/deprotonation might be linked to the direction of formate translocation. Using a formate-responsive -based reporter system we monitored changes in formate levels when H209 in FocA was exchanged for either of the non-protonatable amino acids asparagine or glutamine, which occur naturally in some FNTs. These FocA variants (with N or Q) functioned as highly efficient formate efflux channels and the bacteria could neither accumulate formate nor produce hydrogen gas. Therefore, the data in this study suggest that this central histidine residue within the FocA pore is required for pH-dependent formate uptake into cells. We also address why H209 is evolutionarily conserved and provide a physiological rationale for the natural occurrence of N/Q variants of FNT channels.