Conserved hydrophilic checkpoints tune FocA-1 mediated formate:H+ symport

FocA belongs to the widespread, evolutionarily ancient formate-nitrite transporter 30 (FNT) family of pentameric anion channels and translocates formic acid bidirectionally. 31 Here, we identify compartmentalized polarity distribution across the complete FocA 32 pore structure – resolved at 2.56 Å – mirrored against a two-fold axis with H209 at its 33 center. The FocA-H209N efflux-only variant reveals a density consistent with formic 34 acid located directly at N209, breaking local polarity distribution. Pyruvate formate-35 lyase, generating formate, orients at the cytoplasmic face where formate delivery is 36 regulated by conformational changes in the FocA vestibule. Comparisons with other 37 FNTs suggest a tuning mechanism of formate-specific transport via checkpoints 38 enriched in hydrophilic residues.