Bacterial formate–nitrite transporters (FNTs) regulate the metabolic flow of small, weak mono‐acids. Recently, the eukaryotic PfFNT was identified as the malaria parasite's lactate transporter and novel drug target. Despite crystal data, central mechanisms of FNT gating and transport remained unclear. Here, we show elucidation of the FNT transport mechanism by single‐step substrate protonation involving an invariant lysine in the periplasmic vestibule. Opposing earlier gating hypotheses and electrophysiology reports, quantification of total uptake by radiolabeled substrate indicates a permanently open conformation of the bacterial formate transporter, FocA, irrespective of the pH. Site‐directed mutagenesis, heavy water effects, mathematical modeling, and simulations of solvation imply a general, proton motive force‐driven FNT transport mechanism: Electrostatic attraction of the acid anion into a hydrophobic vestibule decreases substrate acidity and facilitates protonation by the bulk solvent. We define substrate neutralization by proton transfer for transport via a hydrophobic transport path as a general theme of the Amt/Mep/Rh ammonium and formate–nitrite transporters.
Transport via formate–nitrite transporters is proposed to be regulated by pH‐dependent gating. Investigation of the mechanism employed by the bacterial formic acid transporter FocA uncovers a pH‐independent transport of a neutralized substrate, suggesting a common transport mode for ammonium and formate–nitrite transporters.
Radiolabeled substrate allows quantification of uptake via E. coli formate–nitrite transporter FocA.
Formate–nitrite transport is not gated but driven by proton‐motive force.
Electrostatic attraction channels substrate anions into the hydrophobic transporter vestibule.
Decreased substrate acidity allows protonation by bulk solvent, enabling transport of a neutral species.
The same mechanistic principle for substrate selectivity applies to formate–nitrite and ammonium transporters.
The EMBO Journal (2017) 36: 949–958
- Received September 22, 2016.
- Revision received January 25, 2017.
- Accepted January 30, 2017.
- © 2017 The Authors
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