The Inlet, Outlet, and New Ratchet Element for Proton Transfer of the Acinetobacter baumannii F-ATP Synthase and Their Critical Role for Viability

The F1FO-ATP synthase is essential to the aerobe Acinetobacter baumannii. Its FO-domain utilizes the proton motive force to rotate the turbine (c10-ring) inside the stator (a subunit), which generates a torque that is translated to the catalytic F1-domain for adenosine 5'-triphosphate (ATP) synthesis. Here, we investigated key features of the FO-domain, including the proton intake channel, proton donor and acceptor residues, an A. baumannii unique subunit a helix, and the proton exit pathway. By employing a heterologous system, we generated mutants and studied their growth kinetic properties in minimal media, as well as the ATP synthesis activity of their inverted-membrane vesicles. The findings highlight the front entry as the main proton uptake pathway and the key residues involved in proton translocation. Molecular dynamics (MD) simulations confirm the role of these charged residues, which interact with water molecules to facilitate a water-mediated proton transfer in a Grotthuss-like mechanism. Similarly, the exit channel with R224 of subunit a playing a central role is described. Importantly, the sequential flow of proton intake, turbine rotation, and proton release are modulated by the unique a subunit helix, which functions like a molecular ratchet to facilitate effective proton transfer for the final formation of ATP. The importance in function, difference in amino acid content, and uniqueness in regulation by its specific molecular ratchet make the A. baumannii proton pathway an attractive inhibitor target, where a cork-like molecule could prevent proton intake and/or release with the consequence of ATP synthesis and cell growth inhibition.