Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase

The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton-motive force to drive hydride transfer from NADH to NADP⁺ in bacteria and eukaryotes. Here we solved a 2.2-Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular dynamics simulations, we show that transient water flows across a narrow pore and a hydrophobic "dry" region in the middle of the membrane channel, with key residues His42^α2 (chain A) being protonated and Thr214^β (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214^β to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.