Conformational gating at histidine junctions coordinates proton translocation in respiratory complex I

<jats:p>Respiratory complex I, a central enzyme in cellular metabolism, converts the free energy of NADH oxidation into a transmembrane proton-motive force to drive ATP synthesis, but the molecular mechanisms by which it couples redox catalysis to vectorial proton translocation remain unresolved. Here, we present high-resolution cryo-EM structures of complex I from Bos taurus captured under conditions designed to change the protonation states of residues in the membrane domain. Our structures reveal conformational rearrangements at key pathway junctions that reconfigure proton-transfer connections. In ND5, helical rearrangements switch the connectivity of histidine-248 between proton-uptake and proton-output pathways. In ND4, rotameric changes of histidine-220 alternately enable proton uptake or lateral proton transfer along the membrane domain. Combined with molecular simulations, our structures define gating mechanisms that impose directionality on proton transfer reactions and provide a framework for proton-coupled energy transduction in complex I.</jats:p>