Grba, D. N. | Search bioRxiv

bioRxiv2026

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

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.

biochemistry↗

bioRxiv2023

Structural basis of a regulatory switch in mammalian complex I

Respiratory complex I powers oxidative phosphorylation in mammalian mitochondria by using the reducing potential of NADH to reduce ubiquinone-10 and drive protons across the inner mitochondrial membrane. High-resolution cryoEM structures have provided a molecular framework for complex I catalysis, but controversies about how to assign functional properties to the states identified in single-particle analyses are preventing progress on its energy-converting mechanism. Here, we combine precise biochemical definition with high-resolution cryoEM structures in the phospholipid bilayer of coupled vesicles and show that the closed and open states observed in mammalian complex I preparations are components of the deactive transition that occurs during ischaemia. Populations of the cryoEM open state and biochemical deactive state match exactly. Deactivation switches the enzyme off, converting the closed state that is capable of rapid, reversible catalysis into an open, dormant state that is unable to start up in reverse. The deactive state is switched back on by slow priming reactions with NADH and ubiquinone-10. Thus, by developing a versatile membrane system to unite structure and function, we define the role of large-scale conformational transitions in complex I and establish a new gold standard for structure-based investigations of catalysis by energy-coupled proteins.

biochemistry↗