Complex I is a redox-driven proton pump that drives electron transport chains and powers oxidative phosphorylation across all domains of life. Yet, despite recently resolved structures from multiple organisms, it still remains unclear how the redox reactions in Complex I trigger proton pumping up to 200 Å away from the active site. Here, we show that the proton-coupled electron transfer reactions during quinone reduction drive long-range conformational changes of conserved loops and trans-membrane (TM) helices in the membrane domain of Complex I from Yarrowia lipolytica. We find that the conformational switching triggers a π → α transition in a TM helix (TM3^ND6) and establishes a proton pathway between the quinone chamber and the antiporter-like subunits, responsible for proton pumping. Our large-scale (>20 μs) atomistic molecular dynamics (MD) simulations in combination with quantum/classical (QM/MM) free energy calculations show that the helix transition controls the barrier for proton transfer reactions by wetting transitions and electrostatic effects. The conformational switching is enabled by re-arrangements of ion pairs that propagate from the quinone binding site to the membrane domain via an extended network of conserved residues. We find that these redox-driven changes create a conserved coupling network within the Complex I superfamily, with point mutations leading to drastic activity changes and mitochondrial disorders. On a general level, our findings illustrate how catalysis controls large-scale protein conformational changes and enables ion transport across biological membranes. 

The respiratory Complex I is a highly intricate redox-driven proton pump that powers oxidative phosphorylation across all domains of life. Yet, despite major efforts in recent decades, its long-range energy transduction principles remain highly debated. We create here minimal proton-conducting membrane modules by engineering and dissecting the key elements of the bacterial Complex I. By combining biophysical, biochemical, and computational experiments, we show that the isolated antiporter-like modules of Complex I comprise all functional elements required for conducting protons across proteoliposome membranes. We find that the rate of proton conduction is controlled by conformational changes of buried ion-pairs that modulate the reaction barriers by electric field effects. The proton conduction is also modulated by bulky residues along the proton channels that are key for establishing a tightly coupled proton pumping machinery in Complex I. Our findings provide direct experimental evidence that the individual antiporter modules are responsible for the proton transport activity of Complex I. On a general level, our findings highlight electrostatic and conformational coupling mechanisms in the modular energy-transduction machinery of Complex I with distinct similarities to other enzymes.

The alternative oxidase (AOX) is a membrane-bound di-iron enzyme that catalyzes O₂-driven quinol oxidation in the respiratory chains of plants, fungi, and several pathogenic protists of biomedical and industrial interest. Yet, despite significant biochemical and structural efforts over the last decades, the catalytic principles of AOX remain poorly understood. We develop here multi-scale quantum and classical molecular simulations in combination with biochemical experiments to address the proton-coupled electron transfer (PCET) reactions responsible for catalysis in AOX from Trypanosoma brucei, the causative agent of sleeping sickness. We show that AOX activates and splits dioxygen via a water-mediated PCET reaction, resulting in a high-valent ferryl/ferric species and tyrosyl radical (Tyr220˙) that drives the oxidation of the quinol via electric field effects. We identify conserved carboxylates (Glu215, Asp100) within a buried cluster of ion-pairs that act as a transient proton-loading site in the quinol oxidation process, and validate their function experimentally with point mutations that result in drastic activity reduction and pK_a-shifts. Our findings provide a key mechanistic understanding of the catalytic machinery of AOX, as well as a molecular basis for rational drug design against energy transduction chains of parasites. On a general level, our findings illustrate how redox-triggered conformational changes in ion-paired networks control the catalysis via electric field effects.

Mitochondrial membranes harbor the electron transport chain (ETC) that powers oxidative phosphorylation (OXPHOS) and drives the synthesis of ATP. Yet, under physiological conditions, the OXPHOS proteins can operate as higher-order supercomplex (SC) assemblies, although their functional role remains poorly understood and much debated. By combining large-scale atomistic and coarse-grained molecular simulations with analysis of cryo-electron microscopic data and statistical as well as kinetic models, we show here that the formation of the mammalian I/III₂ supercomplex reduces the molecular strain of inner mitochondrial membranes by altering the local membrane thickness and leads to an accumulation of both cardiolipin and quinone around specific regions of the SC. We find that the SC assembly also affects the global motion of the individual ETC proteins with possible functional consequences. On a general level, our findings suggest that molecular crowding and entropic effects provide a thermodynamic driving force for the SC formation, with a possible flux enhancement in crowded biological membranes under constrained respiratory conditions.

The Rnf complex is the primary respiratory enzyme of several anaerobic prokaryotes that transfers electrons from ferredoxin to NAD⁺ and pumps ions (Na⁺ or H⁺) across a membrane, powering ATP synthesis. Rnf is widespread in primordial organisms and the evolutionary predecessor of the Na⁺-pumping NADH-quinone oxidoreductase (Nqr). By running in reverse, Rnf uses the electrochemical ion gradient to drive ferredoxin reduction with NADH, providing low potential electrons for nitrogenases and CO₂ reductases. Yet, the molecular principles that couple the long-range electron transfer to Na⁺ translocation remain elusive. Here, we resolve key functional states along the electron transfer pathway in the Na⁺-pumping Rnf complex from Acetobacterium woodii using redox-controlled cryo-electron microscopy that, in combination with biochemical functional assays and atomistic molecular simulations, provide key insight into the redox-driven Na⁺ pumping mechanism. We show that the reduction of the unique membrane-embedded [2Fe2S] cluster electrostatically attracts Na⁺, and in turn, triggers an inward/outward transition with alternating membrane access driving the Na⁺ pump and the reduction of NAD⁺. Our study unveils an ancient mechanism for redox-driven ion pumping, and provides key understanding of the fundamental principles governing energy conversion in biological systems.