Respiratory complex I is a central enzyme of cellular energy metabolism that couples electron transfer with proton translocation across a biological membrane. In doing so, it powers oxidative phosphorylation that drives energy consuming processes. Mutations in complex I lead to severe neurodegenerative diseases in humans. However, the biochemical consequences of these mutations remain largely unknown. Here, we use the Escherichia coli complex I as a model to biochemically characterize the F124LMT-ND5 mutation found in patients suffering from Leigh syndrome. We show that the mutation drastically perturbs proton translocation and electron transfer activities to the same extent, despite the remarkable 140 Å distance between the mutated position and the electron transfer domain. Our molecular dynamics simulations suggest that the disease-causing mutation induces conformational changes that hamper the propagation of an electric wave through an ion-paired network essential for proton translocation. Our findings imply that malfunction of the proton translocation domain is entirely transmitted to the electron transfer domain underlining the action-at-a-distance coupling in the proton-coupled electron transfer of respiratory complex I.

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.

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.

Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO₂, but the molecular mechanisms have remained enigmatic. The key enzyme responsible for powering these thermodynamically challenging reactions is the electron-bifurcating [FeFe]-hydrogenase HydABC that reduces low-potential ferredoxins (Fd) by oxidizing hydrogen gas (H₂). By combining single-particle cryo-electron microscopy (cryoEM) under catalytic turnover conditions with site-directed mutagenesis experiments, functional studies, infrared spectroscopy, and molecular simulations, we show that HydABC from the acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui employ a single flavin mononucleotide (FMN) cofactor to establish electron transfer pathways to the NAD(P)⁺ and Fd reduction sites by a mechanism that is fundamentally different from classical flavin-based electron bifurcation enzymes. By modulation of the NAD(P)⁺ binding affinity via reduction of a nearby iron–sulfur cluster, HydABC switches between the exergonic NAD(P)⁺ reduction and endergonic Fd reduction modes. Our combined findings suggest that the conformational dynamics establish a redox-driven kinetic gate that prevents the backflow of the electrons from the Fd reduction branch toward the FMN site, providing a basis for understanding general mechanistic principles of electron-bifurcating hydrogenases.

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. 

Aerobic life is powered by membrane-bound enzymes that catalyze the transfer of electrons to oxygen and protons across a biological membrane. Cytochrome c oxidase (CcO) functions as a terminal electron acceptor in mitochondrial and bacterial respiratory chains, driving cellular respiration and transducing the free energy from O₂ reduction into proton pumping. Here we show that CcO creates orientated electric fields around a nonpolar cavity next to the active site, establishing a molecular switch that directs the protons along distinct pathways. By combining large-scale quantum chemical density functional theory (DFT) calculations with hybrid quantum mechanics/ molecular mechanics (QM/MM) simulations and atomistic molecular dynamics (MD) explorations, we find that reduction of the electron donor, heme a, leads to dissociation of an arginine (Arg438)–heme a₃ D-propionate ion-pair. This ion-pair dissociation creates a strong electric field of up to 1 V Å^-1 along a water-mediated proton array leading to a transient proton loading site (PLS) near the active site. Protonation of the PLS triggers the reduction of the active site, which in turn aligns the electric field vectors along a second, “chemical,” proton pathway. We find a linear energy relationship of the proton transfer barrier with the electric field strength that explains the effectivity of the gating process. Our mechanism shows distinct similarities to principles also found in other energy-converting enzymes, suggesting that orientated electric fields generally control enzyme catalysis.

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.

The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme’s catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the "active"-to-"deactive" transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I.