Complex I functions as the initial electron acceptor in aerobic respiratory chains of most organisms. This gigantic redox-driven enzyme employs the energy from quinone reduction to pump protons across its complete approximately 200-Å membrane domain, thermodynamically driving synthesis of ATP. Despite recently resolved structures from several species, the molecular mechanism by which complex I catalyzes this long-range protoncoupled electron transfer process, however, still remains unclear. We perform here large-scale classical and quantum molecular simulations to study the function of the proton pump in complex I from Thermus thermophilus. The simulations suggest that proton channels are established at symmetry-related locations in four subunits of the membrane domain. The channels open up by formation of quasi one-dimensional water chains that are sensitive to the protonation states of buried residues at structurally conserved broken helix elements. Our combined data provide mechanistic insight into long-range coupling effects and predictions for sitedirected mutagenesis experiments.NADH:ubiquinone oxidoreductase | proton pumping | Grotthuss mechanism | multiscale simulation | bioenergetics C omplex I (NADH:ubiquinone reductase) is the largest enzyme of the respiratory chain, generating a proton motive force (pmf) that is used for synthesis of adenosine triphosphate (ATP) and active transport (1, 2). Complex I catalyzes electron transfer (eT) between nicotine adenine dinucleotide (NADH) and quinone (Q), and couples the energy released to pumping of four protons across the membrane (3-9). The distance between the electron and proton transferring modules extends up to approximately 200 Å. It currently remains unclear, however, how complex I catalyzes this remarkable long-range proton-coupled electron transfer (PCET) process. In addition to its central role in biological energy conversion, elucidating the molecular mechanism of complex

Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH 2 ) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions.NADH-quinone oxidoreductase | electron transfer | molecular dynamics simulations | QM/MM simulations | cell respiration C omplex I (NADH-quinone oxidoreductase) is the largest (550-980 kDa) and one of the most enigmatic enzymes of the electron transport chains of mitochondria and bacteria. It catalyzes electron transfer (eT) from reduced nicotinamide adenine dinucleotide (NADH) to quinone (Q) and couples the reaction to translocation of three to four protons across the membrane (1, 2). The established electrochemical proton gradient is further used to synthesize adenosine triphosphate (ATP) for active transport (3). Due to its central role in cellular respiration, elucidating the catalytic mechanism of complex I is crucial for understanding the molecular principles of biological energy transduction and for unveiling the origins of many mitochondrial disorders (4).The electrons donated by NADH to complex I are transferred via flavin mononucleotide (FMN) to Q, bound at the lower edge of the hydrophilic domain at a distance of ∼80 Å from the FMN (Fig. 1). The eT process is mediated by seven to eight iron-sulfur (FeS) clusters, depending on the organism, and takes place in ∼100 μs (5). It is believed that the eT process does not couple to proton translocation, which is likely to occur on millisecond timescales (5, 6), but it is rather the oxidoreduction chemistry of the bound Q molecule that drives the proton pump (2, 5-9; cf. ref. 10).The proton-pumping machinery of complex I is located in the membrane domain of the enzyme (9) and is responsible for pumping three to four protons across the membrane (Fig. 1) (8, 11). Biochemical and structural studies suggest that the reduction of Q activates the proton pump via a conformationaldriven coupling mechanism, accompanied by electrostatic gating (2, 6-8, 12-14). A ...

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.