SignificanceComplex I is the primary energy-converting enzyme of aerobic respiratory chains. By reducing quinone to quinol, this gigantic enzyme pumps protons across its membrane domain, which in turn powers ATP synthesis and active transport. Despite the recently resolved molecular structures of complex I, the quinone dynamics and its coupling to the pumping function remains unclear. Here we show by large-scale molecular simulations that the quinone reduction leads to ejection of the quinol molecule from the active site into a second binding site near the proton-pumping membrane domain of complex I. The identified region has been linked with human mitochondrial disorders. Our work suggests that the quinone dynamics provides a key coupling element in complex I.

Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or mutation of Zn-binding residues blocked a late step of complex I assembly. An accumulating assembly intermediate lacked accessory subunit N7BM (NDUFA12), whereas a paralog of this subunit, the assembly factor N7BML (NDUFAF2), was found firmly bound instead. EPR spectroscopic analysis and metal content determination after chromatographic purification of the assembly intermediate showed that NUMM is required for insertion or stabilization of FeS cluster N4.assembly | metal protein | FeS cluster | NDUFAF2 | NDUFA12 P roton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) is a multisubunit membrane protein complex with a central function in aerobic energy metabolism (1, 2). Fourteen central subunits that harbor the bioenergetic core functions are conserved from bacteria to humans. In addition, eukaryotic complex I comprises around 30 accessory subunits of largely unknown function. The structures of bacterial and mitochondrial complex I were analyzed by X-ray crystallography and electron microscopy (3-6). The central subunits can be assigned to functional modules for NADH oxidation (N-module), ubiquinone reduction (Q-module), and proton pumping (P-module). The subunits forming the N-and Q-module harbor a chain of FeS clusters that connects the NADH oxidation site with the ubiquinone reduction site where the redox energy is released to drive proton translocation.The assembly of complex I subunits is a multistep process that proceeds via defined intermediates and is aided by a number of assembly factors (7). It also requires the concerted insertion of preformed FeS clusters into several subunits of the N-and Q-module (8). Dysfunction of complex I is the most frequent cause of mitochondrial disorders (9). Pathogenic mutations were identified not only in central subunits, encoded by either nuclear or mitochondrial DNA, but also in accessory subunits and assembly factors. The aerobic yeast Yarrowia lipolytica has been established as a yeast genetic model system to study structure and function of eukaryotic complex I, as well as complex I linked diseases (10).In this study, we focused on the accessory subunit NUMM of Y. lipolytica complex I. NUMM belongs to a limited subset of accessory subunits that is already found in α-proteobacteria and harbors a conserved putative Zn-binding motif, comprising three cysteines and one histidine in its C-terminal part (11). Zn binding to complex I was previously reported for bovine complex I but its functional relevance and the position of the Zn site remained elusive (12, 13). A poly...

Na + /H + antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na + /H + antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na + /H + exchanger NHE1, which is an important drug target. Here we resolve the Na + and H + transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.

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