Mitochondrial complex I plays a key role in cellular energy production by transferring electrons from NADH to ubiquinone coupled to proton translocation across the membrane1,2. It is the largest protein assembly of the respiratory chain with total mass of 970 kDa3. Here we present a nearly complete atomic structure of ovine mitochondrial complex I at 3.9 Å resolution, solved by cryo-electron microscopy aided by crosslinking/mass-spectrometry mapping. All 14 conserved core and 31 mitochondria-specific supernumerary subunits are resolved within the L-shaped molecule. The hydrophilic matrix arm harbours FMN and 8 iron-sulphur clusters involved in electron transfer, and the membrane arm contains 78 transmembrane helices, mostly contributed by antiporter-like subunits involved in proton translocation. Supernumerary subunits form an interlinked, stabilizing shell around the conserved core. Tightly bound lipids (including cardiolipins) further stabilize interactions between the hydrophobic subunits. Subunits with possible regulatory roles contain additional cofactors, NADPH and two phosphopantetheine molecules, revealed to be involved in inter-subunit interactions. We observe two different conformations of the complex, which may be related to the conformationally driven coupling mechanism and to the active/deactive transition of the enzyme. Our structure provides insight into complex I mechanism, assembly, maturation and dysfunction, allowing detailed molecular analysis of disease-causing mutations.The electrochemical proton gradient across the inner mitochondrial membrane required by ATP synthase is maintained by the electron transport chain (ETC) proton-pumping complexes I, III and IV1,2. Complex I (CI) is crucial for the entire process and even mildCorrespondence and requests for materials should be addressed to L. S. (sazanov@ist.ac.at). Author Contributions: K.F. purified complex I for grid preparation, prepared cryo-EM grids, acquired and processed EM data, and co-built the models; J.A.L. purified complex I for cross-linking experiments, analysed cross-linking data and co-built the models; G.D. performed cross-linking/mass-spectrometry experiments, K.K. performed model re-building in Rosetta and sequence alignments; G.D. and M.S. analysed cross-linking data; L.A.S. designed and supervised the project, processed and analysed data and wrote the manuscript, with contributions from all authors.The authors declare no competing financial interests.Author Information: The EM maps have been deposited in the EMDataBank under accession codes . The model has been deposited in the PDB under accession code 5LNK. Europe PMC Funders GroupAuthor Manuscript Nature. Author manuscript; available in PMC 2017 April 20. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts complex I deficiencies can cause severe pathologies4. Mammalian CI is built of 45 (44 unique) subunits. Fourteen "core" subunits, conserved from bacteria, comprise the "minimal" form of the enzyme1,5, an L-shaped structure with seven su...

Mitochondrial electron transport chain complexes are organized into supercomplexes responsible for carrying out cellular respiration. Here we present three architectures of mammalian (ovine) supercomplexes determined by cryo-electron microscopy. We identify two distinct arrangements of supercomplex CICIIICIV (the respirasome)-a major 'tight' form and a minor 'loose' form (resolved at the resolution of 5.8 Å and 6.7 Å, respectively), which may represent different stages in supercomplex assembly or disassembly. We have also determined an architecture of supercomplex CICIII at 7.8 Å resolution. All observed density can be attributed to the known 80 subunits of the individual complexes, including 132 transmembrane helices. The individual complexes form tight interactions that vary between the architectures, with complex IV subunit COX7a switching contact from complex III to complex I. The arrangement of active sites within the supercomplex may help control reactive oxygen species production. To our knowledge, these are the first complete architectures of the dominant, physiologically relevant state of the electron transport chain.

In voltage-gated Na ؉ , K ؉ , and Ca 2؉ channels, four voltage-sensor domains operate on a central pore domain in response to membrane voltage. In contrast, the voltage-gated proton channel (Hv) contains only a voltage-sensor domain, lacking a separate pore domain. The subunit stoichiometry and organization of Hv has been unknown. Here, we show that human Hv1 forms a dimer in the membrane and define regions that are close to the dimer interface by using cysteine cross-linking. Two dimeric interfaces appear to exist in Hv1, one mediated by S1 and the adjacent extracellular loop, and the other mediated by a putative intracellular coiled-coil domain. It may be significant that Hv1 uses for its dimer interface a surface that corresponds to the interface between the voltage sensor and pore in Kv channels.membrane protein ͉ voltage-dependent ion channel ͉ voltage sensor V oltage-gated six-transmembrane cation channels (Na ϩ , K ϩ , and Ca 2ϩ ) contain voltage-sensor and pore domains (1). In this class of ion channels voltage-sensor and pore domains carry out voltage sensing and cation conduction, respectively. Four voltage-sensor domains surround a single, centrally located ion conduction pathway. Each voltage sensor is attached to the pore in a specific manner so that conformational changes within the voltage sensors are transmitted to the pore's gate (2). It was originally thought that voltage-sensor domains existed only in the context of voltage-gated cation channels. However, the cloning of voltage-gated proton (Hv) channels and voltagesensor phosphatase enzymes revealed that voltage-sensor domains also exist in other contexts, apparently as independent (of a cation pore) membrane proteins (3-5), corroborating the studies of MacKinnon and coworkers (2, 6, 7) and Lu and coworkers (8) that supported the idea that voltage sensors, even in the context of voltage-dependent cation channels, are rather loosely attached to the central pore.The long-sought molecular identity of Hv1 (9) showed that it contains only a voltage-sensor domain without a separate pore domain in the membrane (3, 4). This observation suggested that in contrast to canonical voltage-dependent cation channels, the voltage-sensor domain of Hv1 is responsible for both H ϩ conduction and voltage sensing. In this study we evaluate the subunit stoichiometry of the Hv1 channel in cell membranes. ResultsHv1 Is a Dimer in the Membrane. To probe the oligomeric state of Hv1, cell membranes isolated from tsA201 cells (HEK293 derivatives) transfected with human Hv1 cDNAs were subjected to the amino-group specific bifunctional cross-linker disuccinimidyl suberate (DSS) and visualized by Western blot analysis using antibodies directed against the Hv1 channel. Amino groupspecific cross-linkers have been successful in defining the oligomeric status of several membrane proteins (10-12). Recombinant Hv1 makes functional channels in HEK293 cells (3, 4). In Fig. 1a, human Hv1 migrates at Ϸ35 kDa in SDS/PAGE under reducing conditions, which is consistent with the molecula...

Highlights d CoQ trapping within isolated respiratory supercomplex I+III 2 limits complex I turnover d CryoEM structures of multiple 3D classes show crosstalk between complex I and III 2 d Key transmembrane helix in complex I rotates upon ''closed'' to ''open'' state transition d CoQ density at only three complex III 2 sites indicates symmetry breaking