@article{12757, abstract = {My group and myself have studied respiratory complex I for almost 30 years, starting in 1994 when it was known as a L-shaped giant ‘black box' of bioenergetics. First breakthrough was the X-ray structure of the peripheral arm, followed by structures of the membrane arm and finally the entire complex from Thermus thermophilus. The developments in cryo-EM technology allowed us to solve the first complete structure of the twice larger, ∼1 MDa mammalian enzyme in 2016. However, the mechanism coupling, over large distances, the transfer of two electrons to pumping of four protons across the membrane remained an enigma. Recently we have solved high-resolution structures of mammalian and bacterial complex I under a range of redox conditions, including catalytic turnover. This allowed us to propose a robust and universal mechanism for complex I and related protein families. Redox reactions initially drive conformational changes around the quinone cavity and a long-distance transfer of substrate protons. These set up a stage for a series of electrostatically driven proton transfers along the membrane arm (‘domino effect'), eventually resulting in proton expulsion from the distal antiporter-like subunit. The mechanism radically differs from previous suggestions, however, it naturally explains all the unusual structural features of complex I. In this review I discuss the state of knowledge on complex I, including the current most controversial issues.}, author = {Sazanov, Leonid A}, issn = {1470-8728}, journal = {The Biochemical Journal}, number = {5}, pages = {319--333}, publisher = {Portland Press}, title = {{From the 'black box' to 'domino effect' mechanism: What have we learned from the structures of respiratory complex I}}, doi = {10.1042/BCJ20210285}, volume = {480}, year = {2023}, } @article{14040, abstract = {Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.}, author = {Zhao, Ziyu and Vercellino, Irene and Knoppová, Jana and Sobotka, Roman and Murray, James W. and Nixon, Peter J. and Sazanov, Leonid A and Komenda, Josef}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis}}, doi = {10.1038/s41467-023-40388-6}, volume = {14}, year = {2023}, } @article{10182, abstract = {The mitochondrial oxidative phosphorylation system is central to cellular metabolism. It comprises five enzymatic complexes and two mobile electron carriers that work in a mitochondrial respiratory chain. By coupling the oxidation of reducing equivalents coming into mitochondria to the generation and subsequent dissipation of a proton gradient across the inner mitochondrial membrane, this electron transport chain drives the production of ATP, which is then used as a primary energy carrier in virtually all cellular processes. Minimal perturbations of the respiratory chain activity are linked to diseases; therefore, it is necessary to understand how these complexes are assembled and regulated and how they function. In this Review, we outline the latest assembly models for each individual complex, and we also highlight the recent discoveries indicating that the formation of larger assemblies, known as respiratory supercomplexes, originates from the association of the intermediates of individual complexes. We then discuss how recent cryo-electron microscopy structures have been key to answering open questions on the function of the electron transport chain in mitochondrial respiration and how supercomplexes and other factors, including metabolites, can regulate the activity of the single complexes. When relevant, we discuss how these mechanisms contribute to physiology and outline their deregulation in human diseases.}, author = {Vercellino, Irene and Sazanov, Leonid A}, issn = {1471-0080}, journal = {Nature Reviews Molecular Cell Biology}, pages = {141–161}, publisher = {Springer Nature}, title = {{The assembly, regulation and function of the mitochondrial respiratory chain}}, doi = {10.1038/s41580-021-00415-0}, volume = {23}, year = {2022}, } @article{11167, abstract = {Complex I is one of the major respiratory complexes, conserved from bacteria to mammals. It oxidises NADH, reduces quinone and pumps protons across the membrane, thus playing a central role in the oxidative energy metabolism. In this review we discuss our current state of understanding the structure of complex I from various species of mammals, plants, fungi, and bacteria, as well as of several complex I-related proteins. By comparing the structural evidence from these systems in different redox states and data from mutagenesis and molecular simulations, we formulate the mechanisms of electron transfer and proton pumping and explain how they are conformationally and electrostatically coupled. Finally, we discuss the structural basis of the deactivation phenomenon in mammalian complex I.}, author = {Kampjut, Domen and Sazanov, Leonid A}, issn = {0959-440X}, journal = {Current Opinion in Structural Biology}, keywords = {Molecular Biology, Structural Biology}, publisher = {Elsevier}, title = {{Structure of respiratory complex I – An emerging blueprint for the mechanism}}, doi = {10.1016/j.sbi.2022.102350}, volume = {74}, year = {2022}, } @article{11551, abstract = {Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance.}, author = {Molina-Granada, David and González-Vioque, Emiliano and Dibley, Marris G. and Cabrera-Pérez, Raquel and Vallbona-Garcia, Antoni and Torres-Torronteras, Javier and Sazanov, Leonid A and Ryan, Michael T. and Cámara, Yolanda and Martí, Ramon}, issn = {23993642}, journal = {Communications Biology}, number = {1}, publisher = {Springer Nature}, title = {{Most mitochondrial dGTP is tightly bound to respiratory complex I through the NDUFA10 subunit}}, doi = {10.1038/s42003-022-03568-6}, volume = {5}, year = {2022}, } @article{12138, abstract = {Complex I is the first enzyme in the respiratory chain, which is responsible for energy production in mitochondria and bacteria1. Complex I couples the transfer of two electrons from NADH to quinone and the translocation of four protons across the membrane2, but the coupling mechanism remains contentious. Here we present cryo-electron microscopy structures of Escherichia coli complex I (EcCI) in different redox states, including catalytic turnover. EcCI exists mostly in the open state, in which the quinone cavity is exposed to the cytosol, allowing access for water molecules, which enable quinone movements. Unlike the mammalian paralogues3, EcCI can convert to the closed state only during turnover, showing that closed and open states are genuine turnover intermediates. The open-to-closed transition results in the tightly engulfed quinone cavity being connected to the central axis of the membrane arm, a source of substrate protons. Consistently, the proportion of the closed state increases with increasing pH. We propose a detailed but straightforward and robust mechanism comprising a ‘domino effect’ series of proton transfers and electrostatic interactions: the forward wave (‘dominoes stacking’) primes the pump, and the reverse wave (‘dominoes falling’) results in the ejection of all pumped protons from the distal subunit NuoL. This mechanism explains why protons exit exclusively from the NuoL subunit and is supported by our mutagenesis data. We contend that this is a universal coupling mechanism of complex I and related enzymes.}, author = {Kravchuk, Vladyslav and Petrova, Olga and Kampjut, Domen and Wojciechowska-Bason, Anna and Breese, Zara and Sazanov, Leonid A}, issn = {1476-4687}, journal = {Nature}, keywords = {Multidisciplinary}, number = {7928}, pages = {808--814}, publisher = {Springer Nature}, title = {{A universal coupling mechanism of respiratory complex I}}, doi = {10.1038/s41586-022-05199-7}, volume = {609}, year = {2022}, } @article{8993, abstract = {N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.}, author = {Abas, Lindy and Kolb, Martina and Stadlmann, Johannes and Janacek, Dorina P. and Lukic, Kristina and Schwechheimer, Claus and Sazanov, Leonid A and Mach, Lukas and Friml, Jiří and Hammes, Ulrich Z.}, issn = {10916490}, journal = {PNAS}, number = {1}, publisher = {National Academy of Sciences}, title = {{Naphthylphthalamic acid associates with and inhibits PIN auxin transporters}}, doi = {10.1073/pnas.2020857118}, volume = {118}, year = {2021}, } @article{9205, abstract = {Cryo-EM grid preparation is an important bottleneck in protein structure determination, especially for membrane proteins, typically requiring screening of a large number of conditions. We systematically investigated the effects of buffer components, blotting conditions and grid types on the outcome of grid preparation of five different membrane protein samples. Aggregation was the most common type of problem which was addressed by changing detergents, salt concentration or reconstitution of proteins into nanodiscs or amphipols. We show that the optimal concentration of detergent is between 0.05 and 0.4% and that the presence of a low concentration of detergent with a high critical micellar concentration protects the proteins from denaturation at the air-water interface. Furthermore, we discuss the strategies for achieving an adequate ice thickness, particle coverage and orientation distribution on free ice and on support films. Our findings provide a clear roadmap for comprehensive screening of conditions for cryo-EM grid preparation of membrane proteins.}, author = {Kampjut, Domen and Steiner, Julia and Sazanov, Leonid A}, issn = {25890042}, journal = {iScience}, number = {3}, publisher = {Elsevier}, title = {{Cryo-EM grid optimization for membrane proteins}}, doi = {10.1016/j.isci.2021.102139}, volume = {24}, year = {2021}, } @article{10146, abstract = {The enzymes of the mitochondrial electron transport chain are key players of cell metabolism. Despite being active when isolated, in vivo they associate into supercomplexes1, whose precise role is debated. Supercomplexes CIII2CIV1-2 (refs. 2,3), CICIII2 (ref. 4) and CICIII2CIV (respirasome)5,6,7,8,9,10 exist in mammals, but in contrast to CICIII2 and the respirasome, to date the only known eukaryotic structures of CIII2CIV1-2 come from Saccharomyces cerevisiae11,12 and plants13, which have different organization. Here we present the first, to our knowledge, structures of mammalian (mouse and ovine) CIII2CIV and its assembly intermediates, in different conformations. We describe the assembly of CIII2CIV from the CIII2 precursor to the final CIII2CIV conformation, driven by the insertion of the N terminus of the assembly factor SCAF1 (ref. 14) deep into CIII2, while its C terminus is integrated into CIV. Our structures (which include CICIII2 and the respirasome) also confirm that SCAF1 is exclusively required for the assembly of CIII2CIV and has no role in the assembly of the respirasome. We show that CIII2 is asymmetric due to the presence of only one copy of subunit 9, which straddles both monomers and prevents the attachment of a second copy of SCAF1 to CIII2, explaining the presence of one copy of CIV in CIII2CIV in mammals. Finally, we show that CIII2 and CIV gain catalytic advantage when assembled into the supercomplex and propose a role for CIII2CIV in fine tuning the efficiency of electron transfer in the electron transport chain.}, author = {Vercellino, Irene and Sazanov, Leonid A}, issn = {1476-4687}, journal = {Nature}, number = {7880}, pages = {364--367}, publisher = {Springer Nature}, title = {{Structure and assembly of the mammalian mitochondrial supercomplex CIII2CIV}}, doi = {10.1038/s41586-021-03927-z}, volume = {598}, year = {2021}, } @article{7788, abstract = {Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4−/− mouse tissues. Ndufs4−/− animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4−/− mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4−/− MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4−/− mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids.}, author = {Adjobo-Hermans, Merel J.W. and De Haas, Ria and Willems, Peter H.G.M. and Wojtala, Aleksandra and Van Emst-De Vries, Sjenet E. and Wagenaars, Jori A. and Van Den Brand, Mariel and Rodenburg, Richard J. and Smeitink, Jan A.M. and Nijtmans, Leo G. and Sazanov, Leonid A and Wieckowski, Mariusz R. and Koopman, Werner J.H.}, issn = {18792650}, journal = {Biochimica et Biophysica Acta - Bioenergetics}, number = {8}, publisher = {Elsevier}, title = {{NDUFS4 deletion triggers loss of NDUFA12 in Ndufs4−/− mice and Leigh syndrome patients: A stabilizing role for NDUFAF2}}, doi = {10.1016/j.bbabio.2020.148213}, volume = {1861}, year = {2020}, } @article{8040, abstract = {The mitochondrial respiratory chain, formed by five protein complexes, utilizes energy from catabolic processes to synthesize ATP. Complex I, the first and the largest protein complex of the chain, harvests electrons from NADH to reduce quinone, while pumping protons across the mitochondrial membrane. Detailed knowledge of the working principle of such coupled charge-transfer processes remains, however, fragmentary due to bottlenecks in understanding redox-driven conformational transitions and their interplay with the hydrated proton pathways. Complex I from Thermus thermophilus encases 16 subunits with nine iron–sulfur clusters, reduced by electrons from NADH. Here, employing the latest crystal structure of T. thermophilus complex I, we have used microsecond-scale molecular dynamics simulations to study the chemo-mechanical coupling between redox changes of the iron–sulfur clusters and conformational transitions across complex I. First, we identify the redox switches within complex I, which allosterically couple the dynamics of the quinone binding pocket to the site of NADH reduction. Second, our free-energy calculations reveal that the affinity of the quinone, specifically menaquinone, for the binding-site is higher than that of its reduced, menaquinol form—a design essential for menaquinol release. Remarkably, the barriers to diffusive menaquinone dynamics are lesser than that of the more ubiquitous ubiquinone, and the naphthoquinone headgroup of the former furnishes stronger binding interactions with the pocket, favoring menaquinone for charge transport in T. thermophilus. Our computations are consistent with experimentally validated mutations and hierarchize the key residues into three functional classes, identifying new mutation targets. Third, long-range hydrogen-bond networks connecting the quinone-binding site to the transmembrane subunits are found to be responsible for proton pumping. Put together, the simulations reveal the molecular design principles linking redox reactions to quinone turnover to proton translocation in complex I.}, author = {Gupta, Chitrak and Khaniya, Umesh and Chan, Chun Kit and Dehez, Francois and Shekhar, Mrinal and Gunner, M. R. and Sazanov, Leonid A and Chipot, Christophe and Singharoy, Abhishek}, issn = {15205126}, journal = {Journal of the American Chemical Society}, number = {20}, pages = {9220--9230}, publisher = {American Chemical Society}, title = {{Charge transfer and chemo-mechanical coupling in respiratory complex I}}, doi = {10.1021/jacs.9b13450}, volume = {142}, year = {2020}, } @misc{9326, abstract = {The mitochondrial respiratory chain, formed by five protein complexes, utilizes energy from catabolic processes to synthesize ATP. Complex I, the first and the largest protein complex of the chain, harvests electrons from NADH to reduce quinone, while pumping protons across the mitochondrial membrane. Detailed knowledge of the working principle of such coupled charge-transfer processes remains, however, fragmentary due to bottlenecks in understanding redox-driven conformational transitions and their interplay with the hydrated proton pathways. Complex I from Thermus thermophilus encases 16 subunits with nine iron–sulfur clusters, reduced by electrons from NADH. Here, employing the latest crystal structure of T. thermophilus complex I, we have used microsecond-scale molecular dynamics simulations to study the chemo-mechanical coupling between redox changes of the iron–sulfur clusters and conformational transitions across complex I. First, we identify the redox switches within complex I, which allosterically couple the dynamics of the quinone binding pocket to the site of NADH reduction. Second, our free-energy calculations reveal that the affinity of the quinone, specifically menaquinone, for the binding-site is higher than that of its reduced, menaquinol forma design essential for menaquinol release. Remarkably, the barriers to diffusive menaquinone dynamics are lesser than that of the more ubiquitous ubiquinone, and the naphthoquinone headgroup of the former furnishes stronger binding interactions with the pocket, favoring menaquinone for charge transport in T. thermophilus. Our computations are consistent with experimentally validated mutations and hierarchize the key residues into three functional classes, identifying new mutation targets. Third, long-range hydrogen-bond networks connecting the quinone-binding site to the transmembrane subunits are found to be responsible for proton pumping. Put together, the simulations reveal the molecular design principles linking redox reactions to quinone turnover to proton translocation in complex I.}, author = {Gupta, Chitrak and Khaniya, Umesh and Chan, Chun and Dehez, Francois and Shekhar, Mrinal and Gunner, M. R. and Sazanov, Leonid A and Chipot, Christophe and Singharoy, Abhishek}, publisher = {American Chemical Society}, title = {{Charge transfer and chemo-mechanical coupling in respiratory complex I}}, doi = {10.1021/jacs.9b13450.s002}, year = {2020}, } @misc{9713, abstract = {Additional analyses of the trajectories}, author = {Gupta, Chitrak and Khaniya, Umesh and Chan, Chun Kit and Dehez, Francois and Shekhar, Mrinal and Gunner, M.R. and Sazanov, Leonid A and Chipot, Christophe and Singharoy, Abhishek}, publisher = {American Chemical Society }, title = {{Supporting information}}, doi = {10.1021/jacs.9b13450.s001}, year = {2020}, } @misc{9878, author = {Gupta, Chitrak and Khaniya, Umesh and Chan, Chun Kit and Dehez, Francois and Shekhar, Mrinal and Gunner, M.R. and Sazanov, Leonid A and Chipot, Christophe and Singharoy, Abhishek}, publisher = {American Chemical Society}, title = {{Movies}}, doi = {10.1021/jacs.9b13450.s002}, year = {2020}, } @article{8318, abstract = {Complex I is the first and the largest enzyme of respiratory chains in bacteria and mitochondria. The mechanism which couples spatially separated transfer of electrons to proton translocation in complex I is not known. Here we report five crystal structures of T. thermophilus enzyme in complex with NADH or quinone-like compounds. We also determined cryo-EM structures of major and minor native states of the complex, differing in the position of the peripheral arm. Crystal structures show that binding of quinone-like compounds (but not of NADH) leads to a related global conformational change, accompanied by local re-arrangements propagating from the quinone site to the nearest proton channel. Normal mode and molecular dynamics analyses indicate that these are likely to represent the first steps in the proton translocation mechanism. Our results suggest that quinone binding and chemistry play a key role in the coupling mechanism of complex I.}, author = {Gutierrez-Fernandez, Javier and Kaszuba, Karol and Minhas, Gurdeep S. and Baradaran, Rozbeh and Tambalo, Margherita and Gallagher, David T. and Sazanov, Leonid A}, issn = {20411723}, journal = {Nature Communications}, number = {1}, publisher = {Springer Nature}, title = {{Key role of quinone in the mechanism of respiratory complex I}}, doi = {10.1038/s41467-020-17957-0}, volume = {11}, year = {2020}, } @article{8581, abstract = {The majority of adenosine triphosphate (ATP) powering cellular processes in eukaryotes is produced by the mitochondrial F1Fo ATP synthase. Here, we present the atomic models of the membrane Fo domain and the entire mammalian (ovine) F1Fo, determined by cryo-electron microscopy. Subunits in the membrane domain are arranged in the ‘proton translocation cluster’ attached to the c-ring and a more distant ‘hook apparatus’ holding subunit e. Unexpectedly, this subunit is anchored to a lipid ‘plug’ capping the c-ring. We present a detailed proton translocation pathway in mammalian Fo and key inter-monomer contacts in F1Fo multimers. Cryo-EM maps of F1Fo exposed to calcium reveal a retracted subunit e and a disassembled c-ring, suggesting permeability transition pore opening. We propose a model for the permeability transition pore opening, whereby subunit e pulls the lipid plug out of the c-ring. Our structure will allow the design of drugs for many emerging applications in medicine.}, author = {Pinke, Gergely and Zhou, Long and Sazanov, Leonid A}, issn = {15459985}, journal = {Nature Structural and Molecular Biology}, number = {11}, pages = {1077--1085}, publisher = {Springer Nature}, title = {{Cryo-EM structure of the entire mammalian F-type ATP synthase}}, doi = {10.1038/s41594-020-0503-8}, volume = {27}, year = {2020}, } @article{8737, abstract = {Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo-electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions.}, author = {Kampjut, Domen and Sazanov, Leonid A}, issn = {10959203}, journal = {Science}, number = {6516}, publisher = {American Association for the Advancement of Science}, title = {{The coupling mechanism of mammalian respiratory complex I}}, doi = {10.1126/science.abc4209}, volume = {370}, year = {2020}, } @article{8284, abstract = {Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.}, author = {Steiner, Julia and Sazanov, Leonid A}, issn = {2050084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter}}, doi = {10.7554/eLife.59407}, volume = {9}, year = {2020}, } @article{6859, abstract = {V (vacuolar)/A (archaeal)-type adenosine triphosphatases (ATPases), found in archaeaand eubacteria, couple ATP hydrolysis or synthesis to proton translocation across theplasma membrane using the rotary-catalysis mechanism. They belong to the V-typeATPase family, which differs from the mitochondrial/chloroplast F-type ATP synthasesin overall architecture. We solved cryo–electron microscopy structures of the intactThermus thermophilusV/A-ATPase, reconstituted into lipid nanodiscs, in three rotationalstates and two substates. These structures indicate substantial flexibility betweenV1and Voin a working enzyme, which results from mechanical competition between centralshaft rotation and resistance from the peripheral stalks. We also describedetails of adenosine diphosphate inhibition release, V1-Votorque transmission, andproton translocation, which are relevant for the entire V-type ATPase family.}, author = {Zhou, Long and Sazanov, Leonid A}, issn = {1095-9203}, journal = {Science}, number = {6455}, publisher = {AAAS}, title = {{Structure and conformational plasticity of the intact Thermus thermophilus V/A-type ATPase}}, doi = {10.1126/science.aaw9144}, volume = {365}, year = {2019}, } @article{7395, abstract = {The mitochondrial electron transport chain complexes are organized into supercomplexes (SCs) of defined stoichiometry, which have been proposed to regulate electron flux via substrate channeling. We demonstrate that CoQ trapping in the isolated SC I+III2 limits complex (C)I turnover, arguing against channeling. The SC structure, resolved at up to 3.8 Å in four distinct states, suggests that CoQ oxidation may be rate limiting because of unequal access of CoQ to the active sites of CIII2. CI shows a transition between “closed” and “open” conformations, accompanied by the striking rotation of a key transmembrane helix. Furthermore, the state of CI affects the conformational flexibility within CIII2, demonstrating crosstalk between the enzymes. CoQ was identified at only three of the four binding sites in CIII2, suggesting that interaction with CI disrupts CIII2 symmetry in a functionally relevant manner. Together, these observations indicate a more nuanced functional role for the SCs.}, author = {Letts, James A and Fiedorczuk, Karol and Degliesposti, Gianluca and Skehel, Mark and Sazanov, Leonid A}, issn = {1097-2765}, journal = {Molecular Cell}, number = {6}, pages = {1131--1146.e6}, publisher = {Cell Press}, title = {{Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk}}, doi = {10.1016/j.molcel.2019.07.022}, volume = {75}, year = {2019}, } @article{6848, abstract = {Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. NNT catalyses the transfer of a hydride between NADH and NADP+, coupled to the translocation of one proton across the membrane. Its main physiological function is the generation of NADPH, which is a substrate in anabolic reactions and a regulator of oxidative status; however, NNT may also fine-tune the Krebs cycle1,2. NNT deficiency causes familial glucocorticoid deficiency in humans and metabolic abnormalities in mice, similar to those observed in type II diabetes3,4. The catalytic mechanism of NNT has been proposed to involve a rotation of around 180° of the entire NADP(H)-binding domain that alternately participates in hydride transfer and proton-channel gating. However, owing to the lack of high-resolution structures of intact NNT, the details of this process remain unclear5,6. Here we present the cryo-electron microscopy structure of intact mammalian NNT in different conformational states. We show how the NADP(H)-binding domain opens the proton channel to the opposite sides of the membrane, and we provide structures of these two states. We also describe the catalytically important interfaces and linkers between the membrane and the soluble domains and their roles in nucleotide exchange. These structures enable us to propose a revised mechanism for a coupling process in NNT that is consistent with a large body of previous biochemical work. Our results are relevant to the development of currently unavailable NNT inhibitors, which may have therapeutic potential in ischaemia reperfusion injury, metabolic syndrome and some cancers7,8,9.}, author = {Kampjut, Domen and Sazanov, Leonid A}, issn = {1476-4687}, journal = {Nature}, number = {7773}, pages = {291–295}, publisher = {Springer Nature}, title = {{Structure and mechanism of mitochondrial proton-translocating transhydrogenase}}, doi = {10.1038/s41586-019-1519-2}, volume = {573}, year = {2019}, } @article{152, abstract = {Complex I has an essential role in ATP production by coupling electron transfer from NADH to quinone with translocation of protons across the inner mitochondrial membrane. Isolated complex I deficiency is a frequent cause of mitochondrial inherited diseases. Complex I has also been implicated in cancer, ageing, and neurodegenerative conditions. Until recently, the understanding of complex I deficiency on the molecular level was limited due to the lack of high-resolution structures of the enzyme. However, due to developments in single particle cryo-electron microscopy (cryo-EM), recent studies have reported nearly atomic resolution maps and models of mitochondrial complex I. These structures significantly add to our understanding of complex I mechanism and assembly. The disease-causing mutations are discussed here in their structural context.}, author = {Fiedorczuk, Karol and Sazanov, Leonid A}, journal = {Trends in Cell Biology}, number = {10}, pages = {835 -- 867}, publisher = {Elsevier}, title = {{Mammalian mitochondrial complex I structure and disease causing mutations}}, doi = {10.1016/j.tcb.2018.06.006}, volume = {28}, year = {2018}, } @article{443, abstract = {Pancreatic cancer has a five-year survival rate of ~8%, with characteristic molecular heterogeneity and restricted treatment options. Targeting metabolism has emerged as a potentially effective therapeutic strategy for cancers such as pancreatic cancer, which are driven by genetic alterations that are not tractable drug targets. Although somatic mitochondrial genome (mtDNA) mutations have been observed in various tumors types, understanding of metabolic genotype-phenotype relationships is limited.}, author = {Hardie, Rae and Van Dam, Ellen and Cowley, Mark and Han, Ting and Balaban, Seher and Pajic, Marina and Pinese, Mark and Iconomou, Mary and Shearer, Robert and Mckenna, Jessie and Miller, David and Waddell, Nicola and Pearson, John and Grimmond, Sean and Sazanov, Leonid A and Biankin, Andrew and Villas Boas, Silas and Hoy, Andrew and Turner, Nigel and Saunders, Darren}, journal = {Cancer & Metabolism}, number = {2}, publisher = {BioMed Central}, title = {{Mitochondrial mutations and metabolic adaptation in pancreatic cancer}}, doi = {10.1186/s40170-017-0164-1}, volume = {5}, year = {2017}, } @inbook{444, abstract = {Complex I (NADH:ubiquinone oxidoreductase) plays a central role in cellular energy generation, contributing to the proton motive force used to produce ATP. It couples the transfer of two electrons between NADH and quinone to translocation of four protons across the membrane. It is the largest protein assembly of bacterial and mitochondrial respiratory chains, composed, in mammals, of up to 45 subunits with a total molecular weight of ∼1 MDa. Bacterial enzyme is about half the size, providing the important “minimal” model of complex I. The l-shaped complex consists of a hydrophilic arm, where electron transfer occurs, and a membrane arm, where proton translocation takes place. Previously, we have solved the crystal structures of the hydrophilic domain of complex I from Thermus thermophilus and of the membrane domain from Escherichia coli, followed by the atomic structure of intact, entire complex I from T. thermophilus. Recently, we have solved by cryo-EM a first complete atomic structure of mammalian (ovine) mitochondrial complex I. Core subunits are well conserved from the bacterial version, whilst supernumerary subunits form an interlinked, stabilizing shell around the core. Subunits containing additional cofactors, including Zn ion, NADPH and phosphopantetheine, probably have regulatory roles. Dysfunction of mitochondrial complex I is implicated in many human neurodegenerative diseases. The structure of mammalian enzyme provides many insights into complex I mechanism, assembly, maturation and dysfunction, allowing detailed molecular analysis of disease-causing mutations.}, author = {Sazanov, Leonid A}, booktitle = {Mechanisms of primary energy transduction in biology }, editor = {Wikström, Mårten}, isbn = {978-1-78262-865-1}, pages = {25 -- 59}, publisher = {Royal Society of Chemistry}, title = {{Structure of respiratory complex I: “Minimal” bacterial and “de luxe” mammalian versions}}, doi = {10.1039/9781788010405-00025}, year = {2017}, } @article{515, abstract = {The oxidative phosphorylation electron transport chain (OXPHOS-ETC) of the inner mitochondrial membrane is composed of five large protein complexes, named CI-CV. These complexes convert energy from the food we eat into ATP, a small molecule used to power a multitude of essential reactions throughout the cell. OXPHOS-ETC complexes are organized into supercomplexes (SCs) of defined stoichiometry: CI forms a supercomplex with CIII2 and CIV (SC I+III2+IV, known as the respirasome), as well as with CIII2 alone (SC I+III2). CIII2 forms a supercomplex with CIV (SC III2+IV) and CV forms dimers (CV2). Recent cryo-EM studies have revealed the structures of SC I+III2+IV and SC I+III2. Furthermore, recent work has shed light on the assembly and function of the SCs. Here we review and compare these recent studies and discuss how they have advanced our understanding of mitochondrial electron transport.}, author = {Letts, James A and Sazanov, Leonid A}, issn = {15459993}, journal = {Nature Structural and Molecular Biology}, number = {10}, pages = {800 -- 808}, publisher = {Nature Publishing Group}, title = {{Clarifying the supercomplex: The higher-order organization of the mitochondrial electron transport chain}}, doi = {10.1038/nsmb.3460}, volume = {24}, year = {2017}, } @article{1209, abstract = {NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies. }, author = {Letts, James A and Degliesposti, Gianluca and Fiedorczuk, Karol and Skehel, Mark and Sazanov, Leonid A}, journal = {Journal of Biological Chemistry}, number = {47}, pages = {24657 -- 24675}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Purification of ovine respiratory complex i results in a highly active and stable preparation}}, doi = {10.1074/jbc.M116.735142}, volume = {291}, year = {2016}, } @article{1226, abstract = {Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) contributes to cellular energy production by transferring electrons from NADH to ubiquinone coupled to proton translocation across the membrane. It is the largest protein assembly of the respiratory chain with a total mass of 970 kilodaltons. Here we present a nearly complete atomic structure of ovine (Ovis aries) mitochondrial complex I at 3.9 Å resolution, solved by cryo-electron microscopy with cross-linking and mass-spectrometry mapping experiments. All 14 conserved core subunits and 31 mitochondria-specific supernumerary subunits are resolved within the L-shaped molecule. The hydrophilic matrix arm comprises flavin mononucleotide and 8 iron-sulfur 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, which are shown 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 the mechanism, assembly, maturation and dysfunction of mitochondrial complex I, and allows detailed molecular analysis of disease-causing mutations.}, author = {Fiedorczuk, Karol and Letts, James A and Degliesposti, Gianluca and Kaszuba, Karol and Skehel, Mark and Sazanov, Leonid A}, journal = {Nature}, number = {7625}, pages = {406 -- 410}, publisher = {Nature Publishing Group}, title = {{Atomic structure of the entire mammalian mitochondrial complex i}}, doi = {10.1038/nature19794}, volume = {538}, year = {2016}, } @article{1232, abstract = {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 CICIII 2 CIV (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 2 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.}, author = {Letts, James A and Fiedorczuk, Karol and Sazanov, Leonid A}, journal = {Nature}, number = {7622}, pages = {644 -- 648}, publisher = {Nature Publishing Group}, title = {{The architecture of respiratory supercomplexes}}, doi = {10.1038/nature19774}, volume = {537}, year = {2016}, } @article{1288, abstract = {Respiratory complex I transfers electrons from NADH to quinone, utilizing the reaction energy to translocate protons across the membrane. It is a key enzyme of the respiratory chain of many prokaryotic and most eukaryotic organisms. The reversible NADH oxidation reaction is facilitated in complex I by non-covalently bound flavin mononucleotide (FMN). Here we report that the catalytic activity of E. coli complex I with artificial electron acceptors potassium ferricyanide (FeCy) and hexaamineruthenium (HAR) is significantly inhibited in the enzyme pre-reduced by NADH. Further, we demonstrate that the inhibition is caused by reversible dissociation of FMN. The binding constant (Kd) for FMN increases from the femto- or picomolar range in oxidized complex I to the nanomolar range in the NADH reduced enzyme, with an FMN dissociation time constant of ~ 5 s. The oxidation state of complex I, rather than that of FMN, proved critical to the dissociation. Such dissociation is not observed with the T. thermophilus enzyme and our analysis suggests that the difference may be due to the unusually high redox potential of Fe-S cluster N1a in E. coli. It is possible that the enzyme attenuates ROS production in vivo by releasing FMN under highly reducing conditions.}, author = {Holt, Peter and Efremov, Rouslan and Nakamaru Ogiso, Eiko and Sazanov, Leonid A}, journal = {Biochimica et Biophysica Acta - Bioenergetics}, number = {11}, pages = {1777 -- 1785}, publisher = {Elsevier}, title = {{Reversible FMN dissociation from Escherichia coli respiratory complex I}}, doi = {10.1016/j.bbabio.2016.08.008}, volume = {1857}, year = {2016}, } @article{1521, abstract = {Complex I (NADH:ubiquinone oxidoreductase) plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. It is the largest protein assembly of respiratory chains and one of the most elaborate redox membrane proteins known. Bacterial enzyme is about half the size of mitochondrial and thus provides its important "minimal" model. Dysfunction of mitochondrial complex I is implicated in many human neurodegenerative diseases. The L-shaped complex consists of a hydrophilic arm, where electron transfer occurs, and a membrane arm, where proton translocation takes place. We have solved the crystal structures of the hydrophilic domain of complex I from Thermus thermophilus, the membrane domain from Escherichia coli and recently of the intact, entire complex I from T. thermophilus (536. kDa, 16 subunits, 9 iron-sulphur clusters, 64 transmembrane helices). The 95. Å long electron transfer pathway through the enzyme proceeds from the primary electron acceptor flavin mononucleotide through seven conserved Fe-S clusters to the unusual elongated quinone-binding site at the interface with the membrane domain. Four putative proton translocation channels are found in the membrane domain, all linked by the central flexible axis containing charged residues. The redox energy of electron transfer is coupled to proton translocation by the as yet undefined mechanism proposed to involve long-range conformational changes. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.}, author = {Berrisford, John and Baradaran, Rozbeh and Sazanov, Leonid A}, journal = {Biochimica et Biophysica Acta - Bioenergetics}, number = {7}, pages = {892 -- 901}, publisher = {Elsevier}, title = {{Structure of bacterial respiratory complex I}}, doi = {10.1016/j.bbabio.2016.01.012}, volume = {1857}, year = {2016}, } @article{1638, abstract = {The mitochondrial respiratory chain, also known as the electron transport chain (ETC), is crucial to life, and energy production in the form of ATP is the main mitochondrial function. Three proton-translocating enzymes of the ETC, namely complexes I, III and IV, generate proton motive force, which in turn drives ATP synthase (complex V). The atomic structures and basic mechanisms of most respiratory complexes have previously been established, with the exception of complex I, the largest complex in the ETC. Recently, the crystal structure of the entire complex I was solved using a bacterial enzyme. The structure provided novel insights into the core architecture of the complex, the electron transfer and proton translocation pathways, as well as the mechanism that couples these two processes.}, author = {Sazanov, Leonid A}, journal = {Nature Reviews Molecular Cell Biology}, number = {6}, pages = {375 -- 388}, publisher = {Nature Publishing Group}, title = {{A giant molecular proton pump: structure and mechanism of respiratory complex I}}, doi = {10.1038/nrm3997}, volume = {16}, year = {2015}, } @article{1683, abstract = {The 1 MDa, 45-subunit proton-pumping NADH-ubiquinone oxidoreductase (complex I) is the largest complex of the mitochondrial electron transport chain. The molecular mechanism of complex I is central to the metabolism of cells, but has yet to be fully characterized. The last two years have seen steady progress towards this goal with the first atomic-resolution structure of the entire bacterial complex I, a 5 Å cryo-electron microscopy map of bovine mitochondrial complex I and a ∼3.8 Å resolution X-ray crystallographic study of mitochondrial complex I from yeast Yarrowia lipotytica. In this review we will discuss what we have learned from these studies and what remains to be elucidated.}, author = {Letts, Jame A and Sazanov, Leonid A}, journal = {Current Opinion in Structural Biology}, number = {8}, pages = {135 -- 145}, publisher = {Elsevier}, title = {{Gaining mass: The structure of respiratory complex I-from bacterial towards mitochondrial versions}}, doi = {10.1016/j.sbi.2015.08.008}, volume = {33}, year = {2015}, } @misc{1981, abstract = {Variation in mitochondrial DNA is often assumed to be neutral and is used to construct the genealogical relationships among populations and species. However, if extant variation is the result of episodes of positive selection, these genealogies may be incorrect, although this information itself may provide biologically and evolutionary meaningful information. In fact, positive Darwinian selection has been detected in the mitochondrial-encoded subunits that comprise complex I from diverse taxa with seemingly dissimilar bioenergetic life histories, but the functional implications of the selected sites are unknown. Complex I produces roughly 40% of the proton flux that is used to synthesize ATP from ADP, and a functional model based on the high-resolution structure of complex I described a unique biomechanical apparatus for proton translocation. We reported positive selection at sites in this apparatus during the evolution of Pacific salmon, and it appeared this was also the case in published reports from other taxa, but a comparison among studies was difficult because different statistical tests were used to detect selection and oftentimes, specific sites were not reported. Here we review the literature of positive selection in mitochondrial genomes, the statistical tests used to detect selection, and the structural and functional models that are currently available to study the physiological implications of selection. We then search for signatures of positive selection among the coding mitochondrial genomes of 237 species with a common set of tests and verify that the ND5 subunit of complex I is a repeated target of positive Darwinian selection in diverse taxa. We propose a novel hypothesis to explain the results based on their bioenergetic life histories and provide a guide for laboratory and field studies to test this hypothesis.}, author = {Garvin, Michael R and Bielawski, Joseph P and Leonid Sazanov and Gharrett, Anthony J}, booktitle = {Journal of Zoological Systematics and Evolutionary Research}, number = {1}, pages = {1 -- 17}, publisher = {Wiley-Blackwell}, title = {{Review and meta-analysis of natural selection in mitochondrial complex I in metazoans}}, doi = {10.1111/jzs.12079}, volume = {53}, year = {2014}, } @article{1980, abstract = {Non-proton pumping type II NADH dehydrogenase (NDH-2) plays a central role in the respiratory metabolism of bacteria, and in the mitochondria of fungi, plants and protists. The lack of NDH-2 in mammalian mitochondria and its essentiality in important bacterial pathogens suggests these enzymes may represent a potential new drug target to combat microbial pathogens. Here, we report the first crystal structure of a bacterial NDH-2 enzyme at 2.5Å resolution from Caldalkalibacillus thermarum. The NDH-2 structure reveals a homodimeric organization that has a unique dimer interface. NDH-2 is localized to the cytoplasmic membrane by two separated C-terminal membrane-anchoring regions that are essential for membrane localization and FAD binding, but not NDH-2 dimerization. Comparison of bacterial NDH-2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non-overlapping binding sites for quinone and NADH in the bacterial enzyme. The bacterial NDH-2 structure establishes a framework for the structure-based design of small-molecule inhibitors.}, author = {Heikal, Adam and Nakatani, Yoshio and Dunn, Elyse A and Weimar, Marion R and Day, Catherine and Baker, Edward N and Lott, Shaun J and Leonid Sazanov and Cook, Gregory}, journal = {Molecular Microbiology}, number = {5}, pages = {950 -- 964}, publisher = {Wiley-Blackwell}, title = {{Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation}}, doi = {10.1111/mmi.12507}, volume = {91}, year = {2014}, } @article{1979, abstract = {NADH-ubiquinone oxidoreductase (complex I) is the first and largest enzyme in the respiratory chain of mitochondria and many bacteria. It couples the transfer of two electrons between NADH and ubiquinone to the translocation of four protons across the membrane. Complex I is an L-shaped assembly formed by the hydrophilic (peripheral) arm, containing all the redox centres performing electron transfer and the membrane arm, containing proton-translocating machinery. Mitochondrial complex I consists of 44 subunits of about 1 MDa in total, whilst the prokaryotic enzyme is simpler and generally consists of 14 conserved “core” subunits. Recently we have determined the first atomic structure of the entire complex I, using the enzyme from Thermus thermophilus (536 kDa, 16 subunits, 9 Fe-S clusters, 64 TM helices). Structure suggests a unique coupling mechanism, with redox energy of electron transfer driving proton translocation via long-range (up to ~200 Å) conformational changes. It resembles a steam engine, with coupling elements (akin to coupling rods) linking parts of this molecular machine.}, author = {Leonid Sazanov}, journal = {Journal of Bioenergetics and Biomembranes}, number = {4}, pages = {247 -- 253}, publisher = {Springer}, title = {{The mechanism of coupling between electron transfer and proton translocation in respiratory complex I}}, doi = {10.1007/s10863-014-9554-z}, volume = {46}, year = {2014}, } @article{1977, abstract = {Complex I (NADH:ubiquinone oxidoreductase) is central to cellular energy production, being the first and largest enzyme of the respiratory chain in mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the inner mitochondrial membrane and is involved in a wide range of human neurodegenerative disorders. Mammalian complex I is composed of 44 different subunits, whereas the 'minimal' bacterial version contains 14 highly conserved 'core' subunits. The L-shaped assembly consists of hydrophilic and membrane domains. We have determined all known atomic structures of complex I, starting from the hydrophilic domain of Thermus thermophilus enzyme (eight subunits, nine Fe-S clusters), followed by the membrane domains of the Escherichia coli (six subunits, 55 transmembrane helices) and T. thermophilus (seven subunits, 64 transmembrane helices) enzymes, and finally culminating in a recent crystal structure of the entire intact complex I from T. thermophilus (536 kDa, 16 subunits, nine Fe-S clusters, 64 transmembrane helices). The structure suggests an unusual and unique coupling mechanism via longrange conformational changes. Determination of the structure of the entire complex was possible only through this step-by-step approach, building on from smaller subcomplexes towards the entire assembly. Large membrane proteins are notoriously difficult to crystallize, and so various non-standard and sometimes counterintuitive approaches were employed in order to achieve crystal diffraction to high resolution and solve the structures. These steps, as well as the implications from the final structure, are discussed in the present review.}, author = {Leonid Sazanov and Baradaran, Rozbeh and Efremov, Rouslan G and Berrisford, John M and Minhas, Gurdeep S}, journal = {Biochemical Society Transactions}, number = {5}, pages = {1265 -- 1271}, publisher = {Portland Press}, title = {{A long road towards the structure of respiratory complex I, a giant molecular proton pump}}, doi = {10.1042/BST20130193}, volume = {41}, year = {2013}, } @article{1978, abstract = {Complex I is the first and largest enzyme of the respiratory chain and has a central role in cellular energy production through the coupling of NADH:ubiquinone electron transfer to proton translocation. It is also implicated in many common human neurodegenerative diseases. Here, we report the first crystal structure of the entire, intact complex I (from Thermus thermophilus) at 3.3 Å resolution. The structure of the 536-kDa complex comprises 16 different subunits, with a total of 64 transmembrane helices and 9 iron-sulphur clusters. The core fold of subunit Nqo8 (ND1 in humans) is, unexpectedly, similar to a half-channel of the antiporter-like subunits. Small subunits nearby form a linked second half-channel, which completes the fourth proton-translocation pathway (present in addition to the channels in three antiporter-like subunits). The quinone-binding site is unusually long, narrow and enclosed. The quinone headgroup binds at the deep end of this chamber, near iron-sulphur cluster N2. Notably, the chamber is linked to the fourth channel by a 'funnel' of charged residues. The link continues over the entire membrane domain as a flexible central axis of charged and polar residues, and probably has a leading role in the propagation of conformational changes, aided by coupling elements. The structure suggests that a unique, out-of-the-membrane quinone-reaction chamber enables the redox energy to drive concerted long-range conformational changes in the four antiporter-like domains, resulting in translocation of four protons per cycle.}, author = {Baradaran, Rozbeh and Berrisford, John M and Minhas, Gurdeep S and Leonid Sazanov}, journal = {Nature}, number = {7438}, pages = {443 -- 448}, publisher = {Nature Publishing Group}, title = {{Crystal structure of the entire respiratory complex i}}, doi = {10.1038/nature11871}, volume = {494}, year = {2013}, } @misc{1976, abstract = {Complex I is a key enzyme of the respiratory chain in many organisms. This multi-protein complex with an intricate evolutionary history originated from the unification of prebuilt modules of hydrogenases and transporters. Using recently determined crystallographic structures of complex I we reanalyzed evolutionarily related complexes that couple oxidoreduction to trans-membrane ion translocation. Our analysis points to the previously unnoticed structural homology of the electron input module of formate dehydrogenlyases and subunit NuoG of complex I. We also show that all related to complex I hydrogenases likely operate via a conformation driven mechanism with structural changes generated in the conserved coupling site located at the interface of subunits NuoB/D/H. The coupling apparently originated once in evolutionary history, together with subunit NuoH joining hydrogenase and transport modules. Analysis of quinone oxidoreduction properties and the structure of complex I allows us to suggest a fully reversible coupling mechanism. Our model predicts that: 1) proton access to the ketone groups of the bound quinone is rigorously controlled by the protein, 2) the negative electric charge of the anionic ubiquinol head group is a major driving force for conformational changes.}, author = {Efremov, Rouslan G and Leonid Sazanov}, booktitle = {Biochimica et Biophysica Acta - Bioenergetics}, number = {10}, pages = {1785 -- 1795}, publisher = {Elsevier}, title = {{The coupling mechanism of respiratory complex i - A structural and evolutionary perspective}}, doi = {10.1016/j.bbabio.2012.02.015}, volume = {1817}, year = {2012}, } @article{1972, abstract = {Outer membrane protein F, a major component of the Escherichia coli outer membrane, was crystallized for the first time in lipidic mesophase of monoolein in novel space groups, P1 and H32. Due to ease of its purification and crystallization OmpF can be used as a benchmark protein for establishing membrane protein crystallization in meso, as a "membrane lyzozyme" The packing of porin trimers in the crystals of space group H32 is similar to natural outer membranes, providing the first high-resolution insight into the close to native packing of OmpF. Surprisingly, interaction between trimers is mediated exclusively by lipids, without direct protein-protein contacts. Multiple ordered lipids are observed and many of them occupy identical positions independently of the space group, identifying preferential interaction sites of lipid acyl chains. Presence of ordered aliphatic chains close to a positively charged area on the porin surface suggests a position for a lipopolysaccharide binding site on the surface of the major E. coli porins.}, author = {Efremov, Rouslan G and Leonid Sazanov}, journal = {Journal of Structural Biology}, number = {3}, pages = {311 -- 318}, publisher = {Academic Press}, title = {{Structure of Escherichia coli OmpF porin from lipidic mesophase}}, doi = {10.1016/j.jsb.2012.03.005}, volume = {178}, year = {2012}, } @article{1975, abstract = {Modern α-proteobacteria are thought to be closely related to the ancient symbiont of eukaryotes, an ancestor of mitochondria. Respiratory complex I from α-proteobacteria and mitochondria is well conserved at the level of the 14 "core" subunits, consistent with that notion. Mitochondrial complex I contains the core subunits, present in all species, and up to 31 "supernumerary" subunits, generally thought to have originated only within eukaryotic lineages. However, the full protein composition of an α-proteobacterial complex I has not been established previously. Here, we report the first purification and characterization of complex I from the α-proteobacterium Paracoccus denitrificans. Single particle electron microscopy shows that the complex has a well defined L-shape. Unexpectedly, in addition to the 14 core subunits, the enzyme also contains homologues of three supernumerary mitochondrial subunits as follows: B17.2, AQDQ/18, and 13 kDa (bovine nomenclature). This finding suggests that evolution of complex I via addition of supernumerary or "accessory" subunits started before the original endosymbiotic event that led to the creation of the eukaryotic cell. It also provides further confirmation that α-proteobacteria are the closest extant relatives of mitochondria.}, author = {Yip, Chui Y and Harbour, Michael E and Jayawardena, Kamburapola G and Fearnley, Ian M and Leonid Sazanov}, journal = {Journal of Biological Chemistry}, number = {7}, pages = {5023 -- 5033}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Evolution of respiratory complex I "Supernumerary" subunits are present in the α-proteobacterial enzyme}}, doi = {10.1074/jbc.M110.194993}, volume = {286}, year = {2011}, } @article{1973, abstract = {Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0 Ã. resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.}, author = {Efremov, Rouslan G and Leonid Sazanov}, journal = {Nature}, number = {7361}, pages = {414 -- 421}, publisher = {Nature Publishing Group}, title = {{Structure of the membrane domain of respiratory complex i}}, doi = {10.1038/nature10330}, volume = {476}, year = {2011}, } @article{1974, abstract = {Complex I is the first enzyme of the respiratory chain and plays a central role in cellular energy production. It has been implicated in many human neurodegenerative diseases, as well as in ageing. One of the biggest membrane protein complexes, it is an L-shaped assembly consisting of hydrophilic and membrane domains. Previously, we have determined structures of the hydrophilic domain in several redox states. Last year was marked by fascinating breakthroughs in the understanding of the complete structure. We described the architecture of the membrane domain and of the entire bacterial complex I. X-ray analysis of the larger mitochondrial enzyme has also been published. The core subunits of the bacterial and mitochondrial enzymes have remarkably similar structures. The proposed mechanism of coupling between electron transfer and proton translocation involves long-range conformational changes, coordinated in part by a long α-helix, akin to the coupling rod of a steam engine.}, author = {Efremov, Rouslan G and Leonid Sazanov}, journal = {Current Opinion in Structural Biology}, number = {4}, pages = {532 -- 540}, publisher = {Elsevier}, title = {{Respiratory complex I: 'steam engine' of the cell?}}, doi = {10.1016/j.sbi.2011.07.002}, volume = {21}, year = {2011}, } @article{1970, abstract = {Complex I is the first enzyme of the respiratory chain and has a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation by an unknown mechanism. Dysfunction of complex I has been implicated in many human neurodegenerative diseases. We have determined the structure of its hydrophilic domain previously. Here, we report the α-helical structure of the membrane domain of complex I from Escherichia coli at 3.9 Å resolution. The antiporter-like subunits NuoL/M/N each contain 14 conserved transmembrane (TM) helices. Two of them are discontinuous, as in some transporters. Unexpectedly, subunit NuoL also contains a 110-Å long amphipathic α-helix, spanning almost the entire length of the domain. Furthermore, we have determined the structure of the entire complex I from Thermus thermophilus at 4.5 Å resolution. The L-shaped assembly consists of the α-helical model for the membrane domain, with 63 TM helices, and the known structure of the hydrophilic domain. The architecture of the complex provides strong clues about the coupling mechanism: the conformational changes at the interface of the two main domains may drive the long amphipathic α-helix of NuoL in a piston-like motion, tilting nearby discontinuous TM helices, resulting in proton translocation.}, author = {Efremov, Rouslan G and Baradaran, Rozbeh and Leonid Sazanov}, journal = {Nature}, number = {7297}, pages = {441 -- 445}, publisher = {Nature Publishing Group}, title = {{The architecture of respiratory complex I}}, doi = {10.1038/nature09066}, volume = {465}, year = {2010}, } @article{1971, abstract = {Complex I plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. The mechanism of this highly efficient enzyme is currently unknown. Mitochondrial complex I is a major source of reactive oxygen species, which may be one of the causes of aging. Dysfunction of complex I is implicated in many human neurodegenerative diseases. We have determined several x-ray structures of the oxidized and reduced hydrophilic domain of complex I from Thermus thermophilus at up to 3.1 Å resolution. The structures reveal the mode of interaction of complex I with NADH, explaining known kinetic data and providing implications for the mechanism of reactive oxygen species production at the flavin site of complex I. Bound metals were identified in the channel at the interface with the frataxin-like subunit Nqo15, indicating possible iron-binding sites. Conformational changes upon reduction of the complex involve adjustments in the nucleotide-binding pocket, as well as small but significant shifts of several α-helices at the interface with the membrane domain. These shifts are likely to be driven by the reduction of nearby iron-sulfur clusters N2 and N6a/b. Cluster N2 is the electron donor to quinone and is coordinated by unique motif involving two consecutive (tandem) cysteines. An unprecedented "on/off switch" (disconnection) of coordinating bonds between the tandem cysteines and this cluster was observed upon reduction. Comparison of the structures suggests a novel mechanism of coupling between electron transfer and proton translocation, combining conformational changes and protonation/deprotonation of tandem cysteines.}, author = {Berrisford, John M and Leonid Sazanov}, journal = {Journal of Biological Chemistry}, number = {43}, pages = {29773 -- 29783}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Structural basis for the mechanism of respiratory complex I}}, doi = {10.1074/jbc.M109.032144}, volume = {284}, year = {2009}, } @article{1967, abstract = {Complex I of respiratory chains transfers electrons from NADH to ubiquinone, coupled to the translocation of protons across the membrane. Two alternative coupling mechanisms are being discussed, redox-driven or conformation-driven. Using "zero-length" cross-linking reagent and isolated hydrophilic domains of complex I from Escherichia coli and Thermus thermophilus, we show that the pattern of cross-links between subunits changes significantly in the presence of NADH. Similar observations were made previously with intact purified E. coli and bovine complex I. This indicates that, upon reduction with NADH, similar conformational changes are likely to occur in the intact enzyme and in the isolated hydrophilic domain (which can be used for crystallographic studies). Within intact E. coli complex I, the cross-link between the hydrophobic subunits NuoA and NuoJ was abolished in the presence of NADH, indicating that conformational changes extend into the membrane domain, possibly as part of a coupling mechanism. Unexpectedly, in the absence of any chemical cross-linker, incubation of complex I with NADH resulted in covalent cross-links between subunits Nqo4 (NuoCD) and Nqo6 (NuoB), as well as between Nqo6 and Nqo9. Their formation depends on the presence of oxygen and so is likely a result of oxidative damage via reactive oxygen species (ROS) induced cross-linking. In addition, ROS- and metal ion-dependent proteolysis of these subunits (as well as Nqo3) is observed. Fe-S cluster N2 is coordinated between subunits Nqo4 and Nqo6 and could be involved in these processes. Our observations suggest that oxidative damage to complex I in vivo may include not only side-chain modifications but also protein cross-linking and degradation.}, author = {Berrisford, John M and Thompson, Christopher J and Leonid Sazanov}, journal = {Biochemistry}, number = {39}, pages = {10262 -- 10270}, publisher = {ACS}, title = {{Chemical and NADH-induced, ROS-dependent, cross-linking between sublimits of complex I from Escherichia coli and Thermus thermophilus}}, doi = {10.1021/bi801160u}, volume = {47}, year = {2008}, } @article{1968, abstract = { Complex I (NADH:ubiquinone oxidoreductase) is the largest protein complex of bacterial and mitochondrial respiratory chains. The first three-dimensional structure of bacterial complex I in vitrified ice was determined by electron cryo-microscopy and single particle analysis. The structure of the Escherichia coli enzyme incubated with either NAD+ (as a reference) or NADH was calculated to 35 and 39 Å resolution, respectively. The X-ray structure of the peripheral arm of Thermus thermophilus complex I was docked into the reference EM structure. The model obtained indicates that Fe-S cluster N2 is close to the membrane domain interface, allowing for effective electron transfer to membrane-embedded quinone. At the current resolution, the structures in the presence of NAD+ or NADH are similar. Additionally, side-view class averages were calculated for the negatively stained bovine enzyme. The structures of bovine complex I in the presence of either NAD+ or NADH also appeared to be similar. These observations indicate that conformational changes upon reduction with NADH, suggested to occur by a range of studies, are smaller than had been thought previously. The model of the entire bacterial complex I could be built from the crystal structures of subcomplexes using the EM envelope described here.}, author = {Morgan, David J and Leonid Sazanov}, journal = {Biochimica et Biophysica Acta - Bioenergetics}, number = {7-8}, pages = {711 -- 718}, publisher = {Elsevier}, title = {{Three-dimensional structure of respiratory complex I from Escherichia coli in ice in the presence of nucleotides}}, doi = {10.1016/j.bbabio.2008.03.023}, volume = {1777}, year = {2008}, } @article{1965, abstract = {Respiratory complex I (NADH:ubiquinone oxidoreductase) is an L-shaped multisubunit protein assembly consisting of a hydrophobic membrane arm and a hydrophilic peripheral arm. It catalyses the transfer of two electrons from NADH to quinone coupled to the translocation of four protons across the membrane. Although we have solved recently the crystal structure of the peripheral arm, the structure of the complete enzyme and the coupling mechanism are not yet known. The membrane domain of Escherichia coli complex I consists of seven different subunits with total molecular mass of 258 kDa. It is significantly more stable than the whole enzyme, which allowed us to obtain well-ordered two-dimensional crystals of the domain, belonging to the space group p22121. Comparison of the projection map of negatively stained crystals with previously published low-resolution structures indicated that the characteristic curved shape of the membrane domain is remarkably well conserved between bacterial and mitochondrial enzymes, helping us to interpret projection maps in the context of the intact complex. Two pronounced stain-excluding densities at the distal end of the membrane domain are likely to represent the two large antiporter-like subunits NuoL and NuoM. Cryo-electron microscopy on frozen-hydrated crystals allowed us to calculate a projection map at 8 Å resolution. About 60 transmembrane α-helices, both perpendicular to the membrane plane and tilted, are present within one membrane domain, which is consistent with secondary structure predictions. A possible binding site and access channel for quinone are found at the interface with the peripheral arm. Tentative assignment of individual subunits to the features of the map has been made. The location of subunits NuoL and NuoM at substantial distance from the peripheral arm, which contains all the redox centres of the complex, indicates that conformational changes are likely to play a role in the mechanism of coupling between electron transfer and proton pumping.}, author = {Baranova, Ekaterina A and Holt, Peter J and Leonid Sazanov}, journal = {Journal of Molecular Biology}, number = {1}, pages = {140 -- 154}, publisher = {Elsevier}, title = {{Projection structure of the membrane domain of Escherichia coli respiratory Complex I at 8 Å resolution}}, doi = {10.1016/j.jmb.2006.11.026}, volume = {366}, year = {2007}, } @article{1969, abstract = {Respiratory complex I catalyses the transfer of electrons from NADH to quinone coupled to the translocation of protons across the membrane. The mechanism of coupling and the structure of the complete enzyme are not known. The membrane domain of the complex contains three similar antiporter-like subunits NuoL/M/N, probably involved in proton pumping. We have previously shown that subunits NuoL/M can be removed from the rest of the complex, suggesting their location at the distal end of the membrane domain. Here, using electron microscopy and single particle analysis, we show that subunits NuoL and M jointly occupy a distal half of the membrane domain, separated by about 10 nm from the interface with the peripheral arm. This indicates that coupling mechanism of complex I is likely to involve long range conformational changes.}, author = {Baranova, Ekaterina A and Morgan, David J and Leonid Sazanov}, journal = {Journal of Structural Biology}, number = {2 SPEC. ISS.}, pages = {238 -- 242}, publisher = {Academic Press}, title = {{Single particle analysis confirms distal location of subunits NuoL and NuoM in Escherichia coli complex I}}, doi = {10.1016/j.jsb.2007.01.009}, volume = {159}, year = {2007}, } @article{1964, abstract = {Complex I of respiratory chains plays a central role in cellular energy production. Mutations in its subunits lead to many human neurodegenerative diseases. Recently, a first atomic structure of the hydrophilic domain of complex I from Thermus thermophilus was determined. This domain represents a catalytic core of the enzyme. It consists of eight different subunits, contains all the redox centers, and comprises more than half of the entire complex. In this review, novel mechanistic implications of the structure are discussed, and the effects of many known mutations of complex I subunits are interpreted in a structural context.}, author = {Leonid Sazanov}, journal = {Biochemistry}, number = {9}, pages = {2275 -- 2288}, publisher = {ACS}, title = {{Respiratory complex I: Mechanistic and structural insights provided by the crystal structure of the hydrophilic domain}}, doi = {10.1021/bi602508x}, volume = {46}, year = {2007}, } @article{1961, abstract = {Respiratory complex I plays a central role in cellular energy production in bacteria and mitochondria. Its dysfunction is implicated in many human neurodegenerative diseases, as well as in aging. The crystal structure of the hydrophilic domain (peripheral arm) of complex I from Thermus thermophilus has been solved at 3.3 angstrom resolution. This subcomplex consists of eight subunits and contains all the redox centers of the enzyme, including nine iron-sulfur clusters. The primary electron acceptor, flavin-mononucleotide, is within electron transfer distance of cluster N3, leading to the main redox pathway, and of the distal cluster Nia, a possible antioxidant. The structure reveals new aspects of the mechanism and evolution of the enzyme. The terminal cluster N2 is coordinated, uniquely, by two consecutive cysteines. The novel subunit Nqo15 has a similar fold to the mitochondrial iron chaperone frataxin, and it may be involved in iron-sulfur cluster regeneration in the complex. }, author = {Leonid Sazanov and Hinchliffe, Philip }, journal = {Science}, number = {5766}, pages = {1430 -- 1436}, publisher = {American Association for the Advancement of Science}, title = {{Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus}}, doi = {10.1126/science.1123809}, volume = {311}, year = {2006}, } @article{1966, abstract = {The hydrophilic domain (peripheral arm) of the proton-translocating NADH:quinone oxidoreductase (complex I) from the thermophilic organism Thermus thermophilus HB8 has been purified and characterized. The subcomplex is stable in sodium dodecyl sulfate up to 80 °C. Of nine iron-sulfur clusters, four to five (one or two binuclear and three tetranuclear) could be detected by EPR in the NADH-reduced enzyme. The preparation consists of eight different polypeptides. Seven of them have been positively identified by peptide mass mapping and N-terminal sequencing as known hydrophilic subunits of T. thermophilus complex I. The eighth polypeptide copurified with the subcomplex at all stages, is strongly associated with the other subunits, and is present in crystals of the subcomplex, used for X-ray data collection. Therefore, it has been identified as a novel complex I subunit and named Nqo15. It is encoded in a locus separate from the nqo operon, containing the 14 other known complex I genes. ORFs encoding Nqo15 homologues are present in the genomes of the closest relatives of T. thermophilus. Our data show that, contrary to previous assumptions, bacterial complex I can contain proteins in addition to a "core" complement of 14 subunits.}, author = {Hinchliffe, Philip and Carroll, Joe D and Leonid Sazanov}, journal = {Biochemistry}, number = {14}, pages = {4413 -- 4420}, publisher = {ACS}, title = {{Identification of a novel subunit of respiratory complex I from Thermus thermophilus}}, doi = {10.1021/bi0600998}, volume = {45}, year = {2006}, } @article{1962, abstract = { Complex I of respiratory chains plays a central role in bioenergetics and is implicated in many human neurodegenerative diseases. An understanding of its mechanism requires a knowledge of the organization of redox centers. The arrangement of iron-sulfur clusters in the hydrophilic domain of complex I from Thermus thermophilus has been determined with the use of x-ray crystallography. One binuclear and six tetranuclear clusters are arranged, maximally 14 angstroms apart, in an 84-angstrom-long electron transfer chain. The binuclear cluster N1a and the tetranuclear cluster N7 are not in this pathway. Cluster N1a may play a role in the prevention of oxidative damage. The structure provides a framework for the interpretation of the large amounts of data accumulated on complex I.}, author = {Hinchliffe, Philip and Leonid Sazanov}, journal = {Science}, number = {5735}, pages = {771 -- 774}, publisher = {American Association for the Advancement of Science}, title = {{Biochemistry: Organization of iron-sulfur clusters in respiratory complex I}}, doi = {10.1126/science.1113988}, volume = {309}, year = {2005}, } @article{1963, abstract = {The mechanism coupling electron transfer and proton pumping in respiratory complex I (NADH-ubiquinone oxidoreductase) has not been established, but it has been suggested that it involves conformational changes. Here, the influence of substrates on the conformation of purified complex I from Escherichia coli was studied by cross-linking and electron microscopy. When a zero-length cross-linking reagent was used, the presence of NAD(P)H, in contrast to that of NAD+, prevented the formation of cross-links between the hydrophilic subunits of the complex, including NuoB, NuoI, and NuoCD. Comparisons using different cross-linkers suggested that NuoB, which is likely to coordinate the key iron-sulfur cluster N2, is the most mobile subunit. The presence of NAD(P)H led also to enhanced proteolysis of subunit NuoG. These data indicate that upon NAD(P)H binding, the peripheral arm of the complex adopts a more open conformation, with increased distances between subunits. Single particle analysis showed the nature of this conformational change. The enzyme retains its L-shape in the presence of NADH, but exhibits a significantly more open or expanded structure both in the peripheral arm and, unexpectedly, in the membrane domain also.}, author = {Mamedova, Aygun A and Holt, Peter J and Carroll, Joe D and Leonid Sazanov}, journal = {Journal of Biological Chemistry}, number = {22}, pages = {23830 -- 23836}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{Substrate-induced conformational change in bacterial complex I}}, doi = {10.1074/jbc.M401539200}, volume = {279}, year = {2004}, } @article{1959, abstract = {The molecular organization of bacterial NADH: ubiquinone oxidoreductase (complex I or NDH-1) is not established, apart from a rough separation into dehydrogenase, connecting and membrane domains. In this work, complex I was purified from Escherichia coli and fragmented by replacing dodecylmaltoside with other detergents. Exchange into decyl maltoside led to the removal of the hydrophobic subunit NuoL from the otherwise intact complex. Diheptanoyl phosphocholine led to the loss of NuoL and NuoM subunits, whereas other subunits remained in the complex. The presence of N,N-dimethyldodecylamine N-oxide or Triton X-100 led to further disruption of the membrane domain into fragments containing NuoL/M/N, NuoA/K/N, and NuoH/J subunits. Among the hydrophilic subunits, NuoCD was most readily dissociated from the complex, whereas NuoB was partially dissociated from the peripheral arm assembly in N,N-dimethyldodecylamine N-oxide. A model of subunit arrangement in bacterial complex I based on these data is proposed. Subunits NuoL and NuoM, which are homologous to antiporters and are implicated in proton pumping, are located at the distal end of the membrane arm, spatially separated from the redox centers of the peripheral arm. This is consistent with proposals that the mechanism of proton pumping by complex I is likely to involve long range conformational changes.}, author = {Holt, Peter J and Morgan, David J and Leonid Sazanov}, journal = {Journal of Biological Chemistry}, number = {44}, pages = {43114 -- 43120}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{The location of NuoL and NuoM subunits in the membrane domain of the Escherichia coli Complex I: implications for the mechanism of proton pumping}}, doi = {10.1074/jbc.M308247200}, volume = {278}, year = {2003}, } @article{1960, abstract = {NADH-ubiquinone oxidoreductase (complex I or NDH-1) was purified from the BL21 strain of Escherichia coli using an improved procedure. The complex was effectively stabilized by addition of divalent cations and lipids, making the preparation suitable for structural studies. The ubiquinone reductase activity of the enzyme was fully restored by addition of native E. coli lipids. Two different two-dimensional crystal forms, with p2 and p3 symmetry, were obtained using lipids containing native E. coli extracts. Analysis of the crystals showed that they are formed by fully intact complex I in an L-shaped conformation. Activity assays and single particle analysis indicated that complex I maintains this structure in detergent solution and does not adopt a different conformation in the active state. Thus, we provide the first experimental evidence that complex I from E. coli has an L-shape in a lipid bilayer and confirm that this is also the case for the active enzyme in solution. This suggests strongly that bacterial complex I exists in an L-shaped conformation in vivo. Our results also indicate that native lipids play an important role in the activation, stabilization and, as a consequence, crystallization of purified complex I from E. coli.}, author = {Leonid Sazanov and Carroll, Joe D and Holt, Peter J and Toime, Laurence J and Fearnley, Ian M}, journal = {Journal of Biological Chemistry}, number = {21}, pages = {19483 -- 19491}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {{A role for native lipids in the stabilization and two dimensional crystallization of the Escherichia coli NADH ubiquinone oxidoreductase (complex I)}}, doi = {10.1074/jbc.M208959200}, volume = {278}, year = {2003}, } @article{1957, abstract = {NADH:ubiquinone oxidoreductase (complex I) is the first and largest enzyme of the mitochondrial respiratory chain. The low-resolution structure of the complex is known from electron microscopy studies. The general shape of the complex is in the form of an L, with one arm in the membrane and the other peripheral. We have purified complex I from beef heart mitochondria and reconstituted the enzyme into lipid bilayers. Under different conditions, several two-dimensional crystal forms were obtained. Crystals belonging to space groups p2221 and c12 (unit cell 488 Å x 79 Å) were obtained at 22°C and contained only the membrane fragment of complex I similar to hydrophobic subcomplex Iβ but lacking the ND5 subunit. A crystal form with larger unit cell (534 Å x 81 Å, space group c12) produced at 4°C contained both the peripheral and membrane arms of the enzyme, except that ND5 was missing. Projection maps from frozen hydrated samples were calculated for all crystal forms. By comparing two different c12 crystal forms, extra electron density in the projection map of large crystal form was assigned to the peripheral arm of the enzyme. One of the features of the map is a deep, channel-like, cleft next to peripheral arm. Comparison with available structures of the intact enzyme indicates that large hydrophobic subunit ND5 is situated at the distal end of the membrane domain. Possible locations of sub-unit ND4 and of other subunits in the membrane domain are proposed. Implications of our findings for the mechanism of proton pumping by complex I are discussed. (C) 2000 Academic Press.}, author = {Sazanov, Leonid A and Walker, John}, issn = {0022-2836}, journal = {Journal of Molecular Biology}, number = {2}, pages = {455 -- 464}, publisher = {Elsevier}, title = {{Cryo-electron crystallography of two sub-complexes of bovine complex I reveals the relationship between the membrane and peripheral arms}}, doi = {10.1006/jmbi.2000.4079}, volume = {302}, year = {2000}, } @article{1958, abstract = { Complex I (NADH:ubiquinone oxidoreductase) purified from bovine heart mitochondria was treated with the detergent N,N-dimethyldodecylamine N-oxide (LDAO). The enzyme dissociated into two known subcomplexes, Iα and Iβ, containing mostly hydrophilic and hydrophobic subunits, and a previously undetected fragment referred to as Iγ. Subcomplex Iγ contains the hydrophobic subunits ND1, ND2, ND3, and ND4L which are encoded in the mitochondrial genome, and the nuclear-encoded subunit KFYL. During size- exclusion chromatography in the presence of LDAO, subcomplex Iα lost several subunits and formed another characterized subcomplex known as Iλ. Similarly, subcomplex Iβ dissociated into two smaller subcomplexes, one of which contains the hydrophobic subunits ND4 and ND5; subcomplex Iγ released a fragment containing ND1 and ND2. These results suggest that in the intact complex subunits ND1 and ND2 are likely to be in a different region of the membrane domain than subunits ND4 and ND5. The compositions of the various subcomplexes and fragments of complex I provide an organization of the subunits of the enzyme in the framework of the known low resolution structure of the enzyme.}, author = {Sazanov, Leonid A and Peak Chew, Sew and Fearnley, Ian and Walker, John}, issn = {0006-2960}, journal = {Biochemistry}, number = {24}, pages = {7229 -- 7235}, publisher = {ACS}, title = {{Resolution of the membrane domain of bovine complex I into subcomplexes: implications for the structural organization of the enzyme}}, doi = {10.1021/bi000335t}, volume = {39}, year = {2000}, } @article{1956, abstract = { The plastid genomes of several plants contain ndh genes-homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria. From sequence similarities with these genes, the ndh gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established. Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant Pisum sativum. The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step. The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS/PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting. The Ndh complex showed an NADH- and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors. This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase. Our results are compatible with a dual role for the Ndh complex in the chloro-respiratory and cyclic photophosphorylation pathways.}, author = {Sazanov, Leonid A and Burrows, Paul and Nixon, Peter}, issn = {0027-8424}, journal = {PNAS}, number = {3}, pages = {1319 -- 1324}, publisher = {National Academy of Sciences}, title = {{The plastid ndh genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes}}, doi = {10.1073/pnas.95.3.1319}, volume = {95}, year = {1998}, } @article{1955, abstract = {The plastid genomes of several plants contain homologues, termed ndh genes, of genes encoding subunits of the NADH:ubiquinone oxidoreductase or complex I of mitochondria and eubacteria. The functional significance of the Ndh proteins in higher plants is uncertain. We show here that tobacco chloroplasts contain a protein complex of 550 kDa consisting of at least three of the ndh gene products: NdhI, NdhJ and NdhK. We have constructed mutant tobacco plants with disrupted ndhC, ndhK and ndhJ plastid genes, indicating that the Ndh complex is dispensible for plant growth under optimal growth conditions. Chlorophyll fluorescence analysis shows that in vivo the Ndh complex catalyses the post-illumination reduction of the plastoquinone pool and in the light optimizes the induction of photosynthesis under conditions of water stress. We conclude that the Ndh complex catalyses the reduction of the plastoquinone pool using stromal reductant and so acts as a respiratory complex. Overall, our data are compatible with the participation of the Ndh complex in cyclic electron flow around the photosystem I complex in the light and possibly in a chloroplast respiratory chain in the dark.}, author = {Burrows, Paul and Sazanov, Leonid A and Sváb, Zóra and Maliga, Pàl and Nixon, Peter}, issn = {0261-4189}, journal = {EMBO Journal}, number = {4}, pages = {868 -- 876}, publisher = {Wiley-Blackwell}, title = {{Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes}}, doi = {10.1093/emboj/17.4.868}, volume = {17}, year = {1998}, } @article{1954, abstract = { We have examined the effects of heat stress on electron transfer in the thylakoid membrane of an engineered plastid ndh deletion mutant, Δ1, incapable of performing the Ndh-mediated reduction of the plastoquinone pool in the chloroplast. Upon heat stress in the dark, the rate of PSII- independent reduction of PSI after subsequent illumination by far-red light is dramatically enhanced in both Δ1 and a wild-type control plant (WT). In contrast, in the dark, only the WT shows an increase in the reduction state of the plastoquinone pool. We conclude that the heat stress-induced reduction of the intersystem electron transport chain can be mediated by Ndh- independent pathways in the light but that in the dark the dominant pathway for reduction of the plastoquinone pool is catalysed by the Ndh complex. Our results therefore demonstrate a functional role for the Ndh complex in the dark. }, author = {Sazanov, Leonid A and Burrows, Paul and Nixon, Peter}, issn = {0014-5793}, journal = {FEBS Letters}, number = {1}, pages = {115 -- 118}, publisher = {Elsevier}, title = {{The chloroplast Ndh complex mediates the dark reduction of the plastoquinone pool in response to heat stress in tobacco leaves}}, doi = {10.1016/S0014-5793(98)00573-0}, volume = {429}, year = {1998}, } @inproceedings{1942, author = {Leonid Sazanov and Burrows, P and Nixon, P J}, pages = {705 -- 708}, publisher = {Kluwer}, title = {{Presence of a large protein complex containing the ndhK gene product and possessing NADH-specific dehydrogenase activity in thylakoid membranes of higher plant chloroplasts}}, volume = {2}, year = {1996}, } @article{1952, abstract = {Two strains of Rhodospirillum rubrum were constructed in which, by a gene dosage effect, the transhydrogenase activity of isolated chromatophores was increased 7-10-fold and 15-20-fold, respectively. The H+/H- ratio (the ratio of protons translocated per hydride ion equivalent transferred from NADPH to an NAD+ analogue, acetyl pyridine adenine dinucleotide), determined by a spectroscopic technique, was approximately 1.0 for chromatophores from the over-expressing strains, but was only approximately 0.6 for wild-type chromatophores. Highly-coupled proteoliposomes were prepared containing purified transhydrogenase from beef-heart mitochondria. Using the same technique, the H+/H- ratio was close to 1.0 for these proteoliposomes. It is suggested that the mechanistic H+/H- ratio is indeed unity, but that a low ratio is obtained in wild-type chromatophores because of inhomogeneity in the vesicle population.}, author = {Bizouarn, Tania and Sazanov, Leonid A and Aubourg, Sébastien and Jackson, Julie}, issn = {0005-2728}, journal = {Biochimica et Biophysica Acta - Bioenergetics}, number = {1}, pages = {4 -- 12}, publisher = {Elsevier}, title = {{Estimation of the H+/H- ratio of the reaction catalysed by the nicotinamide nucleotide transhydrogenase in chromatophores from over-expressing strains of Rhodospirillum rubrum and in liposomes inlaid with the purified bovine enzyme}}, doi = {10.1016/0005-2728(95)00125-5}, volume = {1273}, year = {1996}, } @article{1951, author = {Sazanov, Leonid A and Burrows, Paul and Nixon, Peter}, issn = {0300-5127}, journal = {Biochemical Society Transactions}, number = {3}, pages = {739 -- 743}, publisher = {Portland Press}, title = {{Detection and characterization of a complex I-like NADH-specific dehydrogenase from pea thylakoids}}, doi = {10.1042/bst0240739}, volume = {24}, year = {1996}, } @article{1943, abstract = {Transhydrogenase from beef-heart mitochondria was solubilised with Triton X-100 and purified by column chromatography. The detergent-dispersed enzyme catalysed the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH, but only in the presence of NADP+. Experiments showed that this reaction was cyclic; NADP(H), whilst remaining bound to the enzyme, was alternately reduced by NADH and oxidised by AcPdAD+. A period of incubation of the enzyme with NADPH at pH 6.0 led to inhibition of the simple transhydrogenation reaction between AcPdAD+ and NADPH. However, after such treatment, transhydrogenase acquired the ability to catalyse the (NADPH-dependent) reduction of AcPdAD+ by NADH. It is suggested that this is a similar cycle to the one described above. Evidently, the binding affinity for NADP+ increases as a consequence of the inhibition process resulting from prolonged incubation with NADPH. The pH dependences of simple and cyclic transhydrogenation reactions are described. Though more complex than those in Escherichia coli transhydrogenase, they are consistent with the view [Hutton, M., Day, J.M., Bizouarn, T. and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041–10511] that, also in the mitochondrial enzyme, binding the release of NADP+ and NADP are accompanied by binding and release of a proton. The enzyme was successfully reconstituted into liposomes by a cholate dilution procedure. The proteoliposomes catalysed cyclic NADPH-dependent reduction of AcPdAD+ by NADH only when they were tightly coupled. However, they catalysed cyclic NADP+-dependent reduction of AcPdAD+ by NADH only when they were uncoupled eg. by addition of carbonylcyanide-p-trifluoromethoxyphenyl hydrazone. These observations are evidence that the proton binding and release which accompany NADP+ binding and release, respectively, take place on the inside of the vesicle, and that they are components of the electrogenic processes of the enzyme.}, author = {Sazanov, Leonid A and Jackson, Baz}, issn = {0005-2728}, journal = {Biochimica et Biophysica Acta - Bioenergetics}, number = {3}, pages = {304 -- 312}, publisher = {Elsevier}, title = {{Cyclic reactions catalysed by detergent-dispersed and reconstituted transhydrogenase from beef heart mitochondria; implications for the mechanism of proton translocation}}, doi = {10.1016/0005-2728(95)00096-2}, volume = {1231}, year = {1995}, } @article{1949, abstract = {H+-transhydrogenase (H+-Thase) and NADP-linked isocitrate dehydrogenase (NADP-ICDH) are very active in animal mitochondria but their physiological function is only poorly understood. This is especially so in the case of the heart and muscle, where there are no major consumers of NADPH. We propose here that H+-Thase and NADP-ICDH have a combined function in the fine regulation of the activity of the tricarboxylic acid (TCA) cycle, providing enhanced sensitivy to changes in energy demand. This is achieved through cycling of substrates by NAD-linked ICDH, NADP-linked ICDH and H+-Thase. It is proposed that NAD-ICDH operates in the forward direction of the TCA cycle, but NADP-ICDH is driven in reverse by elevated levels of NADPH resulting from the action of the transmembrane proton electrochemical potential gradient (Δp) on H+-Thase. This has the effect of increasing the sensitivity to allosteric modifiers of NAD-ICDH (NADH, ADP, ATP, Ca2+ etc), potentially giving rise to large changes in the net flux from iso-citrate to α-ketoglutarate. Furthermore, changes in the level of Δp resulting from changes in the demand for ATP would, via H+-Thase, shift the redox state of the NADP pool and this, in turn, would lead to a change in the rate of the reaction catalysed by NADP-ICDH and hence to an additional and complementary effect on the net metabolic flux from isocitrate to α-ketoglutarate. Other consequences of this substrate cycle are, (i) the production of heat at the expense of Δp, which may contribute to thermoregulation in the animal, and (ii) an increased rate of dissipation of Δp (leak).}, author = {Sazanov, Leonid A and Jackson, Julie}, issn = {0014-5793}, journal = {FEBS Letters}, number = {2-3}, pages = {109 -- 116}, publisher = {Elsevier}, title = {{Proton translocating transhydrogenase and NAD- and NADP-linked isocitrate dehydrogenases operate in a substrate cycle which contributes to fine regulation of the tricarboxylic acid cycle activity in mitochondria}}, doi = {10.1016/0014-5793(94)00370-X}, volume = {344}, year = {1994}, } @article{1953, abstract = {The respiratory burst induced by phorbol myristate acetate in mouse macrophages was inhibited by ultra-low doses (10-15 -10-13 M) of an opioid peptide [d-Ala2] methionine enkephalinamide. The effect disappeared at concentrations above and below this range. The inhibition approached 50% and was statistically significant (P < 0.001). Increasing the time of the opioid incubation with cells brought about a shift in the maximal effect to lower concentrations of the opioid (from 10-13 to 5 · 10-15 M) and led to a decrease in the value of the effect, fully in accord with the previously proposed adaptation mechanism of the action of ultra-low doses.}, author = {Efanov, Alexander and Koshkin, Aleksei and Sazanov, Leonid A and Borodulina, O I and Varfolomeev, Sergei and Zaǐtsev, Sergei}, issn = {0014-5793}, journal = {FEBS Letters}, number = {2}, pages = {114 -- 116}, publisher = {Elsevier}, title = {{Inhibition of the respiratory burst in mouse macrophages by ultra-low doses of an opioid peptide is consistent with a possible adaptation mechanism}}, doi = {10.1016/0014-5793(94)01109-5}, volume = {355}, year = {1994}, } @article{1947, abstract = {Mitochondrial transhydrogenase has been reported previously to be inhibited by high, rather non-physiological concentrations (in the range of 2-20 mM) of divalent cations. We show that the enzyme could be activated by low (from about 1 μM to 1 mM) concentrations of Ca2+ and Mg2+, which are within physiological range. These results bring in line the effects observed with mitochondrial enzyme to the findings with bacterial transhydrogenases. The activation of transhydrogenase by divalent cations is interpreted as an increase in affinity of the NADP(H)-binding site of the enzyme-NAD(H) complex. Reported effects of the metal ions could be important for the enzyme function in vivo.}, author = {Sazanov, Leonid A and Jackson, Julie}, issn = {0005-2728}, journal = {Biochimica et Biophysica Acta - Bioenergetics}, number = {2}, pages = {225 -- 228}, publisher = {Elsevier}, title = {{Activation and inhibition of mitochondrial transhydrogenase by metal ions}}, doi = {10.1016/0005-2728(93)90177-H}, volume = {1144}, year = {1993}, } @article{1948, author = {Sazanov, Leonid A and Jackson, Julie}, issn = {0300-5127}, journal = {Biochemical Society Transactions}, number = {3}, pages = {260}, publisher = {Portland Press}, title = {{Possible functions of the NADP-linked isocitrate dehydrogenase and H+ -transhydrogenase in heart mitochondria }}, doi = {10.1042/bst021260s}, volume = {21}, year = {1993}, } @article{1950, author = {Jackson, Julie and Cotton, N P J and Williams, Ross and Bizouarn, Tania and Hutton, Mike and Sazanov, Leonid A and Thomas, Christopher}, issn = {0300-5127}, journal = {Biochemical Society Transactions}, number = {4}, pages = {1010 -- 1013}, publisher = {Portland Press}, title = {{Proton-translocating transhydrogenase in bacteria}}, doi = {10.1042/bst0211010}, volume = {21}, year = {1993}, } @article{1945, abstract = {The effects of ultra-low (10(-18)-10(-14) M) doses (ULD) of biologically active substances have been reviewed in terms of common regularities of ULD effects and peculiarities of action of various groups of compounds. The most common and at the same time paradoxical regularities of ULD action are bi- or polymodal patterns of dose dependence, absence or presence of an inverse effect at higher doses, and instability of ULD effect. Possible mechanisms of ULD action including the mechanism based on the adaptation theory are discussed.}, author = {Sazanov, Leonid A and Zaǐtsev, Sergei}, issn = {0006-2979}, journal = {Biochemistry (Moscow)}, number = {10}, pages = {1443 -- 1460}, publisher = {Izdatel'stvo Nauka}, title = {{Effect of superlow doses (10(-18)-10-(-14) M) of biologically active substances: general rules, features, and possible mechanisms}}, volume = {57}, year = {1992}, } @article{1946, abstract = {An ultra-low dose (10-14 M) of opioid peptide [D-Ala2]methionine enkephalinamide (DAMEA) is found to exert an inhibitory effect on the production of reactive oxygen species (respiratory burst) in human neutrophils. The validity of this phenomenon has been verified in a series of studies that comprised 30 experiments. The inhibition has proved to be statistically significant (P<0.001). The dose-response dependence of the effect (10-15-10-9 M) followed a characteristic biphasic pattern (with the maximum effect at ultra-low doses). An opioid antagonist, naloxone partially blocks the inhibitory effect, which indicates that the DAMEA action is at least partially mediated by opioid receptors.}, author = {Zaǐtsev, Sergei and Sazanov, Leonid A and Koshkin, Aleksei and Sud'Ina, Galina and Varfolomeev, Sergei}, issn = {0014-2956}, journal = {FEBS Letters}, number = {1}, pages = {84 -- 86}, publisher = {Elsevier}, title = {{Respiratory burst inhibition in human neutrophils by ultra-low doses of [D-Ala2] methionine enkephalinamide}}, doi = {10.1016/0014-5793(91)81109-L}, volume = {291}, year = {1991}, } @article{1941, author = {Sazanov, Leonid A and Karavaev, V A and Kukushkin, A K}, issn = {1990-7923}, journal = {Russian Journal of Physical Chemistry B}, pages = {3351 -- 3354}, publisher = {Elsevier}, title = {{Mathematical model of photosynthesis regulation accounts for the effects of changes in external conditions and observed oscillations}}, volume = {52}, year = {1988}, }