Respiratory complex I with charge symmetry in the membrane arm pumps protons.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.

Pulse-chase SILAC-based analyses reveal selective oversynthesis and rapid turnover of mitochondrial protein components of respiratory complexes.

Mammalian mitochondria assemble four complexes of the respiratory chain (RCI, RCIII, RCIV, and RCV) by combining 13 polypeptides synthesized within mitochondria on mitochondrial ribosomes (mitoribosomes) with over 70 polypeptides encoded in nuclear DNA, translated on cytoplasmic ribosomes, and imported into mitochondria. We have previously observed that mitoribosome assembly is inefficient because some mitoribosomal proteins are produced in excess, but whether this is the case for other mitochondrial assemblies such as the RCs is unclear. We report here that pulse-chase stable isotope labeling with amino acids in cell culture (SILAC) is a valuable technique to study RC assembly because it can reveal considerable differences in the assembly rates and efficiencies of the different complexes. The SILAC analyses of HeLa cells indicated that assembly of RCV, comprising F1/Fo-ATPase, is rapid with little excess subunit synthesis, but that assembly of RCI (i.e. NADH dehydrogenase) is far less efficient, with dramatic oversynthesis of numerous proteins, particularly in the matrix-exposed N and Q domains. Unassembled subunits were generally degraded within 3 h. We also observed differential assembly kinetics for individual complexes that were immunoprecipitated with complex-specific antibodies. Immunoprecipitation with an antibody that recognizes the ND1 subunit of RCI co-precipitated a number of proteins implicated in FeS cluster assembly and newly synthesized ubiquinol-cytochrome c reductase Rieske iron-sulfur polypeptide 1 (UQCRFS1), the Rieske FeS protein in RCIII, reflecting some coordination between RCI and RCIII assemblies. We propose that pulse-chase SILAC labeling is a useful tool for studying rates of protein complex assembly and degradation.

Contribution of mitochondrial ND1 3394T>C mutation to the phenotypic manifestation of Leber's hereditary optic neuropathy.

Mitochondrial DNA (mtDNA) mutations have been associated with Leber's hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we investigated the pathophysiology of a LHON susceptibility allele (m.3394T>C, p.30Y>H) in the Mitochondrial (MT)-ND1 gene. The incidence of m.3394T>C mutation was 2.7% in the cohort of 1741 probands with LHON. Extremely low penetrances of LHON were observed in 26 pedigrees carrying only m.3394T>C mutation, while 21 families bearing m.3394T>C, together with m.11778G>A or m.14484T>C mutation, exhibited higher penetrance of LHON than those in families carrying single mtDNA mutation(s). The m.3394T>C mutation disrupted the specific electrostatic interactions between Y30 of p.MT-ND1 with the sidechain of E4 and backbone carbonyl group of M1 of NDUFA1 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 1) of complex I, thereby altering the structure and function of complex I. We demonstrated that these cybrids bearing only m.3394T>C mutation caused mild mitochondrial dysfunctions and those harboring both m.3394T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions than cybrids carrying only m.11778G>A mutation. In particular, the m.3394T>C mutation altered the stability of p.MT-ND1 and complex I assembly. Furthermore, the m.3394T>C mutation decreased the activities of mitochondrial complexes I, diminished mitochondrial ATP levels and membrane potential and increased the production of reactive oxygen species in the cybrids. These m.3394T>C mutation-induced alterations aggravated mitochondrial dysfunctions associated with the m.11778G>A mutation. These resultant biochemical defects contributed to higher penetrance of LHON in these families carrying both mtDNA mutations. Our findings provide new insights into the pathophysiology of LHON arising from the synergy between mitochondrial ND1 and ND4 mutations.

Peculiar combinations of individually non-pathogenic missense mitochondrial DNA variants cause low penetrance Leber's hereditary optic neuropathy.

We here report on the existence of Leber's hereditary optic neuropathy (LHON) associated with peculiar combinations of individually non-pathogenic missense mitochondrial DNA (mtDNA) variants, affecting the MT-ND4, MT-ND4L and MT-ND6 subunit genes of Complex I. The pathogenic potential of these mtDNA haplotypes is supported by multiple evidences: first, the LHON phenotype is strictly inherited along the maternal line in one very large family; second, the combinations of mtDNA variants are unique to the two maternal lineages that are characterized by recurrence of LHON; third, the Complex I-dependent respiratory and oxidative phosphorylation defect is co-transferred from the proband's fibroblasts into the cybrid cell model. Finally, all but one of these missense mtDNA variants cluster along the same predicted fourth E-channel deputed to proton translocation within the transmembrane domain of Complex I, involving the ND1, ND4L and ND6 subunits. Hence, the definition of the pathogenic role of a specific mtDNA mutation becomes blurrier than ever and only an accurate evaluation of mitogenome sequence variation data from the general population, combined with functional analyses using the cybrid cell model, may lead to final validation. Our study conclusively shows that even in the absence of a clearly established LHON primary mutation, unprecedented combinations of missense mtDNA variants, individually known as polymorphisms, may lead to reduced OXPHOS efficiency sufficient to trigger LHON. In this context, we introduce a new diagnostic perspective that implies the complete sequence analysis of mitogenomes in LHON as mandatory gold standard diagnostic approach.

Solvent-Induced Protein Precipitation for Drug Target Discovery on the Proteomic Scale.

High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug-protein interaction by using complex cell lysate directly without any modification of the drugs. In this study, we developed a novel energetics-based proteomics strategy, the solvent-induced protein precipitation (SIP) approach, to profile the interaction of drugs with their target proteins by using quantitative proteomics. The method is easy to use for any laboratory with the common chemical reagents of acetone, ethanol, and acetic acid. The SIP approach was able to identify the well-known protein targets of methotrexate, SNS-032, and a pan-kinase inhibitor of staurosporine in cell lysate. We further applied this approach to discover the off-targets of geldanamycin. Three known protein targets of the HSP90 family were successfully identified, and several potential off-targets including NADH dehydrogenase subunits NDUFV1 and NDUFAB1 were identified for the first time, and the NDUFV1 was validated by using Western blotting. In addition, this approach was capable of evaluating the affinity of the drug-target interaction. The data collectively proved that our approach provides a powerful platform for drug target discovery.

LMAP_S: Lightweight Multigene Alignment and Phylogeny eStimation.

BACKGROUND: Recent advances in genome sequencing technologies and the cost drop in high-throughput sequencing continue to give rise to a deluge of data available for downstream analyses. Among others, evolutionary biologists often make use of genomic data to uncover phenotypic diversity and adaptive evolution in protein-coding genes. Therefore, multiple sequence alignments (MSA) and phylogenetic trees (PT) need to be estimated with optimal results. However, the preparation of an initial dataset of multiple sequence file(s) (MSF) and the steps involved can be challenging when considering extensive amount of data. Thus, it becomes necessary the development of a tool that removes the potential source of error and automates the time-consuming steps of a typical workflow with high-throughput and optimal MSA and PT estimations. RESULTS: We introduce LMAP_S (Lightweight Multigene Alignment and Phylogeny eStimation), a user-friendly command-line and interactive package, designed to handle an improved alignment and phylogeny estimation workflow: MSF preparation, MSA estimation, outlier detection, refinement, consensus, phylogeny estimation, comparison and editing, among which file and directory organization, execution, manipulation of information are automated, with minimal manual user intervention. LMAP_S was developed for the workstation multi-core environment and provides a unique advantage for processing multiple datasets. Our software, proved to be efficient throughout the workflow, including, the (unlimited) handling of more than 20 datasets. CONCLUSIONS: We have developed a simple and versatile LMAP_S package enabling researchers to effectively estimate multiple datasets MSAs and PTs in a high-throughput fashion. LMAP_S integrates more than 25 software providing overall more than 65 algorithm choices distributed in five stages. At minimum, one FASTA file is required within a single input directory. To our knowledge, no other software combines MSA and phylogeny estimation with as many alternatives and provides means to find optimal MSAs and phylogenies. Moreover, we used a case study comparing methodologies that highlighted the usefulness of our software. LMAP_S has been developed as an open-source package, allowing its integration into more complex open-source bioinformatics pipelines. LMAP_S package is released under GPLv3 license and is freely available at https://lmap-s.sourceforge.io/.

The global motion affecting electron transfer in Plasmodium falciparum type II NADH dehydrogenases: a novel non-competitive mechanism for quinoline ketone derivative inhibitors.

With the emergence of drug-resistant Plasmodium falciparum, the treatment of malaria has become a significant challenge; therefore, the development of antimalarial drugs acting on new targets is extremely urgent. In Plasmodium falciparum, type II nicotinamide adenine dinucleotide (NADH) dehydrogenase (NDH-2) is responsible for catalyzing the transfer of two electrons from NADH to flavin adenine dinucleotide (FAD), which in turn transfers the electrons to coenzyme Q (CoQ). As an entry enzyme for oxidative phosphorylation, NDH-2 has become one of the popular targets for the development of new antimalarial drugs. In this study, reliable motion trajectories of the NDH-2 complex with its co-factors (NADH and FAD) and inhibitor, RYL-552, were obtained by comparative molecular dynamics simulations. The influence of cofactor binding on the global motion of NDH-2 was explored through conformational clustering, principal component analysis and free energy landscape. The molecular interactions of NDH-2 before and after its binding with the inhibitor RYL-552 were analyzed, and the key residues and important hydrogen bonds were also determined. The results show that the association of RYL-552 results in the weakening of intramolecular hydrogen bonds and large allosterism of NDH-2. There was a significant positive correlation between the angular change of the key pocket residues in the NADH-FAD-pockets that represents the global functional motion and the change in distance between NADH-C4 and FAD-N5 that represents the electron transfer efficiency. Finally, the possible non-competitive inhibitory mechanism of RYL-552 was proposed. Specifically, the association of inhibitors with NDH-2 significantly affects the global motion mode of NDH-2, leading to widening of the distance between NADH and FAD through cooperative motion induction; this reduces the electron transfer efficiency of the mitochondrial respiratory chain. The simulation results provide useful theoretical guidance for subsequent antimalarial drug design based on the NDH-2 structure and the respiratory chain electron transfer mechanism.

Identification of the Catalytic Ubiquinone-binding Site of Vibrio cholerae Sodium-dependent NADH Dehydrogenase: A NOVEL UBIQUINONE-BINDING MOTIF.

The sodium-dependent NADH dehydrogenase (Na+-NQR) is a key component of the respiratory chain of diverse prokaryotic species, including pathogenic bacteria. Na+-NQR uses the energy released by electron transfer between NADH and ubiquinone (UQ) to pump sodium, producing a gradient that sustains many essential homeostatic processes as well as virulence factor secretion and the elimination of drugs. The location of the UQ binding site has been controversial, with two main hypotheses that suggest that this site could be located in the cytosolic subunit A or in the membrane-bound subunit B. In this work, we performed alanine scanning mutagenesis of aromatic residues located in transmembrane helices II, IV, and V of subunit B, near glycine residues 140 and 141. These two critical glycine residues form part of the structures that regulate the site's accessibility. Our results indicate that the elimination of phenylalanine residue 211 or 213 abolishes the UQ-dependent activity, produces a leak of electrons to oxygen, and completely blocks the binding of UQ and the inhibitor HQNO. Molecular docking calculations predict that UQ interacts with phenylalanine 211 and pinpoints the location of the binding site in the interface of subunits B and D. The mutagenesis and structural analysis allow us to propose a novel UQ-binding motif, which is completely different compared with the sites of other respiratory photosynthetic complexes. These results are essential to understanding the electron transfer pathways and mechanism of Na+-NQR catalysis.

The role of climatic cycles and trans-Saharan migration corridors in species diversification: Biogeography of Psammophis schokari group in North Africa.

Highlands, hydrographic systems and coastal areas have been hypothesised to form corridors across the hyperarid Sahara desert in North Africa, allowing dispersal and gene flow for non-xeric species. Here we aim to provide a genetic test for the trans-Saharan corridor model, and predict the location and stability of ecological-corridors, by combining phylogeography and palaeoclimatic modelling. The model was the Psammophis schokari (Schokari sand racer) group, fast-moving and widely distributed generalist colubrids occurring mostly in arid and semiarid scrublands. We combined dated phylogenies of mitochondrial and nuclear markers with palaeoclimatic modelling. For the phylogeographic analysis, we used 75 samples of P. schokari and P. aegyptius, and Bayesian and Maximum-Likelihood methods. For the ecological models, we used Maxent over the distribution of P. schokari and West African lineages. Models were projected to past conditions (mid Holocene, Last Glacial Maximum and Last Inter-Glacial) to infer climatic stable areas. Climatic stability was predicted to be mostly restricted to coastal areas and not spatially continuous. A putative temporary trans-Saharan corridor was identified in Eastern Sahara, with a more stable one along the Atlantic coast. Six parapatric lineages were identified within P. schokari, four occurring in North Africa. These likely diverged during the Pliocene. The Tamanraset River might have been a vicariant agent. African lineages may have experienced further subsequent diversification during the late Pleistocene. The main P. schokari refugia were probably located along the northern margins of the Sahara, allowing its North-to-South colonization. Trans-Saharan corridors seem to have played a role in P. schokari biogeography, allowing colonization of central Saharan mountains and Sahel. Some might have worked as refugia, and even the most stable corridors may have sections working as filters, depending on each climatic phase. We expect the use of trans-Saharan corridors to decrease for more mesic species or with less dispersal capabilities.

'Tethering' fragment-based drug discovery to identify inhibitors of the essential respiratory membrane protein type II NADH dehydrogenase.

Energy generation is a promising area of drug discovery for both bacterial pathogens and parasites. Type II NADH dehydrogenase (NDH-2), a vital respiratory membrane protein, has attracted attention as a target for the development of new antitubercular and antimalarial agents. To date, however, no potent, specific inhibitors have been identified. Here, we performed a site-directed screening technique, tethering-fragment based drug discovery, against wild-type and mutant forms of NDH-2 containing engineered active-site cysteines. Inhibitory fragments displayed IC50 values between 3 and 110 µM against NDH-2 mutants. Possible binding poses were investigated by in silico modelling, providing a basis for optimisation of fragment binding and improved potency against NDH-2.

Compound heterozygosity for severe and hypomorphic NDUFS2 mutations cause non-syndromic LHON-like optic neuropathy.

BACKGROUND: Non-syndromic hereditary optic neuropathy (HON) has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function. However, the disease causing gene remains unknown in many families. The objective of the present study was to identify the molecular cause of non-syndromic LHON-like disease in siblings born to non-consanguineous parents of French origin. METHODS: We used a combination of genetic analysis (gene mapping and whole-exome sequencing) in a multiplex family of non-syndromic HON and of functional analyses in patient-derived cultured skin fibroblasts and the yeast Yarrowia lipolytica. RESULTS: We identified compound heterozygote NDUFS2 disease-causing mutations (p.Tyr53Cys; p.Tyr308Cys). Studies using patient-derived cultured skin fibroblasts revealed mildly decreased NDUFS2 and complex I abundance but apparently normal respiratory chain activity. In the yeast Y. lipolytica ortholog NUCM, the mutations resulted in absence of complex I and moderate reduction in nicotinamide adenine dinucleotide-ubiquinone oxidoreductase activity, respectively. CONCLUSIONS: Biallelism for NDUFS2 mutations causing severe complex I deficiency has been previously reported to cause Leigh syndrome with optic neuropathy. Our results are consistent with the view that compound heterozygosity for severe and hypomorphic NDUFS2 mutations can cause non-syndromic HON. This observation suggests a direct correlation between the severity of NDUFS2 mutations and that of the disease and further support that there exist a genetic overlap between non-syndromic and syndromic HON due to defective mitochondrial function.

Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I.

Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or mutation of Zn-binding residues blocked a late step of complex I assembly. An accumulating assembly intermediate lacked accessory subunit N7BM (NDUFA12), whereas a paralog of this subunit, the assembly factor N7BML (NDUFAF2), was found firmly bound instead. EPR spectroscopic analysis and metal content determination after chromatographic purification of the assembly intermediate showed that NUMM is required for insertion or stabilization of FeS cluster N4.

Unravelling the Effects of the Mutation m.3571insC/MT-ND1 on Respiratory Complexes Structural Organization.

Mammalian respiratory complex I (CI) biogenesis requires both nuclear and mitochondria-encoded proteins and is mostly organized in respiratory supercomplexes. Among the CI proteins encoded by the mitochondrial DNA, NADH-ubiquinone oxidoreductase chain 1 (ND1) is a core subunit, evolutionary conserved from bacteria to mammals. Recently, ND1 has been recognized as a pivotal subunit in maintaining the structural and functional interaction among the hydrophilic and hydrophobic CI arms. A critical role of human ND1 both in CI biogenesis and in the dynamic organization of supercomplexes has been depicted, although the proof of concept is still missing and the critical amount of ND1 protein necessary for a proper assembly of both CI and supercomplexes is not defined. By exploiting a unique model in which human ND1 is allotopically re-expressed in cells lacking the endogenous protein, we demonstrated that the lack of this protein induces a stall in the multi-step process of CI biogenesis, as well as the alteration of supramolecular organization of respiratory complexes. We also defined a mutation threshold for the m.3571insC truncative mutation in mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1), below which CI and its supramolecular organization is recovered, strengthening the notion that a certain amount of human ND1 is required for CI and supercomplexes biogenesis.

In Silico Discovery of a Substituted 6-Methoxy-quinalidine with Leishmanicidal Activity in Leishmania infantum.

There is an urgent need for the discovery of new antileishmanial drugs with a new mechanism of action. Type 2 NADH dehydrogenase from Leishmania infantum (LiNDH2) is an enzyme of the parasite's respiratory system, which catalyzes the electron transfer from NADH to ubiquinone without coupled proton pumping. In previous studies of the related NADH: ubiquinone oxidoreductase crystal structure from Saccharomyces cerevisiae, two ubiquinone-binding sites (UQI and UQII) were identified and shown to play an important role in the NDH-2-catalyzed oxidoreduction reaction. Based on the available structural data, we developed a three-dimensional structural model of LiNDH2 using homology detection methods and performed an in silico virtual screening campaign to search for potential inhibitors targeting the LiNDH2 ubiquinone-binding site 1-UQI. Selected compounds displaying favorable properties in the computational screening experiments were assayed for inhibitory activity in the structurally similar recombinant NDH-2 from S. aureus and leishmanicidal activity was determined in the wild-type axenic amastigotes and promastigotes of L. infantum. The identified compound, a substituted 6-methoxy-quinalidine, showed promising nanomolar leishmanicidal activity on wild-type axenic promastigotes and amastigotes of L. infantum and the potential for further development.

The plant defensin NaD1 introduces membrane disorder through a specific interaction with the lipid, phosphatidylinositol 4,5 bisphosphate.

Plant defensins interact with phospholipids in bilayers as part of their cytotoxic activity. Solanaceous class II defensins with the loop 5 sequence pattern "S-[KR]-[ILVQ]-[ILVQ]-[KR]-[KR]" interact with PI(4,5)P2. Here, the prototypical defensin of this class, NaD1, is used to characterise the biophysical interactions between these defensins and phospholipid bilayers. Binding of NaD1 to bilayers containing PI(4,5)P2 occurs rapidly and the interaction is very strong. Dual polarisation interferometry revealed that NaD1 does not dissociate from bilayers containing PI(4,5)P2. Binding of NaD1 to bilayers with or without PI(4,5)P2 induced disorder in the bilayer. However, permeabilisation assays revealed that NaD1 only permeabilised liposomes with PI(4,5)P2 in the bilayer, suggesting a role for this protein-lipid interaction in the plasma membrane permeabilising activity of this defensin. No defensins in the available databases have the PI(4,5)P2 binding sequence outside the solanaceous class II defensins, leading to the hypothesis that PI(4,5)P2 binding co-evolved with the C-terminal propeptide to protect the host cell against the effects of the tight binding of these defensins to their cognate lipid as they travel along the secretory pathway. This data has allowed us to develop a new model to explain how this class of defensins permeabilises plasma membranes to kill target cells.

Characterization of clinically identified mutations in NDUFV1, the flavin-binding subunit of respiratory complex I, using a yeast model system.

Dysfunctions in mitochondrial complex I (NADH:ubiquinone oxidoreductase) are both genetically and clinically highly diverse and a major cause of human mitochondrial diseases. The genetic determinants of individual clinical cases are increasingly being described, but how these genetic defects affect complex I on the molecular and cellular level, and have different clinical consequences in different individuals, is little understood. Furthermore, without molecular-level information innocent genetic variants may be misassigned as pathogenic. Here, we have used a yeast model system (Yarrowia lipolytica) to study the molecular consequences of 16 single amino acid substitutions, classified as pathogenic, in the NDUFV1 subunit of complex I. NDUFV1 binds the flavin cofactor that oxidizes NADH and is the site of complex I-mediated reactive oxygen species production. Seven mutations caused loss of complex I expression, suggesting they are detrimental but precluding further study. In two variants complex I was fully assembled but did not contain any flavin, and four mutations led to functionally compromised enzymes. Our study provides a molecular rationale for assignment of all these variants as pathogenic. However, three variants provided complex I that was functionally equivalent to the wild-type enzyme, challenging their assignment as pathogenic. By combining structural, bioinformatic and functional data, a simple scoring system for the initial evaluation of future NDUFV1 variants is proposed. Overall, our results broaden understanding of how mutations in this centrally important core subunit of complex I affect its function and provide a basis for understanding the role of NDUFV1 mutations in mitochondrial dysfunction.

Identification and characterization of the novel point mutation m.3634A>G in the mitochondrial MT-ND1 gene associated with LHON syndrome.

Leber's hereditary optic neuropathy (LHON) is a mitochondrial genetic disease characterized by bilateral acute or subacute progressive central visual loss. Most cases of LHON syndrome are caused by point mutations in the MT-ND1, MT-ND4, and MT-ND6 genes. Here, we report a novel homoplasmic mutation in the MT-ND1 gene (m.3634A>G, p.Ser110Gly) in a patient with the classical clinical features of LHON syndrome. Several observations support the idea that the mutation is pathogenic and involved in the clinical phenotype of the patient: 1) The mutation affected a highly conserved amino acid, 2) A pathogenic mutation in the same amino acid (m.3635G>A, p.Ser110Asn) was previously reported in a patient with LHON syndrome, 3) The mutation is not recorded in the Mitomap or Human Mitochondrial Genome Database, 4) In silico predictors classified the mutation as "probably damaging", and 5) Cybrids carrying the mutation showed decreased Complex I enzyme activity, lower cell proliferation, and decreased mitochondrial membrane potential relative to control cybrids.

Structure of the membrane domain of respiratory complex I.

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 Å 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.

Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I.

Complex I serves as the primary electron entry point into the mitochondrial and bacterial respiratory chains. It catalyzes the reduction of quinones by electron transfer from NADH, and couples this exergonic reaction to the translocation of protons against an electrochemical proton gradient. The membrane domain of the enzyme extends ∼180 Šfrom the site of quinone reduction to the most distant proton pathway. To elucidate possible mechanisms of the long-range proton-coupled electron transfer process, we perform large-scale atomistic molecular dynamics simulations of the membrane domain of complex I from Escherichia coli. We observe spontaneous hydration of a putative proton entry channel at the NuoN/K interface, which is sensitive to the protonation state of buried glutamic acid residues. In hybrid quantum mechanics/classical mechanics simulations, we find that the observed water wires support rapid proton transfer from the protein surface to the center of the membrane domain. To explore the functional relevance of the pseudosymmetric inverted-repeat structures of the antiporter-like subunits NuoL/M/N, we constructed a symmetry-related structure of a possible alternate-access state. In molecular dynamics simulations, we find the resulting structural changes to be metastable and reversible at the protein backbone level. However, the increased hydration induced by the conformational change persists, with water molecules establishing enhanced lateral connectivity and pathways for proton transfer between conserved ionizable residues along the center of the membrane domain. Overall, the observed water-gated transitions establish conduits for the unidirectional proton translocation processes, and provide a possible coupling mechanism for the energy transduction in complex I.

Substrate-Protein Interactions of Type II NADH:Quinone Oxidoreductase from Escherichia coli.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD(+) balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate-protein interaction in NDH-2 from Escherichia coli (EcNDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of EcNDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate.