RESUMO
Respiratory complex I is a redox-driven proton pump. Several high-resolution structures of complex I have been determined providing important information about the putative proton transfer paths and conformational transitions that may occur during catalysis. However, how redox energy is coupled to the pumping of protons remains unclear. In this article, we review biochemical, structural and molecular simulation data on complex I and discuss several coupling models, including the key unresolved mechanistic questions. Focusing both on the quinone-reductase domain as well as the proton-pumping membrane-bound domain of complex I, we discuss a molecular mechanism of proton pumping that satisfies most experimental and theoretical constraints. We suggest that protonation reactions play an important role not only in catalysis, but also in the physiologically-relevant active/deactive transition of complex I.
Assuntos
Complexo I de Transporte de Elétrons , Prótons , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Antiporters/metabolismo , Elétrons , Simulação de Dinâmica Molecular , Oxirredução , BenzoquinonasRESUMO
We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing a strong membrane curvature of â¼100°. Our structure explains the structural basis of cristae formation in mitochondria, a landmark signature of eukaryotic cell morphology.
Assuntos
Proteínas Fúngicas/química , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , ATPases Mitocondriais Próton-Translocadoras/química , Yarrowia/enzimologia , Trifosfato de Adenosina/metabolismo , Catálise , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestrutura , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Subunidades Proteicas , Relação Estrutura-Atividade , Yarrowia/ultraestruturaRESUMO
The mitochondrial acyl carrier protein (ACPM/NDUFAB1) is a central element of the mitochondrial fatty acid synthesis type II machinery. Originally ACPM was detected as a subunit of respiratory complex I but the reason for the association with the large enzyme complex remained elusive. Complex I from the aerobic yeast Yarrowia lipolytica comprises two different ACPMs, ACPM1 and ACPM2. They are anchored to the protein complex by LYR (leucine-tyrosine-arginine) motif containing protein (LYRM) subunits LYRM3 (NDUFB9) and LYRM6 (NDUFA6). The ACPM1-LYRM6 and ACPM2-LYRM3 modules are essential for complex I activity and assembly/stability, respectively. We show that in addition to the complex I bound fraction, ACPM1 is present as a free matrix protein and in complex with the soluble LYRM4(ISD11)/NFS1 complex implicated in Fe-S cluster biogenesis. We show that the presence of a long acyl chain bound to the phosphopantetheine cofactor is important for docking ACPMs to protein complexes and we propose that association of ACPMs and LYRMs is universally based on a new protein-protein interaction motif.
Assuntos
Proteína de Transporte de Acila/genética , Proteínas Fúngicas/genética , Proteínas Ferro-Enxofre/genética , Mitocôndrias/metabolismo , Complexos Multiproteicos/química , Proteína de Transporte de Acila/metabolismo , Sequência de Aminoácidos/genética , Complexo I de Transporte de Elétrons/genética , Ácidos Graxos/biossíntese , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/química , Mitocôndrias/genética , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Yarrowia/metabolismoRESUMO
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.
Assuntos
Mitocôndrias/metabolismo , NADH Desidrogenase/química , Zinco/química , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Complexo I de Transporte de Elétrons/metabolismo , Eletroforese , Deleção de Genes , Humanos , Membranas Mitocondriais/metabolismo , Chaperonas Moleculares/química , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteômica , EspectrofotometriaRESUMO
Mitochondrial complex I is an intricate 1MDa membrane protein complex with a central role in aerobic energy metabolism. The minimal form of complex I consists of fourteen central subunits that are conserved from bacteria to man. In addition, eukaryotic complex I comprises some 30 accessory subunits of largely unknown function. The gene for the accessory NDUFS4 subunit of human complex I is a hot spot for fatal pathogenic mutations in humans. We have deleted the gene for the orthologous NUYM subunit in the aerobic yeast Yarrowia lipolytica, an established model system to study eukaryotic complex I and complex I linked diseases. We observed assembly of complex I which lacked only subunit NUYM and retained weak interaction with assembly factor N7BML (human NDUFAF2). Absence of NUYM caused distortion of iron sulfur clusters of the electron input domain leading to decreased complex I activity and increased release of reactive oxygen species. We conclude that NUYM has an important stabilizing function for the electron input module of complex I and is essential for proper complex I function.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , NADH Desidrogenase/metabolismo , Yarrowia/metabolismo , Elétrons , Metabolismo Energético/fisiologia , Proteínas Fúngicas/metabolismo , Humanos , Mitocôndrias/metabolismo , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Proton-pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of the respiratory chain. Fourteen central subunits represent the minimal form of complex I and can be assigned to functional modules for NADH oxidation, ubiquinone reduction, and proton pumping. In addition, the mitochondrial enzyme comprises some 30 accessory subunits surrounding the central subunits that are not directly associated with energy conservation. Complex I is known to release deleterious oxygen radicals (ROS) and its dysfunction has been linked to a number of hereditary and degenerative diseases. We here review recent progress in structure determination, and in understanding the role of accessory subunits and functional analysis of mitochondrial complex I. For the central subunits, structures provide insight into the arrangement of functional modules including the substrate binding sites, redox-centers and putative proton channels and pump sites. Only for two of the accessory subunits, detailed structures are available. Nevertheless, many of them could be localized in the overall structure of complex I, but most of these assignments have to be considered tentative. Strikingly, redox reactions and proton pumping machinery are spatially completely separated and the site of reduction for the hydrophobic substrate ubiquinone is found deeply buried in the hydrophilic domain of the complex. The X-ray structure of complex I from Yarrowia lipolytica provides clues supporting the previously proposed two-state stabilization change mechanism, in which ubiquinone redox chemistry induces conformational states and thereby drives proton pumping. The same structural rearrangements may explain the active/deactive transition of complex I implying an integrated mechanistic model for energy conversion and regulation. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/ultraestrutura , Proteínas Mitocondriais/química , Proteínas Mitocondriais/ultraestrutura , Bombas de Próton/química , Espécies Reativas de Oxigênio/síntese química , Sequência de Aminoácidos , Transporte de Elétrons , Ativação Enzimática , Modelos Químicos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Oxirredução , Conformação Proteica , Estrutura Terciária de Proteína , Bombas de Próton/ultraestrutura , Relação Estrutura-AtividadeRESUMO
Mitochondrial complex I is a 1MDa membrane protein complex with a central role in aerobic energy metabolism. The bioenergetic core functions are executed by 14 central subunits that are conserved from bacteria to man. Despite recent progress in structure determination, our understanding of the function of the ~30 accessory subunits associated with the mitochondrial complex is still limited. We have investigated the structure of complex I from the aerobic yeast Yarrowia lipolytica by cryo-electron microscopy. Our density map at 7.9Å resolution closely matches the 3.6-3.9Å X-ray structure of the Yarrowia lipolytica complex. However, the cryo-EM map indicated an additional subunit on the side of the matrix arm above the membrane surface, pointing away from the membrane arm. The density, which is not present in any previously described complex I structure and occurs in about 20 % of the particles, was identified as the accessory sulfur transferase subunit ST1. The Yarrowia lipolytica complex I preparation is active in generating H2S from the cysteine derivative 3-mercaptopyruvate, catalyzed by ST1. We thus provide evidence for a link between respiratory complex I and mitochondrial sulfur metabolism.
Assuntos
Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético , Proteínas Fúngicas/metabolismo , Mitocôndrias/enzimologia , Transferases de Grupos de Enxofre/metabolismo , Enxofre/metabolismo , Yarrowia/enzimologia , Catálise , Cisteína/análogos & derivados , Cisteína/metabolismo , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/ultraestrutura , Sulfeto de Hidrogênio/metabolismo , Mitocôndrias/ultraestrutura , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Transferases de Grupos de Enxofre/química , Transferases de Grupos de Enxofre/genética , Transferases de Grupos de Enxofre/ultraestrutura , Yarrowia/genética , Yarrowia/ultraestruturaRESUMO
Mitochondrial complex I is the largest and most complicated enzyme of the oxidative phosphorylation system. It comprises a number of so-called accessory subunits of largely unknown structure and function. Here we studied subunit NB4M [NDUFA6, LYR motif containing protein 6 (LYRM6)], a member of the LYRM family of proteins. Chromosomal deletion of the corresponding gene in the yeast Yarrowia lipolytica caused concomitant loss of the mitochondrial acyl carrier protein subunit ACPM1 from the enzyme complex and paralyzed ubiquinone reductase activity. Exchanging the LYR motif and an associated conserved phenylalanine by alanines in subunit NB4M also abolished the activity and binding of subunit ACPM1. We show, by single-particle electron microscopy and structural modeling, that subunits NB4M and ACPM1 form a subdomain that protrudes from the peripheral arm in the vicinity of central subunit domains known to be involved in controlling the catalytic activity of complex I.
Assuntos
Proteína de Transporte de Acila/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/metabolismo , Biocatálise , Espectroscopia de Ressonância de Spin Eletrônica , Yarrowia/metabolismoRESUMO
Regulation of mtDNA expression is critical for maintaining cellular energy homeostasis and may, in principle, occur at many different levels. The leucine-rich pentatricopeptide repeat containing (LRPPRC) protein regulates mitochondrial mRNA stability and an amino-acid substitution of this protein causes the French-Canadian type of Leigh syndrome (LSFC), a neurodegenerative disorder characterized by complex IV deficiency. We have generated conditional Lrpprc knockout mice and show here that the gene is essential for embryonic development. Tissue-specific disruption of Lrpprc in heart causes mitochondrial cardiomyopathy with drastic reduction in steady-state levels of most mitochondrial mRNAs. LRPPRC forms an RNA-dependent protein complex that is necessary for maintaining a pool of non-translated mRNAs in mammalian mitochondria. Loss of LRPPRC does not only decrease mRNA stability, but also leads to loss of mRNA polyadenylation and the appearance of aberrant mitochondrial translation. The translation pattern without the presence of LRPPRC is misregulated with excessive translation of some transcripts and no translation of others. Our findings point to the existence of an elaborate machinery that regulates mammalian mtDNA expression at the post-transcriptional level.
Assuntos
Deficiência de Citocromo-c Oxidase/genética , Doença de Leigh/genética , Mitocôndrias Cardíacas/fisiologia , Proteínas de Neoplasias/fisiologia , Poliadenilação/fisiologia , Biossíntese de Proteínas/fisiologia , Animais , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/análise , Células HeLa , Humanos , Substâncias Macromoleculares , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Especificidade de Órgãos , Polinucleotídeo Adenililtransferase , Estabilidade de RNA , RNA Mensageiro , Proteínas de Ligação a RNA/metabolismoRESUMO
Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8mΔ) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na(+)/H(+) antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8mΔ. Nevertheless, nb8mΔ still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Mitocondriais/metabolismo , Bombas de Próton/metabolismo , Yarrowia/genética , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Ensaios Enzimáticos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Deleção de Genes , Técnicas de Inativação de Genes , Microscopia Eletrônica , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Peso Molecular , Conformação Proteica , Bombas de Próton/química , Yarrowia/metabolismoRESUMO
We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F(1) heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Substâncias Macromoleculares/metabolismo , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Bovinos , Complexo I de Transporte de Elétrons/ultraestrutura , Fungos/enzimologia , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Multimerização Proteica , Solanum tuberosum/enzimologia , TomografiaRESUMO
Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.
Assuntos
Complexo I de Transporte de Elétrons , Mitocôndrias , Humanos , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Microscopia Crioeletrônica , Mitocôndrias/metabolismoRESUMO
Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, the accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here, we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two large membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with titratable amino acids modeled in their standard protonation (charged) states, the structure diverges far from its starting conformation. In comparison, MD simulations performed with predetermined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results support the notion that it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to the launch of any conventional MD simulations. Furthermore, the combined approach of fast protonation state prediction and MD simulations can provide valuable information about the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states in proteinaceous environments currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions but also the atomic modeling of density data.
Assuntos
Proteínas de Membrana , Simulação de Dinâmica Molecular , Prótons , Aminoácidos/química , Conformação Molecular , Conformação ProteicaRESUMO
Platelets are the critical target for preventing and treating pathological thrombus formation. However, despite current antiplatelet therapy, cardiovascular mortality remains high, and cardiovascular events continue in prescribed patients. In this study, first results were obtained with ortho-carbonyl hydroquinones as antiplatelet agents; we found that linking triphenylphosphonium cation to a bicyclic ortho-carbonyl hydroquinone moiety by a short alkyl chain significantly improved their antiplatelet effect by affecting the mitochondrial functioning. The mechanism of action involves uncoupling OXPHOS, which leads to an increase in mitochondrial ROS production and a decrease in the mitochondrial membrane potential and OCR. This alteration disrupts the energy production by mitochondrial function necessary for the platelet activation process. These effects are responsive to the complete structure of the compounds and not to isolated parts of the compounds tested. The results obtained in this research can be used as the basis for developing new antiplatelet agents that target mitochondria.
Assuntos
Plaquetas , Hidroquinonas , Potencial da Membrana Mitocondrial , Compostos Organofosforados , Inibidores da Agregação Plaquetária , Humanos , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Hidroquinonas/farmacologia , Hidroquinonas/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Compostos Organofosforados/química , Fosforilação Oxidativa/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondrial complex I has a molecular mass of almost 1 MDa and comprises more than 40 polypeptides. Fourteen central subunits harbour the bioenergetic core functions. We are only beginning to understand the significance of the numerous accessory subunits. The present review addresses the role of accessory subunits for assembly, stability and regulation of complex I and for cellular functions not directly associated with redox-linked proton translocation.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexos Multiproteicos/química , Conformação Proteica , Bombas de Próton/química , Metabolismo Energético , Humanos , Oxirredução , Estrutura Terciária de Proteína , Subunidades Proteicas/química , PrótonsRESUMO
Respiratory complex I in mitochondria and bacteria catalyzes the transfer of electrons from NADH to quinone (Q). The free energy available from the reaction is used to pump protons and to establish a membrane proton electrochemical gradient, which drives ATP synthesis. Even though several high-resolution structures of complex I have been resolved, how Q reduction is linked with proton pumping, remains unknown. Here, microsecond long molecular dynamics (MD) simulations were performed on Yarrowia lipolytica complex I structures where Q molecules have been resolved in the ~30 Å long Q tunnel. MD simulations of several different redox/protonation states of Q reveal the coupling between the Q dynamics and the restructuring of conserved loops and ion pairs. Oxidized quinone stabilizes towards the N2 FeS cluster, a binding mode not previously described in Yarrowia lipolytica complex I structures. On the other hand, reduced (and protonated) species tend to diffuse towards the Q binding sites closer to the tunnel entrance. Mechanistic and physiological relevance of these results are discussed.
Assuntos
Complexo I de Transporte de Elétrons , Prótons , Complexo I de Transporte de Elétrons/metabolismo , Quinonas , Benzoquinonas/metabolismoAssuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/ultraestrutura , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Modelos Químicos , Modelos Moleculares , Animais , Complexo I de Transporte de Elétrons/metabolismo , Ativação Enzimática , Humanos , Mitocôndrias/metabolismo , Modelos BiológicosRESUMO
Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.
Assuntos
Ácido Desoxicólico/química , Eletroforese em Gel de Poliacrilamida/métodos , Histidina/química , Proteínas de Membrana/análise , Complexos Multiproteicos/análise , Anabaena/química , Animais , Bovinos , Parede Celular/química , Cloroplastos/química , Cianobactérias/química , Coração , Espectrometria de Massas , Medicago sativa/química , Proteínas de Membrana/isolamento & purificação , Mitocôndrias/química , Complexos Multiproteicos/isolamento & purificação , Músculos/química , Pisum sativum/química , Transporte Proteico , Tilacoides/química , Yarrowia/químicaRESUMO
Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.
Assuntos
Complexo I de Transporte de Elétrons/química , Subunidades Proteicas/química , Bombas de Próton/química , Mitocôndrias/enzimologia , Modelos Moleculares , Subunidades Proteicas/metabolismo , Yarrowia/enzimologiaRESUMO
NADH:ubiquinone oxidoreductase (respiratory complex I) is a redox-driven proton pump with a central role in mitochondrial oxidative phosphorylation. The ubiquinone reduction site of complex I is located in the matrix arm of this large protein complex and connected to the membrane via a tunnel. A variety of chemically diverse compounds are known to inhibit ubiquinone reduction by complex I. Rotenone, piericidin A, and annonaceous acetogenins are representatives of complex I inhibitors from biological sources. The structure of complex I is determined at high resolution, and inhibitor binding sites are described in detail. In this review, we summarize the state of knowledge of how natural inhibitors bind in the Q reduction site and the Q access pathway and how their inhibitory mechanisms compare with that of a synthetic anti-cancer agent.