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1.
Br J Cancer ; 122(3): 421-433, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31772326

RESUMO

BACKGROUND: We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function. METHODS: LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression. RESULTS: LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects. CONCLUSIONS: We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options.


Assuntos
Carcinoma de Células Renais/metabolismo , Ferro/metabolismo , Neoplasias Renais/metabolismo , Lipocalina-2/metabolismo , RNA Mensageiro/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Feminino , Humanos , Técnicas In Vitro , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Lipocalina-2/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Espectrofotometria Atômica , Esferoides Celulares , Células Tumorais Cultivadas
2.
Biochim Biophys Acta Bioenerg ; 1859(5): 366-373, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29501404

RESUMO

The NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory chain and the entry point for most electrons. Generally, the bacterial complex I consists of 14 core subunits, homologues of which are also found in complex I of mitochondria. In complex I preparations from the hyperthermophilic bacterium Aquifex aeolicus we have identified 20 partially homologous subunits by combining MALDI-TOF and LILBID mass spectrometry methods. The subunits could be assigned to two different complex I isoforms, named NQOR1 and NQOR2. NQOR1 consists of subunits NuoA2, NuoB, NuoD2, NuoE, NuoF, NuoG, NuoI1, NuoH1, NuoJ1, NuoK1, NuoL1, NuoM1 and NuoN1, with an entire mass of 504.17 kDa. NQOR2 comprises subunits NuoA1, NuoB, NuoD1, NuoE, NuoF, NuoG, NuoH2, NuoI2, NuoJ1, NuoK1, NuoL2, NuoM2 and NuoN2, with a total mass of 523.99 kDa. Three Fe-S clusters could be identified by EPR spectroscopy in a preparation containing predominantly NQOR1. These were tentatively assigned to a binuclear center N1, and two tetranuclear centers, N2 and N4. The redox midpoint potentials of N1 and N2 are -273 mV and -184 mV, respectively. Specific activity assays indicated that NQOR1 from cells grown under low concentrations of oxygen was the more active form. Increasing the concentration of oxygen in the bacterial cultures induced formation of NQOR2 showing the lower specific activity.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/sangue , Complexo I de Transporte de Elétrons/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
J Med Genet ; 54(5): 346-356, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28031252

RESUMO

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.


Assuntos
Mutação/genética , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber/genética , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Estudos de Casos e Controles , Bovinos , Sequência Conservada/genética , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Feminino , Fibroblastos/metabolismo , Haplótipos/genética , Heterozigoto , Humanos , Masculino , Mitocôndrias/genética , Proteínas Mutantes/metabolismo , NADH Desidrogenase/química , Oftalmoscopia , Linhagem , Fenótipo , Tomografia de Coerência Óptica , Yarrowia/metabolismo
4.
Proc Natl Acad Sci U S A ; 112(18): 5685-90, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25902503

RESUMO

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 , Espectrofotometria
5.
Biochim Biophys Acta Bioenerg ; 1858(2): 175-181, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27871794

RESUMO

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/metabolismo
6.
Proc Natl Acad Sci U S A ; 111(14): 5207-12, 2014 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-24706851

RESUMO

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/metabolismo
7.
Arch Biochem Biophys ; 580: 75-83, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26116786

RESUMO

Manganese-induced toxicity has been recently associated with an increased ROS generation from mitochondrial complex II (succinate:ubiquinone oxidoreductase). To achieve a deeper mechanistic understanding how divalent manganese ions (Mn(2+)) could stimulate mitochondrial ROS production we performed investigations with bovine heart submitochondrial particles (SMP). In succinate fueled SMP, the Mn(2+) induced hydrogen peroxide (H2O2) production was blocked by the specific complex II ubiquinone binding site (IIQ) inhibitor atpenin A5 while a further downstream block at complex III increased the rate markedly. This suggests that site IIQ was the source of the reactive oxygen species. Moreover, Mn(2+) ions also accelerated the rate of superoxide dismutation, explaining the general increase in the measured rates of H2O2 production and an attenuation of direct superoxide detection.


Assuntos
Complexo II de Transporte de Elétrons/metabolismo , Peróxido de Hidrogênio/metabolismo , Manganês/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Partículas Submitocôndricas/efeitos dos fármacos , Animais , Cátions Bivalentes , Bovinos , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Membranas Mitocondriais/metabolismo , Piridonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Partículas Submitocôndricas/metabolismo , Ácido Succínico/metabolismo , Ácido Succínico/farmacologia , Superóxido Dismutase/metabolismo , Ubiquinona/antagonistas & inibidores , Ubiquinona/metabolismo
8.
Proc Natl Acad Sci U S A ; 109(9): 3275-80, 2012 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-22334648

RESUMO

The cytochrome c oxidase Cox2 has been purified from native membranes of the hyperthermophilic eubacterium Aquifex aeolicus. It is a cytochrome ba(3) oxidase belonging to the family B of the heme-copper containing terminal oxidases. It consists of three subunits, subunit I (CoxA2, 63.9 kDa), subunit II (CoxB2, 16.8 kDa), and an additional subunit IIa of 5.2 kDa. Surprisingly it is able to oxidize both reduced cytochrome c and ubiquinol in a cyanide sensitive manner. Cox2 is part of a respiratory chain supercomplex. This supercomplex contains the fully assembled cytochrome bc(1) complex and Cox2. Although direct ubiquinol oxidation by Cox2 conserves less energy than ubiquinol oxidation by the cytochrome bc(1) complex followed by cytochrome c oxidation by a cytochrome c oxidase, ubiquinol oxidation by Cox2 is of advantage when all ubiquinone would be completely reduced to ubiquinol, e.g., by the sulfidequinone oxidoreductase, because the cytochrome bc(1) complex requires the presence of ubiquinone to function according to the Q-cycle mechanism. In the case that all ubiquinone has been reduced to ubiquinol its reoxidation by Cox2 will enable the cytochrome bc(1) complex to resume working.


Assuntos
Proteínas de Bactérias/metabolismo , Citocromos c/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ubiquinona/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/isolamento & purificação , Crescimento Quimioautotrófico , Cobre , Cianetos/farmacologia , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/classificação , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Elétrons , Metabolismo Energético , Heme , Dados de Sequência Molecular , Complexos Multienzimáticos , Oxirredução , Subunidades Proteicas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ubiquinona/metabolismo
9.
PLoS Biol ; 9(8): e1001128, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21886480

RESUMO

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/metabolismo
10.
EMBO J ; 27(12): 1736-46, 2008 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-18497740

RESUMO

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Yarrowia/metabolismo , Cisteína/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Deleção de Genes , Ferro/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Filogenia , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Biochem J ; 437(2): 279-88, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21545356

RESUMO

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/enzimologia
12.
Biochim Biophys Acta ; 1797(12): 1883-90, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20493164

RESUMO

Complex I (NADH:ubiquinone oxidoreductase) has a central function in oxidative phosphorylation and hence for efficient ATP production in most prokaryotic and eukaryotic cells. This huge membrane protein complex transfers electrons from NADH to ubiquinone and couples this exergonic redox reaction to endergonic proton pumping across bioenergetic membranes. Although quinone reduction seems to be critical for energy conversion, this part of the reaction is least understood. Here we summarize and discuss experimental evidence indicating that complex I contains an extended ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron-sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal ß-sheet of the 49-kDa subunit. We discuss the possible functions of a highly conserved HRGXE motif and a redox-Bohr group associated with cluster N2. Resistance patterns observed with a large number of point mutations suggest that all types of hydrophobic complex I inhibitors also act at the interface of the 49-kDa and the PSST subunit. Finally, current controversies regarding the number of ubiquinone binding sites and the position of the site of ubiquinone reduction are discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Thermus thermophilus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Benzoquinonas/química , Sítios de Ligação , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/genética , Modelos Moleculares , Mutação , Oxirredução , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
13.
Biochim Biophys Acta ; 1797(6-7): 625-32, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20117074

RESUMO

Iron-sulfur cluster N2 of complex I (proton pumping NADH:quinone oxidoreductase) is the immediate electron donor to ubiquinone. At a distance of only approximately 7A in the 49-kDa subunit, a highly conserved tyrosine is found at the bottom of the previously characterized quinone binding pocket. To get insight into the function of this residue, we have exchanged it for six different amino acids in complex I from Yarrowia lipolytica. Mitochondrial membranes from all six mutants contained fully assembled complex I that exhibited very low dNADH:ubiquinone oxidoreductase activities with n-decylubiquinone. With the most conservative exchange Y144F, no alteration in the electron paramagnetic resonance spectra of complex I was detectable. Remarkably, high dNADH:ubiquinone oxidoreductase activities were observed with ubiquinones Q1 and Q2 that were coupled to proton pumping. Apparent Km values for Q1 and Q2 were markedly increased and we found pronounced resistance to the complex I inhibitors decyl-quinazoline-amine (DQA) and rotenone. We conclude that Y144 directly binds the head group of ubiquinone, most likely via a hydrogen bond between the aromatic hydroxyl and the ubiquinone carbonyl. This places the substrate in an ideal distance to its electron donor iron-sulfur cluster N2 for efficient electron transfer during the catalytic cycle of complex I.


Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ubiquinona/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência Conservada , Espectroscopia de Ressonância de Spin Eletrônica , Complexo I de Transporte de Elétrons/genética , Proteínas Fúngicas/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxirredução , Ligação Proteica , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Tirosina/química , Yarrowia/genética , Yarrowia/metabolismo
14.
Acc Chem Res ; 43(2): 181-9, 2010 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-19842617

RESUMO

Metalloproteins often contain metal centers that are paramagnetic in some functional state of the protein; hence electron paramagnetic resonance (EPR) spectroscopy can be a powerful tool for studying protein structure and function. Dipolar spectroscopy allows the determination of the dipole-dipole interactions between metal centers in protein complexes, revealing the structural arrangement of different paramagnetic centers at distances of up to 8 nm. Hyperfine spectroscopy can be used to measure the interaction between an unpaired electron spin and nuclear spins within a distance of 0.8 nm; it therefore permits the characterization of the local structure of the paramagnetic center's ligand sphere with very high precision. In this Account, we review our laboratory's recent applications of both dipolar and hyperfine pulsed EPR methods to metalloproteins. We used pulsed dipolar relaxation methods to investigate the complex of cytochrome c and cytochrome c oxidase, a noncovalent protein-protein complex involved in mitochondrial electron-transfer reactions. Hyperfine sublevel correlation spectroscopy (HYSCORE) was used to study the ligand sphere of iron-sulfur clusters in complex I of the mitochondrial respiratory chain and substrate binding to the molybdenum enzyme polysulfide reductase. These examples demonstrate the potential of the two techniques; however, they also highlight the difficulties of data interpretation when several paramagnetic species with overlapping spectra are present in the protein. In such cases, further approaches and data are very useful to enhance the information content. Relaxation filtered hyperfine spectroscopy (REFINE) can be used to separate the individual components of overlapping paramagnetic species on the basis of differences in their longitudinal relaxation rates; it is applicable to any kind of pulsed hyperfine or dipolar spectroscopy. Here, we show that the spectra of the iron-sulfur clusters in complex I can be separated by this method, allowing us to obtain hyperfine (and dipolar) information from the individual species. Furthermore, performing pulsed EPR experiments at different magnetic fields is another important tool to disentangle the spectral components in such complex systems. Despite the fact that high magnetic fields do not usually lead to better spectral separation for metal centers, they provide additional information about the relative orientation of different paramagnetic centers. Our high-field EPR studies on cytochrome c oxidase reveal essential information regarding the structural arrangement of the binuclear Cu(A) center with respect to both the manganese ion within the enzyme and the cytochrome in the protein-protein complex with cytochrome c.


Assuntos
Proteínas de Bactérias/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Proteínas Fúngicas/química , Metaloproteínas/química , Paracoccus denitrificans/química , Yarrowia/química , Cobre/química , Citocromos c/química , Citocromos c/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ferro/química , Metaloproteínas/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
15.
Biochim Biophys Acta ; 1787(6): 584-92, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19366602

RESUMO

Electron Paramagnetic Resonance (EPR) spectroscopy is the method of choice to study paramagnetic cofactors that often play an important role as active centers in electron transfer processes in biological systems. However, in many cases more than one paramagnetic species is contributing to the observed EPR spectrum, making the analysis of individual contributions difficult and in some cases impossible. With time-domain techniques it is possible to exploit differences in the relaxation behavior of different paramagnetic species to distinguish between them and separate their individual spectral contribution. Here we give an overview of the use of pulsed EPR spectroscopy to study the iron-sulfur clusters of NADH:ubiquinone oxidoreductase (complex I). While FeS cluster N1 can be studied individually at a temperature of 30 K, this is not possible for FeS cluster N2 due to its severe spectral overlap with cluster N1. In this case Relaxation Filtered Hyperfine (REFINE) spectroscopy can be used to separate the overlapping spectra based on differences in their relaxation behavior.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Complexo I de Transporte de Elétrons/química , Proteínas Fúngicas/química , Proteínas Ferro-Enxofre/química , Mitocôndrias/enzimologia , Estrutura Molecular , Yarrowia/enzimologia
16.
Biochim Biophys Acta ; 1787(6): 574-83, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19366614

RESUMO

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and remains by far the least understood enzyme complex of the respiratory chain. It consists of a peripheral arm harbouring all known redox active prosthetic groups and a membrane arm with a yet unknown number of proton translocation sites. The ubiquinone reduction site close to iron-sulfur cluster N2 at the interface of the 49-kDa and PSST subunits has been mapped by extensive site directed mutagenesis. Independent lines of evidence identified electron transfer events during reduction of ubiquinone to be associated with the potential drop that generates the full driving force for proton translocation with a 4H(+)/2e(-) stoichiometry. Electron microscopic analysis of immuno-labelled native enzyme and of a subcomplex lacking the electron input module indicated a distance of 35-60 A of cluster N2 to the membrane surface. Resolution of the membrane arm into subcomplexes showed that even the distal part harbours subunits that are prime candidates to participate in proton translocation because they are homologous to sodium/proton antiporters and contain conserved charged residues in predicted transmembrane helices. The mechanism of redox linked proton translocation by complex I is largely unknown but has to include steps where energy is transmitted over extremely long distances. In this review we compile the available structural information on complex I and discuss implications for complex I function.


Assuntos
Complexo I de Transporte de Elétrons/química , Proteínas Fúngicas/química , Domínio Catalítico , Cristalografia por Raios X , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Imageamento Tridimensional , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas , Bombas de Próton/química , Bombas de Próton/metabolismo , Yarrowia/enzimologia
17.
Nat Commun ; 11(1): 6008, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33243981

RESUMO

Respiratory complex I catalyzes electron transfer from NADH to ubiquinone (Q) coupled to vectorial proton translocation across the inner mitochondrial membrane. Despite recent progress in structure determination of this very large membrane protein complex, the coupling mechanism is a matter of ongoing debate and the function of accessory subunits surrounding the canonical core subunits is essentially unknown. Concerted rearrangements within a cluster of conserved loops of central subunits NDUFS2 (ß1-ß2S2 loop), ND1 (TMH5-6ND1 loop) and ND3 (TMH1-2ND3 loop) were suggested to be critical for its proton pumping mechanism. Here, we show that stabilization of the TMH1-2ND3 loop by accessory subunit LYRM6 (NDUFA6) is pivotal for energy conversion by mitochondrial complex I. We determined the high-resolution structure of inactive mutant F89ALYRM6 of eukaryotic complex I from the yeast Yarrowia lipolytica and found long-range structural changes affecting the entire loop cluster. In atomistic molecular dynamics simulations of the mutant, we observed conformational transitions in the loop cluster that disrupted a putative pathway for delivery of substrate protons required in Q redox chemistry. Our results elucidate in detail the essential role of accessory subunit LYRM6 for the function of eukaryotic complex I and offer clues on its redox-linked proton pumping mechanism.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades Proteicas/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/ultraestrutura , Proteínas Fúngicas/genética , Proteínas Fúngicas/ultraestrutura , Mutagênese Sítio-Dirigida , Oxirredução , Subunidades Proteicas/genética , Prótons , Ubiquinona/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
18.
Nat Commun ; 11(1): 1643, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242014

RESUMO

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Animais , Complexo I de Transporte de Elétrons/genética , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mioblastos/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
19.
Biochim Biophys Acta ; 1767(5): 393-400, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17448440

RESUMO

Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron-sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b.


Assuntos
Complexo I de Transporte de Elétrons/química , Yersinia/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavoproteínas/genética , Mutagênese Sítio-Dirigida , Deleção de Sequência
20.
Nat Commun ; 9(1): 4500, 2018 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-30374105

RESUMO

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain and a significant source of reactive oxygen species (ROS). We hypothesized that during energy conversion by complex I, electron transfer onto ubiquinone triggers the concerted rearrangement of three protein loops of subunits ND1, ND3, and 49-kDa thereby generating the power-stoke driving proton pumping. Here we show that fixing loop TMH1-2ND3 to the nearby subunit PSST via a disulfide bridge introduced by site-directed mutagenesis reversibly disengages proton pumping without impairing ubiquinone reduction, inhibitor binding or the Active/Deactive transition. The X-ray structure of mutant complex I indicates that the disulfide bridge immobilizes but does not displace the tip of loop TMH1-2ND3. We conclude that movement of loop TMH1-2ND3 located at the ubiquinone-binding pocket is required to drive proton pumping corroborating one of the central predictions of our model for the mechanism of energy conversion by complex I proposed earlier.


Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/ultraestrutura , Bombas de Próton/química , Ubiquinona/química , Ubiquinona/ultraestrutura , Cristalografia por Raios X , Dissulfetos , Transporte de Elétrons , Complexo I de Transporte de Elétrons/genética , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Cinética , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Bombas de Próton/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
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