Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.5.3 (complex I)
8,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed Yarrowia lipolytica as a model system to study mitochondrial complex I that combines the application of fast and convenient yeast genetics with efficient structural and functional analysis of its very stable complex I isolated by his-tag affinity purification with high yield. Guided by a structural model based on homologies between complex I and [NiFe] hydrogenases mutational analysis revealed that the 49 kDa subunit plays a central functional role in complex I. We propose that critical parts of the catalytic core of complex I have evolved from the hydrogen reactive site of [NiFe] hydrogenases and that iron-sulfur cluster N2 resides at the interface between the 49 kDa and PSST subunits. These findings are in full agreement with the "semiquinone switch" mechanism according to which coupling of electron and proton transfer in complex I is achieved by a single integrated pump comprising cluster N2, the binding site for substrate ubiquinone, and a tightly bound quinone or quinoid group.
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PMID:Exploring the catalytic core of complex I by Yarrowia lipolytica yeast genetics. 1169 28

NADH-quinone (Q) oxidoreductase is a large and complex redox proton pump, which utilizes the free energy derived from oxidation of NADH with lipophilic electron/proton carrier Q to translocate protons across the membrane to generate an electrochemical proton gradient. Although its molecular mechanism is largely unknown, recent biochemical, biophysical, and molecular biological studies have revealed that particular subunits and cofactors play an essential role in the energy-coupling reaction. Based on these latest experimental data, we exhaustively analyzed the sequence information available from evolutionarily related enzymes such as [NiFe] hydrogenases. We found significant and conserved sequence differences in the PSST/Nqo6/NuoB, 49kDa/Nqo4/NuoD, and ND1/Nqo8/NuoH subunit homologs between complex I/NDH-1 and [NiFe] hydrogenases. The alterations, especially in the postulated ligand motif for cluster N2 in the PSST/Nqo6/NuoB subunits, appear to be evolutionarily important in determining the physiological function of complex I/NDH-1. These observations led us to propose a hypothetical evolutionary scheme: during the course of evolution, drastic changes have occurred in the putative cluster N2 binding site in the PSST/Nqo6/NuoB subunit and the progenitors of complex I/NDH-1 have concurrently become to utilize a lipophilic electron/proton carrier such as Q as its physiological substrate. This scheme provides new insights into the structure and function relationship of complex I/NDH-1 and may help us understand its energy-coupling mechanism.
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PMID:The origin of cluster N2 of the energy-transducing NADH-quinone oxidoreductase: comparisons of phylogenetically related enzymes. 1169 31

We have cloned and inactivated, by repeat-induced point mutations, the nuclear gene encoding the 19.3 kDa subunit of complex I (EC 1.6.5.3) from Neurospora crassa, the homologue of the bovine PSST polypeptide. Mitochondria from mutant nuo19.3 lack the peripheral arm of complex I while its membrane arm accumulates. Transformation with wild-type cDNA rescues this phenotype and assembly of complex I is restored. To interfere with assembly of a proposed bound iron-sulphur cluster, site-directed mutants were constructed by introducing cDNA with altered codons for two adjacent cysteines, Cys-101 and Cys-102. The mutant complexes were purified and their enzymic activities and EPR and UV/visible spectra were analysed. Either of the mutations abolishes assembly of iron-sulphur cluster N2, showing that this redox group is bound to the 19.3 kDa protein. We also observed an interference with the reduction of redox group X, suggesting that cluster N2 is the electron donor to this high-potential redox group.
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PMID:Disruption of iron-sulphur cluster N2 from NADH: ubiquinone oxidoreductase by site-directed mutagenesis. 1204 48

The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron-sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this 'catalytic core' region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump.
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PMID:Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism? 1278 86

Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of water-soluble [NiFe] hydrogenases.
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PMID:Two aspartic acid residues in the PSST-homologous NUKM subunit of complex I from Yarrowia lipolytica are essential for catalytic activity. 1293 Aug 34

NADH and Ca2+ have important regulatory functions in cardiomyocytes related to excitation-contraction coupling and ATP production. To elucidate elements of these functions, we examined the effect of NADH on sarcoplasmic reticulum (SR) Ca2+ release and the mechanisms of this regulation. Physiological concentrations of cytosolic NADH inhibited ryanodine receptor type 2 (RyR2)-mediated Ca2+-induced Ca2+ release (CICR) from SR membranes (IC50=120 micromol/L) and significantly lowered single channel open probability. In permeabilized single ventricular cardiomyocytes, NADH significantly inhibited the amplitude and frequency of spontaneous Ca2+ release. Blockers of electron transport prevented the inhibitory effect of NADH on CICR in isolated membranes and permeabilized cells, as well as on the activity of RyR2 channels reconstituted in lipid bilayer. An endogenous NADH oxidase activity from rat heart copurified with SR enriched with RyR2. A significant contribution by mitochondria was excluded as NADH oxidation by SR exhibited >9-fold higher catalytic activity (8.8 micromol/mg protein per minute) in the absence of exogenous mitochondrial complex I (ubiquinone) or complex III (cytochrome c) electron acceptors, but was inhibited by rotenone and pyridaben (IC50=2 to 3 nmol/L), antimycin A (IC50=13 nmol/L), and diphenyleneiodonium (IC50=28 micromol/L). Cardiac junctional SR treated with [3H](trifluoromethyl)diazirinyl-pyridaben specifically labeled a single 23-kDa PSST-like protein. These data indicate that NADH oxidation is tightly linked to, and essential for, negative regulation of the RyR2 complex and is a likely component of an important physiological negative-feedback mechanism coupling SR Ca2+ fluxes and mitochondrial energy production.
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PMID:NADH oxidase activity of rat cardiac sarcoplasmic reticulum regulates calcium-induced calcium release. 1500 40

The H(+)(Na(+))-translocating NADH-quinone (Q) oxidoreductase (NDH-1) of Escherichia coli is composed of 13 different subunits (NuoA-N). Subunit NuoA (ND3, Nqo7) is one of the seven membrane domain subunits that are considered to be involved in H(+)(Na(+)) translocation. We demonstrated that in the Paracoccus denitrificans NDH-1 subunit, Nqo7 (ND3) directly interacts with peripheral subunits Nqo6 (PSST) and Nqo4 (49 kDa) by using cross-linkers (Di Bernardo, S., and Yagi, T. (2001) FEBS Lett. 508, 385-388 and Kao, M.-C., Matsuno-Yagi, A., and Yagi, T. (2004) Biochemistry 43, 3750-3755). To investigate the structural and functional roles of conserved charged amino acid residues, a nuoA knock-out mutant and site-specific mutants K46A, E51A, D79N, D79A, E81Q, E81A, and D79N/E81Q were constructed by utilizing chromosomal DNA manipulation. In terms of immunochemical and NADH dehydrogenase activity-staining analyses, all site-specific mutants are similar to the wild type, suggesting that those NuoA site-specific mutations do not significantly affect the assembly of peripheral subunits in situ. In addition, site-specific mutants showed similar deamino-NADH-K(3)Fe(CN)(6) reductase activity to the wild type. The K46A mutation scarcely inhibited deamino-NADH-Q reductase activity. In contrast, E51A, D79A, D79N, E81A, and E81Q mutation partially suppressed deamino-NADH-Q reductase activity to 30, 90, 40, 40, and 50%, respectively. The double mutant D79N/E81Q almost completely lost the energy-transducing NDH-1 activities but did not display any loss of deamino-NADH-K(3)Fe(CN)(6) reductase activity. The possible functional roles of residues Asp-79 and Glu-81 were discussed.
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PMID:Functional roles of four conserved charged residues in the membrane domain subunit NuoA of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli. 1517 26

With 46 subunits, human mitochondrial complex I is the largest enzyme of the oxidative phosphorylation system. We have studied the assembly of complex I in cultured human cells. This will provide essential information about the nature of complex I deficiencies and will enhance our understanding of mitochondrial disease mechanisms. We have found that 143B206 rho zero cells, not containing mitochondrial DNA, are still able to form complex I subcomplexes. To further address the nature of these subcomplexes, we depleted 143B osteosarcoma cells of complex I by inhibiting mitochondrial protein translation with doxycycline. After removing this drug, complex I formation resumes and assembly intermediates were observed by two-dimensional blue native electrophoresis. Analysis of the observed subcomplexes indicates that assembly of human complex I is a semi-sequential process in which different preassembled subcomplexes are joined to form a fully assembled complex. The membrane part of the complex is formed in distinct steps. The B17 subunit is part of a subcomplex to which ND1, ND6 and PSST are subsequently added. This is bound to a hydrophilic subcomplex containing the 30 and 49 kDa subunits, to which a subcomplex including the 39 kDa subunit is incorporated, and later on the 18 and 24 kDa subunits. At a later stage more subunits, including the 15 kDa, are added and holo-complex I is formed. Our results suggest that human complex I assembly resembles that of Neurospora crassa, in which a membrane arm is formed and assembled to a preformed peripheral arm, and support ideas about modular evolution.
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PMID:Human mitochondrial complex I assembles through the combination of evolutionary conserved modules: a framework to interpret complex I deficiencies. 1531 50

While diagnosis and genetic analysis of mitochondrial disorders has made remarkable progress, we still do not understand how given molecular defects are correlated to specific patterns of symptoms and their severity. Towards resolving this dilemma for the largest and therefore most affected respiratory chain enzyme, we have established the yeast Yarrowia lipolytica as a eucaryotic model system to analyse respiratory chain complex I. For in vivo analysis, eYFP protein was attached to the 30-kDa subunit to visualize complex I and mitochondria. Deletions strains for nuclear coded subunits allow the reconstruction of patient alleles by site-directed mutagenesis and plasmid complementation. In most of the pathogenic mutations analysed so far, decreased catalytic activities, elevated K(M) values, and/or elevated I(50) values for quinone-analogous inhibitors were observed, providing plausible clues on the pathogenic process at the molecular level. Leigh mutations in the 49-kDa and PSST homologous subunits are found in regions that are at the boundaries of the ubiquinone-reducing catalytic core. This supports the proposed structural model and at the same time identifies novel domains critical for catalysis. Thus, Y. lipolytica is a useful lower eucaryotic model that will help to understand how pathogenic mutations in complex I interfere with enzyme function.
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PMID:Application of the yeast Yarrowia lipolytica as a model to analyse human pathogenic mutations in mitochondrial complex I (NADH:ubiquinone oxidoreductase). 1557 52

The obligate aerobic yeast Yarrowia lipolytica has been established as a powerful model system for the analysis of mitochondrial complex I. Using a combination of genomic and proteomic approaches, a total of 37 subunits was identified. Several of the accessory subunits are predicted to be STMD (single transmembrane domain) proteins. Site-directed mutagenesis of Y. lipolytica complex I has provided strong evidence that a significant part of the ubiquinone reducing catalytic core resides in the 49 kDa and PSST subunits and can be modelled using X-ray structures of distantly related enzymes, i.e. water-soluble [NiFe] hydrogenases from Desulfovibrio spp. Iron-sulphur cluster N2, which is related to the hydrogenase proximal cluster, is directly involved in quinone reduction. Mutagenesis of His226 and Arg141 of the 49 kDa subunit provided detailed insight into the structure-function relationships around cluster N2. Overall, our findings suggest that proton pumping by complex I employs long-range conformational interactions and ubiquinone intermediates play a critical role in this mechanism.
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PMID:Structure-function relationships in mitochondrial complex I of the strictly aerobic yeast Yarrowia lipolytica. 1604 11


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