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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)

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) was isolated from Escherichia coli by chromatographic steps performed in the presence of an alkylglucoside detergent at pH 6.0. The complex is obtained in a monodisperse state with a molecular mass of approximately 550,000 Da and is composed of 14 subunits. The subunits were assigned to the 14 genes of the nuo operon, partly based on their N-terminal sequences and partly on their apparent molecular masses. The preparation contains one noncovalently bound FMN/molecule. At least two binuclear (N1b and N1c) and three tetranuclear (N2, N3 and N4) iron-sulfur clusters were detected by EPR in the preparation when reduced with NADH. Their EPR characteristics remained mostly unaltered during the isolation process. After reconstitution in phospholipid membranes, the preparation catalyses piericidin-A-sensitive electron transfer from NADH to ubiquinone-2 with Km values similar to those of complex I in cytoplasmic membranes but with only 10% of the Vmax value. The isolated complex I was cleaved into three fragments when the pH was raised from 6.0 to 7.5 and the detergent exchanged to Triton X-100. One of these fragments is a water-soluble NADH dehydrogenase fragment which is composed of three subunits bearing at least four iron-sulfur clusters (N1b, N1c, N3 and N4) that can be reduced with NADH, one of them bearing FMN. The second, amphipathic, fragment, which is presumed to connect the NADH dehydrogenase fragment with the membrane, contains four subunits and at least one EPR-detectable iron-sulfur cluster whose spectral properties are reminiscent of the eucaryotic cluster N2. The third membrane fragment is composed of seven homologues of the mitochondrially encoded subunits of the eucaryotic complex I. This subunit arrangement coincidences to some extent with the order of the genes on the nuo operon. A topological model of the E. coli complex I is proposed.
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PMID:Isolation and characterization of the proton-translocating NADH: ubiquinone oxidoreductase from Escherichia coli. 760 27

The steady-state kinetics of the NADH dehydrogenase activity of the three-subunit flavo-iron-sulfur protein (FP, Type II NADH dehydrogenase) in the presence of the one-electron acceptor hexammineruthenium(III) (HAR) were studied. The maximal catalytic activities of FP with HAR as electron acceptor calculated on the basis of FMN content were found to be approximately the same for the submitochondrial particles, Complex I and purified FP. This result shows that the protein structure responsible for the primary NADH oxidation by FP is not altered during the isolation procedure and the lower (compared with Complex I) catalytic capacity of the enzyme previously reported was due to the use of inefficient electron acceptors. Simple assay procedures for NADH dehydrogenase activity with HAR as the electron acceptor are described. The maximal activity at saturating concentrations of HAR was insensitive to added guanidine, whereas at fixed concentration of the electron acceptor, guanidine stimulated oxidation of low concentrations of NADH and inhibited the reaction at saturating NADH. The inhibitory effect of guanidine was competitive with HAR. The double-reciprocal plots 1/v vs. 1/[NADH] at various HAR concentrations gave a series of straight lines intercepting on the ordinate. The plots 1/v vs. 1/[HAR] at various NADH concentrations gave a series of straight lines intercepting in the fourth quadrant. The kinetics support the mechanism of the overall reaction where NADH is oxidized by the protein-Ru(NH3)3+(6) complex in which positively charged electron acceptor is bound at the specific site close to FMN, thus stabilizing the flavosemiquinone intermediate.
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PMID:Kinetics of the mitochondrial three-subunit NADH dehydrogenase interaction with hexammineruthenium(III). 761 40

The Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus was extracted from the bacterial membranes and purified by ion exchange chromatographic procedures. The enzyme catalyzed NADH oxidation by suitable electron acceptors, e.g. menadione, and the Na+ and NADH-dependent reduction of ubiquinone-1. Four dominant bands and a number of minor bands were visible on SDS-PAGE that could be part of the enzyme complex. Flavin analyses indicated the presence of FAD but no FMN in the purified enzyme. FAD but no FMN were also present in V. alginolyticus membranes. FAD is therefore a prosthetic group of the Na(+)-translocating NADH:ubiquinone oxidoreductase and FMN is not present in the enzyme. The FAD was copurified with the NADH dehydrogenase. The purified enzyme exhibited an absorption spectrum with a maximum at 450 nm that is typical for a flavoprotein. Upon incubation with NADH this absorption disappeared indicating reduction of the enzyme-bound FAD.
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PMID:The Na(+)-translocating NADH:ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN. 764 53

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first of the respiratory complexes providing the proton motive force which is essential for the synthesis of ATP. Closely related forms of this complex exist in the mitochondria of eucaryotes and in the plasma membranes of purple bacteria. The minimal structural framework common to the mitochondrial and the bacterial complex is composed of 14 polypeptides with 1 FMN and 6-8 iron-sulfur clusters as prosthetic groups. The mitochondrial complex contains many accessory subunits for which no homologous counterparts exist in the bacterial complex. Genes for 11 of the 14 minimal subunits are also found in the plastidial DNA of plants and in the genome of cyanobacteria. However, genes encoding the 3 subunits of the NADH dehydrogenase part of complex I are apparently missing in these species. The possibility is discussed that chloroplasts and cyanobacteria contain a complex I equipped with a different electron input device. This complex may work as a NAD(P)H: or a ferredoxin:plastoquinone oxidoreductase participating in cyclic electron transport during photosynthesis.
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PMID:The proton-pumping respiratory complex I of bacteria and mitochondria and its homologue in chloroplasts. 779 4

Enzymically active subcomplexes were purified from bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I) by sucrose-gradient centrifugation in the presence of detergents. These subcomplexes, named I lambda, IS, and I lambda S, catalyse ferricyanide and ubiquinone-1 (Q-1) reduction by NADH at similar rates to complex I, but do not catalyse the reduction of decylubiquinone. In addition, the Q-1 reductase activity of all the subcomplexes is insensitive to rotenone. Chemical and EPR analyses of the subcomplexes show that FMN and all the Fe-S clusters of complex I are present, but that the line shape of cluster 2 is modified. The smallest subcomplex, I lambda S, contains only approximately 13 subunits, as compared to approximately 22 in the previously described subcomplex I alpha [Finel, M., Skehel, J. M., Albracht, S. J. P., Fearnley, I. M. & Walker, J. E. (1992) Biochemistry 31, 11425-11434], but it retains the 75-, 51-, 49-, 30-, 24-, 23- (TYKY) and 20-kDa (PSST) subunits, which are suggested to form a functional core that comprises the EPR-detectable Fe-S clusters 1-4, and FMN. The structural and functional implications of such an arrangement are discussed.
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PMID:Isolation and characterisation of subcomplexes of the mitochondrial NADH:ubiquinone oxidoreductase (complex I). 795 54

This paper reports the first direct characterization of flavin (noncovalently bound FMN) in energy coupling site I of the mitochondrial respiratory chain. Thermodynamic parameters of its redox reactions were determined potentiometrically monitoring the g = 2.005 signal of its free radical form in isolated bovine heart NADH:ubiquinone oxidoreductase (complex I). The midpoint redox potentials of consecutive one-electron reduction steps are Em1/0 = -414 mV and Em2/1 = -336 mV at pH 7.5. This corresponds to a stability constant of the intermediate flavosemiquinone state of 4.5 x 10(-2). The pK values of the free radical (Fl.-<==>FlH.) and reduced flavin (FlH-<==>FlH2) were estimated as 7.7 and 7.1, respectively. The potentiometrically obtained g = 2.005 flavin free radical EPR signal revealed an unusually broad (2.4 mT) and pH-independent peak-to-peak line width. The spin relaxation of flavosemiquinone in complex I is much faster than that of flavodoxin due to strong dipole-dipole interaction with iron-sulfur cluster N3. Guanidine, an activator of NADH-ferricyanide reductase activity of complex I, was found to have a strong stabilizing effect on the flavin free radical generated both by equilibration with the NADH/NAD+ redox couple and by potentiometric redox titration. The addition of guanidine also leads to a slight modification of the EPR spectrum of iron-sulfur cluster N3. Anaerobic titration of flavosemiquinone free radical with the strictly n = 2 NADH/NAD+ and APADH/APAD+ redox couples revealed that nucleotide binding narrows the EPR signal line width of the flavin free radical to 1.7 mT and changes a shape of the titration curve.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thermodynamic analysis of flavin in mitochondrial NADH:ubiquinone oxidoreductase (complex I). 806 Sep 76

Diphenyleneiodonium (DPI) inhibits the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) on the substrate side of the Fe-S clusters. In the inhibited NADH-supplemented state all of the Fe-S clusters are oxidized, whereas the reduced minus oxidized difference spectrum of the protein-bound FMN can be visualized. It is characterized by troughs at 370 and 450 nm and a small increase of absorbance in the 500-700-nm region. DPI probably reacts irreversibly with FMN, because oxidation of FMN is blocked even after its extraction from the enzyme. Inhibition requires preincubation of enzyme in the presence of NADH and DPI. The lower the NADH/NAD+ ratio or the pH, or the higher the NAD+/DPI ratio, the more DPI is required for inhibition. NAD+ and DPI apparently compete for a common site. Both ubiquinone and dichlorophenolindophenol reductase activities are fully blocked by DPI, whereas the ferricyanide reductase activity is inhibited by 75%. Similar results were found with Complex I and two rotenone-insensitive preparations, subcomplex I lambda and the flavoprotein fraction. DPI also inhibits NADH oxidation by bacterial NADH-ubiquinone oxidoreductase-1 (NDH-1) in membranes of Paracoccus denitrificans and Escherichia coli.
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PMID:Diphenyleneiodonium inhibits reduction of iron-sulfur clusters in the mitochondrial NADH-ubiquinone oxidoreductase (Complex I). 806 22

The electron carriers of the mitochondrial NADH:ubiquinone oxidoreductase (complex I) are contained predominately in two extramembranous subcomplexes, a flavoprotein (FP) and an iron-sulfur protein (IP). FP contains three subunits with molecular masses of 51, 24, and 9 kDa. The 51-kDa subunit carries the NADH binding site and contains FMN and a tetranuclear iron-sulfur cluster. The 24-kDa subunit contains a binuclear iron-sulfur cluster. IP contains seven subunits with molecular masses of 75, 49, 30, 18, 15, 13, and 11 kDa. It contains a tetranuclear and very likely a binuclear iron-sulfur cluster in the 75-kDa subunit. FP and IP make contact through the 51- and the 75-kDa subunits. The remainder of complex I (hydrophobic protein (HP), 31 subunits) is largely membrane-intercalated and contains two iron-sulfur clusters apparently in a 23-kDa subunit and possibly another in a 20-kDa subunit. In this study, the stoichiometries of the FP and IP subunits in complex I were determined by radioimmunoassay. Per mole of complex I, there are 2 mol of the 15-kDa subunit and 1 mol each of the FP and the four largest IP subunits. The stoichiometries of the 13- and the 11-kDa subunits could not be determined separately, because they comigrate upon gel electrophoresis. In addition, the effect of substrates (NADH, NADPH, NAD, and NADH plus potassium ferricyanide to rapidly oxidize NADH via FP) on the cross-linking patterns of FP and IP subunits was investigated, using three different cross-linking reagents of different molecular lengths.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytic sector of complex I (NADH:ubiquinone oxidoreductase): subunit stoichiometry and substrate-induced conformation changes. 816 12

The NADH:ubiquinone oxidoreductase (complex I) is made up of a peripheral part and a membrane part. The two parts are arranged perpendicular to each other and give the complex an unusual L-shaped structure. The peripheral part protrudes into the matrix space and constitutes the proximal segment of the electron pathway with the NADH-binding site, the FMN and at least three iron-sulfur clusters. The membrane part constitutes the distal segment of the electron pathway with at least one iron-sulfur cluster and the ubiquinone-binding site. Both parts are assembled separately and relationships of the major structural modules of the two parts with different bacterial enzymes suggest, that both parts also emerged independently in evolution. This assumption is further supported by the conserved order of bacterial complex I genes, which correlates with the topological arrangement of the corresponding subunits in the two parts of complex I.
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PMID:Attempts to define distinct parts of NADH:ubiquinone oxidoreductase (complex I). 822 14

The 3-subunit iron-sulfur flavoprotein (NADH-artificial electron acceptor oxidoreductase) derived from complex I (EC 1.6.5.3) is rapidly and irreversibly inactivated in the presence of NADH. The rate of inactivation increases with a decrease of the enzyme concentration. The activities with ferricyanide, menadione and cytochrome c were lost synchronously during preincubation of the enzyme in the presence of NADH or dithionite under either aerobic or anaerobic conditions. The titration of the inactivation rate with the NADH/NAD+ pair suggests that reduction of a component with Em' = -325 mV (n = 2) is a prerequisite for a loss of the enzyme activity. Among the compounds tested only FMN and NAD+ were able to protect the enzyme against the reductive inactivation. NADH-induced loss of the enzyme activity in diluted solutions is accompanied with the synchronous appearance of a fluorescence characteristic for free FMN. It is concluded that the reduction of flavin leads to a strong decrease of FMN affinity to its specific binding site, and possible implications of the redox-dependent affinity changes in operation of NADH-ubiquinone reductase are discussed.
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PMID:Reductive inactivation of the mitochondrial three subunit NADH dehydrogenase. 839 15


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