Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.5 (NADH dehydrogenase)
2,135 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 dissimilar subunits which are designated NQO1-14 and contains one noncovalently bound FMN and at least five EPR-visible iron-sulfur clusters (N1a, N1b, N2, N3, and N4) as prosthetic groups. Comparison of the deduced primary structures of the subunits with consensus sequences for the cofactor binding sites has predicted that NQO1, NQO2, NQO3, NQO9, and probably NQO6 subunits are cofactor binding subunits. Previously, we have reported that the NQO2 (25 kDa) subunit was overexpressed as a water-soluble protein in Escherichia coli and was found to ligate a single [2Fe-2S] cluster with rhombic symmetry (gx,y,z = 1.92, 1.95, and 2.00) (Yano, T., Sled', V.D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). In the present study, the NQO3 (66 kDa) subunit, which is equivalent to the 75-kDa subunit of bovine heart Complex I, was overexpressed in E. coli. The expressed NQO3 subunit was found predominantly in the cytoplasmic phase and was purified by ammonium sulfate fractionation and anion-exchange chromatography. The chemical analyses and UV-visible and EPR spectroscopic studies showed that the expressed NQO3 subunit contains at least two distinct iron-sulfur clusters: a [2Fe-2S] cluster with axial EPR signals (g perpendicular, parallel = 1.934 and 2.026, and L perpendicular parallel = 1.8 and 3.0 millitesla) and a [4Fe-4S] cluster with rhombic symmetry (gx,y,z = 1.892, 1.928, and 2.063, and Lx,y,z = 2.40, 1.55, and 1.75 millitesla). The midpoint redox potentials of [2Fe-2S] and [4Fe-4S] clusters at pH 8.6 are -472 and -391 mV, respectively. The tetranuclear cluster in the isolated NQO3 subunit is sensitive toward oxidants and converts into [3Fe-4S] form. The assignment of these iron-sulfur clusters to those identified in the P. denitrificans NDH-1 enzyme complex and the possible functional role of the NQO3 subunit is discussed.
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PMID:Expression and characterization of the 66-kilodalton (NQO3) iron-sulfur subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans. 762 45

EPR spectroscopy was used to investigate the cytochrome P-450-dependent steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafworm (Spodoptera littoralis) and the redox centres associated with membranes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been assigned to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bound) was tentatively assigned to a signal at g = 8.0, which was detectable from membranes as prepared. An EPR signal characteristic of a [2Fe-2S] cluster detected from the soluble mitochondrial matrix fraction has been shown to be distinct from the signals associated with mitochondrial NADH dehydrogenase and succinate dehydrogenase, and has therefore been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 20-hydroxylation comprises both ferredoxin and cytochrome P-450 components, and thus resembles the enzyme systems of adrenocortical mitochondria. EPR signals characteristic of the respiratory chain were also observed from fat-body mitochondria and assigned to the iron-sulphur clusters associated with Complex I (Centres N1, N2), Complex II (Centres S1, S3), Complex III (the Rieske centre), and the copper centre of Complex IV, demonstrating similarities to mammalian mitochondria. The reduced membrane fraction also yielded a major resonance at g = 2.09 and 1.88 characteristic of the [4Fe-4S] cluster of electron-transferring flavoprotein: ubiquinone oxidoreductase. As the fat-body is the major metabolic organ of insects, this protein is presumably required for the beta-oxidation of fatty acids in mitochondria. High-spin haem signals in the low-field region of spectra also demonstrated that the mitochondrial fraction contains relatively high concentrations of catalase.
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PMID:EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm). 774 2

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

Dopamine, due to metabolism by monoamine oxidase or autoxidation, can generate toxic products such as hydrogen peroxide, oxygen-derived radicals, semiquinones, and quinones and thus exert its neurotoxic effects. Intracerebroventricular injection of dopamine into rats pretreated with the monoamine oxidase nonselective inhibitor pargyline caused mortality in a dose-dependent manner with LD50 = 90 micrograms. Norepinephrine was less effective with LD50 = 141 micrograms. The iron chelator desferrioxamine completely protected against dopamine-induced mortality. In the absence of pargyline more rats survived, indicating that the products of dopamine enzymatic metabolism are not the main contributors to dopamine-induced toxicity. Biochemical analysis of frontal cortex and striatum from rats that received a lethal dose of dopamine did not show any difference from control rats in lipid and protein peroxidation and glutathione reductase and peroxidase activities. Moreover, dopamine significantly reduced the formation of iron-induced malondialdehyde in vitro, thus suggesting that earlier events in cell damage are involved in dopamine toxicity. Indeed, dopamine inhibited mitochondrial NADH dehydrogenase activity with IC50 = 8 microM, and that of norepinephrine was twice as much (IC50 = 15 microM). Dopamine-induced inhibition of NADH dehydrogenase activity was only partially reversed by desferrioxamine, which had no effect on norepinephrine-induced inhibition. These results suggest that catecholamines can cause toxicity not only by inducing an oxidative stress state but also possibly through direct interaction with the mitochondrial electron transport system. The latter was further supported by the ability of ADP to reverse dopamine-induced inhibition of NADH dehydrogenase activity in a dose-dependent manner.
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PMID:Dopamine neurotoxicity: inhibition of mitochondrial respiration. 783 65

In order to identify the ligand residues of the [2Fe-2S] cluster of the 25 kDa (NQO2) subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans, we mutated individually all seven cysteine residues (C61, C96, C101, C104, C113, C137, and C141) and one conserved histidine residue (H92) to Ser or Ala and expressed them in E. coli. After purification of the mutated 25 kDa subunits, the effect of mutations on the iron-sulfur cluster were characterized by chemical analyses and UV-visible and EPR spectroscopy. All mutated subunits, especially mutants of conserved cysteines, contained lower amounts of non-heme iron than wild-type. The subunits of three non-conserved cysteine residues (C61, C104, and C113) mutated to Ser and a histidine residue (H92) mutated to Ala exhibited essentially the same spectroscopic properties as those of the wild-type subunit. In contrast, mutation of the four conserved cysteine residues (C96, C101, C137, and C141) to Ser or Ala considerably altered the UV-visible and EPR spectra from the wild-type subunit. These results indicate that the four conserved cysteine residues coordinate the [2Fe-2S] cluster in the P. denitrificans 25 kDa subunit.
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PMID:Identification of amino acid residues associated with the [2Fe-2S] cluster of the 25 kDa (NQO2) subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans. 795 17

Until now ubisemiquinones associated with NADH:ubiquinone oxidoreductase (complex I) have been reported to occur in isolated enzyme and in tightly coupled submitochondrial particles. In this report it is shown that ubisemiquinones are always detectable during steady-state electron transfer from NADH to ubiquinone, independent of the type of inner-membrane preparation used. The EPR signal of the rotenone-sensitive ubisemiquinones could be detected not only in coupled MgATP submitochondrial particles, but also in routine preparations of uncoupled submitochondrial particles and in mitochondria. The ubisemiquinone formation in coupled preparations was completely insensitive to uncouplers. The maximal radical concentration during steady-state electron transfer from NADH to quinone was equal to that of iron-sulphur cluster 2. Experiments with antimycin, myxothiazol and 2-thenoyltrifluoroacetone demonstrated that about half of this radical was associated with complex I, giving a ubisemiquinone concentration of about 0.5 mol semiquinone/mol cluster 2. Uncoupled submitochondrial particles, prepared by extensive sonification, never showed radical signals within 100 ms after mixing with NADH. This was due to the reversible inactivation of the enzyme, caused by elevated temperatures during sonification. In preparations with deliberately heat-inactivated complex I, no radical signals were detected within 200 ms after mixing with NADH; at 1 s, however, radical formation was maximal. Yet, depending on the procedure of reactivation of the complex, in preparations previously treated to inactivate them ubisemiquinone concentrations were always less than in untreated particles. When complex I was in the active state the ubisemiquinone signal was maximal within 40 ms. The results described in this report lead to the conclusion that ubisemiquinones form obligatory intermediates in the reaction of NADH dehydrogenase with ubiquinone.
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PMID:Ubisemiquinones as obligatory intermediates in the electron transfer from NADH to ubiquinone. 802 8

The 25-kDa subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans has been expressed in Escherichia coli and purified to homogeneity. EPR studies of the reduced recombinant protein indicated that the expressed subunit contains a single [2Fe-2S] cluster (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). In this report, the electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center have been investigated by the combination of absorption, circular dichroism, variable-temperature magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopies and compared with a range of simple [2Fe-2S]-containing proteins. The results are consistent with coordination by two cysteinyl residues at both the reducible and nonreducible iron sites and reveal a striking similarity between the properties of the [2Fe-2S] cluster in the P. denitrificans NDH-1 25-kDa subunit and those of the subclass of ferredoxin-type [2Fe-2S] centers typified by Clostridium pasteurianum 2Fe ferredoxin. The four cyteines residues involved in cluster ligation in these proteins have been tentatively identified based on sequence homology considerations.
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PMID:Properties of the iron-sulfur center in the 25-kilodalton subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans. 806 21

We describe the structure and expression of a wheat mitochondrial gene, which codes for a subunit of mitochondrial NADH dehydrogenase. The deduced protein sequence has 70% similarity to the 30-kDa subunit of bovine mitochondrial complex I and 65% similarity to the 31-kDa subunit of Neurospora crassa complex I, components of the iron-sulfur-protein fraction, both nuclear-encoded proteins. We named this wheat mitochondrial gene as nad9. The wheat nad9 gene is transcribed in a single mRNA of 0.9 kb that is edited (C-to-U conversions) in 14 positions. Transcript mapping revealed that the first ATG codon is just 20 nucleotides downstream of the mRNA 5' end and that the 3' end is just 23 nucleotides downstream of the nad9 stop codon. The expression of the nad9 gene in plant mitochondria was studied. Polyclonal antibodies prepared against a wheat NAD9 fusion protein specifically recognise the 30-kDa subunit of bovine mitochondrial complex I and a 27.5-kDa protein in the membrane fractions of wheat, maize and common bean mitochondria, whereas the same serum recognizes a 30-kDa protein in the mitochondria of pea, chickpea and lentil.
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PMID:Higher plant mitochondria encode an homologue of the nuclear-encoded 30-kDa subunit of bovine mitochondrial complex I. 822 39

The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.
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PMID:The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex. 833 28


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