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)

Mitochondrial superoxide (O2.) is an important mediator of ischemia/reperfusion (I/R) injury. The O2. generated in mitochondria also acts as a redox signal triggering cellular apoptosis. The enzyme succinate ubiquinone reductase (SQR or complex II) is one of the major mitochondrial components hosting regulatory thiols. Here the intrinsic protein S-glutathionylation (PrSSG) at the 70-kDa FAD-binding subunit of SQR was detected in rat heart and in isolated SQR using an anti-GSH monoclonal antibody. When rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion, the electron transfer activity (ETA) of SQR in post-ischemic myocardium was significantly decreased by 41.5 +/- 2.9%. The PrSSGs of SQR-70 kDa were partially or completely eliminated in post-ischemic myocardium obtained from in vivo regional I/R hearts or isolated global I/R hearts, respectively. These results were further confirmed by using isolated succinate cytochrome c reductase (complex II + complex III). In the presence of succinate, O2. was generated and oxidized the SQR portion of SCR, leading to a 60-70% decrease in its ETA. The gel band of the S-glutathionylated SQR 70-kDa polypeptide was cut out and digested with trypsin, and the digests were subjected to liquid chromatography/tandem mass spectrometry analysis. One cysteine residue, Cys(90), was involved in S-glutathionylation. These results indicate that the glutathione-binding domain, (77)AAFGLSEAGFNTACVTK(93) (where underline indicates Cys(90)), is susceptible to redox change induced by oxidative stress. Furthermore, in vitro S-glutathionylation of purified SQR resulted in enhanced SQR-derived electron transfer efficiency and decreased formation of the 70-kDa-derived protein thiyl radical induced by O2. . Thus, the decreasing S-glutathionylation and ETA in mitochondrial complex II are marked during myocardial ischemia/reperfusion. This redox-triggered impairment of complex II occurs in the post-ischemic heart and should be useful to identify disease pathogenesis related to reactive oxygen species-induced mitochondrial dysfunction.
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PMID:Mitochondrial complex II in the post-ischemic heart: oxidative injury and the role of protein S-glutathionylation. 1784 55

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.
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PMID:Impact of mutations on the midpoint potential of the [4Fe-4S]+1,+2 cluster and on catalytic activity in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). 1806 58

A novel NADH dehydrogenase (NADH-dh) involving FAD as coenzyme, distinct from NADPH dehydrogenase (NADPH-dh, old yellow enzyme, EC 1.6.99.1), was found in the same cytoplasmic fraction of Gluconobacter strains. Conventional artificial electron acceptors were more effective than molecular oxygen in the NADH-dh reaction. NADH-dh did not appear to be identical with any previously described flavoproteins, although the N-terminal amino acid sequence showed 100% similarity with a non-heme chloroperoxidase. The N-terminal amino acid sequence of NADPH-dh matched 100% a putative oxidoreductase containing the old yellow enzyme-like FMN-binding domain. NADH-dh might function to regenerate NAD coupling with NAD-dependent dehydrogenases in the cytoplasm of Gluconobacter strains.
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PMID:The occurrence of a novel NADH dehydrogenase, distinct from the old yellow enzyme, in Gluconobacter strains. 1817 96

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae is a membrane-bound, respiratory Na+ pump. Its NqrF subunit contains one FAD and a [2Fe-2S] cluster and catalyzes the initial oxidation of NADH. A soluble variant of NqrF lacking its hydrophobic, N-terminal helix (NqrF') was produced in V. cholerae wild type and nqr deletion strain. Under identical conditions of growth and induction, the yield of NqrF' increased by 30% in the presence of the Na+-NQR. FAD-containing NqrF' species with or without the FeS cluster were observed, indicating that assembly of the FeS center, but not insertion of the flavin cofactor, was limited during overproduction in V. cholerae. A comparison of these distinct NqrF' species with regard to specific NADH dehydrogenase activity, pH dependence of activity and thermal inactivation showed that NqrF' lacking the [2Fe-2S] cluster was less stable, partially unfolded, and therefore prone to proteolytic degradation in V. cholerae. We conclude that the overall yield of NqrF' critically depends on the amount of fully assembled, FeS-containing NqrF' in the V. cholerae host cells. The Na+-NQR is proposed to increase the stability of NqrF' by stimulating the maturation of FeS centers.
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PMID:The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae enhances insertion of FeS in overproduced NqrF subunit. 1828 89

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na+/H+ antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na+-resistance on an antiporter-deficient E. coli strain, nor did they confer Na+/H+ antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.
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PMID:Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4. 1835 84

The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD(+) as a specific electron acceptor, and the apparent K(m) values for DLA and NAD(+) were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K(m) values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40 degrees C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60 degrees C.
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PMID:Characterization of a dihydrolipoyl dehydrogenase having diaphorase activity of Clostridium kluyveri. 1839 50

Mitochondrial superoxide (O(2) (-)) production is an important mediator of oxidative cellular injury and pathogenesis of many diseases such as myocardial ischemia/reperfusion. The O(2) (-) generated in mitochondria acts as a redox signal triggering cellular events including apoptosis, proliferation, and senescence. The molecular mechanism of O(2) (-) produced by electron transport chain components isolated from the inner membrane is investigated by the technique of EPR spin trapping with 5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), indicating that FMN/FMN-binding domain (complex I), ubiquinone (complex I and III), FAD/FAD-binding domain (complex II), and cytochrome b (complex III) control the mediation of O(2) (-) production in mitochondria. O(2) (-) generation by ETC also induces oxidative damage with protein radical formation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry are used to define the specific site of oxidative damage, indicating cysteine-206 and tyrosine-177 of complex I/51 kDa FMN-binding subunit and cysteine-655 of complex II/70 kDa FAD-binding subunit are involved in specific protein radical formation caused by O(2) (-) attack.
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PMID:EPR spin-trapping and nano LC MS/MS techniques for DEPMPO/OOH and immunospin-trapping with anti-DMPO antibody in mitochondrial electron transfer system. 1908 40

The Na(+)-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR) is a component of respiratory chain of various bacteria, and it generates a redox-driven transmembrane electrochemical Na(+) potential. Primary steps of the catalytic cycle of Na(+)-NQR from Vibrio harveyi were followed by the ultrafast freeze-quench approach in combination with conventional stopped-flow technique. The obtained sequence of events includes NADH binding ( approximately 1.5 x 10(7) m(-1) s(-1)), hydride ion transfer from NADH to FAD ( approximately 3.5 x 10(3) s(-1)), and partial electron separation and formation of equivalent fractions of reduced 2Fe-2S cluster and neutral semiquinone of FAD ( approximately 0.97 x 10(3) s(-1)). In the last step, a quasi-equilibrium is approached between the two states of FAD: two-electron reduced (50%) and one-electron reduced (the other 50%) species. The latter, neutral semiquinone of FAD, shares the second electron with the 2Fe-2S center. The transient midpoint redox potentials for the cofactors obtained during the fast kinetics measurements are very different from ones achieved during equilibrium redox titration and show that the functional states of the enzyme realized during its turning over cannot be modeled by the equilibrium approach.
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PMID:Primary steps of the Na+-translocating NADH:ubiquinone oxidoreductase catalytic cycle resolved by the ultrafast freeze-quench approach. 1911 49

The Ndi1 enzyme found in the mitochondrial membrane of Saccharomyces cerevisiae is an NDH-2-type alternative NADH-quinone oxidoreductase. As Ndi1 is expected to be a possible remedy for complex I defects of mammalian mitochondria, a detailed biochemical characterization of the enzyme is needed. To identify the ubiquinone (UQ) binding site in Ndi1, we conducted photoaffinity labeling using a photoreactive biotinylated UQ mimic (compound 2) synthesized following a concept of the least possible modification of the substituents on the quinone ring. Cleavage with CNBr of Ndi1 cross-linked by 2 revealed the UQ ring of 2 to be specifically cross-linked to the Phe281-Met410 region (130 amino acids). Digestion of the CNBr fragment with V8 protease and lysylendopeptidase (Lys-C) gave approximately 8 and approximately 4 kDa peptides, respectively. The approximately 8 kDa V8 digest was identified as the Thr329-Glu399 region (71 amino acids) by an N-terminal sequence analysis. Although the approximately 4 kDa Lys-C digest could not be identified by N-terminal sequence analysis, the band was thought to cover the Gly374-Lys405 region (32 amino acids). Taken together, the binding site of the Q ring of 2 must be located in a common region of the V8 protease, and Lys-C digests Gly374-Glu399 (26 amino acids). Superimposition of the Ndi1 sequence onto a three-dimensional structural model of NDH-2 from Escherichia coli suggested that the C-terminal portion of this region is close to the isoalloxazine ring of FAD.
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PMID:Characterization of the ubiquinone binding site in the alternative NADH-quinone oxidoreductase of Saccharomyces cerevisiae by photoaffinity labeling. 2019 60

Complex I is the first and largest enzyme in the respiratory chain and is located in the inner mitochondrial membrane. Complex I deficiency is the most commonly reported mitochondrial disorder presenting in childhood, but the molecular basis of most cases remains elusive. We describe a patient with complex I deficiency caused by mutation of the molecular chaperone FOXRED1. A combined homozygosity mapping and bioinformatics approach in a consanguineous Iranian-Jewish pedigree led to the identification of a homozygous mutation in FOXRED1 in a child who presented with infantile-onset encephalomyopathy. Silencing of FOXRED1 in human fibroblasts resulted in reduced complex I steady-state levels and activity, while lentiviral-mediated FOXRED1 transgene expression rescued complex I deficiency in the patient fibroblasts. This FAD-dependent oxidoreductase, which has never previously been associated with human disease, is now shown to be a complex I-specific molecular chaperone. The discovery of the c.1054C>T; p.R352W mutation in the FOXRED1 gene is a further contribution towards resolving the complex puzzle of the genetic basis of human mitochondrial disease.
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PMID:FOXRED1, encoding an FAD-dependent oxidoreductase complex-I-specific molecular chaperone, is mutated in infantile-onset mitochondrial encephalopathy. 2602 95


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