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:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two cytochrome b respiratory-deficient mutants were sequenced and their DNA base change identified, leading to the replacement of glycine (G137 by valine or glutamic acid. No variation in their cytochrome b content with regard to cytochrome oxidase and cytochrome (c + c1) was found to have occurred. Their cellular respiratory activity with various substrates was partly conserved and was totally inhibited by antimycin A. Their ubiquinol (QH2)-cytochrome c reductase/mole cytochrome b activity decreased by about 50%. Paradoxically their growth on respiratory substrate was abolished. Both mutants retained a high-affinity binding site for antimycin A, and exhibited a myxothiazol-resistance at the mitochondrial level. It seems likely that the mutated position (137), which belongs to the ubiquinol oxidizing domain of the bc1 complex, interferes, directly or indirectly, with the respiratory growth capacity of the cell.
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PMID:Two substitutions at the same position in the mitochondrial cytochrome b gene of S. cerevisiae induce a mitochondrial myxothiazol resistance and impair the respiratory growth of the mutated strains abbeit maintaining a good electron transfer activity. 207 67

The 45 kDa diphenylene iodonium-binding flavoprotein of the human neutrophil superoxide-generating oxidase has been purified by affinity chromatography. The polypeptide was eluted from Blue Memsep or 2',5'-ADP-agarose columns with either NADP or low concentrations of the specific inhibitor diphenylene iodonium. The purified protein was shown to bind FAD at a ratio of 1.09 mol of FAD/mol of protein. The reconstituted flavoprotein had a fluorescence spectrum similar, but not identical, to that of free FAD. It had an isoelectric point of approx. 4.0. The reconstituted flavoprotein displayed no diaphorase activity towards a range of artificial electron acceptors. Polyclonal antibodies raised against the pure protein inhibited superoxide generation by solubilized oxidase in a dose-dependent manner, and inhibited superoxide generation when incubated with either cytosol or membrane fractions in a reconstituted system. These antibodies precipitated the 45 kDa polypeptide together with a haem-containing 23 kDa protein thought to be the small subunit of cytochrome b-245. Antibodies raised against cytochrome P-450 reductase also precipitated these two polypeptides. These results are consistent with the 45 kDa polypeptide being the flavoprotein of the neutrophil superoxide-generating oxidase.
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PMID:Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase. 215 84

Funiculosin is a well-known inhibitor of the mitochondrial respiratory chain, probably acting at the ubiquinone reducing site or center i of QH2-cytochrome c reductase. We report here the isolation, mapping and RNA sequence analysis of yeast apo-cytochrome b mutants resistant to this inhibitor. Funiculosin-resistance was found to be conferred, in 4 independent isolates, upon replacement of a leucine residue by phenylalanine in position 198 of the cytochrome b polypeptide chain.
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PMID:Isolation and RNA sequence analysis of cytochrome b mutants resistant to funiculosin, a center i inhibitor of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae. 215 9

The ubiquinol:cytochrome c reductase activity of Paracentrotus lividus mitochondria is relatively insensitive to the specific inhibitors myxothiazol and mucidin. The I50 of myxothiazol and mucidin are three and two orders of magnitude higher, respectively, in P. lividus than in bovine heart mitochondria. The natural resistance of the P. lividus reductase to these inhibitors can be correlated with a single amino replacement, an alanine for a glycine at position 143, in the sequence of cytochrome b. This position is located in a conserved region of the molecule, believed to be important in the oxidation of ubiquinol by the reductase.
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PMID:The cytochrome b of the sea urchin Paracentrotus lividus is naturally resistant to myxothiazol and mucidin. 215 21

A nuclear gene (QCR9) encoding the 7.3-kDa subunit 9 of the mitochondrial cytochrome bc1 complex from Saccharomyces cerevisiae has been isolated from a yeast genomic library by hybridization with a degenerate oligonucleotide corresponding to nine amino acids proximal to the N terminus of purified subunit 9. QCR9 includes a 195-base pair open reading frame capable of encoding a protein of 66 amino acids and having a predicted molecular weight of 7471. The N-terminal methionine of subunit 9 is removed posttranslationally because the N-terminal sequence of the purified protein begins with serine 2. The ATG triplet corresponding to the N-terminal methionine is separated from the open reading frame by an intron. The intron is 213 base pairs long and contains previously reported 5' donor, 3' acceptor, and TACTAAC sequences necessary for splicing. The splice junctions, as well as the 5' end of the message, were confirmed by isolation and sequencing of a cDNA copy of QCR9. In addition, the intron contains a nucleotide sequence in which 15 out of 18 nucleotides are identical with a sequence in the intron of COX4, the nuclear gene encoding cytochrome c oxidase subunit 4. The deduced amino acid sequence of the yeast subunit 9 is 39% identical with that of a protein of similar molecular weight from beef heart cytochrome bc1 complex. If conservative substitutions are allowed for, the two proteins are 56% similar. The predicted secondary structure of the 7.3-kDa protein revealed a single possible transmembrane helix, in which the amino acids conserved between beef heart and yeast are asymmetrically arranged along one face of the helix, implying that this domain of the protein is involved in a conserved interaction with another hydrophobic protein of the cytochrome bc1 complex. Two yeast strains, JDP1 and JDP2, were constructed in which QCR9 was deleted. Both strains grew very poorly, or not at all, on nonfermentable carbon sources and exhibited, at most, only 5% of wild-type ubiquinol-cytochrome c oxidoreductase activity. Optical spectra of mitochondrial membranes from the deletion strains revealed slightly reduced levels of cytochrome b. When JDP1 and JDP2 were complemented with a plasmid carrying QCR9, the resulting yeast grew normally on ethanol/glycerol and exhibited normal cytochrome c reductase activities and optical spectra. These results indicate that QCR9 encodes a 7.3-kDa subunit of the bc1 complex that is required for formation of a fully functional complex.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Isolation and characterization of QCR9, a nuclear gene encoding the 7.3-kDa subunit 9 of the Saccharomyces cerevisiae ubiquinol-cytochrome c oxidoreductase complex. An intron-containing gene with a conserved sequence occurring in the intron of COX4. 217 27

The properties of the ubiquinol-cytochrome c reductase complex (bc1 complex) have been studied in respiratory defective mutants of Saccharomyces cerevisiae bearing lesions in the core 1 subunit. All the cor1 mutants examined have greatly reduced concentrations of mitochondrial cytochrome b and display succinate-cytochrome c reductase activities near the limits of detection. Two mutants (E576 and C7), however, had 5% of wild type activity when the cells were grown at 23 degrees C, but not at 37 degrees C. The temperature-sensitive phenotype was determined to result from substitution of either Arg or Glu for Gly68 of the core 1 subunit. The respiratory competent revertants E576/R8 and C7/R4 derived from E576 and C7 retain the temperature sensitivity of the original mutants. Both revertants are temperature sensitive in vivo, but only mitochondria isolated from E576/R8 are temperature sensitive in vitro. The bc1 complex of mitochondria isolated from this revertant displays a normal value of the ratio Kcat/Km for cytochrome c and four times higher than the wild type for duroquinol. The succinate-cytochrome c reductase activity of E576/R8 is almost completely abolished after incubation at 37 degrees C for 90 min. It is inferred that the quaternary structure of ubiquinol-cytochrome c reductase complex is more labile at the nonpermissive temperature in the mutant and undergoes an alteration such that cytochrome b is no longer able to receive electrons through either the "o" or the "i" site pathway. The temperature lability and kinetic properties of the mutant enzyme point to a requirement of the core 1 not only for assembly but also for the catalytic activity of the complex.
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PMID:Structure and function of the mitochondrial bc1 complex. Properties of the complex in temperature-sensitive cor1 mutants. 217 74

Our work relating to the role of cytochrome b in the CoQH2-cytochrome c reductase segment of the respiratory chain of S. cerevisiae mitochondria is reviewed here and new results are reported. The results concerning the structure-function relationship of cytochrome b in this complex, analyzed within the framework of the eight transmembrane alpha helice cytochrome b folding model, agree with the following features of the proton motive Q cycle (or SQ cycle): i) the antimycin A and myxothiazol binding domains are located on opposite sides of the inner mitochondrial membrane; and ii) the antimycin A binding domain is associated with the b562 domain, the myxothiazol domain with the b565 domain. These results were obtained from structural data derived from amino-acid sequence studies on mit- mutants and from biochemical studies of these mutants. However, functional studies are reported here that are not in agreement with the following features of the above models: i) the serial arrangement of the two hemes of cytochrome b and ii) the isolation of cytochrome b from redox changes with the couple fumarate/succinate in the presence of antimycin A and myxothiazol.
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PMID:Studies on the CoQH2-cytochrome c reductase segment of the respiratory chain of yeast mitochondria, using mutants of the cytochrome b split gene. 251 75

A strain of yeast lacking the gene for the Rieske iron-sulfur protein (RIP) of the cytochrome b-c1 complex was used to study the assembly of this complex in the mitochondrial membrane. This strain lacks the mRNA for the iron-sulfur protein as evidenced by both Northern hybridization using a probe containing the coding region of the gene plus in vitro translation of total RNA followed by immunoprecipitation with a specific antibody against the iron-sulfur protein. In addition, isolated mitochondria from this strain lacked cytochrome c reductase activity with either succinate or the decyl analog of ubiquinol as substrate. Immunoblotting studies with antiserum against the cytochrome b-c1 complex revealed that mitochondria from the iron-sulfur protein-deficient strain have levels of core protein I, core protein II, and cytochrome c1 equal to those of wild-type mitochondria; however, a decrease in cytochrome b was evident from both immunoblotting and spectral analysis. Moreover, it is evident from the immunoprecipitates of radiolabeled mitochondria that the amounts of the low-molecular-weight subunits (17, 14, and 11 kDa) are decreased 53, 65, and 50%, respectively, in mitochondria lacking the iron-sulfur protein. These results suggest that the iron-sulfur protein is required for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex.
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PMID:The iron-sulfur protein is necessary for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex of yeast mitochondria. 253 36

A ubiquinone derivative, 3-chloro-5-hydroxyl-2-methyl-6-decyl- 1,4-benzoquinone (3-CHMDB), which shows different effects on the mitochondrial cytochrome b-c1 complex and chloroplast cytochrome b6-f complex, has been synthesized and characterized. When the cytochrome b-c1 complex is treated with varying concentrations of 3-CHMDB and assayed at constant substrate (Q2H2) concentration, a 50% inhibition is observed when 2 mol of 3-CHMDB per mol of enzyme are used. The degree of inhibition is dependent on the substrate concentration. When ubiquinol-cytochrome c reductase is treated with 2 mol of 3-CHMDB per mol of enzyme, less inhibition is observed with a lower substrate concentration, suggesting the possible existence of two forms of reductases: one with a high affinity for ubiquinone and another with a low affinity. 2-Chloro-5-hydroxyl-3-methyl-6-decyl-1,4-benzoquinone (2-CHMDB), an isomer of 3-CHMDB, shows much less inhibition of the mitochondrial cytochrome b-c1 complex, suggesting that the quinone binding site in this complex is highly specific. In contrast to the inhibition observed with the cytochrome b-c1 complex, 3-CHMDB causes no inhibition of the plastoquinol-plastocyanin reductase activity of chloroplast cytochrome b6-f complex, regardless of whether plastoquinol-2 or ubiquinol-2 is used as substrate. 3-CHMDB restores the dibromothymoquinone-altered EPR spectra of iron-sulfur protein in both complexes. In the case of the cytochrome b6-f complex, 3-CHMDB also partially restores the dibromothymoquinone-inhibited activity. Reduced form 3- or 2-CHMDB is oxidizable by the cytochrome b6-f complex, but not by the cytochrome b-c1 complex. These results suggest that the quinol oxidizing sites in the cytochrome b6-f complex may differ from those in the mitochondrial cytochrome b-c1 complex.
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PMID:A ubiquinone derivative that inhibits mitochondrial cytochrome b-c1 complex but not chloroplast cytochrome b6-f complex activity. 253 47

Chlamydomonas reinhardtii mitochondrial (mt)DNA was digested with ClaI + HpaI and shotgun cloned into the M13mp19 vector cleaved with AccI + SmaI. One of the recombinant clones, with a 1.8-kb DNA insert, was completely sequenced using the dideoxy chain-termination method. Besides containing part of the cytochrome b (COB)-encoding gene (cob), this DNA fragment encodes subunit 4 of NADH dehydrogenase (NAD4). The deduced amino acid sequence and hydrophilicity plot indicate that NAD4 is highly hydrophobic. The nad4 gene shows a unique preference for certain codons which are also found in other C. reinhardtii mt proteins. Both the genes encoding NAD4 and COB are shown to be transcriptionally active by Northern hybridization. These closely linked genes suggest that RNA-processing events found in vertebrate mt are present in Chlamydomonas mt as well.
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PMID:Nucleotide sequence of cloned nad4 (urf4) gene from Chlamydomonas reinhardtii mitochondrial DNA. 262 73


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