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)

Dibromothymoquinone (DBMIB) inhibits antimycin A-sensitive ubiquinol-cytochrome c reductase activity; the maximal inhibition is 90%. DBMIB alters the EPR spectra of reduced iron-sulfur protein in intact ubiquinol-cytochrome c reductase. The maximal spectral change occurs with 60 mol inhibitor per mol cytochrome c1 in the reductase. DBMIB causes little alteration in the EPR characteristics of iron-sulfur protein when ubiquinol-cytochrome c reductase is delipidated. When delipidated ubiquinol-cytochrome c reductase is replenished with phospholipid, the effect of DBMIB reappears. However, when DBMIB is added to delipidated protein prior to replenishment with phospholipid, very little spectral alteration is observed. DBMIB does not alter the EPR spectra of purified iron-sulfur protein, with or without phospholipid in the preparation. Reduced DBMIB does not alter the EPR characteristics of iron-sulfur protein in intact or delipidated ubiquinol-cytochrome c reductase. Cysteine and other thiol compounds can reverse the spectral alternation caused by DBMIB. This reversal probably results from the reduction of DBMIB.
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PMID:Phospholipid-dependent interaction between dibromothymoquinone and iron-sulfur protein in mitochondrial ubiquinol-cytochrome c reductase. 302 77

The number and type of iron-sulfur clusters present in the NADH dehydrogenase of the mammalian respiratory chain were studied by a combination of low temperature magnetic circular dichroism (MCD) and quantitative electron paramagnetic resonance spectroscopies. MCD was used with the high molecular weight, soluble enzyme, and EPR was used with both the purified enzyme and Complex I (NADH:ubiquinone oxidoreductase). The results of the EPR experiments of the two types of preparations agreed with each other, as well as with the data in the literature for various types of membrane-bound preparations. The two methods gave concordant results showing the presence of one binuclear and of three tetranuclear NADH-reducible iron-sulfur clusters. Earlier studies using the cluster extrusion technique indicated a higher ratio of binuclear to tetranuclear clusters which may be explained by cluster interconversion during the extrusion process.
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PMID:Spectroscopic characterization of the number and type of iron-sulfur clusters in NADH:ubiquinone oxidoreductase. 308 91

We have investigated in detail the effects of dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on the ubiquinol-cytochrome c reductase (cytochrome bc1 complex) from bovine heart mitochondria. The inhibitory action of DBMIB on the steady-state activity of the bc1 complex is related to the specific binding of the quinone to the purified enzymatic complex. At concentrations higher than 10 mol per mol of the enzyme, DBMIB is able to stimulate an antimycin-insensitive reduction of cytochrome c catalyzed by the bc1 complex. In accordance with kinetic data showing a competition by endogenous ubiquinone in the inhibitory action, DBMIB can be considered as a product-like inhibitor of the ubiquinol-cytochrome c reductase activity. The site of specific binding of dibromothymoquinone in the bc1 complex enables it to interact with the iron-sulphur center of the enzyme, as indicated by changes induced in the EPR spectrum of the center. However, the inhibitor also directly interacts with cytochrome b, promoting a fast chemical oxidation of the reduced heme center. In spite of these effects, DBMIB has been found not to exert significant effects on the first turnover of the fully oxidized bc1 complex, as monitored by the rapid reduction of both cytochromes b and c1 by ubiquinol-1. In the presence of antimycin, only a stimulation of cytochrome c1 reduction, in parallel to an enhanced cytochrome b reoxidation, is observed. Moreover, DBMIB does not affect the oxidant-induced extra cytochrome b reduction in the presence of antimycin. On the basis of the evidences suggesting a competition with the endogenous ubiquinone in the redox cycle of the bc1 complex, a model is proposed for the mechanism of DBMIB inhibition. Such model can also explain at the molecular level the redox bypass induced by dibromothymoquinone in the whole respiratory chain (Degli Esposti, M., Rugolo, M. and Lenaz, G. (1983) FEBS Lett. 156, 15-19).
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PMID:Effects of dibromothymoquinone on the structure and function of the mitochondrial bc1 complex. 609 48

Two N-1 type iron-sulfur clusters in NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) were potentiometrically resolved: one was titrated as a component with a midpoint oxidation-reduction potential of -335 mV at pH 8.0, and with an n-value equal to one; the other as an extremely low midpoint potential component (Em 8.0 less than -500 mV). These two clusters are tentatively assigned to N-1b and N-1a, respectively. Cluster N-1b is completely reducible with NADH and has a spin concentration of about 0.8/FMN. Its EPR spectrum can be simulated as a single rhombic component with principal g values of 2.019, 1.937, and 1.922, which correspond to the Center 1 reported earlier by Orme-Johnson, N. R., Hansen, R. E., and Beinert, H. (1974) J. Biol. Chem. 249, 1922-1927. At extremely low oxidation-reduction potentials (less than -450 mV), additional EPR signals emerge with apparent g values of gz = 2.03, gy = 1.95, and gx = 1.91, which we assign to cluster N-1a. It is difficult, however, to simulate the detailed spectral line shape of this component as a single rhombic component, suggesting some degree of protein modification or interaction with a neighboring oxidation-reduction component. EPR spectra of soluble NADH dehydrogenase, containing 5-6 g atoms of non-heme iron and 5-6 mol of acid-labile sulfide/mol of FMN, were examined. Signals from at least two iron-sulfur species could be distinguished in the NADH-reduced form: one of an N-1b type spectrum; the other of a spectrum with g values of 2.045, 1.95, and 1.87 (total of about 0.5 spin equivalents/FMN). This is the first example of an N-1 type signal detected in isolated soluble NADH dehydrogenase.
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PMID:Iron-sulfur N-1 clusters studied in NADH-ubiquinone oxidoreductase and in soluble NADH dehydrogenase. 626 66

The low molecular weight NADH dehydrogenase which can be solubilized from the mitochondrial NADH-ubiquinone oxidoreductase complex with chaotropic agents consists of three subunits in equimolar ratio [Galante, Y. M., & Hatefi, Y. (1979) Arch. Biochem. Biophys. 192, 559]. The largest subunit (subunit I) can be completely separated from the other two (subunits II + III) by treatment with sodium trichloroacetate and ammonium sulfate fractionation. Both the subunit I and subunit II + III fractions contain iron and acid-labile sulfur. From visible and EPR spectroscopy and the iron and acid-labile sulfide content, we propose that the subunit II + III fraction contains a binuclear cluster. The cluster structure present in subunit I is as yet unclear. On separation of the subunits of NADH dehydrogenase, the FMN is lost.
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PMID:Resolution of mitochondrial NADH dehydrogenase and isolation of two iron-sulfur proteins. 627 47

A synthetic quinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), inhibits electron transfer reactions in the cytochrome b-c1 segment of the mitochondrial respiratory chain. Addition of UHDBT to isolated succinate-cytochrome c reductase complex has effects on reduction of the cytochromes b and c1 by succinate similar to those which result from removal of the iron-sulfur protein from the b-c1 complex. Thus, UHDBT inhibits reduction of cytochrome c1 by succinate and, if antimycin is added before succinate, UHDBT inhibits reduction of cytochrome b in addition to c1. UHDBT increases the midpoint potential of the iron-sulfur protein of the b-c1 complex from +280 to +350 mV at pH 7.2. The inhibitor also shifts the gx peak in the EPR spectrum of the iron-sulfur protein from g = 1.80 to 1.76 and shifts the gz peak from g = 2.02 to 2.03. It causes only a slight shift in the central gy = 1.90 signal. The efficacy of inhibition of cytochrome c reductase activity of isolated reductase complex by UHDBT appears to depend on the oxidation-reduction poise of some component(s) in the b-c1 complex. Inhibition is decreased and there is an extensive lag in the onset of inhibition under conditions favoring oxidation of the b-c1 complex; inhibition increases and the lag is eliminated under conditions favoring reduction of the b-c1 complex. The titer for inhibition of cytochrome c reductase activity of isolated reductase complex is one UHDBT per b-c1 complex. With reductase complex from which the iron-sulfur protein of the b-c1 complex is reversibly resolved, the titer for inhibition is proportional to the amount of iron-sulfur protein reconstituted to the complex. These results suggest that UHDBT inhibits mitochondrial respiration by binding to the iron-sulfur protein of the b-c1 complex, possibly at a site which is otherwise involved in binding ubiquinone, and that this binding is enhanced when the iron-sulfur protein is reduced.
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PMID:An analogue of ubiquinone which inhibits respiration by binding to the iron-sulfur protein of the cytochrome b-c1 segment of the mitochondrial respiratory chain. 628 79

In 5,5'-dithiobis(2-nitrobenzoate) (DTNB)-treated succinate: cytochrome c reductase, the electron transfer from duroquinol to cytochrome c is inhibited due to the fact that the Rieske Fe-S cluster and, consequently, cytochrome, c, are no longer reducible by substrate. The finding that, after this treatment, cytochrome b is still reducible by substrate in the absence of antimycin, but not in its presence, is consistent with a Q-cycle mechanism for the electron transfer through QH2:cytochrome c oxidoreductase. The inhibitory effect of DTNB and its effect on the EPR spectrum of the [2Fe-2S] cluster suggest that it prevents either the binding of ubiquinone in the vicinity of this cluster or the interaction between the Fe-S protein and a ubiquinone-binding protein.
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PMID:The site of inhibition by 5,5'-dithiobis(2-nitrobenzoate) in ubiquinol: cytochrome c oxidoreductase. 628 87

Myxothiazol, an antibiotic from Myxococcus fulvus, which inhibits mitochondrial respiration in the bc1 complex of the respiratory chain, has effects on the redox components of isolated succinate-cytochrome c reductase complex which suggest that it interacts with both cytochrome b and the iron-sulfur protein of the bc1 complex. The inhibitor appears to increase the midpoint potentials of cytochromes b-562 and b-566, as indicated by an increase in their reducibility by the succinate/fumarate couple. It also causes a red shift in the optical spectrum of ferrocytochrome b-566, as reported previously (Becker, W. F., Von Jagow , G., Anke , T., Steglisch , W. (1981) FEBS Lett. 132, 329-333). This red shift is enhanced by Triton X-100, and there is no shift in the spectrum of b-562. These results are consistent with evidence that mutations conferring myxothiazol resistance in yeast map to the mitochondrial gene for cytochrome b ( Thierbach , G., and Michaelis, G. (1982) Mol. Gen. Genet. 186, 501-506). In addition, myxothiazol has effects on reduction of the cytochromes b and c1 by succinate or ubiquinol which are identical to those caused by removal of the iron-sulfur protein from the bc1 complex. It blocks reduction of cytochrome c1 during single and multiple turnovers of the bc1 complex, but does not block reduction of the b cytochromes. In the presence of antimycin, it blocks reduction of both cytochromes b and c1. In contrast to antimycin, myxothiazol inhibits oxidant-induced reduction of both b cytochromes and does not inhibit their oxidation by fumarate. Myxothiazol also inhibits reduction of the iron-sulfur protein by ubiquinol and shifts the gx resonance in the EPR spectrum of the iron-sulfur protein from g = 1.79 to 1.76. It does not affect the midpoint potential of the iron-sulfur protein, but does eliminate the increase in midpoint potential which is caused by inhibitory hydroxyquinones which bind to the iron-sulfur protein. The effects of myxothiazol are consistent with a protonmotive Q cycle pathway of electron transfer in which myxothiazol binds to cytochrome b and displaces quinone from the iron-sulfur protein of the bc1 complex. These results suggest either that a myxothiazol-induced conformational change in cytochrome b is transmitted to a quinone binding site on the iron-sulfur protein, or that there is a quinone binding site which consists of peptide domains from both cytochrome b and iron-sulfur protein.
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PMID:An inhibitor of mitochondrial respiration which binds to cytochrome b and displaces quinone from the iron-sulfur protein of the cytochrome bc1 complex. 632 77

The structural nature of the iron-sulfur clusters of NADH dehydrogenase from beef heart mitochondria has been studied by the cluster extrusion technique. Enzyme samples were unfolded anaerobically in 80% (v/v) hexamethylphosphoramide/aqueous buffer in the presence of o-xylyl-alpha,alpha'-dithiol as the displacing agent and the extruded clusters were then reacted with p-trifluoromethylbenzenethiol and analyzed by Fourier transform 19F NMR at 339 MHz. Whenever extrusion was nearly complete, both binuclear and tetranuclear clusters were found at a mole ratio of approximately 2:1. Thus, the dehydrogenase, with 16 g atoms of non-heme iron present/mol of FMN, contains most likely four [2Fe-2S] and two [4Fe-4S] clusters. Because the enzyme contains four or, at the most five, EPR-detectable iron-sulfur centers, it appears that one or more of the clusters are EPR-silent.
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PMID:Structural identification of iron-sulfur clusters of the respiratory chain-linked NADH dehydrogenase. 720 98

Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of > 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2 component contains two binuclear iron centers. In total, the structural, spectroscopic, and catalytic features of toluene 2-monooxygenase are reminiscent of soluble methane monooxygenase obtained from methanotrophic bacteria. The two enzyme systems also differ in many subtle ways; for example, they oxidize toluene with completely different regiospecificity.
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PMID:Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4. 757 4


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