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)

A thenoyl trifluoroacetone-sensitive and antimycin-insensitive ubisemiquinone radical (Qs) is readily detected in purified succinate-cytochrome c reductase. When this reductase is resolved into succinate-Q and ubiquinol-cytochrome c reductases, Qs was not detected in either reductase. The difficulty in detecting such a radical in purified succinate-Q reductase has puzzled investigators for years. A deficiency of Q in the isolated complex is the reason for the failure to detect Qs. Upon addition of exogenous Q, a thenoyl trifluoroacetone-sensitive Q-radical is readily detectable in isolated succinate-Q reductase under a controlled redox potential. Maximum radical concentration is observed when 5 mol of exogenous Q, per mole of flavin, is added. The radical gives an EPR signal with a g-value of 2.005 and a line-width of 12 G. The Em of Qs is 84 mV at pH 7.4, with half-potentials of E1 = 40 mV and E2 = 128 mV. The Qs-radical does not show power saturation, even at 200 mW.
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PMID:Characterization of ubisemiquinone radicals in succinate-ubiquinone reductase. 130 86

The interaction between cytochrome c oxidase complex and adenosine triphosphate synthase (F1F0) complex in the purified, dispersed state and embedded in phospholipid vesicles was studied by differential scanning calorimetry and by spin-label electron paramagnetic resonance. The detergent-dispersed cytochrome oxidase and F1F0 complexes undergo endothermic thermodenaturation. However, when these complexes are embedded in phospholipid vesicles, they undergo exothermic thermodenaturation. The energy released is believed to result from the collapse of a strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by the removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both cytochrome oxidase complex and F1F0 was smaller than that of a mixture of protein-phospholipid vesicles formed from each individual electron transfer complex. This suggests specific interaction between cytochrome oxidase complex and F1F0 in the membrane. Further evidence for interaction between these two complexes is provided by saturation transfer EPR studies in which the rotational correlation time of spin-labeled cytochrome oxidase increases significantly when the complex is mixed with F1F0 prior to being embedded in phospholipid vesicles. From these results, it is concluded that at least a part of cytochrome oxidase and a part of F1F0 form a supermacromolecular complex in the inner mitochondrial membrane. No such supermacromolecular complex is detected between F1F0 and ubiquinol--cytochrome c reductase.
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PMID:Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial cytochrome c oxidase and adenosine triphosphate synthase complex. 131 90

The concentration of the iron-sulphur (Fe-S) cluster 1b, present in complex I or soluble high-molecular-mass NADH dehydrogenase, was determined using different methods. It was found that direct double integration of the EPR signal at temperatures higher than 40 K, as is commonly used in this field of research, results in a considerable overestimation of the concentration of cluster 1b. It is demonstrated that this is caused by contributions from the relaxation-broadened signals of the Fe-S clusters 2-4 in the enzyme. The correct way for determining the intensity of the EPR signal of cluster 1b is by comparison with a simulated line shape. It is concluded that the concentration of cluster 1b is half that of cluster 2. This corroborates our proposal based on presteady-state kinetic and inhibitor-titration studies [Van Belzen, R., Van Gaalen, M. C. M., Cuypers, P. A. & Albracht S. P. J. (1990) Biochim. Biophys Acta 1017, 152-159] that the minimal functional unit of mitochondrial NADH:ubiquinone oxidoreductase must be a heterodimer.
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PMID:On the stoichiometry of the iron-sulphur clusters in mitochondrial NADH: ubiquinone oxidoreductase. 133 May 59

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

Electron paramagnetic resonance spectra obtained during turnover of the Mo center of NADH:nitrate reductase at pH 8 were comprised of two Mo(V) species, signal A (g1 = 1.996, g2 = 1.969, g3 = 1.967, A1H = 1.25 mT, A2H = 1.18 mT, and A3H = 1.63 mT) and signal B (g1 = 1.996, g2 = 1.969, and g3 = 1.967), the former exhibiting superhyperfine interaction due to strong coupling with a single, exchangeable proton. Binding of halides and nitrite to the Mo center increased the proportion of signal A whereas phosphate had no effect on the EPR line shape. Halides decreased and phosphate increased the rates of enzyme activities involving the Mo center (NADH:nitrate reductase and reduced methyl viologen:nitrate reductase), but neither had any effect on activities involving FAD (NADH:ferricyanide reductase) or heme (NADH:cytochrome c reductase), indicating specific binding of halides to the Mo center. Halides were found to be weak, mixed competitive-noncompetitive inhibitors (Cl- KI = 39 mM, mu = 0.2 M, pH 8) of nitrate reductase forming a catalytically inactive ternary halide-nitrate-enzyme complex. Inhibition patterns changed from nearly noncompetitive (F-) to nearly competitive (I-). The weakening of nitrate binding due to halide binding correlated with increased halide electronegativity rather than ionic radius. In contrast, phosphate (Kd = 7.4 mM, mu = 0.2 M, pH 8) and arsenate were determined to be nonessential activators, characterized by a constant value of (Vmax/Km)app, increasing nitrate reductase activity by weakening nitrate binding without affecting the stability of the transition state. Phosphate had no effect on product inhibition by nitrite (KI = 0.33 mM) or the oxidation-reduction midpoint potentials of the Mo center.
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PMID:EPR and kinetic analysis of the interaction of halides and phosphate with nitrate reductase. 255 63

Bovine heart submitochondrial particles were incubated for 2-6 h at 37 degrees C with various concentrations of tetradecanoic acid, and the effects on the activities, the total acid-labile sulphide content and EPR spectra of the electron transfer system were studied. Two distinct time-dependent processes of the slow irreversible inactivation of the electron-transfer system were found. They differ in the concentration of tetradecanoic acid required. The more specific effect, induced by 100-400 nmol tetradecanoic acid per mg protein, consists of a selective blockage of electron transfer between the Fe-S clusters of the NADH dehydrogenase and ubiquinone, without damage to any of the Fe-S clusters. Higher concentrations of tetradecanoic acid caused gradual destruction of all Fe-S clusters of NADH dehydrogenase and of the 3-Fe cluster of succinate dehydrogenase, leading to complete inactivation of both NADH and succinate oxidation.
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PMID:Two modes of irreversible inactivation of the mitochondrial electron-transfer system by tetradecanoic acid. 298 61

Succinate dehydrogenase is a conserved membrane-bound enzyme consisting of two nonidentical subunits: a flavo iron-sulfur protein (Fp) subunit, containing a covalently bound flavin, and an iron-sulfur protein (Ip) subunit. Bacillus subtilis succinate dehydrogenase in wild type bacteria and 12 well characterized succinate dehydrogenase-defective mutants were examined by low temperature EPR spectroscopy to characterize the enzyme and study subunit location and biosynthesis of its iron-sulfur clusters. The wild type B. subtilis enzyme contains iron-sulfur clusters which are analogous to clusters S-1 and S-3 of bovine heart succinate dehydrogenase but with slightly different EPR characteristics. Spins from cluster S-2 were not detectable as in the case of the intact form of bovine heart succinate dehydrogenase. However, dithionite reduction of the B. subtilis enzyme greatly enhanced spin relaxation of the ferredoxin-type cluster S-1, indicating the presence of the cluster S-2. Iron-sulfur cluster S-1 was found to be assembled in soluble succinate dehydrogenase subunits in the cytoplasm, but only if full-length Fp polypeptides and relatively large fragments of Ip polypeptides were present. Cluster S-1 was not detected in mutants with soluble mutated Fp polypeptides or in a mutant totally lacking Ip subunit polypeptide. Iron-sulfur clusters S-1, S-2, and S-3 were assembled also when the covalently bound flavin in the Fp subunit was absent. Clusters S-1 and S-3 in the membrane-bound flavin-deficient succinate dehydrogenase were not reduced by succinate but could be reduced by electron transfer from NADH dehydrogenase via the menaquinone pool.
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PMID:Characterization by electron paramagnetic resonance and studies on subunit location and assembly of the iron-sulfur clusters of Bacillus subtilis succinate dehydrogenase. 298 99

A simple procedure for preparation of highly purified soluble succinate-ubiquinone reductase from bovine heart mitochondrial particles is described. The enzyme exhibits four major bands on sodium dodecyl sulfate gel electrophoresis and contains (nmol per mg protein): covalently bound flavin, 6; non-heme iron, 53; acid-labile sulfur, 50; cytochrome b-560 heme, 1.2. The enzyme catalyzes thenoyltrifluoroacetone, or carboxin-sensitive (pure non-competitive with Q2) reduction of Q2 by succinate with a turnover number close to that in parent submitochondrial particles. The succinate reduced enzyme exhibits ferredoxin-type iron-sulfur center EPR-signal (g = 1.94 species) and a semiquinone signal (g = 2.00). An oxidized preparation shows a symmetric signal centered around g = 2.01. An unusual dissociation of the enzyme in the absence of a detergent is described. When added to the assay mixture from a concentrated protein-detergent solution, the enzyme does not reduce Q2 being highly reactive towards ferricyanide ('low Km ferricyanide reactive site'; Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Commun. 65, 1264-1269). The ubiquinone reductase, not the ferricyanide reductase was observed when the enzyme was added to the assay mixture from the diluted protein-detergent solutions. Thus the dissociation of succinate dehydrogenase from the complex occurs in the absence of a detergent dependent on the concentration of the protein-detergent complex in the stock preparation where the samples for the assay are taken from. An active antimycin-sensitive succinate-cytochrome c reductase was reconstituted by admixing of the soluble succinate-ubiquinone reductase and the cytochrome b-c1 complex, i.e., from the complexes which both contain the ubiquinone reactivity conferring protein (QPs). Cytochrome c reductase was also reconstituted from the succinate-ubiquinone reductase and succinate-cytochrome c reductase containing inactivated succinate dehydrogenase. The reconstitution experiments suggest that there exists a specific protein-protein (or lipid) interaction between QPs and a certain component(s) of the b-c1 complex.
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PMID:Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase. 299 19

Removal of lipid from detergent-solubilized succinate cytochrome c reductase by a mild method leads to a series of changes in the optical and EPR spectra of the b cytochromes. This culminates in a state that resembles purified b cytochromes from the same source and bisimidazole ferriheme model complexes. Reconstitution of the lipid-depleted complex with phospholipid restores the native spectra in a significant fraction of the complexes in the early stages of lipid depletion. Once the final state has been reached, however, reconstitution has so far been incapable of restoring described in this communication can be related to a model for integral membrane cytochromes.
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PMID:Effects of protein-protein and protein-lipid interactions on heme site conformation in the mitochondrial b cytochromes. 300 7

An anaerobic procedure was developed for the purification of the flavin:NADH oxidoreductase (flavoprotein) component of methane monooxygenase to homogeneity. The molecular weight of the flavoprotein determined by gel filtration was about 40,000, and by sedimentation equilibrium analysis, about 38,000. The purified flavoprotein is a monomeric protein with a sedimentation constant (S20,W) value of about 2.1 S. The absorption spectrum of the flavoprotein has a peak at 460 nm and shoulder at 395 nm. The fluorescent excitation and emission spectra of the fluorescent component of flavoprotein had peaks at 450, 370, and 530 nm, respectively. A FAD was identified as a prosthetic group of flavoprotein by thin-layer chromatography. The flavoprotein contained about 1 mol of FAD and 2 mol each of iron and acid-labile sulfide per mole of protein. The flavoprotein was directly reduced by NADH under anaerobic conditions. The formation of neutral flavin semiquinone was detected during anaerobic titration of flavoprotein by NADH and also as a free radical signal at a g value of 2.004 by EPR spectroscopy. The iron sulfur cluster has g values of 2.04, 1.96, and 1.87, yielding a g average of 1.96, characteristic of a Fe2S2 center. Antibody prepared against the flavoprotein reacted with flavoprotein and inhibited methane monooxygenase activity.
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PMID:Methane monooxygenase: purification and properties of flavoprotein component. 302 58


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