Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfate-reducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidized/semiquinone forms at pH 6.7 and 25 degrees C is -40 mV, while the value for the semiquinone/hydroquinone forms (E1), at the same pH, -387 mV. E2 varies linearly with pH, while E1 is independent of pH at high values. However, at low pH (< 7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin [Moura, I., Moura, J.J.G., Bruschi, M. & LeGall, J. (1980) Biochim. Biophys. Acta 591, 1-8]. The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS. Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model [similarity, 48.6%; Watenpaugh, K.D., Sieker, L.C., Jensen, L.H., LeGall, J. & Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185-3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force-field calculations.
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PMID:Primary sequence, oxidation-reduction potentials and tertiary-structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin. 814 52

The production of nitrate (NO3-) and nitrite (NO2-) from macrophage-derived NO was studied using EPR and spin trapping. The formation of NO3- was determined via EPR in reactions involving the iron-binding protein, lactoferrin. The formation of NO2- was determined via EPR/spin trapping in the reaction between NO2- and H2O2. Dissolved nitric oxide (NO.) was reacted with lactoferrin yielding an EPR spectrum (77 degrees K) different from the normal EPR spectrum obtained for lactoferrin, suggesting that NO. interacts with the ferric ions bound to lactoferrin forming a ferric-nitrosyl type complex. The EPR spectrum (77 degrees K) of this ferric-nitrosyl type complex was also observed in the supernatant fluid of macrophage cell suspensions following their stimulation with lipopolysaccharide (LPS). During LPS stimulation of macrophages, these cells generate NO. which in turn produces NO3- and NO2-. The ferric-nitrosyl type complex is formed in a reaction mixture containing apolactoferrin and bicarbonate following the reaction of Fe+2 with NO3-, generated from macrophage-derived NO(.), to produce Fe+3 and NO(.). Furthermore, in an acidic medium, NO2- reacts with H2O2 forming peroxynitrous acid (HOONO) which rapidly decomposes into hydroxyl radicals (.OH) and the nitrogen dioxide (NO2.) radical. In the supernatant fluid of LPS-stimulated macrophage suspensions, the production of .OH was verified by spin trapping using 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) as the spin trap and ethanol as the .OH scavenger. The EPR spectra corresponding to the DMPO-OH and the DMPO-hydroxyethyl adducts were identified.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nitric oxide interaction with lactoferrin and its production by macrophage cells studied by EPR and spin trapping. 828 25

The effect of recombinant gp120 HIV envelope glycoprotein on the generation of free radicals by monocyte-derived macrophages (MDM) was measured by EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). After 1 day in culture, MDM produced a spin trap adduct of DMPO with hyperfine splitting constants superimposable on those of DMPO-OH. The addition of gp120 to MDM increased the production of DMPO-OH and after 1 h, the amount of DMPO-OH produced by 40 micrograms/ml gp120 was about 300% that of untreated MDM. The use of selective inhibitors suggested the participation of the nitric oxide/L-arginine oxidative pathway, but did not provide evidence for trapping of hydroxyl radical or other oxygen free radicals. The specificity of gp120 was proven by two different anti-gp120 antibodies that either inhibited (polyclonal) or increased (monoclonal) the production of free radicals. Dexamethasone inhibited the effect of gp120, suggesting the possible involvement of an inducible nitric oxide (NO) synthase. Moreover, treatment of MDM with gp120 for 15 h increased in a dose-dependent manner the production of NO2-, a stable end product of NO. Soluble CD4 did not modify the intensity of the DMPO-OH adduct, whereas yeast mannan and Ca(2+)-chelators abolished the increase in the DMPO-OH signal induced by gp120. These data suggest the possible involvement of mannose-specific endocytotic lectin of MDM. The reaction of DMPO with sodium nitroprusside, an organic nitrate that releases NO, also produced DMPO-OH. Our findings indicate that gp120 increases free radical production from MDM as detected by spin-trapping methods, and that the spin trap adduct results from a reaction involving NO or closely related oxidized derivatives.
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PMID:gp120 HIV envelope glycoprotein increases the production of nitric oxide in human monocyte-derived macrophages. 830 Dec 14

We have used site-directed mutagenesis to alter the ligands to the iron-sulfur centers of Escherichia coli nitrate reductase A. The beta subunit of this enzyme contains four Cys groups which are thought to accommodate the single [3Fe-4S] center and the three [4Fe-4S] centers involved in the electron-transfer process from quinol to nitrate. The third Cys group (group III) contains a Trp at a site occupied by a Cys residue in typical ferredoxin arrangements or in the DmsB subunit of dimethyl sulfoxide (DMSO) reductase. In an attempt to determine the coordination site of the different iron-sulfur centers in the amino acid sequence, we have changed the Trp of group III to Cys, Ala, Phe, and Tyr and the first Cys residue of groups II-IV to Ala and Ser. Physiological, biochemical, and EPR studies were performed on the mutated enzymes. Substitution of Ala for either Cys184, Cys217, or Cys244 results in the full loss of all four iron-sulfur centers present in the wild-type enzyme. These inactive enzymes still possess the alpha,beta, and gamma polypeptides associated in a membrane-bound complex. These Cys have important structural roles and are very likely involved in the coordination of the iron-sulfur centers. Substitution of Cys184 with a Ser residue produces an enzyme containing the four iron-sulfur centers, but displaying reduced activity. EPR studies suggest that Cys184 is a ligand of the [4Fe-4S] center whose midpoint potential is -200 mV in the native enzyme. All substitutions performed in this study on Trp220 lead to mutant enzymes harboring the four iron-sulfur centers and a nitrate reductase activity close to that of the wild-type. In spite of the high similarity between the NarH and DmsB subunits, the Trp220-->Cys substitution does not allow the conversion of the [3Fe-4S] center of the nitrate reductase into a [4Fe-4S] center. Therefore, Trp220 does not seem to play any major role in the beta subunit.
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PMID:Site-directed mutagenesis of conserved cysteine residues within the beta subunit of Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of the mutated enzymes. 838 31

Isolated rat hepatocytes were examined by EPR spectroscopy after exposure to inflammatory stimuli (interferon-gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], and lipopolysaccharide [LPS]) in vitro, after in vivo immune activation by Corynebacterium parvum, and after exposure to .N = O and to nitroprusside (nitroferricyanide), an NO-donating nitrovasodilator. Hepatocytes exposed to IFN-gamma, TNF-alpha, IL-1 beta, and LPS demonstrated the appearance of a g = 2.04 axial EPR signal indicative of the formation of nonheme iron-nitrosyl complexes. Concurrent incubation with L-NG-monomethylarginine (L-NMMA), a competitive inhibitor of .N = O synthase, prevented the appearance of the signal. The g = 2.04 signal was localized in the cytosolic fraction of hepatocyte extracts. Hepatocytes freshly isolated from C. parvum-treated rats exhibited a modest g = 2.04 signal, which was increased by a factor of approximately 2.5-fold upon subsequent 24-h culture in media without additional stimuli. This increase was prevented by L-NMMA in the culture medium and also by the presence of rat erythrocytes added to the culture. In the presence of erythrocytes, virtually all of the .N = O produced was oxidized by reaction with intracellular hemoglobin within the erythrocyte, as judged by the relative amounts of nitrite and nitrate detected. These results suggest that in this model system .N = O is sufficiently stable and diffusible to escape from the hepatocyte and diffuse into the erythrocyte without first reacting with oxygen or with intracellular iron at the site of its formation within the hepatocyte. Treatment of hepatocytes with exogenous .N = O or nitroprusside generated an identical g = 2.04 signal of much greater intensity than with cytokines plus LPS. Treatment with nitroprusside also caused the appearance of a signal from pentacyanonitrosylferrate ion, verifying the previously reported metabolism of this nitrovasodilator by reduction and liberation of cyanide ion and .N = O. These results indicate significant differences in intracellular nonheme iron nitrosylation in hepatocytes compared to cytotoxic activated macrophages, which may correlate with the differences in physiological function of .N = O in these two systems.
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PMID:Nonheme iron-nitrosyl complex formation in rat hepatocytes: detection by electron paramagnetic resonance spectroscopy. 838 4

The beta-subunit of the nitrate reductase of Escherichia coli contains four groups of Cys residues (I-IV) which are thought to bind the single [3Fe-4S] center and the three [4Fe-4S] centers. The first or second Cys residue of group I was substituted by site-directed mutagenesis with Ala or Ser. Physiological, biochemical, and EPR studies were performed on the mutated enzymes. With small variations, the properties of these mutant enzymes do not differ from one another. They were found to be as abundant and as stably bound to the membrane as the native enzyme, provided the gamma-subunit was present. Although physiological activity was reduced, it was sufficient to allow growth on nitrate. The study of variations in EPR intensity as a function of the redox potential indicated that these enzymes only contained three iron-sulfur centers instead of the usual four in the native enzyme. Spectral EPR analysis showed that the [4Fe-4S] center of high redox potential (center 1, +80 mV) was missing. The loss of this center did not affect the stable integration of the other three centers. The data presented here are in total contrast to those we have reported for each of the other three centers (centers 2-4), the loss of which was detrimental to the integration of all centers and to the integration of the molybdenum cofactor (Augier et al., in press). Taken together, our results demonstrated that the first and second Cys residues of group I are the ligands of the [4Fe-4S] center (center 1, +80 mV) and that this center participates in electron transfer, but is dispensable. On the basis of these results, it is proposed that the [3Fe-4S] center (center 2, +60 mV) also plays a biological role and that in the native enzyme both high-potential centers, centers 1 and 2, contribute independently and in parallel to the electron transfer to the molybdenum cofactor.
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PMID:Removal of the high-potential [4Fe-4S] center of the beta-subunit from Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of site-directed mutated enzymes. 838 53

Assimilatory NADH:nitrate reductase catalyzes the transfer of reducing equivalents from NADH to molecular oxygen. Initial rate studies performed under conditions of optimal pH (8.0) and constant ionic strength (mu = 0.2) revealed that the maximal rate of activity with molecular oxygen was 0.5% (0.44 mumol NADH consumed/min/nmol heme) with a Km for O2 of 586 microM. NADH:molecular oxygen reductase activity exhibited a pH optimum of 9.2, was inhibited by cyanide, and was unaffected by changes in ionic strength or the presence of phosphate ions. Spectroscopic studies indicated NADH:molecular oxygen reductase activity resulted in the production of the superoxide radical, detected as the formation of adrenochrome from epinephrine and by the formation of adrenochrome from epinephrine and by the reduction of nitroblue tetrazolium, both of which could be inhibited by the addition of superoxide dismutase and were unaffected by the addition of catalase. Direct observation of superoxide production using spin-trapping in combination with EPR spectroscopy resulted in the detection of the spin adduct 5.5-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH). The formation of this spin adduct was abolished either in the absence of nitrate reductase, NADH, or DMPO or the the addition of superoxide dismutase or nitrate and was greatly reduced by the presence of cyanide. Inclusion of catalase or ethanol had no effect on the formation of the spin adduct. These results indicate that nitrate reductase can utilize molecular oxygen as an electron acceptor and that the product, O2.(-), is primarily generated via the Mopterin center.
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PMID:Superoxide production during reduction of molecular oxygen by assimilatory nitrate reductase. 861 Oct 27

Formation of peroxynitrite by the fast reaction between nitric oxide and superoxide anion may represent a critical control point in cells producing both species, leading to either down-regulation of the physiological effects of superoxide anion and nitric oxide by forming an inert product, nitrate, or to potentiation of their toxic effects by oxidation of nearby molecules by peroxynitrite. (The term peroxynitrite is used to refer to the sum of all possible forms of peroxynitrite anion and peroxynitrous acid unless otherwise specified.) In this report we demonstrate that, in spite of all the antioxidant defences present in human plasma, its interaction with peroxynitrite leads to generation of free radical intermediates such as (i) the ascorbyl radical, detected by direct EPR, (ii) the albumin-thiyl radical, detected by spin-trapping experiments with both N-tert-butyl-alpha-phenylnitrone and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and (iii) a uric acid-derived free radical, detected as the DMPO radical adduct in plasma whose thiol groups were previously blocked with 5,5-dithiobis-(2-nitrobenzoic acid). The identity of the latter adduct was confirmed by parallel experiments demonstrating that it is not detectable in plasma pretreated with uricase, whereas it is formed in incubations of peroxynitrite with uric acid. Peroxynitrite-mediated oxidations were also followed by oxygen consumption and ascorbate and plasma-thiol depletion. Our results support the view that peroxynitrite-mediated one-electron oxidation of biomolecules may be an important event in its cytotoxic mechanism. In addition, the data have methodological implications by providing support for the use of EPR methodologies for monitoring both free radical reactions and ascorbate concentrations in biological fluids.
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PMID:Peroxynitrite-mediated formation of free radicals in human plasma: EPR detection of ascorbyl, albumin-thiyl and uric acid-derived free radicals. 861 82

The electron paramagnetic resonance linewidth of aquo gadolinium(III) ion changes with the counter-ion identity and concentration in aqueous solutions. The EPR linewidth of 2 mM gadolinium(III) chloride increases from 49.2 to 89.0 mT when carbonate ion is added and decreases to 17.3 mT when nitrite ion is added. These observations suggest association reactions between aquo gadolinium(III) ion and anions that change the electron spin relaxation rates of the aquo ion. The concentration dependence of the gadolinium(III) EPR linewidth is consistent with binding constants for nitrite and nitrate ion with the aquo gadolinium(III) ion of 37 +/- 6 and 2.3 +/- 0.3 L mol-1 respectively. The decreases in the EPR linewidth factors with these association reactions are difficult to understand unless the anion reactions increase the symmetry of the metal center. Although first-coordination reactions may not be ruled out, the decrease in EPR linewidth is more consistent with an outer-sphere association reaction that also reduces the coordination number of the metal center from 9 to 8.
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PMID:Ionic association and electron spin relaxation rates in aquo gadolinium (III) complexes. 866 Dec 68

Platinum complexes PtII(DAPO)X2 with diaminonitroxyl radical-trans-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl (DAPO)-were synthesized by the direct reaction of DAPO with K2PtX4 (X = Cl, I) or by the replacement of chloro ligands in PtII(DAPO)Cl2 by bromo, nitrato, oxalato, malonato, and 1,1-cyclobutanedicarboxylato ligands. The complexes thus obtained were characterized by elemental analysis, infrared,electronic, electron paramagnetic resonance spectroscopic techniques, and high-performance liquid chromatography. The toxicity of compounds in terms of LD50 strongly depends on the nature of X-ligands, and varies between 11 mg/kg (X = NO3) and 400 mg/kg (X2 = 1,1-cyclobutanedicarboxylate). Up to 66% of mice bearing leukemia L1210 survive after the administration of these complexes. This effect is comparable to the effect of cisplatin (50% survive). An increase in the life span of the rest of the animals ranges from 158 to 383%. Complex PtII(DAPO)Cl2 appears to be more efficient than cisplatin against adenocarcinoma 755. Cisplatin, cis-diamminedichloroplatinum(II); CBDCA, 1,1-cyclobutanedicarboxylic acid; DAPO, trans-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl; Mal, malonic acid; Ox, oxalic acid; IR, infrared; EPR, electron paramagnetic resonance; HPLC, high-performance liquid chromatography; Ca755, adenocarcinoma 755; LD50 and LD100, dose of compounds (mg/kg), causing a death of 50 or 100% or treated animals; ILS, increase in life span of mice.
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PMID:Synthesis and antitumor activity of platinum(II) complexes with trans-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl. 883


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