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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The apo-enzymes of porphobilinogen oxygenase and horseradish peroxidase were reconstituted with hemin IX, deuterohemin IX, 2,4-diacetyldeuterohemin IX, 2-vinyl-4-deuterohemin IX and hemin I. The apoproteins did not reconstitute with the dimethyl or diethyl esters of hemin IX. The native enzymes and the synthetic hemoproteins showed similar oxygenase activities toward porphobilinogen in the presence of dithionite and oxygen. They also showed peroxidase activity in the presence of H2O2, which was affected by the side-chain substitution pattern of the hemes. Oxygenase activities, however, were not affected by the heme structure. Iron chelators completely inhibited the oxygenase, but not the peroxidase activities. The EPR spectra of the native and synthetic porphobilinogen oxygenase showed that dithionite reduction produced a rapid disappearance of the high-spin heme-iron signal at g = 6.0. It reappeared 1 min later but the enzyme retained its catalytic activity. The changes in the EPR spectra could be correlated with the biphasic kinetics of the oxygenase reaction which was very fast during the first minute and then decreased to a half-value rate. The oxygenase reaction was inhibited by addition of superoxide dismutase during the fast rate phase, but not during the slower phase. These results could be explained by the formation of a superoxide anion during the first minute of the oxygenase reaction, after which a protein-stabilized radical (g = 2.0) is generated (very likely a tyrosyl radical). The latter then oxidizes the substrate porphobilinogen and facilitates its reaction with O2 to give oxopyrrolenines.
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PMID:Structure/activity relationships in porphobilinogen oxygenase and horseradish peroxidase. An analysis using synthetic hemins. 824 71

The reactions of hydrogen peroxide (H2O2) with nitrite (NO2-) and of superoxide (O2-.) with nitric oxide (NO.) were studied using EPR and spin trapping techniques. These reactions reportedly have a common peroxynitrite (OONO-) intermediate. It has been suggested that this intermediate when protonated rapidly decomposes producing hydroxyl radicals (.OH) and the nitrogen dioxide radical (NO2.). The production of .OH in the reaction between H2O2 and NO2- was confirmed in spin trapping experiments using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). H2O2 and NO2- were mixed at neutral pH and then the pH was decreased to pH 3-3.5 in the presence of DMPO or DMPO and ethanol. In these experiments, the EPR spectrum of the DMPO-OH adduct was obtained in addition to a weak EPR spectrum consisting of a triplet of triplets (a N = 1.415 mT and a N beta = 0.35 mT) indicating the addition of a nitrogen centered radical to DMPO. The formation of .OH was confirmed using ethanol as an .OH scavenger. The DMPO-hydroxyethyl adduct was produced from the reaction of .OH with ethanol. However, in experiments using an excess of ethanol, the formation of DMPO-OH was not prevented. This suggests that the DMPO-OH formed in the decomposition of HOONO does not entirely originate from a direct addition of .OH to DMPO. The reaction of O2-. with NO. was carried out in deaerated and air-saturated solutions at pH 12.3 where the dismutation of O2-. is minimal. The pH was then decreased to pH 3-3.5 in the presence of DMPO or DMPO and ethanol. In these experiments, the most prominent EPR spectrum obtained was a triplet of triplets (aN = 1.415 mT and a beta N = 0.35 mT) suggesting the addition of a nitrogen centered radical to DMPO. The formation of DMPO-OH was minimal and there was no formation of DMPO-hydroxyethyl adducts in the presence of ethanol. The results suggest that NO. in solution yields additional reactive species which act as nitrating agents in the presence of DMPO.
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PMID:Reactions of active oxygen and nitrogen species studied by EPR and spin trapping. 828 10

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

Luteoskyrin is a bis-dihydroanthraquinone mycotoxin produced by Penicillium islandicum Sopp. By using EPR spin-trap techniques we investigated whether .OH is formed in a model system containing ascorbic acid and the toxin. In the presence of DMSO and DMPO, we found signals of DMPO-CH3, a more specific and reliable signature of .OH than DMPO-OH, together with the signals of ascorbyl radical. DMPO-CH3 signals increased with time of incubation up to 5.5 min. The DMPO-CH3 formation depended completely on both luteoskyrin and ascorbic acid, and deferoxamine, an iron-chelator, inhibited its formation. The signals disappeared in the presence of excess amount of catalase whereas SOD showed no effect. These results suggest that .OH is formed from ferrous ion present in the mixture of H2O2 generated from ascorbic acid and luteoskyrin.
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PMID:Luteoskyrin, an anthraquinoid hepatotoxin, and ascorbic acid generate hydroxyl radical in vitro in the presence of a trace amount of ferrous iron. 828 36

Combined stimulation, by superoxide ions generated by the xanthine-xanthine oxidase reaction, and platelet-activating factor (PAF), induced cell differentiation of rat monocytic leukemia cells (c-WRT-LR) to macrophage-like mature cells. Monitoring of cytochrome c reduction revealed that PAF stimulation induced the release of superoxide ions from c-WRT-LR. To further investigate the effect of superoxide ions in the autocrine or paracrine mechanism in cell differentiation, molecular species of the oxygen radicals under PAF stimulation were examined using the EPR spin trap, 5,5'-dimethyl-1-pyrroline N-oxide (DMPO). PAF and/or phorbol myristate acetate caused the formation of EPR spectra, a combination of DMPO/.OOH and DMPO/.OH. Since both spectra were diminished in the presence of superoxide dismutase, it was concluded that DMPO/.OH was derived from superoxide ions. Mannitol and catalase suppressed cell differentiation induced by combined stimulation with PAF and oxygen radicals generated by the xanthine-xanthine oxidase reaction. Taken together, these results suggest that hydroxyl radicals generated by Fenton reaction from H2O2 may be involved in the mechanism of cell differentiation in rat monocytic leukemia cells.
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PMID:A role for oxygen radicals in rat monocytic leukemia cell differentiation under stimulation with platelet-activating factor. 830 1

Histidine-93(F8) in human myoglobin (Mb), which is the proximal ligand of the heme iron, has been replaced with cysteine or tyrosine by site-directed mutagenesis. The resultant proximal cysteine and tyrosine mutant Mbs (H93C and H93Y Mbs, respectively) exhibit the altered axial ligation analogous to P-450, chloroperoxidase, and catalase. Coordination of cysteine or tyrosine to the ferric heme iron is confirmed by spectroscopic measurements including electronic absorption, hyperfine-shifted 1H-NMR, EPR, resonance Raman spectroscopies, and redox potential measurements of ferric/ferrous couple. H93C Mb is five-coordinate ferric high-spin with the proximal cysteine. H93Y Mb bearing the proximal tyrosine ligated to the iron is also in a ferric high-spin, five-coordinate state. The reactions of the mutants with cumene hydroperoxide show that the thiolate ligand enhances heterolytic O-O bond cleavage of the oxidant, while the phenolate ligand hardly affects the heterolysis/homolysis ratio for O-O bond scission in comparison with wild-type Mb. Monooxygenase activities such as epoxidation of styrene and N-demethylation of N,N-dimethylaniline, and catalase activity (dismutation of hydrogen peroxide) by wild-type Mb and the mutants, are examined by using H2O2. The increase of the catalytic activities by the mutation was, at most, 5-fold in the epoxidation reaction.
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PMID:Roles of proximal ligand in heme proteins: replacement of proximal histidine of human myoglobin with cysteine and tyrosine by site-directed mutagenesis as models for P-450, chloroperoxidase, and catalase. 838 Mar 34

We investigated the role of free radicals in hemoglobin (Hb) oxidation and denaturation. To generate free radicals, we used two azocompounds, the hydrophilic 2,2'-azobis(2-amidinopropane hydrochloride and the hydrophobic 2,2'-azobis(2,4-dimethylvaleronitrile) and a drug of the quinone family, phenazine methosulfate. The radical species involved were analyzed by direct EPR and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide, and N-t-butyl-alpha-phenyl-nitrone. The free radicals generated by the azocompounds were carbon radicals and, in the presence of molecular oxygen, peroxyl/alkoxyl radicals. The reaction of phenazine with Hb produced a nitrogen-centered semiquinoid radical detectable by EPR only under N2 and reactive oxygen species (O2-. and H2O2) in the presence of molecular oxygen. Azocompounds oxidized Hb to methemoglobin, hemichromes, and choleglobin while phenazine produced methemoglobin and ferrylhemoglobin. For all three drugs, low oxygen tensions (pO2 = 62 mm Hg) increased the formation of Hb oxidation products, whereas high oxygen tensions (pO2 = 540 mm Hg) reduced Hb oxidation. The formation of irreversible Hb oxidation products (irreversible hemichromes and Hb cross-linking) was observed only with the azocompounds and was reduced at high pO2. Spin traps and thiourea protected Hb from the oxidative damage induced by the azocompounds, whereas enzymes scavenging reactive oxygen species, such as superoxide dismutase and catalase, affected Hb oxidation induced by phenazine and that induced by the hydrophobic azocompound. These results indicate distinct patterns of oxidation and denaturation with each agent. Damage induced by phenazine was dependent on the formation of reactive oxygen species, whereas the damage induced by the azocompounds was due mainly to carbon-centered radicals with some involvement by reactive oxygen species only for the hydrophobic azocompound. The preferential interaction of Hb with drug radicals scavenged by molecular oxygen indicates that this protein may be more reactive under hypoxic conditions and led to the view that a good supply of oxygen can provide an important defense against drug-induced Hb oxidation.
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PMID:Role of oxygen and carbon radicals in hemoglobin oxidation. 838

Although the cytochrome c peroxidase/H2O2 reaction product, compound ES, has been a long-standing subject of research, only recently has its broad EPR signal been proven to arise from a radical at Trp-191. Despite this advance, no model has satisfactorily explained the anomalous breadth and shape of this signal, which is conventionally interpreted as having axial symmetry with g parallel approximately 2.04 > g perpendicular approximately 2.01, contrary to expectations for a planar pi radical. Furthermore, these g values exhibit marked temperature and preparation dependencies as well as an unexplained high-field "tail" extending from the g = 2.01 peak. We have reexamined the EPR and ENDOR spectra of compound ES at 35 GHz, as well as those of compound ES in the mutant D235E. This mutation significantly alters the line shape of the Trp-191 free radical. We present a comprehensive model that successfully accounts for the properties of this unusual protein free radical. We show that the EPR spectra of both proteins can be described in terms of a weak exchange interaction between the S = 1 oxyferryl (Fe = O)2+ moiety and a radical on Trp-191; a distribution in protein conformation leads to a distribution in the coupling, which ranges from ferromagnetic to antiferromagnetic. We also derive, for the first time, explicit expressions for frozen-solution and single-crystal spectra of such spin-coupled systems and show that the model accounts for all the data that previously led to apparent anomalies in the interpretation of the frozen-solution and single-crystal [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 349-355] EPR properties. Finally, we have used the CW EPR and pulsed-EPR saturation-recovery methodology to address reports that the broad signal from the spin-coupled Trp-191 radical is accompanied by a minority (approximately 10%), narrow signal that is associated with a radical site other than Trp-191. We find no evidence for such a species and discuss the earlier reports in light of our model.
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PMID:Comprehensive explanation of the anomalous EPR spectra of wild-type and mutant cytochrome c peroxidase compound ES. 838 47

A peroxidase was purified 98.3-fold from the culture filtrate of Pleurotus ostreatus with an overall yield of 12.4%. The molecular mass determined by gel filtration was found to be approx. 140 kDa. SDS-PAGE revealed that the enzyme consists of two identical subunits with a molecular mass of approx. 72 kDa. The pI value of this enzyme is approx. 4.3. The enzyme contains 41% carbohydrate by weight, and aspartic acid and asparagine (16.8%), and glutamic acid and glutamine (12.0%). The enzyme has the highest affinity toward synaptic acid and affinity towards various phenolic compounds containing methoxyl and p-hydroxyl groups, directly attached to the benzene ring. However, the enzyme does not react with veratryl alcohol and shows no affinity for nonphenolic compounds. The optimal reaction pH and temperature are 4.0 and 40 degrees C, respectively. The catalytic mechanism of the enzymic reaction is of the Ping-Pong type. The activity of the enzyme is competitively inhibited by high concentrations of H2O2 and its Ki value is 1.70 mM against H2O2. This enzyme contains approx. 1 mol of heme per mol of one subunit of the enzyme. The pyridine hemochrome spectrum of the enzyme indicates that the heme of P. ostreatus peroxidase is iron protoporphyrin IX. The EPR spectrum of the native peroxidase shows the presence of a high-spin ferric complex with g values at 6.102, 5.643 and 1.991.
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PMID:Purification and characterisation of an extracellular peroxidase from white-rot fungus Pleurotus ostreatus. 838 25

Oxidation of amidopyrine, analgin and 4-aminoantipyrine by means of H2O2 was studied in presence of peroxidase and hemoglobin. As shown by NMR, EPR and spectrophotometry the oxidation was of single electron type accompanied by free radical formation. The radicals of amidopyrine and analgin were stable, colored, participated in chemical exchange with initial molecules and disproportionated as demonstrated by stoichiometry. Radicals of 4-aminoantipyrine were unstable and dimerized to antipyrine red. Aminopyrazolone radicals were also formed after direct oxidation by Fe3+ in acidified water containing no complexes. The Fe-complexes developed shifted the equilibrium towards reduction of radicals with formation of the initial molecules. Presence of 4-amino groups was responsible for oxidation of pyrazolones under mild conditions. Alkyl derivatives of 4-amino group protons stabilized the radicals and altered their subsequent transformation form dimerization to disproportionation. Proton catalysis of aminopyrazolone oxidation occurred.
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PMID:[Aminopyrazolone free radicals in the hydrogen peroxide oxidation reaction]. 838 98


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