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)

A rat heme oxygenase (HO-1) gene without the sequence coding for the last 23 amino acids has been constructed and expressed behind the pho A promoter in Escherichia coli. The enzyme is expressed at high levels as a soluble catalytically active protein that causes the bacterial cells to accumulate biliverdin. The purified truncated heme-heme oxygenase complex is spectroscopically indistinguishable from the complex with the native enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. Reaction of the recombinant heme-heme oxygenase complex with H2O2 produces a species with the spectroscopic properties of verdoheme. Unidentified products are obtained when this intermediate is directly extracted from the protein, but biliverdin is obtained if the verdoheme-protein complex is exposed to cytochrome P450 reductase and NADPH before the extraction step. In contrast, reaction of the heme-heme oxygenase complex with meta-chloroperbenzoic acid (mCPBA), tert-butylhydroperoxide, or cumene hydroperoxide yields a ferryl (FeIV = O) complex. Reaction of the heme-heme oxygenase complex with mCPBA also produces an EPR-detectable protein radical. In accord with formation of a ferryl intermediate, recombinant heme oxygenase catalyzes the mCPBA- and alkylhydroperoxide-dependent peroxidation of 2-methoxyphenol (guaiacol). Guaiacol oxidation is not observed during turnover of the enzyme by cytochrome P450 reductase/NADPH or H2O2. Conversely, biliverdin is not formed with tert-butylhydroperoxide or mCPBA. H2O2 thus supports the first step of the normal catalytic oxidation of heme by heme oxygenase, but alkyl and acyl hydroperoxides do not. These results suggest that the alpha-meso-hydroxylation required for biliverdin formation is mediated by the distal of the two oxygens in the iron-dioxygen intermediate (Fe-O-O) engendered by reaction with either cytochrome P450 reductase/NADPH or H2O2.
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PMID:Rat liver heme oxygenase. High level expression of a truncated soluble form and nature of the meso-hydroxylating species. 822 46

Anaerobically grown Escherichia coli contain an oxygen-sensitive ribonucleotide reductase. The enzyme requires anaerobic activation by two E. coli fractions with S-adenosylmethionine, NADPH, dithiothreitol, and KCl. We now find that photochemically reduced deazaflavin can substitute for these two fractions and NADPH. The reductase contained roughly equimolar amounts of iron and sulfide, suggesting the presence of an Fe-S complex. The cluster is characterized by a charge transfer band at 420 nm and a low temperature EPR signal centered at g = 2.01 that is difficult to saturate at 14 K, suggested to be a (3Fe-4S)+ cluster. In five different preparations of essentially protein-pure reductase containing widely different amounts of iron, the catalytic activity correlated well with the iron content. The iron signal disappeared during reductive anaerobic activation, with the appearance of a new EPR signal at g = 2.0033 showing a temperature behavior and microwave power saturability consistent with an organic free radical. The signal disappeared after exposure of the activated enzyme to air. We suggest that activation involves generation of a specific amino acid free radical that is dependent on the reduced Fe-S cluster and S-adenosylmethionine. From other work it appears likely that the free radical is localized on glycine 681 of the polypeptide chain.
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PMID:An iron-sulfur center and a free radical in the active anaerobic ribonucleotide reductase of Escherichia coli. 838 2

Molecular properties of superoxide (O2-)-producing cytochrome b558 purified from neutrophils were investigated focusing on the mechanism of the catalytic reaction. The purified cytochrome, which was depleted of FAD, exhibited high O2(-)-generating activity with consumption of NADPH in the presence of microsomal NADPH-cytochrome P-450 reductase. Exogenous additions of CO, CN-, or N3- had no effect on the enzymatic activity. Potentiometric titration of the ferric-ferrous couple of the cytochrome showed that the midpoint reduction potential was -255 mV at pH 7.4. When the reaction of the reduced cytochrome with O2 was analyzed by stopped flow and rapid scanning spectrophotometry, the ferrous form was found to be converted to the ferric form at a rate constant of 9.3 x 10(6) M-1 s-1 at 10 degrees C without showing formation of an oxygenated intermediate. EPR measurement of the ferric cytochrome at 10 K showed that the electronic spin state was in a low spin with g values of 3.2, 2.05, and 1.5. These results suggest that the heme in a six-coordinated low spin state catalyzes one electron reduction of O2 without ligation of O2 to the heme iron during the catalytic cycle.
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PMID:Superoxide-producing cytochrome b. Enzymatic and electron paramagnetic resonance properties of cytochrome b558 purified from neutrophils. 838 86

EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals from a number of organic peroxides and peracids which are known or suspected tumour promoters. All of the compounds when incubated with rat liver microsomal fractions in the presence of NADPH or NADH are metabolised to radical species which can be detected, and in most cases identified definitively, as the corresponding spin adducts; the assignment of particular signals to certain spin adducts has been confirmed by photolytic experiments. In the majority of cases, the predominant species are the arenecarbonyloxyl [RC(O)O.] and hydroxyl radical adducts. The mechanism of formation of the former species is shown to be enzymatic and cytochrome P-450 dependent and requires the presence of reducing equivalents. This type of radical is shown to undergo ready decarboxylation to give aryl radicals in agreement with previous chemical studies. The detection of these radical species, which are known to cause DNA strand breaks and be cytotoxic, with all the compounds tested, provides strong supportive evidence for the theory that it is the generation of radical species from these compounds which is the cause of their tumour-promoting activity.
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PMID:Radical production from peroxide and peracid tumour promoters: EPR spin trapping studies. 838 32

The formation of alpha-hydroxyethyl radical from ethanol by deermouse microsomes supplemented with NADPH has been demonstrated with the EPR technique of spin trapping with alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) as the spin trap, in the presence of deferoxamine mesylate. Superoxide dismutase prevented the formation of the radical adduct of the alpha-hydroxyethyl radical in a dose-dependent fashion, causing complete inhibition of radical formation at a concentration of 20 micrograms/ml, while catalase, azide, or azide and hydrogen peroxide had no effect. Boiling the microsomes or omission of NADPH abolished free radical formation. Metyrapone, cimetidine, isoniazid, octylamine, and p-nitrophenol, inhibitors of cytochrome P450IIE1 activity, decreased free radical formation by 24 to 74% at concentrations ranging from 0.05 to 10 mM. The POBN/.CH(OH)CH3 radical adduct signal was also diminished by formate or mannitol, indicating competition by so-called "hydroxyl radical scavengers. "The involvement of trace transition metals in alpha-hydroxyethyl radical formation by deermouse microsomes was demonstrated by a decrease in free radical adduct signal when metal ions were removed from reagents by treatment with Chelex 100 resin. Chelex treatment was effective even though the metal chelator deferoxamine mesylate, which is generally presumed to render iron catalytically inactive, was present in all incubations. Thus, it is concluded that free radical formation from ethanol in deermouse microsomes is mediated by a transition metal- and cytochrome P450-derived superoxide-dependent oxidizing species even in the presence of deferoxamine mesylate.
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PMID:Role of superoxide and trace transition metals in the production of alpha-hydroxyethyl radical from ethanol by microsomes from alcohol dehydrogenase-deficient deermice. 839 Feb 20

The energy metabolism of the intestinal parasite, Giardia lamblia, involves the iron-sulphur protein, pyruvate:ferredoxin oxidoreductase. Cell fractionation studies showed that this enzyme is associated with the membranes. NADH and NADPH dehydrogenases were found in both the membrane and cytosolic fractions. EPR spectroscopic studies showed the presence of iron-sulphur clusters in the membrane fraction and in the cytosolic fraction, non-sedimentable at 6 x 10(6) g.min. An acidic, soluble protein fraction was separated from the cytosol. It had an EPR spectrum in the reduced state, characteristic of the 2[4Fe-4S] type of ferredoxin, with g-factors at 2.04. 1.93 and 1.89, and the midpoint redox potential was estimated to be -360 mV. This species is probably a ferredoxin, like those of anaerobic bacteria such as Clostridium and Desulfovibrio spp. and also that of Entamoeba histolytica. The protein was readily and irreversibly oxidized to give [3Fe-4S] clusters.
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PMID:Electron transport components of the parasitic protozoon Giardia lamblia. 839 75

During anaerobic growth Escherichia coli uses a specific ribonucleoside triphosphate reductase for the production of deoxyribonucleoside triphosphates. The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha 2) with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain. The radical is formed in an enzymatic reaction involving S-adenosylmethionine, NADPH, a reducing flavodoxin system and an additional 17.5-kDa polypeptide, previously called activase. Here, we demonstrate by EPR spectroscopy that this small protein contains a 4Fe-4S cluster that joins two peptides in a 35-kDa small homodimer (beta 2). A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase. These preparations were contaminated with a small amount of the small protein. The large and the small proteins form a tight complex. From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex. The anaerobic reductase thus has an alpha 2 beta 2 structure. We speculate that the small protein interacts with S-adenosylmethionine and forms a transient radical involved in the generation of the stable glycyl radical in the large protein that participates in the catalytic process.
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PMID:The anaerobic Escherichia coli ribonucleotide reductase. Subunit structure and iron sulfur center. 862 8

Glutamate synthase is a complex iron-sulfur flavoprotein containing one molecule each of FAD and FMN and three distinct iron-sulfur centers/alpha beta protomer. Production of the beta subunit was observed in total extracts of Escherichia coli BL21 (DE) cells harbouring a pT7-7 derivative carrying gltD, the gene encoding the Azospirillum brasilense glutamate synthase beta subunit. The protein was soluble, and the identity of the purified protein with the Azospirillum glutamate synthase beta subunit was confirmed by N-terminal sequence analysis. The kinetic and spectroscopic characterization of the glutamate synthase beta subunit confirmed that it contains the NADPH binding site, but, in contrast with earlier proposals that assigned both FAD and FMN binding sites to the alpha subunit of glutamate synthase, the beta subunit was shown to contain stoichiometric amounts of FAD. No iron-sulfur centers were detected by EPR spectroscopy measurements of the recombinant beta subunit. Under steady-state conditions, the glutamate synthase beta subunit can catalyze the NADPH-dependent reduction of several synthetic electron acceptors but no glutamate synthase or glutamate dehydrogenase reactions in either direction. The results are in agreement with previous data from our laboratory and, together with the absence of amino acid sequence similarity between glutamate synthase beta subunit and glutamate dehydrogenases, are against the hypothesis that glutamate synthase is evolutionarily derived from the association of an ancestral glutamate dehydrogenase (the beta subunit) and an amidotransferase (the alpha subunit). The protein-bound FAD is reduced by NADPH at a rate much faster than turnover with synthetic electron acceptors, leading to formation of a stable reduced flavin-NADP+ charge-transfer complex. The rate of reduction of the bound FAD by NADPH is also similar to the rate at which one of the flavins is reduced in the native glutamate synthase, as measured in a stopped-flow spectrophotometer under pre-steady-state conditions. The ability of FAD bound to the beta subunit of glutamate synthase to react with NADPH and the lack of reactivity with sulfite lead us to conclude that FAD is Flavin 1 of glutamate synthase [Vanoni, M.A., Edmondson, D.E., Zanetti, G. & Curti, B. (1992) Biochemistry 31, 4613-4623].
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PMID:Properties of the recombinant beta subunit of glutamate synthase. 866 16

Several species of marine tunicates store oxygen-sensitive VIII in blood cells. A sensitive colorimetric VIII assay was used to survey the leading candidates for the native reducing agent of vanadate in tunicates (i.e., An-type tunichromes, glutathione, NADPH, and H2S) in reactions with VV or VIV ions under anaerobic, aqueous conditions at acidic or neutral pH. Except for the case of An-1 and VV ions in pH 7 buffer, the assay results for the biogenic reducing agents clearly showed that appreciable quantities of VIII products were not generated under the conditions tested. Therefore, the assay results place new limits on hypothetical mechanisms of VIII formation in vivo. For reactions between An-1 and VV ions in pH 7 buffer, low levels of VIII products could not be ruled out because of an interfering peak in the colorimetric assays. For similar reactions between VV ions and An-1, or an An-1,2 mixture, in mildly to moderately basic media, the product mixtures precipitated as greenish black solids. Analyses of the precipitated V/An mixtures using vanadium K-edge X-ray absorption spectroscopy (XAS) showed that the major products were tris(catecholate)-type VIV complexes (65 +/- 6%) and bis(catecholate)-type VIVO complexes (20 +/- 4%). XAS analysis of the V/An-1 product mixture also provided evidence of a minor VIII component (9 +/- 5% of total V), notable for possible relevance to tunicate biochemistry. The combined results of XAS studies, spectrophotometric studies [Ryan, D. E., et al. (1996) Biochemistry 35, 8640-8650], and EPR studies [Grant, K. B., et al. (1996) J. Inorg. Biochem. (manuscript in preparation)] consistently establish that reactions between tunichromes (Mm-1 or An-1) and VV ions generate predominantly VIV-tunichrome complexes in neutral to moderately basic aqueous media.
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PMID:Reactions between vanadium ions and biogenic reductants of tunicates: spectroscopic probing for complexation and redox products in vitro. 867 27

Rapid events in the processes of electron transfer and substrate binding to cytochrome P-450 BM3 from Bacillus megaterium and its constituent haem-containing and flavin-containing domains have been investigated using stopped-flow spectrophotometry. The formation of a blue semiquinone flavin form occurs during the NADPH-dependent reduction of the flavin domain and a species with a similar absorption maximum is also seen during reduction of the holoenzyme by NADPH. EPR spectroscopy confirms the formation of the flavin semiquinone. The formation of this semiquinone is transient during fatty acid monooxygenation by the holoenzyme, but in the presence of excess NADPH the species reforms once fatty acid is exhausted. Electron transfers through the reductase domain are too rapid to limit the fatty acid monooxygenation reaction. The substrate-binding-induced haem iron spin-state shift also occurs much faster than the Kcat at 25 degrees C. The rate of first electron transfer to the haem domain is also rapid; but it is of the order of 5-10-times larger than the Kcat for the enzyme (dependent on the fatty acid used). Given that two successive electron transfers to haem iron are required for the oxygenation reaction, these rates are likely to exert some control over the rate of fatty acid oxygenation reactions. The presence of large amounts of NADPH also results in decreased rates of electron transfer from flavin to haem iron. In the difference spectrum of the active fatty acid hydroxylase, features indicative of a high-spin iron haem accumulate. These are in accordance with the presence of large amounts of an Fe(3+)-product bound enzyme during turnover and indicate that product release may also contribute to rate limitation. Taken together, these data suggest that the catalytic rate is not determined by the accumulation of a single intermediate in the reaction scheme, but rather that it is controlled in a series of steps.
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PMID:Probing electron transfer in flavocytochrome P-450 BM3 and its component domains. 870 47


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