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

We describe the purification of a H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. The enzyme is a monomeric flavoprotein containing flavin adenine dinucleotide in a 1:1 molar ratio with the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophoresis and N-terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a K(m) value of 4.2 microM for NADH and 16 microM for NADPH (pH 7.8 at room temperature). With oxygen as the primary electron acceptor under aerobic conditions, the pure enzyme did not produce O.-2 nor H2O2 as stoichiometric products of oxygen reduction, implicating H2O as the end product and obviating the need for superoxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (Cu2+ and Zn2+) inhibited this activity. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron acceptors including nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using either NADH or NADPH as electron donors. NADPH, however, was a more efficient electron donor. Cytochrome c was not reduced under any assay conditions used. The enzyme reduced the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pure NADH oxidase did not demonstrate ferredoxin:NAD(P)1 oxidoreductase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore, function as a terminal oxidase, similar to the mitochondrial cytochrome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardia close to the anaerobic bacteria in evolutionary terms.
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PMID:A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. 889 1

Hydroxyl and 1-hydroxyethyl radical adducts of 5,5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenan-throlene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe+2 and K3Fe(CN)6, but Fe+3 alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes from superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe+2, and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.
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PMID:Degradation of DMPO adducts from hydroxyl and 1-hydroxyethyl radicals by rat liver microsomes. 895 20

The role of reactive oxygen species in causing DNA damage through interaction of chromium (III) and hydrogen peroxide was examined using plasmid relaxation assay and EPR spectroscopy. Marked DNA strand breakage was induced by CrCl3 plus H2O2 in a phosphate buffer at pH 6-8.9; whereas, only slight DNA strand breakage was observed during similar treatment at pH less than 4. DNA breakage also increased as the reaction temperature and Cr(III)/H2O2 concentrations increased. Control experiments with Cr(III) or H2O2 alone did not cause DNA breakage. Sodium azide, D-mannitol, Tris-HCl, or catalase completely inhibited Cr(III)/H2O2-induced DNA breakage, but superoxide dismutase did not. The D2O enhancing effect on DNA breaks was not observed. Cr(III) pre-incubated with a 30-fold molar excess of EDTA did not cause any significant DNA breakage in the presence of H2O2. In a phosphate buffer containing Cr(III) and H2O2, singlet oxygen and hydroxyl radicals were detected using EPR spectrometry with the spin traps 2,2,6,6-tetramethyl-4-piperidone and 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), respectively. DMPO/.OH adducts and DNA breakage induced by Cr(III)/H2O2 were markedly higher than those induced by Cr(VI)/H2O2. Furthermore, ascorbate decreased Cr(III)/H2O2-induced DNA breakage. EPR studies revealed that ascorbate (mole ratio to Cr(III) = 0.5:1) attenuated the DMPO/.OH signal generated by Cr(III)/H2O2/DMPO, but a Cr(V) signal and ascorbate radicals were detected. NADPH, GSH, and GSSG also decreased DMPO/.OH generated by Cr(III)/H2O2/DMPO; however, they were less efficient than ascorbate and no Cr(V) signals were detected. This study shows that Cr(III)/H2O2 generates oxidative damage to DNA through a Fenton-like reaction: Cr(III) + H2O2-->Cr(IV) + .OH + OH.
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PMID:Formation of reactive oxygen species and DNA strand breakage during interaction of chromium (III) and hydrogen peroxide in vitro: evidence for a chromium (III)-mediated Fenton-like reaction. 902 Nov 67

Phenoxyl radicals are intermediates of one-electron oxidation of phenolic compounds by various peroxidases. This report describes reactions of phenoxyl radicals with human NADPH-cytochrome P-450 oxidoreductase (OR) and NADPH. Purified truncated OR catalyzed quenching of EPR signal of the phenoxyl radical of a vitamin E homolog, 2,2,5,7,8-pentamethyl-6-hydroxychromane. The quenching required both reductase and NADPH and was not supported by NADH. NADPH quenched directly the EPR signal of phenoxyl radical of a phenolic antitumor drug, etoposide, in the absence of the OR. Quenching of the EPR signal was accompanied by increased rate of NADPH oxidation and decreased rate of etoposide oxidation. Phenoxyl radicals of etoposide did not inactivate the OR. In the absence of NADPH, OR was inhibited irreversibly when exposed to phenoxyl radicals of phenol. The activity of the flavoprotein could not be recovered by dithiothreitol (DTT) but the inhibition was prevented by saturation of OR with NADP+ prior to the exposure to phenoxyl radicals. The OR was also inhibited by 5,5'-dithionitrobenzoic acid (DTNB). The inhibition was reversible by subsequent addition of DTT. OR pretreated with DTNB was protected from inhibition by phenoxyl radicals of phenol. The results indicate that phenoxyl radical of 2,2,5,7,8-pentamethyl-6-hydroxychromane is likely reduced enzymatically by transfer of electrons from NADPH via the FAD/FMN of the OR. Phenoxyl radicals with higher redox potential, e.g., phenoxyl radicals of etoposide, oxidize NADPH directly. Phenoxyl radicals of phenol can also inactivate OR likely by oxidation of cysteine 565 in the NADPH binding region of the enzyme.
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PMID:Reactions of phenoxyl radicals with NADPH-cytochrome P-450 oxidoreductase and NADPH: reduction of the radicals and inhibition of the enzyme. 911 95

Cytochrome P450BM3 is a self-sufficient soluble fatty acid hydroxylase from Bacillus megaterium utilizing tightly bound FAD and FMN cofactors to transfer reducing equivalents from NADPH to the heme active site. Active-inactive transitions of cytochrome P450BM3 were exploited to identify catalytic intermediates of the enzyme. Shortly upon reduction by NADPH, a two-electron reduced active P450BM3 is formed with two flavin semiquinones, anionic and neutral, present simultaneously. P450BM3 inactivated by NADPH has a three-electron reduced flavoprotein domain. NADPH is unable to reduce P450BM3 rapidly unless the flavoprotein domain is fully oxidized. During steady-state hydroxylation of a poor substrate, tetradecanol, the flavoprotein reduction state does not exceed two, with two flavin semiquinones, anionic and neutral, present. Absorbance and EPR spectroscopic characterization of both anionic and neutral flavin semiquinone is presented. NADPH and NADH were compared as electron donors for P450BM3-catalyzed fatty acid hydroxylation and cytochrome c and heme iron reduction. The Km for NADH of 3-5 mM is about 3000 times higher than the Km of 1-1.5 microM for NADPH. Although NADH can support cytochrome c reduction and fatty acid hydroxylation with the rates as high as 22 and 13 s-1, respectively, these turnover numbers are only about 20% of those observed with NADPH. The results suggest that nucleotide binding plays an important role in catalysis by controlling electron-transfer properties of the flavin cofactors. In W574G and G570D mutant P450BM3 enzymes that are deficient in FMN, NADP+ binding stabilizes fully reduced FAD. P450BM3 catalyzes single-turnover and steady-state laurate hydroxylation with near stoichiometric product formation at NADPH concentrations below that of the enzyme. A mechanism of electron transfer by the flavoprotein domain of P450BM3 is proposed with the reduction state of the flavoprotein domain cycling in a 0-2-1-0 sequence. We also propose that an interaction of bound NADP+ with anionic FAD semiquinone is essential for splitting a pair of electrons that are then transferred in two one-electron transfer steps to the heme catalytic site.
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PMID:Functional interactions in cytochrome P450BM3: flavin semiquinone intermediates, role of NADP(H), and mechanism of electron transfer by the flavoprotein domain. 920 88

In previous studies, benzo[a]pyrene-7,8-dione (BPQ), a polycyclic aromatic hydrocarbon (PAH) o-quinone, was found to be 200-fold more potent as a nuclease than (+/-)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene, a suspect human carcinogen. The mechanism of strand scission mediated by naphthalene-1,2-dione (NPQ) and BPQ was further characterized using either phiX174 DNA or poly(dG).poly(dC) as the target DNA. Strand scission was extensive, dependent on the concentration of o-quinone (0-10 microM), and required the presence of NADPH (1 mM) and CuCl2 (10 microM). The production of reactive species, i.e., superoxide anion radical, o-semiquinone anion (SQ) radical, hydrogen peroxide (H2O2), hydroxyl radical (OH.), and Cu(I), was measured in the incubation mixtures. The formation of SQ radicals was measured by EPR spectroscopy under anaerobic conditions in the presence of NADPH. A Cu(II)/Cu(I) redox cycle was found to be critical for DNA cleavage. No strand scission occurred in the absence of Cu(II) or when Cu(I) was substituted, yet Cu(I) was required for OH* production. Both DNA strand scisson and OH. formation were decreased to an equal extent, albeit not completely, by the inclusion of OH. scavengers (mannitol, soduim benzoate, and formic acid) or Cu(I) chelators (bathocuproine and neocuproine). In contrast, although the SQ radical signals of NPQ and BPQ were quenched by DNA, no strand scission was observed. When calf thymus DNA was treated with PAH o-quinones, malondialdehyde (MDA) was released by acid hydrolysis. The formation of MDA was inhibited by OH. scavengers suggesting that OH* cleaved the 2'-deoxyribose moiety in the DNA to produce base propenals. These studies indicate that for PAH o-quinones to act as nucleases, NADPH, Cu(II), Cu(I), H2O2, and OH*, were necessary and that the primary species responsible for DNA fragmentation was OH., generated by a Cu(I)-catalyzed Fenton reaction. The genotoxicity of PAH o-quinones may play a role in the carcinogenicity and mutagenicity of the parent hydrocarbons.
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PMID:DNA strand scission by polycyclic aromatic hydrocarbon o-quinones: role of reactive oxygen species, Cu(II)/Cu(I) redox cycling, and o-semiquinone anion radicals,. 921 11

Porcine recombinant dihydropyrimidine dehydrogenase was purified from Escherichia coli cells using cell disruption, ammonium sulfate fractionation, and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose. The yield was 60% with a specific activity of 14 units/mg protein. On SDS/PAGE the purified dehydrogenase exhibits a single band, indicating that no proteolytic degradation was taking place during purification. In agreement with the native enzyme, all cofactors, FMN, FAD, NADPH, and two iron-sulfur clusters, have been found. EPR spectra of the reduced dehydrogenase obtained at pH 9.5 are characteristic for two [4Fe-4S]1+ cubanes in dipolar interaction. Quantification of the observed signals indicated 0.95 spins per subunit, showing only partially reduced iron-sulfur clusters. The kinetic parameters of the porcine recombinant enzyme are very similar to those of the native enzyme. Thus, it can be concluded that the porcine recombinant enzyme behaves like the native dehydrogenase.
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PMID:Purification, characterization, and kinetics of porcine recombinant dihydropyrimidine dehydrogenase. 922 14

Compound I of Proteus mirabilis and bovine liver catalases (PMC and BLC, respectively) were studied combining EPR spectroscopy and the rapid-mix freeze-quench techniques. Both enzymes, when treated with peroxyacetic acid, form a catalytic intermediate which consists of an oxoferryl porphyrin pi-cation radical. In PMC this intermediate is semistable, and an unexpected reversible equilibrium under pH influence takes place between two forms of compound I with different coupling between the oxoferryl and the porphyrin pi-cation radical. At acid pH, one form has a ferromagnetic character as in Micrococcus luteus compound I. At neutral pH, another form with a much smaller coupling, reminiscent of the horse radish peroxidase compound I, is detected. The approximate midpoint, estimated for these changes in the range 5.3 < pH < 6.0, approaches the pKa value of an histidyl residue. The residues possibly involved in the transformation are discussed in terms of the known structure of PMC compound I. The EPR spectrum of BLC compound I (pH 5.6), obtained in the millisecond time scale (40 ms), also showed a mixture of two forms which, most probably, correspond to two different magnetic exchange interactions, as in the case of PMC. Taken together, the low-temperature electronic absorption and the EPR spectra of BLC compound I formed in the 0.04-15 s range show that the porphyrin pi-cation radical disappears and, instead, a tyrosyl radical is formed. ENDOR experiments confirm our previously estimated hyperfine couplings to the C2,6 and C3,5 ring protons and the beta-methylene protons of the purported tyrosyl radical. Candidates for such a tyrosyl radical are discussed in connection with the possible electron transfer pathways between the heme active site and the NADPH cofactor.
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PMID:EPR investigation of compound I in Proteus mirabilis and bovine liver catalases: formation of porphyrin and tyrosyl radical intermediates. 923 78

The effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems with respect to three cytochrome P-450 isozymes in rat liver microsomes were investigated. FK-506 non-competitively inhibited the aniline p-hydroxylase, p-nitroanisole O-demethylase and lidocaine N-deethylase activities of cytochrome P-450-linked monooxygenase systems, these activities being mainly catalyzed by cytochromes P-450 CYP2E1, CYP2C11 and CYP3A4, respectively, and the Ki values of the activities for FK-506 were determined to be 605, 491 and 97 microM, respectively. The inhibition of cytochrome P-450-linked monooxygenase systems by FK-506 seemed to involve the direct inhibition of cytochromes P-450 because the NADPH-cytochrome c reductase and NADPH-ferricyanide reductase activities of NADPH-cytochrome P-450 reductase were not affected by the presence of 1 mM FK-506 at all. A spectrophotometric study showed that a reverse type I spectral change was induced on the addition of FK-506 to rat liver microsomes, and the Ks value was apparently 125 microM. On the other hand, the EPR spectra of cytochromes P-450 in rat liver microsomes were not affected by 1 mM FK-506. These results suggest direct interaction between FK-506 and cytochrome P-450 apoproteins, except for the heme iron regions of cytochromes P-450, resulting in inhibition of the drug-metabolism activities catalyzed by cytochromes P-450.
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PMID:Effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems in rat liver microsomes. 930 7

The effects of nitric oxide (NO) on superoxide (O-2) generation of the NADPH oxidase in pig neutrophils were studied. NO dose-dependently suppressed O-2 generation of both neutrophil NADPH oxidase and reconstituted NADPH oxidase. Effects of NO on NADPH-binding site and the redox centers including FAD and low spin heme in cytochrome b558 and the electron transfer rates from NADPH to heme via FAD were examined under anaerobic conditions. Both reaction rates and the Km value for NADPH were unchanged by NO. Visible and EPR spectra of cytochrome b558 showed that the structure of heme was unchanged by NO, indicating that NO does not affect the redox centers of the oxidase. In reconstituted NADPH oxidase system, NO did not inhibit O-2 generation of the oxidase when added after activation. The addition of NO to the membrane component or the cytosol component inhibited the activity by 24.0 +/- 5.3 or 37.4 +/- 7.1%, respectively. The addition of NO during the activation process or to the cytosol component simultaneously with myristate inhibited the activity by 74.0 +/- 5.2 or 70.0 +/- 8.3%, respectively, suggesting that cytosol protein(s) treated with myristate becomes susceptible to NO. Peroxynitrite did not interfere with O-2 generation.
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PMID:Nitric oxide inactivates NADPH oxidase in pig neutrophils by inhibiting its assembling process. 940 51


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