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

Calculations of the energy response of an agar-alanine phantom dosimeter (AAPD) to electrons in the energy range of 0.15-20 MeV together with experimental results at 16 MeV are presented. It is shown that the sensitivity of the EPR dosimeter (EPR signal/Gray) is independent of alanine crystal size and varies less than 2%, in the electron energy range indicated. Thus, the measured free radical density distribution may be used directly as an indication of the absorbed dose distribution in an irradiated phantom.
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PMID:Energy response of agar-alanine free radical dosimetry to therapeutic electron beams. 828 28

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

EPR investigations of a variety of irradiated materials have provided the potential for useful dosimetry applications. Herbs and spices imported into Australia have been investigated to establish whether or not they have been irradiated. Post-irradiation studies have shown that there is more than one free radical species in most cases which decay rapidly with time. Changes to transition metal ion signals, e.g., Cu2+ or Fe3+, appear to be permanent against further irradiation. Thus if these signals change upon irradiation, the material almost certainly has not previously been irradiated. Power saturation studies of alanine, a favored dosimetry material, suggest two distinguishable types of behavior consistent with the presence of spin-flip transitions. Irradiation of vanadium doped beryl yields stable VO2+ ions which may provide a useful dosimetry material. Dosimetry applications would appear to demand low cost, user friendly, automated EPR spectrometers. A patented option based on a 2.5 GHz microstrip microwave bridge will be described briefly.
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PMID:Radiation induced EPR centers in foodstuffs and inorganic materials. 838 46

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

This paper reports the first clinical trial of the application of the continuous wave electron paramagnetic resonance (CW-EPR) spectrum of alanine to determine the radiation dose received by a patient. The results of these measurements are compared with those obtained using thermoluminescent dosimetry (TLD) simultaneously. The 9 GHz EPR measurements were made at 13 degrees C. The time stability of the radiation induced radicals in alanine was confirmed. The fractionated radiation doses received in the clinical trial were determined from a calibration curve (linear regression coefficient r = 0.9995) obtained by irradiating L-alanine samples with 60-800 cGy doses using cobalt 60 gamma rays obtained from an Eldorado 8 Cobalt 60 unit at a rate of approximately 60 cGy/min. It is shown that the absorbed dose in tissue-equivalent material can be determined using EPR spectroscopy with an accuracy of approximately 3% at low dose levels (60 cGy) whereas the error using TLD is approximately 5% and that this method of dose determination is preferable to the present TLD method because it is simpler and more accurate.
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PMID:The application of EPR dosimetry for radiotherapy and radiation protection. 839 19

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/sub-unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/Km values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.
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PMID:Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues. 853 44

This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue. Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKred.1 = 5.39 +/- 0.08, pKox = 7.29 +/- 0.14, and pKred.2 = 7.84 +/- 0.14 with Em.7 = -459 +/- 4 mV, and a constant redox potential at high pH of -485 +/- 4 mV. The dependence of the semiquinone/hydroquinone redox potential on temperature is -0.5 +/- 0.1 mV . K(-1), yielding delta H degrees = 28.6 +/- 1.5 kJ . mol(1) and delta S degrees = -50.0 +/- 6.2 J . mol(-1) . K(-1). No significant differences in redox properties of wild-type, C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin II.
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PMID:Redox properties of wild-type, Cys69Ala, and Cys69Ser Azotobacter vinelandii flavodoxin II as measured by cyclic voltammetry and EPR spectroscopy,. 863 24

Growth hormone releasing peptide (GHRP, sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) is a synthetic hexapeptide under consideration for transdermal iontophoretic drug delivery. Cyclic voltammetry, controlled-potential electrolysis, HPLC/UV analysis, LC/MS/MS analysis, and EPR spin-trapping studies indicate that the electrolysis-induced oxidative degradation of GHRP is likely to be mediated by electrogenerated oxygen radicals from the electrolysis of water. Within 2 h and up to 2.5 V versus an Ag/AgCl reference electrode, the peptide backbone remains largely intact. The chemical modifications are selectively on imidazole (histidine) and indole (tryptophan). Strategies for alleviating the electrolysis-induced degradation of GHRP are proposed.
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PMID:Structural study of electrolysis-induced degradation of the growth hormone releasing peptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2. 863 65

The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an oxygen-sensitive organic radical, which is essential for catalysis. The radical was tentatively proposed to be on glycine 681, based on a comparison with the glycyl radical-containing enzyme pyruvate formate-lyase. By EPR spectroscopy of selectively 2H- and 13C-labeled anaerobic ribonucleotide reductase, the radical was now unambiguously assigned to carbon-2 of a glycine residue. The large 1H hyperfine splitting (1.4 millitesla) was assigned to the alpha-proton. Site-directed mutagenesis was used to change glycine 681 into an alanine residue. In separate experiments, the two adjacent residues, cysteine 680 and tyrosine 682, were changed into serine and phenylalanine, respectively. All mutated proteins were retained on dATP-Sepharose, indicating that the mutant proteins had intact allosteric sites. They also contained amounts of iron comparable with the wild type reductase and showed the same iron-sulfur-related spectrum, suggesting that the mutant proteins were properly folded. Of the three mutant proteins only the G681A protein completely lacked the detectable glycyl radical as well as enzyme activity. Our results identify glycine 681 as the stable free radical site in E. coli anaerobic ribonucleotide reductase.
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PMID:The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681. 863 6

The beta subunit of the nitrate reductase A from Escherichia coli contains four groups of cysteine residues (I-IV) which are thought to bind the four iron-sulfur centers (1-4) of the enzyme. The fourth Cys residue of each group was replaced by Ala by site-directed mutagenesis, which led to the C26A, C196A, C227A, and C263A mutants. Physiological and biochemical effects of the mutations were investigated on both the membrane-bound and the soluble forms of the enzyme. In addition, detailed redox titrations of the mutants were monitored by EPR spectroscopy. The C196A and C227A mutations resulted in the full loss of the four Fe-S clusters and of the Mo-cofactor, leading to inactive enzymes. In contrast, the C26A and C263A mutants retained significant nitrate reductase activities. The EPR analysis showed that the highest redox potential [4Fe-4S] cluster (center 1) was selectively removed by the C263A mutation and that the C26A replacement likely eliminated the lowest potential [4Fe-4S] cluster (center 4). In both mutants, the three remaining Fe-S clusters kept the same spectral and redox properties as in the wild type enzyme. These results enabled the determination of the Cys ligands of center 1 to be completed and led to a proposed model for the coordination of the four Fe-S centers by the four Cys groups of the beta subunit. In this model, the four clusters are organized in two pairs, (center 1, center 4) and (center 2, center 3), which is in good agreement with the magnitude of intercenter magnetic interactions observed by EPR and with the stability of the different mutants. The possible implications on the intramolecular electron transfer pathway are discussed.
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PMID:Complete coordination of the four Fe-S centers of the beta subunit from Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of site-directed mutants lacking the highest or lowest potential [4Fe-4S] clusters. 866 73


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