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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NADH-ubiquinone (Q) reductase isolated from beef heart mitochondria exhibited, upon reduction by NADH, a prominent EPR signal at room temperature attributable to stable ubisemiquinone radical(s). The concentration of the ubisemiquinone radical reached as high as 40% of the total Q content in the reductase. The radical was virtually abolished by adding rotenone, whereas rotenone had no effect on the reduction of FMN by NADH. The radical showed an EPR signal of g = 2.0042 at approximately 9.5 GHz with no resolved hyperfine structure and had a line width of 6.8 Gauss at 23 degrees C. The Q-band EPR spectra at 35 GHz showed well resolved g-anisotropy and had a field separation between derivative extrema of 24 Gauss. These results substantiate the fact that this radical was bound to a protein; we call it ubiquinone protein-N (QP-N). The pH dependence of the EPR signals demonstrated that the species of the ubisemiquinone radical(s) consisted of not only an anionic form but also a neutral form. Only about half of the QP-N radical formed by NADH reduction was abolished by p-chloromercuric sulfonate. The microwave power saturation curve of the radical was biphasic; the first phase leveled off at about 5 milliwatts and then at about 20 milliwatts. These results suggested that the ubisemiquinone radical from QP-N was heterogenous, consisting of at least two populations of stable ubisemiquinone radical(s). It is suggested that two kinds of QP-N exist in NADH-Q reductase. Each mole of protein may bind two mol of Q.
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PMID:Evidence of an ubisemiquinone radical(s) from the NADH-ubiquinone reductase of the mitochondrial respiratory chain. 629 5

The structural nature of the iron-sulfur clusters of NADH dehydrogenase from beef heart mitochondria has been studied by the cluster extrusion technique. Enzyme samples were unfolded anaerobically in 80% (v/v) hexamethylphosphoramide/aqueous buffer in the presence of o-xylyl-alpha,alpha'-dithiol as the displacing agent and the extruded clusters were then reacted with p-trifluoromethylbenzenethiol and analyzed by Fourier transform 19F NMR at 339 MHz. Whenever extrusion was nearly complete, both binuclear and tetranuclear clusters were found at a mole ratio of approximately 2:1. Thus, the dehydrogenase, with 16 g atoms of non-heme iron present/mol of FMN, contains most likely four [2Fe-2S] and two [4Fe-4S] clusters. Because the enzyme contains four or, at the most five, EPR-detectable iron-sulfur centers, it appears that one or more of the clusters are EPR-silent.
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PMID:Structural identification of iron-sulfur clusters of the respiratory chain-linked NADH dehydrogenase. 720 98

Addition of HS- enhanced the O(2-)-scavenging activity of bovine erythrocyte Cu,Zn superoxide dismutase (EC 1.15.1.1) by about twofold. The positive effect was measured using a diverse selection of SOD activity assays, and cannot be an artifact restricted to any single technique. Km values for HS- varied in different assay techniques, but we estimate Km approximately 80 microM HS-. In contrast to HS-, other small molecules tested with SOD either had little effect or were inhibitory. Consumption of HS- and O2- occurred in nearly 1:1 mole ratio. The products were H2O2 and sulfane sulfur, such as either elemental sulfur or polysulfide. Binding of HS- to the enzyme was rapid, with k > 10(7) M-1 s-1. The resulting complex exhibited a Cu-to-S charge-transfer absorbance band at 345 nm and an altered Cu(II) EPR spectrum. Taken together, these observations suggest that HS- binds at the catalytic Cu center of SOD and can be a genuine substrate of the enzyme.
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PMID:Interaction of Cu,Zn superoxide dismutase with hydrogen sulfide. 773 52

Peptidylglycine alpha-amidating enzyme catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate in a reaction that requires O2, ascorbate and 2 mol of copper per mole of enzyme [Kulathila et al. (1994) Arch. Biochem. Biophys. 311, 191-195]. Peptides with a C-terminal alpha-hydroxyglycine residue are intermediates in the amidation reaction. Benzylhydrazine inactivates the enzymatic conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 in a time- and concentration-dependent manner. In contrast, the enzymatic conversion of dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 is unaffected by benzylhydrazine. The plot of 1/(inactivation rate) vs 1/[benzylhydrazine] is parabolic, indicating that the inactivation results from the interaction of 2 mol of benzylhydrazine per mole of enzyme. EPR spectra obtained from benzylhydrazine inactivation reactions carried out in the presence of a radical trap, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, show the formation of a carbon-centered benzyl radical. The benzyl radical most likely results from redox chemistry between benzylhydrazine and the enzyme-bound Cu(II) ions because EPR studies show that enzyme-bound Cu(II) is reduced to Cu(I) in the presence of benzylhydrazine. The kinetic constants for benzylhydrazine as a reductant in the amidation reaction were determined at benzylhydrazine concentrations too low to cause significant enzyme inactivation. Mimosine exhibits mixed inhibition vs benzylhydrazine; however, previous results have shown that benzylhydrazine is competitive vs ascorbate [Miller et al. (1992) Arch. Biochem. Biophys. 298, 380-388]. This change in kinetic mechanism coupled with the nonlinear inactivation kinetics have lead to a proposal that the two enzyme-bound Cu(II) atoms are nonequivalent with respect to their reduction by benzylhydrazine.
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PMID:The inactivation of bifunctional peptidylglycine alpha-amidating enzyme by benzylhydrazine: evidence that the two enzyme-bound copper atoms are nonequivalent. 787 9

The Desulfovibrio desulfuricans ATCC 27774 prismane protein was isolated from a Desulfovibrio vulgaris (Hildenborough) strain that contained the gene for this protein in expression vector pSUP104. A redox titration demonstrated that the [Fe-S] cluster in this protein may attain four different redox states, indicated as +3, +4, +5 and +6, with midpoint potentials for the transitions of approx. -220, +50/-25 and +370 mV, respectively. EPR spectra of the protein in the various redox states are reminiscent of those of the D. vulgaris prismane protein (Pierik et al. (1992) Eur. J. Biochem. 206, 705-719), but differ in details. In the +5-state, virtually all the iron is in a S = 9/2 spin state, indicative for a cluster that is more complex than common [4Fe-4S] or [2Fe-2S] clusters. Similarity of the EPR spectrum of the protein in the +3-state with those of inorganic [6Fe-6S] model compounds suggests that the cluster in the protein is also [6Fe-6S]. In the +4-state of the protein a broad signal due to an integer-spin system can be detected with normal-mode EPR. A dramatic sharpening-up and increase of intensity of this band (g = 14.7) is observed with parallel-mode EPR. In accordance with the chemically determined iron content of the protein (6.0 +/- 0.45 moles of iron/mole of protein), the spectroscopic data indicate one [6Fe-6S] cluster in this protein. We did not find evidence for a previous claim (Moura et al. (1992) J. Biol. Chem. 267, 4489-4496) that the D. desulfuricans protein contains two [6Fe-6S] clusters.
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PMID:Overproduction of the prismane protein from Desulfovibrio desulfuricans ATCC 27774 in Desulfovibrio vulgaris (Hildenborough) and EPR spectroscopy of the [6Fe-6S] cluster in different redox states. 800 28

We describe here a new procedure permitting rapid (12-13 h) isolation of a pure oxygen-evolving photosystem II (PSII) core complex from the cyanobacterium Synechocystis PCC 6803. This procedure involves dodecyl maltoside extraction of thylakoid membranes followed by single-step column chromatography using a weak anion-exchanger. SDS-PAGE and immunoblotting show that the complex consists of five intrinsic membrane proteins (CP47, CP43, D1, D1, and cyt b559), one extrinsic protein (MSP), and one unknown protein with a molecular mass of approximately 26 kDa. A chemical and functional analysis, normalized to 2 molecules of pheophytin a, indicates that this PSII core complex contains 1 photoactive plastoquinone, QA, 4 manganese atoms, 38 chlorophyll a molecules, 1 cytochrome b559, 2 plastoquinone-9, and 9-10 beta-carotenes. The complex exhibits high rates of oxygen evolution, typically 2400-2600 mumol of O2 (mg of Chl)-1 h-1 in the presence of 2,5-dichlorobenzoquinone as an artificial electron acceptor with a pH optimum of 6.5. A strong light minus dark multiline EPR signal, arising from the S2 state of the oxygen-evolving complex (OEC), is observed at 10 K following illumination at 198 K. The determination of the absolute oxygen yield per saturating microsecond flash indicates that essentially all of the PSII centers contain functional oxygen-evolving complexes. This point is further supported by the absence of photoaccumulation, upon room temperature illumination, of the immediate oxidant of the OEC, redox-active tyrosine, YZ.. On the basis of EPR spectra, oxidized minus reduced difference spectra, and SDS-PAGE, the preparation contains on a per mole basis with PSII only trace amounts of PSI (approximately 0.04), cytochrome b6/f complex (< or = 0.01), and ATPase (< or = 0.05). All of these results indicate that this PSII preparation is to date the most highly purified oxygen-evolving core complex from Synechocystis 6803 that retains all of the reaction centers active for oxygen evolution. As Synechocystis 6803 is being used extensively for site-directed mutagenesis of PSII, this preparation is particularly valuable for spectroscopic and biochemical analyses of PSII from wild-type and from site-directed mutants.
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PMID:Biochemical and spectroscopic characterization of a new oxygen-evolving photosystem II core complex from the cyanobacterium Synechocystis PCC 6803. 816 15

We have studied the molecular mechanism of Ca-ATPase activation in sarcoplasmic reticulum (SR) by the volatile anesthetic halothane. Using time-resolved phosphorescence anisotropy, we determined the rotational correlation times and mole fractions of different oligomeric states of the enzyme, as a function of halothane and temperature. Lipid fluidity was measured independently, using EPR of spin-labeled lipids. At 4 and 7 degrees C, the principal effects of halothane were to increase the activity of the Ca-ATPase and to promote the formation of monomers and dimers of the enzyme from larger aggregates. At higher temperatures (up to 25 degrees C), halothane activated the enzyme, but to a lesser extent than observed at lower temperatures. While the functional effects of halothane were temperature dependent, the effects of halothane on lipid fluidity and protein aggregation state were similar at all temperatures tested. We conclude that at low temperatures Ca-ATPase activity is dominated by aggregation state, so halothane activates the enzyme primarily by promoting the formation of monomers and dimers of the enzyme from larger aggregates. At higher temperatures, the activity of the enzyme is dominated by lipid fluidity, so halothane activates the enzyme by increasing the lipid fluidity. The physical mechanism of Ca-ATPase activation, dominated by aggregation state at low temperature and lipid fluidity at higher temperature, provides an explanation for the break in the Arrhenius plot of Ca-ATPase activity (in the absence of halothane) at approximately 20 degrees C.
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PMID:Molecular mechanism of Ca-ATPase activation by halothane in sarcoplasmic reticulum. 839 42

In an effort to better understand the structure and function of the metallo-beta-lactamase from Bacteroides fragilis, spectroscopic and metal-binding studies were performed on the native, metal-substituted, and mutant forms of the enzyme. Atomic absorption studies demonstrate that the native B. fragilis enzyme tightly binds 2 mol of Zn(II) and, along with mutagenesis studies, that the presence of both metal ions is required for full catalytic activity. EPR spectroscopy was used to confirm that the Co(II)-substituted beta-lactamase binds 2 mol of Co(II) per mole of enzyme, that the two Co(II)'s are highspin and probably uncoupled, with apparent g values of 6.5, 4.2, and 2.0, and that the coordination number of the Co(II) is 5 or 6. This number of ligands for the Co(II)-substituted enzyme is confirmed by UV-Vis spectra, which demonstrate the presence of very weak d-d transitions between 550 and 650 nm (epsilon approximately 30 M-1.cm-1) and an intense feature at 320 nm (epsilon approximately 1570 M-1.cm-1). The latter is assigned to a cysteine sulfur to Co(II) ligand-to-metal charge transfer band, and this assignment is confirmed by the disappearance of this band in the UV-Vis spectrum of a Co(II)-substituted C168S mutant. H NMR studies on the Co(II)-substituted enzyme suggest the presence of three histidine ligands bound to Co(II). Taken together, these studies support the sequence comparison study of Rasmussen et al., in which there is a catalytic metal-binding site with three histidines and one cysteine (C168). The remaining ligands are postulated to be water molecules involved in catalysis. Mutagenesis studies, in combination with activity assays and metal-binding studies, have been used to identify Asp61, Asp90, Asp152, and Asp183 as possible ligands to the second metal-binding site, with Asp90 and Asp152 having a pronounced effect on kcat. These results are discussed in light of the recent crystal structure of the metallo-beta-lactamase from B. cereus.
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PMID:Characterization of the metal-binding sites of the beta-lactamase from Bacteroides fragilis. 881 Sep 19

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

The molecular properties of a novel membrane quinol oxidase from the marine bacterium Pseudomonas nautica 617 are presented. The protein contains 2b hemes/mole which may be distinguished by EPR spectroscopy but not by optical spectroscopy and electrochemistry. Respiration, though being cyanide insensitive, is not inhibited by carbon monoxide and oxygen reduction is carried out only half-way with production of hydrogen peroxide. The terminal oxidase represents, therefore, a unique example in the large family of terminal oxidases known up to date.
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PMID:The terminal oxidase in the marine bacterium Pseudomonas nautica 617. 933 88


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