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Enrique Cadenas - One of the best experts on this subject based on the ideXlab platform.
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Leucoisoprenochrome-o-Semiquinone formation in freshly isolated adult rat cardiomyocytes.
Chemical research in toxicology, 2004Co-Authors: Fernando Remião, Daniel Rettori, Derick Han, Félix Carvalho, Maria De Lourdes Bastos, Enrique CadenasAbstract:Sustained high levels of circulating catecholamines can lead to cardiotoxicity. There is increasing evidence that this process may result from metal-catalyzed catecholamine oxidation into Semiquinones, quinones, and aminochromes. We have previously shown that Cu2+-induced oxidation of isoproterenol into isoprenochrome induces toxic effects in isolated cardiomyocytes. The aim of this study was to characterize the isoproterenol oxidation process and to locate the formation of Semiquinone radicals in cardiomyocyte suspensions. Freshly isolated rat cardiomyocytes were incubated with 1 or 10 mM isoproterenol and 20 μM Cu2+ for 4 h. The formation of an isoproterenol oxidation radical was detected in the extracellular medium, cells, membranes, and heavy organelles by electron spin resonance spectroscopy. An electron spin resonance signal assigned to leucoisoprenochrome-o-Semiquinone increased in a time-dependent manner in the extracellular medium. A second electron spin resonance signal, characteristic of an imm...
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OXIDATION OF ADRENALINE BY FERRYLMYOGLOBIN
Free radical biology & medicine, 1998Co-Authors: Cecilia R Giulivi, Enrique CadenasAbstract:Abstract The oxidation of adrenaline by ferrylmyoglobin, the product formed by the oxidation of myoglobin with H 2 O 2 , was examined by absorption, fluorescence, and EPR spectroscopy in terms of the formation of intermediate free radicals and stable molecular products and the binding of adrenaline oxidation products to the apoprotein. The reaction of adrenaline with ferrylmyoglobin resulted in reduction of the hemoprotein to metmyoglobin and consumption of adrenaline. Quantification of metmyoglobin formed per adrenaline yielded a ratio of 1.66. The reaction was found first order on adrenaline concentration and second order on ferrylmyoglobin concentration. This, together with the above ratio, suggested a mechanism by which two oxoferryl moieties (ferrylmyoglobin) were reduced by adrenaline yielding metmyoglobin and the o -Semiquinone state of adrenaline. The decay of the o -Semiquinone to adrenochrome was confirmed by an increase in absorbance at 485 nm. The product was nonfluorescent; alkalinization of the reaction mixture resulted in a strong fluorescence at 540 nm ascribed to 3,5,6-trihydroxyindol or adrenolutin. Hence, adrenochrome and its alkali-catalyzed product, adrenolutin, are the major molecular products formed during the oxidation of adrenaline by ferrylmyoglobin. Semiquinones formed during the adrenaline/ferrylmyoglobin interaction were detected by EPR, spin stabilizing these species with Mg 2+ . The six-line EPR spectrum observed (a N = 4.5 G, a N (CH 3 ) = 5.1, and a 2H = 0.91; g = 2.0040) may be assigned to the Semiquinone forms of adrenochrome and/or adrenolutin or a composite of these species. The intensity of the EPR signal increased with time and its subsequent decay followed a second-order kinetics as inferred by the proportionality of the square of the EPR line intensity with H 2 O 2 concentration. Heme destruction and lysine loss, inherent in the reaction of metmyoglobin with H 2 O 2 , were prevented 80 and 34% by adrenaline, respectively. The low protection exerted by adrenaline against lysine loss was possibly due to the formation of Schiff bases between the ϵ-NH 2 group of lysine and the o -quinone oxidation product(s) of adrenaline. The yield of Schiff base formation was 20–25%. The autoxidation of adrenaline at physiological pH is extremely slow or nonexistent. These data provide a rationale for the primary oxidation of adrenaline by the pseudoperoxidatic activity of ferrylmyoglobin and suggest implications of the free radicals thereby formed for the oxidative damage in reperfusion injury.
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Reactions of halogen-substituted aziridinylbenzoquinones with glutathione. Formation of diglutathionyl conjugates and Semiquinones.
Chemico-biological interactions, 1998Co-Authors: Cecilia R Giulivi, Angela Forlin, Stefano Bellin, Enrique CadenasAbstract:Abstract The reaction between glutathione and 2,5-diaziridinyl-1,4-benzoquinones bearing halogen substituents at C 3 and C 6 was examined in terms of the formation of glutathionyl-quinone conjugates and Semiquinones by HPLC with UV detection, mass spectroscopy and EPR. The reactivity of the halogen atoms toward sulfur substitution is the primary reaction leading to the formation of mono- and di-glutathionyl-substituted quinones. The relative formation of these conjugates depended on the GSH/quinone molar ratios. At GSH/quinone molar ratios below unity, the products observed were the reduced form of the parent quinone, a dithioether derivative and GSSG. Disulfide formation accounted for 60–68% of total GSH consumed. EPR analysis of these reaction mixtures showed a 5-line spectrum (1:2:3:2:1 relative intensities) with 2 equivalent N ( a N =1.98 G) and assigned to the Semiquinone form of dichloro- diaziridinylbenzoquinone. Semiquinone quantification by double integration of the EPR signals and interpolation with an adequate standard revealed that the amount of Semiquinone formed per GSH consumed was 0.98. At GSH/quinone molar ratios above unity (4, 10 and 100 molar excess of GSH) a pattern of products emerged consisting of 3,6-diglutathionyl quinones with two, one and no aziridinyl moieties, identified by mass spectral analysis. EPR studies revealed that these compounds were minor components of a composite EPR spectrum (a 3-line signal with 1:1:1 relative intensities, 1 equivalent N ( a N =1.73 G) and 1 H ( a H =1.45 G) or a 3-line signal with 1:2:1 relative intensities and 2 equivalent H ( a H =1.4 G). These minor components were assigned to the diglutathionyl conjugates bearing one- or no aziridinyl moiety, respectively. The major component in the EPR signal showed a 3-line spectrum (1:1:1 relative intensity) with 1 equivalent N ( a N =1.7 G) and a g shift of −0.96 G. This spectrum was assigned to a triglutathionyl conjugate of a monoaziridinylbenzoquinone. This major component was also observed when GSH/quinone mixtures were incubated with the two-electron transfer flavoprotein NAD(P)H:quinone oxidoreductase. The Semiquinone signals were abolished by superoxide dismutase. In the presence of catalase, the contribution of these components to the overall EPR spectrum was equal. These data are discussed in terms of the one-electron transfer steps encompassed by thiol oxidation and Semiquinone formation and the two-electron transfers inherent in sulfur substitution and aziridinyl group loss.
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Enzymic- and thiol-mediated activation of halogen-substituted diaziridinylbenzoquinones: redox transitions of the Semiquinone and Semiquinone-thioether species.
Free radical biology & medicine, 1995Co-Authors: José Goin, Cecilia R Giulivi, John Butler, Enrique CadenasAbstract:Abstract Activation of 2,5-diaziridinyl-1,4-benzoquinones bearing halogen (Cl, Br, or F) substituents at C3 and C6 by NADPH-cytochrome P450 reductase and glutathione nucleophilic substitution was examined in terms of free radical production and DNA strand scission. A Semiquinone species was observed by direct ESR in aerobic conditions during: (a) NADPH-cytochrome P450 reductase-catalyzed reduction of the above quinones. (b) The interaction of these quinones with GSH entailing primarily reactivity of halogen substituents toward sulfur substitution. (c) NADPH-cytochrome P450 reductase-catalyzed activation of products resulting from the quinone/GSH interaction. The Semiquinone ESR signal observed during enzymic catalysis was suppressed by superoxide dismutase and was not affected by catalase. ESR studies in conjunction with the spin trapping technique on the autoxidation of the Semiquinones formed by the above reaction pathways indicated the formation of superoxide radicals. In addition, thiyl radicals were formed during the reactions following glutathione nucleophilic substitution of the above quinones. The ESR signals of both superoxide and thiyl radicals were abolished by superoxide dismutase. No hydroxyl radicals were formed in solution during the redox transitions of these halogen-containing diaziridinylbenzoquinones. Bioreductive activation of these compounds via NADPH-cytochrome P450 reductase or sulfur nucleophilic substitution was associated with the formation of DNA strand breaks. This process was substantially inhibited (74–86%) by superoxide dismutase and to a lesser extent (23–31%) by catalase. It is suggested that DNA strand breakage proceeds in a manner entailing a Semiquinone-dependent reduction of metal-ligands bound at the DNA surface and leading to site-specific, hydroxyl radical production.
Tomoko Ohnishi - One of the best experts on this subject based on the ideXlab platform.
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an escherichia coli mutant quinol fumarate reductase contains an epr detectable Semiquinone stabilized at the proximal quinone binding site
Journal of Biological Chemistry, 1999Co-Authors: Cecilia Hagerhall, Vladimir D Sled, Sergey Magnitsky, Imke Schroder, Robert P Gunsalus, Gary Cecchini, Tomoko OhnishiAbstract:The EPR and thermodynamic properties of Semiquinone (SQ) species stabilized by mammalian succinate:quinone reductase (SQR) in situ in the mitochondrial membrane and in the isolated enzyme have been well documented. The equivalent Semiquinones in bacterial membranes have not yet been characterized, either in SQR or quinol:fumarate reductase (QFR) in situ. In this work, we describe an EPR-detectable QFR Semiquinone using Escherichia coli mutant QFR (FrdC E29L) and the wild-type enzyme. The SQ exhibits a g = 2.005 signal with a peak-to-peak line width of approximately 1.1 milliteslas at 150 K, has a midpoint potential (E(m(pH 7.2))) of -56.6 mV, and has a stability constant of approximately 1.2 x 10(-2) at pH 7.2. It shows extremely fast spin relaxation behavior with a P(1/2) value of >>500 milliwatts at 150 K, which closely resembles the previously described SQ species (SQ(s)) in mitochondrial SQR. This SQ species seems to be present also in wild-type QFR, but its stability constant is much lower, and its signal intensity is near the EPR detection limit around neutral pH. In contrast to mammalian SQR, the membrane anchor of E. coli QFR lacks heme; thus, this prosthetic group can be excluded as a spin relaxation enhancer. The trinuclear iron-sulfur cluster FR3 in the [3Fe-4S](1+) state is suggested as the dominant spin relaxation enhancer of the SQ(FR) spins in this enzyme. E. coli QFR activity and the fast relaxing SQ species observed in the mutant enzyme are sensitive to the inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO). In wild-type E. coli QFR, HQNO causes EPR spectral line shape perturbations of the iron-sulfur cluster FR3. Similar spectral line shape changes of FR3 are caused by the FrdC E29L mutation, without addition of HQNO. This indicates that the SQ and the inhibitor-binding sites are located in close proximity to the trinuclear iron-sulfur cluster FR3. The data further suggest that this site corresponds to the proximal quinone-binding site in E. coli QFR.
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structure function studies of iron sulfur clusters and Semiquinones in the nadh q oxidoreductase segment of the respiratory chain
Biochimica et Biophysica Acta, 1998Co-Authors: Tomoko Ohnishi, Vladimir D Sled, Takahiro Yano, Takao Yagi, Dosymzhan Sh Burbaev, Andrei D VinogradovAbstract:Abstract Our recent experimental data on iron-sulfur clusters and Semiquinones in the complex I segment of the respiratory chain is presented, focusing on the Paracoccus (P.) denitrificans and bovine heart studies. The iron-sulfur cluster N2 has attracted the attention of investigators in the research field of complex I, since the mid-point redox potential of this cluster is the highest among all clusters in complex I, and is pH dependent (60 mV/pH). It is known that this cluster is located either in the NQO6 (NuoB in E. coli /PSST in bovine heart nomenclature) or in the NQO9 (NuoI/TYKY) subunit in the amphipathic domain of complex I. Our preliminary data indicate that the cluster N2 is located in the NuoB rather than the long-advocated NuoI subunit, and may have a unique ligand structure. We previously reported spin-spin interactions between cluster N2 and two distinct species of Semiquinone (designated SQ Nf and SQ Ns ) associated with complex I. A parallel intensity change was observed between the SQ Nf ( g =2.00) signal and the N2 split g ∥ signal, further supporting our proposed interaction between SQ Nf and N2 spins.
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Iron-sulfur clusters/Semiquinones in complex I.
Biochimica et biophysica acta, 1998Co-Authors: Tomoko OhnishiAbstract:NADH-quinone1 oxidoreductase (Complex I) isolated from bovine heart mitochondria was, until recently, the major source for the study of this most complicated energy transducing device in the mitochondrial respiratory chain. Complex I has been shown to contain 43 subunits and possesses a molecular mass of about 1 million. Recently, Complex I genes have been cloned and sequenced from several bacterial sources including Escherichia coli, Paracoccus denitrificans, Rhodobacter capsulatus and Thermus thermophilus HB-8. These enzymes are less complicated than the bovine enzyme, containing a core of 13 or 14 subunits homologous to the bovine heart Complex I. From this data, important clues concerning the subunit location of both the substrate binding site and intrinsic redox centers have been gleaned. Powerful molecular genetic approaches used in these bacterial systems can identify structure/function relationships concerning the redox components of Complex I. Site-directed mutants at the level of bacterial chromosomes and over-expression and purification of single subunits have allowed detailed analysis of the amino acid residues involved in ligand binding to several iron–sulfur clusters. Therefore, it has become possible to examine which subunits contain individual iron–sulfur clusters, their location within the enzyme and what their ligand residues are. The discovery of g=2.00 EPR signals arising from two distinct species of Semiquinone (SQ) in the activated bovine heart submitochondrial particles (SMP) is another line of recent progress. The intensity of Semiquinone signals is sensitive to ΔμH+ and is diminished by specific inhibitors of Complex I. To date, Semiquinones similar to those reported for the bovine heart mitochondrial Complex I have not yet been discovered in the bacterial systems. This mini-review describes three aspects of the recent progress in the study of the redox components of Complex I: (A) the location of the substrate (NADH) binding site, flavin, and most of the iron–sulfur clusters, which have been identified in the hydrophilic electron entry domain of Complex I; (B) experimental evidence indicating that the cluster N2 is located in the amphipathic domain of Complex I, connecting the promontory and membrane parts. Very recent data is also presented suggesting that the cluster N2 may have a unique ligand structure with an atypical cluster-ligation sequence motif located in the NuoB (NQO6/PSST) subunit rather than in the long advocated NuoI (NQO9/TYKY) subunit. The latter subunit contains the most primordial sequence motif for two tetranuclear clusters; (C) the discovery of spin–spin interactions between cluster N2 and two distinct Complex I-associated species of Semiquinone. Based on the splitting of the g signal of the cluster N2 and concomitant strong enhancement of the Semiquinone spin relaxation, one Semiquinone species was localized 8–11 A from the cluster N2 within the inner membrane on the matrix side (N-side). Spin relaxation of the other Semiquinone species is much less enhanced, and thus it was proposed to have a longer distance from the cluster N2, perhaps located closer to the other side (P-side) surface of the membrane. A brief introduction of EPR technique was also described in Appendix Aof this mini-review.
Arnold L. Rheingold - One of the best experts on this subject based on the ideXlab platform.
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Singlet−Triplet Gap in Triplet Ground-State Biradicals Is Modulated by Substituent Effects
Journal of the American Chemical Society, 2002Co-Authors: David A. Shultz, Scot H. Bodnar, Jeff W. Kampf, Christopher D. Incarvito, Arnold L. RheingoldAbstract:Three S = 1 bis(Semiquinone) complexes have been prepared. To ensure ferromagnetic intramolecular exchange coupling, the two Semiquinones are attached 1,3 to a 5-substituted phenylene ring. The biradical complexes differ in their meta-substituents: 1-NMe2, X = N,N-dimethylamino; 1-t-Bu, X = tert-butyl; 1-NO2, X = nitro. All three structures have been determined by X-ray crystallography. Results of structural studies indicate that the biradical ligands of all three complexes have nearly identical conformations with average Semiquinone ring torsions of 32° ± 2° relative to the 5-substituted phenylene ring. The exchange parameter, J (Η = −2JŜ1·Ŝ2), ranges from +31.0 ± 0.6 cm-1 for 1-NO2 to +59.3 ± 1.2 cm-1 for 1-t-Bu, with J = +34.9 ± 0.7 cm-1 for 1-NMe2. Since the conformations are nearly identical, the differences in exchange coupling parameter J are due to substituent effects. The experimental results are supported by Huckel theory arguments and previous computational work.
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singlet triplet gap in triplet ground state biradicals is modulated by substituent effects
Journal of the American Chemical Society, 2002Co-Authors: David A. Shultz, Scot H. Bodnar, Jeff W. Kampf, Christopher D. Incarvito, Arnold L. RheingoldAbstract:Three S = 1 bis(Semiquinone) complexes have been prepared. To ensure ferromagnetic intramolecular exchange coupling, the two Semiquinones are attached 1,3 to a 5-substituted phenylene ring. The biradical complexes differ in their meta-substituents: 1-NMe2, X = N,N-dimethylamino; 1-t-Bu, X = tert-butyl; 1-NO2, X = nitro. All three structures have been determined by X-ray crystallography. Results of structural studies indicate that the biradical ligands of all three complexes have nearly identical conformations with average Semiquinone ring torsions of 32° ± 2° relative to the 5-substituted phenylene ring. The exchange parameter, J (Η = −2JŜ1·Ŝ2), ranges from +31.0 ± 0.6 cm-1 for 1-NO2 to +59.3 ± 1.2 cm-1 for 1-t-Bu, with J = +34.9 ± 0.7 cm-1 for 1-NMe2. Since the conformations are nearly identical, the differences in exchange coupling parameter J are due to substituent effects. The experimental results are supported by Huckel theory arguments and previous computational work.
David A. Shultz - One of the best experts on this subject based on the ideXlab platform.
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Singlet−Triplet Gap in Triplet Ground-State Biradicals Is Modulated by Substituent Effects
Journal of the American Chemical Society, 2002Co-Authors: David A. Shultz, Scot H. Bodnar, Jeff W. Kampf, Christopher D. Incarvito, Arnold L. RheingoldAbstract:Three S = 1 bis(Semiquinone) complexes have been prepared. To ensure ferromagnetic intramolecular exchange coupling, the two Semiquinones are attached 1,3 to a 5-substituted phenylene ring. The biradical complexes differ in their meta-substituents: 1-NMe2, X = N,N-dimethylamino; 1-t-Bu, X = tert-butyl; 1-NO2, X = nitro. All three structures have been determined by X-ray crystallography. Results of structural studies indicate that the biradical ligands of all three complexes have nearly identical conformations with average Semiquinone ring torsions of 32° ± 2° relative to the 5-substituted phenylene ring. The exchange parameter, J (Η = −2JŜ1·Ŝ2), ranges from +31.0 ± 0.6 cm-1 for 1-NO2 to +59.3 ± 1.2 cm-1 for 1-t-Bu, with J = +34.9 ± 0.7 cm-1 for 1-NMe2. Since the conformations are nearly identical, the differences in exchange coupling parameter J are due to substituent effects. The experimental results are supported by Huckel theory arguments and previous computational work.
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singlet triplet gap in triplet ground state biradicals is modulated by substituent effects
Journal of the American Chemical Society, 2002Co-Authors: David A. Shultz, Scot H. Bodnar, Jeff W. Kampf, Christopher D. Incarvito, Arnold L. RheingoldAbstract:Three S = 1 bis(Semiquinone) complexes have been prepared. To ensure ferromagnetic intramolecular exchange coupling, the two Semiquinones are attached 1,3 to a 5-substituted phenylene ring. The biradical complexes differ in their meta-substituents: 1-NMe2, X = N,N-dimethylamino; 1-t-Bu, X = tert-butyl; 1-NO2, X = nitro. All three structures have been determined by X-ray crystallography. Results of structural studies indicate that the biradical ligands of all three complexes have nearly identical conformations with average Semiquinone ring torsions of 32° ± 2° relative to the 5-substituted phenylene ring. The exchange parameter, J (Η = −2JŜ1·Ŝ2), ranges from +31.0 ± 0.6 cm-1 for 1-NO2 to +59.3 ± 1.2 cm-1 for 1-t-Bu, with J = +34.9 ± 0.7 cm-1 for 1-NMe2. Since the conformations are nearly identical, the differences in exchange coupling parameter J are due to substituent effects. The experimental results are supported by Huckel theory arguments and previous computational work.
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SYNTHESIS AND CHARACTERIZATION OF A PLANARIZED, TRIMETHYLENEMETHANE-TYPE BIS(Semiquinone) BIRADICAL
Tetrahedron, 1999Co-Authors: David A. Shultz, Hyoyoung Lee, Rosario M. FicoAbstract:Abstract The synthesis and characterization of a biradical in which two Semiquinone rings are held rigidly co-planar and are attached in a geminal fashion to a carbon-carbon double bond is described. The results of cyclic voltammetry and variable-temperature EPR studies are interpreted in terms of the interaction of the two Semiquinones through the carbon-carbon double bond coupling unit.
Andrei D Vinogradov - One of the best experts on this subject based on the ideXlab platform.
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structure function studies of iron sulfur clusters and Semiquinones in the nadh q oxidoreductase segment of the respiratory chain
Biochimica et Biophysica Acta, 1998Co-Authors: Tomoko Ohnishi, Vladimir D Sled, Takahiro Yano, Takao Yagi, Dosymzhan Sh Burbaev, Andrei D VinogradovAbstract:Abstract Our recent experimental data on iron-sulfur clusters and Semiquinones in the complex I segment of the respiratory chain is presented, focusing on the Paracoccus (P.) denitrificans and bovine heart studies. The iron-sulfur cluster N2 has attracted the attention of investigators in the research field of complex I, since the mid-point redox potential of this cluster is the highest among all clusters in complex I, and is pH dependent (60 mV/pH). It is known that this cluster is located either in the NQO6 (NuoB in E. coli /PSST in bovine heart nomenclature) or in the NQO9 (NuoI/TYKY) subunit in the amphipathic domain of complex I. Our preliminary data indicate that the cluster N2 is located in the NuoB rather than the long-advocated NuoI subunit, and may have a unique ligand structure. We previously reported spin-spin interactions between cluster N2 and two distinct species of Semiquinone (designated SQ Nf and SQ Ns ) associated with complex I. A parallel intensity change was observed between the SQ Nf ( g =2.00) signal and the N2 split g ∥ signal, further supporting our proposed interaction between SQ Nf and N2 spins.
