43 research outputs found

    Porfirinas de manganeso como inactivadores de peroxinitrito: evaluación cinética y efectos sobre blancos moleculares

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    Hemos caracterizado la cinética de reacción de un conjunto de porfirinas de manganeso (MnPorfirinas) solubles en agua frente a peroxinitrito (ONOO–) y radical carbonato (CO3·–). La reacción, entre ONOO– y el complejo de MnIII, tiene constantes de velocidad entre 105 y 107 M-1 s-1 a 37 ºC con un comportamiento sigmoide en función del pH, pKa aparentes cercanos al del par ONOOH/ONOO– y máximos a pH alcalino. La reacción produce el complejo O=MnIV y ·NO2. Los valores de estas constantes de velocidad tienen una relación lineal de energía libre con parámetros fisicoquímicos de la porfirina tales como el potencial redox del par MnIII/MnII o la acidez de Brønsted de los nitrógenos pirrólicos o de las moléculas de agua axiales. La reacción, entre ONOO– y el complejo de MnII, tiene constantes de velocidad <106 M-1 s-1 a pH 7.4 y 37 ºC y produce estequiométricamente O=MnIV y NO2–. La reacción, entre CO3·– y el complejo de MnIII tiene constantes de velocidad extrapoladas a pH neutro entre 2 y 12 x 108 M-1 s-1 con un comportamiento ácido base congruente con la ionización de las moléculas axiales de agua presentes en el complejo. La reacción entre CO3·– y el complejo de MnII tiene constantes de velocidad a pH = 10.4 entre 1 y 5 x 109 M-1 s-1. En ambos casos el CO3·– produce oxidaciones por un electrón.Los complejos de O=MnIV se reducen en forma rápida con ascorbato o urato hasta MnIII en presencia de oxígeno y, con bajas concentraciones de O2, la reducción puede llegar hasta MnII si los reductores son flavoenzimas reducidas, entre ellas succinato deshidrogenasa y NADH deshidrogenasa de la cadena mitocondrial de transporte de electrones. Estas reacciones de reducción completan dos posibles ciclos para catalizar la reducción de ONOO– o CO3·– con una eficiencia suficiente para proteger blancos biológicos en concentraciones micromolares de MnPorfirinas.Se presentan resultados de uso del ciclo catalítico MnIII/MnIV con urato como reductor en la protección de LDL in vitro y del ciclo catalítico MnII/MnIV con succinato/succinato deshidrogenasa como reductor para proteger actividades enzimáticas de partículas submitocondriales in vitro.Los resultados obtenidos indican que la interacción de MnPorfirinas con reductores biológicos y en particular con flavoenzimas mitocondriales podría explicar en parte la actividad protectora de estos compuestos en diversos modelos de patología vinculada con estrés oxidativo y disfunción mitocondri

    Differential parameters between cytosolic 2-Cys peroxiredoxins, PRDX1 and PRDX2

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    Peroxiredoxins are thiol-dependent peroxidases that function in peroxide detoxification and H2O2 induced signaling. Among the six isoforms expressed in humans, PRDX1 and PRDX2 share 97% sequence similarity, 77% sequence identity including the active site, subcellular localization (cytosolic) but they hold different biological functions albeit associated with their peroxidase activity. Using recombinant human PRDX1 and PRDX2, the kinetics of oxidation and hyperoxidation with H2O2 and peroxynitrite were followed by intrinsic fluorescence. At pH 7.4, the peroxidatic cysteine of both isoforms reacts nearly tenfold faster with H2O2 than with peroxynitrite, and both reactions are orders of magnitude faster than with most protein thiols. For both isoforms, the sulfenic acids formed are in turn oxidized by H2O2 with rate constants of ca 2 × 103 M−1 s−1 and by peroxynitrous acid significantly faster. As previously observed, a crucial difference between PRDX1 and PRDX2 is on the resolution step of the catalytic cycle, the rate of disulfide formation (11 s−1 for PRDX1, 0.2 s−1 for PRDX2, independent of the oxidant) which correlates with their different sensitivity to hyperoxidation. This kinetic pause opens different pathways on redox signaling for these isoforms. The longer lifetime of PRDX2 sulfenic acid allows it to react with other protein thiols to translate the signal via an intermediate mixed disulfide (involving its peroxidatic cysteine), whereas PRDX1 continues the cycle forming disulfide involving its resolving cysteine to function as a redox relay. In addition, the presence of C83 on PRDX1 imparts a difference on peroxidase activity upon peroxynitrite exposure that needs further study.Fil: Dalla Rizza, Joaquín. Universidad de la Republica; UruguayFil: Randall, Lía M.. Universidad de la Republica; UruguayFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ferrer Sueta, Gerardo. Universidad de la Republica; UruguayFil: Denicola, Ana. Universidad de la República; Urugua

    Catalysis of Peroxide Reduction by Fast Reacting Protein Thiols

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    Life on Earth evolved in the presence of hydrogen peroxide, and other peroxides also emerged before and with the rise of aerobic metabolism. They were considered only as toxic byproducts for many years. Nowadays, peroxides are also regarded as metabolic products that play essential physiological cellular roles. Organisms have developed efficient mechanisms to metabolize peroxides, mostly based on two kinds of redox chemistry, catalases/peroxidases that depend on the heme prosthetic group to afford peroxide reduction and thiol-based peroxidases that support their redox activities on specialized fast reacting cysteine/selenocysteine (Cys/Sec) residues. Among the last group, glutathione peroxidases (GPxs) and peroxiredoxins (Prxs) are the most widespread and abundant families, and they are the leitmotif of this review. After presenting the properties and roles of different peroxides in biology, we discuss the chemical mechanisms of peroxide reduction by low molecular weight thiols, Prxs, GPxs, and other thiol-based peroxidases. Special attention is paid to the catalytic properties of Prxs and also to the importance and comparative outlook of the properties of Sec and its role in GPxs. To finish, we describe and discuss the current views on the activities of thiol-based peroxidases in peroxide-mediated redox signaling processes.Fil: Zeida, Ari. Universidad de la República; UruguayFil: Trujillo, Madia. Universidad de la Republica; UruguayFil: Ferrer Sueta, Gerardo. Universidad de la Republica; UruguayFil: Denicola, Ana. Universidad de la Republica; UruguayFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Radi, Rafael. Universidad de la República; Urugua

    Kinetic Characterization of Sulfenic Acid Reduction in 1-Cys Peroxiredoxins by Ascorbate

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    Peroxiredoxins (Prxs) are Cys-based peroxidases with\ud remarkable catalytic efficiency that can be divided into 1-Cys or 2-\ud Cys, depending on the number of Cys residues involved in\ud catalysis. Initially, reduction of Prx was described to be strictly\ud dependent on thiols, but later we showed that ascorbate can also\ud reduce the sulfenic intermediate of 1-Cys Prx (1-Cys Prx-SOH) in\ud various organisms [1]. Here, the kinetic characterization of 1-Cys\ud Prx-SOH reduction by ascorbate is described. Reduction of 1-Cys\ud Prx-SOH by ascorbate was initially analyzed using an enzyme\ud from A. fumigatus (AfPrxA) that is 37% similar to PRDX6 (human\ud 1-Cys Prx). H2O2 levels were determined by means of a specific\ud electrode (Free Radical Analyzer 4100), using a steady-state bisubstrate approach. AfPrxA decomposed H2O2 with good\ud efficiency (kcat/KM = 7.4 x103\ud M1\ud .s1\ud ), through a Bi-Bi Ping-Pong\ud mechanism. To further support these findings, a second,\ud independent approach was also employed: competition between\ud dichlorophenolindophenol (DCPIP) and AfPrxA-SOH for\ud ascorbate. DCPIP is a redox sensor, whose blue color is lost\ud when reduced and its second-order rate constant with ascorbate\ud is 718 M1\ud .s1\ud , enabling the determination of the rate constant of the\ud reaction between AfPrxA-SOH and ascorbate: 1.5 x 103\ud M1\ud .s1\ud .\ud Therefore, by two independent approaches, we showed that\ud ascorbate efficiently reduced AfPrxA-SOH. Next, the reductions of\ud 1-Cys Prx SOH in other organisms (bacteria, yeast and plant)\ud were also investigated and again the constants were in the\ud 103\ud M1\ud .s1 range. We conclude that the reduction of 1-Cys PrxSOH\ud by ascorbate is probably relevant in the subcellular\ud compartments in which this reductant is present at millimolar\ud levels. We are currently studying the reduction of 1-Cys Prx-SOH\ud by ascorbate in other proteins, which could open new\ud perspectives in cellular redox processes in vivo.\ud [1] Proc.Natl.Acad.Sci. USA. 2007 104:4886-9

    Aqua(glycinato)(3,4,7,8-tetramethyl-1,10-phenanthroline)copper(II) Nitrate

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    In the title compound, [Cu(C2H4NO2)(C16H16N2)-(H2O)]NO3, CuII displays distorted square-pyramidal coordination where the water molecule is in the apical position and the base is defined by the N and one of the O atoms from the glycinate ligand, and both phenanthroline N atoms. The phenanthroline chelate-ring plane (N1, C12, C11, N2, Cu) is slightly distorted from planarity, whereas the five-membered ring formed by the glycinate ligand (defined by atoms N3, C18, C17, O1 and Cu), presents a distorted half-chair conformation

    Kinetic studies of peroxiredoxin 6 from Arenicola marina: Rapid oxidation by 3 hydrogen peroxide and peroxynitrite but lack of reduction by hydrogen sulfide

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    , respectively, at pH 7.4 and 25°C. Reduction of tert-butyl hydroperoxide was slower. 34 The pK a of the peroxidatic thiol of AmPrx6 was determined as 5

    Acidity and nucleophilic reactivity of glutathione persulfide

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    Persulfides (RSSH/RSS2) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/ GSS2), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS2 presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called a effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the a effect correlated with the Brønsted nucleophilic factor, bnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the a effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.Fil: Benchoam, Dayana. Universidad de la República; UruguayFil: Semelak, Jonathan Alexis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Cuevasanta, Ernesto. Universidad de la República; UruguayFil: Mastrogiovanni, Mauricio. Universidad de la Republica; UruguayFil: Grassano, Juan S.. Universidad de Buenos Aires; ArgentinaFil: Ferrer-Sueta, Gerardo. Universidad de la Republica; UruguayFil: Zeida Camacho, Ari Fernando. Universidad de la Republica; UruguayFil: Trujillo, Madia. Universidad de la Republica; UruguayFil: Möller, Matías N.. Universidad de la Republica; UruguayFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Alvarez, Beatriz. Universidad de la Republica; Urugua

    Reduction of sulfenic acids by ascorbate in proteins, connecting thiol-dependent to alternative redox pathways

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    Sulfenic acids are the primary product of thiol oxidation by hydrogen peroxide and other oxidants. Several aspects of sulfenic acid formation through thiol oxidation were established recently. In contrast, the reduction of sulfenic acids is still scarcely investigated. Here, we characterized the kinetics of the reduction of sulfenic acids by ascorbate in several proteins. Initially, we described the crystal structure of our model protein (Tsa2-C170S). There are other Tsa2 structures in distinct redox states in public databases and all of them are decamers, with the peroxidatic cysteine very accessible to reductants, convenient features to investigate kinetics. We determined that the reaction between Tsa2-C170S-Cys-SOH and ascorbate proceeded with a rate constant of 1.40 ± 0.08 × 103 M−1 s−1 through a competition assay developed here, employing 2,6–dichlorophenol-indophenol (DCPIP). A series of peroxiredoxin enzymes (Prx6 sub family) were also analyzed by this competition assay and we observed that the reduction of sulfenic acids by ascorbate was in the 0.4–2.2 × 103 M−1 s−1 range. We also evaluated the same reaction on glyceraldehyde 3-phosphate dehydrogenase and papain, as the reduction of their sulfenic acids by ascorbate were reported previously. Once again, the rate constants are in the 0.4–2.2 × 103 M−1 s−1 range. We also analyzed the reduction of Tsa2-C170S-SOH by ascorbate by a second, independent method, following hydrogen peroxide reduction through a specific electrode (ISO-HPO-2, World Precision Instruments) and employing a bi-substrate, steady state approach. The was 7.4 ± 0.07 × 103 M−1 s−1, which was in the same order of magnitude as the value obtained by the DCPIP competition assay. In conclusion, our data indicates that reduction of sulfenic acid in various proteins proceed at moderate rate and probably this reaction is more relevant in biological systems where ascorbate concentrations are high

    Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

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    Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.Fil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Vazquez, Diego Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ferrer Sueta, Gerardo. Universidad de la República; UruguayFil: Agudelo Suarez, William Armando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Howard, Eduardo Ignacio. Université de Strasbourg; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rasia, Rodolfo Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Manta, Bruno. Universidad de la República; UruguayFil: Cousido Siah, Alexandra. Université de Strasbourg; FranciaFil: Mitschler, André. Université de Strasbourg; FranciaFil: Podjarny, Alberto Daniel. Université de Strasbourg; FranciaFil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin
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