title: Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation.
abstract: The ascorbate-glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO(-)) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO(-) and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO(-). The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO(-). These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO(-) or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate-glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement of NO and reactive oxygen species metabolism in antioxidant defence against nitro-oxidative stress situations in plants.
authors.affiliation: Group of Biochemistry and Cell Signaling in Nitric Oxide, Biochemistry and Molecular Biology Division, Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Campus 'Las Lagunillas', E-23071 Jaén, Spain.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Begara-Morales
authors.affiliation: Group of Biochemistry and Cell Signaling in Nitric Oxide, Biochemistry and Molecular Biology Division, Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Campus 'Las Lagunillas', E-23071 Jaén, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Sánchez-Calvo
authors.affiliation: Group of Biochemistry and Cell Signaling in Nitric Oxide, Biochemistry and Molecular Biology Division, Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Campus 'Las Lagunillas', E-23071 Jaén, Spain.
authors.forename: Mounira
authors.initials: M
authors.lastname: Chaki
authors.affiliation: Group of Biochemistry and Cell Signaling in Nitric Oxide, Biochemistry and Molecular Biology Division, Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Campus 'Las Lagunillas', E-23071 Jaén, Spain.
authors.forename: Capilla
authors.initials: C
authors.lastname: Mata-Pérez
authors.affiliation: Group of Biochemistry and Cell Signaling in Nitric Oxide, Biochemistry and Molecular Biology Division, Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Campus 'Las Lagunillas', E-23071 Jaén, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Valderrama
authors.affiliation: Group of Biochemistry and Cell Signaling in Nitric Oxide, Biochemistry and Molecular Biology Division, Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Campus 'Las Lagunillas', E-23071 Jaén, Spain.
authors.forename: María N
authors.initials: MN
authors.lastname: Padilla
authors.affiliation: Institute of Biotechnology, University of Granada, E-18071 Granada, Spain jbarroso@ujaen.es javier.corpas@eez.csic.es.
authors.forename: Javier
authors.initials: J
authors.lastname: López-Jaramillo
authors.affiliation: Center for Advanced Studies in Olives and Olive Oil, University of Jaén, Campus 'Las Lagunillas', E-23071 Jaén, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Luque
authors.affiliation: Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, Apartado 419, E-18080 Granada, Spain jbarroso@ujaen.es javier.corpas@eez.csic.es.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Corpas
authors.affiliation: Group of Biochemistry and Cell Signaling in Nitric Oxide, Biochemistry and Molecular Biology Division, Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Campus 'Las Lagunillas', E-23071 Jaén, Spain Center for Advanced Studies in Olives and Olive Oil, University of Jaén, Campus 'Las Lagunillas', E-23071 Jaén, Spain jbarroso@ujaen.es javier.corpas@eez.csic.es.
authors.forename: Juan B
authors.initials: JB
authors.lastname: Barroso
mesh_terms: D002736:Chloroplasts / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D005980:Glutathione Reductase / Q000235:genetics* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017422:Sequence Analysis, DNA
chemical_list: D010940:Plant Proteins; D009569:Nitric Oxide; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH); D005980:Glutathione Reductase
keywords: Glutathione reductase; S-nitrosoglutathione.; S-nitrosylation; monodehydroascorbate reductase; nitration; nitric oxide; peroxynitrite; reactive nitrogen species; salinity