Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration

• Published in: Biochimica et biophysica acta Vol. 1830; no. 11; pp. 4981 - 4989
• Main Authors: Corpas, Francisco J., Leterrier, Marina, Begara-Morales, Juan C., Valderrama, Raquel, Chaki, Mounira, López-Jaramillo, Javier, Luque, Francisco, Palma, José M., Padilla, María N., Sánchez-Calvo, Beatriz, Mata-Pérez, Capilla, Barroso, Juan B.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2013
• Subjects: Amino Acid Sequence; antibodies; Arabidopsis; Arabidopsis - drug effects; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis - metabolism; enzyme activity; enzyme inhibition; Evolution, Molecular; Glutathione - pharmacology; high performance liquid chromatography; hydrogen peroxide; Hydrogen Peroxide - pharmacology; Hydroxypyruvate reductase; Hydroxypyruvate Reductase - antagonists & inhibitors; Hydroxypyruvate Reductase - genetics; Hydroxypyruvate Reductase - metabolism; leaves; Models, Molecular; Molecular Sequence Data; Nitrates - metabolism; Nitration; Nitric oxide; Nitro-proteome; Oxidation-Reduction - drug effects; peas; Peroxisome; peroxisomes; Peroxisomes - drug effects; Peroxisomes - genetics; Peroxisomes - metabolism; Peroxynitrite; Peroxynitrous Acid - genetics; Peroxynitrous Acid - metabolism; photorespiration; physiological regulation; Pisum sativum; Pisum sativum - enzymology; Pisum sativum - genetics; Pisum sativum - metabolism; Plant Leaves - genetics; Plant Leaves - metabolism; post-translational modification; proteins; Proteome - drug effects; Proteome - genetics; Proteome - metabolism; proteomics; site-directed mutagenesis; tandem mass spectrometry; tyrosine; Tyrosine - analogs & derivatives; Tyrosine - genetics; Tyrosine - metabolism; NO; Nitration; Peroxynitrite; PTM; HPR; GSNO; Nitro-proteome; SIN-1; Hydroxypyruvate reductase; RNS; Nitric oxide; Peroxisome; ONOO; GSH; ONOO(−); post-translational modification; reduced glutathione; reactive nitrogen species; 3-morpholinosydnonimine; S-nitrosoglutathione
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.07.002

Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported. We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches. Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO− molecule caused the highest inhibition of activity (51% at 5mM SIN-1), with 5mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite. These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function. This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions. [Display omitted] •Protein tyrosine nitration is a PTM mediated by NO-derived molecules.•Peroxisomal NADH-dependent HPR activity is a target of tyrosine nitration.•Tyr198 of HPR is the primary site of nitration responsible for the inhibition.