Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation Begará Morales, Juan Carlos Sánchez Calvo, Beatriz Chaki, Mounira Valderrama, Raquel Mata-Pérez, Capilla López Jaramillo, Francisco Javier Padilla, María N. Carreras, Alfonso Corpas Aguirre, Francisco Javier Barroso, Juan B. Ascorbate peroxidase Nitration Nitric oxide S-Nitrosoglutathione S-nitrosylation Peroxynitrite Reactive nitrogen species Salinity stress JBM acknowledges a PhD fellowship (F.P.U.) from the Ministry of Science and Innovation. This work was supported by an ERDF-co-financed grant from the Ministry of Science and Innovation (BIO2009-12003-C02-01, BIO2009-12003-C02-02, and BIO2012-33904) and Junta de Andalucia (group BIO286 and BIO192), Spain. LC/MS/MS analyses were carried out at the Laboratorio de Proteomica LP-CSIC/UAB, a member of the ProteoRed network. Technical and human support provided by CICT of Universidad de Jaen (UJA, MINECO, Junta de Andalucia, FEDER) is gratefully acknowledged. We acknowledge Mr Carmelo Ruiz-Torres for his excellent technical support. Post-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H2O2, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation. 2020-07-17T12:10:37Z 2020-07-17T12:10:37Z 2014-02 journal article Juan C. Begara-Morales, Beatriz Sánchez-Calvo, Mounira Chaki, Raquel Valderrama, Capilla Mata-Pérez, Javier López-Jaramillo, María N. Padilla, Alfonso Carreras, Francisco J. Corpas, Juan B. Barroso, Dual regulation of cytosolic ascorbate peroxidase (APX) by tyrosine nitration and S-nitrosylation, Journal of Experimental Botany, Volume 65, Issue 2, February 2014, Pages 527–538, [https://doi.org/10.1093/jxb/ert396] http://hdl.handle.net/10481/63038 10.1093/jxb/ert396 eng http://creativecommons.org/licenses/by/3.0/es/ open access Atribución 3.0 España