Gpxs (glutathione peroxidases) constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms (SeCys-Gpx or Cys-Gpx), which are regenerated by glutathione or Trxs (thioredoxins) respectively. In the present paper we show new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. The present study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M−1·s−1. In PtGpx5 (poplar Gpx5; Pt is Populus trichocarpa), Glu79, which replaces the glutamine residue usually found in the Gpx catalytic tetrad, is likely to be involved in substrate selectivity. Although the redox midpoint potential of the Cys44–Cys92 disulfide bond and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (Kperoxide and kcat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide bond between the Trx catalytic cysteine residue and the Gpx5 resolving cysteine residue. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Also, MS analysis of in-vitro-oxidized PtGpx5 demonstrated that the peroxidatic cysteine residue can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen-peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.
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March 2012
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Research Article|
February 13 2012
Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5: kinetics, catalytic mechanism and oxidative inactivation
Benjamin Selles;
Benjamin Selles
*UMR1136 Nancy University-INRA, Interactions Arbres-Microorganismes, IFR 110, EFABA 54506 Vandoeuvre-lès-Nancy cedex, France
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Martin Hugo;
Martin Hugo
†Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay
‡Centre for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay
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Madia Trujillo;
Madia Trujillo
†Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay
‡Centre for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay
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Vaibhav Srivastava;
Vaibhav Srivastava
§Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå Plant Science Center, 90183 Umeå, Sweden
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Gunnar Wingsle;
Gunnar Wingsle
§Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå Plant Science Center, 90183 Umeå, Sweden
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Jean-Pierre Jacquot;
Jean-Pierre Jacquot
*UMR1136 Nancy University-INRA, Interactions Arbres-Microorganismes, IFR 110, EFABA 54506 Vandoeuvre-lès-Nancy cedex, France
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Rafael Radi;
Rafael Radi
†Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay
‡Centre for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Montevideo, Uruguay
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Nicolas Rouhier
Nicolas Rouhier
1
*UMR1136 Nancy University-INRA, Interactions Arbres-Microorganismes, IFR 110, EFABA 54506 Vandoeuvre-lès-Nancy cedex, France
1To whom correspondence should be addressed (email nrouhier@scbiol.uhp-nancy.fr).
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Publisher: Portland Press Ltd
Received:
July 28 2011
Revision Received:
November 17 2011
Accepted:
November 29 2011
Accepted Manuscript online:
November 29 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 442 (2): 369–380.
Article history
Received:
July 28 2011
Revision Received:
November 17 2011
Accepted:
November 29 2011
Accepted Manuscript online:
November 29 2011
Citation
Benjamin Selles, Martin Hugo, Madia Trujillo, Vaibhav Srivastava, Gunnar Wingsle, Jean-Pierre Jacquot, Rafael Radi, Nicolas Rouhier; Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5: kinetics, catalytic mechanism and oxidative inactivation. Biochem J 1 March 2012; 442 (2): 369–380. doi: https://doi.org/10.1042/BJ20111378
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