Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5: kinetics, catalytic mechanism and oxidative inactivation

Abstract

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.

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

“…In fact, the rate constant of peroxynitrite reduction by Ohr is comparable to the rate constants reported for Prxs and thiolor selenol-dependent Gpxs (10,12,13,(53)(54)(55). Therefore, it is feasible that Ohr plays a relevant role in vivo in the reduction of peroxynitrite.…”

Resveratrol attenuates cisplatin renal cortical cytotoxicity by modifying oxidative stress

“…Another possibility is that cisplatin directly binds to sulfhydryl groups on cysteine in Mn SOD and GSH peroxidase causing diminished activity similar to what occurs following exposure of mice to 2,6-di-tert-butyl-4-methylphenol (Meier et al, 2007). A third possible mechanism for the decline in GSH peroxidase and MnSOD activity may be due to oxidative stress causing oxidation of cysteine groups within each enzyme which has been reported to impair enzyme activity (Selles et al, 2012). …”

The Glutathione Peroxidase Gene Family in Gossypium hirsutum: Genome-Wide Identification, Classification, Gene Expression and Functional Analysis

“…GPX genes from a range of plant species, such as Nicotiana sylvestris 18, Citrus sinensis 1920, Avena fatua 21, Spinacia oleracea 22, Brassica campestris 23, Helianthus annuus 24, Pisum sativum 25, Lycopersicon esculentum 26, Arabidopsis thaliana 272829, Oryza sativa 30, Triticum aestivum 31, Panax ginseng 32, Populus trichocarpa 3334 and Thellungiella salsuginea 35, have been isolated and characterised. Unfortunately very few reports on genome-wide identification and characterisation of GPX family proteins to date have been reported.…”