Post-translational modifications of Medicago truncatula glutathione peroxidase 1 induced by nitric oxide

Plant glutathione peroxidases (Gpx) catalyse the reduction of various peroxides, such as hydrogen peroxide (H₂O₂), phospholipid hydroperoxides and peroxynitrite, but at the expense of thioredoxins rather than glutathione. A main function of plant Gpxs is the protection of biological membranes by scavenging phospholipid hydroperoxides, but some Gpxs have also been associated with H₂O₂ sensing and redox signal transduction. Nitric oxide (NO) is not only known to induce the expression of Gpx family members, but also to inhibit Gpx activity, presumably through the S-nitrosylation of conserved cysteine residues. In the present study, the effects of NO-donors on both the activity and S-nitrosylation state of purified Medicago truncatula Gpx1 were analyzed using biochemical assay measurements and a biotin-switch/mass spectrometry approach. MtGpx1 activity was only moderately inhibited by the NO-donors diethylamine-NONOate and S-nitrosoglutathione, and the inhibition may be reversed by DTT. The three conserved Cys of MtGpx1 were found to be modified through S-nitrosylation and S-glutathionylation, although to different extents, by diethylamine-NONOate and S-nitrosoglutathione, or by a combination of diethylamine-NONOate and reduced glutathione. The regulation of MtGpx1 and its possible involvement in the signaling process is discussed in the light of these results.