Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5: kinetics, catalytic mechanism and oxidative inactivation.
2012 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 442, 369-380 p.Article in journal (Refereed) Published
Glutathione peroxidases constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms ((SeCys- or Cys-Gpxs) which are regenerated by glutathione or thioredoxins, (Trxs) respectively. We present here new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. This study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M-1 s-1. In PtGpx5, Glu79, which replaces the Gln usually found in Gpx catalytic tetrad, is likely involved in substrate selectivity. Although the redox midpoint potential of the Cys44-Cys92 disulfide 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 between the Trx catalytic cysteine and the Gpx5 resolving cysteine. 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. Besides, mass spectrometry analysis of in vitro oxidized PtGpx5 demonstrated that the peroxidatic cysteine 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.
Place, publisher, year, edition, pages
2012. Vol. 442, 369-380 p.
glutathione peroxidase, over-oxidation, peroxynitrite, redox property, site-directed mutagenesis, thiol peroxidase.
IdentifiersURN: urn:nbn:se:kth:diva-79019DOI: 10.1042/BJ20111378ISI: 000301038400013PubMedID: 22122405OAI: oai:DiVA.org:kth-79019DiVA: diva2:495560
QC 201302132012-02-092012-02-082013-02-13Bibliographically approved