Characterization of 1-cys peroxiredoxin from Sulfolobus solfataricus and its involvement in the response to oxidative stress.

Introduction To protect against toxic Reactive oxygen species (ROS), aerobic organisms are equipped with a full array of defence mechanisms. In recent years, much attention has been given to peroxiredoxins (Prxs) [1], a new family of thiol-specific antioxidant proteins. Prxs use a redox-active cysteine to reduce peroxides. In the aerobic hyperthermophilic archaeon Sulfolobus solfataricus the investigation of the mechanisms governing ROS protection is at initial stage. Recent analyses of the genomic sequence of S. solfataricus [2] have revealed the presence of four putative Prxs (Bcp1, Bcp2, Bcp3, Bcp4). We report the involvement of the Bcp2 in oxidative stress in S. solfataricus and the characterization of the recombinant protein (rBcp2) in order to shed light on its role in the detoxification process and on its catalytic mechanism. Material and Methods Purification of rBcp2 by affinity chromatography (HisTrap HP Amersham) and size-exclusion chromatography (HiLoad Superdex 75; Amersham). DNA cleavage assay by the metal ion catalyzed oxidation (MCO) system and assay of peroxidase activity were according to Lim et al. [3]. The mutant rBcp2 C49S was obtained by QuickChange II site directed mutagenesis kit (Stratagene). Results To understand the function of Bcp2 in oxidative stress, we provided evidence of its involvement in response to various oxidant agents. The transcriptional and the western blot analysis have showed the increased level of specific mRNA and Bcp2 respectively in response to Paraquat, tert-butyl hydroperoxide and H2O2. rBcp2, expressed in E.coli, was purified and functionally characterized: rBcp2 removes the H2O2 in a DTT-dependent manner and protects plasmidic DNA against the MCO system (DTT/ Fe3+/O2). Lastly the loss of activity in the Bcp2 mutant (C49S) obtained shows that Cys49 must necessarily be involved in the catalysis.