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Peroxiredoxins are ubiquitous enzymes that are part of the oxidative stress defense system. In the present study, we identified three peroxiredoxins [bacterioferritin comigratory protein (Bcp)1, Bcp3 and Bcp4] in the genome of the aerobic hyperthermophilic archaeon Sulfolobus solfataricus. Based on the cysteine residues conserved in the deduced aminoacidic sequence, Bcp1 and Bcp4 can be classified as 2-Cys peroxiredoxins and Bcp3 as a 1-Cys peroxiredoxin. A comparative study of the recombinant Bcps produced in Escherichia coli showed that these enzymes protect DNA plasmid from oxidative damage and remove both H(2)O(2) and tert-butyl hydroperoxide, although at different efficiencies. We observed that all of them were particularly thermostable and that peak enzymatic activity fell within the range of the growth temperature of S. solfataricus. Furthermore, we discovered an alternative Bcp reduction system whose composition differs from that of the peroxiredoxin reduction system previously characterized in the aerobic hyperthermophilic archaeon Aeropyrum pernix. Whereas the latter uses the thioredoxin/thioredoxin reductase/NADPH system, this alternative Bcp system is formed of the protein disulfide oxidoreducatase, SSO0192, the thioredoxin reductase, SSO2416, and NADPH. The role of Bcps in oxidative stress was investigated using transcriptional analysis. Different northern blot analysis responses suggested that the Bcp antioxidant system of S. solfataricus can both operate at the constitutive level, with Bcp1 and Bcp4 preventing endogenous peroxide formation, and at the inducible level, with Bcp3 and the already characterized Bcp2 protecting cells from the attack of external peroxides.

The detoxification from peroxides in Sulfolobus solfataricus is performed by the Bacterioferritin comigratory proteins (Bcps), Bcp1 (Sso2071), Bcp2 (Sso2121), Bcp3 (Sso2255) and Bcp4 (Sso2613), antioxidant enzymes belonging to one of the subfamilies of the Peroxiredoxins. In this paper we report on the functional, structural and docking analyses of Bcp4, characterized by the CXXXXC motif in the active site. Bcp4 represents the first dimeric Bcp so far investigated. Biochemical studies showed that the protein has a non-covalent dimeric structure and adopts an atypical 2-Cys catalytic mechanism. The X-ray structure of the double mutant C45S/C50S, representative of the fully reduced enzyme state, described the protein dimeric arrangement. Finally, concurrent availability of the crystallographic structure of the monomeric Bcp1 allowed comparative analysis of the interaction with Protein Disulfide Oxidoreductase SsPDO (Sso0192), involved in the reduction of both Bcp1 and Bcp4, through a protein-protein docking approach.

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO) contains three disulphide bridges linking residues C(41)XXC(44), C(155)XXC(158), C(173)XXXXC(178). To get information on the role played by these cross-links in determining the structural and functional properties of the protein, we performed site-directed mutagenesis on Cys residues and investigated the changes in folding, stability and functional features of the mutants and analysed the results with computational analysis. The reductase activity of SsPDO and its mutants was evaluated by insulin and thioredoxin reductase assays also coupled with peroxiredoxin Bcp1 of S. solfataricus. The three-dimensional model of SsPDO was constructed and correlated with circular dichroism data and functional results. Biochemical analysis indicated a key function for the redox site constituted by Cys155 and Cys158. To discriminate between the role of the two cysteine residues, each cysteine was mutagenized and the behaviour of the single mutants was investigated elucidating the basis of the electron-shuffling mechanism for SsPDO. Finally, cysteine pK values were calculated and the accessible surface for the cysteine side chains in the reduced form was measured, showing higher reactivity and solvent exposure for Cys155.

Bcps constitute a group of antioxidant enzymes, belonging to the Prx family, that are widely distributed in bacteria, plants, and fungi. These proteins can contain two conserved cysteines within the CXXXXC motif. Recent studies demonstrated that though the role of the first cysteine is well defined, being the catalytic peroxidatic cysteine in all the members of this protein family, data on the function of the second cysteine are controversial and require further investigation. In this article, we report on the functional and structural characterization of Bcp1, an archaeal Bcp isolated from Sulfolobus solfataricus, which presents two conserved cysteine residues at positions 45 and 50. Functional studies revealed that this enzyme performs the catalytic reaction using an atypical 2-Cys mechanism, where Cys45 is the peroxidatic and Cys50 is the resolving cysteine. The X-ray structure of the double mutant C45S/C50S, representative of the fully reduced enzyme state, was determined at a resolution of 2.15 A ° , showing a Trx fold similar to that of other Prxs. Superposition with a structural homologue in the oxidized state provided, for the first time, a detailed description of the structural rearrangement necessary for a member of the Bcp family to perform the catalytic reaction. From this structural analysis, it emerges that a significant conformational change from a fully folded, to a locally unfolded form is required to form the intramolecular disulfide bond upon oxidation, according to the proposed reaction mechanism. Two residues, namely Arg53 and Asp54, which could play a role in this rearrangement, were also identified.