Insights into the catalytic mechanism of the Bcp family: Functional and structural analysis of Bcp1 from Sulfolobus solfataricus

Bcps constitute a group of antioxidant enzymes, belonging to the Prx family, that are widely distributed in bacteria, plants, and fungi. These proteinscan 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 andrequire further investigation. In this article, we report on the functional and structural characterization of Bcp1, an archaeal Bcp isolated fromSulfolobus solfataricus, which presents two conserved cysteine residues at positions 45 and 50. Functional studies revealed that this enzyme performsthe catalytic reaction using an atypical 2-Cys mechanism, where Cys45 is the peroxidatic and Cys50 is the resolving cysteine. The X-ray structureof 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 similarto that of other Prxs. Superposition with a structural homologue in the oxidized state provided, for the first time, a detailed description of the structuralrearrangement necessary for a member of the Bcp family to perform the catalytic reaction. From this structural analysis, it emerges that a significantconformational change from a fully folded, to a locally unfolded form is required to form the intramolecular disulfide bond upon oxidation, accordingto the proposed reaction mechanism. Two residues, namely Arg53 and Asp54, which could play a role in this rearrangement, were also identified.