Insights into the Alkyl Peroxide Reduction Pathway of Xanthomonas campestris Bacterioferritin Comigratory Protein from the Trapped Intermediate–Ligand Complex Structures

Abstract

Considerable insights into the oxidoreduction activity of the Xanthomonas campestris bacterioferritin comigratory protein (XcBCP) have been obtained from trapped intermediate/ligand complex structures determined by X-ray crystallography. Multiple sequence alignment and enzyme assay indicate that XcBCP belongs to a subfamily of atypical 2-Cys peroxiredoxins (Prxs), containing a strictly conserved peroxidatic cysteine (CP48) and an unconserved resolving cysteine (CR84). Crystals at different states, i.e. Free_SH state, Intra_SS state, and Inter_SS state, were obtained by screening the XcBCP proteins from a double C48S/C84S mutant, a wild type, and a C48A mutant, respectively. A formate or an alkyl analog with two water molecules that mimic an alkyl peroxide substrate was found close to the active site of the Free_SH or Inter_SS state, respectively. Their global structures were found to contain a novel substrate-binding pocket capable of accommodating an alkyl chain of no less than 16 carbons. In addition, in the Intra_SS or Inter_SS state, substantial local unfolding or complete unfolding of the CR-helix was detected, with the CP-helix remaining essentially unchanged. This is in contrast to the earlier observation that the CP-helix exhibits local unfolding during disulfide bond formation in typical 2-Cys Prxs. These rich experimental data have enabled us to propose a pathway by which XcBCP carries out its oxidoreduction activity through the alternate opening and closing of the substrate entry channel and the disulfide-bond pocket.