The hydrogen peroxide hypersensitivity of OxyR2 in Vibrio vulnificus depends on conformational constraints

Abstract

Most Gram-negative bacteria respond to excessive levels of H₂O₂ using the peroxide-sensing transcriptional regulator OxyR, which can induce the expression of antioxidant genes to restore normality. <i>Vibrio vulnificus</i> has two distinct OxyRs (OxyR1 and OxyR2), which are sensitive to different levels of H₂O₂ and induce expression of two different peroxidases, Prx1 and Prx2. Although OxyR1 has both high sequence similarity and H₂O₂ sensitivity comparable with that of other OxyR proteins, OxyR2 exhibits limited sequence similarity and is more sensitive to H₂O₂ To investigate the basis for this difference, we determined crystal structures and carried out biochemical analyses of OxyR2. The determined structure of OxyR2 revealed a flipped conformation of the peptide bond before Glu-204, a position occupied by glycine in other OxyR proteins. Activity assays showed that the sensitivity to H₂O₂ was reduced to the level of other OxyR proteins by the E204G mutation. We solved the structure of the OxyR2-E204G mutant with the same packing environment. The structure of the mutant revealed a dual conformation of the peptide bond before Gly-204, indicating the structural flexibility of the region. This structural duality extended to the backbone atoms of Gly-204 and the imidazole ring of His-205, which interact with H₂O₂ and invariant water molecules near the peroxidatic cysteine, respectively. Structural comparison suggests that Glu-204 in OxyR2 provides rigidity to the region that is important in H₂O₂ sensing, compared with the E204G structure or other OxyR proteins. Our findings provide a structural basis for the higher sensitivity of OxyR2 to H₂O₂ and also suggest a molecular mechanism for bacterial regulation of expression of antioxidant genes at divergent concentrations of cellular H₂O₂.