Structural snapshots of OxyR reveal the peroxidatic mechanism of H₂O₂ sensing.

Abstract

Hydrogen peroxide (H₂O₂) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H₂O₂ One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H₂O₂ In this study, we present crystallographic evidence for the H₂O₂-sensing mechanism and H₂O₂-dependent structural transition of <i>Corynebacterium glutamicum</i> OxyR by capturing the reduced and H₂O₂-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H₂O₂-bound structure, we pinpoint the key residues for the peroxidatic reduction of H₂O₂, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H₂O₂ reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H₂O₂-induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H₂O₂.