Structural snapshots of OxyR reveal the peroxidatic mechanism of H(2)O(2) sensing

Hydrogen peroxide (H(2)O(2)) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H(2)O(2). One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H(2)O(2). In this study, we present crystallographic evidence for the H(2)O(2)-sensing mechanism and H(2)O(2)-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H(2)O(2)-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H(2)O(2)-bound structure, we pinpoint the key residues for the peroxidatic reduction of H(2)O(2), and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H(2)O(2) reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H(2)O(2)-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 H(2)O(2).