FIGURE 6.

Proposed overoxidation mechanism of OxyR. A, modeled structures of OxyR, focusing on the sensing cysteine (C_S). S-Hydroxylated sensing cysteine (C_S-SOH) is oxidized by the bound H₂O₂ (left), resulting in S-sulfinated sensing cysteine (C_S-SO₂H; middle), which is in turn further oxidized to S-sulfonated sensing cysteine (C_S-SO₃H; right). The models were constructed based on the crystal structure of the P. aeruginosa OxyR-C199D variant complexed with a H₂O₂ molecule. The H₂O₂ molecule was bound by the polar interactions with the side chain and the backbone amide group of Thr¹³⁶, the backbone carbonyl group of Glu²⁰⁴, and the backbone amide group of Cys²⁰⁶, as indicated by the dotted lines. The lone pair of electrons are displayed as the sp³ orbital shapes, and the bound water molecules are shown as red spheres. The blue arrows indicate the nucleophilic attack by Sγ of sensing cysteine on an oxygen atom of H₂O₂. B, schematic reaction mechanism of oxidation of sensing cysteine and the concomitant three redox states of OxyR. Sensing cysteine of OxyR in the reduced state is converted to S-hydroxylated sensing cysteine by H₂O₂, as a reaction intermediate (black arrow). S-Hydroxylated sensing cysteine rapidly makes a disulfide with the other redox-sensitive cysteine (C_R), resulting in the disulfide state of OxyR (blue arrow). Alternatively, S-hydroxylated sensing cysteine is further oxidized consecutively by H₂O₂ to the final S-sulfonated sensing cysteine in the presence of excess H₂O₂, resulting in the overoxidized state of OxyR (red arrows).