OxyR2 functions as a three-state redox switch to tightly regulate production of Prx2, a peroxiredoxin of Vibrio vulnificus

The bacterial transcriptional regulator OxyR is known to function as a two-state redox switch. OxyR senses cellular levels of H₂O₂ via a "sensing cysteine" that switches from the reduced to a disulfide state upon H₂O₂ exposure, inducing the expression of antioxidant genes. The reduced and disulfide states of OxyR, respectively, bind to extended and compact regions of DNA, where the reduced state blocks and the oxidized state allows transcription and further induces target gene expression by interacting with RNA polymerase. Vibrio vulnificus OxyR2 senses H₂O₂ with high sensitivity and induces the gene encoding the antioxidant Prx2. In this study, we used mass spectrometry to identify a third redox state of OxyR2, in which the sensing cysteine was overoxidized to S-sulfonated cysteine (Cys-SO₃H) by high H₂O₂ in vitro and in vivo, where the modification deterred the transcription of prx2. The DNA binding preferences of OxyR2_5CA-C206D, which mimics overoxidized OxyR2, suggested that overoxidized OxyR2 binds to the extended DNA site, masking the -35 region of the prx2 promoter. These combined results demonstrate that OxyR2 functions as a three-state redox switch to tightly regulate the expression of prx2, preventing futile production of Prx2 in cells exposed to high levels of H₂O₂ sufficient to inactivate Prx2. We further provide evidence that another OxyR homolog, OxyR1, displays similar three-state behavior, inviting further exploration of this phenomenon as a potentially general regulatory mechanism.