Molecular Insights into Hydrogen Peroxide-sensing Mechanism of the Metalloregulator MntR in Controlling Bacterial Resistance to Oxidative Stresses.

Manganese contributes to anti-oxidative stress particularly in catalase-devoid bacteria, and DtxR family metalloregulators, through sensing cellular Mn(2+) content, regulate its homeostasis. Here, we show that metalloregulator MntR (So-MntR) functions dually as Mn(2+) and H(2)O(2) sensors in mediating H(2)O(2) resistance by an oral streptococcus. H(2)O(2) disrupted So-MntR binding to Mn(2+) transporter mntABC promoter and induced disulfide-linked dimerization of the protein. Mass spectrometry identified Cys-11/Cys-156 and Cys-11/Cys-11 disulfide-linked peptides in H(2)O(2)-treated So-MntR. Site mutagenesis of Cys-11 and Cys-156 and particularly Cys-11 abolished H(2)O(2)-induced disulfide-linked dimers and weakened H(2)O(2) damage on So-MntR binding, indicating that H(2)O(2) inactivates So-MntR via disulfide-linked dimerization. So-MntR C123S mutant was extremely sensitive to H(2)O(2) oxidization in dimerization/oligomerization, probably because the mutagenesis caused a conformational change that facilitates Cys-11/Cys-156 disulfide linkage. Intermolecular Cys-11/Cys-11 disulfide was detected in C123S/C156S double mutant. Redox Western blot detected So-MntR oligomers in air-exposed cells but remarkably decreased upon H(2)O(2) pulsing, suggesting a proteolysis of the disulfide-linked So-MntR oligomers. Remarkably, elevated C11S and C156S but much lower C123S proteins were detected in H(2)O(2)-pulsed cells, confirming Cys-11 and Cys-156 contributed to H(2)O(2)-induced oligomerization and degradation. Accordingly, in the C11S and C156S mutants, expression of mntABC and cellular Mn(2+) decreased, but H(2)O(2) susceptibility increased. In the C123S mutant, increased mntABC expression, cellular Mn(2+) content, and manganese-mediated H(2)O(2) survival were determined. Given the wide distribution of Cys-11 in streptococcal DtxR-like metalloregulators, the disclosed redox regulatory function and mechanism of So-MntR can be employed by the DtxR family proteins in bacterial resistance to oxidative stress.