FIGURE 3.

Metal-dependent DNA binding and H₂O₂-mediated oxidation of PerR_SAin vitro. A, metal-dependent DNA binding activity of PerR_SA. Various concentrations of metal ions (open circle for Fe²⁺ and filled circle for Mn²⁺) are added to samples containing 100 nm DNA and 100 nm active PerR_SA dimer, and metal-dependent DNA-binding of PerR_SA was monitored by fluorescence anisotropy change. B, sensitivity of PerR_SA to H₂O₂ in the presence of Fe²⁺. Protein (100 nm active PerR_SA:Zn dimer), FeSO₄, H₂O₂, and MnCl₂ were sequentially added to buffer A containing 100 nm DNA with an interval of 2 min between each addition, and FA was measured after each addition. 10 μm H₂O₂ rapidly (<20 s) inactivated PerR_SA in the presence of 2 μm Fe²⁺ and the loss of PerR_SA activity was not recovered by the addition of Mn²⁺. C, sensitivity of PerR_SA to H₂O₂ in the presence of Mn²⁺. Protein (100 nm active PerR_SA:Zn dimer), MnCl₂, H₂O₂, and EDTA were sequentially added to buffer A containing 100 nm DNA with an interval of 2 min between each addition except for 10 min between H₂O₂ and EDTA, and FA was measured after each addition. PerR_SA was insensitive to 1 mm H₂O₂ for 10 min but lost DNA-binding activity rapidly by addition of 1 mm EDTA. D, metal-dependent oxidation of PerR_SA. Oxidation of PerR_SA by H₂O₂ in the presence of 10 μm Fe²⁺ or 100 μm Mn²⁺ was monitored by MALDI-TOF MS after tryptic digestion. Note that no cysteine oxidation was observed as judged by fully alkylated T9 peptide (T9*) containing Cys-102 and Cys-105.