PerR is a metal-dependent peroxide sensing transcription factor which controls the expression of genes involved in peroxide resistance. The function of Bacillus subtilis PerR is mainly dictated by the regulatory metal ion (Fe2+ or Mn2+) coordinated by three N-donor ligands (His37, His91, and His93) and two 0 donor ligands (Asp85 and Asp104). While H2O2 sensing by PerR is mediated by Fe2+-dependent oxidation of N-donor ligand (either His37 or His91), one of the O-donor ligands (Asp104), but not Asp85, has been proposed as the key residue that regulates the sensitivity of PerR to H2O2. Here we systematically investigated the relative roles of two O-donor ligands of PerR in metal-binding affinity and H2O2 sensitivity in vivo and in vitro. Consistent with the previous report, in vitro the D104E-PerR could not sense low levels of H2O2 in the presence of excess Fe2+ sufficient for the formation of the Fe2+-bound D104E-PerR. However, the expression of PerR-regulated reporter fusion was not repressed by D104E-PerR in the presence of Fe2+, suggesting that Fe2+ is not an effective corepressor for this mutant protein in vivo. Furthermore, in vitro metal titration assays indicate that D104E-PerR has a significantly reduced affinity for Fe2+, but not for Mn2+, when compared to wild type PerR. These data indicate that the type of O-donor ligand (Asp vs. Glu) at position 104 is an important determinant in providing high Fe2+-binding affinity required for the sensing of the physiologically relevant Fe2+-levels, in addition to its role in rendering PerR highly sensitive to physiological levels of H2O2. In comparison, the D85E-PerR did not show a perturbed change in Fe2+-binding affinity, however, it displayed a slightly decreased sensitivity to H2O2 both in vivo and in vitro, suggesting that the type of O-donor ligand (Asp vs. Glu) at position 85 may be important for the fine-tuning of H2O2 sensitivity. (C) 2018 Elsevier Inc. All rights reserved.