The roles of two O-donor ligands in the Fe-binding and HO-sensing by the Fe-dependent HO sensor PerR.

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 (Fe or Mn) coordinated by three N-donor ligands (His37, His91, and His93) and two O-donor ligands (Asp85 and Asp104). While HO sensing by PerR is mediated by Fe-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 HO. Here we systematically investigated the relative roles of two O-donor ligands of PerR in metal-binding affinity and HO sensitivity in vivo and in vitro. Consistent with the previous report, in vitro the D104E-PerR could not sense low levels of HO in the presence of excess Fe sufficient for the formation of the Fe-bound D104E-PerR. However, the expression of PerR-regulated reporter fusion was not repressed by D104E-PerR in the presence of Fe, suggesting that Fe 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 Fe, but not for Mn, 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 Fe-binding affinity required for the sensing of the physiologically relevant Fe-levels, in addition to its role in rendering PerR highly sensitive to physiological levels of HO. In comparison, the D85E-PerR did not show a perturbed change in Fe-binding affinity, however, it displayed a slightly decreased sensitivity to HO 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 HO sensitivity.