The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum. Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr–ICE complex. The addition of EDTA to the Irr–ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn2+ addition, indicating that Mn2+ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn2+ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn2+ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr–ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29–mutated Irr. These results indicate that Irr requires Mn2+ binding to form the Irr–ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn2+ with heme or by heme binding to Cys-29.
The Structural Environment Around the Heme-Binding Site in Cyclooxygenase and Its Implication
