Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr)

• Nam, Dayeon

• Matsumoto, Yuki

• Uchida, Takeshi

• O'Brian, Mark R.

• Ishimori, Koichiro

Abstract

The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacteriumBradyrhizobium 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 Mn(2+)addition, indicating that Mn(2+)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 Mn(2+)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 Mn(2+)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 Mn(2+)binding to form the Irr-ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn(2+)with heme or by heme binding to Cys-29.

Journal

• Journal of Biological Chemistry (JBC)

  Journal of Biological Chemistry (JBC) 295 (32), 11316-11325, 2020-08-07

  American Society for Biochemistry and Molecular Biology (ASBMB)

Keywords

• iron metabolism
• iron
• DNA binding protein
• metalloprotein
• metal ion-protein interaction
• protein-DNA interaction
• heme
• protein motif
• Bradyrhizobium japonicum
• gene regulation

Details

• CRID: 1050852018416948224
• NII Article ID: 120007126835
• HANDLE: 2115/82428
• ISSN: 00219258
• Text Lang: en
• Article Type: journal article
• Data Source:
  • IRDB
  • CiNii Articles