open access

https://hdl.handle.net/2115/82428

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

英語

gene regulation

2020-08-07

American Society for Biochemistry and Molecular Biology (ASBMB)

Journal of Biological Chemistry (JBC)

295(32)

11316-11325

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.

学術雑誌論文

Version of Record

This research was originally published in the Journal of Biological Chemistry. Dayeon Nam, Yuki Matsumoto, Takeshi Uchida, Mark R. O'Brian and Koichiro Ishimori. Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr) . J. Biol. Chem. 2020; Vol295:pp11316-11325 © the American Society for Biochemistry and Molecular Biology or © Dayeon Nam, Yuki Matsumoto, Takeshi Uchida, Mark R. O'Brian and Koichiro Ishimori.

0021-9258