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

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Please use this identifier to cite or link to this item:http://hdl.handle.net/2115/82428

Title: Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr)
Authors: Nam, Dayeon Browse this author
Matsumoto, Yuki Browse this author
Uchida, Takeshi Browse this author
O'Brian, Mark R. Browse this author
Ishimori, Koichiro Browse this author →KAKEN DB
Keywords: iron metabolism
iron
DNA binding protein
metalloprotein
metal ion-protein interaction
protein-DNA interaction
heme
protein motif
Bradyrhizobium japonicum
gene regulation
Issue Date: 7-Aug-2020
Publisher: American Society for Biochemistry and Molecular Biology (ASBMB)
Journal Title: Journal of Biological Chemistry (JBC)
Volume: 295
Issue: 32
Start Page: 11316
End Page: 11325
Publisher DOI: 10.1074/jbc.RA119.011855
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.
Rights: 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.
Type: article
URI: http://hdl.handle.net/2115/82428
Appears in Collections:理学院・理学研究院 (Graduate School of Science / Faculty of Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 石森 浩一郎

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