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Redox-Dependent Dynamics in Heme-Bound Bacterial Iron Response Regulator (Irr) Protein

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The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
§ Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto 606-8561, Japan
Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo, New York 14214, United States
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan
*E-mail: [email protected]. Telephone: +81-6-6879-8501.
Cite this: Biochemistry 2016, 55, 29, 4047–4054
Publication Date (Web):July 5, 2016
https://doi.org/10.1021/acs.biochem.6b00512
Copyright © 2016 American Chemical Society

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    Abstract

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    The iron response regulator (Irr) protein from Bradyrhizobium japonicum mediates iron-dependent regulation of heme biosynthesis. Irr degrades in response to heme availability through a process that involves the binding of heme to Cys-29 in the heme regulatory motif (HRM) in the presence of molecular oxygen. In this work, we assessed the dynamics of one-electron reduction of heme-bound Irr by monitoring the formation of transient intermediates by pulse radiolysis. Hydrated electrons generated by pulse radiolysis reduced heme iron-bound Irr, facilitating the binding of molecular oxygen to the heme iron in Irr through an initial intermediate with an absorption maximum at 420 nm. This initial intermediate was converted to a secondary intermediate with an absorption maximum at 425 nm, with a first-order rate constant of 1.0 × 104 s–1. The Cys-29 → Ala (C29A) mutant of Irr, on the other hand, did not undergo the secondary phase, implying that ligand exchange of Cys-29 for another ligand takes place during the process. Spectral changes during the reduction of the heme-bound Irr revealed that binding of CO to ferrous heme consisted of two phases with kon values of 1.3 × 105 and 2.5 × 104 M–1 s–1, a finding consistent with the presence of two distinct hemes in Irr. In aerobic solutions, by contrast, oxidation of the ferrous heme to the ferric form was found to be a two-phase process. The C29A mutant was similarly oxidized, but this occurred as a single-phase process. We speculate that a reactive oxygen species essential for degradation of the protein is generated during the oxidation process.

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