title: Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator.
abstract: The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the "active site conversion" from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel "two-step self-oxidative modification" mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion.
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan.|Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Kitatsuji
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Kozue
authors.initials: K
authors.lastname: Izumi
authors.affiliation: Institute of Multidisciplinary Research for Advance Materials, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Shusuke
authors.initials: S
authors.lastname: Nambu
authors.affiliation: Faculty of Advanced Life Science, and Graduate School of Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Kurogochi
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchida
authors.affiliation: Faculty of Advanced Life Science, and Graduate School of Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Nishimura
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan.|CREST, Japan Science and Technology Agency, Tokyo 103-0027, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Iwai
authors.affiliation: Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo, New York 14214, United States.
authors.forename: Mark R
authors.initials: MR
authors.lastname: O'Brian
authors.affiliation: Institute of Multidisciplinary Research for Advance Materials, Tohoku University, Sendai 980-8577, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Ikeda-Saito
authors.affiliation: Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Ishimori
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002374:Catalase / Q000378:metabolism; D020134:Catalytic Domain; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors; C114259:iron response regulator protein, Bacteria; D006418:Heme; D006861:Hydrogen Peroxide; D007501:Iron; D002374:Catalase; D013482:Superoxide Dismutase