Kitatsuji Chihiro, Izumi Kozue, Nambu Shusuke, Kurogochi Masaki, Uchida Takeshi, Nishimura Shin-ichiro, Iwai Kazuhiro, O'Brian Mark R, Ikeda-Saito Masao, Ishimori Koichiro
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.
Sci Rep. 2016 Jan 5;6:18703. doi: 10.1038/srep18703.
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.
慢生根瘤菌转录调节因子Irr（铁反应调节因子）是铁稳态的关键调节因子，它通过一种涉及蛋白质氧化修饰的机制，在与血红素结合时发生降解。在此，我们表明血红素结合的Irr激活O₂形成高活性氧物种（ROS），伴随着从血红素铁到非血红素铁的“活性位点转换”以降解自身。在血红素和还原剂存在的情况下，ROS清除实验表明，Irr与其他血红蛋白一样从O₂生成H₂O₂，但H₂O₂氧化肽的效果较差，提示H₂O₂会进一步激活。有趣的是，我们发现在氧化过程中，Soret带强度随时间下降，并且在g = 4.3处出现特征性EPR信号，表明血红素降解以及非血红素铁位点的相继形成。结合突变研究，我们在此提出一种新的“两步自氧化修饰”机制，在此过程中，O₂在血红素调节基序（HRM）位点被激活形成H₂O₂，并且生成的H₂O₂在由活性位点转换形成的His簇区域的非血红素铁位点进一步转化为更具反应性的物种，如·OH。
