Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-8628, Japan.
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan; Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8530, Japan.
J Inorg Biochem. 2018 May;182:238-248. doi: 10.1016/j.jinorgbio.2018.01.007. Epub 2018 Jan 9.
Iron regulatory proteins (IRPs), regulators of iron metabolism in mammalian cells, control the translation of proteins involved in iron uptake, storage and utilization by binding to specific iron-responsive element (IRE) sequences of mRNAs. Two homologs of IRPs (IRP1 and IRP2) have a typical heme regulatory motif (HRM), a consensus sequence found in "heme-regulated proteins". However, specific heme binding to HRM has been reported only for IRP2, which is essential for oxidative modification and loss of binding to target mRNAs. In this paper, we confirmed that IRP1 also specifically binds two molar equivalents of heme, and found that the absorption and resonance Raman spectra of heme-bound IRP1 were quite similar to those of heme-bound IRP2. This shows that the heme environmental structures in IRP1 are close to those of proteins using heme as a regulatory molecule. Pulse radiolysis experiments, however, clearly revealed an axial ligand exchange from Cys to His immediately after the reduction of the heme iron to form a 5-coordinate His-ligated heme in heme-bound IRP2, whereas the 5-coordinate His-ligated heme was not observed after the reduction of heme-bound IRP1. Considering that the oxidative modification is only observed in heme-bound IRP2, but not IRP1, probably owing to the structural flexibility of IRP2, we propose that the transient 5-coordinate His-ligated heme is a prerequisite for oxidative modification of heme-bound IRP2, which functionally differentiates heme binding of IRP2 from that of IRP1.
铁调节蛋白（IRPs）是哺乳动物细胞中调节铁代谢的调节剂，通过与 mRNAs 中特定的铁反应元件（IRE）序列结合，控制参与铁摄取、储存和利用的蛋白质的翻译。IRPs 的两个同源物（IRP1 和 IRP2）具有典型的血红素调节基序（HRM），这是在“血红素调节蛋白”中发现的一个共识序列。然而，只有 IRP2 被报道具有特定的血红素结合 HRM，这对于氧化修饰和与靶 mRNAs 结合的丧失是必不可少的。在本文中，我们证实 IRP1 也特异性地结合两个摩尔当量的血红素，并发现血红素结合的 IRP1 的吸收和共振拉曼光谱与血红素结合的 IRP2 的非常相似。这表明 IRP1 中的血红素环境结构与将血红素用作调节分子的蛋白质的结构非常相似。然而，脉冲辐射分解实验清楚地表明，在血红素结合的 IRP2 中，血红素铁还原形成 5 配位 His 配体的血红素后，立即从 Cys 到 His 发生轴向配体交换，而在血红素结合的 IRP1 还原后则未观察到 5 配位 His 配体的血红素。考虑到氧化修饰仅在血红素结合的 IRP2 中观察到，而在 IRP1 中未观察到，可能是由于 IRP2 的结构灵活性，我们提出瞬时 5 配位 His 配体的血红素是血红素结合的 IRP2 氧化修饰的前提条件，这使得 IRP2 的血红素结合功能与 IRP1 的不同。
