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（IRP）是哺乳动物细胞中铁代谢的调节剂，通过与mRNA的特定铁反应元件（IRE）序列结合，控制参与铁吸收，存储和利用的蛋白质的翻译。IRP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”中发现的共有序列。然而，仅对IRP2报道了与HRM的特异性血红素结合，这对于氧化修饰和与靶mRNA的结合丧失是必不可少的。在本文中，我们确认了IRP1也特异性结合了两个摩尔当量的血红素，并且发现血红素结合的IRP1的吸收和共振拉曼光谱与血红素结合的IRP2相当。这表明IRP1中的血红素环境结构与使用血红素作为调节分子的蛋白质的环境结构接近。然而，脉冲放射分解实验清楚地表明，在血红素铁还原后，在血红素结合的IRP2中形成了5配位的His-连接的血红素，而Cys向His的轴向配体交换则是，而5配位的His-连接的血红素是血红素结合的IRP1减少后未观察到。考虑到氧化修饰仅在血红素结合的IRP2中观察到，而不是在IRP1中观察到，可能是由于IRP2的结构灵活性，我们建议瞬时5坐标His连接的血红素是血红素结合的IRP2氧化修饰的先决条件，在功能上将IRP2的血红素结合与IRP1的血红素结合区分开。清楚地表明，在血红素铁还原后立即从Cys到His进行轴向配体交换，从而在结合血红素的IRP2中形成5配位的His连接的血红素，而在5配位的Hesp连接的血红素还原后未观察到5配位的His连接的血红素。血红素结合的IRP1。考虑到氧化修饰仅在血红素结合的IRP2中观察到，而不是在IRP1中观察到，可能是由于IRP2的结构灵活性，我们建议瞬时5坐标His连接的血红素是血红素结合的IRP2氧化修饰的先决条件，在功能上将IRP2的血红素结合与IRP1的血红素结合区分开。清楚地表明，在血红素铁还原后立即从Cys到His进行轴向配体交换，从而在结合血红素的IRP2中形成5配位的His连接的血红素，而在5配位的Hesp连接的血红素还原后未观察到5配位的His连接的血红素。血红素结合的IRP1。考虑到氧化修饰仅在血红素结合的IRP2中观察到，而不是在IRP1中观察到，可能是由于IRP2的结构灵活性，我们建议瞬时5坐标His连接的血红素是血红素结合的IRP2氧化修饰的先决条件，在功能上将IRP2的血红素结合与IRP1的血红素结合区分开。