Structure and Function of Heme-regulated Proteins and Their Molecular Mechanisms

在这个研究项目中，我们获得的主要成果如下：1。Irr和IRP2中Cys与铁血红素的连接。基于共振拉曼光谱，我们成功地鉴定了铁血红素结合Irr和IRP2中的Fe-Cys拉伸模式。与传统的cys连接的血红蛋白（如P450cam）相比，Fe-Cys的拉伸模式被降低了。下移的Fe-Cys拉伸模式对应于这些蛋白质对铁血红素的亲和力较低，这也得到了Irr中荧光血红素滴定的支持。这些蛋白对血红素的弱亲和力可能是具有“血红素调控Motif”2的血红素调控蛋白的特征之一。轴向配体的氧化还原依赖性取代。我们在Irr和IRP2中证实了Cys与铁血红素的连接。然而，血红素铁的还原使血红素结合的Irr和IRP2的吸收光谱发生了巨大的变化，产生的光谱与亚铁P450cam有很大的不同，而与双his连接的血红蛋白如细胞色素b_5非常相似。血红素结合的Irr和IRP2的CO加合物与his连接的血红蛋白的光谱相似性更为明显。共振拉曼测量。在铁血红素结合的lrr和IRP2中清晰地显示出Fe-His的拉伸模式，在CO加合物中显示出Fe-C和fe - o的拉伸模式，证实了通过血红素铁的还原，轴向Cys被His取代。考虑到分子氧可以与亚铁血红素结合，而不能与铁血红素结合，因此这些蛋白质中的His结合种将是活性种，产生活性氧，从而导致肽的氧化修饰和蛋白质的降解。

The major results we have obtained in this research project are as follows:1. Ligation of Cys to Ferric Heme in Irr and IRP2. Based on the resonance Raman spectra, we successfully identified the Fe-Cys stretching modes in ferric heme-bound Irr and IRP2. These Fe-Cys stretching modes were downshifted, compared with that in conventional Cys-ligated hemoproteins such as P450cam. The downshifted Fe-Cys stretching modes correspond to the lower affinities of these proteins to ferric heme, which is also supported by fluorescence heme titration in Irr. Such weak affinities of these proteins to heme would be one of the characteristics of heme-regulated proteins having "Heme Regulatory Motif'2. Redox-dependent Replacement of Axial Ligands. We confirmed the ligation of Cys to ferric heme in Irr and IRP2. By reduction of the heme iron, however, the absorption spectra of heme-bound Irr and IRP2 were drastically changed and the resultant spectra were quite different from ferrous P450cam, which were rather similar to bis-His ligated hemoproteins like cytochrome b_5. The spectral similarity to His-ligated hemoprotein was more evident in the CO adducts of heme-bound Irr and IRP2. The resonance Raman measurements. clearly showed the Fe-His stretching modes in ferrous heme bound lrr and IRP2 and the Fe-C and FeC-O stretching modes in the CO adducts, confirming that axial Cys is replaced with His by reduction of the heme iron. Considering that molecular oxygen can bind to ferrous heme, not ferric heme, the His ligated species in these proteins would be active species to generate reactive oxygen species, which leads to the oxidative modification of the peptide and protein degradation.