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

血红素调节蛋白的结构、功能及其分子机制

基本信息

批准号：
15350101
负责人：
ISHIMORI Koichiro
金额：
$ 9.28万
依托单位：
Hokkaido University (2005)Kyoto University (2003-2004)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

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.

本研究项目取得的主要成果如下： 1． Irr 和 IRP2 中半胱氨酸与铁血红素的连接。基于共振拉曼光谱，我们成功识别了铁血红素结合的 Irr 和 IRP2 中的 Fe-Cys 伸缩模式。与传统的 Cys 连接血红蛋白（例如 P450cam）相比，这些 Fe-Cys 拉伸模式降低了。下移的 Fe-Cys 拉伸模式对应于这些蛋白质对铁血红素的较低亲和力，Irr 中的荧光血红素滴定也支持这一点。这些蛋白质与血红素的如此弱的亲和力将是具有“血红素调节基序”2的血红素调节蛋白质的特征之一。轴配体的氧化还原依赖性替换。我们证实了Irr和IRP2中半胱氨酸与铁血红素的连接。然而，血红素铁的还原，血红素结合的 Irr 和 IRP2 的吸收光谱发生了巨大的变化，所得光谱与亚铁有很大不同。 P450cam，与双组氨酸连接的血红素蛋白（如细胞色素 b_5）非常相似，与血红素结合的 Irr 和 IRP2 的 CO 加合物的光谱相似性更明显。共振拉曼测量清楚地显示了 Fe-His。亚铁血红素结合的 lrr 和 IRP2 中的拉伸模式以及 CO 加合物中的 Fe-C 和 FeC-O 拉伸模式，证实轴向 Cys 被替换为考虑到分子氧可以与亚铁血红素结合，而不是铁血红素，这些蛋白质中的组氨酸连接物种将是活性物种，产生活性氧物种，从而导致肽和蛋白质的氧化修饰。降解。