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

本课题的主要研究成果如下：1. Irr和IRP 2中Cys与铁血红素的连接。基于共振拉曼光谱，我们成功地确定了铁血红素结合Irr和IRP 2的Fe-Cys伸缩模式。这些Fe-Cys的伸缩模式，与传统的半胱氨酸连接的血红素蛋白，如P450 cam相比，下移。下移的Fe-Cys伸缩模式对应于这些蛋白质对铁血红素的较低亲和力，这也得到了Irr中荧光血红素滴定的支持。这些蛋白质对血红素的这种弱亲和力将是具有“血红素调节基序”的血红素调节蛋白质的特征之一2。轴向配体的氧化还原依赖性置换。我们证实了Irr和IRP 2中Cys与铁血红素的连接。然而，通过还原血红素铁，血红素结合的Irr和IRP 2的吸收光谱发生了急剧变化，所得光谱与亚铁P450 cam有很大的不同，亚铁P450 cam与bis-His连接的血红素蛋白如细胞色素b_5有很大的相似性。光谱的相似性，以他的连接血红素蛋白是更明显的血红素结合Irr和IRP 2的CO加合物。共振拉曼测量。清楚地显示亚铁血红素结合的Irr和IRP 2中的Fe-His伸缩模式以及CO加合物中的Fe-C和FeC-O伸缩模式，证实轴向Cys通过血红素铁的还原被His取代。考虑到分子氧可以结合亚铁血红素，而不是铁血红素，这些蛋白质中的His连接物种将是产生活性氧的活性物种，这导致肽的氧化修饰和蛋白质降解。

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.