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

本研究取得的主要成果如下：1.IRR和IRP2中半胱氨酸与亚铁血红素的连接。基于共振拉曼光谱，我们成功地确定了铁血红素结合的IRR和IRP2中的Fe-Cys伸缩模式。与P450cam等传统的Cys连接的血红蛋白相比，这些Fe-Cys伸展模式发生了下移。铁-半胱氨酸伸展模式的下移对应于这些蛋白质与铁血红素的低亲和力，这也得到了IRR中的荧光血红素滴定的支持。这些蛋白质与血红素的亲和力很弱，这将是具有“血红素调节基序‘2.依赖氧化还原的轴向配体替换”的血红素调节蛋白质的特征之一。我们证实了IRR和IRP2中半胱氨酸与高铁血红素的连接。然而，通过还原血红素铁，血红素结合的IRR和IRP2的吸收光谱发生了很大的变化，产生的光谱与亚铁P450cam有很大的不同，后者与双His连接的血红素蛋白如细胞色素b_5有很大的相似之处。共振拉曼光谱测量。清晰地显示了亚铁血红素结合的LRR和IRP2中的Fe-His伸缩模式和CO加合物中的Fe-C和FeC-O伸缩模式，证实了轴向Cys被His取代是由于血红素铁的还原。考虑到分子氧可以与亚铁血红素结合，而不是与铁血红素结合，这些蛋白质中的组氨酸连接物种可能是产生活性氧物种的活性物种，从而导致多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.