Insertion of tetrapyrrole cofactors into cytochrome cd1 nitrite reductase

在反硝化过程中催化第二步反应的胞浆细胞色素cd1亚硝酸盐还原酶（NirS）含有血红素c和血红素d1作为必需的假基。虽然血红素c通过细胞色素c成熟系统的作用与蛋白质共价结合，但血红素d1插入的过程几乎完全未知。在我们的工作模型中，血红素d1生物合成的最后一步可能由质周NirF催化。建议外质血红素d1结合蛋白NirN从NirF中吸收辅因子并将其插入近红外光谱中。到目前为止，我们可以证明来自铜绿假单胞菌的NirF是一种膜锚定脂蛋白，并且三种蛋白NirF、NirN和NirS在体内相互作用。在本项目中，我们将详细研究血红素d1从最后一个生物合成酶NirF转移到血红素d1的伴侣蛋白NirN上，并通过NirN的作用将血红素d1插入到近红外光谱中。这些研究包括：(1)详细分析血红素d1与NirN的结合及其相关氨基酸残基；(2)研究NirF、NirN和NirS以及潜在附加蛋白之间的相互作用网络；(3)研究血红素c在NirN中对血红素d1转移的作用；(4)研究NirN和NirF的结晶和x射线结构测定。

The periplasmic cytochrome cd1 nitrite reductase (NirS) which catalyzes the second step during denitrification contains heme c and heme d1 as essential prosthetic groups. Whereas the heme c is covalently attached to the protein by the action of the cytochrome c maturation system, the process of heme d1 insertion is almost completely unknown. In our working model the last step of heme d1 biosynthesis is potentially catalyzed by periplasmic NirF. The periplasmic, heme d1-binding protein NirN is proposed to take up the cofactor from NirF and to insert it into NirS. So far, we could show that NirF from Pseudomonas aeruginosa is a membrane-anchored lipoprotein and that the three proteins NirF, NirN and NirS interact with each other in vivo. In this project we want to study in detail the transfer of heme d1 from the last biosynthesis enzyme NirF to the heme d1 chaperone NirN and the insertion of heme d1 into NirS by the action of NirN. These studies include (1) the detailed analysis of heme d1 binding to NirN and the involved amino acid residues, (2) the investigation of the interaction network between NirF, NirN and NirS and potential additional proteins, (3) the investigation of the role of the heme c in NirN for heme d1 transfer and (4) the crystallization and X-ray structure determination of NirN and NirF.