Insertion of tetrapyrrole cofactors into cytochrome cd1 nitrite reductase

周质细胞色素cd 1亚硝酸盐还原酶（NirS）催化反硝化过程中的第二步含有血红素c和血红素d1作为必要的辅基。而血红素c是通过细胞色素c成熟系统的作用共价连接到蛋白质上的，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周质NirF催化。周质血红素d1结合蛋白NirN被提议从NirF中摄取辅因子并将其插入到NirS中。到目前为止，我们可以表明，来自铜绿假单胞菌的NirF是一种膜锚定脂蛋白，并且三种蛋白质NirF，NirN和NirS在体内相互作用。在这个项目中，我们希望详细研究血红素d1从最后的生物合成酶NirF转移到血红素d1伴侣NirN，以及通过NirN的作用将血红素d1插入到NirS中。这些研究包括（1）详细分析血红素d1与NirN的结合及其所涉及的氨基酸残基;（2）研究NirF、NirN和NirS与潜在的额外蛋白质之间的相互作用网络;（3）研究NirN中血红素c对血红素d1转移的作用;（4）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.