Insertion of tetrapyrrole cofactors into cytochrome cd1 nitrite reductase

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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.