title: Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF.
abstract: The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS-NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV-visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS.
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Tristan
authors.initials: T
authors.lastname: Nicke
authors.forename: Tobias
authors.initials: T
authors.lastname: Schnitzer
authors.forename: Karin
authors.initials: K
authors.lastname: Münch
authors.forename: Julia
authors.initials: J
authors.lastname: Adamczack
authors.forename: Kristin
authors.initials: K
authors.lastname: Haufschildt
authors.forename: Sabine
authors.initials: S
authors.lastname: Buchmeier
authors.forename: Martin
authors.initials: M
authors.lastname: Kucklick
authors.forename: Undine
authors.initials: U
authors.lastname: Felgenträger
authors.forename: Lothar
authors.initials: L
authors.lastname: Jänsch
authors.forename: Katharina
authors.initials: K
authors.lastname: Riedel
authors.forename: Gunhild
authors.initials: G
authors.lastname: Layer
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D003580:Cytochromes / Q000032:analysis / Q000378:metabolism*; D058440:Denitrification; D006418:Heme / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D009572:Nitrite Reductases / Q000032:analysis / Q000378:metabolism*; D060066:Protein Interaction Maps; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D045725:Tetrapyrroles / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003580:Cytochromes; D045725:Tetrapyrroles; D006418:Heme; C029400:heme d1; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming)