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.

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

“…For the final insertion of heme d 1 into NirS, the system uses NirN, a structural homologue of NirS without nitrite reductase activity. NirS, NirF, and NirN form a stable complex during nitrite reductase maturation (26). In agreement, protein interactions of NorB and NorC with NirN were detected in our study (Table 3).…”

“…The strongest interaction of NorBC (bait) for an isolated prey protein was detected for the periplasmic, membrane-attached lipoprotein NirF, the last enzyme in heme d 1 synthesis and a cofactor of nitrite reductases (26). For the final insertion of heme d 1 into NirS, the system uses NirN, a structural homologue of NirS without nitrite reductase activity.…”

“…The two c-type cytochromes NirM and NirC act as electron donors (25). NirN and NirF form a stable complex with the nitrite reductase NirS during enzyme maturation (26). NirF is involved in heme d 1 insertion (27).…”

NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis

“…In line with this proposal it has been shown that NirN from P. aeruginosa interacts with both NirS and NirF in vivo. However, in that study it was also observed by UV-visible absorption spectroscopy of periplasmic protein fractions that the cofactor content of NirS in the P. aeruginosa ⌬nirN strain was different from that in the P. aeruginosa WT strain (15). Later, the same observation was reported for a ⌬nirN strain of Magnetospirillum gryphiswaldense (17).…”

“…Depending on the organism, NirF is either a soluble periplasmic protein or attached to the outer face of the cytoplasmic membrane, most likely by a lipid anchor (14,15). In addition to its periplasmic location, purified recombinant NirF from Paracoccus pantotrophus has been shown to bind heme d 1 in vitro (13).…”