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

Abstract

Heme d1 plays an important role in denitrification as the essential cofactor of the cytochrome cd1 nitrite reductase NirS. At present, the biosynthesis of heme d1 is only partially understood. The last step of heme d1 biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d1. Instead, the NirS purified from this strain contains the heme d1 precursor dihydro-heme d1 lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d1 was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d1 and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d1.

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

“…The solubilized membrane proteins present in the supernatant were mixed for 1 h at 4°C with nickle-nitrilotriacetic acid (Ni-NTA) agarose previously equilibrated with NPI-10 buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole [Protino]; MachereyNagel GmbH, Düren, Germany). The column was washed with imidazole at increasing concentrations (20,30,40, and 50 mM in NPI-20, NPI-30, NPI-40, and NPI-50 buffers consecutively). Recombinantly tagged NosR, NorC, and NorB proteins, along with their potential interaction partners, were eluted from the column by addition of 300 mM imidazole in NPI-300 buffer.…”

“…NirF is involved in heme d 1 insertion (27). The final step of heme d 1 biosynthesis, localized mainly in the periplasm, involves the uroporphyrin III c-methyltransferase NirE (28), the siroheme decarboxylase NirDL/NirGH (29), and a novel electron-bifurcating dehydrogenase NirN (30). Nitrate from the environment gets transported into the cytoplasm by the nitrate/nitrite antiporter NarK2 and converted by the activity of NarGHI into nitrite, which then returns via NarK2 into the periplasm to serve as the substrate for NirS (31).…”

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

“…Following the decarboxylation reaction, didecarboxysiroheme undergoes a loss of the propionate acid side chains in a reaction catalyzed by NirJ (11), which is a member of the radical SAM family (187). Such a reaction would generate a dihydroheme d 1 molecule, which then has to undergo dehydrogenation of the propionate acid side chain attached to C-17 to give the acrylate functionality and generate heme d 1 .In fact, although the NirJ reaction has not yet been demonstrated, the formation of the double bond on the C-17 propionate has been shown to be mediated by NirN within the periplasm (188,189). This was demonstrated when a NirN knockout gave rise to a NirS enzyme containing dihydro-heme d 1 .…”

“…This was demonstrated when a NirN knockout gave rise to a NirS enzyme containing dihydro-heme d 1 . Moreover, NirN was then shown to convert dihydro-heme d 1 into heme d 1 , most likely by employing an electron bifurcation mechanism for this 2-electron oxidation, utilizing both the cytochrome c prosthetic group and the heme d 1 product (188). The role of NirF in this biosynthetic process is still to be elucidated, but interestingly, it too is located within the periplasm (189,190).…”

Bacterial Electron Transfer Chains Primed by Proteomics

“…All in all, enzyme maturation is highly complex, wherein each RMP depends on its dedicated set of assembly factors acting in concert. Assembly processes of the major RMPs, including quinol: cytochrome c oxidoreductases (Hasan, Proctor, Yamashita, Dokholyan, & Cramer, 2014;Hasan, Yamashita, & Cramer, 2013;Smith, Fox, & Winge, 2012), terminal oxidases (Bühler et al, 2010;Ekici, Pawlik, Lohmeyer, Koch, & Daldal, 2012;Gurumoorthy & Ludwig, 2015), nitrite-/nitric oxide-/nitrous oxide reductases (Adamczack et al, 2014;Barth, Isabella, & Clark, 2009;Hatzixanthis, Richardson, & Sargent, 2005;Nicke et al, 2013;Spiro, 2012), hydrogenases (Peters et al, 2015), formate dehydrogenases (Hartmann, Schwanhold, & Leimkühler, 2015) and other molybdopterin-containing RMPs (Arnoux et al, 2015;Magalon & Mendel, 2015), have been studied in more or less detail. Notoriously, the assembly of complex I with its about 45 subunits in unicellular and higher eukaryotes is puzzling (Mimaki et al, 2012;Vartak et al, 2014;Vogel, Smeitink, & Nijtmans, 2007).…”