title: 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.
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Adamczack
authors.affiliation: Institute of Inorganic and Analytical Chemistry and Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Hoffmann
authors.affiliation: Institute of Organic Chemistry, Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany; and.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Papke
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Haufschildt
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Tristan
authors.initials: T
authors.lastname: Nicke
authors.affiliation: Institute of Inorganic and Analytical Chemistry and Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Bröring
authors.affiliation: Institute of Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany.
authors.forename: Murat
authors.initials: M
authors.lastname: Sezer
authors.affiliation: Institute of Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Weimar
authors.affiliation: Institute of Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Kuhlmann
authors.affiliation: Institute of Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany;. Electronic address: g.layer@tu-braunschweig.de.
authors.forename: Gunhild
authors.initials: G
authors.lastname: Layer
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry*; D006418:Heme / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013059:Spectrum Analysis, Raman
chemical_list: D001426:Bacterial Proteins; D006418:Heme; C029400:heme d1; D010088:Oxidoreductases
keywords: Cytochrome c; Dehydrogenase; Denitrification; Enzyme Catalysis; Enzyme Mechanism; Heme; Heme d1; NirN