5 research outputs found
The last step of nitrite reductase cofactor Heme d1 biosynthesis in Pseudomonas aeruginosa
In dieser Arbeit wurde der letzte Schritt der Biosynthese des Nitritreduktase Kofaktors HÀm d1 in P. aeruginosa untersucht. Der biosynthetische VorlÀufer von HÀm d1, DihydrohÀm d1, wurde zum ersten Mal aus der nativ gereinigten Nitritreduktase NirS aus dem P. aeruginosa Stamm RM361 (nirN::tet) isoliert und charakterisiert. Weiterhin wurde gezeigt, dass das periplasmatische Protein NirN die Umsetzung von DihydrohÀm d1 zu HÀm d1 katalysiert, wohingegen NirF sowohl DihydrohÀm d1 als auch HÀm d1 bindet, diese aber nicht weiter umsetzt.
FĂŒr die Isolierung von DihydrohĂ€m d1 wurde zunĂ€chst ein geeignetes Produktions- und Reinigungsprotokoll fĂŒr die native Nitritreduktase NirS aus dem P. aeruginosa Stamm RM361 etabliert. Das proteingebundene DihydrohĂ€m d1 wurde mittels UV/Vis- und Resonanz-Raman Spektroskopie genauer untersucht. ZusĂ€tzlich wurde DihydrohĂ€m d1 aus der gereinigten NirS extrahiert und mittels UV-Vis Spektroskopie, HPLC und Massenspektrometrie charakterisiert. Dabei wurde der Kofaktor zweifelsfrei als DihydrohĂ€m d1 identifiziert. Um zu zeigen, dass NirN den letzten Schritt der HĂ€m d1 Biosynthese katalysiert, wurde ein EnzymaktivitĂ€tsassay mit DihydrohĂ€m d1 als Substrat etabliert. NirN war dabei in der Lage, DihydrohĂ€m d1 zu HĂ€m d1 umzusetzen. Die DurchfĂŒhrung des AktivitĂ€tsassays unter anaeroben Bedingungen zeigte, dass der NirN gebundene HĂ€m c Kofaktor wĂ€hrend der Katalyse reduziert wird. Eine NirN Variante, die keine N-terminale DomĂ€ne und somit keinen HĂ€m c Kofaktor enthielt, war komplett inaktiv. Mit Hilfe von Zyklovoltammetrie wurden die Redoxpotentiale des NirN gebundenen HĂ€m c Kofaktors, sowie die von HĂ€m d1 und DihydrohĂ€m d1 bestimmt.
Das Produktions- und Reinigungsprotokoll fĂŒr NirF aus D. shibae wurde optimiert. Es wurde auĂerdem mittels UV-Vis Spektroskopie gezeigt, dass NirF in der Lage ist, HĂ€m d1 und DihydrohĂ€m d1 zu binden. Die vorliegenden Ergebnisse zeigen jedoch, dass NirF nicht in der Lage ist, DihydrohĂ€m d1 umzusetzen. Durch Kristallisations-experimente konnten Kristalle von NirN und NirF erzeugt werden. Jedoch war es nicht möglich, DatensĂ€tze von den Kristallen aufzunehmen. Anhand der in dieser Arbeit gewonnenen Erkenntnisse konnte das Maturationsmodell der Nitritreduktase NirS in P. aeruginosa verfeinert werden.Heme d1 is the unique cofactor of the nitrite reductase which catalyzes the second step of denitrification. This work aimed to elucidate the last enzymatic step in heme d1 biosynthesis. The biosynthetic heme d1 precursor, dihydroheme d1 was isolated for the first time from the P. aeruginosa mutant RM361 (nirN::tet). It was shown that the periplasmatic potein NirN catalyzes the conversion of dihydroheme d1 to heme d1. NirF binds heme d1 as well as dihydroheme d1 but does not convert the latter to heme d1.
The nitrite reductase NirS was isolated from the P. aeruginosa strain RM361 using a novel protocol. The NirS bound dihydroheme d1 was characterized using UV-Vis spectroscopy as well as Resonance Raman Spectroscopy. Dihydroheme d1 was extracted from NirS and characterized using UV-Vis spectroscopy, HPLC and masspectrometry. Using these methods, the NirS cofactor from the P. aeruginosa mutant RM361 could be identified as the heme d1 precursor dihydroheme d1. NirN was shown to generate heme d1 in an enzymatic activity assay using dihydroheme d1 as a substrate. By perfoming the activity assay under anaerobic conditions, it was shown that the NirN bound heme c cofactor is reduced during the conversion of dihydroheme d1 to heme d1. A NirN variant lacking the heme c binding domain was completely inactive. Using protein film voltammetry, the reduction potential of the NirN bound heme c cofactor could be determined. The results suggest that NirN is an electron bifurcating dehydrogenase that forms its product in the reduced state and transfers the other electron from the reaction to its bound heme c cofactor.
Moreover, the production and purification of NirF from D. shibae was improved. NirF was shown to bind heme d1 and dihydroheme d1. However, NirF was not able to convert dihydroheme d1 to heme d1. Crystals of NirN and NirF could be obtained, yet the crystal structure of both proteins could not be solved. The model for nitrite reductase maturation could be refined using the insights gained from this work
Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF
Crystal Structure of Dihydro-Heme d Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis.
Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called "denitrification" and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd1 nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d1 as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d1. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d1-binding ÎČ-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d1 at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417
Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF.
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 ÎnirN 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
NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis
Heme d(1) plays an important role in denitrification as the essential cofactor of the cytochrome cd(1) nitrite reductase NirS. At present, the biosynthesis of heme d(1) is only partially understood. The last step of heme d(1) 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 d(1). Instead, the NirS purified from this strain contains the heme d(1) precursor dihydro-heme d(1) lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d(1) 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 d(1) 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 d(1)