The nifB gene has been long recognized as crucial for nitrogen fixation because nifB participates in an early synthetic step that is common to the biosyntheses of FeMo-cofactor. It utilizes two equivalents of SAM to insert a carbide atom and fuse two substrate iron-sulfur clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron-molybdenum cofactor (FeMo-co) of nitrogenase.
Using electron paramagnetic resonance spectroscopy Wilcoxen et al. have characterized three [4Fe-4S] clusters, one SAM binding cluster with the canonical radical SAM motif (CxxxCxxC) and two auxiliary clusters probably which probable act as substrates for NifB-co formation. Further, nitrogen coordination to one or more of the auxiliary clusters in NifB was observed.
Curatti L, Ludden PW, Rubio LM
NifB-dependent in vitro synthesis of the iron-molybdenum cofactor of nitrogenase
▸ Abstract
Biological nitrogen fixation, an essential process of the biogeochemical nitrogen cycle that supports life on Earth, is catalyzed by the nitrogenase enzyme. The nitrogenase active site contains an iron and molybdenum cofactor (FeMo-co) composed of 7Fe-9S-Mo-homocitrate and one not-yet-identified atom, which probably is the most complex [Fe-S] cluster in nature. Here, we show the in vitro synthesis of FeMo-co from its simple constituents, Fe, S, Mo, and homocitrate. The in vitro FeMo-co synthesis requires purified NifB and depends on S-adenosylmethionine, indicating that radical chemistry is required during FeMo-co assembly.
Proc Natl Acad Sci U S A.
2006;103(14):5297-5301
| PubMed ID:
16567617
Jared A. Wiig, Yilin Hu, Chi Chung Lee, Markus W. Ribbe
Radical SAM-Dependent Carbon Insertion into the Nitrogenase M-Cluster
▸ Abstract
The active site of nitrogenase, the M-cluster, is a metal-sulfur cluster containing a carbide at its core. Using radiolabeling experiments, we show that this carbide originates from the methyl group of S-adenosylmethionine (SAM) and that it is inserted into the M-cluster by the assembly protein NifB. Our SAM cleavage and deuterium substitution analyses suggest a similarity between the mechanism of carbon insertion by NifB and the proposed mechanism of RNA methylation by the radical SAM enzymes RlmN and Cfr, which involves methyl transfer from one SAM equivalent, followed by hydrogen atom abstraction from the methyl group by a 5′-deoxyadenosyl radical generated from a second SAM equivalent. This work is an initial step toward unraveling the importance of the interstitial carbide and providing insights into the nitrogenase mechanism.
Science
2012;337(6102):1672-1675
| PubMed ID:
23019652
Wiig JA, Hu Y, Ribbe MW
Refining the pathway of carbide insertion into the nitrogenase M-cluster
▸ Abstract
Carbide insertion plays a pivotal role in the biosynthesis of M-cluster, the cofactor of nitrogenase. Previously, we proposed a carbide insertion pathway involving methyltransfer from SAM to a FeS precursor and hydrogen abstraction from this methyl group that initiates the radical-based precursor maturation. Here we demonstrate that the methyl group is transferred to a precursor-associated sulfur before hydrogen abstraction, thereby refining the initial steps of the carbide insertion pathway.
Nat Commun
2015;6(None):803-8034
| PubMed ID:
26259825
Wilcoxen J, Arragain S, Scandurra AA, Jimenez-Vicente E, Echavarri-Erasun C, Pollmann S, Britt RD, Rubio LM
Electron Paramagnetic Resonance Characterization of Three Iron-Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB from Methanocaldococcus infernus
▸ Abstract
NifB utilizes two equivalents of SAM to insert a carbide atom and fuse two substrate [Fe-S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron-molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen Methanocaldococcus infernus is a radical SAM enzyme able to reductively cleave SAM to 5'-deoxyadenosine radical and is competent in FeMo-co matu-ration. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe-4S] clusters, one SAM binding cluster and two auxiliary clusters probably acting as substrates for NifB-co formation. Nitrogen coordination to one or more of the auxiliary clusters in NifB was observed, and its mechanistic implications for NifB-co dissociation from the maturase are discussed.
J Am Chem Soc
2016;None(None):None-None
| PubMed ID:
27268267
Hu Y, Ribbe MW
Biosynthesis of the Metalloclusters of Nitrogenases
▸ Abstract
Nitrogenase is a versatile metalloenzyme that is capable of catalyzing two important reactions under ambient conditions: the reduction of nitrogen (N2) to ammonia (NH3), a key step in the global nitrogen cycle; and the reduction of carbon monoxide (CO) and carbon dioxide (CO2) to hydrocarbons, two reactions useful for recycling carbon waste into carbon fuel. The molybdenum (Mo)- and vanadium (V)-nitrogenases are two homologous members of this enzyme family. Each of them contains a P-cluster and a cofactor, two high-nuclearity metalloclusters that have crucial roles in catalysis. This review summarizes the progress that has been made in elucidating the biosynthetic mechanisms of the P-cluster and cofactor species of nitrogenase, focusing on what is known about the assembly mechanisms of the two metalloclusters in Mo-nitrogenase and giving a brief account of the possible assembly schemes of their counterparts in V-nitrogenase, which are derived from the homology between the two nitrogenases.
Annu Rev Biochem
2016;85(None):455-483
| PubMed ID:
26844394
Guo Y, Echavarri-Erasun C, Demuez M, Jiménez-Vicente E, Bominaar EL, Rubio LM
The Nitrogenase FeMo-Cofactor Precursor Formed by NifB Protein: A Diamagnetic Cluster Containing Eight Iron Atoms
▸ Abstract
The biological activation of N2 occurs at the FeMo-cofactor, a 7Fe-9S-Mo-C-homocitrate cluster. FeMo-cofactor formation involves assembly of a Fe6-8 -SX -C core precursor, NifB-co, which occurs on the NifB protein. Characterization of NifB-co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe-4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB-co and deliver it to the NifEN protein, upon which FeMo-cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB-co-loaded form provided an opportunity for Mössbauer analysis of NifB-co. The results indicate that NifB-co is a diamagnetic (S=0) 8-Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the (57) Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB-co is either a 4Fe(2+) -4Fe(3+) or 6Fe(2+) -2Fe(3+) cluster having valence-delocalized states.
Angew Chem Int Ed Engl
2016;55(41):12764-12767
| PubMed ID:
27611968
