The active site of nitrogenase, 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 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 significance of the interstitial carbide and providing insights into the nitrogenase mechanism.

NifEN plays an essential role in the biosynthesis of the nitrogenase FeMo cofactor (M-cluster). It is an α2β2 tetramer that is homologous to the catalytic MoFe protein (NifDK) component of nitrogenase. NifEN serves as a scaffold for the conversion of an iron-only precursor to a matured form of the M-cluster before delivering the latter to its target location within NifDK. Here, we present the structure of the precursor-bound NifEN of Azotobacter vinelandii at 2.6 Å resolution. From a structural comparison of NifEN with des-M-cluster NifDK and holo NifDK, we propose similar pathways of cluster insertion for the homologous NifEN and NifDK proteins.

Assembly of nitrogenase FeMoco is one of the key processes in bioinorganic chemistry. NifB and NifEN are two essential elements immediately adjacent to each other along the biosynthetic pathway of FeMoco. Previously, an 8Fe-precursor of FeMoco was identified on NifEN; however, the identity of the biosynthetic intermediate on NifB has remained elusive to date. Here, we present a combined biochemical and spectroscopic investigation of a His-tagged NifEN-B fusion protein of Azotobacter vinelandii. Our data from the EPR and activity analyses confirm the presence of the 8Fe-precursor in the NifEN entity of NifEN-B; whereas those from the metal, EPR, and UV/Vis experiments reveal the presence of additional ½Fe 4 S 4 -type cluster species in the NifB entity of NifEN-B. EPR-, UV/Vis-and metal-based quantitative analyses suggest that the newly identified cluster species in NifEN-B consist of both SAM-motif (CXXXCXXC)-and non-SAM-motif-bound ½Fe 4 S 4 -type clusters. Moreover, EPR and activity experiments indicate that the non-SAM-motif ½Fe 4 S 4 cluster is a NifB-bound intermediate of FeMoco assembly, which could be converted to the 8Fe-precursor in a SAM-dependent mechanism. Combined outcome of this work provides the initial insights into the biosynthetic events of FeMoco on NifB. More importantly, the full capacity of NifEN-B in FeMoco biosynthesis demonstrates the potential of this fusion protein as an excellent platform for further investigations of the role of NifB and its interaction with NifEN during the process of FeMoco assembly.

Nitrogenase biosynthesis protein NifB catalyzes the radical S-adenosyl-L-methionine (SAM)-dependent insertion of carbide into the M cluster, the cofactor of the molybdenum nitrogenase from Azotobacter vinelandii. Here, we report the identification and characterization of two naturally "truncated" homologs of NifB from Methanosarcina acetivorans (NifB Ma ) and Methanobacterium thermoautotrophicum (NifB Mt ), which contain a SAM-binding domain at the N terminus but lack a domain toward the C terminus that shares homology with NifX, an accessory protein in M cluster biosynthesis. NifB Ma and NifB Mt are monomeric proteins containing a SAM-binding [Fe 4 S 4 ] cluster (designated the SAM cluster) and a [Fe 4 S 4 ]-like cluster pair (designated the K cluster) that can be processed into an [Fe 8 S 9 ] precursor to the M cluster (designated the L cluster). Further, the K clusters in NifB Ma and NifB Mt can be converted to L clusters upon addition of SAM, which corresponds to their ability to heterologously donate L clusters to the biosynthetic machinery of A. vinelandii for further maturation into the M clusters. Perhaps even more excitingly, NifB Ma and NifB Mt can catalyze the removal of methyl group from SAM and the abstraction of hydrogen from this methyl group by 5′-deoxyadenosyl radical that initiates the radical-based incorporation of methyl-derived carbide into the M cluster. The successful identification of NifB Ma and NifB Mt as functional homologs of NifB not only enabled classification of a new subset of radical SAM methyltransferases that specialize in complex metallocluster assembly, but also provided a new tool for further characterization of the distinctive, NifB-catalyzed methyl transfer and conversion to an iron-bound carbide.nitrogenase | NifB | methanogens | radical SAM | homologs N itrogenase biosynthesis protein NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that plays an essential role in the biosynthesis of the M cluster, a [MoFe 8 S 9 C-homocitrate] cluster that serves as the cofactor of the molybdenum (Mo) nitrogenase from Azotobacter vinelandii (1-7). Carrying a signature CxxxCxxC motif at its N terminus that houses the SAM-binding [Fe 4 S 4 ] cluster (designated the SAM cluster), NifB also contains a number of additional ligands that could accommodate coordination of the entire complement of iron (Fe) atoms of the M cluster (Fig. S1). Moreover, it shares sequence homology with NifX, an accessory protein in M-cluster biosynthesis (8), toward its C terminus (Fig. S1). Characterization of the NifB protein from A. vinelandii had long been hampered by the instability of NifB in aqueous solutions until this protein was expressed as part of a NifEN-B fusion protein, wherein NifB was fused with and protected by NifEN, the biosynthetic apparatus immediately downstream of NifB along the M-cluster assembly pathway (9). Expression of the NifEN-B fusion protein in A. vinelendii was "modeled" after a naturally occurring NifEN-B fusion protein in Clostridium pasteurianum, which has the N terminus o...

Mo-nitrogenase catalyzes the reduction of dinitrogen to ammonia at the cofactor (i.e., FeMoco) site of its MoFe protein component. Biosynthesis of FeMoco involves NifEN, a scaffold protein that hosts the maturation of a precursor to a mature FeMoco before it is delivered to the target location in the MoFe protein. Previously, we have shown that the NifEN-bound precursor could be converted, in vitro, to a fully complemented “FeMoco” in the presence of 2 mM dithionite. However, such a conversion was incomplete and Mo was only loosely associated to the NifEN-bound “FeMoco”. Here we report the optimized maturation of NifEN-associated precursor in 20 mM dithionite. Activity analyses show that, upon the optimal conversion of precursor to “FeMoco”, NifEN is capable of activating a FeMoco-deficient form of MoFe protein to the same extent as the isolated FeMoco. Further, EPR and XAS/EXAFS analyses reveal the presence of a tightly organized Mo site in NifEN-bound “FeMoco”, which allows the observation of a FeMoco-like, S = 3/2 EPR signal and the modeling of a NifEN-bound “FeMoco” that adopts a very similar conformation to that of the MoFe protein-associated FeMoco. The sensitivity of FeMoco maturation to dithionite concentration suggests an essential role of redox chemistry in this process, and the optimal potential of dithionite solution could serve as a guideline for future identification of in vivo electron donors for FeMoco maturation.

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 prior to hydrogen abstraction, thereby refining the initial steps of the carbide insertion pathway.

NifEN is a key player in the biosynthesis

Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are Class I enzymes, structural studies to date have focused primarily on the Class II KARIs, which arose through domain duplication. Here, we present five new crystal structures of Class I KARIs. These include the first structure of a KARI with a 6-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate binding geometry distinct from that of the 7- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in Class I KARIs upon binding of cofactor and metal ions. The Class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the Class II KARIs of rice and spinach and different from the opening of the active site observed in the Class II KARI of E. coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide binding site.