Jared A. Wiig

Radical SAM-Dependent Carbon Insertion into the Nitrogenase M-Cluster

A Radical Mechanism The bacterial enzyme nitrogenase plays a key role in the global nitrogen cycle by catalyzing the reduction of N2 to ammonia. At the heart of the enzyme is a metal-sulfur cluster that contains an interstitial light atom recently identified as a carbide. The identification raised questions concerning the role of the carbide in the enzyme mechanism and how it is inserted into the metal cluster. Wiig et al. (p. 1672; see the Perspective by Boal and Rosenzweig) now show that the carbide derives from S-adenosylmethionine (SAM) and is inserted into the core by the radical SAM enzyme NifB. The carbon atom in the middle of a large metal cluster originates from the one-carbon donor S-adenosylmethionine. 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.

Structure of precursor-bound NifEN: a nitrogenase FeMo cofactor maturase/insertase.

A metalloprotein structure involved in nitrogen fixation offers insight into metal-cluster insertion in nitrogenase. NifEN plays an essential role in the biosynthesis of the nitrogenase iron-molybdenum (FeMo) cofactor (M cluster). It is an α2β2 tetramer that is homologous to the catalytic molybdenum-iron (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 angstrom 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.

NifEN-B complex of Azotobacter vinelandii is fully functional in nitrogenase FeMo cofactor assembly

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 [Fe4S4]-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 [Fe4S4]-type clusters. Moreover, EPR and activity experiments indicate that the non-SAM-motif [Fe4S4] 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.

Tracing the interstitial carbide of the nitrogenase cofactor during substrate turnover.

The fate of the interstitial atom of the nitrogenase cofactor during substrate turnover has remained a topic of interest since the discovery of this atom more than a decade ago. In this study, we labeled the interstitial carbide atom with (14)C and (13)C isotopes and traced the fate of the isotope under turnover conditions. Our results show that the interstitial carbide cannot be exchanged upon turnover, nor can it be used as a substrate and incorporated into the products. These observations point to a role of the interstitial carbide in stabilizing the cofactor structure, although a function of this atom in indirectly modulating the reactivity of the cofactor or directly interacting with the substrate cannot be excluded.

Catalytic activities of NifEN: Implications for nitrogenase evolution and mechanism

NifEN is a key player in the biosynthesis of nitrogenase MoFe protein. It not only shares a considerable degree of sequence homology with the MoFe protein, but also contains clusters that are homologous to those found in the MoFe protein. Here we present an investigation of the catalytic activities of NifEN. Our data show that NifEN is catalytically competent in acetylene (C2H2) and azide (N3−) reduction, yet unable to reduce dinitrogen (N2) or evolve hydrogen (H2). Upon turnover, C2H2 gives rise to an additional S = 1/2 signal, whereas N3− perturbs the signal originating from the NifEN-associated FeMoco homolog. Combined biochemical and spectroscopic studies reveal that N3− can act as either an inhibitor or an activator for the binding and/or reduction of C2H2, while carbon monoxide (CO) is a potent inhibitor for the binding and/or reduction of both N3− and C2H2. Taken together, our results suggest that NifEN is a catalytic homolog of MoFe protein; however, it is only a “skeleton” version of the MoFe protein, as its associated clusters are simpler in structure and less versatile in function, which, in turn, may account for its narrower range of substrates and lower activities of substrate reduction. The resemblance of NifEN to MoFe protein in catalysis points to a plausible, sequential appearance of the two proteins in nitrogenase evolution. More importantly, the discrepancy between the two systems may provide useful insights into nitrogenase mechanism and allow reconstruction of a fully functional nitrogenase from the “skeleton” enzyme, NifEN.

Refining the Pathway of Carbide Insertion into the Nitrogenase M-cluster

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.

Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens

Significance Nitrogenase biosynthesis protein NifB catalyzes the radical S-adenosyl-L-methionine (SAM)-dependent insertion of carbide into the nitrogenase cofactor, M cluster, in a chemically unprecedented and biologically important reaction. The observation that two naturally “truncated” NifB homologs from Methanosarcina acetivorans (NifBMa) and Methanobacterium thermoautotrophicum (NifBMt) are functional equivalents of NifB from the diazotrophic organism, Azotobacter vinelandii, establishes the minimum sequence requirement for a functional NifB protein and reveals the species-dependent difference between members of this protein family; more importantly, it leads to the categorization of a distinct class of radical SAM methyltransferases that function in complex metallocluster assembly while opening up new avenues to study the structure and mechanism of NifB. 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 (NifBMa) and Methanobacterium thermoautotrophicum (NifBMt), 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. NifBMa and NifBMt are monomeric proteins containing a SAM-binding [Fe4S4] cluster (designated the SAM cluster) and a [Fe4S4]-like cluster pair (designated the K cluster) that can be processed into an [Fe8S9] precursor to the M cluster (designated the L cluster). Further, the K clusters in NifBMa and NifBMt 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, NifBMa and NifBMt 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 NifBMa and NifBMt 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.

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases

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. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- 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 Escherichia 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.

P+ state of nitrogenase p-cluster exhibits electronic structure of a [Fe4S4]+ cluster.

Mo nitrogenase consists of two component proteins: the Fe protein, which contains a [Fe(4)S(4)] cluster, and the MoFe protein, which contains two different classes of metal cluster: P-cluster ([Fe(8)S(7)]) and FeMoco ([MoFe(7)S(9)C·homocitrate]). The P-cluster is believed to mediate the electron transfer between the Fe protein and the MoFe protein via interconversions between its various oxidation states, such as the all-ferrous state (P(N)) and the one- (P(+)) and two-electron (P(2+)) oxidized states. While the structural and electronic properties of P(N) and P(2+) states have been well characterized, little is known about the electronic structure of the P(+) state. Here, a mutant strain of Azotobacter vinelandii (DJ1193) was used to facilitate the characterization of the P(+) state of P-cluster. This strain expresses a MoFe protein variant (designated ΔnifB β-188(Cys) MoFe protein) that accumulates the P(+) form of P-cluster in the resting state. Magnetic circular dichroism (MCD) spectrum of the P-cluster in the oxidized ΔnifB β-188(Cys) MoFe protein closely resembles that of the P(2+) state in the oxidized wild-type MoFe protein, except for the absence of a major charge-transfer band centered at 823 nm. Moreover, magnetization curves of ΔnifB β-188(Cys) and wild-type MoFe proteins suggest that the P(2+) species in both proteins have the same spin state. MCD spectrum of the P(+) state in the ΔnifB β-188(Cys) MoFe protein, on the other hand, is associated with a classic [Fe(4)S(4)](+) cluster, suggesting that the P-cluster could be viewed as two coupled 4Fe clusters and that it could donate either one or two electrons to FeMoco by using one or both of its 4Fe halves. Such a mode of action of P-cluster could provide energetic and kinetic advantages to nitrogenase in the complex mechanism of N(2) reduction.

Dual functions of NifEN: insights into the evolution and mechanism of nitrogenase

Nitrogenase catalyzes the nucleotide-dependent conversion of dinitrogen to ammonia at the iron-molybdenum cofactor (FeMoco) center of its molybdenum-iron (MoFe) protein component. Biosynthesis of FeMoco is arguably one of the most complex processes in the field of bioinorganic chemistry, which involves the participation of a number of nif (nitrogen fixing) gene products. One key player in this process, NifEN (encoded by nifE and nifN), is homologous to the MoFe protein with regard to both the primary sequences and the types of the metal centers. Recently, an all-iron precursor has been identified on NifEN, which closely resembles the Fe/S core structure of the mature cofactor. Such a precursor-bound form of NifEN has not only served as an excellent platform for the investigation of FeMoco assembly, but also facilitated the examination of the capacity of NifEN as a catalytic homolog of MoFe protein. This perspective will focus on the recent advances toward elucidating the dual functions of NifEN in nitrogenase assembly and catalysis, and the insights afforded by these advances into the evolution and mechanism of nitrogenase.