Markus W. Ribbe

X-ray Emission Spectroscopy Evidences a Central Carbon in the Nitrogenase Iron-Molybdenum Cofactor

A central light atom in a cofactor at the nitrogenase active site is identified as a carbon. Nitrogenase is a complex enzyme that catalyzes the reduction of dinitrogen to ammonia. Despite insight from structural and biochemical studies, its structure and mechanism await full characterization. An iron-molybdenum cofactor (FeMoco) is thought to be the site of dinitrogen reduction, but the identity of a central atom in this cofactor remains unknown. Fe Kβ x-ray emission spectroscopy (XES) of intact nitrogenase MoFe protein, isolated FeMoco, and the FeMoco-deficient ∆nifB protein indicates that among the candidate atoms oxygen, nitrogen, and carbon, it is carbon that best fits the XES data. The experimental XES is supported by computational efforts, which show that oxidation and spin states do not affect the assignment of the central atom to C4–. Identification of the central atom will drive further studies on its role in catalysis.

Vanadium Nitrogenase Reduces CO

An enzyme that reduces nitrogen to ammonia can also reduce carbon monoxide to hydrocarbons. Vanadium nitrogenase not only reduces dinitrogen to ammonia but also reduces carbon monoxide to ethylene, ethane, and propane. The parallelism between the two reactions suggests a potential link in mechanism and evolution between the carbon and nitrogen cycles on Earth.

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.

Extending the carbon chain: hydrocarbon formation catalyzed by vanadium/molybdenum nitrogenases.

The molybdenum nitrogenase enzyme can reduce carbon monoxide, albeit inefficiently, in addition to its native substrate, nitrogen. In a small-scale reaction, vanadium-dependent nitrogenase has previously been shown to catalyze reductive catenation of carbon monoxide (CO) to ethylene, ethane, propylene, and propane. Here, we report the identification of additional hydrocarbon products [α-butylene, n-butane, and methane (CH4)] in a scaled-up reaction featuring 20 milligrams of vanadium-iron protein, the catalytic component of vanadium nitrogenase. Additionally, we show that the more common molybdenum-dependent nitrogenase can generate the same hydrocarbons from CO, although CH4 was not detected. The identification of CO as a substrate for both molybdenum- and vanadium-nitrogenases strengthens the hypothesis that CO reduction is an evolutionary relic of the function of the nitrogenase family. Moreover, the comparison between the CO-reducing capacities of the two nitrogenases suggests that the identity of heterometal at the active cofactor site affects the efficiency and product distribution of this reaction.

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.

Structural insights into a protein-bound iron-molybdenum cofactor precursor

The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein is a highly complex metallocluster that provides the catalytically essential site for biological nitrogen fixation. FeMoco is assembled outside the MoFe protein in a stepwise process requiring several components, including NifB-co, an iron- and sulfur-containing FeMoco precursor, and NifEN, an intermediary assembly protein on which NifB-co is presumably converted to FeMoco. Through the comparison of Azotobacter vinelandii strains expressing the NifEN protein in the presence or absence of the nifB gene, the structure of a NifEN-bound FeMoco precursor has been analyzed by x-ray absorption spectroscopy. The results provide physical evidence to support a mechanism for FeMoco biosynthesis. The NifEN-bound precursor is found to be a molybdenum-free analog of FeMoco and not one of the more commonly suggested cluster types based on a standard [4Fe–4S] architecture. A facile scheme by which FeMoco and alternative, non-molybdenum-containing nitrogenase cofactors are constructed from this common precursor is presented that has important implications for the biosynthesis and biomimetic chemical synthesis of FeMoco.

Unique features of the nitrogenase VFe protein from Azotobacter vinelandii

Nitrogenase is an essential metalloenzyme that catalyzes the biological conversion of dinitrogen (N2) to ammonia (NH3). The vanadium (V)-nitrogenase is very similar to the “conventional” molybdenum (Mo)-nitrogenase, yet it holds unique properties of its own that may provide useful insights into the general mechanism of nitrogenase catalysis. So far, characterization of the vanadium iron (VFe) protein of Azotobacter vinelandii V-nitrogenase has been focused on 2 incomplete forms of this protein: αβ2 and α2β2, both of which contain the small δ-subunit in minor amounts. Although these studies provided important information about the V-dependent nitrogenase system, they were hampered by the heterogeneity of the protein samples. Here, we report the isolation and characterization of a homogeneous, His-tagged form of VFe protein from A. vinelandii. This VFe protein has a previously-unsuspected, α2β2δ4-heterooctameric composition. Further, it contains a P-cluster that is electronically and, perhaps, structurally different from the P-cluster of molybdenum iron (MoFe) protein. More importantly, it is catalytically distinct from the MoFe protein, particularly with regard to the mechanism of H2 evolution. A detailed EPR investigation of the origins and interplays of FeV cofactor- and P-cluster-associated signals is presented herein, which lays the foundation for future kinetic and structural analysis of the VFe protein.

Characterization of Isolated Nitrogenase FeVco

The cofactors of the Mo- and V-nitrogenases (i.e., FeMoco and FeVco) are homologous metal centers with distinct catalytic properties. So far, there has been only one report on the isolation of FeVco from Azotobacter chroococcum. However, this isolated FeVco species did not carry the full substrate-reducing capacity, as it is unable to restore the N(2)-reducing ability of the cofactor-deficient MoFe protein. Here, we report the isolation and characterization of a fully active species of FeVco from A. vinelandii. Our metal and activity analyses show that FeVco has been extracted intact, carrying with it the characteristic capacity to reduce C(2)H(2) to C(2)H(6) and, perhaps even more importantly, the ability to reduce N(2) to NH(3). Moreover, our EPR and XAS/EXAFS investigations indicate that FeVco is similar to, yet distinct from FeMoco in electronic properties and structural topology, which could account for the differences in the reactivity of the two cofactors. The outcome of this study not only permits the proposal of the first EXAFS-based structural model of the isolated FeVco but also lays a foundation for future catalytic and structural investigations of this unique metallocluster.

FeMo cofactor maturation on NifEN

FeMo cofactor (FeMoco) biosynthesis is one of the most complicated processes in metalloprotein biochemistry. Here we show that Mo and homocitrate are incorporated into the Fe/S core of the FeMoco precursor while it is bound to NifEN and that the resulting fully complemented, FeMoco-like cluster is transformed into a mature FeMoco upon transfer from NifEN to MoFe protein through direct protein–protein interaction. Our findings not only clarify the process of FeMoco maturation, but also provide useful insights into the other facets of nitrogenase chemistry.

Biosynthesis of Nitrogenase Metalloclusters

Nitrogenase is a complex metalloenzyme that is best known for its function in biological nitrogen fixation.1,2 Harbored in a group of microbes called diazotrophs, nitrogenase catalyzes the reduction of nitrogen (N2) to ammonia (NH3) in a reaction that is usually depicted as N2 + 8H+ + 16MgATP + 8e− → 2NH3 + H2 + 16MgADP + 16Pi. This reaction not only represents a key step in the global nitrogen cycle, but also embodies the formidable chemistry of breaking the exceptionally stable N≡N triple bond. Recently, nitrogenase was shown to reduce carbon monoxide (CO) to hydrocarbons under the same reaction conditions of biological nitrogen fixation,3–6 defining it as a versatile metalloenzyme that is capable of activating N2 and CO and converting them into products of agronomic and economic values. Interestingly, the reactions of N2- and CO-reduction by nitrogenase parallel two important processes in industry: the Haber-Bosch process, which is used for ammonia production from N2 and hydrogen (H2);7 and the Fischer-Tropsch process, which is used for carbon fuel production from CO and H2.8 However, contrary to the industrial processes, the nitrogenase-catalyzed reactions occur under ambient conditions, making this enzyme a fascinating subject from the perspective of chemical energy. Three homologous nitrogenases, namely, the molybdenum (Mo), vanadium (V) and iron (Fe)-only nitrogenases, have been identified to date.9,10 The best studied among them is the Mo nitrogenase from Azotobacter vinelandii, which consists of two component proteins. One, designated the Fe protein (NifH), is a γ2-dimer that contains a subunit-bridging [Fe4S4] cluster per dimer and an ATP binding site within each subunit. The other, designated the MoFe protein (NifDK), is an α2β2-tetramer that contains two complex metalloclusters per αβ-dimer: a P-cluster ([Fe8S7]) at the α/β-subunit interface and an M-cluster ([MoFe7S9C-homocitrate]) within the α-subunit.11–14 Catalysis by the Mo nitrogenase involves the formation of a complex between NifH and NifDK15,16 and the inter-protein transfer of electrons from the [Fe4S4] cluster of NifH, via the P-cluster, to the M-cluster of NifDK, where substrate reduction eventually occurs (Figure 1). Such an electron pathway highlights the functions of the P- and M-clusters in substrate reduction. Both are high-nuclearity metalloclusters with unusual structures not recognized in other biological systems, and both have evaded successful chemical synthesis so far. The unique properties of the P- and M-clusters of nitrogenase will be discussed below (section 1.1), followed by an overview of proteins involved in the biosynthesis of these clusters (section 1.2). Figure 1 Crystal structure of the ADP•AlF4−-stabilized NifH/NifDK complex (A) and the relative positions of components involved in the transfer of electrons (B). The two subunits of NifH are colored gray and light brown, and the α- and ... 1.1. Properties of the metal clusters in nitrogenase The P-cluster is bridged between the α- and β-subunits of NifDK at a position that is 10 A below the surface of the protein.11–13 Structurally, it can be viewed as two [Fe4S3] partial cubanes bridged by a μ6-sulfide (Figure 2A and B); whereas chemically, it can exist in three oxidation states (designated the PN, P1+ and POX state, respectively). In the presence of excess dithionite, the P-cluster exists in an all-ferrous, diamagnetic state (designated the PN-cluster). Following the treatment of a dye oxidant [e.g., indigodisulfonate (IDS)], the PN-cluster can be two-electron oxidized to a stable S = integer (3 or 4) state (designated the POX-cluster), which displays a characteristic, parallel-mode electron paramagnetic resonance (EPR) signal at g =11.817–19. Both the PN- and the POX-clusters (Figure 2A and B) are covalently coordinated by six cysteinyl ligands in NifDK, three from the α-subunit (Cysα62, Cysα88 and Cysα154) and three from the β-subunit (Cysβ70, Cysβ95 and Cysβ153). Each of the Cysα62, Cysα154, Cysβ70 and Cysβ153 ligands coordinates one Fe atom, and each of the Cysα88 and Cysβ95 ligands coordinates two Fe atoms of the P-cluster.20,21 However, the core structures of the PN- and POX-clusters are different, with one half of the POX-cluster present in a more open conformation (Figure 2B). Such a structural rearrangement is accompanied by a change in the ligation pattern, as the POX-cluster is coordinated by two more protein ligands than the PN-cluster.21 One of these ligands is Serβ188, which coordinates an Fe atom through an Oγ ligand together with the cysteinyl group of Cysβ153; the other ligand is Cysα88, which coordinates an Fe atom through a backbone amide nitrogen ligand and a cysteinyl group (Figure 2B). Figure 2 Crystal structures of the PN (A) and POX (B) states of the P-cluster and the M-cluster (C). The clusters are shown as ball-and-stick models. The atoms are shown as transparent balls and colored as those in Figure 1; and the ligands are shown as sticks. ... The M-cluster (also called FeMoco or cofactor) is buried within the α-subunit of NifDK, 14 A away from the P-cluster. Structurally, the M-cluster can be viewed as [Fe4S3] and [MoFe3S3] partial cubanes bridged by three μ2-sulfides (Figure 2C). In addition to its metal-sulfur core, the M-cluster also contains an organic homocitrate moiety attached through its 2-hydroxy and 2-carboxyl groups to the Mo atom and a μ6-interstitial carbide coordinated in the central cavity.11–14 The interstitial carbide cannot be exchanged upon turnover, nor can it be used as a substrate and incorporated into the products, suggesting a role of the interstitial carbide in stabilizing the structure of the M-cluster.22,23 However, a function of this atom in indirectly modulating the reactivity of the M-cluster or directly interacting with the substrate cannot be excluded.24 The M-cluster is coordinated by only two ligands in NifDK: Cysα275, which coordinates the terminal Fe atom; and Hisα442, which coordinates the opposite Mo atom. A third residue, Lysα426, provides an additional hydrogen-bonded anchor for homocitrate at the Mo end of the cluster.11–14 In addition to the covalent ligands, the M-cluster is held within NifDK through direct and water-bridged hydrogen bonds. The apparently “simple” coordination pattern of the M-cluster permits extraction of this cluster as an intact entity into organic solvents, such as N-methylformamide (NMF).25–27 The extracted M-cluster was shown to be anionic26 despite a proposed charge of +1 or +3 for the metal-sulfur core of this cluster in the resting state.28,29 The overall negative charge of the M-cluster is believed to originate from its endogenous homocitrate entity, which is −4 if the hydroxyl (-OH) group is deprotonated. The extracted M-cluster can bind CO and cyanide (CN−) at certain oxidation states.30,31 Moreover, it can catalyze the ATP-independent reduction of CO and CN− to hydrocarbons in the presence of a strong reductant, europium(II) diethylenetriaminepentaacetate [Eu(II) DTPA],32 although conditions are yet to be defined for N2 reduction by the extracted M-cluster. Both the solvent-extracted and the protein-bound M-clusters display a characteristic, S = 3/2 EPR signal at g = 4.7, 3.7 and 2.0 in the presence of excess dithionite; however, the signal displayed by the extracted M-cluster is broader in line-shape than that displayed by its protein-bound counterpart.26,33 Moreover, the M-cluster can undergo a reversible one-electron oxidation and reduction process, which is reflected by the disappearance of the S = 3/2 signal upon oxidation and the re-appearance of this signal upon re-reduction.1