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