How many metals does it take to fix N2? A mechanistic overview of biological nitrogen fixation

Abstract

During the process of biological nitrogen fixation, the enzyme nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia. Nitrogenase consists of two component metalloproteins, the iron (Fe) protein and the molybdenum-iron (MoFe) protein; the Fe protein mediates the coupling of ATP hydrolysis to interprotein electron transfer, whereas the active site of the MoFe protein contains the polynuclear FeMo cofactor, a species composed of seven iron atoms, one molybdenum atom, nine sulfur atoms, an interstitial light atom, and one homocitrate molecule. This Perspective provides an overview of biological nitrogen fixation and introduces three contributions to this special feature that address central aspects of the mechanism and assembly of nitrogenase.

Scheme 1.

Kinetic scheme depicting ATP-dependent electron transfer between the component proteins of nitrogenase. Av1 and Av2 denote the MoFe protein and Fe protein, respectively, from Azotobacter vinelandii. The superscripts R and Ox denote reduced and oxidized states of Av2, and the superscripts N and N-1 indicate the oxidation levels of Av1 before and after electron transfer from Av2. Fld, flavodoxin.

Fig. 1.

Ball-and-stick representation of the nitrogenase metalloclusters. Shown are the Fe-protein [4Fe:4S] cluster (A) and the two clusters of the MoFe protein, FeMo cofactor, and the P cluster (B) assigned to the oxidation state P^N (C). The radii of all non-protein atoms have been set to 0.7 Å, and the protein ligands are presented as black bonds. Iron, molybdenum, sulfur, carbon, oxygen, and nitrogen atoms are colored burgundy, orange, yellow, gray, red, and blue, respectively. This figure was generated with the program MolScript (74) from Protein Data Bank entries 2NIP, 1M1N, and 3MIN.

Fig. 2.

Complex of the nitrogenase proteins stabilized by ADP-AIF₄⁻. (Left) ADP-AlF₄⁻-stabilized half-complex between a Fe-protein dimer and an αβ-subunit pair of the MoFe protein. The subunits are depicted as Cα traces with the MoFe α- and β-subunits colored red and blue, respectively, and the individual subunits of each Fe protein colored green and yellow. Non-protein groups are shown in a space-filling representation using the color scheme of Fig. 1, with fluorine and magnesium colored orange and green, respectively. (Right) Transduction pathway coupling the nucleotide and cofactor sites in the nitrogenase complex. This view represents a slice through the complex that includes the ADP-AlF₄⁻, [4Fe:4S]-cluster, P-cluster, and FeMo-cofactor sites. The side chains of Asp-129 of each Fe-protein subunit are depicted as space-filling models to illustrate the locations of these critical residues adjacent to both the nucleotide and cluster sites.

Fig. 3.

Correlation between hinge angle (bars) and the distance between centroids (filled triangles) of the [4Fe:4S] and P cluster of the nitrogenase proteins in a series of states ordered along a potential reaction coordination for nucleotide hydrolysis. (Inset) The hinge angle is defined by the rotation angle about an axis along the dimer interface required to superimpose one subunit of a specified Fe-protein structure onto a subunit of the ADP-AlF₄⁻-stabilized Fe protein, after initially superimposing the other subunits in these structures.

Fig. 4.

Space-filling representation of the FeMo-cofactor (Left) and [4Fe:4S] cluster of aconitase (Right) using chemically appropriate radii for their atoms, including the protein side-chain ligands and homocitric acid. Dinitrogen is included for size comparison. The radii used to generate this figure were as follows: C, 1.5 Å; O, 1.4 Å; interstitial ligand (N³⁻), 1.4 Å; N in N₂, 1.6 Å; S, 1.7 Å; Fe, 0.7 Å; and Mo, 0.8 Å (56, 57).

Scheme 2.

Generalized Chatt-type mechanism for the reduction of dinitrogen to ammonia catalyzed at a single metal accommodating oxidation states n to n + 3. For the case of MMo and Fe, n = +3 and +1, respectively.