Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase

Abstract

The enzyme nitrogenase uses a suite of complex metallocofactors to reduce dinitrogen (N₂) to ammonia. Mechanistic details of this reaction remain sparse. We report a 1.83-angstrom crystal structure of the nitrogenase molybdenum-iron (MoFe) protein captured under physiological N₂ turnover conditions. This structure reveals asymmetric displacements of the cofactor belt sulfurs (S2B or S3A and S5A) with distinct dinitrogen species in the two αβ dimers of the protein. The sulfur-displaced sites are distinct in the ability of protein ligands to donate protons to the bound dinitrogen species, as well as the elongation of either the Mo-O5 (carboxyl) or Mo-O7 (hydroxyl) distance that switches the Mo-homocitrate ligation from bidentate to monodentate. These results highlight the dynamic nature of the cofactor during catalysis and provide evidence for participation of all belt-sulfur sites in this process.

Fig. 1.. The oxidized P-clusters in Av1*.

Structures of P-clusters at (A–C) Chain-A/B interface (P-cluster^(A/B)) and (D–F) Chain-C/D interface (P-cluster^(C/D)) of Av1*. Chain-A and Chain-C are the α-subunits, and Chain-B and Chain-D are the β-subunits of the two αβ-dimers of Av1*. (A, D) The P-clusters are shown in ball-and-stick presentation, and the key residues interacting with the P-clusters are indicated as sticks. Chain-A and Chain-C are shown as wheat ribbons, and Chain-B and Chain-D are shown as light-blue ribbons. (B, C) P-cluster^(A/B) and (E, F) P-cluster^(C/D) superimposed with (B, E) the anomalous density maps calculated at 7100 eV at a resolution of 2.18 Å and contoured at 4.0 σ, showing the position of sulfur atoms (mint-blue mesh); and with (C, F) the anomalous density maps calculated at 7141 eV at a resolution of 2.1 Å and contoured at 15.0 σ, showing the position of iron atoms (red mesh). Color code of atoms: Fe, orange; S, yellow; O, red; N, blue.

Fig. 2.. The nitrogen ligand-bound M-clusters in Av1*.

Structures of M-clusters in (A–C) Chain-A (M-cluster^(A)) and (D–F) Chain-C (M-cluster^(C)) refined at a resolution of 1.83 Å. View along the Fe1-C-Mo direction of (A) M-cluster^(A) and (D) M-cluster^(C) superimposed with the anomalous density maps (mint-blue mesh) calculated at 7100 eV at a resolution of 2.18 Å and contoured at 4.0 σ (A) and 5.3 σ (D), respectively, showing the displacement of sulfur at the S2B site in M-cluster^(A) (A) and at the S3A and S5A sites in M-cluster^(C) (D). Electron densities of the belt sulfurs (S^Belt) relative to the average density of cluster sulfurs (S^Cluster) in (B) Chain-A and (E) Chain-C, respectively, expressed in sigma values. The average sigma values of the cluster sulfurs in Chain-A and Chain-C are 4.8 and 5.5, respectively (B, E). Side view of (C) M-cluster^(A) and (F) M-cluster^(C) with key residues interacting with the clusters and the bound nitrogen ligands indicated as sticks. M-cluster^(A) and M-cluster^(C) are superimposed with the F_o-F_c omit map of the nitrogen ligands contoured at 10 σ (mint-blue mesh). The peptides and atoms are colored as those in Fig. 1.

Fig. 3.. The reduced P-clusters in Av1*(TOD).

Structures of P-clusters at (A, B) Chain-A/B interface (P-cluster^(A/B)) and (C, D) Chain-C/D interface (P-cluster^(C/D)) of Av1*(TOD). Chain-A and Chain-C are the α-subunits, and Chain-B and Chain-D are the β-subunits of the two αβ-dimers of Av1*(TOD). (B, D) P-clusters superimposed with the anomalous density maps calculated at 7100 eV at a resolution of 2.17 Å and contoured at 4.5 σ, showing the position of sulfur atoms (mint-blue mesh). The peptides and atoms are colored as those in Fig. 1.

Fig. 4.. The M-clusters in Av1*(TOD).

Structures of M-clusters in (A, B) Chain-A (M-cluster^(A)) and (C, D) Chain-C (M-cluster^(C)) refined at a resolution of 1.73 Å. Side view of (A) M-cluster^(A) and (C) M-cluster^(C) with key residues interacting with the clusters and the bound nitrogen ligands indicated as sticks. M-cluster^(A) and M-cluster^(C) are superimposed with the F_o-F_c omit maps of the belt sulfurs contoured at 13 σ (mint-blue mesh). View along the Fe1-C-Mo direction of (B) M-cluster^(A) and (D) M-cluster^(C) superimposed with the anomalous density maps calculated at 7100 eV at a resolution of 2.17 Å and contoured at 4.0 σ, showing the presence of the anomalous sulfur density (mint-blue mesh) at all belt sulfur positions (S2B, S3A and S5A) in (B) M-cluster^(A) and (D) M-cluster^(C). The peptides and atoms are colored as those in Fig. 1.