Vanadium nitrogenase: a two-hit wonder?

Abstract

Nitrogenase catalyzes the biological conversion of atmospheric dinitrogen to bioavailable ammonia. The molybdenum (Mo)- and vanadium (V)-dependent nitrogenases are two homologous members of this metalloenzyme family. However, despite their similarities in structure and function, the characterization of V-nitrogenase has taken a much longer and more winding path than that of its Mo-counterpart. From the initial discovery of this nitrogen-fixing system, to the recent finding of its CO-reducing capacity, V-nitrogenase has proven to be a two-hit wonder in the over-a-century-long research of nitrogen fixation. This perspective provides a brief account of the catalytic function and structural basis of V-nitrogenase, as well as a short discussion of the theoretical and practical potentials of this unique metalloenzyme.

Fig. 1. X-ray crystal structures of the component proteins and the complex of Mo-nitrogenase

(A, B) The γ₂-dimeric Fe protein (A) and the α₂β₂-tetrameric MoFe protein (B) of A. vinelandii. (C, D) Half of the ADP•AlF₄⁻-stabilized Fe protein/MoFe protein complex (C) and the relative positions of components that are involved in the electron flow during catalysis (D). The two subunits of Fe protein are colored yellow and orange, and the α- and β-subunits of MoFe protein are colored red and blue, respectively. The positions of [Fe₄S₄] cluster, P-cluster and FeMoco are indicated. All clusters and ADP•AlF₄⁻ are shown as space-filling models, with the atoms colored as follows: Fe, orange; S, yellow; Mo, cyan; O, red; C, gray; N, dark blue; X (C, N or O), light gray; Mg, green; Al, beige; F, light blue. PYMOL was used to create the figure (PDB entries 1NIP, 1M1N and 1N2C).

Fig. 2. Catalytic units of Mo- and V-nitrogenases

Schematic presentations of the catalytic unit of Mo-nitrogenase (left), which consists of the γ₂-dimeric Fe protein (encoded by nifH) and one half of the α₂β₂-tetrameric MoFe protein (encoded by nifDK); and the catalytic unit of V-nitrogenase (right), which consists of the γ₂-dimeric Fe protein (encoded by vnfH) and one half of the α₂β₂δ₄-octameric VFe protein (encoded by vnfDGK). It is hypothesized that, during catalysis, the Fe protein forms a functional complex with the MoFe or VFe protein, in which electrons are sequentially transferred from the [Fe₄S₄] cluster (of the Fe protein), through the P-cluster, to the FeMoco (of MoFe protein) or the FeVco (of VFe protein), where substrate reduction eventually occurs.

Fig. 3. Comparison between the primary sequences of Mo- and V-nitrogenases

(A) Sequence alignment of the Fe proteins of A. vinelandii Mo-nitrogenase (Av-NifH), A. vinelandii V-nitrogenase (Av-VnfH) and A. chroococcum V-nitrogenase (Ac-VnfH). The two conserved Cys residues, which serve as the [Fe₄S₄] cluster ligands, are highlighted in green; and the Gly-X-Gly-X-X-Gly consensus nucleotide-binding motifs are highlighted in yellow. (B) Sequence alignment of the α-subunits of A. vinelandii MoFe protein (Av-NifD), A. vinelandii VFe protein (Av-VnfD) and A. chroococcum VFe protein (Ac-VnfD). (C) Sequence alignment of the β-subunits of A. vinelandii MoFe protein (Av-NifK), A. vinelandii VFe protein (Av-VnfK) and A. chroococcum VFe protein (Ac-VnfK). The VFe protein contains an additional δ-subunit (Av- or Ac-VnfG), which is absent from the MoFe protein. The six conserved Cys residues, which serve as the P-cluster ligands, are highlighted in orange; and the conserved Cys and His residues, which serve as the FeMoco ligands, are highlighted in blue.

Fig. 4. Specific activities of CO reduction by V- and Mo-nitrogenases

(A) Total activities of product formation by the two nitrogenases in the presence of H₂O (solid) or D₂O (striped). (B, C) Specific activities of individual product formation by V (B)- or Mo (C)-nitrogenase.

Fig. 5. Product distributions of CO reduction by V- and Mo-nitrogenases

(A, B) Product profile of V-nitrogenase in the presence of H₂O (A) or D₂O (B). (C, D) Product profile of Mo-nitrogenase in the presence of H₂O (C) or D₂O (D). The total amounts of hydrocarbons detected in V (A, B)- and Mo (C, D)-nitrogenase-catalyzed reactions were set as 100%, respectively, and the percentages of individual products in these reactions were determined accordingly for each nitrogenase.

Fig. 6. Ratios between alkenes and alkanes in reactions catalyzed by V- and Mo-nitrogenases

Shown are alkene/alkane ratios of the two (red)-, three (yellow)- and four (pink)-carbon products in the presence of H₂O (solid) or D₂O (striped).

Fig. 7. [Fe₄S₄] clusters of the Fe proteins of Mo-nitrogenase (NifH) and V-nitrogenase (VnfH)

(A, B) Dithionite-reduced EPR spectra of NifH (A) and VnfH (B). (C–E) XAS/EXAFS-derived structures of the [Fe₄S₄] clusters in NifH (C) and VnfH (D), and the structural overlay of the two [Fe₄S₄] clusters (E). The clusters are shown as ball-and-stick models, with the atoms colored as those in Fig. 1. PYMOL was used to create the figure.

Fig. 8. P-clusters of the MoFe and VFe proteins

(A, B) Indigo disulfonate (IDS)-oxidized EPR spectra of MoFe (A) and VFe (B) proteins. (C, D) Dithionite-reduced EPR spectra of MoFe (C) and VFe (D) proteins. The EPR feature associated with the P-cluster in MoFe protein is highlighted in blue shade, and those associated with the P-cluster in VFe protein are highlighted in red shade. The g values are indicated. (E, F) Crystal structure of the P-cluster in MoFe protein (E) and proposed structure of the P-cluster in VFe protein (F). The clusters are shown as ball-and-stick models, with the atoms colored as those in Fig. 1. PYMOL was used to create the figure.

Fig. 9. Cofactors of the MoFe and VFe proteins

(A, C) Dithionite-reduced EPR spectra of MoFe protein (A) and NMF-extracted FeMoco (C). (B, D) Dithionite-reduced EPR spectra of VFe protein (B) and NMF-extracted FeVco (D). The corresponding EPR features in the protein-bound and NMF-extracted states are highlighted in blue shade for the FeMoco (A, C) and in red shade for the FeVco (B, D). The g values are indicated. (E, F) The XAS/EXAFS-derived structures of NMF-extracted FeMoco (E) and FeVco (F). The clusters are shown as ball-and-stick models, whereas the NMF molecules are represented by sticks, with the atoms colored as those in Fig. 1. PYMOL was used to create the figure.

Fig. 10. Specific activities of the “standard” substrate reduction by reconstituted and native nitrogenases

Specific activities of FeVco (A)- and FeMoco (B)-reconstituted MoFe proteins and native VFe (C) and MoFe (D) proteins in H₂ formation under Ar (black), C₂H₄ (red) and C₂H₆ (green) formation under C₂H₂, and NH₃ (yellow) and H₂ (blue) formation under N₂. ①, characteristic catalytic features of V-nitrogenase; ②, characteristic catalytic features of Mo-nitrogenase.