The structure of vanadium nitrogenase reveals an unusual bridging ligand

Abstract

Nitrogenases catalyze the reduction of dinitrogen (N₂) gas to ammonium at a complex heterometallic cofactor. This most commonly occurs at the FeMo cofactor (FeMoco), a [Mo-7Fe-9S-C] cluster whose exact reactivity and substrate-binding mode remain unknown. Alternative nitrogenases replace molybdenum with either vanadium or iron and differ in reactivity, most prominently in the ability of vanadium nitrogenase to reduce CO to hydrocarbons. Here we report the 1.35-Å structure of vanadium nitrogenase from Azotobacter vinelandii. The 240-kDa protein contains an additional α-helical subunit that is not present in molybdenum nitrogenase. The FeV cofactor (FeVco) is a [V-7Fe-8S-C] cluster with a homocitrate ligand to vanadium. Unexpectedly, it lacks one sulfide ion compared to FeMoco, which is replaced by a bridging ligand, likely a μ-1,3-carbonate. The anion fits into a pocket within the protein that is obstructed in molybdenum nitrogenase, and its different chemical character helps to rationalize the altered chemical properties of this unique N₂- and CO-fixing enzyme.

Figure 1. Structure of vanadium nitrogenase.

(a) Cartoon representation of the A. vinelandii VnfD₂K₂G₂ heterohexamer. Two copies of VnfG (blue) are located apically and are in exclusive contact with the adjacent VnfD subunits (yellow). The positions of the active site FeV cofactor and electron-transferring P-cluster, as well as the stabilizing Mg²⁺ cation are indicated in one monomer. (b) A schematic surface representation of VFe protein highlights the subunit arrangement and the positions of the VnfD, VnfK and VnfG peptides. (c) Molybdenum nitrogenase (NifD₂K₂) from the same organism shares the architecture of the core subunits, but differs markedly in aspects such as the prominent N termini of the NifK subunits. (d) Stereo representation of VnfG. The subunit is a bundle of four α-helices without substantial similarities to other known structures.

Figure 2. Electron-transferring P-cluster of vanadium nitrogenase.

(a) Stereo image of the [8Fe:7S] P-cluster in the reduced P^N state. Three of the six cysteine ligands to the cluster originate from VnfD, and the others from VnfK, where S153^K is the conserved ligand for Fe6 in the oxidized state of the cluster. (b) Detail of the lower half cubane of P-cluster, highlighting Fe5 and Fe6 that in molybdenum nitrogenase relocate upon oxidation to the P^Ox state. The 2F_o–F_c electron density map (blue mesh) is contoured at the 2σ level. The green mesh shows a F_o–F_c electron density map contoured at the +6σ level, indicating the presence of a low proportion of a second conformation for Fe6. Contrary to the MoFe protein, no equivalent feature is visible at or near Fe5. The oxidized form thus likely represents a P¹⁺ state as predicted by spectroscopy. (c) Arrangement of the subunits in a VnfDKG heterotrimer and location of the FeV cofactor (V) and the P-cluster (P). The P-cluster is centered precisely on the pseudo-twofold symmetry axis connecting VnfD and VnfK, while the VnfG subunit is in exclusive contact with VnfD.

Figure 3. Structure of FeV cofactor.

(a) FeVco is a [V:7Fe:8S:C] cluster with a homocitrate ligand to the vanadium ion and a carbonate bridging Fe4 and Fe5. Iron ions (grey) and sulfide ions (yellow) are numbered. The cluster is coordinated to the protein via the amino acids C257 and H423 of VnfD. (b) Coordination of vanadium (stereo image). The coordinating atoms of H423 and the homocitrate moiety are labelled. (c) Ligation of Fe4 and Fe5 by carbonate. Average bond distances are given in Å.

Figure 4. The carbonate ligand to FeV cofactor.

(a) Stereo representation of the protein environment of the carbonate ligand, CO₃^2–. The cluster is coordinated to the VnfD subunit of the enzyme via Fe1 to C257 and V to H423. (b) Detail of the binding pocket for the carbonate anion, coordinated by a short loop region present in both VFe protein (black) and MoFe protein (green). The swapping of a proline and a leucine in this loop leads to a slightly smaller pocket in the MoFe protein, where residue P360 would clash with the ligand.