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Iron-only Fe-nitrogenase underscores common catalytic principles in biological nitrogen fixation

Nature Catalysis volume 6, pages 415–424 (2023)Cite this article

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Abstract

Nitrogenases uniquely reduce atmospheric N2 to bioavailable ammonium. They group into three isoforms that primarily differ in the architecture of their active-site cofactors. A molybdenum or vanadium ion is introduced into a common precursor cluster to form Mo- and V-dependent nitrogenases, respectively. In contrast, the third class of the enzyme only utilizes abundant iron to reduce N2 under ambient conditions and is consequently of high interest for mechanistic studies and catalyst design. Here we report the three-dimensional structure of Fe-nitrogenase from Azotobacter vinelandii and its FeFe cofactor, a [8Fe:9S:C] cluster with an interstitial carbide and an organic homocitrate ligand at the apical iron that substitutes for Mo or V in the other isoforms. The structure reveals lability of sulfide S2B, the proposed binding site for substrate in other nitrogenases, further supporting a general mechanism of proton and electron transfer for all nitrogenases and all their substrates.

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Fig. 1: Architecture of the FeFe protein of Fe-only nitrogenase.
Fig. 2: The metal clusters of FeFe protein.
Fig. 3: Conformations of FeFe cofactor.
Fig. 4: Hydride formation and mechanistic implications.
Fig. 5: Possible E4 state structures and relevant model complexes.

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Data availability

The atomic coordinates and structure factors for A. vinelandii Fe-only nitrogenase FeFe protein have been deposited with the Protein Data Bank at http://www.pdb.org with accession code 8BOQ. All other data are available from the authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the European Research Council (grant no. 310656) and Deutsche Forschungsgemeinschaft (PP 1927, project ID 311061829, and RTG 1976, project ID 235777276). We thank P. dos Santos, M. Rohde, K. Parison, J. Gies-Elterlein, F. Schneider, S.L.A. Andrade and P. Franke for helpful discussions and the beamline staff at the Swiss Light Source, Villigen, Switzerland, for excellent assistance with data collection.

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  1. Institute for Biochemistry, University of Freiburg, Freiburg, Germany

    Christian Trncik, Franka Detemple & Oliver Einsle

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  1. Christian Trncik
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Contributions

C.T. and O.E. designed the experiments. C.T. and F.D. produced protein and generated crystals. C.T. and O.E. built and refined the crystal structure. C.T., F.D. and O.E. analysed data and wrote the paper.

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Correspondence to Oliver Einsle.

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Extended data

Extended Data Fig. 1 Isolation and characterization of Fe nitrogenase from Azotobacter vinelandii.

a) Analytical size exclusion chromatography of FeFe protein on Superdex S200 (Cytiva), using a triple detector array with UV absorbance (purple), right-angle light scattering (green) and refractive index (red). The derived absolute molecular mass is shown in black. b) SDS-PAGE of preparations of the three Fe proteins of A. vinelandii (left) and the three dinitrogenases (right). c) N2 reduction activities of the three A. vinelandii nitrogenase isoenzymes with their respective Fe proteins. Data from three technical replicates from the protein batch used for structure determination, presented as mean values + /– standard deviation.

Source data

Extended Data Fig. 2 Structural comparison of the dinitrogenase core subunits of A. vinelandii.

Stereo images of the D- and K-subunits of Fe-, V-, and Mo-nitrogenases (top to bottom) in identical orientation, colored from blue at the N-terminus to red on the C-terminus. The respective cofactors are shown in the D-subunits, P-clusters are shown in both subunits to emphasize their position in the interface. Note the extended C-terminus of AnfD and N-terminus of NifK that in part occupy the same position within the quaternary structure of the respective dinitrogenases (Fig. 1a, Extended Data Fig. 3).

Extended Data Fig. 3 Structural Differences and Similarities between Mo- and Fe-nitrogenase.

a) Schematic representation of Fe-nitrogenase (left) and Mo-nitrogenase (right) from A. vinelandii. Although the surfaces of both enzymes appear distinct, the extended C-terminus of AnfD (red) occupies a very similar position on the protein surface as the C-terminus of NifK (blue). b) Detail views of A) with surface representations for AnfD with its C-terminus (left) and NifK with its N-terminus.

Extended Data Fig. 4 The P-cluster in A. vinelandii Fe nitrogenase.

The [8Fe:7S] cluster in the structure is observed in the all-ferrous PN state. The labelled Fe6 that moves towards the conserved serine S143K upon oxidation to the P+1 state does not show the dual conformation that is frequently observed in crystal structures. The stereo image shows the 2Fo–Fc electron density map contoured at the 2σ level (grey) and the 6σ level (blue), as well as an anomalous difference electron density map collected at the Fe K-edge contoured at the 5σ level (orange).

Extended Data Fig. 5 Homocitrate synthase from A. vinelandii.

a) Structure of the NifV gene product as predicted by Alphafold2. Like other known enzymes of this type, NifV forms a homodimer with a TIM-barrel domain that coordinates a Zn2+ ion in its centre as the active site. The metal ion and a homocitrate ligand were modelled based on the structure of R-homocitrate synthase from Thermus thermophilus (PDB 2ZTK)84. Homocitrate is formed by the condensation of 2-oxoglutarate with acetyl-CoA. b) The modelled metal site of NifV. Zn2+ is coordinated by two histidine and one aspartate residue. During catalysis, 2-oxoglutarate binds as a bidentate ligand to the metal and is then reacted with the acetyl group of acetyl-CoA.

Extended Data Fig. 6 Kinetic Scheme for N2 reduction by nitrogenases according to Lowe and Thorneley.

Catalysing an eight-electron process, the enzyme cycles through eight states E0–E7, in which each electron transfer (blue arrows) is accompanied by a protonation event for charge compensation. In an initial charging phase (grey), the enzyme must be reduced from its resting state E0 to at least E3, more likely E4, to gain the ability to bind and activate N2. From E2 on, unproductive H2 release is observed, which is commonly interpreted as the (unwanted) protonation of a surface hydride on the cofactor, turning the enzyme back two states in the cycle. In contrast, the H2 released in E4 is the result of the reductive elimination of H2 from two hydrides, leaving the cofactor in a 2-electron-reduced state that is uniquely capable of N2 binding and activation. The remaining steps of substrate reduction (E5–E7) are then facile. Lowe and Thorneley did not observe H2 formation from these states, indicating that no further surface hydrides are formed, and reduction occurs directly on the bound intermediates.

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Source data

Source Data Extended Data Fig. 1

Unprocessed SDS–PAGE for Extended Data Fig. 1B.

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Trncik, C., Detemple, F. & Einsle, O. Iron-only Fe-nitrogenase underscores common catalytic principles in biological nitrogen fixation. Nat Catal 6, 415–424 (2023). https://doi.org/10.1038/s41929-023-00952-1

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