The mechanism of nitrogenase remains enigmatic, with a major unresolved issue concerning how inhibitors and substrates bind to the active site. We report a crystal structure of carbon monoxide (CO) inhibited nitrogenase MoFe-protein at 1.50 Å resolution, revealing a CO molecule bridging Fe2 and Fe6 of the FeMo-cofactor. The μ2 binding geometry is achieved by replacing a belt-sulfur atom (S2B) and highlights the generation of a reactive iron species uncovered by the displacement of sulfur. The CO inhibition is fully reversible as established by regain of enzyme activity and reappearance of S2B in the 1.43 Å resolution structure of the reactivated enzyme. The substantial and reversible reorganization of the FeMo-cofactor accompanying CO binding was unanticipated and provides insights into a catalytically competent state of nitrogenase.
Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.DOI:
http://dx.doi.org/10.7554/eLife.11620.001
As an approach towards unraveling the nitrogenase mechanism, we have studied the binding of CO to the active‐site FeMo‐cofactor. CO is not only an inhibitor of nitrogenase, but it is also a substrate, undergoing reduction to hydrocarbons (Fischer–Tropsch‐type chemistry). The C−C bond forming capabilities of nitrogenase suggest that multiple CO or CO‐derived ligands bind to the active site. Herein, we report a crystal structure with two CO ligands coordinated to the FeMo‐cofactor of the molybdenum nitrogenase at 1.33 Å resolution. In addition to the previously observed bridging CO ligand between Fe2 and Fe6 of the FeMo‐cofactor, a new ligand binding mode is revealed through a second CO ligand coordinated terminally to Fe6. While the relevance of this state to nitrogenase‐catalyzed reactions remains to be established, it highlights the privileged roles for Fe2 and Fe6 in ligand binding, with multiple coordination modes available depending on the ligand and reaction conditions.
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