Reduction of N by nitrogenases occurs at an organometallic iron cofactor that commonly also contains either molybdenum or vanadium. The well-characterized resting state of the cofactor does not bind substrate, so its mode of action remains enigmatic. Carbon monoxide was recently found to replace a bridging sulfide, but the mechanistic relevance was unclear. Here we report the structural analysis of vanadium nitrogenase with a bound intermediate, interpreted as a μ-bridging, protonated nitrogen that implies the site and mode of substrate binding to the cofactor. Binding results in a flip of amino acid glutamine 176, which hydrogen-bonds the ligand and creates a holding position for the displaced sulfide. The intermediate likely represents state E or E of the Thorneley-Lowe model and provides clues to the remainder of the catalytic cycle.

Nitrogenases are responsible for biological nitrogen fixation, a crucial step in the biogeochemical nitrogen cycle. These enzymes utilize a two-component protein system and a series of iron–sulfur clusters to perform this reaction, culminating at the FeMco active site (M = Mo, V, Fe), which is capable of binding and reducing N2 to 2NH3. In this review, we summarize how different spectroscopic approaches have shed light on various aspects of these enzymes, including their structure, mechanism, alternative reactivity, and maturation. Synthetic model chemistry and theory have also played significant roles in developing our present understanding of these systems and are discussed in the context of their contributions to interpreting the nature of nitrogenases. Despite years of significant progress, there is still much to be learned from these enzymes through spectroscopic means, and we highlight where further spectroscopic investigations are needed.

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The FeMoco cluster of Mo nitrogenase undergoes minor distortions upon reduction to E1, supporting iron-based reduction and belt sulfide protonation.