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 reduce N2, the most abundant element in Earth's atmosphere that is otherwise resistant to chemical conversions due to its stable triple bond. Vanadium nitrogenase stands out in that it additionally processes carbon monoxide, a known inhibitor of the reduction of all substrates other than H+. The reduction of CO leads to the formation of hydrocarbon products, holding the potential for biotechnological applications in analogy to the industrial Fischer–Tropsch process. Here we report the most highly resolved structure of vanadium nitrogenase to date at 1.0 Å resolution, with CO bound to the active site cofactor after catalytic turnover. CO bridges iron ions Fe2 and Fe6, replacing sulfide S2B, in a binding mode that is in line with previous reports on the CO complex of molybdenum nitrogenase. We discuss the structural consequences of continued turnover when CO is removed, which involve the replacement of CO possibly by OH−, the movement of Q176D and K361D, the return of sulfide and the emergence of two additional water molecules that are absent in the CO‐bound state.

Nitrogenase enzymes are the only biological catalysts able to convert N 2 to NH 3 . Molybdenum-dependent nitrogenase consists of two proteins and three metallocofactors that sequentially shuttle eight electrons between three distinct metallocofactors during the turnover of one molecule of N 2 . While the kinetics of isolated nitrogenase has been extensively studied, little is known about the thermodynamics of its cofactors under catalytically relevant conditions. Here, we employ a recently described pyrenemodified linear poly(ethylenimine) hydrogel to immobilize the catalytic protein of nitrogenase onto an electrode surface. The resulting electroenzymatic interface enabled direct measurement of reduction potentials associated with each metallocofactor of the nitrogenase complex, illuminating the role of nitrogenase reductase in altering the potential landscape in the active site of nitrogenase and revealing the endergonic nature of electron-transfer steps associated with the conversion of N 2 to NH 3 under physiological conditions.

The binary vanadium nitrogenase system (VnfHDKG) is a homolog of the well‐characterized molybdenum nitrogenase system. Vanadium nitrogenase catalyzes the ATP‐dependent reduction of dinitrogen to ammonia, but is also able to reduce other substrates such as carbon monoxide. The Fe protein (VnfH) serves as an electron donor for the VFe protein (VnfDKG) of vanadium nitrogenase. Electrons are transferred from the Fe protein via the P‐cluster to the catalytic center at the FeV cofactor, where substrate binding and reduction occurs. While the Fe protein of vanadium nitrogenase shows no significant deviations from its molybdenum nitrogenase homolog, VFe protein comprises an additional subunit VnfG with unknown function, and its metal clusters, P‐cluster and FeV cofactor, exhibit structural features that previously have not been observed in the homologous molybdenum nitrogenase system.

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