The binding of reducible N 2 in the reaction domain of nitrogenase.

The binding of N ₂ to FeMo-co, the catalytic site of the enzyme nitrogenase, is central to the conversion to NH ₃ , but also has a separate role in promoting the N ₂ -dependent HD reaction (D ₂ + 2H ⁺ + 2e ^- → 2HD). The protein surrounding FeMo-co contains a clear channel for ingress of N ₂ , directly towards the exo -coordination position of Fe2, a position which is outside the catalytic reaction domain. This led to the hypothesis [I. Dance, Dalton Trans ., 2022, 51 , 12717] of 'promotional' N ₂ bound at exo -Fe2, and a second 'reducible' N ₂ bound in the reaction domain, specifically the endo -coordination position of Fe2 or Fe6. The range of possibilities for the binding of reducible N ₂ in the presence of bound promotional N ₂ is described here, using density functional simulations with a 486 atom model of the active site and surrounding protein. The pathway for ingress of the second N ₂ through protein, past the first N ₂ at exo -Fe2, and tumbling into the binding domain between Fe2 and Fe6, is described. The calculations explore 24 structures involving 6 different forms of hydrogenated FeMo-co, including structures with S2BH unhooked from Fe2 but tethered to Fe6. The calculations use the most probable electronic states. End-on (η ¹ ) binding of N ₂ at the endo position of either Fe2 or Fe6 is almost invariably exothermic, with binding potential energies ranging up to -18 kcal mol ^-1 . Many structures have binding energies in the range -6 to -14 kcal mol ^-1 . The relevant entropic penalty for N ₂ binding from a diffusible position within the protein is estimated to be 4 kcal mol ^-1 , and so the binding free energies for reducible N ₂ are suitably negative. N ₂ binding at endo -Fe2 is stronger than at endo -Fe6 in three of the six structure categories. In many cases the reaction domain containing reducible N ₂ is expanded. These results inform computational simulation of the subsequent steps in which surrounding H atoms transfer to reducible N ₂ .