The binding of reducible N₂ in the reaction domain of nitrogenase

The binding of N₂ to FeMo-co, the catalytic site of the enzyme nitrogenase, is central to the conversion to NH₃, but also has a separate role in promoting the N₂-dependent HD reaction (D₂ + 2H⁺ + 2e^- → 2HD). The protein surrounding FeMo-co contains a clear channel for ingress of N₂, 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' N₂ bound at exo-Fe2, and a second 'reducible' N₂ bound in the reaction domain, specifically the endo-coordination position of Fe2 or Fe6. The range of possibilities for the binding of reducible N₂ in the presence of bound promotional N₂ 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 N₂ through protein, past the first N₂ 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 (η¹) binding of N₂ 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 N₂ binding from a diffusible position within the protein is estimated to be 4 kcal mol^-1, and so the binding free energies for reducible N₂ are suitably negative. N₂ 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 N₂ is expanded. These results inform computational simulation of the subsequent steps in which surrounding H atoms transfer to reducible N₂.