The Mechanism of Nitrogenase: formation and release of the second NH3 and completion of the cycle

The enzyme nitrogenase catalyses the reaction N₂ + 8e^– + 8H⁺ = 2NH₃ + H₂. Two prior papers in this series report a computed mechanism for the first and second phases of this catalysis. In the first phase H₂ is formed, the H₂/N2 exchange occurs, N₂ is captured in a concerted step forming the bound HNHH intermediate. The second phase breaks the N-N bond and converts this intermediate to bound NH plus the first NH₃, which dissociates. This third paper describes the final phase, which forms and then releases the second NH₃, and recovers the resting state. The mechanism is supported by density functional calculations with a 483+ atom quantum model of the active site, FeMo-co, and relevant surrounding amino acids and water. Calculated reaction trajectories and potential energy profiles generate five mechanistic pathways through this NH₃ formation phase of the reaction. These pathways are evaluated with incorporation of entropic components and possible kinetic contributions by H atom tunneling, leading to the identification of the most favourable pathway for generation of NH₃ and its subsequent dissociation. The steps regenerating the resting state and completing the mechanism cycle are described. All steps in this third and final phase of the mechanism are thermodynamically and kinetically feasible. Atom S2B of FeMo-co, whose retention during enzyme turnover is experimentally controversial, remains intact as a bridge between Fe2 and Fe6 and is an essential H transfer agent in the proposed mechanism. The architecture of the active site and its surrounds that promote the chemical choreography of nitrogenase in its performance space are outlined, and key features and principles of the proposed complete mechanism are summarised.