The Mechanism of Mo-Nitrogenase: from N2 capture to first release of NH3

Mo-nitrogenase hydrogenates N₂ to NH₃. This report continues from the previous paper [Dalton Transactions 53, 14193 (2024)] that described how the active site FeMo-co of the enzyme is uniquely able to capture and activate N₂, forming a key intermediate with Fe-bound HNNH. Density functional simulations with a 485+ atom model of the active site and surrounds are used to describe here the further reactions of this HNNH intermediate. The first step is hydrogenation to form HNNH₂ bridging Fe2 and Fe6. Then a single step reaction breaks the N-N bond, generating an Fe2-NH-Fe6 bridge and forming NH₃ bound to Fe6. Then NH₃ dissociates from Fe6. Reaction potential energies and kinetic barriers for all steps are reported for the most favourable electronic states of the system. The sequence of steps that follow the Fe2-NH-Fe6 intermediate, forming and dissociating the second NH₃, are outlined, including regeneration of the resting state of the enzyme. These results provide an interpretation of the recent steady-state kinetic data and analysis by Harris et al, [Biochemistry 61, 2131 (2022)] who found a slow step after the formation of the HNNH intermediate. The calculated potential energy barriers for the HNNH₂ → NH + NH3 reaction (30 - 36 kcal mol-1), are larger than the potential energy barriers for the N₂ → HNNH reaction (19 to 29 kcal mol-1). I propose that the post-HNNH slow step identified kinetically is the key HNNH₂ → NH + NH₃ reaction described here. This step and the N₂-capture step are the most difficult in the conversion of N₂ to 2NH₃. The steps in the complete mechanism still to be computationally detailed are relatively straightforward.