The mechanism of Mo-nitrogenase: from N capture to first release of NH.

Mo-nitrogenase hydrogenates N to NH. This report continues from the previous paper [I. Dance, , 2024, , 14193-14211] that described how the active site FeMo-co of the enzyme is uniquely able to capture and activate N, forming a key intermediate with Fe-bound HNNH. Density functional simulations with a 485+ atom model of the active site and its surroundings are used to describe here the further reactions of this HNNH intermediate. The first step is hydrogenation to form HNNH bridging Fe2 and Fe6. Then a single-step reaction breaks the N-N bond, generating an Fe2-NH-Fe6 bridge and forming NH bound to Fe6. Then NH dissociates from Fe6. Reaction potential energies and kinetic barriers for all steps are reported for the most favourable electronic states of the system. The steps that follow the Fe2-NH-Fe6 intermediate, forming and dissociating the second NH, and regenerating the resting state of the enzyme, are outlined. These results provide an interpretation of the recent steady-state kinetics data and analysis by Harris ., [, 2022, , 2131-2137] who found a slow step after the formation of the HNNH intermediate. The calculated potential energy barriers for the HNNH → NH + NH reaction (30-36 kcal mol) are larger than the potential energy barriers for the N → HNNH reaction (19-29 kcal mol). I propose that the post-HNNH slow step identified kinetically is the key HNNH → NH + NH reaction described here. This step and the N-capture step are the most difficult in the conversion of N to 2NH. The steps in the complete mechanism still to be computationally detailed are relatively straightforward.