Nitrogenase is the only enzyme that can convert N₂ to NH₃. Crystallographic structures have indicated that one of the sulfide ligands of the active‐site FeMo cluster, S2B, can be replaced by an inhibitor, like CO and OH⁻, and it has been suggested that it may be displaced also during the normal reaction. We have investigated possible proton transfer pathways within the FeMo cluster during the conversion of N₂H₂ to two molecules of NH₃, assuming that the protons enter the cluster at the S3B, S4B or S5A sulfide ions and are then transferred to the substrate. We use combined quantum mechanical and molecular mechanical (QM/MM) calculations with the TPSS and B3LYP functionals. The calculations indicate that the barriers for these reactions are reasonable if the S2B ligand remains bound to the cluster, but they become prohibitively high if S2B has dissociated. This suggests that it is unlikely that S2B reversibly dissociates during the normal reaction cycle.

We have used combined quantum mechanical and molecular mechanical (QM/MM) calculations to study the reaction mechanism of nitrogenase, assuming that none of the sulfide ligands dissociates. To avoid the problem that there is no consensus regarding the structure and protonation of the E₄ state, we start from a state where N₂ is bound to the cluster and is protonated to N₂H₂, after dissociation of H₂. We show that the reaction follows an alternating mechanism with HNNH (possibly protonated to HNNH₂) and H₂NNH₂ as intermediates and the two NH₃ products dissociate at the E₇ and E₈ levels. For all intermediates, coordination to Fe6 is preferred, but for the E₄ and E₈ intermediates, binding to Fe2 is competitive. For the E₄, E₅ and E₇ intermediates we find that the substrate may abstract a proton from the hydroxy group of the homocitrate ligand of the FeMo cluster, thereby forming HNNH₂, H₂NNH₂ and NH₃ intermediates. This may explain why homocitrate is a mandatory component of nitrogenase. All steps in the suggested reaction mechanism are thermodynamically favourable compared to protonation of the nearby His‐195 group and in all cases, protonation of the NE2 atom of the latter group is preferred.

Nitrogenase is the only enzyme that can cleave the triple bond in N₂, making nitrogen available for all lifeforms. Previous computational studies have given widely diverging results regarding the reaction mechanism of the enzyme. For example, some recent studies have suggested that one of the μ₂-bridging sulfide ligands (S2B) may dissociate from one of the Fe ions when protonated in the doubly reduced and protonated E₂ state, whereas other studies indicated that such half-dissociated states are unfavorable. We have examined how the relative energies of 26 structures of the E₂ state depend on details of combined quantum mechanical and molecular mechanical (QM/MM) calculations. We show that the selection of the broken-symmetry state, the basis set, relativistic effects, the size of the QM system, relaxation of the surroundings, and the conformations of the bound protons may affect the relative energies of the various structures by up to 12, 22, 9, 20, 37, and 33 kJ/mol, respectively. However, they do not change the preferred type of structures. On the other hand, the choice of the DFT functional strongly affects the preferences. The hybrid B3LYP functional strongly prefers doubly protonation of the central carbide ion, but such a structure is not consistent with experimental EPR data. Other functionals suggest structures with a hydride ion, in agreement with the experiments, and show that the ion bridges between Fe2 and Fe6. Moreover, there are two structures of the same type that are degenerate within 1-5 kJ/mol, in agreement with the observation of two EPR signals. However, the pure generalized gradient approximation (GGA) functional TPSS favors structures with a protonated S2B also bridging Fe2 and Fe6, whereas r²SCAN (meta-GGA) and TPSSh (hybrid) prefer structures with S2B dissociated from Fe2 (but remaining bound to Fe6). The energy difference between the two types of structure is so small (7-18 kJ/mol) that both types need to be considered in future investigations of the mechanism of nitrogenase.

We study the binding of N₂to nitrogenase in the E₀–E₄state with QM/MM and four DFT functionals. In general, structures with two hydride ions bridging the same pair of Fe ions and a sulfide ion dissociated from one Fe ion are low in energy.

Monophenolase and diphenolase activities of the [Cu₂O₂]²⁺active site ofAspergillus oryzaecatechol oxidase are revealed by hybrid QM/MM calculations.