QM/MM Study of Partial Dissociation of S2B for the E₂ Intermediate of Nitrogenase

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.