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

Abstract

Nitrogenase is the only enzyme that can cleave the triple bond in N 2 , 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 μ 2 -bridging sulfide ligands (S2B) may dissociate from one of the Fe ions when protonated in the doubly reduced and protonated E 2 state, whereas other studies indicated that such half-dissociated states are unfavorable. We have examined how the relative energies of 26 structures of the E 2 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 r 2 SCAN (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.

Understanding the Electronic Structure Basis for N2 Binding to FeMoco: A Systematic Quantum Mechanics/Molecular Mechanics Investigation

“…Both models are shown in Figure 1 . As discussed both in ref ( 12 ) and more recently in ref ( 35 ), E 2 isomers are, however, quite sensitive to the DFT method used with TPSS, e.g., giving opposite trends to r 2 SCAN and TPSSh. A saddlepoint leading to H 2 formation from E 2 -hyd → E 0 was also reported in ref ( 12 ), with a fairly high activation barrier of ∼20 kcal/mol, which is consistent with a recent kinetics study, 9 indicating the E 2 → E 0 step to be slower than other H 2 formation pathways.…”

“…Such opening of protonated belt sulfides has also been discussed in computational work by Dance, 40 Raugei, 13 and recently by Ryde and co-workers. 35 Kinetic and spectroscopic studies 7,37,41−44 indicate that dinitrogen will bind to the E 4 state, while no binding has been directly observed in earlier E n states (with the role of E 3 unclear). Alternative substrates and inhibitors like hydrazine, diazene, acetylene, and CO likely bind on the other hand to the E 1 or E 2 states.…”

“…The E 2 -hyd with a terminal SH – on Fe6, named as E 2 -hyd-SH – @Fe6 , is more stable than the one with a SH – on Fe2, as also seen in a recent QM/MM study. 35 The E 2 -nonhyd model contains a doubly reduced Fe environment and two protons located on S2B and S5A. Both models are shown in Figure 1 .…”

“…Such opening of protonated belt sulfides has also been discussed in computational work by Dance, 40 Raugei, 13 and recently by Ryde and co-workers. 35 …”

The binding of reducible N2 in the reaction domain of nitrogenase

“…28–30 A feasible variation of the reaction domain is the unhooking of S2B–H from one of its two Fe atoms. 31–38 I have analysed the factors that influence this unhooking, and described the unhooking of S2B (or S2BH) from Fe2, tethered to Fe6, and the rehooking reaction. 39 2H + + 2e − → H 2 N 2 + 6H + + 6e − → 2NH 3 D 2 + 2H + + 2e − → 2HD…”