Proton Transfer Pathways in Nitrogenase with and without Dissociated S2B

Abstract

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 N₂ H₂ 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.

Keywords: QM/MM; S2B dissociation; nitrogenase; proton transfer; reaction mechanisms.

Figure 1

The FeMo cluster with atom names indicated. All structures in the figures use the same perspective.

Figure 2

Reaction and activation energies, as well as structures for proton transfers in the E₅ state without S2B bound to the cluster, from S5A, S4B or S3B to the NNH₂ intermediate bound to Fe6 and Fe2, leading to a HNNH₂ product. Energies in black and red indicate that the BS10‐147 and BS7‐235 states are most stable, respectively. S4B(7) differs from S4B in that the proton points towards Fe7, rather than towards Mo. Fe7(2) differs from Fe7 in that the proton points towards Fe2, rather than towards S4B. In S3B(7) and S3B(6), the proton points towards Fe7 or Fe6, respectively. Fe6(Mo) differs from Fe6 in that the proton points towards Mo, rather than towards Fe2 (sometimes called the exo and endo positions, respectively).[ ³³ , ³⁵ , ³⁶ , ³⁷ ] HNNH₂(3) differs from HNNH₂(5) in that the added proton points towards S5A rather than S3A. A final prime in the name of the structures (e.g. S3B(6)′) indicates that the NNH₂ substrate has changed its conformation so that the NH₂ group binds to Fe6 (while the other N atom still bridges Fe2 and Fe6).

Figure 3

Reaction and activation energies, as well as structures for proton transfers in the E₆ state without S2B bound to the cluster. Energies in black and red indicate that BS10‐147 and BS7‐235 states are most stable, respectively. Fe7(5) differs from Fe7 in that the proton points towards S5A.

Figure 4

Reaction and activation energies, as well as structures for proton transfers in the E₇ state without S2B bound to the cluster. Energies in black and red indicate that BS10‐147 and BS7‐235 states are most stable, respectively.

Figure 5

Reaction and activation energies, as well as structures for proton transfers in the E₈ state without S2B bound to the cluster. Energies in black and red indicate that BS10‐147 and BS7‐235 states are most stable, respectively.

Figure 6

Reaction and activation energies, as well as structures for proton transfers in the E₅ state with S2B remaining bound to the cluster.

Figure 7

Reaction and activation energies, as well as structures for proton transfers in the E₆ state with S2B remaining bound to the cluster, from S5A, S4B or S3B to the H₂NNH₂ intermediate bound to Fe6, leading to H₂NNH₂ or NH₂ product. In the H₂NNH₂ structure, the extra proton has been transferred to homocitrate.

Figure 8

Reaction and activation energies, as well as structures for proton transfers in the E₇ state with S2B remaining bound to the cluster, from S5A, S4B or S3B to the H₂NNH₂ intermediate bound to Fe6, leading to a NH₂ product.

Figure 9

Reaction and activation energies, as well as structures for proton transfers in the E₇ state with S2B remaining bound to the cluster, from S5A, S4B or S3B to the NH₂ intermediate bound to Fe6, leading to a NH₃ product.

Figure 10

Reaction and activation energies, as well as structures for proton transfers in the E₈ state with S2B remaining bound to the cluster. In the S3B(HCA) structure, the extra proton points towards homocitrate.

Figure 11

Relative energies for the proton‐transfer reactions at the various E _n states with and without S2B bound. As shown in Figures 2–10, there are often multiple possible paths, but this figure shows only the most favourable ones (those with the lowest net barriers). Alternative paths, inspired by the results for the other E _n states were also tested, but sometimes failed owing to subtle differences in the structures.