Thermodynamically Favourable States in the Reaction of Nitrogenase without Dissociation of any Sulfide Ligand

Abstract

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 N₂ H₂ , after dissociation of H₂ . We show that the reaction follows an alternating mechanism with HNNH (possibly protonated to HNNH₂ ) and H₂ NNH₂ 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 , H₂ NNH₂ 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.

Keywords: QM/MM; alternating mechanism; homocitrate; nitrogen fixation; nitrogenase.

Figure 1

Structure of the FeMo cluster (with trans‐HNNH bound to Fe6), illustrating also the QM system used in all calculations, as well as the names of the nearby residues (a). (b) shows only the FeMo cluster with atom names indicated.

Figure 2

The best E₄ structures: (a) Fe6‐tHNNH, (b) Fe6‐HNNH₂ and (c) Fe2‐tHNNH(3), all with the HIE state of His‐195.

Figure 3

The best E₅ structures: (a) Fe6‐H₂NNH₂(3) and (b) Fe6‐HNNH₃(5) both with the HIE state of His‐195.

Figure 4

The best E₆ structures: (a) Fe6‐H₂NNH₂(3), (b) Fe6‐H₂NNH₂(5) and (c) Fe2‐H₂NNH₂(3), all with the HIE state of His‐195.

Figure 5

The best E₇ structures: (a) Fe6‐H₂NNH₃(5), (b) Fe6‐NH₂+NH₃, (c) H₂NNH₃ dissociated and (d) Fe2/6‐NH₂(5)‐Fe6‐NH₃, all with the HIE state of His‐195.

Figure 6

The best structures with only one N atom: (a) E₅‐Fe6‐N, (b) E₅‐Fe2‐N, (c) E₆‐Fe6‐NH₂, (d) E₇‐Fe6‐NH₃, all with the HIE state of His‐195.

Figure 7

The E₈ structures: (a) Fe6‐NH₃, (b) NH₄ dissociated and (c) Fe2‐NH₃, all with the HIE state of His‐195.

Figure 8

Suggested reaction mechanism for nitrogenase, assuming that S2B remains bound to the cluster.