Does the crystal structure of vanadium nitrogenase contain a reaction intermediate? Evidence from quantum refinement

Abstract

Recently, a crystal structure of V-nitrogenase was presented, showing that one of the µ2 sulphide ions in the active site (S2B) is replaced by a lighter atom, suggested to be NH or NH2, i.e. representing a reaction intermediate. Moreover, a sulphur atom is found 7 Å from the S2B site, suggested to represent a storage site for this ion when it is displaced. We have re-evaluated this structure with quantum refinement, i.e. standard crystallographic refinement in which the empirical restraints (employed to ensure that the final structure makes chemical sense) are replaced by more accurate quantum–mechanical calculations. This allows us to test various interpretations of the structure, employing quantum–mechanical calculations to predict the ideal structure and to use crystallographic measures like the real-space Z-score and electron-density difference maps to decide which structure fits the crystallographic raw data best. We show that the structure contains an OH−-bound state, rather than an N2-derived reaction intermediate. Moreover, the structure shows dual conformations in the active site with ~ 14% undissociated S2B ligand, but the storage site seems to be fully occupied, weakening the suggestion that it represents a storage site for the dissociated ligand. Graphic abstract

Critical evaluation of a crystal structure of nitrogenase with bound N2 ligands

“…1 ), bridging Fe2 and Fe6 [ 6 ]. A similar replacement was also observed for a crystal structure of a turnover state, which was originally interpreted as showing a N 2 -derived reaction intermediate [ 7 ], but later studies have shown that it probably contains an OH − ion instead [ 8 , 9 ]. This has inspired computational investigations of reaction mechanisms involving the exchange of S2B with the N 2 substrate [ 10 , 11 ].…”

Calculating the chemical mechanism of nitrogenase: new working hypotheses

“…[20][21][22][23][24] It remains to be determined whether these disrupted forms of FeMo-co are mechanistically valid or artefactual. [25][26][27] Another possibility is that S2B reversibly unhooks one of its bonds to Fe, changing from doubly-bridging to terminal S2B-H. This has been proposed and analysed using density functional calculations: [28][29][30][31][32][33][34] and in a forthcoming paper I analyse the factors that influence the unhooking and rehooking of S2B.…”

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

“…This forms a binding site, where N 2 binds in an end‐on fashion, bridging two Fe ions and it is then sequentially protonated on the outer N atom. The dissociation of the sulfide ion was inspired by several crystallographic studies of both Mo and V nitrogenase, showing that the S2B group can be replaced by several other ligands, for example CO, OH − and Se [8,52–56] …”

“…The dissociation of the sulfide ion was inspired by several crystallographic studies of both Mo and V nitrogenase, showing that the S2B group can be replaced by several other ligands, for example CO, OH À and Se. [8,[52][53][54][55][56] Recently, we studied a similar mechanism, involving dissociation of S2B. [57] We used a larger and more realistic model system, which was studied with the combined quantum mechanical and molecular mechanics (QM/MM) approach.…”

“…The dissociation of the sulfide ion was inspired by several crystallographic studies of both Mo and V nitrogenase, showing that the S2B group can be replaced by several other ligands, for example CO, OH − and Se. [ 8 , 52 , 53 , 54 , 55 , 56 ]…”