The HD Reaction of Nitrogenase: a Detailed Mechanism

Abstract

Nitrogenase is the enzyme that converts N₂ to NH₃ under ambient conditions. The chemical mechanism of this catalysis at the active site FeMo-co [Fe₇ S₉ CMo(homocitrate)] is unknown. An obligatory co-product is H₂ , while exogenous H₂ is a competitive inhibitor. Isotopic substitution using exogenous D₂ revealed the N₂ -dependent reaction D₂ +2H⁺ +2e^- →2HD (the 'HD reaction'), together with a collection of additional experimental characteristics and requirements. This paper describes a detailed mechanism for the HD reaction, developed and elaborated using density functional simulations with a 486-atom model of the active site and surrounding protein. First D₂ binds at one Fe atom (endo-Fe6 coordination position), where it is flanked by H-Fe6 (exo position) and H-Fe2 (endo position). Then there is synchronous transfer of these two H atoms to bound D₂ , forming one HD bound to Fe2 and a second HD bound to Fe6. These two HD dissociate sequentially. The final phase is recovery of the two flanking H atoms. These H atoms are generated, sequentially, by translocation of a proton from the protein surface to S3B of FeMo-co and combination with introduced electrons. The first H atom migrates from S3B to exo-Fe6 and the second from S3B to endo-Fe2. Reaction energies and kinetic barriers are reported for all steps. This mechanism accounts for the experimental data: (a) stoichiometry; (b) the N₂ -dependence results from promotional N₂ bound at exo-Fe2; (c) different N₂ binding K_m for the HD reaction and the NH₃ formation reaction results from involvement of two different sites; (d) inhibition by CO; (e) the non-occurrence of 2HD→H₂ +D₂ results from the synchronicity of the two transfers of H to D₂ ; (f) inhibition of HD production at high pN₂ is by competitive binding of N₂ at endo-Fe6; (g) the non-leakage of D to solvent follows from the hydrophobic environment and irreversibility of proton introduction.

Keywords: HD reaction; density functional calculations; enzyme catalysis; nitrogenases; reaction mechanisms.

Figure 1

(a) FeMo‐co, the active site of nitrogenase, an Fe₇MoS₉C^c cluster with bidentate coordination of Mo by homocitrate and ligation by His442 at Mo and Cys275 at Fe1. C^c is a central carbon atom bonded equally to six Fe atoms in the resting state. Fe magenta, Mo brown, C atoms of homocitrate are dark green. The positions of the significant amino acids Val70, Arg96, Gln191 and His195 surrounding FeMo‐co are marked. Residue numbering is from the α‐subunit of Azotobacter vinelandii, crystal structure PDB 3 U7Q. Hydrogen bonds are striped. (b) Atom labels for the cluster, and identification of the potential coordination positions at Fe2 and Fe6, exo or endo relative to the Fe−C^c bond. S2B, S5A and S3A are often referred to as ‘belt’ atoms.

Figure 2

(a) The pathways proposed for N₂ and H₂ accessing the active site. His383 (blue surface) is at the channel mouth on the surface of the protein. (b) A working hypothesis, in which the role of exo‐Fe2−N₂ (yellow) is to expand the reaction zone (violet) between Fe2 and Fe6, thereby promoting other reactions including binding of the N₂ that is subsequently hydrogenated to NH₃, in competition with the binding of H₂ at the same site.

Figure 3

When the spin populations of Fe2 and Fe6 are diminished due to ligation, there are two pairs of negative spin, red or blue, that yield four opposite (green) spin pairs along the shorter edges of the Fe3, Fe4, Fe5, Fe7 face where the spin populations have largest magnitude.

Scheme 1

First stages of the mechanism, showing the binding of D₂ (2) and the synchronous double hydrogenation of D₂ forming two bound HD (3). The sketches show only the essential components. The triangular symbols report electronic(S) states and calculated energies (kcal mol⁻¹): the reaction energy is on the horizontal arrow, and barrier energies are oblique to the transition state (TS).

Scheme 2

Two routes for the sequential dissociation of two Fe‐bound HD.

Scheme 3

Reaction sequences for the introduction of the H atom at exo‐Fe6 (upper panel) and subsequently introduction of the second H atom and its movement to the endo‐Fe2 position (lower panel). Both H atoms begin at S3B in the 3b5 conformation. The sketches show only essential components, and the triangular symbols report electronic(S) states and calculated energies (kcal mol⁻¹): the reaction energy is on the horizontal arrow, and barrier energies are oblique to the transition state (TS).

Figure 4

Geometric details of the synchronous conversion of D₂ to 2HD. D atoms black, distances in Å.

Figure 5

The last three stages in the mechanism for proton translocation along the water chain (red) and protonation of S3B (C). Extracted from Ref. [32c].

Figure 6

The conformations of S3B−H and their labels (modified from Ref [32c]). The four yellow highlighted structures are energy minima with pyramidal stereochemistry at S3B, and the other four structures are the transition states between them. Distances (Å) and energies (kcal mol⁻¹) are those calculated for unligated FeMo‐co. ^[32c]

Figure 7

Movement of endo‐Fe2−H when the exo‐Fe2 position contains H or is void.

Scheme 4

An alternative mechanism that allows the reaction 2HD→D₂+H₂. This cannot occur with the proposed mechanism because the essential intermediate, yellow highlight, is absent.