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 [Fe7S9CMo(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 Km 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.

The binding of reducible N2 in the reaction domain of nitrogenase

“…In that paper I reported one calculation of the end-on binding of reducible-N 2 also at endo-Fe6, and indicated how N 2 could diffuse towards this site. 51 I now explore more fully the possibilities for the inward diffusion and binding of the reducible N 2 , in the presence of promotional N 2 at exo-Fe2. These calculations are precursors to the development of mechanisms for the hydrogenation of reducible N 2 .…”

“…the trans Fe2-C c bond. This enlargement facilitates further reactivity in this space, such as the binding of H 2 /D 2 at the endo-Fe site not occupied by reducible N 2 , 51 the conversion of end-on reducible N 2 to a bridging bonding mode, and the transfer of H atoms from the surrounding gallery to reducible N 2 .…”

“…Previous calculations of the HD reaction did not explore options involving the unhooking of S2B. 51 The framework for these continuing investigations of the various reaction steps is shown in Scheme 1. These are not intended to represent the E n (nH) intermediates in the kinetically derived reaction scheme: 46,50,89 my calculations aim to derive an understanding of the fundamental chemistry of reaction steps, as needed for reliable formulation of the intermediates.…”