The HD Reaction of Nitrogenase: a Detailed Mechanism

Nitrogenase is the enzyme that converts N-2 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 H-2, while exogenous H-2 is a competitive inhibitor. Isotopic substitution using exogenous D-2 revealed the N-2-dependent reaction D-2+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 D-2 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 D-2, 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 N-2-dependence results from promotional N-2 bound at exo-Fe2; (c) different N-2 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 -> H-2+D-2 results from the synchronicity of the two transfers of H to D-2; (f) inhibition of HD production at high pN(2) is by competitive binding of N-2 at endo-Fe6; (g) the non-leakage of D to solvent follows from the hydrophobic environment and irreversibility of proton introduction.

Automated summary

This study investigates the mechanism of the conversion of N-2 to NH3 catalyzed by nitrogenase. The authors propose a detailed mechanism for the reaction, where D-2 first binds to a Fe atom and then two H atoms transfer synchronously to D-2 to form HD. The HD molecules dissociate sequentially and the flanking H atoms are recovered through proton translocation and electron combination. This mechanism is consistent with experimental observations and provides insights into the catalytic process of nitrogenase.