The HD Reaction of Nitrogenase: a Detailed Mechanism

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.