The HD Reaction of Nitrogenase: a Detailed Mechanism

Nitrogenase is the enzyme that converts N(2) to NH(3) under ambient conditions. The chemical mechanism of this catalysis at the active site FeMo‐co [Fe(7)S(9)CMo(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 NH(3) 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.