Understanding the Electronic Structure Basis for N₂ Binding to FeMoco: A Systematic Quantum Mechanics/Molecular...

The FeMo cofactor (FeMoco) of Mo nitrogenase is responsible for reducing dinitrogen to ammonia, but it requires the addition of 3-4 e^-/H⁺ pairs before N₂ even binds. A binding site at the Fe2/Fe3/Fe6/Fe7 face of the cofactor has long been suggested based on mutation studies, with Fe2 or Fe6 nowadays being primarily discussed as possibilities. However, the nature of N₂ binding to the cofactor is enigmatic as the metal ions are coordinatively saturated in the resting state with no obvious binding site. Furthermore, the cofactor consists of high-spin Fe(II)/Fe(III) ions (antiferromagnetically coupled but also mixed-valence delocalized), which are not known to bind N₂. This suggests that an Fe binding site with a different molecular and electronic structure than the resting state must be responsible for the experimentally known N₂ binding in the E state of FeMoco. We have systematically studied N₂ binding to Fe2 and Fe6 sites of FeMoco at the broken-symmetry QM/MM level as a function of the redox-, spin-, and protonation state of the cofactor. The local and global electronic structure changes to the cofactor taking place during redox events, protonation, Fe-S cleavage, hydride formation, and N₂ coordination are systematically analyzed. Localized orbital and quasi-restricted orbital analysis via diamagnetic substitution is used to get insights into the local single Fe ion electronic structure in various states of FeMoco. A few factors emerge as essential to N₂ binding in the calculations: spin-pairing of d and d orbitals of the N₂-binding Fe ion, a coordination change at the N₂-binding Fe ion aided by a hemilabile protonated sulfur, and finally hydride ligation. The results show that N₂ binding to E, E, and E models is generally unfavorable, likely due to the high-energy cost of stabilizing the necessary spin-paired electronic structure of the N₂-binding Fe ion in a ligand environment that clearly favors high-spin states. The results for models of E, however, suggest a feasible model for why N₂ binding occurs experimentally in the E state. E models with two bridging hydrides between Fe2 and Fe6 show much more favorable N₂ binding than other models. When two hydrides coordinate to the same Fe ion, an increased ligand-field splitting due to octahedral coordination at either Fe2 or Fe6 is found. This altered ligand field makes it easier for the Fe ion to acquire a spin-paired electronic structure with doubly occupied d and d orbitals that backbond to a terminal N₂ ligand. Within this model for N₂ binding, both Fe2 and Fe6 emerge as possible binding site scenarios. Due to steric effects involving the His195 residue, affecting both the N₂ ligand and the terminal SH^- group, distinguishing between Fe2 and Fe6 is difficult; furthermore, the binding depends on the protonation state of His195.