Unravelling Catalytic Divergence in Mo- and Fe-Only Nitrogenases: The Role of the Heterometal-Site and Protein Environment from QM/MM Insights

Two key characteristics differentiate molybdenum-dependent (MoFe) and iron-only (FeFe) nitrogenases: their efficacy for N2 fixation and their product distributions for CO2 reduction, yielding HCO2– and CH4, respectively. Despite their divergent properties, prior research argues that the distinct cofactors share a common mechanism and equivalent structures with the addition of “n” electrons and protons at their En catalytic states. The proposed equivalence between the cofactors was foundational in the assignment of an Fe-hydride at their E1 states based on interpretation of FeFeco’s photolabile and thermolabile E1 isomers (Inorg. Chem. 2022, 61, 5459–5464). However, the E0 state crystal structures of FeMoco and FeFeco have sharp, previously unaddressed distinctions in their metal–metal distances and proximal residue identities at their respective octahedral M-sites (M = Mo or Fe). Herein, we study QM/MM models of the E0 and E1 states of FeMoco and FeFeco to distinguish their geometric and electronic structures. Our analysis shows diminished metal–metal bonding and increased hydrogen bonding at FeFeco’s M-site, supporting the presence of two energetically low-lying E1 states differentiated by protonation of a μ3- or μ2-sulfide. The calculated thermodynamic and kinetic properties of FeFeco’s sulfide-protonated states agree with experimental data, without invoking Fe-hydride formation. Unlike FeFeco, FeMoco’s E1 state strictly favors μ2-sulfide protonation. FeFeco’s distinct μ3-sulfide-protonated E1 isomer has a five-coordinate, reduced M-site that could explain its divergent reactivity relative to FeMoco.