Mechanistic Insights into Nitrogenase-Catalyzed Nitrogen Fixation: A QM/MM Study

Nitrogenase catalyzes the reduction of dinitrogen (N2) to ammonia via proton-coupled electron transfer at the FeMo-cofactor (FeMo-co). However, the sequence of H2 evolution and N2 activation at the key E4 intermediate remains highly debated. To address this, we employ a multiscale computational approach combining broken-symmetry DFT (BS-DFT, TPSSh), QM/MM, and molecular dynamics (MD) simulations to investigate the electronic structure and reactivity of the E4 state. Our analysis reveals the lowest-energy electronic configuration, showing that protonation localizes two hydrides on the FeMo-co. MD simulations indicate that N2 preferentially prebinds near the Fe2–Fe6 edge, stabilized by residues α-Val70 and α-His195. A quantitative comparison of mechanistic pathways demonstrates that reductive H2 elimination (barrier = 7.4 kcal mol–1) preceding N2 binding (8.0 kcal mol–1) is strongly favored over direct N2 binding first (barrier = 17.0 kcal mol–1), supporting an “H2-first” (Janus) model. Subsequent N2 hydrogenation proceeds through distinct η1- and μ2-bound intermediates that channel reactivity toward distal (kinetically favored) or alternating (thermodynamically favored) pathways. These results provide a unified framework for E4-state reactivity, establishing that H2 evolution is crucial for efficient N2 binding and activation at the Fe2 site.