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.