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

Nitrogenase catalyzes the reduction of dinitrogen (N 2 ) to ammonia via proton-coupled electron transfer at the FeMo-cofactor (FeMo-co). However, the sequence of H 2 evolution and N 2 activation at the key E 4 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 E 4 state. Our analysis reveals the lowest-energy electronic configuration, showing that protonation localizes two hydrides on the FeMo-co. MD simulations indicate that N 2 preferentially prebinds near the Fe2–Fe6 edge, stabilized by residues α-Val70 and α-His195. A quantitative comparison of mechanistic pathways demonstrates that reductive H 2 elimination (barrier = 7.4 kcal mol –1 ) preceding N 2 binding (8.0 kcal mol –1 ) is strongly favored over direct N 2 binding first (barrier = 17.0 kcal mol –1 ), supporting an “H 2 -first” (Janus) model. Subsequent N 2 hydrogenation proceeds through distinct η 1 - and μ 2 -bound intermediates that channel reactivity toward distal (kinetically favored) or alternating (thermodynamically favored) pathways. These results provide a unified framework for E 4 -state reactivity, establishing that H 2 evolution is crucial for efficient N 2 binding and activation at the Fe2 site.