Origin of Nitric Oxide Reduction Activity in Flavo–Diiron NO Reductase: Key Roles of the Second Coordination Sphere

The second coordination sphere constitutes a distinguishing factor in the active site to modulate enzymatic reactivity. To unravel the origin of NO‐to‐N₂O reduction activity of non‐heme diiron enzymes, herein we report a strong second‐coordination‐sphere interaction between a conserved Tyr₁₉₇ and the key iron–nitrosyl intermediate of Tm FDP (flavo–diiron protein), which leads to decreased reaction barriers towards N–N formation and N–O cleavage in NO reduction. This finding supports the direct coupling of diiron dinitrosyl as the N–N formation mode in our QM/MM modeling, and reconciles the mechanistic controversy of external reduction between FDPs and synthetic biomimetics of the iron–nitrosyls. This work highlights the application of QM/MM ⁵⁷Fe Mössbauer modeling in elucidating the structural features of not only first, but also second coordination spheres of the key transient species involved in NO/O₂ activation by non‐heme diiron enzymes.