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 Tyr(197) 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 Fe-57 Mossbauer modeling in elucidating the structural features of not only first, but also second coordination spheres of the key transient species involved in NO/O-2 activation by non-hence diiron enzymes.