Daichi Yamada

Nitric oxide (NO) reductase from the fungus <italic>Fusarium oxysporum</italic> is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N₂O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate (<italic><underline>I</underline></italic>), a key state to promote N–N bond formation and N–O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of <italic><underline>I</underline></italic>. TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe–NO coordination in <italic><underline>I</underline></italic>, with an elongated Fe–NO bond length (Fe–NO = 1.91 Å, Fe–N–O = 138°) in the absence of NAD⁺. TR-infrared (IR) spectroscopy detects the formation of <italic><underline>I</underline></italic> with an N–O stretching frequency of 1,290 cm⁻¹ upon hydride transfer from NADH to the Fe³⁺–NO enzyme via the dissociation of NAD⁺ from a transient state, with an N–O stretching of 1,330 cm⁻¹ and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of <italic><underline>I</underline></italic> is characterized by a singly protonated Fe³⁺–NHO^•− radical. The current findings provide conclusive evidence for the N₂O generation mechanism via a radical–radical coupling of the heme nitroxyl complex with the second NO molecule.