Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum 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 (I), 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 I. TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe–NO coordination in I, 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 I 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 I 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.