부산대학교 도서관

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide ([N.sub.2]O) 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 [Angstrom], Fe-N-O = 138[degrees]) in the absence of [NAD.sup.+]. TR-infrared (IR) spectroscopy detects the formation of I with an N-O stretching frequency of 1,290 [cm.sup.-1] upon hydride transfer from NADH to the [Fe.sup.3+]-NO enzyme via the dissociation of [NAD.sup.+] from a transient state, with an N-O stretching of 1,330 [cm.sup.-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 [Fe.sup.3+]-NHO*- radical. The current findings provide conclusive evidence for the [N.sub.2]O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule. nitric oxide | NOR | time-resolved IR spectroscopy | XFEL-based crystallography | in crystallo microspectroscopy