Short-lived intermediate in N 2 O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography

• Published in: Proceedings of the National Academy of Sciences - PNAS 2021-05, Vol.118 (21)
• Main Authors: Nomura, Takashi, Kimura, Tetsunari, Kanematsu, Yusuke, Yamada, Daichi, Yamashita, Keitaro, Hirata, Kunio, Ueno, Go, Murakami, Hironori, Hisano, Tamao, Yamagiwa, Raika, Takeda, Hanae, Gopalasingam, Chai, Kousaka, Ryota, Yanagisawa, Sachiko, Shoji, Osami, Kumasaka, Takashi, Yamamoto, Masaki, Takano, Yu, Sugimoto, Hiroshi, Tosha, Takehiko, Kubo, Minoru, Shiro, Yoshitsugu
• Format: Article
• Language: English
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2101481118

Nitric oxide (NO) reductase from the fungus is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N 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 ( ), 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 TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe-NO coordination in , 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 with an N-O stretching frequency of 1,290 cm upon hydride transfer from NADH to the Fe -NO enzyme via the dissociation of NAD from a transient state, with an N-O stretching of 1,330 cm 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 is characterized by a singly protonated Fe -NHO radical. The current findings provide conclusive evidence for the N O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule.