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 sho...

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Published in:Proceedings of the National Academy of Sciences - PNAS 2021-05, Vol.118 (21), p.1
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
Subjects:
Biological Sciences
Coordination
Coupling (molecular)
Crystallography
Electronic structure
Enzymes
Free electron lasers
Fusarium oxysporum
Heme
Hydrides
Infrared spectroscopy
Iron
NAD
NADH
Nicotinamide adenine dinucleotide
Nitric oxide
Nitrogen
Nitrogen cycle
Nitrous oxide
Physical Sciences
Quantum mechanics
Reductases
Spectrum analysis
Stretching
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2101481118