7DVO

Structure of Reaction Intermediate of Cytochrome P450 NO Reductase (P450nor) Determined by XFEL

Summary for 7DVO

• Entry DOI: 10.2210/pdb7dvo/pdb
• Descriptor: NADP nitrous oxide-forming nitric oxide reductase, PROTOPORPHYRIN IX CONTAINING FE, NITRIC OXIDE, ... (5 entities in total)
• Functional Keywords: heme enzyme, oxidoreductase, metal binding protein
• Biological source: Fusarium oxysporum (Fusarium vascular wilt)
• Total number of polymer chains: 2
• Total formula weight: 90686.93

Authors

Nomura, T.,Kimura, T.,Kanematsu, Y.,Yamashita, K.,Hirata, K.,Ueno, G.,Murakami, H.,Hisano, T.,Yamagiwa, R.,Takeda, H.,Gopalasingam, C.,Yuki, K.,Kousaka, R.,Yanagasawa, S.,Shoji, O.,Kumasaka, T.,Takano, Y.,Ago, H.,Yamamoto, M.,Sugimoto, H.,Tosha, T.,Kubo, M.,Shiro, Y. (deposition date: 2021-01-14, release date: 2021-05-19, Last modification date: 2023-11-29)

Primary citation

Nomura, T.,Kimura, T.,Kanematsu, Y.,Yamada, D.,Yamashita, K.,Hirata, K.,Ueno, G.,Murakami, H.,Hisano, T.,Yamagiwa, R.,Takeda, H.,Gopalasingam, C.,Kousaka, R.,Yanagisawa, S.,Shoji, O.,Kumasaka, T.,Yamamoto, M.,Takano, Y.,Sugimoto, H.,Tosha, T.,Kubo, M.,Shiro, Y.
Short-lived intermediate in N 2 O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography.
Proc.Natl.Acad.Sci.USA, 118:-, 2021

PubMed Abstract: Nitric oxide (NO) reductase from the fungus is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (NO) 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 NO generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule.

PubMed: 34001620
DOI: 10.1073/pnas.2101481118

Experimental method

X-RAY DIFFRACTION (1.8 Å)