Trapping of a Mononitrosyl Nonheme Intermediate of Nitric Oxide Reductase by Cryo-Photolysis of Caged Nitric Oxide

• Published in: The journal of physical chemistry. B 2023-02, Vol.127 (4), p.846-854
• Main Authors: Takeda, Hanae, Shimba, Kanji, Horitani, Masaki, Kimura, Tetsunari, Nomura, Takashi, Kubo, Minoru, Shiro, Yoshitsugu, Tosha, Takehiko
• Format: Article
• Language: English
• Subjects: B: Biophysical and Biochemical Systems and Processes; Metalloproteins; Nitric Oxide - chemistry; Oxidoreductases - chemistry; Photolysis
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/acs.jpcb.2c05852

Characterization of short-lived reaction intermediates is essential for elucidating the mechanism of the reaction catalyzed by metalloenzymes. Here, we demonstrated that the photolysis of a caged compound under cryogenic temperature followed by thermal annealing is an invaluable technique for trapping of short-lived reaction intermediates of metalloenzymes through the study of membrane-integrated nitric oxide reductase (NOR) that catalyzes reductive coupling of two NO molecules to N2O at its heme/nonheme FeB binuclear center. Although NO produced by the photolysis of caged NO did not react with NOR under cryogenic temperature, annealing to ∼160 K allowed NO to diffuse and react with NOR, which was evident from the appearance of EPR signals assignable to the S = 3/2 state. This indicates that the nonheme FeB-NO species can be trapped as the intermediate. Time-resolved IR spectroscopy with the use of the photolysis of caged NO as a reaction trigger showed that the intermediate formed at 10 μs gave the NO stretching frequency at 1683 cm–1 typical of nonheme Fe-NO, confirming that the combination of the cryo-photolysis of caged NO and annealing enabled us to trap the reaction intermediate. Thus, the cryo-photolysis of the caged compound has great potential for the characterization of short-lived reaction intermediates.