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

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 similar 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 its 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.

Automated summary

The characterization of short-lived reaction intermediates is crucial for understanding the mechanism of metalloenzyme-catalyzed reactions. In this study, we demonstrated that the photolysis of a caged compound followed by thermal annealing is an effective technique for trapping short-lived reaction intermediates of metalloenzymes. By applying this technique to the study of membrane-integrated nitric oxide reductase (NOR), we were able to trap and characterize the intermediate species involved in the reductive coupling of NO molecules by NOR at its heme/nonheme FeB binuclear center.