Short-lived intermediate in N <sub>2</sub> O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography
Takashi Nomura, Tetsunari Kimura, Yusuke Kanematsu, Daichi Yamada, Keitaro Yamashita, Kunio Hirata, Go Ueno, Hironori Murakami, Tamao Hisano, Raika Yamagiwa, Hanae Takeda, Chai C. Gopalasingam, Ryota Kousaka, Sachiko Yanagisawa, Osami Shoji, Takashi Kumasaka, Masaki Yamamoto, Yu Takano, Hiroshi Sugimoto, Takehiko Tosha, Minoru Kubo, Yoshitsugu Shiro
Abstract
Significance The short-lived intermediate formed during the reduction of nitric oxide (NO) to nitrous oxide (N 2 O) in denitrification, microbial anaerobic respiration, is a key state for understanding the generation mechanism of N 2 O, known not only as a greenhouse gas but also as an ozone-depleting substance on the global level. This paper combined state-of-the-art, time-resolved techniques, such as flow-flash infrared spectroscopy and X-ray free electron laser-based crystallography, and captured the intermediate of a P450-type NO reductase at the atomic and electronic levels. The intermediate was identified as a singly protonated Fe 3+ –NHO •− radical, offering insights into a radical–radical coupling mechanism for the N–N bond formation in N 2 O generation.