Nitric oxide (NO) plays pivotal roles in various biological processes although it shows high cytotoxicity. In microbial denitrification, in which nitrate is reduced to dinitrogen in a step-wise manner, NO is produced as an intermediate product. To eliminate the cytotoxic effect of NO, microorganisms utilize NO reductase (NOR),which decomposes two NO molecules to nitrous oxide (N2O) (2NO + 2H+ + 2e- → N2O + H2O). A fungus, Fusarium oxysporum, utilizes soluble NOR (P450nor) whose active site is a thiolate-coordinated heme to decompose NO. Although several spectroscopic studies proposed the catalytic mechanism for P450nor, lack of the structures of the reaction intermediates do not allow us to draw the complete picture of the P450nor-catalyzed reaction. Here, we applied recently developed time-resolved crystallography and spectroscopies for the characterization of the reaction intermediates of P450nor. Photosensitive caged NO which releases two equivalent of NO upon UV irradiation was utilized as a reaction trigger for time-resolved measurements. Time-resolved X-ray crystallography using X-ray free electron laser at SACLA provided the molecular structures of ferric NO-bound form (1st reaction intermediate) and 2nd reaction intermediate which was produced by hydride transfer from NADH to the 1st intermediate [1,2]. In addition, time-resolved infrared measurements showed that the NO stretching frequency of 1st and 2nd reaction intermediates were 1852 and 1307 cm-1, respectively [2]. From these results, we can conclude that a NO molecule binds to ferric P450nor to form ferric NO-bound intermediate, followed by hydride transfer from NADH to yield a Fe3+-NHO●- state. This information suggests that the reaction intermediate of the Fe3+-NHO●- state reacts with another NO molecule via radical coupling to produce N-N bond for N2O formation.
References
[1] Takehiko Tosha, Takashi Nomura, Takuma Nishida, Naoya Saeki, Kouta Okubayashi, Raika Yamagiwa, Michihiro Sugahara, Takanori Nakane, Keitaro Yamashita, Kunio Hirata, Go Ueno, Tetsunari Kimura, Tamao Hisano, Kazumasa Muramoto, Hitomi Sawai, Hanae Takeda, Eiichi Mizohata, Ayumi Yamashita, Yusuke Kanematsu, Yu Takano, Eriko Nango, Rie Tanaka, Osamu Nureki, Osami Shoji, Yuka Ikemoto, Hironori Murakami, Shigeki Owada, Kensuke Tono, Makina Yabashi, Masaki Yamamoto, Hideo Ago, So Iwata, Hiroshi Sugimoto, Yoshitsugu Shiro, Minoru Kubo, Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate, Nat. Commun, 8, 1585 (2017). https://doi.org/10.1038/s41467-017-01702-1.
[2] Takashi Nomura, Tetsunari Kimura, Yusuke Kanematsu, Daichi Yamada, Keitaro Yamashita, Kunio Hirata, Go Ueno, Hironori Murakami, Tamao Hisano, Raika Yamagiwa, Hanae Takeda, Chai Gopalasingam, Ryota Kousaka, Sachiko Yanagisawa, Osami Shoji, Takashi Kumasaka, Masaki Yamamoto, Yu Takano, Hiroshi Sugimoto, Takehiko Tosha, Minoru Kubo, Yoshitsugu Shiro, Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography, Proc. Natl. Acad. Sci. USA, 118, e2101481118 (2021). https://doi.org/10.1073/pnas.2101481118.
Takehiko Tosha is Professor of graduate school of science, University of Hyogo, Japan. He received his Ph.D. from Kyoto University in 2003. As a postdoctoral researcher, he worked at the Okazaki Institute of Integrative Bioscience, at the Children’s Hospital Oakland Research Institute, USA and at RIKEN SPring-8 center, where he studied in the mechanism of enzymatic nitric oxide reduction using time-resolved X-ray crystallography at XFEL facility, SACLA, and time-resolved spectroscopic techniques, as a senior research scientist. His research interests include molecular mechanisms of metallo-protein-catalyzed reactions and structural dynamics of proteins related to metals in biology.
