Serendipitous discovery can convert toxic nitric oxide into nitrogen gas!

Nitrogen is a crucial component of proteins and nucleic acids, the fundamental building blocks of all living things, and thus is essential to life on Earth. Gaseous N₂ from the atmosphere can be “fixed” by soil bacteria capable of converting N₂ to ammonia or nitrates (NO₃). Nitrifying bacteria in the soil then convert ammonia into NO₃, which plants utilize for growth. Animals that consume plants put the N₂ back into the soil in the form of ammonia when they die or excrete waste. The NO₃ in the soil is converted back into N₂ and released into the atmosphere by the activity of denitrifying microbes in the soil. This native nitrogen cycle regulates the N₂ levels in the atmosphere and on Earth and is vital for sustaining life.

Farmers have long taken advantage of this knowledge and supplemented soil with fertilizers rich in NO₃ to boost agricultural yield. Unfortunately, prolonged and unchecked use of fertilizers has led to excess N₂ in our water bodies, harming the aquatic ecosystem. To protect the environment, immense research efforts have been directed towards the denitrification of polluted water and industrial emissions.

Now, a chance observation by Professor Hiroaki Kitagishi’s research team at the Department of Molecular Chemistry and Biochemistry, Doshisha University, Japan, presents a new potential denitrification process in the native nitrogen cycle. Their research reports a unique reaction between Nitric oxide (NO) and glycine on synthetic heme-mimetics to produce N₂ gas in water at room temperature. This groundbreaking study was made available online in the Journal of the American Chemical Society on November 24, 2025.

“Our study is an example of how careful observation is important for breakthrough discoveries,” comments Prof. Kitagishi.

In the industry, denitrification, or direct conversion of NO to N₂, is done by using metal-based catalysts that bind to NO. These complexes have a delocalized electron that is vulnerable to be attacked by nucleophiles like ammonia. However, this metal-nitrosyl chemistry that helps in generating the N−N bond is typically conducted at a high temperature of 200−300 degrees Celsius. Development of newer and cleaner methods for the denitrification of water and air is a significant unmet need.

Prior to stumbling onto this discovery, Prof. Kitagishi’s group was developing synthetic heme models in water to mimic the natural iron-containing heme molecule that binds oxygen. They had developed synthetic heme-like porphyrin-iron complexes, hemoCDs, where the porphyrin-iron is encapsulated in cyclodextrin (CD) dimers bearing nitrogenous-ligand moiety  like pyridine (P) or imidazole (I).

It was during these experiments that a Ph.D. course student (3rd  year), Mr. Atsuki Nakagami from the Graduate School of Science and Engineering, Doshisha University, introduced NO into a solution of hemoCD-P and -I in an acidic glycine-containing buffer. To their surprise, they observed appearance of bubbles within minutes, indicating the production of significant amounts of insoluble gas.

To understand this chemical reaction, Prof. Kitagishi and Mr. Nakagami teamed up with Associate Professor Yoshihito Shiota from the Institute for Materials Chemistry and Engineering, Department of Fundamental Organic Chemistry, Kyushu University, Japan.

Together, they performed advanced chemical analyses, including gas chromatography (GC) analysis and nuclear magnetic resonance spectroscopy, to identify the end products N₂ and α-hydroxyacid. They also conducted isotope-labeling experiments with ¹⁵NO and ¹⁵N-glycine to track the trajectory of the N₂ molecules in the chemical reaction.

They observed that this unique chemical reaction, which led to N₂ release, was facilitated by highly stable ferric nitrosyl complexes supported by hemoCD and the presence of excess glycine. The hemoCD system is the very first report of the direct conversion of NO to N₂ occurring on a heme iron.

Sharing the long-term implications of their study, Prof. Kitagishi said, “Our study reveals new means of clean conversion of toxic NO to non-toxic N₂. The hemoCD has the potential to detoxify NO to N₂ under mild conditions and can be utilized for denitrification processes in the industry.”

This biomimetic study unveils the different mechanisms of new N-N bond formation in the native nitrogen cycle and advances our understanding of this process. This novel transformation reaction will also benefit industrial processes while protecting the environment.

About Professor Hiroaki Kitagishi from Doshisha University, Japan
Professor Hiroaki Kitagishi is a Professor at the Department of Molecular Chemistry and Biochemistry, Doshisha University, Kyoto, Japan. In 2006, he received his Doctorate in Engineering from Doshisha University and later joined Osaka University as a Postdoctoral fellow before returning to Doshisha University as an Assistant Professor in 2008. He has over 117 publications in leading scientific journals, covering his research in organic synthesis, bioinorganic chemistry, and supramolecular chemistry. In recognition of his work, Prof. Kitagishi received the BMRC Award (Doshisha University Biomimetics Research Center) in 2004 and the 15^th Cyclodextrin Society Encouragement Award in 2011.
Read more at: https://kitagishi-lab.doshisha.ac.jp/dr_hiroaki_kitagishi/

Funding information
This work was financially supported by JSPS KAKENHI (24K01640 to Hiroaki Kitagishi, 25KJ2204 to Atsuki Nakagami), AMED (24ym0126808j to Hiroaki Kitagishi), and JST (JPMJSF2305 to Hiroaki Kitagishi).

Media contact:
Organization for Research Initiatives & Development.
Doshisha University
Kyotanabe, Kyoto 610-0394, JAPAN
E-mail: jt-ura@mail.doshisha.ac.jp