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Nitrous oxide (N₂O) is a potent greenhouse gas that both traps heat and destroys ozone, reducing Earth's protection from solar radiation.

"Small increases in N₂O have disproportionate contributions to global temperature increases," said Frank Loeffler, the Goodrich Chair of Excellence in Civil Engineering and a professor in the Department of Civil and Environmental Engineering.

Atmospheric N₂O is also at an all-time planetary high thanks to the industrial synthesis and use of nitrogen fertilizers. For most of Earth's history, molecular nitrogen (N₂) from the atmosphere was transformed into ammonium by certain N₂-fixing soil microbes. Plants could then use some of this ammonium to fuel their growth.

Early in the 20th century, chemists learned how to perform this reaction, and the production of synthetic fertilizer has been increasing ever since. Unfortunately, plants can't take up all the nitrogen applied to agricultural fields, and the leftover ammonia gets metabolized by soil microbes into huge amounts of N₂O.

"Our global population is still increasing, so we continue to need more food; it will not be realistic to reduce the use of nitrogen fertilizer," said Guang He, Ph.D., a postdoctoral researcher in Loeffler's lab. "Instead, the global objective is to reduce emissions by maximizing the soil microbiome's ability to convert (reduce) N₂O back to climate-neutral N₂."

Last year, He and Loeffler of a novel N₂O-consuming bacterial microbiome in acidic soil where N₂O reduction had previously not been detected.

He later discovered that the enzymes that some of those bacteria use to conduct the reaction were also novel to science—and in fact, represent a new class of important proteins. The discovery, last month, has already gained international attention, recontextualizing previous research studies and potentially impacting scientific models of both the nitrogen cycle and greenhouse gas emissions.

"The processes of the nitrogen cycle are currently not balanced—we have much more production than consumption of N₂O," Loeffler said. "When you have a better understanding of the microbes that consume N₂O, you can generate opportunities to increase that consumption, so we have a better balance and fewer emissions."

Describing a new protein family

There is a pattern in biology called convergent evolution, where proteins with similar functions often have similar structures and are encoded by similar gene sequences. When scientists search the environment for microbes with certain abilities, they can compare microbes' genetic sequences with libraries of sequences that have known functions—encoding N₂O reductase proteins (N₂ORs), for example.

N₂ORs are generally categorized into two canonical groups, one of which was discovered nearly a century ago and the other in 2012—in , in fact.

So when He found no canonical N₂ORs in the genetic code of the microbes that converted N₂O to N₂, he had an inkling of what might be going on.

That doesn't mean he was immediately confident in the result.

"I thought, 'How can it be? Is something wrong with my bioinformatics skills, or is this a real phenomenon?'" He recalled.

After going back over his results numerous times, and even confirming his methodology with the bioinformatics program's developer, He reached out to Loeffler's longtime collaborator at the Georgia Institute of Technology, Kostas Konstantinidis.

"Professor Konstantinidis, a world-renowned bioinformatics expert, confirmed every detail," He said. "That finally convinced me that I did everything correctly—that it was a fact that the canonical N₂OR genes were not present."

Within the microbiome culture, He was able to find a gene that was a 30% match for canonical N₂OR genes—far below the generally accepted matching threshold of 40%—but the sequence was so different that the protein couldn't belong to either of the canonical N₂OR groups.

Jerry Parks, the group leader for the Molecular Biophysics Group in the Biosciences Division at Oak Ridge National Laboratory (ORNL), helped He learn state-of-the-art AI approaches to predict protein function based on 3D renders of protein structure. Together, they identified critical clues supporting the hypothesis that the new protein is indeed an N₂OR.

The leader of ORNL's Bioanalytical Mass Spectrometry Group, Robert Hettich, then led the team in measuring the novel protein in the bacterial culture and demonstrating its function as an N₂OR.

"The data analysis required cutting-edge bioinformatics computation of DNA and protein sequences," Loeffler said. "Doctors Konstantinidis, Parks, and Hettich provided guidance and expertise to help Guang complete these analyses very efficiently and using the newest tools."

Implications for the past and future

Within days of their publication, He got requests from labs across the US, Europe, and Asia asking for his methodology so they could apply it to their own studies. Some older publications have already surfaced showing similar sequences which were not recognized as N₂ORs at the time.

"The impacts of this study are very immediate," He said. "These new sequences will surely account for certain examples where people have found robust N₂OR activity but could not find the genetic evidence to support it."

The new protein's inclusion in genomic reference libraries also means that other N₂ORs from the previously unrecognized lineage will be properly identified going forward.

"There's an entire group of N₂ORs that weren't on the radar. Guang's discovery can solve some longstanding questions about nitrogen cycling and N₂O emissions," Loeffler said. "The new information will help other researchers to develop a more comprehensive picture of microbial diversity and the genes and proteins involved in N₂O reduction."