PNNL

A team of researchers led by PNNL computational scientist Simone Raugei have revealed new insights about how this complex enzyme does its job, finding that the seemingly wasteful formation of hydrogen has an essential purpose. Their paper, "Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N₂ reduction," was published in the Proceedings of the National Academy of Sciences in November 2018. Raugei's co-authors are Lance Seefeldt, who holds a joint appointment at PNNL and Utah State University, and Brian Hoffman of Northwestern University.

Why it matters: Nitrogenase can convert nitrogen to ammonia at room temperature and atmospheric pressure. Industry, on the other hand, relies on the Haber-Bosch process, a century-old technique using high temperature and pressure. Fossil fuels typically provide the energy for this process, which is why industrial ammonia production alone accounts for more than 1% of the world's total energy-related carbon emissions. Understanding what lends nitrogenase its bond-breaking muscle can lead to new, stimulating ideas for the design of synthetic catalysts to make ammonia.

Summary: For every molecule of nitrogen transformed to ammonia, nitrogenase makes at least one molecule of hydrogen (H₂), which is "one of the most puzzling mysteries of nitrogenase," Raugei said. "Instead of just producing ammonia, you also produce this byproduct. Why is that necessary?"

The researchers found that this phenomenon actually helps nitrogenase tackle nitrogen's strong bonds. "Nature found a solution by coupling the production of hydrogen, which releases energy, with cleaving nitrogen, which requires energy," Raugei said. "It's total balance."

To arrive at the results, the team used a blend of theoretical and experimental methods. Raugei conducted quantum chemical calculations on models of the core of the enzyme, relying on guidance from Seefeldt and Hoffman, who are experts on the biochemistry of nitrogenase. Their experimental data helped inform the computations, and vice versa.

The researchers focused on the nitrogenase catalytic core, composed of iron, molybdenum and sulfur (FeMo-co). During the catalytic event, when FeMo-co has acquired a critical number of electrons and protons (H⁺) in the form of two bridging hydrides (Fe-H-Fe) in its peripherical belt, generation of an H₂ bound to FeMo-co and its displacement by N₂ provides the give and take of energy to spark nitrogen reduction, the researchers found.

"We were very well positioned to make to this breakthrough because we combined experimental information on nitrogenase with the computational information," Raugei said. "That was key."

What's next? The researchers seek to extend the research by examining the fine details of electron- and proton-accumulation at the active site of nitrogenase and exactly how the N₂ bond is broken to form ammonia.

Acknowledgments: The Pacific Northwest National Laboratory (PNNL) Enzymatic Energy Conversion program aims to provide a better understanding of the core principles employed by enzymes in order to control the flow of energy and matter to achieve remarkable specificities, efficiencies, and catalytic rates. Our program integrates state-of-the-art theory and computation with experimental efforts across the U.S. Department of Energy's (DOE's) Basic Energy Sciences (BES) Physical Biosciences community to fill critical gaps in knowledge about how enzymes orchestrate spatial and temporal events to direct electrons, protons, and substrates for selective conversions and allosteric regulation. These advances will contribute to the design of next-generation synthetic catalysts.

This work directly addresses two Basic Research Needs Priority Research Directions for BES: "Design Catalysts Beyond the Binding Site" and "Drive New Catalyst Discoveries by Coupling Data Science, Theory, and Experiment," both listed in Basic Research Needs: Catalysis for Energy.

Sponsor: The work by Simone Raugei and Lance Seefeldt was managed for the US Department of Energy (DOE), Office of Science, Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Bio-Sciences under Award DE-AC05-76RL01830/FWP66476. The work by Brian Hoffman was supported by the National Institutes of Health Award GM 111097 and by National Science Foundation Award MCB 1515981.

Research Area: Computational Chemistry and Biophysics

User Facilities: Computer resources were provided by the W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science User Facility located at Pacific Northwest National Laboratory and managed for DOE's Office of Biological and Environmental Research.

Research Team: Simone Raugei (Pacific Northwest National Laboratory); Lance C. Seefeldt (Utah State University and Pacific Northwest National Laboratory; and Brian M. Hoffman (Northwestern University)

Reference: Simone Raugei, Lance C. Seefeldt, Brian M. Hoffman. "Critical computational analysis illuminates the reductive-elimination mechanism that activates nitrogenase for N₂ reduction." Proceedings of the National Academy of Sciences, November 6, 2018. DOI: 10.1073/pnas.1810211115.