The catalytic reduction of atmospheric dinitrogen into ammonia (i.e., reductive nitrogen fixation) is a key process for the biosynthesis of life’s building blocks like amino and nucleic acids. Approximately, half of fixed nitrogen in our biosphere is provided by the industrial Haber-Bosch process and the other half by nitrogenases produced by diazotropic bacteria. Despite its remarkable efficiency, the Haber-Bosch process requires extreme conditions (>400 oC, >250 atm H2/N2), is highly energy intensive (accounting for ~2% of all global energy consumption), and a major contributor to greenhouse gas emissions. In contrast, nitrogenase catalysis occurs at ambient conditions of bacterial growth, albeit at the expense of a large number of ATP molecules required to control the flow of electrons and protons to enable nitrogen reduction by nitrogenase.1
Although nitrogenase has been studied extensively for over six decades, the key details of its catalytic mechanism, biosynthesis, and cellular activities are yet to be elucidated: (a) what is the mechanism of nitrogen reduction by the catalytic cofactor (FeMo-co) of nitrogenase? (b) how does ATP hydrolysis control/gate the flow of multiple electrons and protons to FeMoco? (c) how are the complex cofactors of nitrogenase synthesized and incorporated into nitrogenase, (d) how does nitrogenase interact with other cellular components? For answering the first two questions, our group previously developed strategies for light-driven systems for ATP-independent nitrogenase catalysis2,3 and elucidated key roles of dynamic/multi-modal protein-protein interactions in ATP-mediated electron transfer and redox-dependent conformational gating events.4-7 Yet, these studies also highlighted the need for new methods for monitoring the structure of nitrogenase during catalytic turnover. Toward this end, we recently joined forces with the Herzik Group at UCSD Chemistry/Biochemistry to capture the structural dynamics of nitrogenase in action by anaerobic cryoEM and detected new conformational states and cofactor dynamics of nitrogenase that could not be visualized by other methods.8 Using these findings as a springboard, the current goals of the nitrogenase sub-group are to visualize catalytic reaction intermediates of nitrogenase at atomic resolution, understand the nature of ATP-directed electron and proton transfer, and characterize the dynamics interactions of nitrogenase with other protein components involved in its biosynthesis and its cellular functions.
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1. Rutledge, H. L. & Tezcan, F. A. Electron transfer in nitrogenase. Chem. Rev. 120, 5158-5193 (2020).
2. Roth, L. E., Nguyen, J. C. & Tezcan, F. A. ATP- and iron-protein-independent activation of nitrogenase catalysis by light. J. Am. Chem. Soc. 132, 13672-13674 (2010).
3. Roth, L. E. & Tezcan, F. A. ATP-Uncoupled, Six-Electron Photoreduction of Hydrogen Cyanide to Methane by the Molybdenum-Iron Protein. J. Am. Chem. Soc. 134, 8416-8419 (2012).
4. Owens, C. P., Katz, F. E., Carter, C. H., Luca, M. A. & Tezcan, F. A. Evidence for functionally relevant encounter complexes in nitrogenase catalysis. J. Am. Chem. Soc. 137, 12704-12712 (2015).
5. Owens, C. P., Katz, F. E. H., Carter, C. H., Oswald, V. F. & Tezcan, F. A. Tyrosine-Coordinated P-Cluster in G. diazotrophicus Nitrogenase: Evidence for the Importance of O-Based Ligands in Conformationally Gated Electron Transfer. J. Am. Chem. Soc. 138, 10124-10127 (2016).
6. Rutledge, H. L., Field, M. J., Rittle, J., Green, M. T. & Tezcan, F. A. Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase P-Cluster. J. Am. Chem. Soc. 144, 22101-22112 (2022).
7. Rutledge, H. L. et al. Redox-Dependent Metastability of the Nitrogenase P-Cluster. J. Am. Chem. Soc. 141, 10091-10098 (2019).
8. Rutledge, H. L., Cook, B. D., Nguyen, H. P. M., Herzik, M. A. & Tezcan, F. A. Structures of the nitrogenase complex prepared under catalytic turnover conditions. Science 377, 865-869 (2022).