Towards electrochemical control over nitrogenase crystals: structure-spectroscopy-reactivity Shoba Laxmi 1 , Stephen B. Carr 1,2 , Zhiyong Yang 3 , Lance Seefeldt 3 , Kylie A. Vincent 1 1 University of Oxford, UK , 2 Research Complex at Harwell, UK, 3 Utah State University, USA Nitrogen fixation is one of the most important biological reactions, and is catalysed by the enzyme nitrogenase. Dinitrogen is reduced under ambient conditions by certain microbes, to form ammonia through an efficient redox cycle. However, the intricate catalytic mechanism employed by nitrogenase has remained elusive. This is largely due to the overlapping presence of various redox and protonation states with nitrogenase (labelled E 0 -E 7 ) at steady state, and the difficulty in generating the N 2 -binding level during experiments. 1 Recent advances in spectroscopy and structural biology have improved our understanding of how substrate reduction occurs, bringing to light evidence for hydride and nitrogen intermediates 2 , and establishing the reversibility of changes in the sulfur belt of the active site cofactor during binding of exogenous ligands 2 . It has been postulated that the active site features multiple substrate binding sites, formed in situ in the presence of specific substrates under turnover conditions. Studies done with carbon monoxide show evidence of multiple binding modes which influence the reaction pathway 3 . To study these states, precise redox control over the active site is required, yet it has proven to be a challenge because of indirect reduction of nitrogenase by the reductase partner protein known as Fe protein. Herein, we seek to apply a multidisciplinary approach to unravel nitrogenase’s black box by marrying structural characterisation with electrochemistry and spectroscopy, by extending spectroelectrochemical methodologies 4-5 developed in the Vincent Group. By applying electrochemical control to protein crystals, the Vincent group was able to access specific intermediates of the nickel-iron hydrogenase, Hyd1 from E. coli, 5 and to characterise them using IR microspectroscopy and X-ray diffraction. Spurred by the success with Hyd1, we seek to translate this to selectively access individual states within nitrogenase to give insight into substrate binding and transformation. As such, we report preliminary results on molybdenum-iron nitrogenase and a plan for future studies. References 1. D. Threatt and D. C. Rees, FEBS Letters , 2023, 597 , 45-58. O. Einsle and D. C. Rees, Chem. Rev., 2020, 120, 4969-5004. 2. T. M. Buscagan, K. A. Perez, A. O. Maggiolo, D. C. Rees and T. Spatzal, Angew. Chem. Int. Ed., 2021, 60, 5704-5707. 3. P. A. Ash, S. E. T. Kendall-Price, R. M. Evans, S. B. Carr, A. R. Brasnett, S. Morra, J. S. Rowbotham, R. Hidalgo, A. J. Healy, G. Cinque, M. D. Frogley, F. A. Armstrong and K. A. Vincent, Chem. Sci., 2021, 12, 12959-12970. 4. S. Morra, J. Duan, M. Winkler, P. A. Ash, T. Happe and K. A. Vincent, Dalton Trans., 2021, 50, 12655-12663.
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