Examination of Selenium Incorporation and Product Formation in the Nitrogenase FeMo-Cofactor

Citation

Arias, Renee Justine (2018) Examination of Selenium Incorporation and Product Formation in the Nitrogenase FeMo-Cofactor. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5WZV-R440. https://resolver.caltech.edu/CaltechTHESIS:05252018-140505343

Abstract

Nitrogenase is the only known enzyme to convert the triply bonded atmospheric dinitrogen (N₂) to bioavailable ammonia (NH₃) in an ambient environment, breaking one of the strongest chemical bond in nature in the process. Industrially, the Haber-Bosch process is also capable of reducing dinitrogen to ammonia, and is essential for worldwide food production ^1,2. Due to the high temperatures and pressures required for the Haber-Bosch process (between 300-550ºC and 15-25 MPa) and its requirement for molecular hydrogen, it has become paramount to scientifically investigate the biological processes of nitrogen fixation to ultimately develop more efficient methods to produce bioavailable ammonia. Nitrogenase utilizes two component proteins, the Fe-protein and the MoFe-protein, to reduce ammonia in an ATP-hydrolysis dependent and electron-intensive reaction. Besides the canonical dinitrogen reduction reaction, nitrogenase can reduce a variety of other substrates including: acetylene (C₂H₂), carbon dioxide (CO₂), carbon monoxide (CO), carbonyl sulfide (COS), nitrous oxide (N₂O), diazene (N₂H₂), and more ^3-11. CO has long been of interest to the study of the mechanism of nitrogenase, owing to its isoelectronic identity to N₂, and its potent inhibitor properties at well as its ability to serve as a weak substrate ^12,13. Like CO, cyanide compounds (X-CN) are also of interest to the study of nitrogenase due to the isoelectronic nature of CN^- to N₂. However, cyanide compounds serve as particularly interesting spectroscopic and crystallographic tools, because X in X-CN can be substituted for more significant sulfur or selenium (Se). In this study, we investigate the substrate properties of SeCN^-, with Se-incorporation into the active site FeMo-cofactor and concurrent reduction of SeCN^- to methane (CH₄). This study serves as yet another link between substrate reduction in nitrogenase. Part of this work describes the incorporation of Se into the cofactor as a vehicle for high-resolution study of nitrogenase under turnover using spectroscopy and crystallography, while another part describes a proposal for future work on the trapping of enzyme intermediates by fast-growing crystallography.

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