Study of Nitrogenase Cofactors bound to Nitrogenase Carrier Proteins

Abstract

Nitrogenases employ Fe-S clusters as catalytic cofactors to perform the kinetically challenging reduction of N₂ to NH₃. The unique reactivity of nitrogenase cofactors is derived in part from the complexity of their cluster composition, featuring eight metal sites, at least seven of which are Fe. Understanding how these cofactors operate requires knowledge of their electronic structure in the resting state, and how they change during the catalysis. However, spectroscopically studying nitrogenase cofactors is challenging since it is difficult to resolve the signal arising from the large number of Fe sites. To overcome this challenge, we have developed a post-biosynthetic modification method to selectively label specific sites of nitrogenase cofactors with ⁵⁷Fe. As a test case, we showed that the terminal Fe sites of nitrogenase L-cluster can be selectively labeled with ⁵⁷Fe in a near quantitative yield. Using this technology together with site-directed mutagenesis, we obtained the first concrete evidence that the L-cluster binds to the nitrogenase carrier protein NifX through His³⁵ residue. In addition, we extended the use of both site-selective labeling strategy and NifX as a protein platform to conduct a comparative electronic structure study of two nitrogenase catalytic cofactors (FeMo-co and FeV-co). We found that the valence distribution across the Fe₇ fragment is very similar for both cofactors in the dithionite-reduced state. Overall, this thesis reports the development and utilization of the site-selective ⁵⁷Fe-labeling in conjunction with the use of carrier protein NifX to study electronic structure of nitrogenase cofactors.