Soutenance de thèse : Assembly machinery of the nitrogenase active site

Par Tu Quynh NGUYEN (IBS/Groupe Métalloprotéines)

Reduction of nitrogen in the form of ammonia (NH₃) is fundamental to all life, arousing huge industrial interest. On an industrial scale, the Haber-Bosch process is commonly used to convert atmospheric dinitrogen (N₂) into NH₃. However, this process is extremely energy-demanding as it requires high temperature and pressure, largely driven by fossil-fuel and leaves a massive carbon footprint throughout the production. In contrast, nature has devised a more sustainable solution using nitrogenase, an enzyme able to catalyze this transformation under ambient conditions. The FeMo-co active site of nitrogenase is a [Mo-7Fe-9S-C-R-homocitrate] center – one of the most sophisticated metalloclusters that exist in nature. The assembly of FeMo-co involves a series of complex steps orchestrated by the nitrogen fixation (NIF) machinery. My doctoral thesis focuses on understanding the structure-function relationship of two central components in this machinery : the radical S-adenosyl-L-methionine (SAM) enzyme NifB responsible for the synthesis of a [8Fe-9S-C] cluster termed NifB-co – a core precursor to FeMo-co, and the scaffold protein NifE₂N₂, which receives NifB-co and facilitates its conversion to FeMo-co.
NifB is responsible for the fusion of two [4Fe-4S] centers, combined with a carbide ion insertion and the addition of a sulfide ion to produce NifB-co. Using X-ray crystallography, we have confirmed that the N-terminal and the C-terminal regions of NifB displayed a flexibility depending on the binding of its iron-sulfur clusters. This flexibility likely plays a role in the housing and conversion of the substrates as well as the transfer of the product NifB-co for further processing. Moreover, studies combining site-directed mutagenesis and electron paramagnetic resonance (EPR) spectroscopy suggested the formation of an 8-Fe intermediate before methyl transfer and carbide insertion. The precise nature of this intermediate is a focus of our ongoing investigations. Furthermore, our investigations on the dynamic interaction between NifE₂N₂ and NifB provided important insights into the nitrogen fixation machinery. Their interaction was suggested to be involved in the transfer of NifB-co from NifB to NifE₂N₂. However, our studies have suggested that this process likely depends on the NifX protein or the C-terminal NifX-like domain present in many NifB proteins, rather than the NifB radical SAM domain itself. Furthermore, our structural and functional studies of NifE₂N₂ identified two key cysteine residues directly involved in the transfer and binding of NifB-co. Preliminary cryo-EM results also showed a new environment for NifB-co binding in NifE₂N₂, reminiscent to that of FeMo-co binding in NifD₂K₂. These findings shed light on the structural changes that NifE₂N₂ undergoes to house complex cofactors.
This study provides a deeper understanding of the biosynthesis of the nitrogenase active site and its unique chemistry. This in turn may help inspire the development of more efficient catalysts for the production of ammonia under ambient conditions.