Nitrogen fixation, the process of converting atmospheric nitrogen (N2) into ammonia (NH3) that can be readily used by plants and other organisms, is a fundamental process for life on Earth. It provides the necessary nitrogen for the synthesis of essential cellular components such as proteins and nucleic acids. On an industrial scale, the Haber-Bosch process is commonly used to produce ammonia. However, this method is energy-intensive and significantly contributes to global greenhouse gas emissions. In contrast, nature has devised a more sustainable solution using nitrogenase, an enzyme able to catalyze this transformation under ambient conditions. Nitrogenase is a complex enzyme that consists of two main components: the MoFe protein or NifD2K2, and the Fe protein or NifH2. NifD2K2 contains the active site, known as FeMo-cofactor (FeMo-co), a [7Fe-9S-C-Mo-(R)-homocitrate] center, where the actual reduction of N2 to NH3 takes place.
The assembly of FeMo-co involves a series of complex steps orchestrated by the nitrogen fixation (NIF) machinery, which ensures the precise assembly of different precursors leading to the active site. My PhD 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.
The function of NifB is complex and involves the coordination of multiple [4Fe-4S] clusters: a radical SAM cluster and two substrate clusters termed K1 and K2. Using X-ray crystallography, we have confirmed that the N-terminal and the C-terminal regions displayed a flexibility depending on the binding of K1 and K2, respectively. This flexibility likely plays a role in the housing and conversion of these substrate clusters as well as the transfer of the product NifB-co for further processing. Moreover, using site-directed mutagenesis and electron paramagnetic resonance (EPR) spectroscopy, we have shown that K1 and/or K2 clusters underwent modifications upon reduction, suggesting the formation of an intermediate before methyl transfer and carbide insertion. The precise nature of this intermediate is a focus of our ongoing investigations. In addition, our analyses of NifB sequences and structures, combined with EPR spectroscopy, revealed a glutamate and a cysteine as potential shared ligands between K1 and K2 prior to their fusion.
​ 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. In summary, while many challenges remain, the study of the structure-function relationship of NifB and NifE2N2 proteins contributes to our understanding of the assembly of the nitrogenase active site, which in turn may help inspire the development of more efficient catalysts for the conversion of N2 into NH3 under ambient conditions.​ ​