Nitrogen (N) plays a crucial role in the biosynthesis of amino acids and nucleotides. The reduction of atmospheric nitrogen (N2) and protons to ammonia and hydrogen is exclusively catalyzed by nitrogenase, an enzyme of great importance (Burgess and Lowe, 1996; Thorneley and Lowe, 1996). Three homologous nitrogenases, namely molybdenum-dependent, vanadium-dependent, and iron-only nitrogenases, have been discovered. Among them, molybdenum (Mo) nitrogenase has received the great attention (Jasniewski et al., 2020). The Mo-nitrogenase complex consists of two proteins. The first is the reductase component, commonly known as iron protein (FeP). FeP is a γ2-homodimer that contains a subunit-bridging (Fe4S4) cluster and an adenosine triphosphate (ATP)-binding site within each subunit (Einsle and Rees, 2020). The second component is the catalytic component called the molybdenum iron protein (MoFeP), MoFeP is an α2β2-tetramer that harbors two complex metalloclusters per αβ-dimer: A P-cluster (Fe8S7) and an M-cluster [(R-homocitrate)MoFe7S9C], also known as the iron–molybdenum cofactor (Einsle and Rees, 2020). During substrate turnover, the two components of nitrogenase form a complex that facilitates electron transfer from the (Fe4S4) cluster of FeP to the P-cluster of MoFeP through ATP hydrolysis in each FeP subunit (Rutledge and Tezcan, 2020). These electrons are subsequently transported to the M-cluster of MoFeP, where the reduction of N2 occurs (Rutledge and Tezcan, 2020).

Understanding the biological mechanism of nitrogenase is hindered by the small size of N2 and the challenge associated with isolating N2-bound M-clusters (Threatt and Rees, 2023). Furthermore, interpreting biochemical and spectroscopic data under turnover conditions presents difficulties due to the presence of multiple conformational states (Threatt and Rees, 2023).

In a recent study by Rutledge et al. (2022), cryo-electron microscopy (cryo-EM) was employed to analyze nitrogenase complex at near-atomic resolution catalytic turnover conditions. Due to the complex and heterogeneous nature of the nitrogenase complex, the researchers gathered a substantial dataset comprising 4.5 million usable particles obtained from over 15,000 movies. To prevent activity loss caused by exposure to oxygen, a protein solution containing adequate levels of ATP and dithionite (5 mM each) was prepared and rapidly frozen in liquid nitrogen in an anaerobic chamber. The cryo-EM grid was manually prepared at 277 K using a plunge freezer developed by the Herzik Lab. The entire process, from turnover to the grid preparation, was completed in less than 30 s, allowing the system to reach a steady state without depleting ATP and dithionite (Rutledge et al., 2022).

Due to their extensive efforts, nitrogenase complexes (FeP and MoFeP) constituted approximately 35% of the total usable particles detected on the cryo-EM grid, while the remaining particles correspond to single proteins consisting of either FeP or MoFeP. Under turnover conditions, two structures of the nitrogenase complex were resolved at a resolution of 2.3 Å, while the cryo-EM structure of resting state of MoFeP was determined at a resolution of 1.8 Å under the same conditions. Although the resting state structure reported in this study exhibited similarity to the earlier crystal structure (Protein Data Bank ID 3U7Q with a root-mean-square deviation = 0.29), distinct nitrogenase complexes were observed under catalytic turnover conditions (Rutledge et al., 2022; Spatzal et al., 2011; Tezcan et al., 2005). The crystal structures of the nitrogenase complex comprised one MoFeP and two homodimeric FePs, resulting in a stoichiometry of 2:1 for the FeP to MoFeP ratio. In contrast, the cryo-EM structures contained one MoFeP and one homodimeric FeP, accompanied by either two nonhydrolyzable ATP analogs or one nonhydrolyzable ATP analog and one ADP (adenosine diphosphate) (Rutledge et al., 2022; Tezcan et al., 2015). The cryo-EM structures revealed that the two αβ halves of MoFeP function independently. Furthermore, these structures demonstrated that the observed partial reactivity and negative cooperativity in nitrogenase arise from the FeP molecule coupled to MoFeP throughout the turnover process. The asymmetric binding of FeP to MoFeP has implications for prolonging the lifespan of the FeP and MoFeP nitrogenase complex, while simultaneously reducing the subsequent electron transfer step from FeP to the P-cluster and M-cluster in MoFeP.

In their groundbreaking study, Rutledge et al. (2022) presented the first cryo-EM structures that unveiled the dynamic nature of the nitrogenase complex under turnover conditions, revealing insights that were not previously attainable through X-ray crystallography. These structures offer compelling evidence supporting the catalytic model of half-reactivity and negative cooperativity in nitrogenase.

Further structural studies coupled with time-resolved studies hold the key to unraveling the precise conformational rearrangements of the M-cluster during turnover in nitrogenase. By capturing dynamic snapshots of the enzyme at different stages of its catalytic cycle, we can gain crucial insights into the intricate structural changes that occur during ATP hydrolysis and dinitrogen reduction. Furthermore, integrating experimental methods with advanced computational modeling and simulation techniques can provide a comprehensive understanding of the nitrogenase mechanism. This integrative approach will enable us to decipher the conformational changes of the cofactors, and the dynamics of the active site, shedding light on the fine details of nitrogenase’s mechanism. Ultimately, these in-depth structural biology studies have the potential to inspire the design of innovative strategies for developing new crops that can fix nitrogen by themselves, potentially providing a sustainable alternative to the energy–consuming Haber–Bosch process of nitrogen fixation and revolutionizing agricultural practices worldwide.