Atmospheric nitrogen (N₂) is, for the most part, an inert gas for living beings on Earth. That is, we cannot directly utilize this vital compound. Therefore, the biochemical nitrogen cycle, and especially the role played by bacteria and plants, is essential to sustaining life on our planet. In some microorganisms, a process known as biological nitrogen fixation occurs, in which nitrogenases, oxygen-sensitive proteins, transform atmospheric nitrogen into forms usable for life. To function, nitrogenases depend on a metal cofactor, a complex molecular component that must be carefully constructed through a series of steps involving numerous proteins. One of the key players in this process is the protein NifEN, which acts as a scaffold to complete the final stages of cofactor assembly before incorporation into nitrogenase, or NifDK. Until now, the structural basis that allows this protein to fulfill its key role in biological nitrogen fixation was unknown.

In this study, published in Nature Chemical Biology, researchers reveal how the NifEN protein performs this function. The researchers used cryo-electron microscopy (cryo-EM) to perform a high-resolution structural analysis of the protein. This cutting-edge technique allowed them to capture unprecedented images of the nitrogenase cofactor assembly. These images revealed a surprisingly dynamic process in which the scaffolding protein NifEN opens and closes like a gate, with parts of the protein moving and rearranging to facilitate the movement of a precursor from the surface to its internal cavity. The researchers were able to draw this conclusion thanks to the crucial discovery of intermediates that show the precursor cofactor in transit between both locations. These findings reveal that the transformation of the precursor may not occur on the surface of the protein, as previously suggested, but within its internal cavity. This discovery not only changes our understanding of nitrogenase cofactor biosynthesis but also sheds light on the evolutionary split between NifEN, which specializes in cofactor construction, and NifDK, which performs nitrogen fixation.

 

​Cartoon representation of the structure of NifEN determined by cryo-electron microscopy. © CEA-Irig/IBS/Y. Nicolet​

Metallocofactor*: molecule that contains at least one metal ion, necessary for an enzyme to catalyze a given reaction.