Abstract
Nitrogenase catalyses small-molecule activation, and has great relevance to agronomy, environment and energy. Understanding the assembly of the complex nitrogenase cofactor is a decades-long goal in the field, and structural insights into this process remain scarce. Here we report a cryogenic electron microscopy (cryo-EM) study of heterologously expressed NifEN, a key player converting the precursor (L-cluster) to a mature cofactor (M-cluster). Structural analyses of apo- and holo-NifEN demonstrate major conformational changes triggered by L-cluster incorporation. Further examinations of NifEN structures with inwardly and outwardly bound L-clusters, coupled with supporting mutational studies, AlphaFold 3 predictions and negative-stain EM analyses of NifEN complexed with upstream (NifB) and downstream (NifH) assembly partners, reveal a tunnel linking NifEN with NifB and NifH, with NifEN serving as a dynamic hub that coordinates L-cluster reception, maturation and delivery via conformation-gated metallocluster trafficking.