Gain‐of‐Function Engineering of the NifEN Scaffold Into a Self‐Assembling Nitrogenase

ABSTRACT Biological nitrogen fixation by Mo‐nitrogenase is a complex process requiring coordinated electron transfer through specialized metal clusters. NifEN, an α 2 β 2 heterotetrameric homolog of the nitrogenase catalytic component NifDK, naturally serves as a maturation scaffold but exhibits only trace catalytic activity due to its “imperfect” cluster environments. In this study, we systematically engineered NifEN to reconstruct the structural features of NifDK, specifically targeting the P→M cluster electron relay and the active‐site M‐cluster environment. Specifically, we developed a series of variants, culminating in NifEN P/M_S2B , which incorporates a restored P‐cluster species and a refined M‐cluster coordination site and S2B‐associated belt region. Spectroscopic analysis confirmed the successful installation of the target clusters. Moreover, activity assays and nanoSIMS analysis demonstrated that NifEN P/M_S2B supported ATP‐dependent N 2 reduction both in vitro and in vivo. Structural modeling further identified remaining catalytic bottlenecks, particularly those in the S3A and S5A regions, highlighting the importance of precise cofactor belt organization for substrate reduction. Together, these findings establish NifEN as a self‐assembling, catalytically competent nitrogenase scaffold that bypasses the need for complex cofactor transfer steps, providing a streamlined platform for mechanistic studies and engineering efforts of biological nitrogen fixation.