Evolution-guided engineering of an ancient nitrogenase interface enhances enzyme activity and stability.

Nitrogenase is essential for biological nitrogen fixation and a key target for improving sustainable agriculture. The structural and functional complexity of nitrogenase makes it difficult to identify regions that can be engineered without disrupting activity, stability, or complex assembly. To address this challenge, we used an evolution-guided approach to identify lineage-specific regions with potential engineering tolerance, revealing a NifK N-terminal extension as a tunable interface. By generating variant libraries exceeding 9,000 members and evaluating them through diazotrophic growth assays and in-vitro characterization, we show that NifK extension is required for nitrogenase activity while remaining broadly tolerant to mutation, revealing a sequence-function landscape that is both flexible and constrained. A subset of residues at the NifD-NifK interface are critical for maintaining complex stability. Structural analyses indicate that the extension stabilizes the MoFe complex via co-evolved electrostatic interactions, and targeted mutations can improve both enzymatic activity and thermostability. These findings identify the NifK extension as a tunable interface, providing a strategy for engineering more robust nitrogenase variants.