Mutagenic Survey of Key Residues of NifB Involved in Radical SAM‐Dependent Nitrogenase Cofactor Assembly

] modules-RS, K1, and K2-that mediate fusion of K1 and K2 via radical SAM chemistry at RS, but the identities and functions of cluster ligands and SAM-binding residues have remained unclear. Here, we report a systematic mutagenic analysis of key residues of NifB. Combining EPR spectroscopy with biochemical assays, we verify C18, H31, and C115 as ligands of the K1-cluster, and C260 and C263 as ligands of the K2-cluster. We further reveal a functional asymmetry in which the K1-module controls the initial sensing and orientation of SAM, whereas the K2-module acts as the catalytic center for radical-driven cluster fusion. Mutations in SAM-binding residues (T139, N194, P225) uncouple substrate binding from catalysis, while alteration of a surface residue (C240) enhances activity, implicating conformational gating in catalysis. Together, these findings define a functionally differentiated RS-K1-K2 triad and establish a mechanistic framework for radical SAM-dependent L-cluster assembly.