NifB, a radical SAM enzyme, catalyzes the formation of a distinct Fe 8 S 9 C core (L‐cluster) of the nitrogenase cofactor. Prior studies have led to the proposal of three Fe 4 S 4 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.
Duffin et al. (Sun,) studied this question.