Probing the coordination and function of FeS modules in nitrogenase assembly protein NifB.

NifB is an essential radical S-adenosylmethionine (SAM) enzyme for nitrogenase cofactor assembly. Previous studies show that NifB couples a putative pair of [FeS] modules (designated K1 and K2) into an [FeSC] cofactor precursor concomitant with radical SAM-dependent carbide insertion through the action of its SAM-binding [FeS] module. However, the coordination and function of the NifB cluster modules remain unknown. Here, we use continuous wave and pulse electron paramagnetic resonance spectroscopy to show that K1- and K2-modules are 3-cysteine-coordinated [FeS] clusters, with a histidine-derived nitrogen serving as the fourth ligand to K1 that is lost upon K1/K2-coupling. Further, we demonstrate that coexistence of SAM/K2-modules is a prerequisite for methyltransfer to K2 and hydrogen abstraction from the K2-associated methyl by a 5'-deoxyadenosyl radical. These results establish an important framework for mechanistic explorations of NifB while highlighting the utility of a synthetic-cluster-based reconstitution approach employed herein in functional analyses of iron-sulfur (FeS) enzymes.