An EPR and VTVH MCD spectroscopic investigation of the nitrogenase assembly protein NifB

NifB, a radical SAM enzyme, catalyzes the biosynthesis of the L cluster (Fe8S9C), a structural homolog and precursor to the nitrogenase active-site M cluster ([MoFe7S9C center dot R-homocitrate]). Sequence analysis shows that NifB contains the CxxCxxxC motif that is typically associated with the radical SAM cluster ([Fe4S4](SAM)) involved in the binding of S-adenosylmethionine (SAM). In addition, NifB houses two transient [Fe4S4] clusters (K cluster) that can be fused into an 8Fe L cluster concomitant with the incorporation of an interstitial carbide ion, which is achieved through radical SAM chemistry initiated at the [Fe4S4](SAM) cluster upon its interaction with SAM. Here, we report a VTVH MCD/EPR spectroscopic study of the L cluster biosynthesis on NifB, which focuses on the initial interaction of SAM with [Fe4S4](SAM) in a variant NifB protein (MaNifB(SAM)) containing only the [Fe4S4](SAM) cluster and no K cluster. Titration of MaNifB(SAM) with SAM reveals that [Fe4S4](SAM) exists in two forms, labeled Fe4S4SAMA+ and Fe4S4SAMB2+. It is proposed that these forms are involved in the synthesis of the L cluster. Of the two cluster types, only Fe4S4SAMB2+ initially interacts with SAM, resulting in the generation of Z, an S = 1/2 paramagnetic [Fe4S4](SAM)/SAM complex. Graphic abstract

Automated summary

NifB, a radical SAM enzyme, is involved in the biosynthesis of the L cluster and contains the characteristic motifs associated with SAM binding. The study of NifB reveals the mechanism of L cluster biosynthesis through interaction with SAM.