Abstract
NifB, a radical SAM enzyme, catalyzes the biosynthesis of the L cluster (Fe(8)S(9)C), a structural homolog and precursor to the nitrogenase active-site M cluster ([MoFe(7)S(9)C·R-homocitrate]). Sequence analysis shows that NifB contains the CxxCxxxC motif that is typically associated with the radical SAM cluster ([Fe(4)S(4)](SAM)) involved in the binding of S-adenosylmethionine (SAM). In addition, NifB houses two transient [Fe(4)S(4)] 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 [Fe(4)S(4)](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 [Fe(4)S(4)](SAM) in a variant NifB protein (MaNifB(SAM)) containing only the [Fe(4)S(4)](SAM) cluster and no K cluster. Titration of MaNifB(SAM) with SAM reveals that [Fe(4)S(4)](SAM) exists in two forms, labeled [Formula: see text] and [Formula: see text]. It is proposed that these forms are involved in the synthesis of the L cluster. Of the two cluster types, only [Formula: see text] initially interacts with SAM, resulting in the generation of Z, an S = ½ paramagnetic [Fe(4)S(4)](SAM)/SAM complex.