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

Abstract

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·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 \(\left[ {{\text{Fe}}_{{4}} {\text{S}}_{{4}} } \right]_{{{\text{SAM}}^{{\text{A}}} }}^{ + }\) and \(\left[ {{\text{Fe}}_{{4}} {\text{S}}_{{4}} } \right]_{{{\text{SAM}}^{{\text{B}}} }}^{{2 + }}\). It is proposed that these forms are involved in the synthesis of the L cluster. Of the two cluster types, only \(\left[ {{\text{Fe}}_{{4}} {\text{S}}_{{4}} } \right]_{{{\text{SAM}}^{{\text{B}}} }}^{{2 + }}\) initially interacts with SAM, resulting in the generation of Z, an S = ½ paramagnetic [Fe₄S₄]_SAM/SAM complex. Graphic abstract