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 (MaNifBSAM) containing only the [Fe4S4]SAM cluster and no K cluster. Titration of MaNifBSAM 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.
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This work was supported by NIH-NIGMS Grant GM67626 (to MWR and YH)
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All authors contributed to the study conception and design. Material preparations were performed by LR, KT, JGR, MWR and YH. Data collection and analysis were performed by KR and BH. The first draft of the manuscript was written by BH and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Rupnik, K., Rettberg, L., Tanifuji, K. et al. An EPR and VTVH MCD spectroscopic investigation of the nitrogenase assembly protein NifB. J Biol Inorg Chem 26, 403–410 (2021). https://doi.org/10.1007/s00775-021-01870-y
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DOI: https://doi.org/10.1007/s00775-021-01870-y