An EPR and VTVH MCD spectroscopic investigation of the nitrogenase assembly protein NifB
- 27 April 2021
- journal article
- research article
- Published by Springer Science and Business Media LLC in JBIC Journal of Biological Inorganic Chemistry
- Vol. 26 (4), 403-410
- https://doi.org/10.1007/s00775-021-01870-y
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. Graphic abstract
Keywords
Funding Information
- NIH-NIGMS (GM67626)
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