Dataset Information

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 [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 [Fe₄S₄]_SAM/SAM complex.

SUBMITTER: Rupnik K

PROVIDER: S-EPMC8403491 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Rupnik Kresimir K Rettberg Lee L Tanifuji Kazuki K Rebelein Johannes G JG Ribbe Markus W MW Hu Yilin Y Hales Brian J BJ

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20210427 4

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 [Fe4< ...[more]

PMID: 33905031

Similar Datasets

Project description:Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct nifB sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandii nifB mutant. Twenty of these variants rescued the nifB mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, nifB functional modules composed of a nifB variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe4S4] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (Methanosarcina acetivorans and Methanocaldococcus infernus) or mitochondria (M. infernus and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe4S4] clusters from NifU and were functional in FeMo-co synthesis in vitro. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants. IMPORTANCE Biological nitrogen fixation is the conversion of inert atmospheric dinitrogen gas into nitrogen-reactive ammonia, a reaction catalyzed by the nitrogenase enzyme of diazotrophic bacteria and archaea. Because plants cannot fix their own nitrogen, introducing functional nitrogenase in cereals and other crop plants would reduce our strong dependency on N fertilizers. NifB is required for the biosynthesis of the active site cofactors of all nitrogenases, which arguably makes it the most important protein in global nitrogen fixation. NifB functionality is therefore a requisite to engineer a plant nitrogenase. The expression of nifB genes from a wide range of prokaryotes into the model diazotroph Azotobacter vinelandii shows a surprising level of genetic complementation suggestive of plasticity in the nitrogenase biosynthetic pathway. In addition, we obtained NifB proteins from both mitochondria and chloroplasts of tobacco that are functional in vitro after reconstitution by providing [Fe4S4] clusters from NifU, paving the way to nitrogenase cofactor biosynthesis in plants.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets