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Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O 2 -sensitive  S -adenosyl methionine (SAM)–radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium  Azotobacter vinelandii  and the thermophilic methanogen  Methanocaldococcus infernus  in  Saccharomyces cerevisiae  and in  Nicotiana benthamiana . NifB proteins were targeted to the mitochondria, where O 2  consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe–S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the  A. vinelandii  version appeared to form aggregates. Similarly, NifB from  M. infernus  was expressed at higher levels in leaves of  Nicotiana benthamiana  and accumulated as a soluble protein while  A. vinelandii  NifB was mainly associated with the non-soluble cell fraction. Soluble  M. infernus  NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the  in vitro  FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening  nif  genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.

Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB