The major part of biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which carries at its active site the most complex metallocluster known in biology, the iron-molybdenum cofactor (FeMo-co). It is composed of 7 Fe, 9 S, 1 Mo, 1homocitrate, and 1 unidentified central atom. The genetic and biochemical analysis of nitrogen-fixation (nif) genes and proteins in Azotobacter vinelandii and Klebsiella pneumoniae during the pre-genomic era led to the identification of at least 11 genes(nifUS-BQ-ENX-V-H-Y and naJY) proposed to be involved in the biosynthesis and insertion of FeMo-co into apo-dinitrogenase (Rubio and Ludden, 2005; Dos Santos et al., 2004). The importance of the product of the nifB gene in this biosynthetic pathway haslong been recognized because its metabolic product (NifB-co) is an intermediate of the FeMo-co biosynthetic pathway, and also of the biosynthetic pathways of the cofactors of the alternative nitrogenases. NifB-co is an iron-sulfur cluster of unknown structure that accumulates in cells of mutant strains lacking nifE, nifN or nifH (Shah et al., 1994). The inability to isolate the NifB protein has unpaired progress in the understanding of the biochemical aspects of the NifB-catalyzed reaction for more thantwo decades. Here, we describe the purification and characterization of the A. vinelandii NifB protein and the development of a NifB-activity assay. The reaction catalyzed by NifB requires S-adenosylmethionine (SAM) and allows, for the first time, the synthesis of FeMo-co from Fe, S, Mo and homocitrate.
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