Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB.

One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases.

Electron Paramagnetic Resonance Characterization of Three Iron-Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB from Methanocaldococcus infernus.

NifB utilizes two equivalents of S-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe-S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron-molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen Methanocaldococcus infernus is a radical SAM enzyme able to reductively cleave SAM to 5'-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe-4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation.

Expression and purification of NifB proteins from aerobic and anaerobic sources.

NifB is the key protein in the biosynthesis of nitrogenase iron-molybdenum cofactor. Due to its extreme sensitivity to O2 and inherent protein instability, NifB proteins must be purified under strict anaerobic conditions by using affinity chromatography methods. We describe here the methods for NifB purification from cells of the strict aerobic nitrogen-fixing bacterium Azotobacter vinelandii, the facultative anaerobic nitrogen-fixing bacterium Klebsiella pneumoniae, and the facultative anaerobic non-nitrogen fixing bacterium Escherichia coli recombinantly expressing a nifB gene of thermophilic origin.