Characterization and site-directed mutagenesis of NifU from Azotobacter vinelandii

In order to elucidate the function of the nifU gene product in nitrogenase maturation in Azotobacter vinelandii. the gene product has been hyperexpressed in Escherichia coli and characterized by various biophysical techniques. Following the initial characterization, site-directed mutagenesis of conserved cysteinyl residues was performed in order to gain further insight into the structure/function relationship of NifU.

Both the Fe protein and the MoFe protein of nitrogenase require processing by additional nif genes including nifM (Fe protein), and nifE, N, B, H, V, and Q (MoFe protein). Two additional genes, nifU and nifS, are required for the maturation of both nitrogenase component proteins. It has been proposed that they may somehow be involved in metallocluster biosynthesis (Jacobson et al., 1989b). Our laboratory has determined that the nifS gene product (Nifs) is a pyridoxal-phosphate containing enzyme capable of catalyzing the desulfurization of L-cysteine and can provide the inorganic sulfide necessary for in vitro metallocluster biosynthesis of the Fe protein (Zheng, et al., 1993: Zheng, et al., 1994).