Modular organization and identification of a mononuclear iron-binding sitewithin the NifU protein

The NifS and NifU nitrogen fixation-specific gene products are required for the full activation of both the Fe-protein and MoFe-protein of nitrogenase from Azotobacter vinelandii. Because the two nitrogenase component protein s both require the assembly of [Fe-S]-containing clusters for their activat ion, it has been suggested that NifS and NifU could have complementary func tions in the mobilization of sulfur and iron necessary for nitrogenase-spec ific [Fe-S] cluster assembly. The NifS protein has been shown to have cyste ine desulfurase activity and can be used to supply sulfide for the in vitro catalytic formation of [Fe-S] clusters. The NifU protein was previously pu rified and shown to be a homodimer with a [2Fe-2S] cluster in each subunit. In the present work, primary sequence comparisons, amino acid substitution experiments, and optical and resonance Raman spectroscopic characterizatio n of recombinantly produced NifU and NifU fragments are used to show that N ifU has a modular structure. One module is contained in approximately the N -terminal third of NifU and is shown to provide a labile rubredoxin-like fe rric-binding site. Cysteine residues Cys(35), Cys(62), and Cys(106) are nec essary for binding iron in the rubredoxin-like mode and visible extinction coefficients indicate that up to one ferric ion can be bound per NifU monom er. The second module is contained in approximately the C-terminal half of NifU and provides the [2Fe-2S] cluster-binding site. Cysteine residues Cys( 137), Cys(139), Cys(172), and Cys(175) provide ligands to the [2Fe-2S] clus ter. The cysteines involved in ligating the mononuclear Fe in the rubredoxi n-like site and those that provide the [2Fe-2S] cluster ligands are all req uired for the full physiological function of NifU. The only two other cyste ines contained within NifU, Cys(272) and Cys(275), are not necessary for ir on binding at either site, nor are they required for the full physiological function of NifU. The results provide the basis for a model where iron bou nd in labile rubredoxin-like sites within NifU is used for [Fe-S] cluster f ormation. The [2Fe-2S] clusters contained within NifU are proposed to have a redox function involving the release of Fe from bacterioferritin and/or t he release of Fe or an [Fe-S] cluster precursor from the rubredoxin-like bi nding site.