Purification and characterization of NafY (apodinitrogenase gamma subunit) from Azotobacter vinelandii

The formation of an active dinitrogenase requires the synthesis and the insertion of the iron-molybdenum cofactor (FeMo-co) into a presynthesized apodinitrogenase. In Azotobacter vinelandii, NafY (also known as gamma protein) has been proposed to be a FeMo-co insertase because of its ability to bind FeMo-co and apodinitrogenase. Here we report the purification and biochemical characterization of NafY and reach the following conclusions. First, NafY is a 26-kDa monomeric protein that binds one molecule of FeMo-co with very high affinity (K(d) approximately equal to 60 nm); second, the NafY-FeMo-co complex exhibits a S = 3/2 EPR signal with features similar to the signals for extracted FeMo-co and the M center of dinitrogenase; third, site-directed mutagenesis of nafY indicates that the His(121) residue of NafY is involved in cofactor binding; and fourth, NafY binding to apodinitrogenase or to FeMo-co does not require the presence of any additional protein. In addition, we have obtained evidence that suggests the ability of NafY to bind NifB-co, an FeS cluster of unknown structure that is a biosynthetic precursor to FeMo-co.