Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii

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Abstract

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 γ 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 (Kd ≈ 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 His121 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.

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Rubio, L. M., Singer, S. W., & Ludden, P. W. (2004). Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii. Journal of Biological Chemistry, 279(19), 19739–19746. https://doi.org/10.1074/jbc.M400965200

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