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.
Department of Plant and Microbial Biology, College of Natural Resources, University of California-Berkeley, Berkeley, CA 94720, USA.
Luis M
LM
Rubio
Steven W
SW
Singer
Paul W
PW
Ludden
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry; D002845:Chromatography; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000737:chemistry* / Q000302:isolation & purification*; D016011:Normal Distribution; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D008983:Molybdoferredoxin; C092498:NifB protein, Bacteria; D009591:Nitrogenase; D005982:Glutathione Transferase