title: Apodinitrogenase: purification, association with a 20-kilodalton protein, and activation by the iron-molybdenum cofactor in the absence of dinitrogenase reductase.
abstract: The Azotobacter vinelandii mutant strain UW45 contains a mutation in the nifB gene and produces an inactive dinitrogenase protein that can be activated by the addition of purified iron-molybdenum cofactor (FeMoco). This FeMoco-deficient dinitrogenase (Apo I) has now been purified 96-fold to greater than 95% purity and is FeMoco-activatable to 2200 nmol of C2H2 reduced/(min.mg of protein). The Apo I complex was found to contain two molecules of a 20-kDa protein, in addition to the alpha 2 beta 2 tetramer found for isolated holodinitrogenase (Holo I). The Apo I complex contained 15 +/- 2 mol of Fe per mole, but no Mo. While the presence of dinitrogenase reductase caused a 2-fold stimulation in the activation of the purified Apo I complex by FeMoco, this enhancement resulted from the stabilization of Apo I by dinitrogenase reductase to the denaturing effects of N-methylformamide. When the activation was performed in the absence of N-methylformamide, there was no enhancement by dinitrogenase reductase alone or by dinitrogenase reductase-Mg-ATP complex. The Apo I complex is more sensitive to O2 than Holo I, with a half-life in air of 6 min; however, the addition of dithiothreitol to Apo I during the exposure to air (or after exposure) resulted in a half-life very similar to that seen for Holo I. This suggests that sulfhydryl(s) is (are) important for the FeMoco-activation reaction.
authors.affiliation: Department of Bacteriology, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
authors.forename: T D
authors.initials: TD
authors.lastname: Paustian
authors.forename: V K
authors.initials: VK
authors.lastname: Shah
authors.forename: G P
authors.initials: GP
authors.lastname: Roberts
mesh_terms: D001051:Apoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001395:Azotobacter / Q000201:enzymology* / Q000235:genetics; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D017037:Dinitrogenase Reductase; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D005288:Ferredoxins / Q000378:metabolism*; D005798:Genes, Bacterial; D006358:Hot Temperature; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D008983:Molybdoferredoxin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D009591:Nitrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D001051:Apoenzymes; D001059:Apoproteins; D017037:Dinitrogenase Reductase; D005288:Ferredoxins; D006882:Hydroxyapatites; D008983:Molybdoferredoxin; D017886:Durapatite; D009591:Nitrogenase