Properties of the MgATP and MgADP binding sites on the Fe protein of nitrogenase from Azotobacter vinelandii.
Flow dialysis was used to study the binding of MgATP and MgADP to the nitrogenase proteins of Azotobacter vinelandii. Both reduced and oxidized Av2 bind two molecules of MgADP, with the following dissociation constants: reduced Av2, K1 = 0.091 +/- 0.021 mM and K2 = 0.044 +/- 0.009 mM; oxidized Av2, K1 = 0.024 +/- 0.015 mM and K2 = 0.039 +/- 0.022 mM. Binding of MgADP to reduced Av2 shows positive co-operativity. Oxidized Av2 binds two molecules of MgATP with dissociation constants K1 = 0.049 +/- 0.016 mM and K2 = 0.18 +/- 0.05 mM. Binding data of MgATP to reduced Av2 can be fitted by assuming one binding site, but a better fit was obtained by assuming two binding sites on the protein with negative co-operativity and with dissociation constants K1 = 0.22 +/- 0.03 mM and K2 = 1.71 +/- 0.50 mM. It was found that results concerning the number of binding sites and the dissociation constants of MgATP-Av2 and MgADP-Av2 complexes depend to a great extent on the specific activity of the Av2 preparation used, and that it is difficult to correct binding data for inactive protein. No binding of MgADP to Av1 could be demonstrated. Binding studies of MgADP to a mixture of Av1 and Av2 showed that Av1 did not affect the binding of MgADP to either oxidized or reduced Av2. Inhibition studies were performed to investigate the interaction of MgATP and MgADP binding to oxidized and reduced Av2. All the experimental data can be explained by the minimum hypothesis, i.e. the presence of two adenine nucleotide binding sites on Av2. MgATP and MgADP compete for these two binding sites on the Fe protein.
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001395:Azotobacter / Q000201:enzymology*; D001665:Binding Sites; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D008274:Magnesium / Q000378:metabolism*; D008433:Mathematics; D008667:Metalloproteins / Q000302:isolation & purification*; D009591:Nitrogenase; D010088:Oxidoreductases
D008667:Metalloproteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D008274:Magnesium