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 Av 2 bind two molecules of MgADP, with the following dissociation constants: reduced Av 2 , K 1 = 0.091 ± 0.021 mM and K 2 = 0.044 ± 0.009 mM; oxidized Av 2 , K 1 = 0.024 ± 0.015 mM and K 2 = 0.039 ± 0.022 mM. Binding of MgADP to reduced Av 2 shows positive co‐operativity. Oxidized Av 2 binds two molecules of MgATP with dissociation constants K 1 = 0.049 ± 0.016 mM and K 2 = 0.18 ± 0.05 mM. Binding data of MgATP to reduced Av 2 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 K 1 = 0.22 ± 0.03 mM and K 2 = 1.71 ± 0.50 mM. It was found that results concerning the number of binding sites and the dissociation constants of MgATP‐Av 2 and MgADP‐Av 2 complexes depend to a great extent on the specific activity of the Av 2 preparation used, and that it is difficult to correct binding data for inactive protein. No binding of MgADP to Av 1 could be demonstrated. Binding studies of MgADP to a mixture of Av 1 and Av 2 showed that Av 1 did not affect the binding of MgADP to either oxidized or reduced Av 2 . Inhibition studies were performed to investigate the interaction of MgATP and MgADP binding to oxidized and reduced Av 2 . All the experimental data can be explained by the minimum hypothesis, i.e. the presence of two adenine nucleotide binding sites on Av 2 . MgATP and MgADP compete for these two binding sites on the Fe protein.