Properties of the MgATP and MgADP binding sites on the Fe protein of nitrogenase from Azotobacter vinelandii

Abstract

Flow dialysis was used to study the binding of MgATP and MgADP to the nitrogenase proteins of A. vinelandii. Both reduced and oxidized Fe protein (Av2) bind 2 molecules of MgADP, with the following Kd: 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 cooperativity. Oxidized Av2 binds 2 molecules of MgATP with Kd 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 1 binding site, but a better fit was obtained by assuming 2 binding sites on the protein with negative cooperativity and with Kd 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 Kd 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 Kd (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 2 adenine nucleotide binding sites on Av2. MgATP and MgADP compete for these 2 binding sites on the Fe protein.