On the Binding of MgATP and MgADP to the Nitrogenase Proteins from Azotobacter Vinelandii

Abstract

To gain insight on the role of MgATP and MgADP in nitrogenase function and regulation, we investigated the interaction of both nucleotides with the nitrogenase proteins from A. vinelandii. The method used for these binding studies was the flow dialysis technique. Fig. 1 shows that reduced Av2 binds one molecule of MgATP (Kd = 0.33 mM) and one molecule of MgADP (Kd= 0.15 mM). Inhibition studies were performed to establish whether the binding site for MgATP on Av2 is the same as the one for MgADP. Up to concentrations of 0.2 mM, MgADP does not have any effect on the binding of MgATP to reduced Av2. At higher concentrations of MgADP there is an increasing inhibition of the MgATP binding. A completely different inhibition pattern is obtained when MgADP binding is measured in the presence of MgATP. By increasing the concentration of MgATP, the hyperbolic binding curve of MgADP becomes sigmoidal. These results indicate that there are different binding sites for MgATP and MgADP present on the reduced protein, but that these two binding sites have interaction with each other. Fig. 2 shows that oxidized Av2 binds two molecules of MgATP (Kd = 0.14 mM) and one molecule of MgADP (Kd=0.14 mM). The binding of MgATP is strongly inhibited by MgADP. MgATP, on the other hand, has only a small effect on the binding of MgADP. No binding of MgATP and MgADP to Av1 could be demonstrated. Tso and Burris (1) found two MgATP binding sites and one MgADP binding site for (reduced) Cp2. It was suggested (2) that these two molecules of MgATP are hydrolyzed during the one electron transfer from the Fe protein to the MoFe protein. Braaksma et al. (3)found that Av2 is a two electron donor. Together with our binding results, this would mean that only one molecule of MgATP is hydrolyzed for each pair of electrons transferred. Since a minimum of 4 ATP per 2 electrons is found for the overall nitrogenase reaction, there should be MgATP hydrolysis after electron transfer.