Binding of MgATP to the nitrogenase proteins from Azotobacter vinelandii.

Abstract

Binding of MgATP to the MoFe and Fe proteins from Azotobacter vinelandii has been studied. By means of the flow dialysis technique it was demonstrated that one molecule of reduced Fe protein binds one molecule of MgATP, with a dissociation constant of 0.56 +/- 0.11 mM. The oxidized Fe protein binds two molecules of MgATP, with identical intrinsic dissociation constants of 0.29 +/- 0.05 mM. The binding of MgATP to the Fe protein was also studied by equilibrium dialysis. It was found that during dialysis of reduced Fe protein in the presence of MgATP, dithionite was oxidized. Moreover, in the presence of MgATP both reduced and oxidized Fe protein were inactivated during the dialysis. These observations demonstrate that binding of MgATP to the Fe protein can only be measured by a relatively fast method. With the same methods as used for the Fe protein, no binding of MgATP to the MoFe protein of A. vinelandii could be demonstrated. The redox properties of the Fe protein in the presence and absence of MgATP are discussed with respect to the observed binding properties of MgATP for the Fe protein. The implications of these results are discussed with respect to the present models for the interactions between the Fe and MoFe proteins of nitrogenase.