Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase

Publisher Website
Google Scholar
Abstract
The pre-steady-state ATPase activity of nitrogenase from Azotobacter vinelandii was investigated. By using a rapid-quench technique, it has been demonstrated that with the oxidized nitrogenase complex the same burst reaction of MgATP hydrolysis occurs as observed with the reduced complex, namely 6-8 mol orthophosphate released/mol MoFe protein. It is concluded that the pre-steady-state ATPase activity is independent of electron transfer from Fe protein to MoFe protein. Results obtained from gel centrifugation experiments showed that during the steady state of reductant-independent ATP hydrolysis there is a slow dissociation of one molecule of MgADP from the nitrogenase proteins (koff .ltoreq. 0.2 s-1); the second MgADP molecule dissociates much faster (koff .gtoreq. 0.6 s-1). Under the same conditions orthophosphate was found to be associated with the nitrogenase proteins. The rate of dissociation of orthophosphate from the nitrogenase complex, as estimated from the gel centrifugation experiments, is the same order of magnitude as the steady-state turnover rate of the reductant-independent ATPase activity (0.6 mol Pi formed .cntdot. s-1 .cntdot. mol .**GRAPHIC**. at 22.degree. C). These data are consistent with dissociation of orthophosphate or MgADP being rate-limiting during nitrogenase-catalyzed reductant-independent ATP hydrolysis.