Hydrolysis of nucleoside triphosphates other than ATP by nitrogenase

The hydrolysis of ATP to ADP and P-i is an integral part of all substrate r eduction reactions catalyzed by nitrogenase. In this work, evidence is pres ented that nitrogenases isolated from Azotobacter vinelandii and Clostridiu m pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nu cleoside diphosphates at rates comparable to those measured for MgATP hydro lysis. The reactions were dependent on the presence of both the iron (Fe) p rotein and the molybdenum-iron (MoFe) protein. The oxidation state of nitro genase was found to greatly influence the nucleotide hydrolysis rates. MgAT P hydrolysis rates were 20 times higher under dithionite reducing condition s (similar to 4000 nmol of MgADP formed per min/mg of Fe protein) as compar ed with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed p er min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis r ates were significantly higher under oxidizing conditions (1400-2000 nmol o f MgNDP formed per min/mg of Fe protein) as compared with reducing conditio ns (80-230 nmol of MgNDP formed per min/mg of Fe protein). The K-m values f or MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330- 540 mu M) for both the reduced and oxidized states of nitrogenase, Incubati on of Fe and MoFe proteins with each of the MgNTP molecules and AlF4- resul ted in the formation of non-dissociating protein-protein complexes, presuma bly with trapped AlF4-. MgNDP. The implications of these results in underst anding how nucleotide hydrolysis is coupled to substrate reduction in nitro genase are discussed.