Role of magnesium adenosine 5'-triphosphate in the hydrogen evolution reaction catalyzed by nitrogenase from Azotobacter vinelandii

Abstract

The role of MgATP in the reaction catalyzed by nitrogenase from A. vinelandii. There is a rapid burst of ATP hydrolysis in the pre-steady-state reaction that occurs on the same time scale as the electron transfer from dinitrogenase reductase to dinitrogenase. This burst corresponds to 2 ATP hydrolyzed/electron transferred between the 2 proteins. Two MgATP molecules are bound to dinitrogenase reductase with Kd of 430 .mu.M and 220 .mu.M. Investigation of the effect of MgATP concentration on the pre-steady-state kinetics of electron transfer from dinitrogenase reductase to dinitrogenase showed that there are 2 MgATP required for this reaction, and the Km values are 220 .mu.M and 970 .mu.M. These values are similar to the Kd for MgATP from dinitrogenase reductase and indicate that electron transfer between the 2 proteins is substantially slower than the binding and dissociation of MgATP from dinitrogenase reductase. The Km values for MgATP in steady-state H2 evolution were 390 .mu.M and 30 .mu.M. The decrease in value of the 2nd Km indicates that a slow, irreversible step occurs after the electron transfer from dinitrogenase reductase to dinitrogenase. It is possible to predict quantitatively the steady-state kinetics from the pre-steady-state kinetics; this shows that the MgATP dependence of electron transfer is sufficient to account for effects of MgATP concentration on the steady-state H2 evolution catalyzed by nitrogenase. The hydrolysis of 2 ATP molecules when an electron is transferred between the 2 proteins of the nitrogenase system is sufficient to account for all of the ATP hydrolysis occurring in the steady-state reaction. The simplified scheme proposed to account for the MgATP dependency of the nitrogenase reaction indicates that the only role of MgATP is in support of the electron transfer from dinitrogenase reductase to dinitrogenase.