Kinetics of dithionite ion utilization and ATP hydrolysis for reactions catalyzed by the nitrogenase complex from Azotobacter vinelandii

Abstract

The kinetics of S2O42-utilization and ATP hydrolysis during the nitrogenase-catalyzed H2 evolution and acetylene and nitrogen-reducing reactions were studied using a polarographic technique to monitor-S2O42-concentration. Rate constants for both S2O42-utilization and ATP hydrolysis were determined as a function of temperature and corresponding activation energies determined. The activation energy for ATP hydrolysis differs from that for product formation or S2O42-utilization by 5 kcal/mol above 20 degrees C and by 25 kcal/mol below 20 degrees C. The rate law for S2O42-utilization was determined and describes the enzyme catalyzed rate over a 1000-fold variation in S2O42-concentration and at least a 100-fold change in ATP concentration. The rate law for S2O42-utilization under N2-reducing conditions at 25 degrees C is given by -d([S2O42-]/dt = (2.3 x 10(-3) ET[S2O42-]1/2-[ATP]2)/([ATP]2 + K1[ATP] + K2), where ET is total enzyme concentration in mg/ml and K1 and K2 are equilibrium constants for ATP binding to nitrogenase. The half-order dependence of the rate on S2O42-concentration in interpreted in terms of the equilibrium S2O42- = 2SO2-, in which SO2- is the actual electron donor to nitrogenase. A partial mechanism incorporating these results is presented.