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

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Abstract
The kinetics of S2O42- utilization and ATP hydrolysis during the nitrogenase-catalyzed H2 evolution and acetylene and N2-reducing reactions were studied using a polarographic technique to monitor S2O42- concentration. Rate constants for S2O42- utilization and ATP hydrolysis were determined as a function of a 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 > 20.degree. C and by 25 kcal/mol > 20.degree. C. The rate law for S2O42- utilization was determined; it described 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.degree. C is given by -d([S2O42-])/dt = (2.3 .times. 10-3 ET[S2O42-]1/2-[ATP]2)/([ATP]2 + K1[ATP] + K2), where ET is total enzyme concentration in milligrams per milliliter and K1 and K2 are equilibrium constants for ATP binding to nitrogenase. The half-order dependence of the rate on S2O42- concentration is 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.

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