The Effect of Reductant in Inorganic Phosphate Release from Adenosine 5'-Triphosphate by Purified Nitrogenase of Clostridium pasteurianum

Abstract

The two components of nitrogenase of Clostridium pasteurianum, azoferredoxin and molybdoferredoxin, together with an electron donor, and energy source, and Mg++ are required for ATP-dependent H2 evolution and reductant-dependent ATP utilization. ATP is also utilized by this N2-fixing system without H2 evolution and without the need for an electron donor. For this activity there is still an absolute requirement for both azoferredoxin and molybdoferredoxin. The ratio of ATP utilized in the absence of reductant to that in the presence of reductant was approximately 1:3 at pH 6.6, whereas at pH 5.2 there was little difference between the two ATP-consuming activities. The ratio of ATP consumed to H2 evolved by the N2-fixing system at pH values between 6.0 and 7.6 varied between 3 and 8. At pH 6.6 a minimum ratio of 3 was obtained. No H2 was evolved in the absence of an electron donor even though ATP was consumed. When the amount of ATP used for each H2 evolved at all pH conditions was corrected for the amount of ATP used in the absence of the reductant, a constant ratio of 2 ATP per H2 evolved was obtained. The visible spectrum of molybdoferredoxin between 300 to 400 nm also changes with pH, suggesting that a certain form of the chromophore of molybdoferredoxin may be required for the reductant-dependent utilization of ATP.