Stoichiometry of the Adenosine Triphosphate Requirement for N2 Fixation and H2 Evolution by a Partially Purified Preparation of Clostridium pasteurianum

Abstract

Abstract Cold ethanol or acetone precipitated about 75% of the N2-fixing enzyme complex from extracts of Clostridium pasteurianum freed of nucleic acids. This simple preparation fixes about 45 nmoles of N2 per mg of protein per min and is adequate for many studies of N2 fixation. The preparation exhibited a dilution effect in N2 fixation supported by Na2S2O4 and an ATP-generating system. H2 was evolved vigorously through an ATP-requiring, CO-insensitive reaction; the preparation evolved H2 less vigorously in the absence of ATP through the CO-sensitive pathway unless ferredoxin was added. The N2-fixing complex required 4.0 to 4.6 molecules of ATP per H2 evolved or per third of a molecule of N2 fixed throughout the time course of the reactions. At pH 6.0 the ATP/2e- ratio approached 4.0, and it was near 4.6 at pH 8.0. Both N2 fixation and ATP-dependent H2 evolution had pH optima between pH 6.5 and 6.6.