The Enzymology of Nitrogen Fixation in Cell-free Extracts of Clostridium Pasteurianum

Extracts of Clostridium pasteurianum have been separated into five protein fractions, which are necessary for nitrogen fixation when pyruvate is used as the source of energy and electrons. Pyruvate oxidase is shown to be heat-stable and to have gel filtration properties corresponding to a molecular weight of 100,000 or greater. The function of ferredoxin as an electron carrier for nitrogen fixation is verified. In addition, it is shown here that reduced ferredoxin can act as the sole source of electrons for nitrogen fixation and for acetylene reduction. Fraction C1S1, which is necessary for both nitrogen fixation and acetylene reduction, has gel filtration properties corresponding to a molecular weight of 100,000 or greater. Its activity is stable at 0° but not at 60°, and is found in nitrogen-grown cells but not in ammonium sulfate-grown cells. Fraction IVn, which is necessary for nitrogen fixation but not for acetylene reduction, is of a size corresponding to a molecular weight of 40,000. Its activity is present only in cells grown on nitrogen gas, and this activity is stable at 0° as well as at 60°. Fraction IVs is necessary for both nitrogen fixation and acetylene reduction, and its size corresponds to a molecular weight of about 45,000. Its activity, which is found in cells grown on either nitrogen gas or ammonium sulfate, is stable at 60° but not at 0°. The properties of these fractions are compared with those of similar fractions previously described.