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The requirement for both an electron donor and ATP for the enzymatic reduction of N2 to ammonia was demonstrated with extracts of Clostridium pasteurianum,1-4 Azotobacter vinelandii,5 7 and Rhodospirillum rubrum.' 8 With hydrosulfite as the electron donor and an ATP-generating system (creatine phosphokinase), N2-reducing preparations from A. vinelandii and R. rubrum were shown to catalyze a concomitant ATP-dependent H2-evolution reaction.6-9 This reaction was subsequently demonstrated with extracts of C. pasteurianum10' 11 by inhibiting the classical hydrogenase with carbon monoxide. The use of hydrosulfite as the electron donor, in combination with an ATP-generating system, provides a useful assay reaction for enzyme purification since both required components can be added exogenously. We reported previously7 that the N2-reducing and ATP-dependent H2-evolving activities could be obtained from azotobacter extracts by precipitation with protamine followed by pH fractionation, and that these fractions contained a high level of nonheme iron as well as molybdenum. Procedures have now been developed for the isolation of two enzyme fractions, both of which are required for N2 reduction, ATP-dependent H2 evolution, and the related release of inorganic phosphate. The first enzyme contains both nonheme iron and molybdenum; the second contains nonheme iron but no molybdenum. The purification and some properties of these two enzymes are described in this report. A preliminary report was presented previously.12 Mlortenson'3 14 recently reported evidence suggesting the requirement for at least two enzymes from C. pasteurianum extracts for the catalysis of N2 reduction and related reactions. Materials and Methods.-Azotobacter vinelandii 0 was cultured and harvested as previously described,6 and the cell paste either used when harvested or stored frozen in an argon atmosphere. Cells (100-120-gm cell paste) were ruptured in a French pressure cell, and the S1441/2 supernatant fraction (obtained by centrifugation at 144,000 X g for 30 min), containing 90-95% of the activity of crude extracts, was prepared as previously described.7 Since a sensitivity to oxygen developed during purification, all buffers and solutions were saturated with argon and contained 0.1 mg of dithiothreitol per ml. Protamine sulfate fractionation: Protamine was used both to remove nucleic acids and to precipitate the enzymatically active components. A 2% solution of the reagent (Sigma Chemical Co., Grade II) was prepared in water at room temperature, the pH adjusted to 6.0 with 1 N NaOH, and the precipitate removed by centrifugation. The supernatant solution was decanted and stirred mechanically under a stream of argon to remove dissolved oxygen. The S144-1/2 fraction, allowed to cool during ultracentrifugation, was held at 00 during protamine fractionation. Protamine sulfate solution was added at the rate of 5 ml per gm of protein, and the precipitated nucleic acids were removed by centrifugation. The decanted supernatant solution was adjusted to pH 6.5 with 0.5 N acetic acid, and the active components were precipitated upon the further addition of 1.2 ml of protamine sulfate solution per gm of S,44-i/, protein. After centrifugation, the precipitate was suspended in 0.01 M potassium phosphate, pH 7.0, at room temperature and stirred with purified7 cellulose phosphate (Sigma Chemical Co.) added at the rate of 100 mg per gm of S44-i/, protein. Cellulose phosphate with its bound protamine was removed on a fritted

The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis.