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

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Two fluorescent phenolic compounds were isolated from iron-deficient cultures of Azotobacter vinelandii. One was identified as 2,3-dihydroxybenzoic acid, an infrequently encountered microbial metabolite not previously reported from Azotobacter. The second compound was shown to be the previously unknown 2-N,6-N-di-(2,3-dihydroxybenzoyl)-~-lysine, with final proof of structure established by synthesis. The

The analyses reported in Table 1 indicate that the fragment I used by us had 7 or 8, rather than 6 disulfide bonds, leading to an even lower probability for return to the native structure by random reoxidation.

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