Oxidative activity and nitrogen fixation in cell-free preparations from Azotobacter vinelandii.

Ammonia is generally conceded to be the "key intermediate" in biological nitrogen fixation-that is, the compound which reacts to form the first stable organic compound, glutamic acid (Wilson and Burris, 1953). However, there is virtually no information concerning the reactions functioning in the conversion of N2 to ammonia. Reproducible nitrogen fixation by a cell-free preparation from bacteria would be most helpful in elucidating the enzymatic mechanism of the intermediate steps. Bach et al. (1934) reported fixation by cellfree preparations from Azotobacter vinelandii, but these results could be repeated neither in Bach's laboratory nor elsewhere. Wilson and Roberts (1954) have presented a brief account of their negative results with cell-free preparations. Hamilton et al. (1953) used N215 as a tracer and reported the detection of limited fixation of nitrogen by cell-free preparations from A. vinelandii. As nitrogen fixation requires energy, it is important to study the respiration of the azotobacter as a potential source of that energy. Lee et al. (1942) investigated cell-free preparations of A. vinelandii and found succinic, lactic and malic dehydrogenases and hydrogenase by coupling the reactions through methylene blue. They also demonstrated oxalacetic and a-ketoglutaric acid decarboxylases. For a time it was believed that the citric acid cycle was inoperative in these organism, because they utilized citrate very slowly and exhibited a marked lag in the oxidation of succinate and malate. Recently, Stone and Wilson (1952a, 1952b, 1952c) have shown an active citric acid