Properties of the nitrogenase system in cell-free extracts of bacteroids from soybean root nodules.

The capacity of excised nodules from soybean plants to fix atmospheric N2 was demonstrated convincingly in 19521 by use of the sensitive '5N technique. Although the specific inhibitory effect of H2 on N2 fixation by nodulated red clover was discovered thirty years ago,2' 3 an understanding of the biochemistry of N2 fixation in symbionts has been delayed as a result of difficulties in obtaining N2-fixing activity in fractions of macerated nodules. Bergerson4 in 1966 utilized a specially designed press and anaerobic conditions for the preparation of an active brei of soybean nodules. Fixation of N2 was detected by the 15N assay and was shown to be dependent upon 02. More recently, Bergerson and Turner' (manuscript kindly supplied by Dr. Bergerson) have reported that washed suspensions of bacteroids from soybean nodules retained a capacity to reduce N2, and that fixation was stimulated by the addition of oxidizable substrates including succinate, fumarate, and pyruvate. The rate of NH3 formation was about 0.4 m,4mole per minute per mg of protein. After the discovery6' 7 that Clostridium pasteurianum extracts would catalyze the reduction of acetylene in addition to that of N2, our laboratory8 9 approached the problem of N2 fixation in symbionts by assaying capacities of nodules and nodule fractions to catalyze acetylene reduction. By use of a method in which anaerobic conditions, a buffered ascorbate medium, and insoluble polyvinylpyrrolidone were employed to remove endogenous phenolics,'0 washed bacteroid suspensions with excellent acetylene-reducing activities were obtained. Cell-free extracts of bacteroids prepared by this technique consistently catalyzed N2 reduction8 and provided the first opportunity for the definition of the biochemical events in N2 fixation by legumes. The quantity of NH3 produced was sufficient for measurement by direct titration. In addition to active extract, the reduction of N2 required an adenosine 5'-triphosphate (ATP) generating system, Na2S204 as an electron donor, and anaerobic conditions. The requirements appeared to be similar to those for the Nrfixing system from Azotobacter vinelandii.1' In the initial experiments fractionation of extracts to remove endogenous compounds of low molecular weight could not be accomplished without loss of N2-fixing activity. Now active extracts relatively free of small molecules are prepared routinely. The purpose of this communication is to describe some properties of the nitrogenase system in bacteroid extracts. Such information is required before embarking upon the tedious task of purifying the nitrogenase system under anaerobic conditions. Materials and Methods.-Source and preparation of extracts: Soybean plants (Glycine max Merr. var. Alerrit) inoculated with a commercial preparation of Rhizobium japonicuim were cultured in the greenhouse or in growth chambers in pots of perlite supplied with a nitrogen-free niutrieiit solutionl.'2 When the plants were about 40 days old, samples, approximately 120 gm each, of nodules were removed from 35 cultures of 10 plants each, washed, and a suspension of bacteroids was prepared.8 All preparative operations to be described were carried out in an