An electron donor system for nitrogenase-dependent acetylene reduction by extracts of soybean nodules.

The catalysis of nitrogen reduction by extracts of nitrogen-fixing organisms requires an ATPgenerating system and a source of electrons (1, 2, 5, 11, 13). The phosplhoroclastic reaction provides botlh ATP anid electrons for the reduction of nitrogen by cell-free extracts of Clostridium1t pasteurianumn (2, 5, 13) and ferredoxin functions as the natural electron carrier from the phlosphoroclastic reaction to nitrogenase ('12). Some evidence has been reported (2) that boro,hydride, hydrogen, NADH or NAiDPH, in presence of ferredoxin, will serve as a reductant for nitrogen fixation by the clostridial system. Cell-free extracts of Azotobacter vinelandiz (1) and bacteroids from soybean nodules (11) also catalvze the reduction of N. to NH, but an exogenous souirce of both reductant and ATP must be furnisihed. Probablv the phvsiological source of reductant and ATP for nitrogen fixation by leguime nodules and azotobacter is the respiratory electron transport chain with its associated oxidative phosphorvlation. All efforts however to coutple nitrogen fixation by cell-free extracts of these organisms to naturally occutrring oxidation-redutction reactions have failed. There are no reports of the identification in azotobacter or legume nodules of a low potential electron carrier analogous to ferredoxin. Using the reduction of acetylene to etlhy-lene as a measure of nitrogenase activity (3, 14), evidence has been obtaiined that NADH genierated by the fl-hydroxybutyrate dehvdrogenase reaction or high concentrations of NADH supplied directly function as a source of electrons for acetvlene redutction by extracts of nodule bacteroids or azotobacter. Activity also is dependent 1110pon an ATP-generating system and a suiitable dye. Cell-free extracts of soybean nodlules were prepared by the nmetho(d of Kochi, Evan,s, and Ruissell (10, 11) with minor changes to improve the nitrogenase activity. Ethyrlene production from acetylene was measuired by a method described by Kelly,