Electron allocation to H+ and N2 by nitrogenase in Rhizobium leguminosarum bacteroids.

Electron allocation to H+ and N2 by nitrogenase in intact Rhizobium leguminosarum bacteroids has been studied. Nitrogenase activity was measured in intact cells with succinate and oxygen substrates. When whole cell nitrogenase activity was inhibited by oxygen-limitation or by the addition of the H+-conducting ionophore carbonylcyanide m-chlorophenylhydrazone, both inducing a low intracellular ATP/ADP ratio, the electron allocation to H+ was favoured over that to N2. When whole cell nitrogenase activity was inhibited by excess oxygen or by the addition of the K+-conducting ionophore valinomycin, both inhibiting electron transport to nitrogenase without affecting the intracellular ATP/ADP ratio, no effect upon the electron allocation to H+ and N2 was observed. The whole cell experiments could be confirmed by experiments with bacteroids treated with hexadecyltrimethylammonium bromide. Nitrogenase is highly active in these preparations with Na2S2O4 and MgATP as substrates. No effect was observed upon electron allocation to H+ and N2 when nitrogenase was inhibited by limitation of reductant (Na2S2O4) or MgATP. Only when nitrogenase was inhibited by MgADP, electron allocation to H+ was favoured. The amount of nitrogenase component 1 and 2 in bacteroids was estimated with protein blotting, followed by an immunological detection. It was found that 17% +/- 3% of total bacteroid protein is component 1 and 12% +/- 2% is component 2. The specific nitrogenase activity of bacteroids treated with hexadecyltrimethylammonium bromide is 178 +/- 62 nmol C2H4 formed X min-1 X mg total protein-1. Despite the high protein concentrations, nitrogenase is not inhibited. With cell-free extracts or with purified nitrogenase components isolated from R. leguminosarum bacteroids, electron allocation to H+ was favoured over that to N2, independently of the mechanism of inhibition. The discrepancies between the whole cell studies and those with isolated enzyme will be discussed with respect to the present mechanism of action of nitrogenase.