Haaker H, Wassink H
Eur J Biochem. 1984 Jul 2;142(1):37-42. doi: 10.1111/j.1432-1033.1984.tb08247.x.
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.
已对完整的豆科根瘤菌类菌体中固氮酶将电子分配给H⁺和N₂的情况进行了研究。在以琥珀酸和氧气为底物的完整细胞中测定了固氮酶活性。当全细胞固氮酶活性因氧气限制或添加传导H⁺的离子载体羰基氰化物间氯苯腙而受到抑制时，二者均会导致细胞内ATP/ADP比值降低，此时电子分配更倾向于H⁺而非N₂。当全细胞固氮酶活性因氧气过量或添加传导K⁺的离子载体缬氨霉素而受到抑制时，二者均会抑制电子向固氮酶的传递且不影响细胞内ATP/ADP比值，此时未观察到对电子分配给H⁺和N₂有任何影响。用十六烷基三甲基溴化铵处理类菌体的实验能够证实全细胞实验的结果。在这些以连二亚硫酸钠和MgATP为底物的制剂中，固氮酶具有高活性。当固氮酶因还原剂（连二亚硫酸钠）或MgATP受限而受到抑制时，未观察到对电子分配给H⁺和N₂有任何影响。只有当固氮酶因MgADP而受到抑制时，电子分配才更倾向于H⁺。用蛋白质印迹法，随后进行免疫检测，估算了类菌体中固氮酶组分1和2的含量。结果发现，类菌体总蛋白的17%±3%是组分1，12%±2%是组分2。用十六烷基三甲基溴化铵处理的类菌体的比固氮酶活性为178±62 nmol乙烯每分钟每毫克总蛋白生成量。尽管蛋白质浓度很高，但固氮酶并未受到抑制。对于从豆科根瘤菌类菌体中分离得到的无细胞提取物或纯化的固氮酶组分，无论抑制机制如何，电子分配均更倾向于H⁺而非N₂。将结合固氮酶目前的作用机制讨论全细胞研究与分离酶研究之间的差异。
