缺失nifE的棕色固氮菌突变种MoFe蛋白的纯化及体外激活

经DEAE纤维素、Sephacryl S-300和Q-Sepharose柱层析分离纯化,从缺失nifE的棕色固氮菌(Azotobactervinelandii Lipmann)突变种(DJ35)的无细胞粗提物中得到△nifE MoFe蛋白(△nifE Av1).SDS凝胶电泳分析表明,△nifE Av1的亚单位种类和分子量分别与棕色固氮菌野生型(OP)MoFe蛋白(Av1)的α和β亚单位相似.当与固氮酶Fe蛋白(Av2)活性互补时,△nifE Av1不具有还原质子的能力,但从OP Av1中抽提的FeMoco却可使其激活.经过量的邻菲啰啉(o-phen)厌氧处理并经Sephadex G-25柱层析分离后,便得到△nifE Av1 .在同时存在Av2和MgATP发生系统的条件下,△nifE Av1 ,而不是△nifE Av1,可为由KMnO4、高柠檬酸铁、Na2S、Na2S2O4和二硫苏糖醇组成的含Mn重组液(RS-Mn)显著激活.但在缺少MgATP或Av2的条件下,RS-Mn则不能激活△nifE Av1 .这就表明,RS-Mn对△nifE Av1 的激活需要o-phen的预先处理及同时存在Av2和MgATP的这二个条件.

Purification and Activation In Vitro of MoFe Protein from a nifE Deleted Mutant Strain of Azotobacter vinelandii

TheΔ nif E MoFe protein (Δ nif E Av1) was obtained by a chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from the unheated crude extract of nifE-deleted mutant strain (DJ35) of Azotobacter vinelandii Lipmann. The analysis by SDS-PAGE showed that theΔ nif E Av1 was similar to OP MoFe protein (Av1) of A. vinelandii in the kinds and molecular weights of subunits (ɑand β subunit). When complemented with nitrogenase Fe protein (Av2), theΔ nif E Av1 had hardly any proton-reduction activity, but could be significantly activated by FeMoco extracted from OP Av1. After theΔ nif E Av1 was treated with an excess o-phenanthroline (o-phen) and chromatographied on Sephadex G-25 column under atmosphere of Ar, Δ nif E Av1 was obtained. In the presence of both Av2 and MgATP regeneration system, theΔ nif E Av1 , rather thanΔ nif E Av1, was significantly activated in vitro by a reconstituent solution containing Mn which composed of KMnO4, ferric homocitrate, Na2S, Na2S2O4 (DT) and dithiothreitol (DTT). But in the absence of MgATP or Av2, the activation ofΔ nifE Av1 did not happen. It indicates that activation ofΔ nif E Av1 by RS-Mn requires the pretreatment with o-phen and the simultaneous presence of Av2 and MgATP.