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经DEAE52,Q-Sepharose和SephacrylS-200柱的厌氧层析,从缺失nifZ基因的棕色固氮菌突变株(DJ194)中纯化得到固氮酶钼铁蛋白(?nifZAv1).厌氧天然电泳后的Westernblotting和SDS-变性凝胶电泳显示,?nifZAv1的电泳迁移率、分子量和亚基组成等与野生种钼铁蛋白(OPAv1)相似;而且?nifZAv1的Mo含量、EPR信号(g≈4.3,3.65和2.01)及660nm附近的圆二色摩尔消光系数(?ε)也都与OPAv1较相似,表明?nifZAv1除具有α2β2四聚体结构组成外,还含有与OPAv1数量相当的、具有3/2自旋态的还原FeMoco.然而,?nifZAv1的Fe含量和对底物(C2H2,H+和N2)的还原活性都较低,分别约为OPAv1的74%和46%~50%,而反映P-cluster状况的450nm附近的?ε也明显低于OPAv1,说明?nifZAv1与OPAv1的差别在于P-cluster,而不在于FeMoco,而且是在于?nifZAv1中P-cluster数目的减少(约50%),而其组成或氧还状态并未改变.据此推测?nifZAv1(DJ194)的结构为:一个αβ亚基对含有一个FeMoco和一个P-cluster,而另一个αβ亚基对只含FeMoco,无P-cluster.由于nifZ基因的缺失只造成了钼铁蛋白中2个P-cluster中的一个不能组装,因此推测P-cluster的组装可能不是受单一基因产物的影响并由此提出了NifZ的可能作用机理.
After anaerobic chromatography on DEAE52, Q-Sepharose and Sephacryl S-200 columns, the nitrogenase fern ferritin (? NifZAv1) was purified from a strain of Azotobacter vinelandii mutant lacking the nifZ gene (DJ194). Westernblotting And SDS-denaturing gel electrophoresis showed that the nifZAv1 electrophoretic mobility, molecular weight and subunit composition were similar to the wild type molybdenum ferritin (OPAv1); and the content of Mo of nifZAv1, EPR signal (g≈4.3, 3.65 and 2.01 ) And the circular dichroism molar extinction coefficient (? Ε) around 660 nm are also similar to those of OPAv1, indicating that? NifZAv1 contains, in addition to the α2β2 tetramer structure composition, a number of OPAv1-equivalent exocycles with a 3/2 spin state However, nifZAv1 has lower Fe content and lower reductive activities for substrates (C2H2, H + and N2), which are about 74% and 46% ~ 50% of OPAv1, respectively, and reflect the state of P-cluster The ε in the vicinity of 450 nm is also significantly lower than that of OPAv1, indicating that the difference between nifZAv1 and OPAv1 lies in P-cluster but not in FeMoco, but also in the reduction of the number of P-clusters in nifZAv1 (about 50%), Therefore, the structure of nifZAv1 (DJ194) is as follows: one αβ subunit pair contains one FeMoco and one P-cluster, while the other αβ subunit pair contains only FeMoco and no P-cluster.Because the deletion of the nifZ gene causes only one of the two P-clusters in the molybdenum ferritin to fail to assemble, it is speculated that P-cluster The assembly may not be affected by a single gene product and thus suggests a possible mechanism of action of NifZ.