固氮酶钼铁蛋白铁钼辅因子的抽提研究进展

固氮酶由两种铁硫蛋白(钼铁蛋白和铁蛋白)组成。由还原剂提供电子经铁(Fe)蛋白传递给钼铁(MoFe)蛋白,在MoFe蛋白的活性中心部位进行N_2、C_2H_2等多种底物的还原[10,11,20]。MoFe蛋白中的Mo、Fe原子和酸不稳定性硫原子(S~*)组成2个M簇(FeM-oco)、3—4个P簇(P-cluster)及1—2个S(2Fe)簇。在底物还原过程中,这些原子簇都可能参与电子的传递。铁钼辅因子(FeMoco)已被认为是络合和还原底物的重要部位。因此,要阐明MoFe蛋白的作用机理就得研究FeMoco的结构和功     

棕色固氮菌固氮酶钼铁蛋白纯净铁钼辅因子的制备和性质

建立了以挥发性酸碱(TFA/NH_4OH)和NH_4OH碱化的NMF从棕色固氮菌固氮酶钼铁蛋白分离提取纯净铁钼辅因子(FeMo—co)的方法。得到了纯净FeMo-co的NMF制剂,从而证明了Tris、ClNa~ 、柠檬酸、HPO_4~(-2)、S_2O_4~(2-)以及氨基酸残基不为FeMo—co的有效分离和结构稳定所必需。制备的纯净FeMo—co的Fe/Mo比为8,比活为230nMC_2H_4/min/MoMn。由于分离制备的FeMo—co制剂除抽提溶剂NMF外不含任何其他成分,所以是进行FeMo—co结晶和结构研究的理想材料。     

棕色固氮菌不固氮变种UW45与固氮酶钼铁蛋白铁钼辅因子重组的某些特性

以UW45抽提液的DEAE-纤维素柱层析的0.15M NaCl或0.25M NaCl洗出液与Smith法或Shah法制备的铁铝辅因子重组,则0.25 M NaCl洗出液的重组对乙炔还原的活性远较0.15 M NaGl的为高。0.15 M NaGl的洗出液较0.25 M NaCl洗出液的组分明显不纯。不全钼铁蛋白与铁钼辅因子和铁蛋白重组后能还原基质氰化钾,但对分子氮或迭氮化钠的还原却较微弱甚至不还原。铁钼辅因子按Smith和Shah法制备,其分子量范围分别为低于1000D和接近1500D。由于Smith和Shah法两种铁钼辅因子还原乙炔和氰化钾的比活不同,电泳图谱有差异、分子量又有大小,因此这两种铁钼辅因子的分子结构可能不尽相同。     

铁钼辅因子

某些固氮生物(如棕色固氮菌)的变种,它们的固氮酶中铁蛋白有催化活性,但由于钼铁蛋白处于不活化状态,所以整个固氮酶不能显示出固氮活性,这种不能固氮的固氮酶可为从野生型固氮菌固氮酶钼铁蛋白中分离得到的物质(或因子)所活化或恢复固氮活性,此种活化因子叫钼铁辅因子。这种钼铁辅因子已从多种固氮生物中分离到,其分子量为1000～3000左右。按钼铁蛋白分子量为270,000计算,其中铝、铁、硫三种原     

铁钼辅因子激活氧钝化钼铁蛋白的初步研究

棕色固氮菌(Azotobacter vinelandii)固氮酶钼铁蛋白氧钝化后,未断裂出任何含钼、铁原子的断片。用有机溶剂从钼铁蛋白中提取得到的铁钼辅因子(FeMoCo)可以激活被氧钝化了的钼铁蛋白,使其还原乙炔能力得到部分或完全恢复。这种激活作用的效率随着钼铁蛋白氧钝化程度的加深而降低。初步结果表明,氧钝化的钼铁蛋白中最先受到损伤的可能是 FeMoCo。如果氧钝化程度进一步加剧,其它部分也可能受到损伤。     

钼铁蛋白铁钼辅因子的有机组分对其功能的影响

棕色固氮菌(Azotobacter vinelandii)固氮酶的钼铁蛋白经邻菲啰啉在厌氧或有氧环境中处理后,变为 P-cluster 单一缺失或 P-cluster 和 FeMoco 同时缺失的失活钼铁蛋白。含柠檬酸盐或高柠檬酸盐的重组液都使这两种失活蛋白能恢复固氮酶重组的 H~ 和 C_2H_2还原活性,活性恢复程度随反映钼铁蛋白中金属原子簇含量变化的圆二色和磁圆二色谱及金属含量的恢复程度的提高而提高,但它们固 N_2能力的恢复程度则不相同:P-cluster 单一缺失的蛋白用两种重组液重组后均可恢复其固 N_2能力,而 P-cluster 和 FeMoco 同时缺失的蛋白,只有用含高柠檬酸盐的重组液重组才恢复其固 N_2能力,表明含不同有机组分的重组液所组装的 P-cluster 均与天然状态相同,只有含高柠檬酸盐的重组液所组装的 FeMoco 才与天然状态相同,从而证明高柠檬酸盐是 FeMoco 的必需的有机组分。     

蓝藻固氮酶钼铁蛋白的研究

改进了蓝藻固氮酶的分离、提纯方法。首次用小型厌氧聚丙烯酰胺凝胶制备电泳法,代替常用的层析法,获取了电泳纯的蓝藻固氮酶钼铁蛋白,简化了程序,缩短了实验周期。SDS凝胶电泳和分子筛凝胶过滤测定分子量结果表明,钼铁蛋白分子量为360,000,由4个分子量为90,000的同一类型亚单位构成。每个钼铁蛋白分子含1个钼,18个铁和3290个氨基酸残基。其中酸性氨基酸占优势。研究了柱孢鱼腥藻(Anabaena cylindrica)固氮酶粗提物和钼铁蛋白的某些特性,其结果是:米氏常数为3.33×10~(-3)大气压乙炔,等电点为5—5.5。紫外、可见光谱与其它固氮生物的类似。盐对蓝藻固氮酶较之对其它固氮生物的固氮酶有更大的抑制作用。     

固氮酶钼铁蛋白羧基的化学修饰

在室温,pH6.4的条件下,钼铁蛋白迅速被大过量的Woodward试剂K所失活。紫外吸收光谱跟踪表明：随着时间延长,修饰程度不断加深。结合氨基酸分析结果算出当修饰2分钟时,活力丧失96%,有2—5个羧基被修饰。     

铁氰化钾氧化固氮酶钼铁蛋白与铁、钼、硫化合物重组的研究

棕色固氮菌固氮酶钼铁蛋白经铁氰化钾氧化后变为“漂白蛋白”,其活性损失约75％,同时释放出80％的钼和50％左右的铁。与铁、钼、硫重组溶液重组,金属组成和乙炔还原活性均得到明显恢复。穆斯鲍尔(M／issbauer)谱表明,重组溶液中的Fe、Mo、S已形成FeMc6原于簇的类似物,“重组蛋白”的M6ssbauer谱和天然MoFe蛋白相似。     

The Effect of Manganese on the Reconstitution of Partial Metallocluster-deficient Nitrogenase Molybdenum-Iron Protein

By treating the reduced MoFe protein of nitrogenase from Azotobacter vinelandii with O-phenanthroline (O-phen) and O2, inactive MoFe protein which was partialy deficient in both P-cluster and FeMoco could be obtained. After incubating the inactive protein with a reconstituent solution containing KMnO4, ferric homocitrate, Na2S and dithiothreitol, a reconstituted protein could be obtained. The absorption spectrum and C2H2, H+ and N2 reduction activity of the reconstituted protein could be well restored to the state of the reduced MoFe protein. However, the α-helix and CD spectrum at 380—550 nm and at 620—670 nm of the reconstituted protein were somewhat different from those of the reduced MoFe protein. The results showed that: (1) the reconstituted protein was composed of the assembled protein which might be a MnFe protein due to the reconstitution of the metalloclusterdeficient MoFe protein with Mn-containing solution and MoFe protein in which metalloclusters were still intact after the treatment with O-phen and O2; (2) It might be possible that the MnFe protein and MoFe protein were similar in the ability of nitrogen fixation, but were somewhat different in the structure from each other.     

固氮酶晶体学研究进展

生物大分子的功能取决于它的空间结构.X射线衍射分析是获得生物大分子结构信息的常用方法.本文对固氮酶晶体的生长及其X射线衍射分析的主要进展简要地进行了介绍和评论.最后展望了今后的发展及问题.     

固氮酶晶体学研究进展

生物大分子的功能取决于它的空间结构。X射线衍射分析是获得生物大分子结构信息的常用方法。本文对固氮酶晶体的生长及其X射线衍射分析的主要进展简要地进行了介绍和评论。最后展望了今后的发展及问题。     

The Relationship Between the Metal Clusters and the Conformation of Molybdenum-Iron Protein from Azotobacter vinelandii

The ultraviolet CD spectrum of nitrogenase MoFe protein from Azotobacter vinelandii had a negative trough with double peaks at 208 nm and 222 nm, respectively, and the shape of the trough was similar to those of other proteins with a-helix structure. After treatment with o-phenanthroline under an aerobic or anaerobic condition, the height of the peak at 222 nm (h222 nm) decreased with the decrease of the C2H2-reduction activity, Fe content and CD spectra at both 450 nm and 660 nm, or at 450 nm of the treated proteins. However, after reconstituting with a reconstituent solution containing Na2MoO4, Na2S, dithiothreitol and either ferric homocitrate or ferric citrate, the h222 nm Of the reconstituted proteins could be restored as well as the activity, Fe content and CD spectra at both of 450 nm and 660 nm. The results show that there is a significant relationship between the metal clusters (FeMoco and P-cluster) and the conformation of MoFe protein.     

Studies on the Circular Dichroism (CD) Spectra of Iron-Molyb-denum Protein from Azotobacter vinelandii Nitrogenase

Since the unsymmetry of metal cluster(P-cluster), which was under different redox states, in FeMo protein of A. vinelandii nitrogenase could be observed by circular dichroism, the numbers of the redox equivalent for the P-cluster was obtained by CD titration of DT-stripped FeMo protein with oxidant, i.e. plotting △δ450nm against the number of equivalent of oxidant added. After exposure to air, at the beginning P-cluster was reversibly oxidized, then followed by irreversible oxygen-damage. Dithiothreitol (DTT) was able to increase the C2H2-reduction activity of FeMo protein, which was not severely damaged by O2, or by other stronger oxidants, but could not change their CD spectra. It seems that the reactivation may be not due to the restoration of the P-cluster to its reducing state.     

Studies on the Reactivation of Aerated Nitrogenase MoFe Protein from Azotobacter vinelandii by the Compounds of iron Molybdenum and Sulfur

After the exposure to air, the crystalline nitrogenase MoFe protein from Azotobacter vinelandii was resulted in the remarkable increase in its absorption (ABS) and the significant decrease in its activity and circular dichroism (CD). However, when the aerated MoFe protein was incubated with the reconstituting solution which consisted of Na2MoO4, ferric citrate, Na2S and dithiothreitol, the ABS and CD of the aerated. MoFe protein both were completely restored, simultaneously with the significant restoration of acetylene reduction. It is shown that the P-cluster and other parts related to the protein activity which was damaged by O2 are able to be repaired to a certain extent by the reconstituting solution.     

几种微生物杀虫蛋白基因研究进展

在过去几年中,已经鉴定并克隆出一些新的苏云金芽孢杆菌杀虫晶体蛋白基因和其他类型的微生物杀虫蛋白基因。其中来自嗜虫沙雷氏菌,双酶梭状芽孢杆菌,球形芽孢杆菌,嗜线虫致病杆菌,发光杆菌和金龟子绿僵菌的新型杀虫蛋白基因在抗虫遗传工程中具有良好的应用前景     

固氮酶铁钼辅基理化特性的研究

在紫外可见光谱区内,固氮酶铁钼辅基〔（Ｍｏ_２Ｆｅ_（１２）Ｓ_（１２））４－〕均无特征吸收峰,不含高柠檬酸盐。含双钼的铁硫簇〔（Ｍｏ_２Ｆｅ_（６～１２）Ｓ_（６～１２））~（１～４）－〕的电荷数、颜色与该金属簇中的亚铁量成对应关系,并都有较高的生物重组活性．