锰对部分缺失金属原子簇的固氮酶钼铁蛋白的重组作用

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白经邻菲口罗啉和Ｏ２ 处理后,变为部分缺失Ｐ－ｃｌｕｓｔｅｒ和ＦｅＭｏｃｏ 的失活蛋白,经与由ＫＭｎＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,重组蛋白的吸收光谱和对Ｃ２Ｈ２、Ｈ＋ 和Ｎ２ 的还原活性都恢复至与还原钼铁蛋白相似的状态,而它的α－螺旋度和在３８０—５５０ ｎｍ 、６２０—６７０ ｎｍ 的ＣＤ谱虽有明显的恢复,但仍与还原钼铁蛋白有所不同。表明：（１）重组蛋白液既含有在缺失金属原子簇的ＭｏＦｅ蛋白与含Ｍｎ 重组液重组过程中可能组装的ＭｎＦｅ 蛋白,又含有在邻菲口罗啉和Ｏ２ 处理后金属原子簇仍旧完整的ＭｏＦｅ蛋白;（２）ＭｎＦｅ蛋白和ＭｏＦｅ蛋白在固氮能力上可能是相似的,而在结构上却可能略有差异     

含铬重组液激活部分缺陷失金属原子族的钼铁蛋白的研究

棕色固氮菌固氮酶相铁蛋白经邻菲罗啉和Ｏ２处理,变为部分缺失ＦｅＭｏｃｏ和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白。与由Ｋ２ＣｒＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显恢复,然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也话存在功能与钼铁蛋白相似,而结构则有所差异的含铬蛋白。     

锰对部分缺失金属原子族的固氮酶钼铁蛋白的重组作用

棕色固氮菌固氮酶钼铁蛋白经邻菲罗啉的Ｏ２处理后,变为部分缺失Ｐ－ｃｌｕｓｔｅｒ和ＦｅＭｏｃｏ的失活蛋白、经与由ＫＭｎＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,重组蛋白的吸收光谱和对Ｃ２Ｈ２、Ｈ＋和Ｎ２的还原活性都恢复至与还原钼铁蛋白相似的状态,而它的α－螺旋度和在３８０－５５０ｎｍ,６２０－６７０ｎｍ的ＣＤ谱虽有明显的恢复,但仍与还原钼铁蛋白有所不同。表明：（１）重组蛋白液既含有在     

对与缺失铁钼辅基的固氮酶钼铁蛋白重组的原子簇的结构要求

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）钼铁蛋白与邻菲口罗啉和Ｏ２ 保温并经凝胶柱层析后,成为部分缺失Ｐ－ｃｌｕｓｔｅｒ和ＦｅＭｏｃｏ 的失活蛋白。由３ 个－ ＯＣＨ３－ 连结于Ｍｏ原子间的２ 个１Ｍｏ∶３Ｆｅ∶４Ｓ结构单位组成的原子簇与４Ｆｅ∶４Ｓ原子簇的混合液,以及由ＫＭｎＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液均可使这种失活蛋白明显恢复Ｃ２Ｈ２ 还原活性,但都不能使可被ＦｅＭｏｃｏ 激活的ｎｉｆＥ和ｎｉｆＨ 基因缺失突变种及ＵＷ ４５的Ａｐｏ－ＭｏＦｅ 蛋白得到激活。表明,不能合成ＦｅＭｏｃｏ 的固氮菌突变种蛋白只能与具有ＦｅＭｏｃｏ相似或基本相似结构的原子簇进行体外重组,而部分缺失金属原子簇的钼铁蛋白能与具有一定结构和组成的原子簇进行体外重组,变为具有催化氮还原能力的各种固氮酶组分蛋白。     

锰,铬和钼重组液及其与部分缺金属原子簇钼铁蛋白重组的比较研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒｖｉｎｅｌａｎｄｉ）钼铁蛋白经邻菲口罗啉和Ｏ２处理后,缺失一部分ＦｅＭｏｃｏ和Ｐｃｌｕｓｔｅｒ,并失去大部分活性。高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇（ＤＴＴ）分别与Ｋ２ＣｒＯ４、ＫＭｎＯ４和Ｎａ２ＭｏＯ４按一定比例混合的具有不同颜色的重组液,均可显著激活失活的钼铁蛋白;然而,除ＤＴＴ可略提高失活蛋白的活性外,其它化合物或缺一、二种化合物的混合液均无激活能力。含９９Ｍｏ重组液与失活蛋白重组后的微分扰动角关联测定显示,该蛋白中的４１％的９９Ｍｏ的状态与克氏肺炎杆菌９９ＭｏＦｅ蛋白中的９９Ｍｏ相似,表明这种激活主要是以金属原子簇含量恢复为基础的。由此可推论出,含Ｍｎ或Ｃｒ的重组液,通过与失活钼铁蛋白重组得到含Ｍｎ或Ｃｒ的固氮酶是可能的。     

棕色固氮菌钼铁蛋白的金属原子簇与其构象的关系

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白的紫外ＣＤ谱在２０５—２３５ ｎｍ 出现由峰位分别为２０８ 和２２２ ｎｍ 的双负峰组成的负槽;经邻菲口罗啉在厌氧或有氧环境中处理后,在２２２ ｎｍ 的负峰随该蛋白中Ｐ－ｃｌｕｓｔｅｒ 和ＦｅＭｏｃｏ 含量的降低而减少。在分别由Ｎａ２ＭｏＯ４、Ｎａ２Ｓ、二硫苏糖醇和高柠檬酸铁或柠檬酸铁组成的重组液重组后,上述两种处理蛋白在２２２ ｎｍ 的负峰又随其Ｐ－ｃｌｕｓｔｅｒ和ＦｅＭｏｃｏ含量的恢复而恢复。表明,钼铁蛋白的金属原子簇与蛋白质构象间存在明显的相关性     

钒铁蛋白的体外组装研究

棕色固氮菌固氮酶ＭｏＦｅ蛋白经邻菲罗啉在厌氧和有氧条件下处理后,成为部分缺失ＦｅＭｏｃｏ和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白,用由钒酸钠,高柠檬酸铁、Ｎａ２Ｓ和二硫硫苏糖醇组成的溶液保温后,可使这失活蛋白变为重组蛋白。重组蛋白的ＣＤ谱除５５０－６５０ｎｍ与ＭｏＦｅ蛋白存在差异外,也能与其吸收光谱和对质子还原活性一样得到基本恢复,但对Ｃ２Ｈ２的还原活性却未得到明显恢复。表明,这种重组蛋白与ＭｏＦｅ蛋白有所     

固氮酶内部结构揭秘

固氮酶由钼铁蛋白和铁蛋白组成 ,钼铁蛋白包含ＦｅＭｏｃｏ和Ｐ Ｃｌｕｓｔｅｒ。为解释固氮机理 ,Ｋｉｍ等 ( 1 992 )、Ｐｅｔｅｒｓ等 ( 1 997)和Ｍａｙｅｒ等 ( 1 999)分别解析出分辨率为 2 8 、2 .0 和1 .6 的钼铁蛋白晶体结构。Ｅｉｎｓｌｅ等 ( 2 0 0 2 )又获得了 1 .1 6 的钼铁蛋白结晶 ,在对其进行结构解析后发现 :ＦｅＭｏｃｏ的内部有一个轻的原子与六个铁原子键连接。此轻原子不可能是硫 ,最可能是氮 ,但不排除是碳或氧的可能。已有极好的证据表明ＦｅＭｏｃｏ是固氮酶的底物结合位点和还原中心 ,但底物是如何结合到ＦｅＭｏｃ…     

高度缺失金属原子簇的钼铁蛋白的制备及其重组

棕色固氮菌固氮酶钼铁蛋白与邻菲罗啉厌氧保温时,尿素可使钼铁蛋白丢失的Ｆｅ原子数目显著增加,在有过量Ｎａ２Ｓ２Ｏ４存在时,每个蛋白分子失去的Ｆｅ原子数猛然增至２６－２８个,经Ｓｅｐｈａｄｅｘ Ｇ－２５柱层析得到的蛋白已失去８５％的乙炔还原活性,而蛋白回收率仍可达６０％。与含Ｍｏ重组液重组后,这种失活蛋白的紫外－可见圆二色谱、凝胶电泳图谱和乙炔还少在性均有所恢复,结果表明,尿素可使厌氧钼铁蛋白的ＦｅＭ     

尿素对钼铁蛋白的活性和金属原子簇稳定性的影响

当尿素浓度高于０．５ ｍ ｏＬ／Ｌ时,棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白的乙炔还原活性呈指数下降;而经厌氧缓冲系统稀释并保温后,又可得到明显恢复。尿素对邻菲口罗啉从还原的或部分缺失Ｐ－ｃｌｕｓｔｅｒ 的ＭｏＦｅ 蛋白中螯合金属原子簇的Ｆｅ 原子均有较大的促进作用。还原ＭｏＦｅ 蛋白在尿素梯度凝胶电泳中的迁移率：在０～１．５ ｍ ｏｌ／Ｌ内,无明显变化;在１．５～５．０ ｍ ｏｌ／Ｌ内,线性变小;在５．０～８．０ ｍ ｏｌ／Ｌ内,则呈平缓状态。结果表明：（１）尿素对不同状态的ＭｏＦｅ 蛋白金属原子簇的影响程度不尽相同,而对蛋白质的变构作用是尿素影响ＭｏＦｅ 蛋白的活性和金属原子簇稳定性的主要原因;（２）ＭｏＦｅ 蛋白的构象与金属原子簇密切相关;（３）ＭｏＦｅ 蛋白在变性过程中,活性下降可能先于分子整体构象的变化     

Reactivation of Partially Metallocluster-deficient MoFe Protein by Chromium-Containing Reconstituent Solution

By treating the reduced MoFe protein from Azotobacter vinelandii with o-phenanthroIi e and O2, partially deficient in both FeMoco and P-cluster and inactive protein could be o rained. After incubating the treated protein with a reconstituent solution containing K2CrO4, ferric homocitrate, Na2S and dithiothreitol, a reactivated protein could be obtained. The absorption spectrum, circular dichroism spectrum, and the C2H2 and proton reduction activities of the reactivated protein were remarkably recovered. However, the spectra were somewhat different from those of the reduced MoFe protein. The results showed that some of the reactivated protein might be Cr-containing protein (CrFe protein) which were similar in function, but somewhat different in structure from MoFe protein.     

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.     

Comparative Studies on Mn,Cr and Mo Contained Reconstituent Solutions and Proteins Reconstituted with Partially Metallocluster-deficient MoFe Protein

By treating the reduced MoFe protein from Azotobacter vinelandii with o-phenanthroline and O2, an inactive protein partially deficient in both FeMoco and P-cluster could be obtained. The inactive protein could be reactivated by the reconsfituent solutions of different colours which were prepared from mixture in different proportions of ferric homocitrate, Na2S and dithiothreitol (DTT) with K2CrO4, KMnO4 and Na2MoO4, respectively, the inactive protein could be somewhat reactivated by DTF but not by the other compounds or their mixtures which were deficient in one or two of the above compounds. 99Mo was found in the reconstituted protein of the inactive MoFe protein with a reconstituent solution containing 99Mo. Its measurement of differential perturbed angular correlation indicated that 41% of 99Mo in the reconstituted protein were in a state similar to that of 99Mo in 99MoFe protein from K. pneumoniae. The results showed that the reactivation of the inactive MoFe rotein was mainly based on the restoration of its metallocluster content. It seemed to be reasonable to postulate that Mn- or Cr-containing nitrogenase could be obtained likewise by the reconstitution of the inactive protein with the Mn- or Cr-containing reconstituent solution.     

Studies on Assembly of Vanadium-iron Protein in Vitro

By treating the reduced MoFe protein of nitrogenase from Azotobacter vinelandii with ophenanthroline under anaerobic or aerobic condition,inactive MoFe protein which was partialy deficient in both P-cluster and FeMoco could be obtained. After incubating the inactive MoFe protein with a reconstituent solution containing NaVO3,ferric homocitrate, Na2S and dithiothreitol, a reconstituted protein could be obtained. The proton reduction activity and absorption spectrum of the reconstituted protein could be well restored, but its C2H2-reduction activity could not be recovered. Its CD spectrum could be recovered except for the 550nm to 650nm region which differed from that of the reduced MoFe protein. The results showed that the reconstituted protein was different from MoFe protein,but was similar to vanadium-iron protein.     

含铼重组液对部分缺失金属原子簇的钼铁蛋白的激活作用

棕色固氮菌(AzotobactervinelandiiLipmann)固氮酶MoFe蛋白经邻菲啉和空气处理后,成为部分缺失P_cluster和FeMoco的失活蛋白。与由Re2O7、高柠檬酸铁、Na2S和二硫苏糖醇(DTT)组成的无圆二色(CD)谱信号的重组液保温后,保温蛋白对乙炔和质子还原的活性都得以显著恢复;紫外和可见光CD谱虽有明显恢复,但仍与还原MoFe蛋白有所差异。这表明:1)保温的蛋白液中除含有未被邻菲啉等处理而破坏的完整MoFe蛋白外,还可能存在新组装的含Re的固氮酶;2)新组装的ReFe蛋白和MoFe蛋白可能在固氮能力上相似,而在结构上有所差别     

Reactivation of Partially Metallocluster deficient MoFe Protein by Rhenium-containing Reconstituent Solution

When the reduced MoFe protein from Azotobacter vinelandii Lipmann was treated with ophenanthroline and air, an inactive protein partially deficient in both FeMoco and P-cluster could be obtained. After incubating the treated protein with a reconstituent solution containing Re2OT, ferric homocitrate, Na2S and dithiothreitol, which had no circular dichroism (CD) signal, the ultraviolet and visible CD spectra, the C2H2 and H+ -reduction activity of the incubated protein were significantly recovered. However, the spectra were somewhat different from those of the reduced MoFe protein. The results showed that: 1) in the incubated protein solution there was possibly a new recombined ReFe protein besides the intact MoFe protein which was not destroyed by the treatment with o-phenanthroline and air; 2) it might be possible that both ReFe protein and MoFe protein exhibited similar ability of nitrogen fixation, although they were somewhat different in structure.     

Structural Requirement for Clusters to be Reconstituted with the FeMoCo-Deficient Molybdenum-Iron Protein

By incubating the reduced MoFe protein from Azotobacter vinelandii with O-phenanthroline under air and chromatographying the incubated solution on Sephadex G-25 column, inactive MoFe protein could be obtained. Its acetylene-reduction activity was remarkably recovered not only by incubation with the reconstituent solution composed of KMnO4, ferric homoeitrate, Na2S and dithiothreitol, but also with a mixture of 4Fe : 4S clusters and another cluster which had two structure units of 1Mo : 3Fe : 4S-bridged by three -OCH3 at the Mo atoms. Neither the reconstituent solution nor the mixture could reactivate apo-MoFe proteins from the mutants deleting nile and nifH genes and from the mutant UW45, which could be reactivated by the FeMoeo extracted from the MoFe protein. The results indicated that the FeMoeo-defieient MoFe proteins from these mutants seemed to be reconstituted only by the clusters which were probably structures only similar to FeMoeo. The partially metalloeluster-deficient MoFe protein could be reconstituted by the clusters with a certain kind of structure and composition; and was changed into different nitrogenase proteins with the ability to fix nitrogen.     

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。     

Characteristics and Crystallization of Nitrogenase MoFe Protein from a nif Z Deleted Strain of Azotobacter vinelandii

△nifZ MoFe protein purified from a nifZ deleted strain of Azotobacter vinelandii (DJ194) was shown to be pure by SDS-Polyacrylamide gel electrophoresis. The protein contained 1.5 Mo atoms and 15.9 Fe atoms per molecule, the ratio of Fe to Mo was lower than that of the MoFe protein purified from the wild type strain of A. vinelandii; and Call2, H+ -reduction activity and their ratio (C2H4/H2 (Ar)) were 16.6%, 21.7% and 77.2% of those of the wild type MoFe protein, respectively. Under a somewhat different condition from that for the crystallization of the wild type MoFe protein dark brown rhombohedron crystals of △nifZ MoFe protein were obtained. It indicated that the deletion of the △nif Z resulted in the decrease of number or change in the structure of P-cluster in the mutant MoFe protein, which caused the significant structured and function of change of the protein.     

Purification and Characteristics of Mn-containing Nitrogenase Component

A mutant UW3, which is unable to fix N2 in the presence of Mo (Nif-) but undergo phenotypic reversal to Nif+ under Mo deficiency, was able to grow in Mo- and NH3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C2H2- and H+-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰprotein.