The "nitrogenase-protective" FeSII protein of Azotobacter vinelandii: overexpression, characterization, and crystallization.
The "nitrogenase-protective" FeSII protein of Azotobacter vinelandii: overexpression, characterization, and crystallization.
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维氏固氮菌的“固氮酶保护性”FeSII 蛋白：过表达、表征和结晶。
DOI：
10.1021/bi00040a007
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Maier,RJ
中科院分区：
文献类型：
--
作者：
Moshiri,F;Crouse,BR;Johnson,MK;Maier,RJ
摘要：Azotobactervinelandii FeSII蛋白在氧化应激时期通过与MoFe和Fe蛋白结合形成非活性但o2稳定的三方复合物，从而为氮酶提供构象保护。在这项工作中，FeSII蛋白在大肠杆菌中过表达，重组蛋白被纯化到均匀性，结晶，并在功能，光谱和氧化还原特性方面进行了表征。重组蛋白为同型二聚体，在每个亚基中表达为一个[2Fe-2S] 2++簇的全蛋白。它在体外重建一个02-稳定的氮酶复合体中显示出功能。紫外-可见吸收、CD、变温MCD、EPR和共振拉曼光谱研究表明，[2Fe-2S] 2+ ' +团簇完全由半胱氨酸残基协调。[2Fe-2S]+簇（g=2.04, 1.95, 1.88）的一级配位半胱氨酸排列和EPR性质与叶绿体铁氧化还原蛋白非常相似。然而，变温MCD、共振拉曼和氧化还原特性（基于染料介导的EPR氧化还原滴定，Em= -262±10 mV）更具有羟酶型铁氧化素（如肾上腺素）的特征。与叶绿体型铁氧化还原蛋白相比，FeSII蛋白中[2Fe-2S] 2+，+簇的振动特性表明，半胱氨酸Fe-S-C-C二面角均不接近180，且簇未暴露于溶剂。初步的x射线衍射分析表明该蛋白呈正交晶型结晶
Revised Manuscript Received July 6, 1995® abstract: The Azotobactervinelandii FeSII protein confers conformational protection to nitrogenase by binding to the MoFe and Fe proteins under periods of oxidative stress to create an inactive but O2-stabilized tripartite complex. In this work the FeSII protein has been overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity, crystallized, and characterized in terms of its functional, spectroscopic, and redox properties. The recombinant protein is a homodimer and is expressed as a holoprotein with one [2Fe-2S] 2++ cluster in each subunit. It is shown to be functional in reconstituting an 02-stable nitrogenase complex in vitro. Spectroscopic studies using the combination of UV—visible absorption, CD, and variable temperature MCD, EPR, and resonance Raman indicate that the [2Fe-2S] 2+’+ cluster is coordinated exclusively by cysteineresidues. The arrangement of coordinating cysteines in the primary sequence and the EPR properties of the [2Fe-2S]+ cluster (g=2.04, 1.95, 1.88) are very similar to those of chloroplast ferredoxins. However, the variable-temperature MCD, resonance Raman, and redox properties (Em=—262±10 mV based on dye-mediated EPR redox titrations) are more characteristic of hydroxylase-type ferredoxins such as adrenodoxin. In contrast to chloroplast-type ferredoxins, the vibrational properties of the [2Fe-2S] 2+,+ cluster in the FeSII protein indicate that none of the cysteinyl Fe—S—C—C dihedral angles are close to 180 and that the cluster is not exposed to solvent. Preliminary X-ray diffraction analysis indicates that the protein crystallizesin an orthorhombic