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Structure and Function of heme related proteins involved in intracellular signal transduction

参与细胞内信号转导的血红素相关蛋白的结构和功能

基本信息

批准号：
12480176
负责人：
IKEDA-SAITO Masao
金额：
$ 1.41万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480176/
关键词：
heme oxygenase heme X-ray crystal structure resonance Raman spectra intermediate of enzyme reaction 結晶化共鳴ラマンスペクトル一酸化炭素

项目摘要

In this work, we studied about heme oxygenase, a heme degradation enzyme. First, our research project was focused on constitutive isoform of mammalian heme oxygenase, HO-2. Recombinant HO-2 overexpressed in E colli. was successfully purified with new method to exclude impurity such as degradated or denatured enzymes. We used this highly purified HO-2 for crystal screening to search an appropriate buffer condition of crystallization, but we could not find the condition. On the other hand, we had already made crystal of HmuO, a bacterial heme oxygenase, and tried to elucidate crystal structure of HmuO. On the basis of spectroscopic data, optical absorption and resonance Raman spectra, and enzymological analysis of HO-2 and HmuO, their heme environment and reaction mechanism were found to be identical to those of HO-1 , inducible form of mammalian heme oxygenase. X-ray crystal structure analysis revealed that the crystal of HmuO belonged to P21 space group and a unit cell of the crystal was composed of three molecules. We could reveal structure of oxidized form and reduced form, the first and second intermediate of the enzyme reaction. The reduced form of crystal was made by the addition of sodium dithionite as an electron donor to the oxidized form crystal. Resolutions of the crystal structures were 1.4 and 1.7 angstroms for oxidized form and reduced form, respectively. From the comparison of crystal structures in oxidized and reduced forms, substantial amount of structural changes provoked by changing of heme iron electronic state were discovered for the first time. Additionally, presence of an hydrogen bonding network, which is assumed to work as a proton donor in the heme-degradation reaction, was pointed out here. Now our efforts are thrusting to realize the structure of CO adduct of HmuO as well as other intermediate, alpha-hydroxyheme form, verdoheme form, and billiverdin form.

本文对血红素加氧酶进行了研究。首先，我们的研究项目集中在哺乳动物血红素加氧酶，HO-2的组成异构体。重组HO-2在大肠杆菌中的高效表达。采用新的纯化方法成功地除去了降解或变性酶等杂质。我们用这种高纯度的HO-2进行晶体筛选，以寻找合适的结晶缓冲条件，但我们找不到该条件。另一方面，我们已经制备了细菌血红素加氧酶HmuO的晶体，并试图阐明HmuO的晶体结构。根据HO-2和HmuO的光谱数据、光学吸收和共振拉曼光谱以及酶学分析，发现它们的血红素环境和反应机理与哺乳动物血红素加氧酶的诱导型HO-1相同。X射线晶体结构分析表明，HmuO晶体属于P21空间群，晶胞由3个分子组成。我们可以揭示酶反应的第一和第二中间产物氧化态和还原态的结构。通过向氧化型晶体中加入作为电子供体的连二亚硫酸钠来制备还原型晶体。氧化形式和还原形式的晶体结构的分辨率分别为1.4和1.7埃。从氧化态和还原态晶体结构的比较中，首次发现了血红素铁电子态变化引起的大量结构变化。此外，氢键网络的存在下，这是假定作为一个质子供体在血红素降解反应中，这里指出。现在我们的努力是推动实现的CO加合物的HmuO以及其他中间体，α-羟基血红素形式，绿血红素形式，和胆绿素形式的结构。