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The study of the heme degradation mechanism by heme oxygenase based on ESR and NMR spectroscopied and crystal structures

基于ESR和NMR波谱和晶体结构的血红素加氧酶降解血红素机制的研究

基本信息

批准号：
12670125
负责人：
NOGUCHI Masato
金额：
$ 1.92万
依托单位：
KURUME UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Rudi Schimd and his coworkers in the middle of 1960s discovered heme oxygenase, an enzyme that is in charge of the physiological degradation of heme. Since then, a number of researchers in the whole world have extensively devoted their efforts in the study of this enzyme. In this project, we have pursued the mechanism of the heme oxygenase reaction from the viewpoint of structural biology and obtained the following outcome. (1) We succeeded in establishing an expression system of a truncated soluble version of heme oxygenase-1, in which the C-terminal membrane-binding stretch consisting of 22 hydrophobic amino acid residues are removed. With this system we are able to routinely obtain ca. 170 mg of purified enzyme from 10-L culture (Kurume med. J. 43, 313, 1996). (2) With a home-made titration device for dioxygen and electron, we found that α -hydroxyheme can be converted to verdoheme by dioxygen in the absence of added reducing equivalents (J. Biol. Chem. 274, 18196, 1999). (3) We obtained a diffraction data with high-resolution (2.4 ^^゜__A) of heme oxygenase-1(Acta. Cryst. D54, 1017, 1998). Then, with selenomethionine mutants of heme oxygenase-1, we determined the crystal structure of rat heme oxygenase-1 (FEBS Lett. 471, 61, 2000). (4) We succeeded in preparing four verdoheme isomaers (J. Inorg. Biochem. 82, 113, 2000). (5) We reconfirmed that α -hydroxyheme can be converted to verdoheme only by dioxygen without exogenous electrons. Further we clarified the discrepancies between researchers concerning the requirement for electron are due to the preparation methods of the complex of heme oxygenase with α -hydroxyheme and also due to the reducing systems employed (Eur. J. Biochem. 269, 5231-5239, 2002). (6) We determined the crystal structures of the apo form of heme oxygenase-1 (Biochemistry 41, 7293-7300, 2002) and the azide-bound form of heme-heme oxygenase complex (J. Biol. Chem. 277, 45086-45090, 2002 )

20世纪60年代中期，Rudi Schimd等人发现了血红素加氧酶，这是一种负责血红素生理降解的酶。从那时起，全世界的许多研究人员都广泛投入了他们的努力，在这种酶的研究。本研究从结构生物学的观点出发，对血红素加氧酶反应的机理进行了探讨，取得了以下成果。(1)我们成功地建立了血红素加氧酶-1的截短的可溶性版本的表达系统，其中去除了由22个疏水氨基酸残基组成的C-末端膜结合延伸。有了这个系统，我们就能常规地获得CA。从10-L培养物中获得170 mg纯化的酶（Kurlemed.J.43，313，1996）。(2)利用自制的分子氧和电子滴定装置，我们发现α -羟基血红素可以在没有添加还原当量的情况下被分子氧转化为绿血红素（J. Biol. Chem. 274，18196，1999）。(3)我们获得了血红素加氧酶-1（Acta. D54，1017，1998）。然后，用血红素加氧酶-1的硒代蛋氨酸突变体，我们确定了大鼠血红素加氧酶-1（FEBS Lett. 471，61，2000）。(4)我们成功地制备了四种绿血红素异构体（J.Inorg.Biochem.82，113，2000）。(5)我们再次证实了α -羟基血红素在没有外源电子的情况下，只能被分子氧转化为绿血红素。此外，我们澄清了研究人员之间关于电子需求的差异是由于血红素加氧酶与α -羟基血红素的复合物的制备方法以及所采用的还原系统（Eur. 269，5231-5239，2002）。(6)我们确定了血红素加氧酶-1的apo形式（Biochemistry 41，7293-7300，2002）和血红素-血红素加氧酶复合物的叠氮化物结合形式（J.Biol.Chem.277，45086-45090，2002）的晶体结构。