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How different biological species make heme oxygenase suit their own needs?_Elucidation of the dependence of heme catabolism on species

不同生物物种如何使血红素加氧酶适应自身需求？_阐明血红素分解代谢对物种的依赖性

基本信息

批准号：
18570130
负责人：
MIGITA Taiko
金额：
$ 2.32万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

1. Characterization of Soybean HO-1 (Gm HO1) and determination of the heme degradation mechanism. We obtained purified proteins of heme oxygenase-1 of plant-origin for the first time and clarified following matters :(1)Stoichiometry of the heme binding, heme coordination numbers and ligand species, spin state of heme, characteristics of the ligand field of heme, pKa of the ligand water, have been determined. (2) The electron-donation system for the heme conversion to be NADPH/FNR/Fd, as well as ascorbate. Chemistry of heme conversion by Gm HO-1 is similar to that of known HOs but interaction of the distal residues with one of the intermediate, verdoheme, is unique. (3) The first detection of the ferric low-spin state of verdoheme-HO intermediate by EPR, during the reaction processes. (4) Estimation of contribution of the distal residues of Gm HO-1 to the heme degradation reaction by use of mutants made by site-specific mutagenesis. (1, 2 : reported on FEBS J, 2006 ; 3, submitted for BB … More RC, 2008 ; 4, in preparation for submission)2. Elucidation of causes discriminating the heme degradation velocity under ascorbate between rat and cyanobacterial HO-ls.Site-specific mutagenesis of ratHO-1 at the proximal helices yielded eight mutants. Structural and kinetic analyses in the presence of ascorbate were performed on the heme-mutant complexes. As a result, it has been evidenced that the whole molecular movement of the heme-distal helix connection coupled with modulation of the redox potential of heme and regulates the heme degradation rate (submitted to Biochemistry, 2008).3. Searches on the electron transmission pathways in the heme-rat HO-1 complexes. Twenty aromatic amino-acid residues were mutated with leucine and kinetics of each heme complexes were examined in detail. On the other hand, binding of the HO complexes to the electron donor, CPR, was measured by the quenching of Ravine fluorescence from CPR. As a result, the inlet of electrons was identified (in preparation for submission). Less

1.大豆HO-1(GMHO1)的特性及血红素降解机理的确定我们首次获得了植物来源的血红素加氧酶-1的纯化蛋白，并澄清了以下问题：(1)测定了血红素结合的化学计量、血红素的配位数和配位体的种类、血红素的自旋状态、血红素的配位场特征、配位水的pKa。(2)血红素转化的供电子体系为NADPH/FNR/FD以及抗坏血酸。GMHO-1转化血红素的化学成分与已知的HOS相似，但远端残基与中间体之一马鞭铁血红素的相互作用是独特的。(3)首次用电子顺磁共振检测反应过程中铁的低自旋态。(4)利用定点突变获得的突变体估计GM-HO-1远端残基对血红素降解反应的贡献。(1、2：FEBS J，2006年报道；3，提交BB…更多RC，2008；4，准备提交)2.阐明在抗坏血酸下区分大鼠和蓝藻HO-1之间的血红素降解速度的原因。Ratho-1在近端螺旋的定点特异突变产生了8个突变。在抗坏血酸存在的情况下，对血红素-突变络合物进行了结构和动力学分析。因此，已有证据表明，血红素-远端螺旋连接的整个分子运动与血红素的氧化还原电位的调制相结合，并调节血红素的降解速率(提交给生物化学，2008)。血红素-大鼠HO-1复合体中电子传递途径的研究。用亮氨酸对20个芳香族氨基酸残基进行了突变，并对每个亚铁血红素络合物的动力学进行了详细研究。另一方面，HO络合物与电子供体CPR的结合是通过CPR的Ravine荧光猝灭来测量的。结果，确定了电子入口处(准备提交)。较少