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Micro-environment of Fe-Cu binuclear center in Cytoobrome Oxidase Studied by Electron Paramagnetic Resonance Spectroscopy

电子顺磁共振波谱研究细胞溴氧化酶Fe-Cu双核中心的微环境

基本信息

批准号：
13680741
负责人：
HORI Hiroshi
金额：
$ 2.11万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680741/
关键词：
Cytocbrome c oxidase bo-type ubipuinol oxidase Heme-Cu_B binuclear center P-intermediate F-intermediate Electron Paramagnetic Resonance Double mode EPR cavity Spin-spin exchange interaction

项目摘要

Cytochrome bo-type ubiquinol oxidase from Eschenchia coli is a member of heme-copper terminal oxidase superfamily. It contains four redox centers, heme b, heme o, Cu_B, and ubiquinol-8(Q_H). Heme o and Cu_B constitute a binuclear center and reduce dioxygen to water. To elucidate the mechanism of dioxygen reduction, it is inevitable to detennine the electronic states of the redox centers in the reaction intermediates.1. The fully oxidized cytochrome bo contains a spin-coupled pair of high-spin heme o (S_<Fe>=5/2) and Cu_B (S_<Cu>=1/2) with an effective spin of S'=2. Integer-spin EPR signals at g=3.6 were investigated by a newly developed bimodal X-band cavity. No signal was observed with the parallel mode cavity due to its low sensitlvty.2. P intermediate can be obtained by addition of low concentrations of hydrogen peroxide to the oxidized enzyme (pulsed form). The reaction of fully oxidized cytochrome bo with hydrogen peroxide was studied by EPR. In the presence of 1 molar equiv of hydrogen peroxide, two EPR signals were observed at g〜2 typical of organic radicals. The broader signal might be a cross-linked tyrosine radical that is magnetically coupled to Cu_B, but it is uncertain at this moment. It would seem quite possible that this radical is an intermediate in the mechanism of dioxygen reduction.3. The reaction of fully reduced cytochrome be with dioxygen was investigated by the direct mixing method with EPR spectroscopy combined with the rapid-freeze quenching device. While no EPR signal derived from the binuclear center and heme b was observed in the time domain from 0.2 to 1 ms, the signals derived from the ferric hydroxide-bound form of heme o and the oxidized form of heme b were simultaneously observed after 1 ms. We interpret that the enzyme is in the F and hydroxy intermediates at 0.2 and 1 ms, respectively.

大肠杆菌细胞色素bo型泛醇氧化酶是血红素铜末端氧化酶超家族的成员。它含有四个氧化还原中心，血红素B、血红素o、Cu_B和泛醇-8（Q_H）。血红素o和Cu_B构成双核中心，将分子氧还原成水。为了阐明分子氧还原反应的机理，确定反应中间体中氧化还原中心的电子态是不可避免的.完全氧化的细胞色素bo含有一对高自旋血红素o（S_<Fe>=5/2）和Cu_B（S_<Cu>=1/2），有效自旋S '= 2。利用新研制的X带双模腔研究了g=3.6的整数自旋EPR信号。由于平行模腔灵敏度低，没有观察到信号. P中间体可以通过向氧化酶（脉冲形式）中加入低浓度的过氧化氢来获得。用电子顺磁共振（EPR）研究了完全氧化的细胞色素bo与过氧化氢的反应。在1摩尔当量的过氧化氢的存在下，观察到两个EPR信号在g = 2典型的有机自由基。更广泛的信号可能是与Cu_B磁耦合的交联酪氨酸自由基，但目前尚不确定。这似乎很有可能，这个自由基是一个中间体的机制，分子氧还原。采用EPR直接混合法结合快速冷冻淬灭装置研究了完全还原的细胞色素be与分子氧的反应。虽然没有EPR信号来自双核中心和血红素B在时域中观察到从0.2至1毫秒，来自铁的氢氧化物结合形式的血红素o和血红素B的氧化形式的信号，同时观察后1毫秒。我们解释说，酶是在F和羟基中间体在0.2和1毫秒，分别。