ELECTRON-TRANSFER FLAVOPROTEIN-UBIQUINONE OXIDOREDUCTASE FROM PIG-LIVER - PURIFICATION AND MOLECULAR, REDOX, AND CATALYTIC PROPERTIES
ELECTRON-TRANSFER FLAVOPROTEIN-UBIQUINONE OXIDOREDUCTASE FROM PIG-LIVER - PURIFICATION AND MOLECULAR, REDOX, AND CATALYTIC PROPERTIES
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DOI：
10.1021/bi00336a016
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
FRERMAN, FE
中科院分区：
文献类型：
--
作者：
BECKMANN, JD;FRERMAN, FE
从猪肝亚线粒体颗粒中提纯了电子转移黄素蛋白-泛醌氧化还原酶(ETF-QO)。在分子量和一般性质上与牛心ETF-QO相当[Ruzicka，F.J.和Beinert，H.(1977)J.Biol]。化学。252,8440-8445]，还原的离子-硫团簇的ESR信号基本相同。ETF-QO催化电子从电转移黄素蛋白(ETF)转移到硝基蓝四氮唑，反应迟滞的周转数为.apprx。10-30分钟-1。相比之下，该酶迅速与ETF半喹酮不成比例，成交量为200亿S-1。氧化和对苯二酚ETF的逆反应提供了一种具有皮摩尔灵敏度的ETF-QO的酶分析方法。平衡光度滴定表明，ETF-QO最多接受ETF的2个电子，接受3个来自二亚硫酸盐或光化学还原的电子当量。所有来自酶或化学还原的蛋白质的电子都可以转移到2，3-dimethoxy-5-methyl-6-pentyl-1，4-benzoquinone(PB)上，而且这个反应很容易就能逆转。2，3-dimethoxy-5-methyl-6-pentyl-1，4-benzohydroquinone对ETF-QO的还原具有pH依赖性，表明该酶具有氧化还原电位，每pH单位降低47 mV。因此，ETF-QO在还原时结合了1到2个质子。PH为7.3时，E0‘’为38 mV。ETF-QO催化ETF氧化还原态平衡的能力被用来评估2ETFsq+NH+.dblarw的平衡。ETFox+ETFhq.平衡的pH依赖关系表明n=1，与ETF半对苯二酚(ETFsq)和对苯二酚黄素物种为真阴离子的指认一致。预测氧化ETF的1电子还原电位与pH无关。
Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) was purified to homogeneity from pig liver submitochondrial particles. It is comparable in MW and general properties to ETF-QO from beef heart [Ruzicka, F.J., and Beinert, H. (1977) J. Biol. Chem. 252, 8440-8445], and the ESR signals of the reduced ion-sulfur cluster are essentially identical. ETF-QO catalyzes the transfer of electrons from electro-transfer flavoprotein (ETF) to nitro blue tetrazolium, with a sluggish reaction turnover number of .apprx. 10-30 min-1. In contrast, the enzyme rapidly disproportionates ETF semiquinone, with a turnover number of 200 s-1. The reverse reaction, comproportionation of oxidized and hydroquinone ETF, provides an enzymatic assay for ETF-QO with picomolar sensitivity. Equilibrium spectrophotometric titrations show that ETF-QO accepts a maximum of 2 electrons from ETF and accepts 3 electron equivalents from dithionite or by photochemical reduction. All electrons from the enzymatically or chemically reduced protein can be transferred to 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone (PB), and this reaction is readily reversible. Reduction of ETF-QO by 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzohydroquinone is pH dependent and indicates the enzyme to have a redox potential that decreases by 47 mV per pH unit. Therefore, ETF-QO binds 1 to 2 protons upon reduction. E0'' at pH 7.3 is 38 mV. The ability of ETF-QO to catalyze the equilibration of ETF redox states has been used to evaluate the equilibrium 2ETFsq + nH+ .dblarw. ETFox + ETFhq. The pH dependence of the equilibrium indicates that n = 1 and is consistent with the assignment of ETF semiquinone (ETFsq) and hydroquinone flavin species as true anions. The 1-electron reduction potential of oxidized ETF is predicted to be independent of pH.