Analysis of inhibitor binding to the mitochondrial cytochrome c reductase by fluorescence quench titration. Evidence for a 'catalytic switch' at the Qo center.
Analysis of inhibitor binding to the mitochondrial cytochrome c reductase by fluorescence quench titration. Evidence for a 'catalytic switch' at the Qo center.
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通过荧光淬灭滴定分析抑制剂与线粒体细胞色素 c 还原酶的结合。
DOI：
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
G. Jagow
中科院分区：
文献类型：
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作者：
Ulrich Brandt;G. Jagow
用荧光猝灭滴定法研究了线粒体细胞色素c还原酶抑制剂的结合特性。基于标准结合方程，所应用的数值方法允许通过拟合Kd、结合位点的数目以及游离和结合的抑制剂的特异性荧光来解释在线记录的滴定曲线。对于Qi中心，使用2-正壬基-4-羟基喹啉N-氧化物和对于Qo中心，使用（E）-β-甲氧基丙烯酸酯-芪（MOA-芪）作为荧光抑制剂。通过竞争分析，实验可以扩展到其他非荧光抑制剂。使用这种方法，我们能够使用相同的实验设置，在酶的不同氧化还原状态下比较Qi和Qo中心抑制剂的结合行为。我们研究了代表所有亚组的细胞色素c还原酶抑制剂之间的竞争，并证明至少有三个抑制剂结合位点存在，两个位于Qo中心，一个位于Qi中心。的氧化，部分还原和完全还原的酶的解离常数的测定表明，在齐中心的抑制剂结合是不氧化还原依赖性的。与此相反，MOA-芪的Qo中心的结合减少后，减少铁-硫中心和细胞色素C1，而这种氧化还原变化增加了Qo中心抑制剂的羟基萘醌型，3-n-十一烷基-2-羟基萘醌的亲和力。根据这些结果，意识到Qo中心的抑制机制是非竞争性的，我们提出了“催化开关”的假设来解释电子流的分叉和Qo中心的抑制。两种构象状态之一的位阻可以作为抑制剂的结构变异性和抑制剂对氧化还原中心的差异效应的有力解释。此外，建议的“开关”提供了一些深入了解其他实验结果，这是很难解释的泛醌循环，目前制定的。
The binding characteristics of inhibitors of the mitochondrial cytochrome c reductase were studied by fluorescence quench titration. Based on the standard binding equation, the applied numerical method allowed the online recorded titration curves to be interpreted by fitting the Kd, the number of binding sites, and the specific fluorescence of the free and the bound inhibitor. For the Qi center, 2-n-nonyl-4-hydroxyquinoline N-oxide and for the Qo center (E)-beta-methoxyacrylate-stilbene (MOA-stilbene) were used as fluorescing inhibitors. The experiments could be extended to other, non-fluorescing inhibitors by competition analysis. Using this method we were able to compare the binding behaviour of Qi and Qo center inhibitors under different redox states of the enzyme using the same experimental set up. We studied the competition between inhibitors of the cytochrome c reductase representative for all subgroups and demonstrated that at least three inhibitor binding sites exist, two located in the Qo center, one located in the Qi center. Determination of the dissociation constants of the oxidized, the partially reduced and the fully reduced enzyme showed that inhibitor binding at the Qi center is not redox-dependent. In contrast, the binding of MOA-stilbene to the Qo center is decreased after reduction of the iron-sulfur center and cytochrome c1, whereas this redox change increases the affinity for a Qo center inhibitor of the hydroxynaphthoquinone type, 3-n-undecyl-2-hydroxynaphthoquinone. From these results, aware of the fact that the inhibitory mechanism at the Qo center is a non-competitive one, we made the hypothesis of a 'catalytic switch' to explain both the bifurcation of electron flow and the inhibition at the Qo center. A steric blockage of one of two conformational states could serve as a cogent explanation for the great structural variability of the inhibitors and differential effects on the redox centers exerted by the inhibitors. Moreover, the proposed 'switch' gives some insight into other experimental results which are difficult to explain with the ubiquinone cycle as currently formulated.
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通过酵母线粒体细胞色素 b-c1 复合物中心 o 的电子转移涉及 VII 亚基，即泛醌结合蛋白。
DOI：
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发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Japa,S;Beattie,DS
通讯作者：
Beattie,DS
泛醌类似物，通过与线粒体呼吸链细胞色素 b-c1 片段的铁硫蛋白结合来抑制呼吸。
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Bowyer,JR;Edwards,CA;Ohnishi,T;Trumpower,BL
通讯作者：
Trumpower,BL
抗霉素存在下细胞色素 c1 的快速减少及其对线粒体呼吸链细胞色素 b-c1 片段中电子转移机制的影响。
DOI：
--
发表时间：
1981
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Bowyer,JR;Trumpower,BL
通讯作者：
Trumpower,BL
一种线粒体呼吸抑制剂，可与细胞色素 b 结合并取代细胞色素 bc1 复合物铁硫蛋白中的醌。
DOI：
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发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
vonJagow,G;Ljungdahl,PO;Graf,P;Ohnishi,T;Trumpower,BL
通讯作者：
Trumpower,BL
哺乳动物线粒体细胞色素 b 突变体抑制剂抗性的分子基础。
DOI：
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发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Howell,N;Appel,J;Cook,JP;Howell,B;Hauswirth,WW
通讯作者：
Hauswirth,WW