A Self-Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone-10 Cycling between Complex I and the Alternative Oxidase
A Self-Assembled Respiratory Chain that Catalyzes NADH Oxidation by Ubiquinone-10 Cycling between Complex I and the Alternative Oxidase
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通过泛醌-10 在复合物 I 和替代氧化酶之间循环催化 NADH 氧化的自组装呼吸链
DOI：
10.1002/ange.201507332
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发表时间：
2015
影响因子：
--
通讯作者：
Jones A
中科院分区：
文献类型：
--
作者：
Jones A
Complex I 是一种重要的呼吸酶，在质子跨膜传输过程中，它可以保存泛醌-10 (Q10) 氧化 NADH 所产生的能量。 Q10 的极端疏水性阻碍了对其能量转换机制的研究，迄今为止，他们依赖于具有多种成分的天然膜或亲水性的 Q10 类似物，这些类似物会分成膜并发生副反应。在此，我们提出了一种没有这些限制的自组装系统：含有哺乳动物复合物 I、Q10和对苯二酚氧化酶（替代氧化酶，AOX）的蛋白脂质体，用于将Q10H2回收为Q10。 AOX 过量存在，因此复合物 I 完全是速率决定因素，并且 Q10pool 在稳态催化下保持氧化状态。该系统用于测量 Q10 的完全定义的 KM 值。该策略适用于任何具有疏水性醌/醌醇底物的酶，并且可用于表征具有药物、杀虫剂或杀菌剂等潜在应用的疏水性抑制剂。
Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone‐10 (Q10) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10, and they have so far relied on native membranes with many components or on hydrophilic Q10analogues that partition into membranes and undergo side reactions. Herein, we present a self‐assembled system without these limitations: proteoliposomes containing mammalian complex I, Q10, and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10H2to Q10. AOX is present in excess, so complex I is completely rate determining and the Q10pool is kept oxidized under steady‐state catalysis. The system was used to measure a fully‐definedKMvalue for Q10. The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.