Mitochondrial respiratory complexes I to IV and the F 1 F o -ATP synthase (complex V) are large protein assemblies producing the universal cellular energy currency adenosine triphosphate (ATP). Individual complexes have been extensively studied in vitro, but functional co-reconstitution of several mammalian complexes into proteoliposomes, in particular, the combination of a primary pump with the ATP synthase, is less well understood. Here, we present a generic and scalable strategy to purify mammalian respiratory complexes I, III and the ATP synthase from enriched mitochondria in enzymatically fully active form, and procedures to reassemble the complexes into liposomes. A robust functionality can be shown by in situ monitoring of ATP synthesis rates and by using selected inhibitors of the respiratory chain complexes. By inclusion of cytochrome c oxidase, our procedures allowed us to reconstruct the entire mitochondrial respiratory chain (complexes I, III, IV, and V) in ubiquinone Q 10 containing liposomes, demonstrating oxidative phosphorylation by nicotinamide adenine dinucleotide hydrogen driven ATP synthesis. The system was fully coupled at all levels and was used to probe cardiolipin as an essential component to activate the mammalian respiratory chain. Structural characterization using electron cryomicroscopy allowed us to resolve apo-state complex III and complex V at high and medium resolution, respectively, using in silico particle sorting, confirming the presence of all protein subunits and cofactors in native stoichiometry and conformation. The reported findings will facilitate future endeavors to characterize or modulate these key bioenergetic processes.

中文翻译：

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一个出色的分子工厂：哺乳动物呼吸链的去解体和重建


线粒体呼吸复合物 I 至 IV 和 F 1 F o -ATP 合酶（复合物 V）是产生通用细胞能量货币三磷酸腺苷 （ATP） 的大型蛋白质组装体。单个复合物已在体外进行了广泛研究，但几种哺乳动物复合物在功能上共重构为蛋白脂质体，特别是初级泵与 ATP 合酶的组合，尚不清楚。在这里，我们提出了一种通用且可扩展的策略，以酶促完全活性形式从富集的线粒体中纯化哺乳动物呼吸复合物 I、III 和 ATP 合酶，以及将复合物重新组装成脂质体的程序。通过原位监测 ATP 合成速率和使用呼吸链复合物的选定抑制剂，可以显示强大的功能。通过加入细胞色素 c 氧化酶，我们的程序使我们能够在含有脂质体的泛醌 Q 10 中重建整个线粒体呼吸链（复合物 I、III、IV 和 V），证明烟酰胺腺嘌呤二核苷酸氢驱动的 ATP 合成进行氧化磷酸化。该系统在所有水平上都是完全耦合的，用于探测心磷脂作为激活哺乳动物呼吸链的重要成分。使用电子冷冻显微镜进行结构表征使我们能够分别在高分辨率和中分辨率下分辨载脂蛋白态复合物 III 和复合物 V，使用计算机颗粒分选，确认天然化学计量和构象中存在所有蛋白质亚基和辅因子。报告的结果将有助于未来努力表征或调节这些关键的生物能量过程。