The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, The Keith Peters Building, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, UK.
Sci Rep. 2021 May 12;11(1):10143. doi: 10.1038/s41598-021-89575-9.
Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a crucial metabolic enzyme that couples the free energy released from NADH oxidation and ubiquinone reduction to the translocation of four protons across the inner mitochondrial membrane, creating the proton motive force for ATP synthesis. The mechanism by which the energy is captured, and the mechanism and pathways of proton pumping, remain elusive despite recent advances in structural knowledge. Progress has been limited by a lack of model systems able to combine functional and structural analyses with targeted mutagenic interrogation throughout the entire complex. Here, we develop and present the α-proteobacterium Paracoccus denitrificans as a suitable bacterial model system for mitochondrial complex I. First, we develop a robust purification protocol to isolate highly active complex I by introducing a His-tag on the Nqo5 subunit. Then, we optimize the reconstitution of the enzyme into liposomes, demonstrating its proton pumping activity. Finally, we develop a strain of P. denitrificans that is amenable to complex I mutagenesis and create a catalytically inactive variant of the enzyme. Our model provides new opportunities to disentangle the mechanism of complex I by combining mutagenesis in every subunit with established interrogative biophysical measurements on both the soluble and membrane bound enzymes.
线粒体复合物 I（NADH：泛醌氧化还原酶）是一种至关重要的代谢酶，它将 NADH 氧化和泛醌还原释放的自由能与穿过线粒体内膜的四个质子的转运偶联起来，为 ATP 合成产生质子动力势。尽管最近在结构知识方面取得了进展，但能量的捕获机制以及质子泵的机制和途径仍然难以捉摸。由于缺乏能够将功能和结构分析与整个复合物的靶向诱变研究结合起来的模型系统，进展受到了限制。在这里，我们开发并提出了 Paracoccus denitrificans（一种α-变形菌）作为线粒体复合物 I 的合适细菌模型系统。首先，我们通过在 Nqo5 亚基上引入 His 标签，开发了一种有效的纯化方案来分离高活性的复合物 I。然后，我们优化了将酶重组到脂质体中的方法，证明了其质子泵活性。最后，我们开发了一种适合于复合物 I 诱变的 P. denitrificans 菌株，并创建了该酶的催化失活变体。我们的模型为通过将每个亚基的诱变与对可溶性和膜结合酶的既定询问生物物理测量相结合来阐明复合物 I 的机制提供了新的机会。
