Real-Time Observation of Conformational Changes and Translocation of Endogenous Cytochrome c within Intact Mitochondria
Real-Time Observation of Conformational Changes and Translocation of Endogenous Cytochrome c within Intact Mitochondria
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DOI：
10.1021/jacs.3c10216
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发表时间：
2024-02-09
影响因子：
15
通讯作者：
Zhang，Xu
中科院分区：
文献类型：
--
作者：
Zhan，Jianhua;Zeng，Danyun;Zhang，Xu
细胞色素c（cyt c）是一种具有多种构象的多功能蛋白质。然而，cyt c在其天然环境线粒体中的构象仍不清楚。在这里，我们应用核磁共振波谱法研究天然同位素丰度的完整线粒体内内源性细胞色素C的构象和位置，主要使用广泛的甲基作为探针。通过监测随时间变化的化学位移扰动，我们观察到大多数细胞色素c位于线粒体内膜和部分未折叠，这是不同于其在溶液中的天然构象。当遭受氧化应激时，由于活性氧（ROS）增加，细胞色素c发生氧化修饰，与膜的静电相互作用减弱，并逐渐移位到线粒体的内膜空间。同时，氧化修饰后的细胞色素C对细胞的杀伤力较正常细胞色素C降低。我们的研究结果显着提高线粒体中的细胞色素c的ROS的调节的分子机制的理解。此外，它突出了NMR在完整细胞或细胞器内以天然同位素丰度监测高浓度分子的潜力。
Cytochrome c (cyt c) is a multifunctional protein with varying conformations. However, the conformation of cyt c in its native environment, mitochondria, is still unclear. Here, we applied NMR spectroscopy to investigate the conformation and location of endogenous cyt c within intact mitochondria at natural isotopic abundance, mainly using widespread methyl groups as probes. By monitoring time-dependent chemical shift perturbations, we observed that most cyt c is located in the inner mitochondrial membrane and partially unfolded, which is distinct from its native conformation in solution. When suffering oxidative stress, cyt c underwent oxidative modifications due to increasing reactive oxygen species (ROS), weakening electrostatic interactions with the membrane, and gradually translocating into the inner membrane spaces of mitochondria. Meanwhile, the lethality of oxidatively modified cyt c to cells was reduced compared with normal cyt c. Our findings significantly improve the understanding of the molecular mechanisms underlying the regulation of ROS by cyt c in mitochondria. Moreover, it highlights the potential of NMR to monitor high-concentration molecules at a natural isotopic abundance within intact cells or organelles.