Real-Time Observation of Conformational Changes and Translocation of Endogenous Cytochrome c within Intact Mitochondria

Abstract

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.

An Encyclopedic Compendium on Chemosensing Supramolecular Metal‐Organic Gels

“…Cytochrome C (Cyt C): Detection of Cyt C levels and its release from mitochondria provides insights into cellular health, apoptosis induction, and disease progression, aiding in research on cancer, neurodegenerative disorders, and mitochondrial dysfunction. [240,241] In the domain of ECL systems, the peroxydisulfate-oxygen (S 2 O 8 2À À O 2 ) system is distinguished by its capacity to preserve luminescence integrity during both detection and storage. However, the utilization of this system has been curtailed due to the absence of a suitable enhancer.…”