Disruption of Redox Homeostasis by Enzymatic Activation of a Triakylphosphine Probe in Mitochondria

Redox homeostasis is essential for cell function and its disruption is associated with cancer, metabolic and neurodegenerative diseases. Redox balance is largely regulated by the relative concentrations of reduced and oxidized glutathione. In eukaryotes, this ratio is different in each cell compartment, and disruption of the mitochondrial redox balance has been specifically linked to pathologies such as obesity and type II diabetes. Although reduced glutathione can be scavenged using electrophiles, there is a lack of probes that can produce it. In this study, we report an organellespecific reducing agent based on tributylphosphine that selectively reduces oxidized glutathione in mitochondria. This probe is activated by endogenous nitroreductases and subsequently releases tributylphosphine, as well as a fluorescent reporter, within the organelle. Confocal imaging and biological assays in human cells revealed that, counterintuitively, increased reduced glutathione induced oxidative stress through accumulation of superoxide. Transcriptomic analysis was used to establish that mitochondrial redox stress activates a cellular response orchestrated by transcription factor ATF4, which upregulates genes involved in glutathione metabolism.