Mitochondrial H₂O₂ Generation Using a Tunable Chemogenetic Tool To Perturb Redox Homeostasis in Human Cells and Induce Cell Death

Abstract

Among reactive oxygen species (ROS), H₂O₂ alone acts as a signaling molecule that promotes diverse phenotypes depending on the intracellular concentration. Mitochondria have been suggested as both sources and sinks of cellular H₂O₂, and mitochondrial dysfunction has been implicated in diseases such as cancer. A genetically-encoded H₂O₂ generator, D-amino acid oxidase (DAAO), was targeted to the mitochondria of human cells, and its utility in investigating cellular response to a range of H₂O₂ doses over time was assessed. Organelle-specific peroxiredoxin dimerization and protein S-glutathionylation were measured as indicators of increased H₂O₂ flux due to the activity of DAAO. Cell death was observed in a concentration- and time-dependent manner, and protein oxidation shifted in localization as the dose increased. This work presents the first systematic study of H₂O₂-specific perturbation of mitochondria in human cells, and it reveals a marked sensitivity of this organelle to increases in H₂O₂ in comparison with prior studies that targeted the cytosol. Keywords: hydrogen peroxide; redox regulation; mitochondria; chemogenetic tools; peroxiredoxin