Mitochondrial H 2 O 2 Generation Using a Tunable Chemogenetic Tool To Perturb Redox Homeostasis in Human Cells and Induce Cell Death

Among reactive oxygen species (ROS), H O 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 H O , and mitochondrial dysfunction has been implicated in diseases such as cancer. A genetically encoded H O 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 H O doses over time was assessed. Organelle-specific peroxiredoxin dimerization and protein S-glutathionylation were measured as indicators of increased H O 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 H O -specific perturbation of mitochondria in human cells, and it reveals a marked sensitivity of this organelle to increases in H O in comparison with prior studies that targeted the cytosol.