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Hydrogen peroxide (H2O2) functions as a second messenger to signal metabolic distress through highly compartmentalized production in mitochondria. The dynamics of ROS generation and diffusion between mitochondrial compartments and into the cytosol govern oxidative stress responses and pathology, though our understanding of these processes remains limited. Here, we couple the H2O2biosensor, HyPer7, with optogenetic stimulation of the ROS-generating protein KillerRed targeted into multiple mitochondrial microdomains. Single mitochondrial photogeneration of H2O2demonstrates the spatiotemporal dynamics of ROS diffusion and transient hyperfusion of mitochondria due to ROS. Measurement of microdomain-specific H2O2diffusion kinetics reveals directionally selective diffusion through mitochondrial microdomains. All-optical generation and detection of physiologically-relevant concentrations of H2O2between mitochondrial compartments provide a map of mitochondrial H2O2diffusion dynamicsin situ. These kinetic details of spatiotemporal ROS dynamics and inter-mitochondrial spreading forms a framework to understand the role of ROS in health and disease.

C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.