Making sense of subcellular H2O2 in mammals

Hydrogen peroxide (H 2 O 2 ) is a non-radical reactive oxygen species (ROS) that plays a crucial role in maintaining physiological cellular functions. Acting as a redox signaling molecule, H 2 O 2 selectively oxidizes reactive thiol groups of protein cysteines, enabling dynamic regulation of protein function through reversible redox modifications. These redox-sensitive modulations are tightly controlled by compartmentalized oxidoreductase systems, which continuously maintain a balance between H 2 O 2 production from endogenous sources such as NADPH oxidases and mitochondria, and its elimination by compartment-specific antioxidant enzymes. Importantly, intracellular H 2 O 2 concentrations are highly heterogeneous and vary across subcellular locales, reflecting distinct redox environments and organelle-specific functional demands within the cell. Physiological, compartment-specific regulation of H 2 O 2 supports redox signaling and adaptive metabolic responses, whereas disruption of spatially confined H 2 O 2 homeostasis results in localized oxidative distress that contributes to cellular dysfunction and disease. Recent advances in genetically encoded biosensors, particularly improved roGFP- and HyPer-based designs, have enabled sensitive, real-time, and compartment-specific detection of H 2 O 2 dynamics in living cells. These tools provide unprecedented insight into localized oxidative signaling and the spatial organization of redox stress under both physiological and pathological conditions. In this review, we summarize current strategies for monitoring H 2 O 2 dynamics using genetically engineered biosensors, with a focus on compartment-specific H 2 O 2 homeostasis in health and disease.