The dynamic equilibrium between the generation of reactive oxygen species (ROS) and the antioxidative defense mechanisms within the cellular environment is a critical determinant of cell fate and function. Hydrogen peroxide (H2O2) is a physiological product of aerobic metabolism, and there is now evidence that there are pleiotropic effects of ROS and H2O2, that depend on the concentration [1]. Pico -to-nanomolar range concentrations of H2O2 are considered oxidative “eustress” [1]. In these lower intracellular concentrations, H2O2 functions as a vital second messenger in signal transduction of oxidant-modulated responses to physiological stimuli that include cell proliferation, migration, angiogenesis, or gene regulation that occur during normal growth and development, physical and mental exercise. In the low nanomolar range, H2O2 signaling is associated with stress responses and adaptations that happen during short-term events like wound healing or in long-term events like aging, inactivity, or malnutrition that can progress to disease states if prolonged. Further increases of H2O2 levels beyond the 100 nM range are oxidative stressful. These include processes of inflammation, fibrogenesis, tumor growth, and finally, at very high concentrations, H2O2 leads to cellular injury and death [3], these elevated levels have been found in conditions like exposure to radiation, ischemia-reperfusion injury, alcoholism, or smoking. The recognition of this dichotomous response to low vs. high concentrations of H2O2, termed “hormesis”, has spurred a considerable research interest in its precise biological functions, particularly within the context of pathogenesis in diseases such as cancer, diabetes, cardiovascular and neurodegenerative disorders.

In this intricate interplay, chemogenetic approaches have emerged as an informative method for dynamically modulating oxidative stress both in vitro and in vivo [1,4]. The term ‘chemogenetics’ has been used to describe model systems where recombinant proteins can be reversibly activated by providing (or withdrawing) a specific and unique ligand or substrate to the biological system. By employing the yeast enzyme D-amino acid oxidase (DAAO), it is possible to generate H₂O₂ by providing its D-amino acid substrate. The DAAO enzyme and its cofactor flavin adenine dinucleotide (FAD) catalyze the oxidation of D- amino acids which results in the generation of its corresponding α-keto acid, plus equimolar quantities of ammonia and H2O2 (Figure 1). The yeast DAAO is highly stereospecific and does not recognize L-amino acids as substrates [5]. Since most mammalian tissues contain L-amino acids and lack appreciable levels of D-amino acids [6], the recombinant DAAO is quiescent in mammalian cells until D-amino acids are provided. It should be noted that the levels of ammonia and keto acid levels that are generated by recombinant DAAO are nominal compared to the much higher intracellular levels of these important metabolites. By contrast, intracellular levels of H2O2 are much lower, and are estimated to increase ∼2-fold after addition of D-amino acids to cells expressing recombinant DAAO [7]. Most of the studies utilizing DAAO have been performed using D-alanine, as a substrate, mainly due to its availability and lower price compared to other D-amino acids. However, studies have used successfully other D-amino acids, including D-proline [8] and D-norvaline [9].

DAAO can be targeted to specific subcellular compartments to spatially and temporally produce H₂O₂ in diverse locales, including the cell cytoplasm, plasma membrane, plasmalemmal caveolae, mitochondria, and nucleus [10]. Moreover, the incorporation of H₂O₂ biosensors with DAAO has provided real-time insights into the dynamics of redox signaling, metabolism, and transport. The recently developed H2O2 biosensor Hyper7 is an ultrasensitive and highly specific genetically encoded probe that facilitates the study of intracellular H2O2 dynamics in cultured cells with great temporal and spatial resolution [11].