Over time, organisms have evolved strategies to cope with the abundance of dioxygen on Earth. Oxygen-utilizing enzymes tightly control the reactions involving O₂ mostly by modulating the reactivity of their cofactors. Flavins are extremely versatile cofactors that are capable of undergoing redox reactions by accepting either one electron or two electrons, alternating between the oxidized and the reduced states. The physical and chemical principles of flavin-based chemistry have been investigated widely. In the following pages we summarize the state of the art on a key area of research in flavin enzymology: the molecular basis for the activation of O₂ by flavin-dependent oxidases and monooxygenases. In general terms, oxidases use O₂ as an electron acceptor to produce H₂O₂, while monooxygenases activate O₂ by forming a flavin intermediate and insert an oxygen atom into the substrate. First, we analyze how O₂ reaches the flavin cofactor embedded in the protein matrix through dedicated access pathways. Then we approach O₂ activation from the perspective of the monooxygenases, their preferred intermediate, the C(4a)–(hydro)peroxyflavin, and the cases in which other intermediates have been described. Finally, we focus on understanding how the architectures developed in the active sites of oxidases promote O₂ activation and which other factors operate in its reactivity.

Quantitative analysis of Ca ²⁺ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca ²⁺ -dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca ²⁺ binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca ²⁺ release kinetics with an off-rate estimated at around 700 s ⁻¹ and appropriate Ca ²⁺ binding affinity, likely attributable to local Ca ²⁺ -induced conformational changes around the designed Ca ²⁺ binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca ²⁺ dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca ²⁺ in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca ²⁺ release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca ²⁺ homeostasis.