Significance Vitamins are often precursors for the biosynthesis of organic enzyme cofactors, as exemplified by the ubiquitous vitamin B2-derived flavins. Enzymes employ flavins, e.g., to oxygenate organic substrates with the help of covalent flavin–oxygen adducts that serve as oxygenating species. However, details of the preceding reaction of O 2 with the reduced flavin cofactor that gives rise to these oxygenating species remain scarce. We have now shown how a flavoenzyme interacts with O 2 and controls the formation of an oxygenating species as key to oxidative catalysis. This knowledge will be useful for the bioengineering of flavoenzymes and fine-tuning of their O 2 reactivity.