Suppress, Stimulate, or Steer: Dioxygen Activation by Flavoenzymes and Their Oxygenation Strategies

The ubiquitous flavoenzymes typically function as oxidoreductases that comprise several distinct main types, including flavoprotein oxidases (FPOs), flavoprotein dehydrogenases (FPDs), and flavoprotein monooxygenases (FPMOs). FPOs and FPDs catalyze two-electron oxidation reactions of organic substrates, typically dehydrogenations, thereby converting oxidized flavin (Flox) into its fully reduced state (Flred). Prior to the next catalytic cycle, molecular oxygen (=dioxygen or O2) or (protein-bound) cofactors facilitate the required Flred reoxidation for FPOs and FPDs. Remarkably, members of these two flavoenzyme types can be homologous with highly similar amino acid compositions and overall structures, as minor protein alterations, particularly in the vicinity of the flavin cofactor, can drastically affect O2 reactivity. Finally, FPMOs incorporate one O2-derived oxygen atom into their substrate. To this end, required electrons for Flred formation and O2 activation either come from NAD(P)H (external FPMOs) or, more rarely, the substrate itself (internal FPMOs). External FPMOs steer O2 reactivity toward the formation of covalent flavin-oxygen adducts primarily at the C4a atom of the flavin's isoalloxazine ring or, in some cases, at the adjacent N5. In contrast, typical internal FPMOs forego the formation of covalent oxygen adducts entirely, although an exception in the form of a flavin-N5-oxide-forming enzyme has been reported. Consequently, natural selection has led to three distinct O2 reactivity patterns in flavoenzymes, which either suppress (FPDs), stimulate (FPOs), or steer (FPMOs) this challenging process. In this review, current knowledge on the relationship between flavoenzymes and O2 is summarized, emphasizing strategies to insert oxygen into organic substrates and counteract uncoupling, while also highlighting open questions and future challenges.