Enzymatic control of dioxygen binding and functionalization of the flavin cofactor

The reactions of enzymes and cofactors with gaseous molecules such as dioxygen (O₂) are challenging to study and remain among the most contentious subjects in biochemistry. To date, it is largely enigmatic how enzymes control and fine-tune their reactions with O₂, as exemplified by the ubiquitous flavin-dependent enzymes that commonly facilitate redox chemistry such as the oxygenation of organic substrates. Here we employ O₂-pressurized X-ray crystallography and quantum mechanical calculations to reveal how the precise positioning of O₂ within a flavoenzyme's active site enables the regiospecific formation of a covalent flavin-oxygen adduct and oxygenating species (i.e., the flavin-N5-oxide) by mimicking a critical transition state. This study unambiguously demonstrates how enzymes may control the O₂ functionalization of an organic cofactor as prerequisite for oxidative catalysis. Our work thus illustrates how O₂ reactivity can be harnessed in an enzymatic environment and provides crucial knowledge for future rational design of O₂-reactive enzymes.