Structural methods for probing the interaction of flavoenzymes with dioxygen and its surrogates

As a rare feature among organic cofactors, reduced flavins (Fl_red) can efficiently react with dioxygen (O₂). As a consequence, many flavin-dependent enzymes may serve as either oxidases that use O₂ as an electron acceptor or as monooxygenases that transfer one oxygen atom derived from O₂ to an organic substrate. For the latter functionality, covalent flavin: oxygen adducts are formed that function as oxygenating species. Remarkably, despite intensive research, many open questions remain how flavoenzymes control the reaction with O₂. Here, we describe O₂-pressurized protein crystallography in detail as a structural approach to gain insight into the interactions between the protein scaffold, the flavin cofactor and O₂. This may allow to further our understanding of how flavoenzymes can steer the formation of different oxygenating species and thus provide missing puzzle pieces for rational flavoenzyme design.