New insight into cofactor-free oxygenation from combined experimental and computational approaches

[Display omitted] •Some oxygenases (and oxidases) do not require metals and/or organic cofactors for catalysis.•α/β-Hydrolase fold bacterial cofactor-free 2,4-dioxygenases rely on a His-Asp catalytic dyad for substrate deprotonation/activation.•A mechanism of direct O2 attack rather than charge transfer might be employed by these enzymes to generate a peroxide intermediate.•An enzyme–peroxide intermediate complex and an in situ-generated enzyme–substrate–O2 complex have been trapped. Molecular oxygen (O2), in spite being a potentially strong oxidant, typically displays very poor reactivity with organic molecules. This is largely due to quantum chemical reasons as O2 in its ground state is a diradical (3O2) whilst common organic substrates are in a singlet state. For this reason catalysis involving O2 as a reactant is typically mediated by enzymes containing redox metal and/or organic co-factors. Cofactor-independent oxygenases (and oxidases) are therefore intriguing enzymes from a fundamental viewpoint. This review looks at recent advances that have been made in understanding of this class of intriguing biocatalysts highlighting the power of an inter-disciplinary approach involving structural biology, spectroscopy and theoretical methods.