Cofactor-free ActVA-Orf6 monooxygenase catalysis via proton-coupled electron transfer: a QM/MM study

Uncovering the comprehensive catalytic mechanism for the activation of triplet O₂ through metal-free and cofactor-free oxidases and oxygenases remains one of the most challenging problems in the area of enzymatic catalysis. Herein, we performed multiscale simulation with molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) techniques to reveal the detailed mechanism of ActVA-Orf6 monooxygenase catalyzed oxygenation of phenols to quinones from Streptomyces coelicolor, such as the oxidation of 6-deoxydihydrocarafungin (DDHK) to dihydrocarafungin (DHK). The entire catalytic mechanism consists of three steps: (1) proton-coupled electron transfer (PCET) from the substrate DDHK to triplet O₂ with the aid of an explicit water molecule, (2) the formation of a C-O bond via an open-shell singlet diradical complexation pathway, and (3) dehydration via a six-membered ring mode assisted by one water molecule. The complete energetic profiles show that the rate-determining step is the dehydration with an energy barrier of 20.7 kcal mol^-1, which is close to that of 19.7 kcal mol^-1 derived from experimental kinetic data. Our mechanistic study not only helps to deeply understand the fundamental mechanism of metal-free and cofactor-free oxidase and oxygenase catalyzed different reactions, but also discloses a new route that proceeds through the processes of PCET and the open-shell singlet transition state.