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

Uncovering the comprehensive catalytic mechanism for the activation of triplet O 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 , 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 O with the aid of an explicit water molecule, (2) the formation of a C-O bond an open-shell singlet diradical complexation pathway, and (3) dehydration 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, which is close to that of 19.7 kcal mol 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.