Cleavage mechanism of the aliphatic C–C bond catalyzed by 2,4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP: a QM/MM study

Abstract

2,4′-Dihydroxyacetophenone dioxygenase (DAD) is a bacterial non-heme enzyme responsible for the oxygenative cleavage of the aliphatic C–C bond, which catalyzes the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid. On the basis of the crystal structure and studies on two synthesized biomimetic model compounds, two possible reaction pathways that involve a dioxacyclic or alkylperoxo intermediate have been previously suggested. However, little is currently known about the mechanistic detail and the proposed intermediates have not been experimentally confirmed yet. To elucidate the reaction mechanism at the atomistic level, on the basis of the recently obtained crystal structure, the reactant enzyme–substrate complex has been constructed, and the reaction details have been studied using a quantum mechanics/molecular mechanics (QM/MM) approach. Our calculations reveal the triplet of the iron(III)-superoxide radical complex as the ground state, but the quintet state which is higher than the triplet by 11.2 kcal mol⁻¹ corresponds to a lower energy barrier in the first step. Thus, the reactant complex may firstly undergo a triplet–quintet crossing to initiate the reaction and then the subsequent chemistry mainly occurs on the quintet state surface. The previously proposed key dioxacyclic or alkylperoxo intermediate was calculated to be energetically unreachable, and the corresponding mechanism has been revised, which contains eight elementary steps, and the key C–C bond cleavage is accompanied by an insertion reaction of the adjacent oxygen radical. Two elementary steps are calculated to be possible rate-determining steps. Our results may provide useful information for further understanding the cleavage mechanism of the aliphatic C–C bond catalyzed by DAD and other dioxygenase enzymes.