Monophenolase and catecholase activity ofAspergillus oryzaecatechol oxidase: insights from hybrid QM/MM calculations

Catechol oxidase fromAspergillus oryzae(AoCO4) can not only catalyze oxidation ofo-diphenols too-quinones, but can also catalyze monooxygenation of small phenolics. To gain insight into the catecholase and monophenolase activities of AoCO4, the reaction mechanism of catechol oxidation was investigated by means of hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. The oxy-form of AoCO4 was found to be a mu-eta(2):eta(2)side-on peroxo dicopper(ii) complex, which can undergo a proton coupled electron transfer from the substrate rather than a proton transfer from the nearby Ser302 residue to generate a hydroperoxide. The mu-1,1-OOH Cu-2(i,ii) complex is thermodynamically more stable than the mu-eta(1):eta(2)hydroperoxide. Moreover, the cleavage of the O-O bond in the mu-1,1-OOH Cu-2(i,ii) intermediate has a much lower barrier than that in the mu-eta(1):eta(2)hydroperoxide species. In both cases, the O-O bond cleavage is the rate-limiting step, generating the reactive (mu-O-center dot)(mu-OH) dicopper(ii) complex. In addition, our results demonstrated that the oxidation of catechol to quinone is much more preferred than the hydroxylation reaction. These findings may provide useful information for understanding the reactivity of the Cu(2)O(2)active site of coupled binuclear copper enzymes.