The mechanism of the action of copper-dependent quercetin 2,3-dioxygenase(2,3QD) has been investigated by means of hybrid density functional theory.The 2,3QD enzyme cleaves the O-heterocycle of a quercetin by incorporation of both oxygen atoms into the substrate and releases carbon monoxide.The calculations show that dioxygen attack on the copper complex is energetically favorable.The adduct has a possible near-degeneracy of states between [Cu 2+-(substrate-H +)] and [Cu +-(substrate-H).],and in addition the pyramidalized C 2 atom is ideally suited for forming a dioxygen-bridged structure.In the next step,the C 3-C 4 bond is cleaved and intermediate Int 5 is formed via transition state TS 4.Finally,the O a-O b and C 2-C 3 bonds are cleaved,and CO is released in one concerted transition state(TS 5) with the barrier of 63.25 and 61.91 kJ/mol in the gas phase and protein environments,respectively.On the basis of our proposed reaction mechanism,this is the rate-limiting step of the whole catalytic cycle and is strongly driven by a relatively large exothermicity of 100.86 kJ/mol.Our work provides some valuable fundamental insights into the behavior of this enzyme. The mechanism of the action of copper-dependent quercetin 2,3-dioxygenase (2,3QD) has been investigated by means of hybrid density functional theory. 2,3QD enzyme cleaves the O-heterocycle of a quercetin by incorporation of both oxygen atoms into the substrate and releases carbon monoxide. These calculations show that dioxygen attack on the copper complex is energetically favorable. The adduct has a possible near-degeneracy of states between [Cu 2 + - (substrate-H +)] and [Cu + - substrate-H.], and in addition to the pyramidalized C 2 atom is ideally suited for forming a dioxygen-bridged structure. In the next step, the C 3-C 4 bond is cleaved and the intermediate Int 5 is formed via transition state TS 4.Finally, the O aO b and C 2 -C 3 bonds are cleaved, and CO released in one concerted transition state (TS 5) with the barrier of 63.25 and 61.91 kJ / mol in the gas phase and protein environments, respectively.On the basis of our proposed reaction mechanism, this is the rate-limiting step of the whole c atalytic cycle and is strongly driven by a relatively large exothermicity of 100.86 kJ / mol. Our work provides some valuable fundamental insights into the behavior of this enzyme.