DFT study of the mechanism of manganese quercetin 2,3-dioxygenase: quest for origins of enzyme unique nitroxygenase activity and regioselectivity.

Quercetin 2,3-dioxygenase (QDO) is an enzyme which accepts various transition metal ions as cofactors, and cleaves the heterocyclic ring of quercetin with consumption of dioxygen and release of carbon monoxide. QDO from B. subtilis that binds Mn(II) displays an unprecedented nitroxygenase activity, whereby nitroxyl (HNO) is incorporated into quercetin cleavage products instead of dioxygen. Interestingly, the reaction proceeds with high regiospecificity, i.e., nitrogen and oxygen atoms of HNO are incorporated into specific fragments of the cleavage product. A nonenzymatic base-catalyzed reaction, which occurs in pH above 7.5, yields the same reaction products. Herein, we report results of quantum chemical studies on the mechanisms of the nitroxygenase reaction of Mn-QDO. Density functional method with dispersion correction (B3LYP-D3) was applied to the Mn-QDO active site model and the reactants of the nonenzymatic reaction. Co(II)- and Fe(II)-variants of the active site were also considered. Analysis of reaction energy profiles suggests that the regiospecificity of the reaction is an inherent property of the reactants, whereas the unique reactivity of Mn-QDO, as opposed to Co- or Fe-QDO that do not catalyze nitroxygenation, stems from weak HNO binding and lack of strong preference for coordination of HNO through the nitrogen atom. Moreover, the enzyme activates quercetin through deprotonation and the proton acceptor-Glu69 needs to reorient for the reaction to proceed.