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

Abstract

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.

On the Structure and Reaction Mechanism of Human Acireductone Dioxygenase

“…The oxidative cleavage reaction catalysed by M–ARD (M=Ni, Co or Mn), which involves C1−C2 and C2−C3 bond cleavage and CO release, bears close resemblance to the reaction catalysed by quercetin 2,3‐dioxygenase (QDO). In both cases the substrates are electron‐rich enolates with a keto group conjugated to the enol group, two adjacent C−C bonds are cleaved with release of CO and various divalent metal ions can support the enzymatic reaction . Thus, it is not surprising that the reaction mechanisms for h ARD and QDO found with the use of computational methods are very much alike (compare Figure (black arrows) and Figure S3 in the Supporting Information).…”

“…The positions of the first two maxima are fairly wellr eproduced, and in the region where there are two shoulders in the experimental spectrat he computational model shows two weak maxima. [21,22] Thus,i ti sn ot surprising that the reactionm echanisms for hARD and QDO found with the use of computational methods are very much alike (compare Figure 7 (black arrows) and Figure S3 in the Supporting Information). Unfortunately,d ue to the high dissociation constant, we were not able to prepare samples with ag reater contento ft he Fe-hARD-ACI complex, which could reveal more differences in the spectra and possibly allow for discrimination between the various models for the complex (suggested by simulationso nly).…”

“…In both cases the substrates are electron-rich enolates with ak eto group conjugated to the enol group,t wo adjacent CÀCb onds are cleaved with release of CO and various divalent metal ions can support the enzymatic reaction. [21,22] Thus,i ti sn ot surprising that the reactionm echanisms for hARD and QDO found with the use of computational methods are very much alike (compare Figure 7 (black arrows) and Figure S3 in the Supporting Information). Acireductone, which exists as am onoanion at physiological pH, binds to the divalentm etal ion in the ARD actives ite as a dianion, [5] and presumably delivers ap rotont ot he His88 residue, which most likely dissociates from the metal upon substrate binding.…”

Computational Understanding of the Selectivities in Metalloenzymes

“…Relative energies are given in kcal/mol relative to the separated complex. (B3LYP-D3/6-311G (d,p)) (Wojdyła and Borowski, 2016). Copyright 2016 Springer Nature.…”

“…Wojdyla and Borowski have performed quantum chemical cluster calculations to elucidate the reactivity and regiospecificity of the Mn-QDO (Wojdyła and Borowski, 2016). The uncatalyzed reaction was first considered with a model containing a quercetin anion and a HNO molecule.…”