Unraveling the Crucial Role of Single Active Water Molecule in the Oxidative Cleavage of Aliphatic C–C Bond of 2,4′-Dihydroxyacetophenone Catalyzed by 2,4′-Dihydroxyacetophenone Dioxygenase Enzyme: A Quantum Mechanics/Molecular Mechanics Investigation

Abstract

2,4′-Dihydroxyacetophenone dioxygenase (DAD), a nonheme dioxygenase enzyme, shows exquisite selectivity in the aliphatic C–C bond cleavage of 2,4′-dihydroxyacetophenone (DHAP) in the presence of molecular oxygen (O2). Molecular dynamics simulations revealed the presence of a single water molecule at the active site of the enzyme. This lone water molecule is pivotal for facilitating the oxidative cleavage of the aliphatic C–C bond of 2,4′-DHAP catalyzed by DAD enzyme, as evident from the findings of our hybrid quantum mechanics/molecular mechanics (QM/MM) studies. 2,4′-DHAP is initially deprotonated through a relay proton transfer mechanism with the aid of the active site water molecule. This water molecule also actively participates in the O–O and C–C bond cleavage steps. The activated water molecule acts as catalytic acid–base species. The O–O cleavage step has been predicted to be the rate-determining step with an associated barrier of 20.3 kcal/mol calculated at the uB3LYP-D3/def2-TZVP/OPLS level of theory on the quintet spin surface. Multiple sequence alignment of the bacterial DAD enzyme has shown the evolutionary importance of the Tyr93 and Glu108 residues, in which Tyr93 acts as proton carrier and Glu108 acts as reservoir, during the relay proton transfer. Our study demonstrates the active role of water in the catalytic cycle of DAD enzyme and additionally unearthed important indirect roles of the two amino acids (Tyr93 and Glu108) in the enzymatic cycle.

Density Functional Theory Study into the Reaction Mechanism of Isonitrile Biosynthesis by the Nonheme Iron Enzyme ScoE

“…The Fe-O distance has elongated to 1.796 Å and the axial Fe-N bond to 2.204 Å. These changes mimic previous calculations on analogous reaction mechanisms well [26][27][28][29][30][31][32][33][34][35][36]. The transition state has a modest imaginary frequency of i619 cm −1 , which is well lower than typical hydrogen atom abstraction transition states that usually have values well over i1000 cm −1 [72][73].…”

“…For cysteine dioxygenase a combination of UV-Vis absorption and electron paramagnetic resonance studies implicated a short-lived oxygen-bound intermediate, which was tentatively identified as either the iron(III)-superoxo species or the bicyclic ring structure [24]. Many computational studies investigated the structure and properties of the iron(IV)-oxo species of nonheme iron dioxygenases in detail using either density functional theory model complexes or quantum mechanics/molecular mechanics methods [25][26][27][28][29][30][31][32][33][34][35][36]. These studies identified the iron(IV)-oxo as a quintet spin ground state showed it to be an efficient oxidant of substrate hydroxylation reactions.…”

Reaction Mechanism of Histone Demethylation in αKG-dependent Non-Heme Iron Enzymes

“…Notably, the oxo rotation is a continuous process in the decarboxylation starting from the Fe(III)-OO The change in the free energy of the reaction ∆Gr from IM2 to IM4 is associated with a value of -41.1 kcal/mol, indicating the overall decarboxylation reaction process is significantly exergonic ( Figure 2H), which is consistent with previous studies on non-heme iron αKG dependent enzymes 6,40 . The IM4 exhibits a 5C trigonal bipyramid geometry, consistent with the results The dominant spin states of iron in non-heme enzymes remains a long controversy 40,43,44, 64-66 , although quintet state has recently been suggested to be the ground state for the resting state and the Fe (IV)=O intermediate in other non-heme enzymes 45,46,55,56,57 , Since the ground state may change along the sequential reaction steps, here we calculated the different spin states (namely, triplet, quintet and septet spin states) of all the species involved in the decarboxylation process using QM/MM optimization. It is shown that the quintet state is consistently the ground state for all the species in the reaction process (Table S2).…”

Selective Hydrogen Atom Abstraction from Dihydroflavonol by a Nonheme Iron Center Is the Key Step in the Enzymatic Flavonol Synthesis and Avoids Byproducts

“…Optimized geometries of the triplet and quintet spin reactant complexes ( 3,5 Re) are shown in Figure 2. Similarly to previous computational studies on analogous iron(IV)-oxo complexes of nonheme iron enzymes [68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84] and experimental EPR and Mössbauer measurements of related nonheme iron dioxygenases, 30,31,[85][86][87] the quintet spin state is the ground state. Because of this molecular orbital occupation, the Fe-O distance is short in 5 Re, i.e.…”