Insights into the Mechanism of Aromatic Ring Cleavage of

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Insights into the Mechanism of Aromatic Ring-Cleavage of Noncatecholic Compound 2-Aminophenol by Aminophenol Dioxygenase: A QM/MM Study Geng Dong, Jiarui Lu, and Wenzhen Lai ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b00372 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 9, 2016

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Insights into the Mechanism of Aromatic RingCleavage of Noncatecholic Compound 2Aminophenol by Aminophenol Dioxygenase: A QM/MM Study Geng Dong, Jiarui Lu, and Wenzhen Lai* Department of Chemistry, Renmin University of China, Beijing, 100872, China.

AUTHOR INFORMATION Corresponding Author *Author to whom correspondence should be addressed. Email: [email protected]

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ABSTRACT: 2-Aminophenol 1,6-dioxygenase (APD) is an extradiol dioxygenase responsible for the ring cleavage of 2-aminophenol (2AP) at the position ortho to the hydroxyl substituent. To elucidate the reaction mechanism, the quantum mechanical/molecular mechanical (QM/MM) calculations were carried out. The substrate-binding mode (monodentately or bidentately) to the iron center was found to have a crucial role in the dioxygen activation. The Fe-O2 adducts with 2AP bound bidentately has a quintet ground state having a FeIII-superoxo character, while the FeO2 adducts with a monodentately bound substrate has been characterized as a substrate-radicalFeII-superoxide. Unlike other extradiol dioxygenases that cleavage catechol analogues using the superoxo moiety of the Fe-O2 adducts to attack the substrate, we found here an FeII-O(H)O intermediate formed through two sequential proton-coupled electron transfer (PCET) steps from the initial FeIII-superoxo species is responsible for the attack. Importantly, the second sphere His195 residue acts as an acid-base catalyst to mediate proton transfer (associated with electron transfer). The present study expands our understanding of the extradiol dioxygenases, especially those catalyzing the ring cleavage of noncatecholic substrates.

KEYWORDS: non-heme; oxygen activation; extradiol dioxygenase; 2-His-1-carboxylate facial triad; QM/MM

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INTRODUCTION Dioxygen activation by non-heme iron-containing oxygenases has attracted much attention due to its importance in a great variety of biological reactions.1-8 Non-heme iron enzymes containing the 2-His-1-carboxylate facial triad (two histidine residues and one aspartate or glutamate residue arranged at the vertices of one face of the iron-coordinating octahedron) represent many of the mechanistic strategies used in nature for O2-dependent metabolic processes.4,9,10 As the archetypal member of 2-His-1-carboxylate facial triad superfamily, extradiol dioxygenases that catalyze the oxidative ring cleavage of aromatic compounds have been extensively studied. By combination of structural, spectroscopic and computational approaches, the understanding of oxygen activation and reaction mechanism for extradiol dioxygenases has been significantly advanced for catecholic substrates in recent years, especially for homoprotocatechuate that cleavaged by homoprotocatechuate 2,3-dioxygenase (HPCD) (Scheme 1a). However, the mechanism for the ring opening of noncatecholic substrates that lack vicinal hydroxyl substituents remains unclear. Whether their ring opening utilizes a similar mechanism as catecholic substrates is still an open question. 2-aminophenol 1,6-dioxygenase (APD) is a key enzyme in the microbial biodegradation of nitrobenzene and p-chlornitrobenzene.11 It catalyzes the ring cleavage of 2-aminophenol (2AP) at the position ortho to the hydroxyl substituent (Scheme 1b).12,13 Recently, a significant breakthrough has been made for this enzyme by Liu et al. for solving the crystal structure of its lactone intermediate and product-bound complexes.13 The crystal structures show that the activesite iron is coordinated by two histidines (His13 and His62) and one glutamate (Glu251), which forms the so-called 2-His-1-carboxylate facial triad (see Figure 1a), as in other extradiol dioxygenases. Note that the arrangement of ligand binding to the iron center is distinctively to

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those in all previously reported structures of the extradiol dioxygenases apart from protocatechuate 4,5-dioxygenase (4,5-PCD). The deprotonated hydroxyl of bound substrate is positioned trans to a Glu in APD (see Figure 1b)13 and 4,5-PCD, rather than a His as in other extradiol dioxygenases. Another interesting feature of the crystal structures is that a secondsphere histidine (His195) is positioned close to the O2-binding site and thus has a potential to serve as an acid-base catalyst, like His200 in HPCD14,15.

Scheme 1. Reaction Catalyzed by (a) HPCD, (b) APD, and (c) 3HAO

Based on the crystal structures of APD and previous studies of HPCD, Liu et al.13 proposed a reaction mechanism for the ring cleavage of 2AP. The first step of the reaction is the substrate binding to the iron center through displacement of two coordinated water molecules. Then dioxygen is bound at the sixth coordination site (Figure 1c). Electron transfer from the substrate

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to dioxygen occurs through iron, leading to a substrate radical-FeII-superoxide (Figure 1d). After the attack of superoxo on the substrate, an FeII-alkylperoxo species (Figure 1e) is formed. A concerted Criegee rearrangement would results in O-O bond fission and C-C bond cleavage to generate a lactone intermediate (Figure 1f), which would proceed to the ring-open product (Figure 1g). Although the lactone intermediate and product-bound complexes have been captured for APD, the mechanism of dioxygen activation and the reactive oxygen species remains unknown.

Figure 1. The reaction mechanism proposed by Liu et al.13 for the ring cleavage of 2AP by APD. Contrary to the formation of the substrate radical-FeII-superoxide (Figure 1d) after dioxygen binding proposed for APD with 2AP,13 a recent theoretical study of the ring cleavage of a 2AP 5


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analogue 3-hydroxyanthrannilic acid (HAA) by an extradiol dioxygenase 3-hydroxyanthranilate 3,4-dioxygenase (3HAO) (Scheme 1c) showed that the FeIII-superoxide formed upon dioxygen binding is the active species for the attack on substrate.16 HAA differs from 2AP by having an electron withdrawing group (COO). It should be pointed out that none of the amino acids in the distal pocket of 3HAO could act as the acid-base catalysts. Our previous theoretical studies17-19 for HPCD demonstrated that the second-sphere His residue is crucial for controlling the electronic configuration of the Fe-O2 adducts and the substrate plays important roles in the dioxygen activation. Therefore, it is very interesting to explore the nature of dioxygen activation by APD and the role of second sphere His195 residue in the catalytic reaction. To elucidate the reaction mechanism of ring cleavage of 2AP, we used here hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, which are known to be able to provide important insight into mechanistic details that may not be possible via experimental means.20-27 As should be demonstrated, the substrate-binding mode (monodentately or bidentately) to the iron center has a crucial role in the dioxygen activation. The FeII-O(H)O intermediate formed through proton transfer from the substrate to proximal oxygen of the dioxygen ligand mediated by His195 (associated with electron transfer) is the reactive species for the attack on substrate. His195 plays important roles in the catalytic reaction by acting as an acid-base catalyst.

COMPUTATIONAL METHODOLOGY Setup of the System. The initial geometry of the enzyme APD was constructed from the experimental X-ray structure of a product-bound monomer (PDB file 3VSJ, subunit D)13 by replacing the product with the original substrate 2AP. The protonation states of the titratable residues were determined by the combination of pKa values predicted by the PROPKA28 program

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and visual inspection. The histidines, His13, His62, His195, His197, His199 and His261 were protonated at  position, while His16, His59, His69, His70, and His136 were protonated at  position. Glutamates (Glu) and aspartates (Asp) were used as negatively charged, while arginines (Arg) and lysines (Lys) were used as positively charged. The missing hydrogen atoms were added via the HBUILD29 module and optimized by the CHARMM force field as implemented in the CHARMM program30 (3600 steps of steepest descent for all hydrogen atoms). Then, a 16Åthick water solvent layer was constructed around the enzyme. The inner 8Å of the solvent layer was processed through a procedure involving (i) MM optimization for 2400 steps, (ii) heating to 300 K, (iii) equilibration for 3 ps, and (iv) a second optimization for 2400 steps. This procedure was repeated twice to ensure not more than 100 additional water molecules were added. The solvated system was then relaxed via energy minimization and subjected to molecular dynamics (MD) simulations at the MM level using the CHARMM force field as implemented within the CHARMM program.30 During the classical energy minimizations and MD simulations, the coordinates of the entire iron-ligating residues as well as the outer 8Å of the solvent layer were kept frozen. A random snapshot from the classical MD trajectory (1 ns) was used as starting point for the QM/MM calculations. QM/MM Methodology. QM/MM calculations were carried out using ChemShell,31 combining Turbomole32 and DL_POLY.33 An electronic embedding scheme34 was applied to include the polarizing effect of the enzymatic environment on the QM region. Hydrogen link atoms35 with the charge shift model were used to treat the QM/MM boundary. The CHARMM30 force field was used for the MM part. The QM part was treated by density functional theory (DFT) with the B3LYP functional, which has successfully been applied to study extradiol dioxygenases.17,18,36-39 Geometry optimization were performed by the hybrid delocalized internal

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coordinates (HDLC) optimizers40 a double-ζ Def-SV(P)41 basis set (B1). The energies were corrected by single point calculations using a larger all-electron basis set B2, which is DefTZVP42 for all the atoms. All minima were fully optimized without any symmetry restraints. The transition states (TSs) were located by an initial potential energy surface scans followed by full TS optimizations using the partitioned rational function optimization (P-RFO) algorithm implemented in HDLC code.40 The frequency calculations were then carried out to confirm the TSs which have a single imaginary vibrational frequency. To ascertain a contiguous energy profile of the reaction, the coordinates were scanned forward and backward until convergence was achieved for each reaction step. Our QM/MM calculations are based on a well-tested procedure, which has been found to be able to give reliable results for iron-containing metalloenzymes.18,20,43-46 The QM region comprises of the first coordination sphere residues, which are two histidines (His13 and His62), one glutamic acid (Glu251), substrate 2-aminophenol and dioxygen, and the second coordination sphere residues His195. In addition, crystal water Wat503, Wat507, Wat578, Wat617 and Wat701 are included in the QM region. Histidines were modeled as methylimidazole, Glu251 as CH3COO.

RESULTS AND DISCUSSION Based on our QM/MM calculations, the reaction mechanism for APD with 2AP was proposed in Scheme 2. We found that the reaction takes place on the quintet potential energy surface. Unlike other extradiol dioxygenases, in which the Fe-O2 adducts were suggested to be the reactive species, reaction of APD with 2AP uses an FeII-O(H)O species (4) formed through two proton coupled electron transfer (PCET) steps to attack the substrate. After that, the reaction proceeds

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via O-O bond breaking, opening of the epoxide ring, nucleophilic attack of the Fe-bound OH group on the carbon, and the scission of ring to result in a semialdehyde intermediate (9). Finally, proton transfer from His195 back to imino group occurs to form the product 2-aminomuconic 6semialdehyde (10). Significantly, the second-sphere residue His195 acts as an acid-base catalyst in the catalytic reaction of APD. The detailed discussion of the mechanism is presented in the following subsections.

Scheme 2. Suggested Mechanism for the Catalytic Reaction of APD.

The Nature of the Fe-O2 Adducts. The coupling of triplet O2 with quintet FeII gives rise to three possible spin states, triplet, quintet, and septet. Here, we considered all the three spin states. The QM/MM-optimized structures of the Fe-O2 adducts were shown in Figure 2, while the relative energies and spin densities were summarized in Table 1. Two conformations were considered here. In the first one (1 in Figure 2a), the substrate coordinates to iron in a bidentate fashion by the hydroxyl oxygen and amino nitrogen with a hydrogen bond between the NH2 group and the second-sphere His195 residue. The quintet state was found to be the most stable one, while the septet state is only 0.1 kcal/mol higher in energy. The small energy difference

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between the quintet and septet states was also found in other iron-containing extradiol dioxygenase.17,18,39 The dioxygen ligand in quintet/septet state has an O-O bond length of 1.30/1.31 Å, which points to the superoxide species (O2). The electronic structure analysis shows that two closely lying quintet and septet states arise from antiferromagnetic and ferromagnetic coupling modes between the sextet electrons in the FeIII center and sixth electron on dioxygen (see Figure 3a). The low-spin triplet state having a shorter O-O bond length of 1.24 Å lies 6.6 kcal/mol above the quintet state. Molecular orbital analysis showed that it is an FeII-O2 species with the S=2 FeII center antiferromagnetic coupled to the triplet O2 (Figure S1, SI).

Figure 2. Optimized structures for the Fe-O2 adducts. (a) 1 and (b) 1′. Key bond lengths are given in Å.

Table 1. Relative Energies and Spin Densities of the Fe-O2 Adducts of APD with 2AP. E species (kcal/mol) 5 1 0.0 7 1 0.1 3 1 6.6 5 1′ 8.3 7 1′ 5.2 3 1′ 6.5

spin densities Fe O2 2AP 4.17 -0.82 0.44 4.15 1.14 0.52 3.60 -1.73 0.07 3.88 -0.93 0.90 3.89 1.06 0.93 3.79 -0.98 -0.90

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Figure 3. Molecular orbital picture for (a) 5,71 and (b) 5,71′.

In the other conformation (1′ in Figure 2b), the substrate is monodentately bound to iron through the hydroxyl oxygen. The amino group lies in the plane of the substrate ring. The hydrogen bonding to His195 is then absent. As shown in Table 1, in 71′, the spin on Fe, dioxygen and substrate are 3.89, 1.07 and 0.93, respectively. 71′ can be described as S-FeII-O2 since all of the unpaired electrons have parallel spins (see Figure 3b). In 51′, the spin on Fe, dioxygen and substrate are 3.88, -0.93 and 0.90 respectively. The opposite spin on the substrate and dioxygen in 51′ (S-FeII-O2, as shown in Figure 3b) suggested that it could be the reactive state for further reaction. In 31′, the spins on dioxygen and substrate are both antiferromagnetically coupled with that on the iron (S-FeII-O2). Our results clearly showed that all three spin states of 1′ are hexaradicaloids having a substrate radical-FeII-superoxo (S-FeII-O2) character, in contrary to the FeIII-superoxo character of

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1. It seems possible that the monodentate binding

mode destabilizes the  orbital of substrate (sub in Figure 3b) due to a weaker interaction 11


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between the substrate and the iron center, as compared to the bidentate binding. As such, the electron transfer from the substrate to the iron center may easily to occur and results in a substrate-radical FeII-superoxo species. Energetically, 71′/51′/31′ lies 5.2/8.3/6.5 kcal/mol above 5

1. Therefore, the FeIII-superoxo with bidentately bound substrate (51) is most stable. The

different electronic structure of the Fe-O2 adducts with monodentately and bidentately bound substrate suggested that the substrate-binding mode has an important role in the dioxygen activation of APD.

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Figure 4. Energy profiles for formation of the alkylperoxo intermediates (a) 2 and 2′; (b) 5; and (c) 11 on the quintet state surface. Relative energies (in kcal/mol) are presented at the UB3LYP(B2)/MM level.

Finding the Reactive Species. To study the reactivity of the Fe-O2 adducts, the attack of the distal oxygen (Od) of its dioxygen ligand on the hydroxyl carbon (C1) of 2AP was considered using Od-C1 bond as the reaction coordinate. We found here that only the quintet state is catalytically relevant for APD. This finding is similar with previous theoretical studies for many other iron-containing extradiol dioxygenase.16-18,38,39 Starting from 31 and 71, the energies keep increasing when shortening the Od-C1 bond and the energy scan (Figure S3 in the SI) could not reach any alkylperoxo species, suggesting that these two states are unreactive. 31′ and 71′ could not be active species since the electron on the substrate and dioxygen have parallel spins. Since the quintet state is the reactive state, we therefore only considered the quintet state in the subsequent calculations. The energy profiles for the formation of various alkylperoxo intermediates on the quintet surface were summarized in Figure 4. In the case of 51 (FeIII-O2), the attack of the superoxo group on C1 of substrate ring results in an alkylperoxo bridge species (52) with the high spin (S=5/2) FeIII antiferromagnetic coupled to the substrate radical. However, our QM/MM calculations showed this process is very sluggish due to a high energy barrier of 35.3 kcal/mol and a large endothermicity of 29.3 kcal/mol (see Figure 4a). Starting from 51′ (S-FeII-O2), the attack of the superoxo on the substrate leads to a bridge species (52′), in which the spin on the substrate and dioxygen are quenched. The computed barrier for this attack (5TS1′) with respect to the reactant (51′) is 14.2 kcal/mol and increases to 22.5 kcal/mol with respect to the most stable Fe-O2 adducts (51).

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Although 51′ has higher reactivity than 51 (Figure 4a), we found it is not necessary for substrate to change its bidentate binding mode to the O-coordinate monodentate mode for the subsequent attack on the substrate. An alternative pathway for the formation of the alkylperoxo species was found (1 → 3 → 4 → 5 in Scheme 2), in which the reactive oxygen species is a quintet Fe-O(H)O species (4). The formation of 4 involves proton transfer from the NH2 group of 2AP to the proximal oxygen of the O2 ligand (mediated by His195) as well as the shifts of two electrons to two different receptors. This process was found to occur stepwise via two proton coupled electron transfer (PCET) steps. Firstly, the proton transfer from the NH2 group of the substrate to His195 is coupled with electron transfer from substrate to Fe, leading to the formation of a substrate radical-FeII-superoxo species (3). Subsequently, the proton transfer from His195 to the proximal oxygen of the O2 ligand occurs concerted with electron transfer from the substrate to the dioxygen ligand, resulting in 4. Clearly, His195 plays an important role by acting as an acid-base catalyst in these two steps. As shown in Figure 4b, the first PCET step has a barrier of 10.0 kcal/mol while the second one has a lower barrier of 2.3 kcal/mol. In 4, the spin population is 3.94 on iron, a typical value for FeII. The spin on dioxygen and substrate is very small, being -0.22 and 0.15, respectively, suggesting that electron transfer from the substrate to the dioxygen is not fully complete. To get more insight into the electronic structure of 4, a detailed analysis of the frontier molecular orbitals was made. Figure 5 shows the molecular orbital (MO) diagram of 4. The combination of the Fe dxz orbital with the π*ip (inplane orbital defined by the Fe-O-O plane) is observed, forming two new orbitals dxz ± π*ip. An interesting observation is that the HOMO of the substrate π system is mixed with the dxz + π*ip orbital (πsub in Figure 5), resulting from the covalent interaction among the substrate, iron, and the O(H)O moiety. This is consistent with a significantly shorter Od-C1 bond distance in 4 as

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compared to that in 1 and 1′ (2.62 Å vs. 3.61 and 3.81 Å). Electronic natural of 4 seems being between the two limiting cases, FeII-O(H)O and SQ-FeII-[O(H)O]. However, the calculated spin contamination is very small (less than 4%). The small spin densities on the substrate and dioxygen as well as a small natural population of the substrate πsub orbital (with a occupancy number of 0.11) suggested that SQ-FeII-[O(H)O] only has a small contribution to the electronic structure. Therefore, 4 can be assigned as FeII-O(H)O.

Figure 5. Molecular orbital diagram of quintet 4. Occupancy numbers are marked in red.

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Figure 6. Optimized structures for the transition states involved in the formation of alkylperoxo species on the quintet surface. Key bond lengths are given in Å.

Starting from 4, the attack on the substrate ring has a tiny energy barrier of 0.1 kcal/mol to generate alkyl hydroperoxo species (5). The optimized transition state (TS4) is shown in Figure 6. From the bond lengths one can notice that this transition state has a very early character, e.g. the Od-C1 bond length is 2.44 Å compared to a value of 1.67 Å in TS1. The distance between the iron and proximal oxygen increases to 2.43 Å, indicating that the Od-C1 bond formation is accompanied by the Fe-Op bond breaking during the process 4 → 5. In addition, the nucleophilic attack on the substrate by 3 was studied. However, the energy scan from 3 shows the energy drops abruptly when the Od-C1 bond distance reaches 2.5 Å (Figure S6). This drop was ascertained to result from proton transfer from His195 to the superoxo moiety, and then the energy scan collapse to the potential energy surface of 4, indicating that the PT precedes the OdC1 bond formation. This behavior is different from what had been found for the widely studied extradiol dioxygenase HPCD, in which the formation of Od-C1 bond occurs concerted with a proton transfer from the second sphere His residue to the proximal oxygen.17,18,39 Inspection of Figure 4b shows that the formation of alkyl hydroperoxo species 5 has a total barrier of 10.0 kcal/mol, which is much lower than that for 2 and 2′ (35.3 and 22.5 kcal/mol). 16


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Figure 7. Spin natural orbital (SNO) that has a negative occupancy (Occ) number in 11.

On the other hand, the formation of the hydroperoxo species (Fe-OOH) by proton transfer from His195 to the distal oxygen was also considered, since it has been found to be more stable than Fe-O(H)O in both heme47-49 and non-heme17,18,39 iron enzymes. Our calculations show that the formation of the Fe-OOH (11) species has a higher barrier (4.8 kcal/mol in Figure 4c), as compared to that for Fe-O(H)O formation (2.3 kcal/mol for 3 → 4 in Figure 4b). The Fe-OOH (11) is indeed very stable in APD, being 9.6 kcal/mol lower in energy than the Fe-O(H)O species. The spin population in 11 is 4.17, 0.19, -0.55 for iron, dioxygen and the substrate, respectively. To gain more insight into the electronic configuration of Fe-OOH, the spin natural orbitals (SNOs) were calculated from symmetry broken calculation using Gaussian09.50 The orbital with an occupancy number of -0.83 (see Figure 7a) clearly shows that 11 has a substrate radical character, while other five SNOs with positive occupancy numbers (Figure S3 in the SI) suggested that all iron d orbitals were singly occupied. Therefore, 11 can be described as FeIIIOOH with a substrate radical. Such species has been trapped in the His200Cys mutant of HPCD

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with the native substrate and was proposed to be involved in the catalytic pathway.51 As such, the reactivity of 11 was examined. However, the calculated reaction barrier for the attack on the substrate is of 26.1 kcal/mol, significantly higher than the preferred pathway from the Fe-O(H)O species. Therefore, in APD, the Fe-OOH 11 is unlikely to be involved in the reaction mechanism.

Figure 8. Superposition of the QM/MM optimized semialdehyde product (red) and the crystal structure of product complex (blue).

Reaction Mechanism for Semialdehyde Product Formation. After formation of 5, the O-O bond is cleaved to form a substrate epoxide (6) via the transition state TS5, which lies 16.9 kcal/mol above the zero level (see Figure 9). 6 is an epoxide-bound FeIII-OH species, having high-spin (S=5/2) FeIII antiferromagnetically coupled to a substrate-based radical. Subsequently, the epoxide ring of 6 is cleaved, and an intermediate (7) that contains a seven-membered ring (lactone) is formed. 7 was found to contain high-spin FeIII antiferromagnetically coupled to a lactone radical. Such species was also found in some other extradiol dioxygenases, e.g., 18


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HPCD17,18,38 and 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)36. It should be pointed out that 7 could not be viewed as the product of Baeyer-Villiger reaction due to the radical character on the lactone. The lactone radical can be attacked by FeIII-bound hydroxyl anion to generate 8, in which the metal center has now re-reduced to its initial Fe(II) oxidation state suggested by the spin population of 3.81. This step is an energy-favored process with a barrier of 7.7 kcal/mol and an exothermicity of 16.5 kcal/mol. The ring-opening of the seven-membered ring, accompanied by the proton transfer from the hydroxyl group to His195 proceeds with a barrier of 10.7 kcal/mol to yield a semialdehyde intermediate (9) having a deprotonated amino group. This ring opening is endothermic by 3.5 kcal/mol. Finally, proton transfer (PT) from His195 to the NH group occurs with a small barrier of 1.7 kcal/mol and an exothermicity of 12.7 kcal/mol, resulting in the formation of the 2-aminomuconic 6-semialdehyde product (10). It can be seen from Figure 8 that our QM/MM optimized 10 overlaps with the X-ray structure very well, except that the hydroxyl oxygen of the optimized semialdehyde product is somehow out of the Fe-N2APOGlu251-NHis62 plane. This may be because the QM/MM optimized product is a pentacoordinated Fe complex, while in the crystal structure of the final product, the additional site is filled by a water molecule. From Figures 4b and 9, it can be seen that the rate-limiting step is the O-O bond cleavage step having an accumulated barrier of 16.9 kcal/mol with respect to 51.

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Figure 9. Energy profiles for formation of the semialdehyde product complex 10 on the quintet surface. Relative energies (in kcal/mol) are presented at the UB3LYP(B2)/MM level.

CONCLUSION The reaction mechanism of oxidative cleavage of 2-aminophenol by 2-aminophenol 1,6dioxygenase (APD) was investigated using the QM/MM approaches. The substrate-binding mode (monodentately or bidentately) was found to have a crucial role in the dioxygen activation. The Fe-O2 adducts with 2AP bound bidentately has an FeIII-superoxo character. The monodentate binding (via the hydroxyl oxygen) facilitates the electron transfer from the substrate to the iron center. A substrate radical-FeII-superoxide was then formed. Importantly, our calculations demonstrated that the FeII-O(H)O intermediate (4) is responsible for the attack on

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the substrate, where the substrate is two-electron oxidized and the dioxygen is two-electron reduced. This finding is different from other extradiol dioxygenases, in which the Fe-O2 adducts (FeIII-superoxo or substrate radical-FeII-superoxo) is the reactive species. The formation of 4 from the initial Fe-O2 adducts involves two-step PCET processes: (i) proton transfer from the amnio group of the substrate to His195 coupled with electron transfer from the substrate to Fe; (ii) proton transfer from His195 to the proximal oxygen of the dioxygen ligand coupled with electron transfer from the substrate to the dioxygen. Subsequent O-O bond cleavage was found to be the rate-limiting step with an accumulated barrier of 16.9 kcal/mol in the proposed mechanism, leading to an epoxide intermediate. After the epoxide intermediate is formed, the reaction proceeds with C1-C6 bond cleavage, attack of Fe-bound OH on C1, ring-opening through C1-Od bond breaking, and proton transfer from His195 to the NH group, to yield the final semialdehyde product (see Figure 9). During the catalytic reaction of APD, a distal residue His195 was found to play important roles by acting as an acid-base catalyst. Undoubtedly the mechanistic insight provided will help us to better understand the extradiol dioxygenases, especially those catalyzing the ring cleavage of noncatecholic substrates.

ASSOCIATED CONTENT Supporting Information QM/MM optimized region, QM/MM energy profiles for the reactions studied, energies and Cartesian coordinates of the key species. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION

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Corresponding Author *Email: [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work is supported by the Fundamental Research Funds for the Central Universities, the Research Funds of Renmin University of China (program No. 16XNLQ04) and the National Natural Science Foundation of China (No. 21203245). REFERENCES (1)

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TOC GRAPHICS

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Insights into the Mechanism of Aromatic Ring Cleavage of

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