Reaction Mechanism of Homoprotocatechuate 2,3-Dioxygenase with

Article pubs.acs.org/JPCB

Reaction Mechanism of Homoprotocatechuate 2,3-Dioxygenase with 4‑Nitrocatechol: Implications for the Role of Substrate Geng Dong and Wenzhen Lai* Department of Chemistry, Renmin University of China, Beijing 100872, China S Supporting Information *

ABSTRACT: The reaction mechanism of the dioxygen activation by homoprotocatechuate 2,3-dioxygenase (HPCD) with the substrate 4nitrocatechol was investigated by quantum mechanical/molecular mechanical calculations. Our results demonstrated that the experimentally determined side-on iron−oxygen complex in crystallo is a semiquinone substrate radical (SQ•)−FeIII−hydroperoxo species, which could not act as the reactive species. In fact, the FeIII−superoxo species with a hydrogen bond between His200 and the proximal oxygen is the reactive oxygen species. The second-sphere His200 residue was found to play an important role in manipulating the orientation of the superoxide in the Fe−O2 adduct for the further reaction. The rate-limiting step is the attack of the superoxo group on the substrate with a barrier of 17.2 kcal/mol, in good agreement with the experimental value of 16.8 kcal/mol. The reaction mechanism was then compared with the one for HPCD with its native substrate homoprotocatechuate studied recently by the same methods, in which a hybrid SQ•−FeII−O2•−/FeIII− O2•− was suggested to be the reactive species. Therefore, our studies suggested that the substrate plays important roles in the dioxygen activation by HPCD.

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(DHPA),12,15 and 4-sulfonylcatechol (4SC),16 were used to trap and characterize the reaction intermediates to gain insights into the reaction mechanism of the extradiol dioxygenase. Notably, the X-ray crystal structure of three intermediates, an Fe−O2 adduct with a side-on conformation, an alkylperoxo bridge species, and the Fe−semialdehyde ring-opened product, were trapped in crystallo reaction of wild-type (WT) HPCD with 4NC.17 In particular, the side-on Fe−O2 adduct was assigned to the semiquinone substrate radical−FeII−superoxo species, SQ•−FeII−O2•−, due to the distortion of the substrate ring. However, spectroscopic evidence shows that short-lived FeIII−superoxo species is likely formed in the reaction of O2 with the enzyme−substrate complex for the His200Asn (H200N) mutant of Fe-HPCD with 4NC.9 To rationalize these experimental observations, it was proposed that the FeIII− superoxo species was initially formed upon oxygen binding, followed by electron transfer from the substrate to the metal center to generate the reactive oxygen species, SQ•−FeII− O2•−.9,18 Several theoretical studies have been carried out for better understanding the oxygen activation mechanism of HPCD.19−21 An early DFT study by Siegbahn et al. found that SQ•−FeII−O2•− (with ferromagnetically coupling between the metal and the superoxo radical) is reactive species for HPCD with native substrate HPCA.19 However, a DFT study

INTRODUCTION Dioxygen activation by mononuclear nonheme iron enzymes (MNHEs) has attracted much attention due to its importance in a wide variety of metabolically important reactions.1−5 Homoprotocatechuate 2,3-dioxygenase (HPCD), an MNHE from Brevibacterium f uscum,6 is one of the most studied nonheme FeII-containing extradiol dioxygenase. It opens the aromatic ring of its substrate and incorporates two atoms of dioxygen into the product (Scheme 1). The FeII active site is Scheme 1. Overall Reaction Catalyzed by HPCD

ligated by two histidines (His155 and His214) and one glutamate (Glu267), which forms a so-called 2-His-1carboxylate facial triad occupying one face of the metal coordination sphere, a motif found in many MNHEs.7,8 Dioxygen activation mechanism of HPCD has long been the subject of controversy. Because of the very rapid reaction of the native enzyme using its native substrate, homoprotocatechuate (HPCA), the Fe−O2 adduct, has not yet been detected and characterized. Instead, the slow reacting substrate analogues, such as 4-nitrocatechol (4NC),9−14 dihydroxyphenylacetate © 2014 American Chemical Society

Received: December 2, 2013 Revised: January 15, 2014 Published: January 27, 2014 1791

dx.doi.org/10.1021/jp411812m | J. Phys. Chem. B 2014, 118, 1791−1798

The Journal of Physical Chemistry B

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by Neese et al. did not find the SQ•−FeII−O2•− species but found that FeIII−O2•− is the reactive species for HPCD with substrate HPCA or 4NC.20 Furthermore, our recent quantum mechanical/molecular mechanical (QM/MM) study21 indicated that the hybrid FeIII−O2•−/SQ•−FeII−O2•− species, formed from the FeIII−O2•− species by a mere hydrogenbond reorientation of the His200 from interacting with the oxygen atom of the substrate ring to hydrogen bonding with the proximal oxygen of the dioxygen moiety, is the reactive oxygen species for HPCD with HPCA, which agrees quite well with the experimental proposals.9,18 Although the reaction of HPCD with native substrate is widely studied theoretically, the mechanism is still not very clear when alternative substrate 4NC was used. Some questions remain unanswered: Does SQ•−FeII−O2•− species exist when HPCD reacts with the electron-poor substrate 4NC? What is the reactive oxygen species? What is the role of active site His200? To answer these questions, we investigated the reaction mechanism of HPCD with 4NC by means of the QM/MM approach.

B2 on the B3LYP/B1-optimized structures using a variety of functionals with different fraction of HF, including BLYP(0%), 30,31 TPSSh(10%), 40 B3LYP*(15%), 35 and BHLYP(50%).41 These calculations were done with Gaussian 09,42 which allows us to use various DFT methods. QM Region. The QM region, shown in Figure 1, comprises the first-coordination sphere residues, which are two histidines

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COMPUTATIONAL METHODOLOGY Setup of the System. The initial coordinates were taken from the X-ray crystal structure (PDB code: 2IGA,17 subunit C) wherein HPCD was bound with 4NC and dioxygen. The protonation states of the titratable residues were determined by the combination of the pKa values predicted by the PKOPKA22 program and visual inspection (for the detailed protonation scheme, see the Supporting Information (SI)). The missing hydrogen atoms were added via the HBUILD23 module and optimized by the CHARMM force field, as implemented in the CHARMM program24 (3600 steps of steepest descent for all hydrogen atoms). Then, a 16 Å thick water solvent layer was constructed around the enzyme. The inner 8 Å 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. QM/MM Methodology and Software. QM/MM calculations were carried out using ChemShell,25 combining Turbomole26 and DL_POLY.27 An electronic embedding scheme28 was applied to include the polarizing effect of the enzymatic environment on the QM region. Hydrogen link atoms29 with the charge shift model were used to treat the QM/MM boundary. Density functional theory (DFT) was used for the QM part, while the CHARMM24 force field was used for the MM part. Geometry optimization was preformed using the hybrid B3LYP30−32 functional in combination with a double-ζ SV(P)33 basis set (B1). The energies were corrected by single-point calculations using a larger all-electron basis set B2, which is Def-TZVP34 for all atoms. All minima were fully optimized without any symmetry restraints. The transition states (TSs) were determined as the highest point on the potential energy surface along the reaction coordinates, which were scanned with a small increment of 0.02 Å near the TSs. Being attentive to the often expressed difficulty in predicting the energetic ordering of the spin-states in transition-metal complexes with DFT,35−39 we tested the effect of other functionals on the relative energies of the Fe−O2 adducts. It has been proposed that the reliability of the DFT methods in describing the relative energies could be sensitive to the amount of exact Hartree−Fock (HF) exchange included.35 Therefore, we performed the single-point calculations with larger basis set

Figure 1. QM region used in this work.

(His155 and His214), one glutamic acid (Glu267), and 4NC, and the second-coordination sphere residues (His200, His248, Trp192, Asn157, Tyr257, and Arg243). Histidines were modeled as methylimidazole, Glu267 as CH3COO−, Trp192 as indole, Asn157 as CH3CONH2, Arg243 as methylguanidinium, and Tyr257 as phenol. The dianionic form of the 4NC substrate was adopted, as suggested by spectroscopic absorption data.9 Optimized QM/MM Region. During the QM/MM geometry optimizations, the active region to be optimized included the core region (iron−oxygen and its first-coordination sphere) and all residues and water molecules within 6 Å from the core region (see the SI for details). Mössbauer Calculations. The Mössbauer isomer shift (δ) and quadrupole splitting (ΔEQ) were calculated with the program ORCA43 using single-point B3LYP calculations on the corresponding QM/MM-optimized geometries with the inclusion of the MM point charges. In these calculations, iron was described by the triply polarized core properties basis set CP(PPP),44 while the other atoms were described by the defSV(P)33 basis set with the inner s functions left uncontracted. The isomer shift was evaluated from the electron density at the iron nucleus.45

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RESULTS AND DISCUSSION Nature of the Fe−O2 Adducts: FeIII−O2•− or SQ•−FeII− O2•−? It is well known that the coupling of triplet O2 with quintet FeII gives rise to three possible spin states, triplet, quintet, and septet. Here all three spin states are considered. Table 1 summarizes the electronic structure, relative energies, and spin densities of the various Fe−O2 adducts. Three Fe−O2 adducts (A1−A3) having different hydrogen-bonding orientations were located. In A1, the second-sphere His200 residue forms a hydrogen bond with the OC2 atom of the 4NC. While in A2, His200 forms two hydrogen bonds with the substrate OC2 and the proximal oxygen of the Fe−O2 moiety. In A3, His200 is hydrogen bonded to the distal oxygen of the Fe−O2 1792



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Table 1. Electronic Structure, Relative Energies, and Spin Densities of the Fe−O2 Adducts of HPCD with the Substrate 4NC spin densities species 5

A1 A1 3 A1 3 A1′ 5 A2 7 A2 3 A2 3 A2′ 3 A2″ 5 A3 5 A3′ 7 A3 3 A3 3 A3′ 7

configuration

ΔE (kcal/mol)

Fe

O1

O2

OC1 + OC2

sub

dxy1dyz1dxz1σ*z21σ*x2−y21π*op1 dxy1dyz1dxz1σ*z21σ*x2−y21π*op1 dxz1π*op1 1 dyz dxz1σ*z21π*op1 dxy1dyz1dxz1σ*z21σ*x2−y21π*ip1 dxy1dyz1dxz1σ*z21σ*x2−y21π*op1 dxz1π*op1 1 dyz dxz1σ*z21π*op1 ϕ4NC1dyz1dxz1σ*z21σ*x2−y21π*op1 dxy1dyz1dxz1σ*z21σ*x2−y21π*op1 ϕ4NC1dyz1dxz1σ*z21σ*x2−y21π*op1 dxy1dyz1dxz1σ*z21σ*x2−y21π*op1 dxz1π*op1 dyz1dxz1σ*z21π*op1

0.0 −0.5 9.3 9.0 2.3 1.5 12.3 13.5 13.4 0.2 12.9 −0.6 9.3 8.7

4.20 4.18 1.03 3.34 4.15 4.19 0.98 3.06 3.71 4.21 3.96 4.18 1.02 3.37

−0.29 0.61 0.41 −0.73 −0.28 0.48 0.34 −0.48 −0.37 −0.32 0.30 0.60 0.41 −0.74

−0.59 0.62 0.54 −0.76 −0.57 0.61 0.57 −0.69 −0.56 −0.52 0.50 0.61 0.54 −0.78

0.19 0.28 0.02 0.07 0.34 0.33 0.07 0.07 −0.33 0.30 −0.34 0.29 0.03 0.07

0.34 0.33 0.04 0.07 0.41 0.43 0.12 0.06 −0.81 0.35 −0.84 0.35 0.05 0.07

Figure 2. Optimized structures of the quintet Fe−O2 adducts for HPCD with 4NC. All distances are in angstroms while angles are in degrees.

found to lie at least 8.5 kcal/mol higher than the corresponding quintet states (see Table 1). Besides the FeIII−O2•− species, two SQ•−FeII−O2•− species were also located (5A3′ and 3A2″ in Table 1). They are both hexaradicaloids. For 5A3′, the S = 2 FeII center is AF coupled to the semiquinone radical but ferromagnetically coupled to the superoxo radical, while for 3 A2″, the S = 2 FeII center is AF coupled to the semiquinone radical and also the superoxo radical. However, these two states are much higher (>11 kcal/mol) in energy than the corresponding quintet states. It is also interesting to note that in 5A1 and 5A3, the Fe−O−O−C2 dihedral angle is close to 0°, while in 5A2, the Fe−O−O−C1 dihedral angle is close to 0° (see Figure 2). As should be demonstrated later, the dioxygen

moiety. Figure 2 shows the QM/MM-optimized geometries of the quintet Fe−O2 adducts. It was found that the lowest quintet state (5A1−5A3 in Figure 2a−c) has FeIII−O2•−↓ character with a high-spin S = 5/2 FeIII center antiferromagnetically (AF) coupled to a superoxo radical (see Figure 3). 5A1 has the lowest energy among all quintet states, being 2.3/0.2 kcal/mol more stable than 5A2/5A3. In addition, 5A1−5A3 can convert to each other very easily by reorientation of the His200. The barriers for 5A1 → 5A2 and 5A2 → 5A3 are 2.7 and 0.6 kcal/mol, respectively. The septet states (7A1, 7A2, and 7A3), which all have FeIII−O2•−↑ character with a high-spin S = 5/2 FeIII center ferromagnetically coupled to a superoxo radical, are only 0.5, 0.8, and 0.8 kcal/mol lower than the corresponding quintet states (5A1, 5A2, and 5A3). The low-spin triplet states were 1793



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BLYP.21,46 As such, BLYP is not be able to describe the electronic structure of the Fe−O2 adducts well, as pointed out in our previous QM/MM study for HPCD with HPCA.21 Therefore, we still chose the widely used B3LYP functional in the subsequent parts of this paper. It should be noted that all of the previously mentioned Fe− O2 adducts have end-on conformation. However, the X-ray structure of HPCD with 4NC showed a side-on bonding mode of the O2 moiety to the Fe center. In fact, when searching the possible side-on Fe−O2 adduct, we found that the proton transfer from His200 to the dioxygen occurs spontaneously to form a side-on semiquinone substrate radical-Fe-hydroperoxo species (5H1). Fe-Hydroperoxo Species. As mentioned above, a side-on semiquinone substrate radical-Fe-hydroperoxo species (5H1 in Figure 4a) was located. It was found to be 12.3 kcal/mol higher in energy than 5A2. In 5H1, the spin population is 4.17, 0.44, and −0.69 for iron, dioxygen, and the substrate, respectively. It can be seen from Figure 4b that our QM/MM-optimized 5H1 overlaps with the X-ray structure of the Fe−O2 adduct very well. Therefore, our results suggested that the experimentally determined side-on iron−oxygen complex is a SQ•−FeIII− hydroperoxo species rather than a SQ•−FeII−O2•− species. In addition, we found that an end-on hydroperoxo species 5 H2 (Figure 4c) can be formed via proton transfer from His200 to the distal oxygen from 5A3′ with a barrier of 1.8 kcal/mol. In 5 H2, the spin population is 4.19, 0.35, and −0.56 for iron, dioxygen, and the substrate, respectively. Energetically, 5H2 is 6.6 kcal/mol lower than 5H1. What is the Reactive Oxygen Species? In previous theoretical studies of HPCD, only the quintet surface was found to be catalytically relevant.20,21 As such, we focus here on the quintet-state mechanism. To find out the reactive oxygen species, we studied the attack of the O2 moiety on the C1 of the substrate starting from the quintet Fe−O2 adducts and hydroperoxo species. It was found that starting from 5A1, 5 A3, 5A3′, and 5H2, the energy scans collapse to the potential energy surface of 5A2 before reaching the TS (see Figure S2 in the SI). The geometric analysis shows that at each point of such collapse there is a hydrogen bond between His200 and the proximal O as in 5A2. In the case of 5H1, we found that the O1−Fe bond was broken when shortening the O2−C1 bond, and no bridge species was formed. Therefore, 5A2 has a proper orientation for the further attack on the substrate. The protonated His200 plays a crucial role in manipulating the orientation of the superoxide to attack the substrate. The TS for this attack (5TS1) lies 17.2 kcal/mol higher than 5A1. The so-generated 5B is a tetraradicaloid FeII-alkylperoxo bridge

Figure 3. Singly occupied spin natural orbitals for the lowest quintet state of the Fe−O2 adducts (5A1−5A3).

moiety in 5A2 has perfect orientation to react with the substrate at the correct ring carbon (C1). It should be pointed out that the present DFT(B3LYP)/MM study for HPCD with 4NC predicted the high-spin septet state to be the ground state, although the energy differences between the septet and quintet states are within 1.0 kcal/mol. Similar behavior was also found in the case of HPCA from the previous theoretical studies.19−21 However, EPR and Mössbauer experiments indicated that the Fe−O2 adduct in the H200N mutant of HPCD with 4NC has a quintet ground state.9 That is to say, B3LYP fails to predict the correct spin ground state for the Fe− O2 adduct of HPCD. We therefore examined the quintet-septet gap of the Fe−O2 adducts by using four different functionals (BLYP, TPSSh, B3LYP*, and BHLYP). The energetics results are summarized in Table 2. It was found that B3LYP* and BHLYP functionals incorrectly predict a septet ground state, as B3LYP did. TPSSh predicts a quintet ground state for A1 and A2 and a septet ground state for A3. Among all DFT functionals examined, only BLYP predicts a quintet ground state for all three Fe−O2 adducts, consistent with experimental results. Although the BLYP data are our “best” predictions for the spin-state energetics of the Fe−O2 adducts, BLYP significantly underestimates the spin density on both Fe and the O2 moiety for the quintet Fe−O2 adducts with values of 3.62 to 3.70 and −0.19∼−0.43, respectively (see Table S2 in the SI). Such spin density values have been previously concluded to be indicative of a peroxo-like species in

Table 2. Relative Energies (ΔErel) and Quintet−Septet Gaps (ΔESQ) for A1, A2, and A3 Species species ΔErel (kcal/mol)

5

A1 A1 5 A2 7 A2 5 A3 7 A3 A1 A2 A3 7

ΔESQ (kcal/mol)a

a

BLYP

TPSSh

B3LYP*

B3LYP

BHLYP

0.0 3.2 −0.1 4.6 −2.1 3.2 3.2 4.7 5.3

0.0 0.1 1.2 1.8 0.4 0.1 0.1 0.6 −0.3

0.0 −0.4 1.6 1.5 0.3 −2.1 −0.4 −0.1 −2.4

0.0 −0.5 2.3 1.5 0.2 −0.6 −0.5 −0.8 −0.8

0.0 −0.1 4.4 2.4 0.2 −0.2 −0.1 −2.0 −0.4

ESQ = E(septet) − E(quintet). 1794



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Figure 4. (a) Optimized structure of 5H1. (b) Superposition of the QM/MM-optimized 5H1 (red) and the crystal structure of the Fe−O2 adduct (blue). (c) Optimized structure of 5H2. All distances are in angstroms, while angles are in degrees.

species with four unpaired electrons on iron. A similar FeIIalkylperoxo bridge species was also found when HPCA was used as the substrate in the previous studies.19−21 Moreover, the optimized geometry of 5B (Figure 5) is in agreement with the experimental X-ray structure (PDB code: 2IGA,17 subunit B).

deficient substrate 4NC, while for electron-rich substrate HPCA it is the hybrid SQ•−FeII−O2•−/FeIII−O2•− species. We also noted that the calculated barrier for the attack of the substrate 4NC by the reactive species is much higher than that for HPCA (1.3 kcal/mol in ref 21). This makes sense because the oxidation of the electron-deficient substrate is more difficult and the partial electron transfer already occurred before the reaction for HPCA. Second, proton transfer from His200 to the proximal oxygen atom of superoxo moiety occurs simultaneously during the attack on the substrate HPCA, but this did not happen in the case of 4NC, where the proton transfer occurs in the latter step, that is, the O−O bond breaking of the alkylperoxo species. Third, for 4NC, the O−O breaking results in the formation of an epoxide species rather than the unprotonated gem-diol (with oxo radical)-bound FeIII−OH species, which is formed for HPCA. Fourth, for 4NC, the process of L → P proceeds via a concerted mechanism (the attack of OH radical on C1 coupled to ring-opening), while for HPCA it is stepwise. The analysis of the mechanism differences derived from the usage of different substrates indicated that the substrate plays key roles in dioxygen activation and reaction cycle of HPCD. Mössbauer Parameters. The Mössbauer isomer shift and quadrupole splitting were calculated for the intermediates in the reaction cycle of HPCD with 4NC. The results are summarized in Table 3 and compared with the available experimental data9,47 for better understanding the reaction mechanism. The calculated δ for 5A1−5A3 are quite close to the experimental value of 0.50 mm/s for the Int-19 in the H200N mutant with 4NC substrate (H200N-4NCInt‑1), which was assigned as a quintet FeIII-superoxo. Although the corresponding ΔEQ values of 5A1 and 5A2 are ∼1 mm/s larger, this is considered to be acceptable for a nonheme system.48 The calculated δ value of 0.99 mm/s for 5B is close to the measured WT-HPCAInt‑1 for the WT HPCD with HPCA, whose δ is 1.08 mm/s.47 Moreover, the calculated ΔEQ and δ values of other species (5H1, 5H2, 5E, 5L, 5P) for WT-4NC are quite close to the corresponding values for WT-HPCA obtained in our previous QM/MM study.21

Figure 5. Optimized structure of 5B. The experimental values of the selected bond lengths were taken from the X-ray structure.17 All distances are in angstroms.

Complete Energy Profile. The complete mechanism was shown in Figure 6a. After the formation of the FeII-alkylperoxo species 5B, the O−O bond is cleaved to form the epoxidebonded FeIII−OH species (5E) with a barrier of 8.4 kcal/mol. Subsequently, the C1−C6 bond of the epoxide was broken to generate lactone (5L). Finally, the attack of OH radical on C1 occurs, accompanied by ring-opening of the lactone ring (C1− O1 bond breaking), to yield the semialdehyde product. It can be seen from Figure 6b that the formation of the bridge species is the rate-limiting step. The calculated barrier for this step (17.2 kcal/mol relative to 5A1) agrees well with the estimated one (16.8 kcal/mol) from the experiment (rate constant of 0.31 ± 0.03 s−1 at 4 °C).14 Comparison of the Reaction Mechanism (4NC versus HPCA). By comparison with our previous QM/MM study for HPCA,21 the differences in the reaction mechanism of HPCD with the native substrate HPCA and alternative substrate 4NC were found in the following four aspects. First, the reactive species is different, which is FeIII−O2•− for the electron-

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CONCLUSIONS Our QM/MM study demonstrated that the FeIII-superoxo species is the reactive oxygen species in HPCD with electrondeficient substrate 4NC. The protonated His200 was found to 1795



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Figure 6. (a) Full reaction mechanism and (b) the energy profile for the quintet surface. All energies (kcal/mol) are given at the B3LYP(defTZVP)/MM level.

constant. In addition, the experimentally determined side-on iron−oxygen complex in crystallo is a semiquinone substrate radical (SQ•)−FeIII−hydroperoxo species, which could not act as the reactive species. It is particularly interesting to note that in our previous study for HPCD with HPCA,21 the electron-rich aromatic substrate HPCA was found to be able to donate partial electron density to produce a hybrid FeIII−O2•−/SQ•−FeII−O2•− species to advance the reaction cycle. The differences in reaction mechanism of HPCD by using different substrate (4NC vs HPCA) can be rationalized by the key role played by the substrate in the dioxygen activation.

Table 3. Calculated and Experimental Mössbauer Parameters system calc.

WT-4NC

expt.

H200N-4NC WT-HPCA

species 5

A1 5 A2 5 A3 5 A3′ 5 H1 5 H2 5 B 5 E 5 L 5 P Int-1 Int-1

ΔEQ (mm/s)

δ (mm/s)

0.63 0.68 0.59 2.99 1.37 −1.73 3.11 −1.77 −1.99 3.08 −0.33 2.33

0.53 0.54 0.52 0.83 0.67 0.54 0.99 0.46 0.47 0.93 0.50 1.08

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ASSOCIATED CONTENT

S Supporting Information *

Complete citations for refs 24, 25, and 42, QM/MM optimized region, QM/MM energy profiles for the reactions studied, energies, spin densities, and Cartesian coordinates of the key species. This material is available free of charge via the Internet at http://pubs.acs.org.

play a crucial role in manipulating the orientation of the superoxide in the Fe−O2 adducts. The rate-limiting step for the whole reaction is the formation of the bridge species with a barrier of 17.2 kcal/mol relative to 5A1, in good agreement with the value of 16.8 kcal/mol estimated from the experimental rate 1796



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-10-82681829. Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS This work is supported by the Fundamental Research Funds for the Central Universities, the Research Funds of Renmin University of China (program no. 12XNLJ04), and the National Natural Science Foundation of China (no. 21203245).

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Reaction Mechanism of Homoprotocatechuate 2,3-Dioxygenase with

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