Insights into the Reaction Mechanism of Aromatic Ring Cleavage by

Subscriber access provided by BALL STATE UNIV

Article

Insights into the Reaction Mechanism of Aromatic RingCleavage by Homogentisate Dioxygenase: A QM/MM Study Yue Qi, Jiarui Lu, and Wenzhen Lai J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b03006 • Publication Date (Web): 27 Apr 2016 Downloaded from http://pubs.acs.org on April 28, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Insights into the Reaction Mechanism of Aromatic Ring-Cleavage by Homogentisate Dioxygenase: A QM/MM Study Yue Qi, 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]

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

ABSTRACT: To elucidate the reaction mechanism of the ring-cleavage of homogentisate by homogentisate dioxygenase, the quantum mechanical/molecular mechanical (QM/MM) calculations were carried out by using two systems in different protonation states of the substrate C2 hydroxyl group. When the substrate C2 hydroxyl group is ionized (the ionized pathway), the superoxo attack on the substrate is the rate-limiting step in the catalytic cycle with a barrier of 15.9 kcal/mol. Glu396 was found to play an important role in stabilizing the bridge species and its O-O cleavage product by donating a proton via the hydrogen-bonded water molecule. When the substrate C2 hydroxyl group is not ionized (the non-ionized pathway), the O-O bond cleavage of the bridge species is the rate-limiting step with a barrier of 15.3 kcal/mol. The QM/MMoptimized geometries for the dioxygen- and alkylperoxo- complexes using the non-ionized model (for the C2 hydroxyl group) are in agreement with the experimental crystal structures, suggesting that the C2 hydroxyl group is more likely to be non-ionized.

2


Page 3 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction Homogentisate 1,2-dioxygenase (HGDO) is a crucial enzyme involved in the catabolism of homogentisate (2,5-dihydroxy-phenylacetate, HG), a central metabolite in the degradation of phenylalanine (Phe) and tyrosine (Tyr) in aerobic organisms.1-2 It catalyzes the oxidative ring cleavage of HG to yield maleylacetoacetate using molecular oxygen as the oxidant (Scheme 1).34

The deficiency of this enzyme in human body causes the metabolic disease alkaptonuria.5-6

Scheme 1. Reaction Catalyzed by Homogentisate 1,2-Dioxygenase (HGDO).

HGDO could be grouped into a class of extradiol dioxygenases containing the cupin fold.7 However, unlike extradiol-cleaving catecholic dioxygenases,8-14 which cleave the C-C bond adjacent to the catechol oxygens, it cleaves an aromatic ring between ortho carbon atoms substituted with acetate and hydroxyl groups. The first crystallographic model of this enzyme was resolved from human (HGDOHs) but in the absence of a substrate.15 The crystal structure of HGDOHs in the Fe-bound state shows that the active site iron is coordinated by two histidines and one glutamate (Figure 1a), which form the so-called 2-His-1-carboxylate facial triad. The facial triad is an emerging structural motif in mononuclear non-heme enzymes, and is a common platform for the extradiol-type dioxygenases, such as 2,3-dihydroxybiphenyl 1,2-dioxygenase

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 36

(BphC),8-10 catechol 2,3-dioxygenase (2,3-CTD),11-12 protocatechuate 4,5-dioxygenae (4,5PCD)13 and homoprotocatechuate 2,3-dioxygenase (HPCD)14. Based on the first reported crystal structure of HGDOHs in which the substrate is absent, Borowski et al.16 predicted a bidentate binding mode of the substrate via an acetate oxygen and a hydroxyl oxygen (see Figure 1b) by using density functional theory (DFT) calculations.

Figure 1. Comparsion of HGDO from human and P. putida. (a) The resting state of HGDOHs (PDB code: 1EY2) (b) The substrate-bonded HGDOHs predicted by DFT calculations. (c) The enzyme-substrate complex from P. putida (PDB code: 3ZDS, chain B).

However, the recently reported crystal structure of HGDO from P. putida (HGDOPp) by Dobbek et al.7 showed that the substrate binds to iron in a monodentate fashion with a hydroxyl oxygen, while the first-sphere glutamic acid (Glu) residue binds as a bidentate ligand (see Figure 1c). Notably, in other extradiol dioxygenases, the substrate binds in a bidentate fashion while the first-sphere Glu coordinates iron as a monodentate ligand. This different binding mode of the substrate and the first-sphere Glu suggested that the way of FeII-substrate interaction in HGDOPp may be different from other extradiol dioxygenases. Note also that in other extradiol

4


Page 5 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dioxygenases, such as HPCD, the second-sphere His200 was found to play important roles during the catalytic cycle.17-18 However, none of the second-sphere amino acids in HGDOPp could act as an acid-base catalyst. Instead, a water molecule is located nearby the dioxygen bonding site of HGDOPp with a short hydrogen-bonding distance to Glu396 (see Figure 1c). These distinct structural features call for revisiting the reaction mechanism of HGDO in order to get more insights into the aromatic ring-cleaving dioxygenases. It has been suggested that the iron-bound substrate could transfer an electron to activate dioxygen in the extradiol dioxygenase.18-24 As such, the protonation state of the substrate is mechanistically important, since it plays a key role in determining the one-electron redox potential of the catechols and other substituted aromatic compounds.25 In early studies of the extradiol dioxygenases, the bidentate-bound catecholic substrate is usually thought to adopt a monoanionic ionization state based on crystal-graphically determined bond lengths, which indicated that the substrate is asymmetrically bound (one Fe-O bond is shorter than the other).10 However, in the case of the most studied extradiol dioxygenase HPCD, our previous QM/MM study24 found that a proton transfer from the monoanionic binding of the nature substrate to the nearby His200 could occur spontaneously to give the dianionic substrate-bound Fe-O2 adduct. The dianionic forms of the enediol unit of substrate (two Fe–O bond lengths are both around 2.0 Å) is consistent with the calorimetry experiments.26-27 We noted here that for HGDO, the bond length between iron and the substrate C2 hydroxyl oxygen is in a range of 1.96 – 2.30 Å in the crystal structure of the enzyme-substrate (ES) complex.7 It seems that the ionized and nonionized forms of the Fe-bound hydroxyl group are both possible. As such, quantum mechanical/molecular mechanical (QM/MM) calculations has been carried out by considering two different protonation state of the Fe-bound hydroxyl group of the substrate.

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 36

2. Computational Methodology Setup of the System. The initial geometry of the Fe-O2 adduct was prepared from the recently determined crystal structure of HGDOPp, wherein the substrate and dioxygen were bound (PDB code: 3ZDS, subunit C, resolution 1.7 Å).7 The protonation states of the titratable residues were determined on the basis of pKa values predicted by the PROPKA28 program in combination with careful visual inspection of local hydrogen-bonded networks. The histidines, His10, His73, His202, His249, His256, His265, His288, His304, His361, His367, and His385 were protonated at the δ position, while His231 and His331 were protonated at the ε position. One glutamic acid residue Glu396 was protonated, while other glutamates and aspartates (Asp) were used as negatively charged. All 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. The resulting protein was then solvated with a 16Å-thick water solvent layer. To attain equilibrium of the inner solvent layer, the inner 8Å of the solvent layer was processed through a procedure involving (i) a 2400-step minimization using adopted basis Newton-Raphson (ABNR) algorithm, (ii) heating to 300 K in 15 ps, (iii) equilibration MD simulation for 3 ps with a timestep of 1 fs, and (iv) a second 2400step ABNR minimization. This procedure was repeated twice to ensure not more than 100 additional water molecules were added. QM/MM Methodology. The QM/MM methodology is analogous to that used in previous studies for the extradiol dioxygenase HPCD from our group.24, 31 All QM/MM calculations were carried out using ChemShell,32 combining Turbomole33 and DL_POLY.34 An electronic embedding scheme35 was applied to include the polarizing effect of the enzymatic environment on the QM region. Hydrogen link atoms36 with the charge shift model were employed to treat the

6


Page 7 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


QM/MM boundary. The CHARMM30 force field was used for the MM region. The pure QM part was treated by density functional theory (DFT) with the B3LYP functional, which has successfully been applied to the aromatic ring-cleaving dioxygenases.16, 22, 24, 31, 37-44 Geometry optimization were performed by the hybrid delocalized internal coordinates (HDLC) optimizers45 using a double-ζ Def-SV(P)46 basis set, labeled B1. 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.45 The energies were then corrected by single point calculations using a larger all-electron basis set, which is Def-TZVP47 for all the atoms (labeled B2). It is well-known that predicting spin-state energetic correctly is a challenging task in the transitional-metal systems. Inclusion of dispersion has been found to be important in theoretical calculations on enzyme-catalyzed reactions.48-50 As such, the empirical dispersion correction was considered by using the Grimme’s DFT-D3 program.51 Dispersion was found to have only a minor effect on the relative energies in the present study. Then, the B3LYPD3 results are relegated to the supporting information (SI) document. It should be pointed that in some dioxygenases,52-55 B3LYP was shown to overestimate the reaction barriers. Several recent studies suggest that the TPSSh56 functional with 10% exact exchange is optimal for describing the energetic of a wide range of first-row transition metal systems.57-59 Therefore, we also employed TPSSh functional to evaluate the energetic of the reaction pathways by performing single-point calculations on the B3LYP-optimized geometries with the larger basis set B2. As shall be shown, the reaction barrier predicted by TPSSh/MM for the rate-limiting step are close to the experimental value while B3LYP/MM tends to overestimate the reaction barrier. Unless stated otherwise, the TPSSh/MM energies are reported.

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 36

The QM region comprises of the first coordination sphere residues, which are two histidines (His331 and His367), one glutamic acid (Glu337), substrate HG and dioxygen, and His288, Asn333, Tyr346, and Glu396. In addition, crystal water Wat412, Wat414, Wat419, Wat428 and Wat481 are included in the QM region. Both ionized and non-ionized forms of Fe-bound hydroxyl group of HG are considered. The former is called “the ionized system” while the latter is “the non-ionized system”. In both cases, the other hydroxyl group of the substrate is in its nonionized state and is hydrogen-bonded to His288. The QM part of the ionized system has a charge of -1, while the QM part of the non-ionized system has a neutral charge. The total charge of the ionized and non-ionized systems is -8 and -7, respectively. To test the effect of the net charge on the energetic of the catalysis, the more charged ionized system was neutralized by protonating the titratable residues on the surface of the protein. The neutralization was found to have little effect on the barrier of the rate-determining step (see Figure S6 in the SI). As such, the net charge of the studied systems does not make much of a difference in the final QM/MM results.

3. Results The mechanism of the ring cleavage of HG by HGDO was investigated starting from the FeO2 adducts, in which two protonation states of the Fe-bound substrate hydroxyl were considered: the ionized and the non-ionized forms. Two reaction pathways (the ionized and non-ionized pathways) then proposed. The study explored many alternative mechanisms and generated plenty of results. To save space, some of these data are presented in the SI, while here we focus on the feasible mechanisms only.

8


Page 9 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 2. The Proposed Ionized Pathway Starting from the Fe-O2 Adduct (I-1).

3.1. Ionized Pathway The ionized pathway was depicted in Scheme 2. The reaction starts with the Fe-O2 adducts (I1 in Scheme 2) in which the distal oxygen (Od) attacks the substrate to form an alkeylperoxo bridge species (I-2 in Scheme 2). After the formation of I-2, the proton transfer (PT) from Glu396 to the proximal oxygen (Op) of the O2 ligand occurs very easily via the hydrogen-bonded water, yielding I-3. The subsequent O-O bond cleavage results in the formation of the arene oxide radical, I-5. Alternatively, the direct O-O bond cleavage of I-2 to form I-4 was found to have a higher barrier than that for I-3, and hence to be discarded. After the formation of I-5, the reaction proceeds with the ring-closure, the attack of the FeIII-bound OH on the ring, and the

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 36

final scission of the lactone ring, to yield the product (I-8). The detailed discussion of the mechanism was presented in the following subsections. The Nature of the Fe-O2 Adducts. The coupling of a triplet O2 with a quintet FeII gives rise to three possible spin states, triplet, quintet, and septet. In this work, all these spin states were considered. All attempts to find the Fe-O2 adducts having the side-on conformation are failed. Five end-on Fe-O2 adducts with different electronic configures are located. Figure 2 shows the QM/MM-optimized structures of the Fe-O2 adduct, while Table 1 summarizes the relative energies and spin density distribution on key atoms or groups. It can be seen from Figure 2 that the Fe-OS (hydroxyl oxygen atom of the substrate) bond length is 1.95 ± 0.05 Å, which is very close to the lower limit value (1.96 Å) found in the crystal structure of the ES complex.7 The OO bond lengths of the dioxygen ligand in the five optimized Fe-O2 adducts are around 1.3 Å, which point to the superoxide species (O2−). We noted that the absolute values of the spin density on the substrate are around half in all the optimized Fe-O2 adducts except 3I-1b, which suggesting the partial electron provided by the substrate to the Fe-O2 moiety. The spin population is 1.14, 0.94, -0.04 for iron, dioxygen and the substrate, respectively in 3I-1b. As such, 3I-1b can be assigned as an FeIII-O2− species with the low-spin (S=1/2) FeIII center ferromagnetic coupled to the doublet superoxo. Energetically, the septet state was found to be the most stable one at the B3LYP(B2)/MM level, while the lowest quintet and triplet (5I-1a and 3I-1a) states are only 0.4 and 0.5 kcal/mol higher in energy, respectively. At the TPSSh(B2)/MM level, the gap between 5

I-1a and 7I-1 decreased. The small energy difference between the quintet and septet states was

also found in other iron-containing extradiol dioxygenase.24, 31, 38 In addition, 5I-1b and 3I-1b lie 6.6 and 3.5 kcal/mol (TPSSh(B2)/MM) above 5I-1a, respectively.

10


Page 11 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Optimized structures of the Fe-O2 adducts in the ionized pathway. Key bond lengths are given in Å while the bond angles are given in degree.

Table 1. Relative Energies and Spin Densities of the Fe-O2 Adducts in the Ionized Pathway. Spin densities ΔEa Fe Op Od sub (kcal/mol) 5 I-1a 0.0 (0.0) 4.11 -0.30 -0.54 0.48 5 I-1b 6.6 (6.1) 3.77 0.32 0.33 -0.54 7 I-1 0.0 (-0.4) 4.09 0.58 0.61 0.48 3 I-1a 0.3 (0.1) 3.60 -0.58 -0.70 -0.42 3 I-1b 3.5 (13.9) 1.14 0.41 0.53 -0.04 a Relative energies were calculated at the TPSSh(B2)/MM level. Values Species

Lb others 0.21 0.04 0.11 0.01 0.22 0.02 0.08 0.02 -0.05 0.01 in parentheses were

obtained at the B3LYP(B2)/MM. b The ligated N atoms of His331 and His367 and O atoms of Glu337.

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 36

In the previous theoretical studies on the extraidol dioxygenases, the hydroperoxide species formed through a proton transfer from the nearby Histidine residue to the distal or proximal oxygen of the FeO2 adducts was found to be stable38 or to be involved in the reaction cycle16. Here, a water molecule in the distal pocket of HGDOPp forms a hydrogen bonding with the dioxygen ligand and the protonated Glu396 (see Figure 2). As such, Glu396 could donate a proton via the hydrogen-bonded water. However, our QM/MM calculations showed that the energies keep increasing during the proton transfer from the water molecule to distal or proximal oxygen (see Figure S12-S13 in the SI). All attempts to locate the hydroperoxide species are failed. As should be shown below, this proton transfer occurs after the formation of the bridge species. Superoxo Radical Attack on Substrate. The attack of the superoxo on the substrate to form an alkylperoxo bridge species is a common step in the reaction catalyzed by the extradiol dioxygenases. To get insights on this important step, all above mentioned Fe-O2 adducts were considered as the reactants. The QM/MM-optimized structures of the TSs and bridge species were shown in Figure 3. Table 2 summarizes the spin density distribution on key atoms or groups. The corresponding energy profiles for this attack are depicted in Figure 4. It can be seen from Figure 4 that the quintet 5I-TS1b is the one having the lowest energy among the five I-TS1 species, laying 15.9 kcal/mol (B3LYP(B2)/MM: 18.2 kcal/mol) above the lowest quintet reactant 5

I-1a. 5I-TS1a/7I-TS1 is 6.1/2.4 kcal/mol higher in energy than 5I-TS1b. It can be also seen that

the profiles of the triplet states nascent from 3I-1a and 3I-1b lie higher than the profiles of the quintet and septet states, to suggest that the triplet states should not be important in the reactivity. The bridge species I-2 has a quintet ground state, in which the spin on the substrate and dioxygen are quenched (see 5I-2 in Table 2). By contrast, in the septet and triplet states of I-2, substantial

12


Page 13 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


spin densities on the substrate are observed (see Table 2). Energetically, 7I-2/3I-2a/3I-2b is 2.9/2.2/7.5 kcal/mol higher than 5I-2. Note that on the quintet surface, the proton transfer from the para hydroxyl group of the substrate to His288 occurs simultaneously during the attack to generate a FeII-alkylperoxo bridge species (see 5I-2 in Figure 3).

Figure 3. Optimized structures for the I-TS1 and I-2 species in the ionized pathway. Key bond lengths are given in Å while the bond angles are given in degree. 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 36

Table 2. Spin Densities of the TSs and Products for the First Step in the Ionized Pathway. Species I-TS1a 5 I-TS1b 7 I-TS1 3 I-TS1a 3 I-TS1b 5 I-2 7 I-2 3 I-2a 3 I-2b 5

Fe 3.81 3.97 4.08 3.06 0.70 3.80 4.16 2.84 1.09

Op -0.26 0.30 0.39 -0.17 0.26 0.05 0.30 -0.03 0.01

Od -0.31 0.15 0.25 -0.21 0.28 -0.00 0.10 -0.06 0.04

sub 0.65 -0.58 1.07 -0.76 0.79 0.04 1.18 -0.84 0.91

L 0.08 0.15 0.19 0.05 -0.04 0.08 0.23 0.06 -0.07

others 0.03 0.01 0.02 0.03 0.01 0.03 0.03 0.02 0.02

Figure 4. The energy profile for the formation of the bridge species in the ionized pathway. The relative energies are given in kcal/mol at the TPSSh(B2)/MM level. Values in parentheses were obtained at the B3LYP(B2)/MM level.

It is widely accepted that only the quintet surface is catalytically relevant in the extradiol dioxygenases.16,

24, 31, 37-38, 60

As mentioned above, we found that the attack of the dioxygen

ligand on the substrate prefers to occur on the quintet surface in HGDO. The so-generated

14


Page 15 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


alkylperoxo species has a quintet ground state, with the triplet and septet states being much higher in energy. Therefore, only the quintet state was considered in the following studies.

Figure 5. The energy profile of the ionized pathway on the quintet surface. The blue line shows the preferred pathway. The relative energies are given in kcal/mol at the TPSSh(B2)/MM level. Values in parentheses were obtained at the B3LYP(B2)/MM level.

The Complete Energy Profile. The computed energy profile on the quintet surface was described in Figure 5. As already noted the initial attack on the substrate proceeds through 5ITS1b with a barrier of 15.9 kcal/mol (with respect to the lowest quintet Fe-O2 adduct). After formation of the bridge species (I-2), the direct O-O bond cleavage proceeds via 5I-TS3 with a barrier of 4.2 kcal/mol and an endothermicity of 3.5 kcal/mol. While the proton transfer from the Glu396 to the proximal oxygen of the dioxygen ligand mediated by the hydrogen-bonded water molecule was found to have a small barrier of 1.4 kcal/mol and an exothermicity of 3.6 kcal/mol. The subsequent O-O bond cleavage proceeds through 5I-TS4, which is 5.6 kcal/mol lower in

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 36

energy than 5I-TS3. And the so-generated 5I-5 is 8.7 kcal/mol more stable than the direct O-O cleavage product 5I-4. Hence, protonation of the peroxo group is very important in stabilizing the bridge species and its O-O cleavage product. The remaining steps of the catalytic cycle will be described only briefly. As shown in Scheme 2, from 5I-5, the ring-closure coupled with the C1-C2 bond breaking would give a sevenmembered lactone radical (5I-6). The reaction is highly exothermic (42.0 kcal/mol) and the calculated barrier is only 2.0 kcal/mol (see Figure 5). The next step involves a nucleophilic attack of the Fe-bound OH group on the carbonyl carbon of the lactone radical. This attack occurs with a barrier of 8.6 kcal/mol, leading to 5I-7, in which the iron center was changed back to its ferrous state. Finally, the opening of the seven-membered ring is coupled with the proton transfer back to Glu396 via the hydrogen-bonded water molecule, leading to the formation of the 2-hydroxymuconaldehyde acid product (5I-8). It can be seen from Figure 5 that the first step, i.e., the attack of the superoxo radical on the substrate, is the rate-limiting step with a barrier of 15.9 kcal/mol in the ionized pathway.

3.2. Non-Ionized Pathway The reaction mechanism for HGDO starting from N-1 was depicted in Scheme 3, which represents all the possible reaction paths. The first step is the attack of the distal oxygen of the O2 ligand on the substrate C2, forming the alkylperoxo bridge species (N-2). The subsequent O-O bond cleavage leads to the arene oxide radical (N-3). The formation of the lactone species (N-5) involves the C-C bond breaking and the proton transfer from Glu396 to the Fe-bound oxygen via the hydrogen-bonded water molecule. These two steps occur in a stepwise manner through two possible paths: (i) the C-C bond breaking followed by the PT (5N-3 → 5N-4 → 5N-5), and (ii) the

16


Page 17 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PT followed by the C-C bond cleavage (5N-3 → 5N-6 → 5N-5). The last phase of the catalytic cycle involves a nucleophilic attack of the Fe-bound OH group on the substrate C2 (5N-TS7 and 5

N-7) and the final scission of the ring (5N-TS9 and 5N-8). The alternative path (5N-TS8) has a

higher barrier than the competing reactions (see below). The detailed discussion of the mechanism and calculated energetic was presented in the following subsections.

Scheme 3. The Computed Non-Ionized Pathway Starting from the Fe-O2 Species (N-1).

The Nature of the Fe-O2 Adducts. When the substrate hydroxyl oxygen atoms are both not ionized, six Fe-O2 adducts were obtained. All of the optimized structures were depicted in Figure 6. The triplet state (3N-1), wherein O2 is bound end-on, was found to be the most stable one at

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 36

the B3LYP(B2)/MM level. Molecular orbital analysis showed that it is an FeII-O2 species with the S=2 FeII center antiferromagnetic coupled to the triplet O2 (Figure S5 in the SI). Three quintet states were located, two of which are end-on species while the other is a side-on one. All three quintet states have six unpaired electrons. The electronic structure of 5N-1a is schematically shown in Figure 7. It is seen that O2 is a superoxide, having a singly occupied π*ip (in-plane orbital defined by the Fe-O-O plane), while the other doubly occupied (π*op, out-of-plane orbital) forms a 3-electon (3-e) bond with the iron dxz orbital. Hence, it can be described as an FeIII-O2•− species. Noted that at the TPSSh(B2)/MM level, 5N-1a is the one having the lowest energy. The side-on complex 5N-1b was found to also have an FeIII-O2•− character, in which the dioxygen π*op is singly occupied (see Figure 8a) while doubly occupied dioxygen π*ip forms a 3-e bond with iron dyz orbital. In 5N-1c, the spin population is 3.68, 0.40 and -0.22 for iron, dioxygen and the substrate, respectively. The electronic natural of 5N-1c is best described as being between the two limiting cases, FeII-O2 and substrate radical (SQ•)-FeII-O2•−. Singly occupied molecular orbital with beta electron is mainly a dioxygen π*ip with a small component of substrate radical character (Figure 8b). The small spin density on the substrate suggested that (SQ•)-FeII-O2•− only has a small contribution to the electronic structure of 5N-1c. Energetically, 5N-1b/5N-1c lies 3.7/3.6 kcal/mol above 5N-1a. In addition, two septet states were obtained. One is an end-on species (7N-1a) while the other is a side-on one (7N-1b). 7N-1a/7N-1b is 2.1/0.2 kcal/mol less stable than 5N-1. In HPCD, the Fe-O2 adduct has been spectroscopic characterized and was found to have a quintet ground state. However, little is known about HGDO. Being attentive to the often expressed difficulty in predicting the energetic ordering of the spin-states, it is hard to draw a conclusion from our calculations about which one is the ground state. Since the quintet state is of main interest here, we set 5N-1a as the reference state.

18


Page 19 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Optimized structures for the Fe-O2 adducts (N-1) in the non-ionized pathway. Key bond lengths are given in Å while the bond angles are given in degree.

Figure 7. Molecular orbital picture for 5N-1a.

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 36

Figure 8. Singly occupied molecular orbital with beta electron for (a) 5N-1b and (b) 5N-1c. The occupancy numbers were given in the parentheses.

Moreover, it can be also found that the spin on the substrate in N-1 is smaller than that in I-1, suggesting that the electron transfer from the non-ionized substrate is more difficult than from the ionized substrate. Hence, the protonation state of the substrate plays a key role in the dioxygen activation. Superoxo Attack on the Substrate. The formation of the alkylperoxo bridge species prefers the quintet surface, as was found in the non-ionized pathway. The reaction on triplet and septets has a much higher reaction barrier. Hence, the corresponding results have been relegated to the SI. We then focus here only the quintet paths. The distal oxygen attack on the substrate from 5N1a/5N-1b/5N-1c was found to occur with concomitant proton transfer from the unbound hydroxyl group of the substrate to His288, resulting in the formation of the same alkylperoxo bridge species (5N-2). The transition states (TSs) for this attack, 5N-TS1a and 5N-TS1c, nascent from the end-on bonded Fe-O2 adducts, are close in energy, lying 9.4 and 9.7 kcal/mol (B3LYP(B2)/MM) higher than the lowest quintet reactant 5N-1a, while 5N-TS1b has a much higher energy (17.1 kcal/mol above the zero level, see Figure 9). The optimized quintet TSs are shown in Figure 10.

20


Page 21 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The critical C-O distance at the 5N-TS1b is 1.76 Å, while it is 1.94/1.96 Å at 5N-TS1a/5N-TS1c, suggesting the attack from the side-on bonded Fe-O2 attack has a late TS compared with those from the end-on ones. This is consistent with the fact that the former reaction has a higher reaction barrier than the latter two. At the TPSSh(B2)/MM level. 5N-TS1a, 5N-TS1b and 5N-TS1c lie 12.7, 15.8 and 14.8 kcal/mol above 5N-1a, respectively (see Figure 9).

Figure 9. The energy profile of the non-ionized pathway from N-1 to N-3 on the quintet surface. The relative energies are given in kcal/mol at the TPSSh(B2)/MM level. Values in parentheses were obtained at the B3LYP(B2)/MM level.

Figure 10. The optimized TSs for the distal oxygen attack on the substrate starting from 5N-1.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

O-O Bond Cleavage and Formation of the Epoxide. The O-O bond cleavage from 5N-2 was found to couple with the ring-closure (through the Od-C1 bond formation) to generate a substrate epoxide (5N-3). The process has a barrier of 15.3 kcal/mol and an endothermicity of 12.2 kcal/mol (B3LYP/MM predicts a barrier of 22.6 kcal/mol, see Figure 9). In the ionized pathway, protonation of the peroxo group was found to play an important role in stabilizing the bridge species and its O-O cleavage product. However, in the non-ionized pathway, this protonation could not occur since the energy is keep increasing during the proton transfer from the distal water molecule (Figure S27 in the SI).

Figure 11. The energy profile of the non-ionized pathway starting from N-3. The relative energies are given in kcal/mol at the TPSSh(B2)/MM level.

22


Page 23 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Formation of the Lactone Species. The Fe-OH bound lactone species was widely thought to be involved in the aromatic ring cleavage catalyzed by the extradiol dioxygenases. Starting from the epoxide 5N-3, the formation of the lactone bound FeIII-OH species involves the C1-C2 bond breaking and a proton transfer from Glu396 to the Fe-bound oxygen (Op) atom via a water molecule. As shown in Scheme 3, two possible paths were investigated: (i) the C-C bond breaking followed by the PT (5N-3 → 5N-4 → 5N-5), and (ii) the PT followed by the C-C bond cleavage (5N-3 → 5N-6 → 5N-5). It can be seen from Figure 11 that both two paths have very small barriers (less than 2.5 kcal/mol). Therefore, they both are equally probable. Completion Steps of the Reaction. After the formation of the lactone species, the reaction proceeds with the attack of the Fe-bound OH group on the substrate and the final ring-opening to yield the maleylacetoacetate product (5N-5 → 5N-7 → 5N-8). Both steps were found to have a small barrier. In addition, the oxygen attack on the substrate from 5N-4 was also studied, but has been discarded due to the higher barrier compared with the PT process (5N-TS8 vs. 5N-TS4 in Figure 11). But, if no any residue or molecule in the distal pocket could donate a proton, the ring cleavage of HG would proceed along 5N-1 → 5N-2 → 5N-3 → 5N-4 → 5N-7 → 5N-8.

4. Discussion It has been proposed that the substrate binding to the iron center could lead to the deprotonation of the C2 hydroxyl group.7 Taking account of the wide range of Fe-O(C2) bond length (1.96 – 2.30 Å) in the crystal structure of the ES complex, we have studied here the mechanism of the catalytic reaction of HGDO, using QM/MM methodology for two systems in different protonation states of the substrate C2 hydroxyl group (the ionized and the non-ionized pathways). As shown above, both pathways are energetically feasible. In the ionized pathway,

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 36

the superoxo attack on the substrate is the rate-limiting step with a barrier of 15.9 kcal/mol (B3LYP(B2)/MM: 18.2 kcal/mol), while in the non-ionized pathway, the O-O bond cleavage is the rate-limiting step with a barrier of 15.3 kcal/mol (B3LYP(B2)/MM: 22.6 kcal/mol). The calculated barriers at the TPSSh(B2)/MM level for the two reaction pathways are both in good agreement with the estimated value of 14.9 kcal/mol form experiment7 (the rate constant of 79.5 s-1).

Figure 12. Comparison of the Fe-dioxygen complex of HGDOPp and HPCD. (a) The FeO2 adduct of HGDO. (b) The FeO2 adduct of HPCD. (c) Superposition of the QM/MM-optimized 5

N-1b and the crystal structure of the FeO2 adduct of HGDO.

In the recent in crystallo study of HGDO, three different intermediates along the reaction pathway were trapped: the dioxygen-, alkylperoxo-, and product-bound complexes.7 Notably, the same three reaction intermediates were also found in an earlier in crystallo study of HPCD with the electron-poor substrate 4-nitrocatechol.14 The crystal structure of the Fe-dioxygen complex shows that the dioxygen bound to the iron in a side-on fashion for both enzymes (see Figure 12a and 12b). It should be pointed out that the side-on iron-dioxygen complex of HPCD was

24


Page 25 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


suggested to be a SQ•-FeIII-hydroperoxo species rather than the SQ•-FeII-superoxo by our previous QM/MM studies.31 However, for the HGDO, the Fe-hydroperoxo species was failed to be located either in the ionized pathway or in the non-ionized pathway. A side-on Fe-O2 (5N-1b) species obtained in the non-ionized pathway was found to overlap very well with the corresponding crystal structure (Figure 12c). Furthermore, Tables 3 and 4 compare the experimental and QM/MM-optimized geometric parameters of the Fe-O2 adducts and alkylperoxo species, respectively. The calculated Fe-O bond lengths of the side-on Fe-O2 (2.13 and 2.22 Å) are shorter than those in the crystal structure (2.32 and 2.50 Å),7 but close to the reported values for the synthetic nonheme ferric-superoxo complexes (2.0 ~ 2.2 Å).61-62 More importantly, the end-on FeO2 adducts were found to be more reactive than the side-on one in the present work. This is consistent with the finding in the previous studies for extradiol dioxygenases that the end-on FeO2 species is the reactive oxygen species.16, 22, 24, 31, 37-38 To get more accurate spin-state energetic of HGDO, further studies using high level ab initio theory may be necessary.

Table 3. Experimental and QM/MM-Optimized Geometrical Parameters of the Fe-O2 Species. parameter Op-Od Fe-Op Fe-Od Fe-OS Fe-O1(Glu337) Fe-O2(Glu337) Fe-N(His331) Fe-N(His367) OS-C2

5

I-1a/5I-1b 1.30/1.28 2.06/2.09 2.87/2.94 1.90/2.00 2.14/2.19 2.28/2.26 2.22/2.24 2.13/2.14 1.33/1.31

5

N-1a/5N-1b 1.25/1.28 2.00/2.13 2.93/2.22 2.05/2.07 2.18/2.24 2.18/2.11 2.17/2.23 2.12/2.17 1.40/1.39

Exptl. 1.35 2.32 2.50 2.20 2.30 2.43 2.24 2.26 1.40

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 36

Table 4. Experimental and QM/MM-Optimized Geometrical Parameters of the Alkylperoxo Intermediate. parameter Op-Od Fe-Op Fe-OS Fe-O1(Glu337) Fe-O2(Glu337) Fe-N(His331) Fe-N(His367) OS-C2

5

I-2 1.46 2.05 2.08 2.23 2.30 2.26 2.16 1.36

5

N-2 1.46 2.03 2.22 2.23 2.22 2.24 2.16 1.42

Exptl. 1.53 2.43 2.29 2.47 2.27 2.23 2.05 1.44

It can be seen from Tables 3 and 4 that the optimized Fe-O2 adducts and alkylperoxo species in two reaction pathways (the ionized and non-ionized pathways) are quite similar. However, the Fe-OS and OS-C2 bond lengths in the Fe-O2 and alkylperoxo species in the nonionized pathway are more close to the experimental values compared with those in the ionized pathway. Therefore, the C2 hydroxyl group is more likely to be non-ionized and the reaction would proceed along the non-ionized pathway.

5. Conclusion In the present work, we have carried out a detailed computational investigation of the catalytic mechanism of HGDO by means of hybrid QM/MM calculations. Both ionized and non-ionized forms of the substrate C2 hydroxyl group were considered. Two reaction pathways, namely the ionized and the non-ionized pathways were proposed. Both pathways were found to be energetically feasible. When the substrate C2 hydroxyl group is ionized (the ionized pathway), the superoxo attack on the substrate is an endothermic reaction and is the rate-limiting step in the catalytic cycle with a barrier of 15.9 kcal/mol. Glu396 was found to play an important role in stabilizing the bridge species and its O-O cleavage product by donating a proton via the

26


Page 27 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydrogen-bonded water molecule. When the substrate C2 hydroxyl group is not ionized (the nonionized pathway), the superoxo attack on the substrate is an exothermic reaction. The subsequent O-O bond cleavage of the bridge species is the rate-limiting step with a barrier of 15.3 kcal/mol. In these two steps, Glu396 remains intact. Comparison of the QM/MM-optimized geometries with the experimental structures trapped in crystallo reaction suggested that the C2 hydroxyl group is likely to be non-ionized. To elucidate the protonation state of the bound substrate, further study may be needed by using some experimental approaches, such as ultraviolet resonance Raman spectroscopy and electronic absorption spectroscopy.10 ASSOCIATED CONTENT Supporting Information QM/MM optimized region, QM/MM energy profiles for the each reaction step, energies, spin distribution and Cartesian coordinates of the key species. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Phone: +86-10-82681829. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 36

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) Arias-Barrau, E.; Olivera, E. R.; Luengo, J. M.; Fernandez, C.; Galan, B.; Garcia, J. L.; Diaz, E.; Minambres, B. The Homogentisate Pathway: A Central Catabolic Pathway Involved in the Degradation of L-Phenylalanine, L-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas Putida. J. Bacteriol. 2004, 186, 5062–5077. (2) Kim, Y. H.; Cho, K.; Yun, S. H.; Kim, J. Y.; Kwon, K. H.; Yoo, J. S.; Kim, S. I. Analysis of Aromatic Catabolic Pathways in Pseudomonas Putida KT 2440 Using A Combined Proteomic Approach: 2-DE/MS and Cleavable Isotope-Coded Affinity Tag Analysis. Proteomics 2006, 6, 1301–1318. (3) Amaya, A. A.; Brzezinski, K. T.; Farrington, N.; Moran, G. R. Kinetic Analysis of Human Homogentisate 1,2-Dioxygenase. Arch. Biochem. Biophys. 2004, 421, 135–142. (4) Veldhuizen, E. J. A.; Vaillancourt, F. H.; Whiting, C. J.; Hsiao, M. M. Y.; Gingras, G.; Xiao, Y. F.; Tanguay, R. M.; Boukouvalas, J.; Eltis, L. D. Steady-State Kinetics and Inhibition of Anaerobically Purified Human Homogentisate 1,2-Dioxygenase. Biochem. J 2005, 386, 305–314. (5) Rodriguez, J. M.; Timm, D. E.; Titus, G. P.; de Bernabe, D. B. V.; Criado, O.; Mueller, H. A.; de Cordoba, S. R.; Penalva, M. A. Structural and Functional Analysis of Mutations in Alkaptonuria. Hum. Mol. Genet. 2000, 9, 2341–2350. (6) Zatkova, A. An Update on Molecular Genetics of Alkaptonuria (AKU). J. Inherit. Metab. Dis. 2011, 34, 1127–1136.

28


Page 29 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7) Jeoung, J. H.; Bommer, M.; Lin, T. Y.; Dobbek, H. Visualizing the Substrate-, Superoxo-, Alkylperoxo-, and Product-Bound States at the Nonheme Fe(II) Site of Homogentisate Dioxygenase. Proc. Nat. Acad. Sci. U.S.A. 2013, 110, 12625–12630. (8) Uragami, Y.; Senda, T.; Sugimoto, K.; Sato, N.; Nagarajan, V.; Masai, E.; Fukuda, M.; Mitsui, Y. Crystal Structures of Substrate Free and Complex Forms of Reactivated BphC, An Extradiol Type Ring-Cleavage Dioxygenase. J. Inorg. Biochem. 2001, 83, 269–279. (9) Sato, N.; Uragami, Y.; Nishizaki, T.; Takahashi, Y.; Sazaki, G.; Sugimoto, K.; Nonaka, T.; Masai, E.; Fukuda, M.; Senda, T. Crystal Structures of the Reaction Intermediate and Its Homologue of An Extradiol-Cleaving Catecholic Dioxygenase. J. Mol. Biol. 2002, 321, 621–636. (10) Vaillancourt, F. H.; Barbosa, C. J.; Spiro, T. G.; Bolin, J. T.; Blades, M. W.; Turner, R. F. B.; Eltis, L. D. Definitive Evidence for Monoanionic Binding of 2,3-Dihydroxybiphenyl to 2,3Dihydroxybiphenyl 1,2-Dioxygenase from UV Resonance Raman Spectroscopy, UV/Vis Absorption Spectroscopy, and Crystallography. J. Am. Chem. Soc. 2002, 124, 2485–2496. (11) Kita, A.; Kita, S.; Fujisawa, I.; Inaka, K.; Ishida, T.; Horiike, K.; Nozaki, M.; Miki, K. An Archetypical Extradiol-Cleaving Catecholic Dioxygenase: the Crystal Structure of Catechol 2,3Dioxygenase (Metapyrocatechase) from Pseudomonas Putida Mt-2. Structure 1999, 7, 25–34. (12) Cho, J.-H.; Jung, D.-K.; Lee, K.; Rhee, S. Crystal Structure and Functional Analysis of the Extradiol Dioxygenase LapB from a Long-chain Alkylphenol Degradation Pathway in Pseudomonas. J. Biol. Chem. 2009, 284, 34321–34330. (13) Sugimoto, K.; Senda, T.; Aoshima, H.; Masai, E.; Fukuda, M.; Mitsui, Y. Crystal Structure of an Aromatic Ring Opening Dioxygenase LigAB, A Protocatechuate 4,5-Dioxygenase, Under Aerobic Conditions. Structure 1999, 7, 953–965.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

(14) Kovaleva, E. G.; Lipscomb, J. D. Crystal Structures of Fe2+ Dioxygenase Superoxo, Alkylperoxo, and Bound Product Intermediates. Science 2007, 316, 453–457. (15) Titus, G. P.; Mueller, H. A.; Burgner, J.; de Cordoba, S. R.; Penalva, M. A.; Timm, D. E. Crystal Structure of Human Homogentisate Dioxygenase. Nat. Struct. Biol. 2000, 7, 542–546. (16)

Borowski, T.; Georgiev, V.; Siegbahn, P. E. M. Catalytic Reaction Mechanism of

Homogentisate Gioxygenase: A Hybrid DFT Study. J. Am. Chem. Soc. 2005, 127, 17303–17314. (17) Fielding, A. J.; Lipscomb, J. D.; Que, L. A Two-Electron-Shell Game: Intermediates of the Extradiol-Cleaving Catechol Dioxygenases. J. Biol. Inorg. Chem. 2014, 19, 491–504. (18) Kovaleva, E. G.; Rogers, M. S.; Lipscomb, J. D. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine 200. Biochemistry 2015, 54, 5329–5339. (19)

Groce, S. L.; Miller-Rodeberg, M. A.; Lipscomb, J. D. Single-Turnover Kinetics of

Homoprotocatechuate 2,3-Dioxygenase. Biochemistry 2004, 43, 15141–15153. (20)

Vaillancourt, F. H.; Bolin, J. T.; Eltis, L. D. The Ins and Outs of Ring-Cleaving

Dioxygenases. Crit. Rev. Biochem. Mol. Biol. 2006, 41, 241–267. (21) Lipscomb, J. D. Mechanism of Extradiol Aromatic Ring-Cleaving Dioxygenases. Curr. Opin. Struct. Biol. 2008, 18, 644–649. (22) Siegbahn, P. E. M.; Haeffner, F. Mechanism for Catechol Ring-Cleavage by Non-Heme Iron Extradiol Dioxygenases. J. Am. Chem. Soc. 2004, 126, 8919–8932. (23) Deeth, R. J.; Bugg, T. D. H. A Density Functional Investigation of the Extradiol Cleavage Mechanism in Non-Heme Iron Catechol Dioxygenases. J. Biol. Inorg. Chem. 2003, 8, 409–418.

30


Page 31 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(24)


Dong, G.; Shaik, S.; Lai, W. Z. Oxygen Activation by Homoprotocatechuate 2,3-

Dioxygenase: A QM/MM Study Reveals the Key Intermediates in the Activation Cycle. Chem. Sci. 2013, 4, 3624–3635. (25) Wardman, P. Reduction Potentials of One‐Electron Couples Involving Free Radicals in Aqueous Solution. J. Phys. Chem. Ref. Data 1989, 18, 1637–1755. (26) Henderson, K. L.; Le, V. H.; Lewis, E. A.; Emerson, J. P. Exploring Substrate Binding in Homoprotocatechuate 2,3-Dioxygenase Using Isothermal Titration Calorimetry J. Biol. Inorg. Chem. 2012, 17, 991–994. (27) Henderson, K. L.; Francis, D. H.; Lewis, E. A.; Emerson, J. P. Thermodynamics of Substrate Binding to the Metal Site in Homoprotocatechuate 2,3-Dioxygenase: Using ITC Under Anaerobic Conditions to Study Enzyme–Substrate Interactions. Biochim. Biophys. Acta 2016, 1860, 910–916. (28) Li, H.; Robertson, A. D.; Jensen, J. H. Very Fast Empirical Prediction and Rationalization of Protein pKa Values. Proteins: Struct., Func., Bioinf. 2005, 61, 704–721. (29) Brunger, A. T.; Karplus, M. Polar Hydrogen Positions in Proteins: Empirical Energy Placement and Neutron Diffraction Comparison. Proteins: Struct., Funct., Genet. 1988, 4, 148– 156. (30) MacKerell, A. D.; Bashford, D.; Bellott, M.; Dunbrack, R. L.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S., et al. All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins. J. Phys. Chem. B 1998, 102, 3586–3616. (31) Dong, G.; Lai, W. Z. Reaction Mechanism of Homoprotocatechuate 2,3-Dioxygenase with 4-Nitrocatechol: Implications for the Role of Substrate. J. Phys. Chem. B 2014, 118, 1791–1798.

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

(32) Sherwood, P.; de Vries, A. H.; Guest, M. F.; Schreckenbach, G.; Catlow, C. R. A.; French, S. A.; Sokol, A. A.; Bromley, S. T.; Thiel, W.; Turner, A. J., et al. QUASI: A General Purpose Implementation of the QM/MM Approach and Its Application to Problems in Catalysis. J. Mol. Struc. (THEOCHEM) 2003, 632, 1–28. (33) Ahlrichs, R.; Bär, M.; Häser, M.; Horn, H.; Kölmel, C. Electronic Structure Calculations on Workstation Computers: The Program System Turbomole. Chem. Phys. Lett. 1989, 162, 165-169. (34)

Smith, W.; Forester, T. R. DL_POLY_2.0: A General-Purpose Parallel Molecular

Dynamics Simulation Package. J. Mol. Graphics 1996, 14, 136–141. (35)

Bakowies, D.; Thiel, W. Hybrid Models for Combined Quantum Mechanical and

Molecular Mechanical Approaches J. Phys. Chem. 1996, 100, 10580–10594. (36) de Vries, A. H.; Sherwood, P.; Collins, S. J.; Rigby, A. M.; Rigutto, M.; Kramer, G. J. Zeolite Structure and Reactivity by Combined Quantum-Chemical−Classical Calculations. J. Phys. Chem. B 1999, 103, 6133–6141. (37) Georgiev, V.; Borowski, T.; Blomberg, M. R. A.; Siegbahn, P. E. M. A Comparison of the Reaction Mechanisms of Iron- and Manganese-Containing 2,3-HPCD: An Important Spin Transition for Manganese. J. Biol. Inorg. Chem. 2008, 13, 929–940. (38)

Christian, G. J.; Ye, S. F.; Neese, F. Oxygen Activation in Extradiol Catecholate

Dioxygenases - A Density Functional Study. Chem. Sci. 2012, 3, 1600–1611. (39) Hupert-Kocurek, K.; Wojcieszynska, D.; Guzik, U.; Borowski, T. A Single Amino Acid Substitution within Catalytically Non-Active N-Terminal Domain of Catechol 2,3-Dioxygenase (C23O) Increases Enzyme Activity towards 4-Chlorocatechol. J. Mol. Catal. B: Enzym. 2015, 122, 64–71.

32


Page 33 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(40)


Brkic, H.; Kovačević, B.; Tomić, S. Human 3-Hydroxyanthranilate 3,4-Dioxygenase

(3HAO) Dynamics and Reaction, a Multilevel Computational Study. Mol. Biosyst. 2015, 11, 898–907. (41)

Nakatani, N.; Hitomi, Y.; Sakaki, S. Multistate CASPT2 Study of Native Iron(III)-

Dependent Catechol Dioxygenase and Its Functional Models: Electronic Structure and Ligandto-Metal Charge-Transfer Excitation. J. Phys. Chem. B 2011, 115, 4781–4789. (42) Borowski, T.; Wojcik, A.; Milaczewska, A.; Georgiev, V.; Blomberg, M. R. A.; Siegbahn, P. E. M. The Alkenyl Migration Mechanism Catalyzed by Extradiol Dioxygenases: A Hybrid DFT Study. J. Biol. Inorg. Chem. 2012, 17, 881–890. (43) Borowski, T.; Siegbahn, P. E. M. Mechanism for Catechol Ring Cleavage by Non-Heme Iron Intradiol Dioxygenases: A Hybrid DFT Study. J. Am. Chem. Soc. 2006, 128, 12941–12953. (44) Wójcik, A.; Borowski, T.; Broclawik, E. The Mechanism of the Reaction of Intradiol Dioxygenase with Hydroperoxy Probe A DFT Study. Catal. Today 2011, 169, 207–216. (45)

Billeter, S. R.; Turner, A. J.; Thiel, W. Linear Scaling Geometry Optimisation and

Transition State Search in Hybrid Delocalised Internal Coordinates. Phys. Chem. Chem. Phys. 2000, 2, 2177–2186. (46) Schäfer, A.; Horn, H.; Ahlrichs, R. Fully Optimized Contracted Gaussian Basis Sets for Atoms Li to Kr. J. Chem. Phys. 1992, 97, 2571–2577. (47) Schäfer, A.; Huber, C.; Ahlrichs, R. Fully Optimized Contracted Gaussian Basis Sets of Triple Zeta Valence Quality for Atoms Li to Kr. J. Chem. Phys. 1994, 100, 5829–5835. (48) Lonsdale, R.; Harvey, J. N.; Mulholland, A. J. Inclusion of Dispersion Effects Significantly Improves Accuracy of Calculated Reaction Barriers for Cytochrome P450 Catalyzed Reactions. J. Phys. Chem. Lett. 2010, 1, 3232–3237.

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

(49) Lonsdale, R.; Harvey, J. N.; Mulholland, A. J. Effects of Dispersion in Density Functional Based Quantum Mechanical/Molecular Mechanical Calculations on Cytochrome P450 Catalyzed Reactions. J. Chem. Theory Comput. 2012, 8, 4637–4645. (50) Kaiyawet, N.; Lonsdale, R.; Rungrotmongkol, T.; Mulholland, A. J.; Hannongbua, S. HighLevel QM/MM Calculations Support the Concerted Mechanism for Michael Addition and Covalent Complex Formation in Thymidylate Synthase. J. Chem. Theory Comput. 2015, 11, 713–722. (51) Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. A Consistent and Accurate Ab Initio Parametrization of Density Functional Dispersion Correction (DFT-D) for the 94 Elements H-Pu. J. Chem. Phys. 2010, 132, 154104. (52) Georgiev, V.; Noack, H.; Borowski, T.; Blomberg, M. R. A.; Siegbahn, P. E. M. DFT Study on the Catalytic Reactivity of a Functional Model Complex for Intradiol-Cleaving Dioxygenases. J. Phys. Chem. B 2010, 114, 5878–5885. (53)

Pabis, A.; Geronimo, I.; Paneth, P. A DFT Study of the cis-Dihydroxylation of

Nitroaromatic Compounds Catalyzed by Nitrobenzene Dioxygenase. J. Phys. Chem. B 2014, 118, 3245–3256. (54) Geronimo, I.; Paneth, P. A DFT and ONIOM Study of C-H Hydroxylation Catalyzed by Nitrobenzene 1,2-Dioxygenase. Phys. Chem. Chem. Phys. 2014, 16, 13889–13899. (55)

Cao, L. L.; Dong, G.; Lai, W. Z. Reaction Mechanism of Cobalt-Substituted

Homoprotocatechuate 2,3-Dioxygenase: A QM/MM Study. J. Phys. Chem. B 2015, 119, 4608– 4616.

34


Page 35 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(56)


Tao, J. M.; Perdew, J. P.; Staroverov, V. N.; Scuseria, G. E. Climbing the Density

Functional Ladder: Nonempirical Meta-Generalized Gradient Approximation Designed for Molecules and Solids. Phys. Rev. Lett. 2003, 91, 146401. (57) Jensen, K. P. Bioinorganic Chemistry Modeled with the TPSSh Density Functional. Inorg. Chem. 2008, 47, 10357–10365. (58) Jensen, K. P.; Cirera, J. Accurate Computed Enthalpies of Spin Crossover in Iron and Cobalt Complexes. The Journal of Physical Chemistry A 2009, 113, 10033–10039. (59)

Kepp, K. P. The Ground States of Iron(III) Porphines: Role of Entropy-Enthalpy

Compensation, Fermi Correlation, Dispersion, and Zero-Point Energies. J. Inorg. Biochem. 2011, 105, 1286–1292. (60) Georgiev, V.; Borowski, T.; Siegbahn, P. E. M. Theoretical Study of the Catalytic Reaction Mechanism of MndD. J. Biol. Inorg. Chem. 2006, 11, 571–585. (61) Chen, H.; Cho, K. B.; Lai, W. Z.; Nam, W.; Shaik, S. Dioxygen Activation by a Non-Heme Iron(II) Complex: Theoretical Study toward Understanding Ferric-Superoxo Complexes. J. Chem. Theory Comput. 2012, 8, 915–926. (62) Cho, K. B.; Chen, H.; Janardanan, D.; de Visser, S. P.; Shaik, S.; Nam, W. Nonheme IronOxo and -Superoxo Reactivities: O2 Binding and Spin Inversion Probability Matter. Chem. Commun. 2012, 48, 2189–2191.

35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 36

TOC GRAPHICS

36


Insights into the Reaction Mechanism of Aromatic Ring Cleavage by

Recommend Documents