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Unraveling the Crucial Role of Single Active Water Molecule in the Oxidative Cleavage of Aliphatic C-C bond of 2,4#dihydroxyacetophenone Catalyzed by DAD enzyme: A QM/MM Investigation Rabindra Nath Manna, Tanmay Malakar, Biman Jana, and Ankan Paul ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b03201 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 25, 2018

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Unraveling the Crucial Role of Single Active Water Molecule in the Oxidative Cleavage of Aliphatic C-C bond of 2,4ʹDihydroxyacetophenone Catalyzed by DAD Enzyme: A QM/MM Investigation Rabindra Nath Manna1#, Tanmay Malakar2#, Biman Jana1* and Ankan Paul2* 1

Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India Raman Center for Atomic, Molecular and Optical Science, Indian Association for the Cultivation of Science, 2A & 2B Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, India 2

ABSTRACT: 2,4ʹ-dihydroxyacetophenone dioxygenase (DAD), a non-heme dioxygenase enzyme, shows exquisite selectivity in the aliphatic C−C bond cleavage of 2,4ʹ-dihydroxyacetophenone (DHAP) in presence of molecular oxygen (O2). Molecular dynamics simulations revealed the presence of a single water molecule at the active site of the enzyme. This lone water molecule is pivotal for facilitating the oxidative cleavage of the aliphatic C−C bond of 2,4ʹ-DHAP catalyzed by DAD enzyme, as evident from the findings of our hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) studies. The DHAP is initially deprotonated through a relay proton transfer mechanism with the aid of the active site water molecule. This water molecule also actively participates in the O−O and C−C bond cleavage steps. The activated water molecule acts as catalytic acid-base species. The O−O cleavage step has been predicted to be the rate determining step with an associated barrier of 20.3 kcal/mol calculated at the uB3LYP-D3/def2-TZVP/OPLS level of theory on the quintet spin surface. Multiple sequence alignment of the bacterial DAD enzyme has shown the evolutionary importance of the Tyr93 and Glu108 residues which acts as proton carrier and reservoir, respectively, during the relay proton transfer. Our study demonstrates the active role of water in the catalytic cycle of DAD enzyme and additionally unearthed important indirect roles of the two amino acids (Tyr93 and Glu108) in the enzymatic cycle. KEYWORDS: QM/MM Method, C−C Bond Cleavage, Dioxygenase, Non Heme Iron Complexes, Oxygen Activation. 2,4ʹ-dihydroxyacetophenone dioxygenase (DAD), a non-heme iron dioxygenase enzyme, has recently come into prominence as an effective catalyst in the aerobic catabolism of toxic compounds.1-15 Unlike other known dioxygenases,16-20 DAD is unique in its activity as it selectively cleaves the aliphatic C−C bond of the 2,4ʹ-dihydroxyacetophenone (DHAP), a αhydroxyketone intermediate in the biodegradation of the natural aromatic pollutant lignin in the presence of dioxygen (O2) to form environment benign 4-hydroxybenzoic acid and formic acid.21-22 Furthermore, it has been widely used for commercial production of unusual amino acids and manufacture of biodegradable plastics.4 Keegan et al. solved the X-ray crystal structure of DAD enzyme from bacterium Alcaligenes sp. 4HAP 21-22 and they reported that in the active site of DAD an iron center is directly coordinated to three histidine residues (His76, His78, and His114) and in the close vicinity of the active structure there exists one tyrosine (Tyr93) and one glutamate (Glu108) residue. However, due to the lack of strong experimental evidence, information regarding the actual oxidation state of the catalytic iron center was vague until extensive experimental studies were carried out by Paine and co-workers on the biomimetic models of DAD.23-27 An oxidation state of +2 of iron was first suggested by Paria et al.23 which was further affirmed through refinement in crystal structure determination at a higher resolution in 2015.24 Biomimetic studies involving small transition-metal model compounds provide an alternative route to obtain crucial insights for understanding complex mechanism of enzymatic reactions. Paine’s group24 has synthesized and isolated a series of new biomimetic iron(II)-α-hydroxy ketone complexes to elucidate the mechanism of oxidative cleavage reactions of the aliphatic C−C bond of the α-hydroxyketone complexes.25 The X-ray crystal structure of their synthesized iron complex reveals that

the α-hydroxyketone substrate binds to the iron center in a bidentate manner where the hydroxyl group gets deprotonated by the triethylamine, as was originally employed by them during the preparation of the aforementioned iron(II)-α-hydroxyketone coordination compounds. This plausibly has a substantial implication in understanding the binding of DHAP at the catalytic iron center present in the actual DAD enzyme. Moreover, their thorough labeling experiments with 18O2 followed by mass spectrometry study involving simple model complexes indicated that the incorporation of one labeled oxygen atom from 18O2 into each product (40 % incorporation) representing a typical scenario of dioxygenase reactivity. Contrastingly, decades back, Hopper et al. reported higher incorporation (over 90%) of 18O solely from 18O2 (and not from H218O) in the product obtained from the reaction between DAD and (4-hydroxybenzoyl)methanol.28 In a follow-up study, Paine and his co-workers have suggested that the remarkably low incorporation of labeled oxygen atom into the final product is primarily due to the involvement of an iron(II)-hydroxide intermediate which exchanges its oxygen atom with water during the reaction. Moreover, they have pointed out that there might be further loss of labeled oxygen at the stage of the acidic workup of the obtained carboxylic acid derivative product.24 Additionally, based on their extensive experimental observations, they have further proposed that an iron(III)-superoxo intermediate generated from dioxygen binding at the iron(II) center initiates the oxidative C−C bond cleavage transformation and the reaction proceeds through an alkyl-peroxo intermediate following the intradiol mechanism. Interestingly, the mechanism put forward by them involves a catalytic base implicated to play a crucial role in abstracting a proton from the alkyl-peroxo intermediate through acid-base catalysis.24 However, the identity of the catalytic base is still shrouded in mystery.

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Scheme 1: Proposed mechanism for the oxidative aliphatic C−C bond cleavage reaction of 2,4ʹ-DHAP in the active site of DAD enzyme. Tyr93, Glu108 and active site water molecules are considered explicitly. We initiated our investigations by conducting docking studies of the 2,4ʹ-DHAP substrate in the active site of DAD enzyme. In our model, the active site of the enzyme consists of 2,4ʹ-DHAP substrate, one catalytic iron metal ion, three histidine residues (His76, His78, His114), Tyr93 and Glu108, respectively. Three

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imidazole rings of the aforementioned histidine residues are coordinated to the iron. 2,4ʹ-DHAP substrate coordinates iron in a loosely bound bidentate mode using its carbonyl and neutral hydroxyl groups (See Supporting Information for computational details). Our results from a classical molecular dynamics (MD) simulation of the whole enzyme disclosed a sharp peak in the radial distribution function (g(r)) between iron and water at ~4.5 Å distance (Figure S3A). Thus, it strongly suggested that the active center of the enzyme is hydrated. The coordination number plot (Figure S3B) evidently shows that a single water molecule is present in the close vicinity (~4.5 Å) of the iron center. Equilibrium simulations revealed that with the progression of the simulations, the inter-atomic distances of Fe–Ow(H2O) and (2,4ʹDHAP)Hd–Ow(H2O) are decreased to 4.5 Å and 2.2 Å, respectively (Figure S4). Note that, Keegen et al. proposed that Tyr93 forms a hydrogen bond with the α-hydroxy unit of the 2,4ʹ-DHAP substrate to provide stabilization of the intermediates of the reaction.21 We observed that a single water molecule resides in between the Tyr93 residue and 2,4ʹ-DHAP substrate. Interestingly, this water molecule forms hydrogen bonds with the 2,4ʹ-DHAP substrate as well as the Tyr93 residue. Recently, QM/MM studies on Glycoside Hydrolases enzyme showed that the presence of water in active site of the enzyme can influence the barrier of a reaction. Such presence of water in the enzyme pocket was indicated by classical MD simulations.29 Furthermore, Guo et al. proposed that Glu108 residue plays a major role in shuttling oxygen into the active site of DAD.22 Our simulations unearthed that Tyr93 forms hydrogen bond with the carboxylate part of Glu108 and with the specific water molecule. Here, it should be noted that in the biomimetic model complex synthesized by Paria et al., the α-hydroxyketone substrate ligates to the Fe2+ center in the anionic form.23 However, whether the substrate DHAP coordinates to the active metal center in anionic or neutral form is still an open question. In the lone theoretical study, recently Zhang et al. have investigated the mechanisms of the aliphatic C−C bond cleavage of 2,4ʹ-DHAP by the DAD enzyme employing QM/MM methodology.30 Zhang et al. considered the binding of DHAP in the neutral form. The QM region of their investigation is composed of only iron, dioxygen, the substrate (2,4′-DHAP), three histidine residues (His76, His78, His114 respectively), and Tyr93. As mentioned earlier Guo et al. have earlier indicated that Glu108 might play a critical role in the binding of the oxygen molecule at the active iron site.22 Zhang et al. did not take into account glutamate (Glu108) amino acid residue in the QM region of their calculation.30 Their QM/MM studies revealed that an iron(III)-superoxo species is formed upon coordination of O2 at the iron center and the ground state of the resulting intermediate is predicted to be in a triplet state. Following this, they suggested that along the reaction path the complex may undergo triplet-quintet crossing and the subsequent reactions occur mainly on the quintet spin state. Their calculated activation energy barrier for the rate determining step associated with the insertion of oxygen radical into the C–C bond effectuating the rupture of the C–C bond was predicted to be 24.9 kcal/mol. However, they did not conduct any computations to verify whether the proposed mechanism of Paine and co-workers involving an alkylperoxo intermediate in the case of the biomimetic model complex is still valid or not for the actual DAD enzyme. As there is no active catalytic base in their QM/MM model, they concluded that the intradiol pathway suggested by Paine’s group is not operational in the case of the DAD enzyme; rather it may be relevant for the biomimetic complexes only. Recent theoretical investigations of the several enzymatic reactions suggested that water in the primary solvation sphere plays a crucial role in enzymatic catalysis.29,31-37 Encouraged by these findings we investigated the prospective presence of

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water molecule in the DAD active site. The lack of proper description of the active site of the enzyme and the contrasting results obtained by Zhang et al. prompted us to investigate further to look for any conceivable role of water molecules which might be present in the active site in facilitating the C−C bond cleavage of 2,4ʹ-DHAP by the DAD enzyme. In the present venture through combined QM/MM calculations, we have tried to explore the possibility of the binding of the 2 ,4 ʹ - DHAP to the active site of DAD enzyme in the deprotonated form as noticed in the biomimetic studies carried out by Paine’s group. The mechanistic picture emerging from our extensive calculations suggested that the single active water molecule present in the close vicinity of the active site of the DAD enzyme has crucial implication in both the binding of the substrate in the anionic form and the subsequent C–C bond cleavage reaction through acid-base catalysis (Scheme 1).

moved our attention to the binding of dioxygen to the active metal center. For 1A the septet and the quintet states are predicted to be the low lying ones with the quintet state 5.3 kcal/mol higher in energy with respect to the septet state. It must be noted that the B3LYP functional due to the presence of high percentage of exact exchange often erroneously tends to overestimate the stability of high spin complexes of Fe2+ compared to the lower spin species.38 We found that the quintet state becomes more stable than the high spin septet state by ~2 kcal/mol if a modified version of B3LYP (uB3LYP*-D3/def2-TVZP/OPLS) with reduced exchange is used, as has been suggested earlier in similar cases 39-41 (Table S2).

Figure 2. Potential energy (in kcal/mol) profile for the C–C bond cleavage reaction of 2,4ʹ-DHAP catalyzed by DAD enzyme obtained at the uB3LYP-D3/def2-TVZP/OPLS level of theory on the quintet spin state.

Figure 1. Transition state structure (TS12) for the deprotonation of DHAP substrate is presented. Key distances are given in Å. The coupling between triplet O2 and a quintet Fe2+ can give rise to three possible spin states, such as triplet, quintet, and septet, we took into consideration of all the three possible energy profiles for the aliphatic C–C bond cleavage reaction of the 2,4ʹ-DHAP. The relative potential energies for all the stationary points of this catalytic reaction at the quintet, triplet and septet spin states are shown in Figures S7, S13, and S15. Active water not only stabilizes the native structure of biological macromolecules but also dictates their functions. Herein, we report that indeed a single active water molecule has an immense importance in the biodegradation of 2,4ʹ-DHAP by the DAD enzyme. We witnessed that the proton (Hd) of t h e α-hydroxyl unit of the 2,4ʹ-DHAP substrate is transferred to carboxylic unit of Glu108 through a relay proton transfer mechanism via TS12 (Figure 1) involving a paltry activation barrier of 1.6 kcal/mol on the quintet spin state and forms the intermediate (1A) (Figure 2 and Figure S7). The transformation of ( 1 ) to ( 1A) is predicted to be stabilized by 27.7 kcal/mol on the septet spin surface. Our calculations clearly suggested that the binding of DHAP to the Fe2+ center is preferred in the anionic form compared to the neutral form which is consistent with the results obtained from the biomimetic studies by Paine’s group.2326 In (1A), Fe is still not bound with the dioxygen (Fe–Oa: ~6 Å). After resolving the coordination preference of 2,4ʹ-DHAP we

After deprotonation of 2,4ʹ- DHAP substrate, dioxygen binds to the sixth coordination site of catalytic iron in an end-on fashion to form iron(III)-superoxo radical complex (2). Fe(III)-superoxo radical complex (2) has slightly higher energy compared to intermediate (1A) at the uB3LYP-D3/def2-TVZP/OPLS level of theory42-45 using the micro-macro iteration scheme46-47 implemented in the fDYNAMO library48 which was successfully used to study several complex enzymatic reactions.49-52 The optimized structure of (2) in the quintet spin state displays a Fe– Oa inter-atomic distance of 2.02 Å which is slightly lower than the corresponding triplet state (2.04 Å) (Figure S7). Interestingly, the structure ( 2) is not a stable minimum on the septet spin state surface. Moreover, we observed that the O2 is far away from iron (Fe–Oa: 3.44 Å), which clearly indicates that t h e catalytic iron is not competent to bind the dioxygen in the septet spin state in accordance with earlier findings.30 However, contrary to the observation of Zhang et al., our computational results indicate that the Fe(III)-superoxo intermediate (2) is 6.5 kcal/mol more stable in the quintet state compared to the triplet state. Our computed triplet-quintet gap of Fe–O2 species in the case of DAD enzyme follows the similar trend with other non-heme iron dioxygenases, for instance, HPCD,53-54 CDO55 and SDO.17,56-58 Here, it should be mentioned again that Zhang et al. considered the Fe(III)-superoxo 2,4ʹDHAP (in the protonated form). Therefore, the choice of a proper computational model, that is the incorporation of single active site water molecule, Glu108 and the coordination of DHAP in the anionic form have a significant impact on the determination of the electronic structure of the species generated in the due course of the aliphatic C–C bond cleavage reaction of the 2,4ʹ-DHAP by DAD enzyme. It has been earlier shown that the hydrogen bond interactions exert significant influence on the energetic of the spin states. Shaoo et al. reported that hydrogen bonding interactions can play an important role to trigger the spin state change of iron(III)-porphyrin complexes.59 In agreement with the previous findings, we found that the participation of the hydrogen bond dictates the spin states of the reacting

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species and we found that in the DAD system the reaction exclusively happens on the quintet surface after binding of dioxygen by the iron center. Following this, we found that the distal oxygen radical of (2) abstracts a hydrogen atom from the α-carbon of the substrate and converts into Fe(III)-hydroperoxide radical species (3). The formation of similar radical species (such as 2 and 3) in the reaction pathway of 2,4ʹ-DHAP was also proposed by Paria et al. based on their experimental observations.23 The activation energy barriers for the formation of radical Fe(III)hydroperoxide complex (3) is estimated to be 17.7 in the quintet spin state at the uB3LYP-D3/def2-TVZP/OPLS level of theory (Figure 2). The C1–H1 and Ob–H1 bond distances at the TS23 structure are 1.32 and 1.31 Å respectively, which implicate that H1 atom is shifted from C1 to Ob. (Figure S7 and Figure S12) The formation of Fe(III)-hydroperoxo intermediate (3) was predicted to be exothermic in both the quintet (-26.7 kcal/mol) and the triplet (-20.0 kcal/mol) spin states. Recently, Rahaman et al. based on their biomimetic studies involving six biomimetic iron(II)-α-hydroxyketone complexes proposed a mechanism where they pointed out that a catalytic base is required to abstract a proton from the Fe–OOH intermediate; however, they did not shed any light on the identity of the catalytic base.24,30

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again gets deprotonated (Figure S13 and Figure S14). Thus, the single active water molecule is engaged in acid-base catalysis on three different occasions to facilitate the catalytic event. During this step, the Fe–O bond is cleaved completely and the coordination number of iron changes from six to five. Next, the Oa–Ob bond cleaves homolytically via TS56 where, Oa–Ob bond distances are 2.06, 1.87 and 2.24 Å, respectively, in the quintet, triplet, and septet spin states (Figure 4 and Figure S13). Water molecule forms two hydrogen bonds in the TS56 (H1–Ow: 1.85 and Hw–O1: 1.78 Å) and thus, stabilizes the transition state structure. The activation energy barrier for TS56 is predicted to be 20.3 kcal/mol and the asso ciated Oa–Ob bond cleavage process is exothermic by 44.3 kcal/mol (Figure 2).

Figure 4. Snapshot of the transition state structure (TS56) for the O–O bond cleavage event is displayed. Key distances are given in Å.

Figure 3. Obtained TS34 structure for the formation of alkylperoxo intermediate (4) with key distance (Å) is shown here. Our QM/MM computations discloses that indeed, the single active molecule present in the vicinity of the active structure can act as catalytic base to abstract a proton from the Fe–OOH moiety and simultaneously shuttles a proton to the deprotonated αhydroxy unit of DHAP to generate the alkylperoxo intermediate (4) through TS34 associated with an activation energy barrier of 16.8 kcal/mol (Figure 2). The formation of alkylperoxo intermediate was also proposed by Rahaman et al.24 The formation of ( 4) is 38.5 kcal/mol downhill in the potential energy landscape of the quintet surface and involves a new bond formation between the radical α carbon (C1) atom of the 2,4ʹDHAP substrate and distal oxygen (Ob). In the TS34, the Oa−Ob bond is elongated to 1.49 Å in the quintet surface (Figure 3). Subsequently, we found that the single active water molecule assists in the complete shift of the –OOH moiety to the α carbon (C1) of the 2,4ʹ-DHAP substrate to form the intermediate (5) in which the α-hydroxy unit of DHAP substrate

In the intermediate (6), Fe is attached with −OH unit (bond length ~ 1.89 Å) and the coordination number of iron thus again becomes six. However, we did not observe the intradiol pathway for the O−O bond cleavage process. Next, the Ob atom of the intermediate (6) is inserted between C1−C2 bond in the 2,4ʹ-DHAP via transition state TS67 associated with an energy barrier of 7.1 kcal/mol and results into the formation of the intermediate (7). The obtained barrier of Ob atom insertion between C1−C2 bond is significantly lower than the value obtained by Zhang et al. (24.9 kcal/mol).30 The optimized structure of TS67 depicted in Figure 5 shows that the C1−C2 bond, Ob−C2 bond, and Ob−C1 bond distances are 1.77, 1.81 and 1.33 Å, respectively, which clearly indicates that C1−C2 bond is broken and Ob−C2 bond is simultaneously formed. Similar threemembered ring transition state structure for C−C bond cleavage has recently been reported by Zhang et al.30 Subsequently, −OH group from Fe(III) atom is transferred to the C1 atom through TS78 to generate the intermediate (8) (Figure S15 and Figure S16). The estimated activation energy barrier for TS78 is gratifyingly low (2.5 kcal/mol) and the formation of species (8) is extremely exothermic (-134.6 kcal/mol). Next, C1−Ob bond of the intermediate (8) is cleaved to generate the intermediate (9) via transition state TS89. The complex (9) is composed of formic acid and 4-hydroxybenzoate, respectively. The bond

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distance of Ob−C1 is 2.13 Å in the TS89 which indicates that the Ob−C1 bond is ruptured (Figure S17). In the potential energy profile, the species ( 9) lies slightly above in energy compared to its forerunner ( 8). Hence, the activation barrier for the Oa−Ob bond cleavage step in the quintet state is 20.3 kcal/mol at the uB3LYP-D3/def2-TZVP/OPLS level of theory which is the possible rate determining step for the oxidative cleavage of the aliphatic C−C bond of 2,4ʹ-DHAP catalyzed by DAD enzyme (Figure 2). Furthermore, Zhang et al. predicted the insertion of oxygen radical into the C–C bond fostering the cleavage of the C–C bond with the activation energy barrier of 24.9 kcal/mol is the rate determining step.30 However, our finding asserted that the presence of water molecule in the active site of DAD enzyme can alter the rate determining step. Thus, the predicted rate constant (kcat) for this reaction from our calculations is three order times faster than the obtained value from Zhang et al. (see Supporting information). Our study also revealed that the quintet state is the most favorable spin surface for the entire reaction. During the whole reaction, Tyr93 forms hydrogen bonds with Glu108 and single active water, which plays a crucial role in helping the catalytic reaction through the stabilization of the stationary and transition state complexes. More precisely, Tyr93 and single water molecule act as proton carriers and Glu108 behave as a proton reservoir during the reaction. Here it has to be mentioned that the Tyr93 and Glu108 residues are found to be highly conserved among several bacteria species (Figure S6), evident from the multiple sequence alignments (MSA). The detailed procedure of MSA analysis was described in our earlier study on conformational transition in Apo-Adenylate Kinase.60 This result indicates that those two residues have a crucial functional role in the catalysis. This is in accordance with our proposed mechanism.

ly accentuates the role of water in the enzyme active site in facilitating the oxidation reactions. Herein, we underscore the importance of the non-innocent role of water molecules in the metalloenzyme active site, which was only revealed after we conducted a classical molecular dynamics investigation to understand the actual nature of the active site in the presence of solvent water molecules. Moreover, new mechanistic channels were unraveled, which would have remained concealed if our QM/MM studies would have relied solely on the crystal structural data. Thus our findings re-emphasize the crucial requirement of carrying out such classical MD simulations in presence of solvent molecules prior to conducting QM/MM studies.29,35,49-52,61-64 Essentially, our findings spell out the definitive requirement for investigating the hydration state of the active site of the metalloenzymes before conducting theoretical mechanistic investigations as water molecules if present can exert a non-trivial impact on the reaction pathways. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Computational methodology, plots of radial distribution, inter atomic distances, Potential energy profiles on the quintet, triplet and septet spin states, snapshots of the transition state structures, multiple sequence alignment (MSA) analysis and Cartesian coordinates of QM region of all optimized structures. AUTHOR INFORMATION Corresponding Authors *Email: [email protected] (A. Paul) *Email: [email protected] (B. Jana) Author Contributions #

RNM and TM contributed equally to this work.

Funding Sources RNM is thankful to Indian Association for the Cultivation of Science for providing the fellowship. TM thanks to IACS for providing fellowship. ABBREVIATIONS DHAP, dihydroxyactophenone; DAD, 2,4ʹ-dihydroxyacetophenone dioxygenase; QM/MM, Quantum Mechanics/Molecular Mechanics. ACKNOWLEDGMENT The authors gratefully acknowledge the central supercomputing facility at Indian Association for the Cultivation of Science, Kolkata, for providing computational resources. REFERENCES Figure 5. Transition state structure (TS67) for the formation complex (7) is obtained at the uB3LYP-D3/def2-TVZP/OPLS level of theory. Key distances are given in Å. We have found that even the presence of single water molecule can have a telling effect on most of the reaction steps, such as the deprotonation of 2,4ʹ-DHAP substrate, Oa−Ob, and C1−C2 bond cleavage steps. Thus, our QM/MM investigation unraveled an alternate mechanistic perspective from the previous theoretical study on the mechanism of the oxidative cleavage of the aliphatic C−C bond of 2,4ʹ-DHAP catalyzed by DAD enzyme and strong-

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[24] Rahaman, R.; Paria, S.; Paine, T. K. Aliphatic C–C Bond Cleavage of α-Hydroxy Ketones by Non-Heme Iron(II) Complexes: Mechanistic Insight into the Reaction Catalyzed by 2,4′Dihydroxyacetophenone Dioxygenase. Inorg. Chem. 2015, 54, 10576-10586. [25] Chatterjee, S.; Paine, T. K. Hydroxylation versus Halogenation of Aliphatic C−H Bonds by a Dioxygen‐Derived Iron–Oxygen Oxidant: Functional Mimicking of Iron Halogenases. Angew. Chem. Int. Ed. 2016, 55, 7717-7722. [26] Bhattacharya, S.; Rahaman, R.; Chatterjee, S.; Paine, T. K. Aliphatic C−C Bond Cleavage in α‐Hydroxy Ketones by a Dioxygen‐Derived Nucleophilic Iron–Oxygen Oxidant. Chem.-Eur. J. 2017, 23, 3815 –3818. [27] Sheet, D.; Paine, T. K. Aerobic alcohol oxidation and oxygen atom transfer reactions catalyzed by a nonheme iron(II)–α-keto acid complex. Chem. Sci. 2016, 7, 5322–5331. [28] Hopper, D. J. Oxygenase properties of the (4-hydroxybenzoyl) methanol-cleavage enzyme from an Alcaligenes sp. Biochem. J. 1986, 239, 469-472. [29] Borišek, J.; Pintar, S.; Ogrizek, M.; Turk, D.; Perdih, A.; Novič. M. A Water-Assisted Catalytic Mechanism in Glycoside Hydrolases Demonstrated on the Staphylococcus aureus Autolysin E. ACS Catal. 2018, 8, 4334−4345. [30] Zhang, S.; Wang, X.; Liu, Y. Cleavage mechanism of the aliphatic C– C bond catalyzed by 2,4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP: a QM/MM study. Catal. Sci. Technol. 2017, 7, 911-922. [31] Vohringer-Martinez, E. V.; Toro-Labbe, A. The role of water in the proton transfer reaction mechanism in tryptophan. J. Comput. Chem. 2010, 15, 2642-2649. [32] Chaplin, M. Do we underestimate the importance of water in cell biology? Nat. Rev. Mol. Cell. Biol. 2006, 7, 861-866. [33] Adkar, B. V.; Jana, B.; Bagchi, B. Role of Water in the Enzymatic Catalysis: Study of ATP + AMP → 2ADP Conversion by Adenylate Kinase. J. Phys. Chem .A 2011, 115, 3691–3697. [34] Duarte, F.; Barrozo, A.; Åqvist, J.; Williams, N. H.; Kamerlin, S. C. L. The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis. J. Am. Chem. Soc. 2016, 138, 10664–10673. [35] Mondal, D.; Warshel, A. EF-Tu and EF-G are activated by allosteric effects. Proc Natl Acad Sci U S A. 2018, 115, 3386-3391. [36] Krewald, V.; Retegan, M.; Cox, N.; Messinger, J.; Lubitz, W.; DeBeer S.; Neese, F.; A Pantazis, D. A. Metal oxidation states in biological water splitting. Chem. Sci. 2015, 6, 1676-1695. [37] Lohmiller, T.; Krewald, V.; Sedoud, A.; Rutherford, A. V.; Neese, F.; Lubitz, W.; Pantazis, D. A.; Cox, N. The First State in the Catalytic Cycle of the Water-Oxidizing Enzyme: Identification of a Water-Derived µ-Hydroxo Bridge. J. Am. Chem. Soc. 2017,139, 14412-14424. [38] Reiher, M. Theoretical Study of the Fe(phen)2(NCS)2 SpinCrossover Complex with Reparametrized Density Functionals. Inorg. Chem. 2002, 41, 6928-6935. [39] 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. [40] Borgogno, A.; Rastrellia, F.; Bagno, A. Predicting the spin state of paramagnetic iron complexes by DFT calculation of proton NMR spectra. Dalton Trans. 2014, 43, 9486-9496. [41] Domarus, M.; Kuznetsov, M. L.; MarÅalo, J.; Pombeiro, A. J. L.; da Silva, J. A. L. Amavadin and Homologues as Mediators of Water Oxidation. Angew Chem Int Ed. 2016, 55, 1489-1492. [42] Grimme, S.; Ehrlich, S.; Goerigk, L. Effect of the damping function in dispersion corrected density functional theory. J. Comput. Chem. 2011, 32, 1456-1465. [43] Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, S. 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, 154104-154123.

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ACS Catalysis 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

Proposed mechanism for the oxidative aliphatic C−C bond cleavage reaction of 2,4ʹ-DHAP in the active site of DAD enzyme. Tyr93, Glu108 and active site water molecules are considered explicitly. 262x645mm (300 x 300 DPI)

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Page 9 of 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

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Transition state structure (TS12) for the deprotonation of DHAP substrate is presented. Key distances are given in Å. 84x95mm (200 x 200 DPI)

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ACS Catalysis 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

Potential energy (in kcal/mol) profile for the C–C bond cleavage reaction of 2,4ʹ-DHAP catalyzed by DAD enzyme obtained at the uB3LYP-D3/def2-TVZP/OPLS level of theory on the quintet spin state. 137x77mm (200 x 200 DPI)

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Page 11 of 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

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Obtained TS34 structure for the formation of alkylperoxo intermediate (4) with key distance (Å) is shown here. 87x98mm (200 x 200 DPI)

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ACS Catalysis 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

Snapshot of the transition state structure (TS56) for the O–O bond cleavage event is displayed. Key distances are given in Å. 101x100mm (200 x 200 DPI)

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Page 13 of 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

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Transition state structure (TS67) for the formation complex (7) is obtained at the uB3LYP-D3/def2-TVZP/OPLS level of theory. Key distances are given in Å. 102x101mm (200 x 200 DPI)

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