Mechanism of tungsten-dependent acetylene hydratase from quantum chemical calculations

Rong-Zhen Liaoa,b, Jian-Guo Yub, and Fahmi Himoa,1

aDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-10691 Stockholm, Sweden; and bCollege of Chemistry, Beijing Normal University, Beijing, 100875, People’s Republic of China

Edited* by Arieh Warshel, University of Southern California, Los Angeles, CA, and approved November 12, 2010 (received for review September 20, 2010)

Acetylene hydratase is a tungsten-dependent enzyme that cata- lyzes the nonredox hydration of acetylene to acetaldehyde. Density functional theory calculations are used to elucidate the reaction mechanism of this enzyme with a large model of the active site devised on the basis of the native X-ray crystal structure. Based Scheme 1. Reaction catalyzed by AH. on the calculations, we propose a new mechanism in which the acetylene substrate first displaces the W-coordinated water mole- The catalytic mechanism of AH is presently poorly under- cule, and then undergoes a nucleophilic attack by the water mole- stood. The mechanistic proposals can be divided into two groups, cule assisted by an ionized Asp13 residue at the active site. This is first- and second-shell mechanisms. Based on the crystal struc- followed by proton transfer from Asp13 to the newly formed vinyl ture, Seiffert et al. proposed a second-shell mechanism in which anion intermediate. In the subsequent isomerization, Asp13 shut- the W-bound water functions as an electrophile to deliver a pro- tles a proton from the hydroxyl group of the vinyl alcohol to the ton to the acetylene substrate (9). In this mechanism, Asp13 is α-carbon. Asp13 is thus a key player in the mechanism, but also W assumed to be protonated and donate a hydrogen bond to the is directly involved in the reaction by binding and activating water molecule, which would stabilize the negative charge on the acetylene and providing electrostatic stabilization to the transition water/hydroxide during the proton transfer. On the other hand, states and intermediates. Several other mechanisms are also con- using density functional theory (DFT) calculations on a small BIOPHYSICS AND sidered but the energetic barriers are found to be very high, ruling model consisting of the tungsten and its first-shell ligands, Antony out these possibilities. and Bayse have shown that the displacement of water by an COMPUTATIONAL BIOLOGY acetylene molecule is exothermic by more than 20 kcal∕mol (13). enzyme catalysis ∣ metalloenzyme ∣ cluster approach Based on this, they suggested that the water molecule performs a nucleophilic attack on the W-bound acetylene, assisted by the ungsten is the heaviest metal in biology and plays prominent neutral Asp13 residue. Most recently, Vincent et al. showed, also roles in carbon, nitrogen, and sulfur metabolisms (1–6). In using DFT calculations, that both these mechanisms have very

T CHEMISTRY all known tungstoenzymes, the tungsten ion coordinates to the high barriers, more than 40 kcal∕mol, ruling them out (14). In- dithiolene group of two pterin cofactors and has oxidation num- stead, they speculated that the reaction could proceed through a ═ ═ bers ranging from þ4 to þ6. Three families of enzymes have been W C CH2 vinylidene intermediate. However, the barrier for the discovered to be tungsten-dependent, namely aldehyde oxidore- formation of this intermediate and also the barriers for the fol- 34 0 ∕ ductase, formate dehydrogenase, and acetylene hydratase (AH) lowing steps were found to be quite high (28.1 and . kcal mol, (1). The former two are involved in oxidative reactions, whereas respectively), suggesting that some other mechanism is in action. the third one catalyzes the nonredox hydration of acetylene In recent years, quantum chemical methods have proven (Scheme 1) in the anaerobic unsaturated hydrocarbon metabo- very successful in the study of enzymatic reaction mechanisms – – lism (7). This reaction is exothermic by about 27 kcal∕mol, and (15 19). In particular, the mechanisms of Mo- (20 24) and W-de- the resulting acetaldehyde can be further oxidized to provide a pendent (13, 14) enzymes have been investigated and a wealth of significant source of carbon and energy for certain bacteria (8). mechanistic insight has been obtained, especially on the role of The crystal structure of AH from Pelobacter acetylenicus has the metal ions in the reactions. Here, density functional calcula- been solved by Seiffert et al. at 1.26-Å resolution, and it reveals tions are employed to elucidate the mechanism of AH. On the a mononuclear tungsten center in the active site and a nearby basis of the crystal structure, a large model of the active site is iron–sulfur cubane cluster (9). In the active site, the tungsten designed, and the hybrid functional B3LYP (25) is used to calcu- ion is ligated by the four sulfur atoms of the two pterin dithiolene late the potential energy profiles for several mechanistic scenar- moieties, Cys141, and an oxygen species, which was assigned to ios. We propose a previously undescribed mechanism involving be a water molecule. A second-shell residue, Asp13, is found to five steps, which all have feasible energy barriers. All other hydrogen-bond to the oxygen species and was suggested to be in mechanisms considered are found to be associated with much the protonated form, based on pH titration calculations using higher barriers. continuum electrostatics and statistical thermodynamics (9). Results and Discussion However, the ionized form of Asp13 cannot be ruled out because Active Site Model. the crystal structure shows that it has two additional hydrogen A model of the active site (Fig. 1) was designed on the basis of the crystal structure of native AH (PDB ID bonds, to the peptide bond of Cys12-Asp13 and the side chain K code 2E7Z) (9). The model comprises the tungsten ion with of Trp179, which would lower the p a significantly. An arginine residue (Arg606) is also located close to the two pterins, provid- ing electrostatic stabilization to the cofactors. Based on studies of Author contributions: R.-Z.L., J.-G.Y., and F.H. designed research; R.-Z.L. performed biomimetic complexes of AH (10) and redox titration (11), it has research; R.-Z.L. and F.H. analyzed data; and R.-Z.L. and F.H. wrote the paper. been demonstrated that WIV participates in the catalysis, whereas The authors declare no conflict of interest. VI W is inactive. Molybdenum, tungsten’s lighter congener, can *This Direct Submission article had a prearranged editor. also be incorporated into the active site, but it results in 60% 1To whom correspondence should be addressed. E-mail: [email protected]. – activity (12). The iron sulfur cluster is not required for enzyme This article contains supporting information online at www.pnas.org/lookup/suppl/ activity if strong reducing agents are available (11). doi:10.1073/pnas.1014060108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1014060108 PNAS Early Edition ∣ 1of5 Downloaded by guest on September 30, 2021 its first-shell ligands, including two pterin molecules, Cys141, and At React, the water molecule coordinates to the tungsten ion a water molecule. The peptide chains formed between Cys141 with a distance of 2.26 Å. The coordination of the WIV ion is close and Met140 as well as between Cys141 and Ile142 were also to octahedral, which reproduces the X-ray structure (9). At included to prevent rotation of Cys141. Several second-shell re- Inter1, the acetylene molecule binds to the tungsten ion with sidues, Cys12, Asp13, Trp179, and Arg606 were also included. two equal W-C distances (2.04 Å). Acetylene acts as a 4e donor The amino acid residues and the pterin cofactors were truncated through its two π-orbitals interacting with two empty d orbitals so that in principle only the functional groups, extended by one or of tungsten (29). In addition, the occupied d orbital of tungsten π two carbons, were kept in the model; see Fig. 1. Hydrogen atoms donates two electrons to one of two unoccupied *-orbitals of ─ were added manually. The catalytically important Asp13 residue acetylene (13). This back donation weakens the C C bond, was modeled in the ionized form. It is engaged in three hydrogen resulting in an increase of the bond length, from 1.21 Å in React ─ ─ ca. bonds, with the peptide bond of Cys12-Asp13, Trp179, and the to 1.30 Å in Inter1. Also, the two H C C angles are bent to 140° in Inter1. Furthermore, we can see that one of the remaining tungsten-bound water molecule. These interactions will lower d the pK of Asp13 considerably, which could render the residue unoccupied orbitals of tungsten interacts with the other unoc- a cupied π*-orbital of acetylene to form a δ-like orbital, which can in the ionized form. Rough pK estimation using PROPKA (26) a facilitate the electron transfer to this empty molecular orbital gives a value of 6.3, further corroborating this idea. As will be during a nucleophilic attack on the acetylene (next step). This discussed in the final section of the paper, we have also consid- analysis shows that the d2 configuration of the metal is required ered a number of mechanisms involving a neutral Asp13, but they to bind and weaken the triple bond and also to facilitate the nu- were all associated with much higher barriers. To keep the opti- cleophilic attack, which could explain why only WIV and MoIV mized structures resembling the experimental one, the truncation can be used in enzymatic acetylene activation. Detailed orbital atoms were kept fixed at their X-ray positions during the geome- interaction diagram for Inter1 is given in SI Appendix. try optimizations. This is a standard procedure in the cluster At Inter1 the substrate is thus set up for a nucleophilic attack approach for modeling enzymes (27, 28). The locked atoms are by the displaced water molecule, which can be activated by a indicated by asterisks in the figures below. The total size of the proton transfer to Asp13. The optimized structures of the con- model is 116 atoms and the overall charge is −1 certed nucleophilic attack-proton transfer transition state (TS1) and the resulting vinyl anion intermediate (Inter2) are shown in Reaction Mechanism. To arrive at a mechanism that has plausible Fig. 2. The barrier for this step (Step 2) is calculated to be energies and barriers, we examined a number of mechanistic 16.9 kcal∕mol (19.8 kcal∕mol without inclusion of solvation scenarios. We first describe the results concerning the preferred effects) relative to Inter1, whereas Inter2 is 17.0 kcal∕mol reaction mechanism (summarized in Scheme 2). Some of the (17.8 kcal∕mol without solvation) higher than Inter1, i.e., essen- other alternative mechanisms that are shown to have higher tially equienergetic with TS1. At TS1, the critical O-C1 distance is barriers will be discussed at the end. 1.88 Å and the proton is transferring from water to Asp13 with The mechanism starts with a ligand exchange step (Step 1, H-Owater and H-OAsp13 distances of 1.05 and 1.43 Å, respectively. Scheme 2), in which the acetylene substrate displaces the It is very interesting to note that the nucleophilic attack takes tungsten-bound water molecule and binds to the metal in an η2 place nearly perpendicularly to the W─C─C plane, indicating fashion (see Fig. 2 for optimized structures). In the current active the involvement of the empty δ-like orbital resulting from the site model, this step (from React to Inter1) is calculated to be d-π interaction discussed above. exothermic by 5.4 kcal∕mol, already here suggesting an energetic Because of the negative charge developed at the vinyl anion preference for the first-shell mechanism. We have not specifically intermediate in Inter2, the two carbons now are bound asymme- calculated the transition state for this ligand exchange step, trically to the tungsten ion, with W-C1 and W-C2 distances of 1─ 2 but the barrier is expected to be quite low because preliminary 2.17 and 1.93 Å, respectively. The C C bond is elongated calculations show that full dissociation of the water molecule re- from 1.30 Å in Inter1 to 1.35 Å in TS1 and 1.40 Å in Inter2. quires less than 9 kcal∕mol. From Inter2, the protonated Asp13 can now function as a general acid to deliver a proton to the C2 center of the vinyl anion, creating a vinyl alcohol intermediate, Inter3 (Step 3, Scheme 2). This turns out to be the rate-limiting step of the reaction. The barrier is 6.0 kcal∕mol relative to Inter2, and the accumulated barrier relative to Inter1 is thus 23.0 kcal∕mol. Step 3 is very exothermic, by 28.5 kcal∕mol. At the transition state, TS2, the key distances of the proton to OAsp13 and C2 are 1.10 and 1.58 Å, respectively. The proton transfer neutralizes the negative charge on C2, weakening thus the W─C2 bond, which is elongated from 1.93 Å in Inter2 to 2.02 Å in TS2 and 2.33 Å in Inter3. At Inter3, the vinyl alcohol coordinates weakly to the tungsten ion, with only one of the two carbon atoms, and the hydroxyl group forms a rather short hydrogen bond to the ionized Asp13 residue (O⋯H distance of 1.53 Å). To complete the reaction, the vinyl alcohol now needs only to tautomerize to acetaldehyde. At this point, the vinyl alcohol can be released and the interconversion can take place outside the active site. However, we have here also examined how it could be affected at the tungsten center. The tautomerization can be achieved with the help again of Asp13 over two steps (Steps 4 and 5, Scheme 2), first a proton transfer to Asp13 via TS3 to form an enolate (Inter4) and then the back delivery of the proton to Fig. 1. Optimized structure of the active site model of AH. Atoms marked C2 via TS4 to form the acetaldehyde (Prod). The barrier for Step with asterisks were fixed at their X-ray structure positions during the geome- 4is9.3 kcal∕mol, whereas the barrier for Step 5 is 14.1 kcal∕mol. try optimizations. Distances are given in angstrom (Å). We note that once the proton is transferred to the aspartate, the

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Scheme 2. Suggested mechanism for acetylene hydratase based on the present calculations. COMPUTATIONAL BIOLOGY

enolate binds to the tungsten ion with the oxygen atom instead of the carbons (W─O distance of 2.10 Å at Inter4). The final pro- duct complex Prod is calculated to be 13.0 kcal∕mol lower than Inter1. The overall potential energy graph for all steps is given CHEMISTRY in Fig. 3. In contrast to previous mechanistic suggestions, this mechan- ism has thus feasible barriers. It requires the Asp13 residue to be in the ionized form, and as discussed above, this is quite reason- able considering that it is hydrogen-bonded to three other groups in the active site.

Alternative Mechanisms. In this section we briefly discuss some other mechanisms that we also investigated and that turned out to have high barriers. The mechanism proposed above (Scheme 2) requires that the Asp13 is in the ionized form to

Fig. 2. Optimized structures of the intermediates and transition states along the reaction pathway. For clarity, only the core of the model is shown. For full Fig. 3. Calculated potential energy profile for the mechanism proposed in model, see Fig. 1. this study.

Liao et al. PNAS Early Edition ∣ 3of5 Downloaded by guest on September 30, 2021 Conclusions We have in this work designed a large model of the active site of tungsten-dependent acetylene hydratase and used density func- tional theory calculations to explore its reaction mechanism. Based on these calculations, we have proposed a previously un- described, energetically feasible reaction mechanism. The me- chanism assumes that the important active site residue Asp13 is in the ionized form and can act as a general base in the reaction. The reaction starts with the displacement of the water mole- cule bound to the WIV ion by the acetylene substrate. Asp13 then deprotonates the water molecule, which performs a nucleophilic attack on the acetylene, resulting in a vinyl anion stabilized by Fig. 4. Potential energy profile and optimized structures for electrophilic coordination to the metal. The proton is then transferred to attack by neutral Asp13. For full active site model, see Fig. 1. the vinyl anion, generating a vinyl alcohol intermediate. This step is calculated to be rate-limiting, associated with a barrier of start with. All previous suggestions considered Asp13 to be 23.0 kcal∕mol. The two final steps involve tautomerization of neutral. We have examined three different mechanisms with a the vinyl alcohol to aldehyde, with help again of Asp13. In this neutral Asp. mechanism, both Asp13 and the tungsten metal have thus central First, in the case of neutral Aps13, the initial ligand exchange roles. The aspartate is essential for activating the nucleophilic step (React’ to Inter1’; see Fig. 4), in which the acetylene sub- water and for shuttling the proton, whereas the metal is essential strate displaces the tungsten-bound water, is now exergonic by for binding and activating the acetylene substrate and for stabi- as much as 21.2 kcal∕mol (19.9 kcal∕mol without solvation). This lizing the anionic intermediates and transition states. We have 2 is quite close to the result of Antony and Bayse (23.7 kcal∕mol) discussed that the d configuration of the metal is of importance (13), but quite different from the 8.8 kcal∕mol found by Vincent for this mechanism, which could explain why only WIV or MoIV et al. (14). The difference could originate from the size of the can be used for enzymatic acetylene activation. Finally, all other model employed in the calculations. This large driving force mechanisms examined assuming a protonated Asp13, both first- for substrate binding at the metal indicates a first-shell mechan- and second-shell variants, have too high barriers. ism also here. When Asp13 now is protonated, and cannot func- Methods tion as a general base as in our suggested mechanism, it could instead act as an electrophile to protonate the tungsten-bound The quantum chemical calculations presented herein were accomplished acetylene, as suggested by Vincent et al. (14). With their model, with the B3LYP functional as implemented in the Gaussian03 program 28 0 ∕ package (31). For geometry optimizations, the LANL2TZ(f) (32) pseudopoten- the barrier was calculated to be . kcal mol for the first step. tial was used for W, 6-311 þ GðdÞ for S, and 6-31Gðd;pÞ for the C, N, O, H Here, we also considered this possibility to investigate whether elements. Based on these optimized geometries, single-point calculations the model size can affect the energetics. We have located the were performed using larger basis sets, where all elements, except W, were transition state for the proton transfer from Asp13 to C1 (TS’, described by 6-311 þ Gð2d;2pÞ. Fig. 4). It turns out that concertedly with this proton transfer, The polarization effects of the protein surrounding on the active site the Cys141 ligand performs a nucleophilic attack on C2, and thus model were taken into account by performing single-point calculations on no WVI-vinyl anion (or WIV-vinyl cation) intermediate could the optimized structures using the conductor-like polarizable continuum be located using the larger active site model employed here. The model method (33). The dielectric constant was set to 4. Recent studies on barrier is calculated to be 31.6 kcal∕mol, providing additional several different classes of enzymes from our group have shown that the dielectric effects from the environment decline as the model size increases evidence that this pathway is not a viable option. (34–36). When the chosen model contains ca. 150–200 atoms, the solvation Another option is that the W-bound acetylene undergoes ═ ═ effects almost vanish and the choice of the dielectric constant becomes less a [1,2] hydride shift to directly form the W C CH2 vinylidene significant. intermediate. In a theoretical study on the tungsten-catalyzed Analytic frequency calculations were carried out at the same level of the- cycloisomerization of 4-pentyn-1-ol, the barrier for a similar ory as the geometry optimizations to obtain zero-point energies (ZPE) and to 0 hydride shift catalyzed by W ðCOÞ5 was calculated to be establish the nature of the various stationary points. Several atoms were kept 31.0 kcal∕mol (30). In our active site model (with an WIV ion) fixed to their crystal structure positions during the geometry optimizations, we found that the barrier for this step is as high as 48.7 kcal∕mol, to mimic the steric constraints by the protein matrix. This coordinate locking SI Appendix procedure gives rise to a few small imaginary frequencies, normally on the ruling out this possibility as well. See for optimized 10–40i −1 structures of the transition state and the vinylidene intermediate. order of cm . These frequencies contribute little to the ZPE and can be disregarded. One final alternative discussed here is based on the second- To examine the sensitivity of the results to the choice of functional, we shell mechanism suggested by Seiffert et al. in which the W-bound have performed single-point calculations (with the larger basis sets) for our water protonates the substrate to give a vinyl cation intermediate proposed mechanism using the B3LYP* (with 15% exact exchange) (37), TPSS (9). As mentioned in the Introduction, later calculations showed (38), and BB1K (39) methods based on the B3LYP-optimized geometries. For that it has a high barrier (14). In our model, we found that con- the rate-limiting step, the calculated barriers are 21.7, 18.0, and 19.5 kcal∕ certedly with the proton transfer, a nucleophilic attack by the mol using B3LYP*, TPSS, and BB1K, respectively. The values are stable and thiolate moiety of the Cys141 ligand takes place on the other car- quite close to the B3LYP barrier (23.0 kcal∕mol), further corroborating the bon of the substrate. According to our calculations, the barrier is adequacy of the B3LYP functional. 24.2 kcal∕mol relative to React’, which might sound reasonable. However, considering that React’ is 21.2 kcal∕mol higher than ACKNOWLEDGMENTS. F.H. gratefully acknowledges financial support from ’ 45 4 ∕ the Swedish Research Council (Grants 621-2009-4736 and 622-2009-371) Inter1 , the total barrier becomes . kcal mol and this mechan- and computer time from the Center for Parallel Computing. This work ism can be ruled out as well. The optimized stationary points for was also supported by grants from the National Natural Science Foundation this mechanism are also given in SI Appendix. of China (Grants 20733002 and 20873008).

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