A Rationally Designed Myoglobin Exhibits a Catalytic Dehalogenation

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A Rationally Designed Myoglobin Exhibits a Catalytic Dehalogenation Efficiency More than 1000-Fold that of a Native Dehaloperoxidase Lu-Lu Yin, Hong Yuan, Can Liu, Bo He, Shu-Qin Gao, Ge-Bo Wen, Xiangshi Tan, and Ying-Wu Lin ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b02979 • Publication Date (Web): 17 Sep 2018 Downloaded from http://pubs.acs.org on September 17, 2018

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ACS Catalysis

A Rationally Designed Myoglobin Exhibits a Catalytic Dehalogenation Efficiency More than 1000-Fold that of a Native Dehaloperoxidase Lu-Lu Yin1†, Hong Yuan2†, Can Liu1†, Bo He1, Shu-Qin Gao3, Ge-Bo Wen3, Xiangshi Tan2 and Ying-Wu Lin1,3* 1

School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China; 2 Department of Chemistry & Institute of Biomedical Science, Fudan University, Shanghai 200433, China; 3Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China. † These authors contributed equally. ABSTRACT: The design of functional metalloenzymes is attractive but few designs can exceed the catalytic rate of native enzymes. In order to create an effective artificial dehaloperoxidase (DHP), we redesigned the heme center of myoglobin (Mb) by introducing a distal Tyr43 and replacing the distal His64 with an Asp, which combine the structural features of chloroperoxidase (a distal Asp) with DHP (a distal Tyr). The rationally designed F43Y/H64D Mb was shown to exhibit a remarkable dehalogenation activity, with a catalytic efficiency more than one thousand folds higher than that of a native DHP from A. ornata. To the best of our knowledge, this is the highest activity reported to date for an artificial DHP. Moreover, X-ray structures of F43Y/H64D Mb and in complex with a typical substrate, 2,4,6-trichlorophenol (TCP), revealed a distal substrate binding site and crucial roles of both distal Tyr43 and Asp64. This study not only provides insights into the structure and function relationship of native and artificial DHPs, but also suggests potential applications in bioremediation of toxic halophenols. KEYWORDS: artificial metalloenzyme, dehaloperoxidase, protein design, myoglobin, X-ray structure, EPR

The rational design of artificial metalloenzymes has received much attention in the last decade, since it can provide not only insights into the structure and function relationship of native enzymes, but also abilities to create biocatalysts for potential applications.1-20 Although tremendous progress has been made in the design of functional metalloenzymes, the catalytic performance is usually limited and few designs can achieve or exceed the catalytic rate of native enzymes. For example, Lu and co-workers engineered an artificial oxidase in myoglobin (Mb) with an O2 reduction rate (52 s-1) comparable to that of a native cytochrome cbb3 oxidase (50 s-1), by redesign of both the heme center and protein surface.21 In an earlier study, Watanabe and co-workers replaced the distal His64 of Mb with an Asp to mimic the active site of chloroperoxidase, and the H64D Mb mutant showed a peroxidase activity 50~70-fold higher than that of wild-type (WT) Mb.22 Later on, Matsuo et al. reconstituted the apo-protein of H64D Mb with a modified heme group, and the constructed artificial enzyme

exhibited an overall catalytic activity ~120% that of native horseradish peroxidase (HRP).23 Recently, we constructed an intramolecular disulfide bond in Mb to mimic that in cytoglobin, as well as a hydrogen(H)-bond network in the heme distal pocket. The designed V21C/V66C/F46S Mb mutant exhibited an activity of dehalogenation, with a catalytic efficiency ~6.3-fold higher than that of a native dehaloperoxidase (DHP).24 DHP was first discovered by Lovell and co-workers in 1996 from a sea worm, A. ornata,25 which can catalyze dehalogenation of mono-, di, and trisubstituted halophenols to the corresponding non-toxic quinone products by using H2O2 as an oxidant.26 Since halophenols are highly harmful to mammalian liver and immune systems,27, 28 it is of practical importance for remediation of halophenols. Meanwhile, the application of the native DHP for bioremediation was hindered by a low catalytic ability.25, 26 To investigate the structure and function relationship of DHP, Lebioda, Dawson and co-workers constructed an HRP-like M86E DHP mutant that exhibited a turnover number (kcat) ~9-fold higher than that of WT DHP.29 Moreover, Lebioda, Dawson and co-workers introduced a large side chain of Ile at position 65 (G65I) in Mb that pushed the distal His64 0.8 Å further away from the heme iron, resulting in ~46-fold and ~16-fold enhancement in kcat over WT Mb and native DHP (pH 7.0, 4 oC), respectively, without report of the Michaelis constant (Km).29 Motivated by these achievements, we were interested in design of an artificial dehaloperoxidase to further improve the catalytic performance, toward the goal of exceeding that of a native enzyme. For this purpose, it is important to begin with a good starting platform. Mb has been demonstrated to be an ideal protein scaffold for design of functional heme enzymes.1-24, 30, 31 In a recent study, we discovered a novel Tyr-heme cross-link, a new post-translational modification of heme proteins, in sperm whale F43Y Mb.32 This mutant was found to exhibit a dramatically enhanced ability of H2O2 activation, which is ~100-fold higher than that of WT Mb. Therefore, we envisaged that it is an ideal starting platform to design an artificial dehaloperoxidase by further modification of the heme active site of F43Y Mb. Although both Mb and DHP have a distal histidine, the distal His55 in DHP has a longer Nε-Fe distance (5.5 Å, PDB code 1EW633) compared to that in Mb (4.4 Å, PDB code 1JP634). Moreover, His55 adopts both open and closed conformations, which facilitate the binding of substrate and the activation of

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H2O2 via an acid-base mechanism.26 In an earlier study, Watanabe and co-workers designed a double mutant of F43H/H64L Mb by relocating the distal His at position 43, resulting in a similar Nε-Fe distance (5.4 Å) to that in native DHP.35 Du et al. showed that this double mutant has an enhanced dehalogenation activity, with kcat ~6-fold higher than that of the native DHP.29 Meanwhile, a distal His cannot be generated at the same position of 43 in F43Y Mb. Alternatively, an acidic amino acid such as Asp may act as an acid-base catalyst for activation of H2O2, as in native chloroperoxidase,36 which was also confirmed in H64D Mb mutant by Watanabe and co-workers.22 Therefore, we envisaged that the introduction of a distal Asp at position 64 in F43Y Mb may combine the properties of the single mutants to generate a more effective artificial DHP. Moreover, the relatively small side chain of Asp may facilitate the binding of substrate to the heme center. To test our speculation, we constructed a double mutant of F43Y/H64D Mb. After expression and purification using the same procedure as that for F43Y Mb,32 the obtained protein showed UV-Vis spectra (ferric, 404, 500, 629 nm; ferrous, 430, 555 nm, Supporting Information, Figure S1A) slightly different from those of F43Y Mb (ferric, 403, 498, 625 nm; ferrous, 429, 553 nm).32 ESI-MS spectrum showed a major (~80%) mass of 17325.5 ± 0.5 Da and a minor (~20%) mass of 17939.0 ± 0.5 Da (Figure S2), which is corresponding to the calculated apo-protein (17325 Da) and holo-protein (17940 Da), respectively. An X-ray crystal structure of F43Y/H64D Mb in the ferric met form was solved at a resolution of 1.60 Å (Figure 1 and Table S1). The structure revealed that instead of forming a Tyr-heme cross-link, Tyr43 points to the heme center and interacts with the heme axial water by an H-bond. This conformation was also observed in the X-ray structure of a double mutant of F43Y/F46Y Mb in our recent study,37 which suggests that further alteration of the heme distal pocket of F43Y Mb may disrupt the Tyr-heme cross-link. Note that the Tyr-heme cross-link has only a slight effect on the activation of H2O2, as shown by a double mutant of L29H/F43Y Mb with and without the cross-link.38 Moreover, the structure revealed that the distal Asp64 forms an H-bond network with several water molecules in the heme distal pocket, as well as the heme propionate-6 group. As a result, a water channel is formed from the solvent to the heme iron center (Figure 1). One O atom of Asp64 has a distance of 6.1 Å to the heme iron, which is close to that of the distal Asp183 in native chloroperoxidase (5.1 Å, PDB code 1CPO), where Asp183 also forms an H-bond network with both an axial water molecule and a distal His105.36 As shown by the overlaid X-ray structures (Figure S3), Asp64 mimics the distal Asp183, together with the H-bond network, reproducing the features of native chloroperoxidase.

Figure 1. X-ray crystal structure of F43Y/H64D Mb in the ferric met form (PDB code 5ZZF), showing the conformation of Tyr43 and Asp64, and the Hbond network in the heme center.

To evaluate the dehalogenation activity of F43Y/H64D Mb, we first collected the UV-Vis spectra using a typical substrate, 2,4,6-trichlorophenol (TCP). As shown in Figure 2A, the absorbance of TCP (244 and 311 nm) decreased rapidly upon addition of H2O2, producing a characteristic peak at 272 nm of the product, 2,6-dichloroquinone (DCQ),39 which was also confirmed by mass spectroscopy (Figure S4). Note that the change of the protein Soret band at 404 nm suggested the activation of H2O2.32 The time-dependent change of A272 nm further indicated that TCP was almost completely converted to DCQ within 90 sec (Figure 2A, inset), suggesting at least 50 turnover numbers for the conversion. We then determined the kinetic parameters for steady-state reactions at various TCP concentrations. A single mutant of H64D Mb (Figures S1B and S5) and F43Y Mb32, and a double mutant of F43Y/H64A Mb (Figures S1C and S6) and F43H/H64A Mb40, together with WT Mb, were prepared for control experiments. Moreover, the G65I Mb mutant was reconstructed for comparison (Figures S1D and S7). The absorption increase at 272 nm due to the formation of product DCQ was monitored by using stoppedflow spectroscopy (Figure S8A-G), and the initial rate was calculated by using a molar absorption coefficient of 12 mM-1·cm-1.41 As shown in Figure 2B, the plots were fitted to the MichaelisMenten equation for WT Mb and the mutants, whereas substrate inhibition was observed for both F43Y/H64D Mb and F43Y/H64A Mb (Figure S8H), in agreement with that for native DHP.42

Figure 2. (a) UV-Vis spectra of F43Y/H64D Mb (2 µM) in presence of TCP (0.1 mM) in 50 mM potassium phosphate (pH 7.0), upon addition of H2O2 (0.5 mM). The spectra were collected each 5 sec for 120 sec at 25 ºC. Timedependent change of absorption at 272 nm was shown as an inset. (B) Steadystate rates of DCQ formation as a function of TCP concentrations (0-0.1 mM), with 1 µM Mbs and 10 mM H2O2. The data were fitted to the MichaelisMenten equation, and substrate inhibition was observed for both F43Y/H64D Mb and F43Y/H64A Mb.

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ACS Catalysis

Table 1. Kinetic Parameters for H2O2-dependent Oxidation of TCP Catalyzed by the Designed Artificial Dehaloperoxidases, with those of WT Mb, a De novo Enzyme and a Native DHP Shown for Comparison. Enzymes

kcat (s-1)

Km (mM)

WT Mb F43Y Mb H64D Mb F43Y/H64D Mb

0.41 ± 0.02 15.6 ± 1.5 9.7 ± 1.4 27.4 ± 0.9

F43Y/H64A Mb

1.52 ± 0.15

F43H/H64A Mb G65I Mb F46S Mb 24 V21C/V66C/F46S Mb 24 De novo enzyme C45 43 DHP (A. ornata) 44

7.5 ± 0.5 25.0 ± 2.5 28.0 ± 2.3 25.6 ± 1.7 0.50 13.67

0.80 ± 0.08 0.34 ± 0.03 0.11 ± 0.03 0.0035 ± 0.0003 0.0010 ± 0.0002 0.10 ± 0.01 0.22 ± 0.03 1.81 ± 0.20 0.61 ± 0.07 0.012 2.07

kcat/Km (M-1·s-1) 510 45 880 88 180 7 828 570 1 520 000 75 000 113 640 15 470 41 970 42 000 6 640

The kinetic parameters were presented in Table 1. The results showed that F43Y/H64D Mb has a kcat (27.4 s-1) ~67-fold higher than that of WT Mb, which is larger than both of the two single mutants F43Y Mb and H64D Mb, suggesting that both mutations are attributed to the activation of H2O2, as observed in previous studies.22, 23, 32 Stopped-flow studies further showed that Compound I (an oxoferryl heme π-cation radical) species was readily formed (within 0.1 sec) when ferric H64D Mb reacted with two equivalents of m-chloroperbenzoic acid (m-CPBA), with a characteristic absorbance at 648 nm (Figure S9A), as observed previously by Watanabe and co-workers.22 Compound I was then rapidly converted to Compound II (oxoferryl heme), with visible bands (548 and 585 nm) resembling those observed for oxoferryl Mb (546 and 581 nm)45. Meanwhile, only Compound II was detected for F43Y Mb (within 0.5 sec, Figure S9B), and also for F43Y/H64D Mb (within 0.1 sec) that was rapidly converted back to the ferric state (within 0.5 sec, Figure S9C), suggesting an ultrafast generation of the catalytic intermediates. Moreover, the Km (3.5 µM) of F43Y/H64D Mb was found to be much lower than those of WT Mb and the single mutants, suggesting a heme pocket with micro-environments more favorable for substrate binding. As a result, F43Y/H64D Mb exhibited a remarkable catalytic efficiency (kcat/Km), which is ~15,400-fold and ~1,180-fold enhancement over that of WT Mb and a native DHP from A. ornata,44 respectively. The important roles of Tyr43 and Asp64 were further supported by control experiments. For example, the double mutant of F43Y/H64A Mb showed a ~18fold lower kcat compared to that of F43Y/H64D Mb, albeit with a very low Km (Table 1). These results indicate that the combination of structural features of chloroperoxidase (a distal Asp) with DHP (a distal Tyr) generated a very effective artificial DHP. Furthermore, the catalytic efficiency of F43Y/H64D Mb is ~104-fold higher than that of the F43H/H64A Mb double mutant, ~185-fold higher than that of the recently designed artificial DHPs, V21C/V66C/F46S Mb24 and de novo enzyme C45,43 and is ~68fold higher than that of G65I Mb, as redetermined in this study under the same reaction conditions (Table 1), respectively. To the best of our knowledge, the catalytic efficiency observed for F43Y/H64D Mb is the highest reported to date for an artificial DHP. After discovering the remarkable activity for F43Y/H64D Mb with a low Km value, we were further interested in revealing the substrate binding mode. By soaking the crystal of F43Y/H64D Mb with the substrate TCP, we successfully solved an X-ray

structure of F43Y/H64D Mb in complex with TCP at a resolution of 1.80 Å (Figure 3 and Table S1). The structure showed that an H-bond is formed between the hydroxyl group of Tyr43 and that of TCP in the heme distal pocket, which presumably controls the binding model of TCP. The binding of TCP was found to displace the heme axial and distal water molecules, where the distance between the Cl4 atom of TCP and the heme Fe is 3.91 Å, resulting in a five-coordinate heme state. This was further confirmed by electron paramagnetic resonance (EPR) studies of F43Y/H64D Mb upon TCP binding. As shown in Figure 4, the signal (g⊥= 6.06) of a six-coordinate high-spin (6cHS) ferric heme (line a) in the low-field region slightly decreased, and a more rhombic g tensor (g1 = 6.63, g2 = 5.40) appeared by addition of TCP (line b). This was similar to those of the ferric heme in native DHP upon binding of inhibitor 4-iodophenol (g1 = 6.22, g2 = 5.50),46 and a resting state recovered from an oxoferryl state (g1 = 6.39, g2 = 5.42).47 These signals were assigned to a five-coordinate high-spin (5cHS) ferric heme where the distal His55 adopted an open conformation.46, 47 Control studies of H64D Mb showed a shoulder signal with a less rhombic g tensor (g1 = 6.55, g2 = 5.62) (line d), which suggests a weak binding of TCP to the internal binding site of H64D Mb, due to the lack of an H-bonding interaction with Tyr43. The structure also showed that the distal Asp64 in the TCPF43Y/H64D Mb complex adopts an open conformation, which is stabilized by H-bonding interactions with Thr67 through a bridging water molecule (Figure 3). This is distinct from the closed conformation of Asp64 in the absence of TCP (Figure 1). It is interesting to note that the distal substrate binding mode revealed in this structure shares some key features of native DHP in complex with the substrate, as shown by the overlaid complex structures (Figure S10). For example, Dawson, Lebioda and coworkers revealed an external substrate binding site for DHP from A. ornata, where TCP occupies a position similar to that in the TCP-F43Y/H64D Mb complex, albeit with a different conformation (Figure S10, orange).48 The hydroxyl group of TCP was shown to form an H-bond with the hydroxyl group of Tyr38 in the heme distal pocket, with a distance of 3.78 Å between the Cl4 atom and heme iron.48 Moreover, similar to the observation for the distal Asp64, a position switch of the distal His55 occurred for A. ornata DHP upon TCP binding to the external site (Figure S10), which is also responsible for the inhibition effect at high substrate concentration.48

Figure 3. X-ray crystal structure of ferric F43Y/H64D Mb in complex with TCP (PDB code 5ZZG), showing the conformation of Tyr43 and TCP, and the H-bonding interactions in the heme center. The distance between the Cl4 atom and the heme Fe (3.91 Å) is also indicated.

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3. 4. 5. 6. 7. 8. 9. 10.

Figure 4. X-band EPR spectra of (a, b) F43Y/H64D Mb and (c, d) H64D Mb (0.5 mM) in the absence (a, c) and presence (b, d) of TCP (0.5 mM) in 50 mM potassium phosphate buffer (pH 7.0), showing the low-field region.

In conclusion, we rationally constructed a very effective artificial DHP of F43Y/H64D Mb by combining the structural features of chloroperoxidase (a distal Asp) with DHP (a distal Tyr) in the same heme active center of Mb. X-ray crystallographic studies revealed that Tyr43 controls the binding of TCP to the distal substrate binding site, and the distal Asp64 plays a similar role to that of the distal histidine in native DHP by conformational changes. Kinetic studies showed that this artificial enzyme exhibits a remarkable catalytic efficiency, which exceeds that of the native DHP from A. ornata by more than one thousand folds, and is the highest activity reported to date for an artificial DHP. Therefore, in addition to providing insights into structure and function relationship of native and artificial DHPs, this study suggests potential applications of the designed artificial DHP in bioremediation of toxic halophenols. Supporting Information: Detailed description of the experimental procedures, supporting UV-Vis, ESI-MS and stopped-flow spectra, overlaid X-ray structures, and crystallography data. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding author: *E-mail: [email protected]; [email protected]

11. 12. 13. 14. 15. 16.

17. 18. 19. 20.

21. 22.

Acknowledgment: It is a pleasure to acknowledge Prof. S. G. Sligar and Prof. Y. Lu of University of Illinois at UrbanaChampaign, for the kind gift of sperm whale Mb gene. X-ray diffraction data were collected at Shanghai Synchrotron Radiation Facility (SSRF), China. This work was supported by the National Natural Science Foundation of China (31370812; 21701081) and the double first class construct program of University of South China.

23.

24.

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