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Research Article Cite This: ACS Catal. 2018, 8, 7016−7024

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Designed Self-Assembly of Peptides with G‑Quadruplex/Hemin DNAzyme into Nanofibrils Possessing Enzyme-Mimicking Active Sites and Catalytic Functions Zhen-Gang Wang,*,†,# Hui Wang,†,# Qing Liu,†,# Fangyuan Duan,† Xinghua Shi,*,†,‡ and Baoquan Ding*,†,‡

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†

CAS Key Laboratory of Nanosystem and Hierarchial Fabrication, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Enzymes fold into three-dimensional structures to arrange their active groups exquisitely for the remarkable catalytic properties. We are inspired to design and assemble Glncontaining peptides with G-quadruplex DNA/hemin complexes to form the catalytic nanofibrils that possess the horseradish peroxidase-mimicking active sites and catalytic functions. Theoretical simulation results revealed that the intermolecular association of Gln peptide may result in local enrichment and proper orientation of carboxamide groups, which provide potential multivalent hydrogen bonds for enhancing H2O2 affinity to hemin and may behave similarly to distal Arg in a natural heme pocket. The self-folded DNA can provide a guanine base as the axial ligand, and a supramolecular scaffold for supporting and orienting hemin. The β-sheet forming capability of the Q peptides is found to significantly affect the catalytic synergy between the G-DNA and the peptide. The role of hydrogen bonds network provided by self-assembled Gln peptides is illustrated by solvent kinetic isotope effects and H2O2-induced degradation of hemin. The assembly of the Q peptide with GDNA/hemin DNAzyme also stimulates the chiroselective oxidization of L-DOPA vs D-DOPA. The incorporation of Hiscontaining peptide into the hemin system via self-assembly, which was demonstrated by confocal colocalization images and fluorescence resonance electron transfer results, further enhanced the catalytic activity of the G-DNA/Q peptide/hemin complex. It is hypothesized that the assembly of the triple components allows mimicking the configuration and the function of the catalytic His-Arg-His triad in horseradish peroxidase. Compared to DNA/hemin or peptide/hemin, the catalytic efficiency of the most active complex shows 10-fold enhanced activity. This work opens an avenue to mimic the catalytic residues and their spatial distribution and may provide a primitive enzyme model for the evolution of modern enzymes. Our results may also have implications for the mechanisms of some cell dysfunctions, which are triggered by catalytic aggregation of biomolecules. KEYWORDS: DNAzyme, peptide, hemin, enzyme mimics, self-assembly

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increase biological defense. At their active sites, the major amino acid residues are located at proximal and distal sides of the heme cofactor and synergistically reduce the activation energy barrier of ferric iron of heme.10,11 The threedimensional cleft of the heme pocket is formed as a result of folding the tertiary structure, which is difficult to achieve through the artificial assembly of simple molecules. A supramolecular scaffold that can support hemin is favored so that the arrangement of the essential groups around hemin can be facilitated. On the other hand, a planar guanidinium group held by arginine, a common residue in the active site of the peroxidases, assists in the cleavage of the O−O bond to form a reactive intermediate compound I11,12 by providing mobile

nzymes harness noncovalent interactions to fold and catalyze biotransformations efficiently, which promotes the rapid development of supramolecular catalysis.1−5 Compared to enzymes, supramolecular catalysts are structurally simpler and easier to modify. However, it has been a challenge to reproduce the structure and chemical environment of the enzymatic active site, where substrates bind and reactions take place, in supramolecular systems to compete with the catalytic proficiency of natural enzymes. The active sites of the early enzymes have been suggested to be formed through association of short polymers,6,7 which resulted in enrichment and proper orientation of the catalytic groups. For example, short β-sheet peptides have a strong tendency to self-assemble into nanostructures to show biological functions.8,9 Peroxidases are a family of enzymes that catalyze one- or two-electron oxidization by hydrogen peroxide or lipid peroxide to prevent the cells’ damage or © XXXX American Chemical Society

Received: March 6, 2018 Revised: June 13, 2018

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Scheme 1. Assembly of Gln-Containing Peptides with G-Quadruplex DNA and Hemin into Catalytic Fibrils to Arrange the Neighboring Gln Residues and Guanine at Different Sides of Hemin, Which Is Proposed According to the Molecular Characters of G-Quadruplex DNA and the Peptidesa

a

Such distribution may allow DNA and peptide to synergistically facilitate H2O2 adsorption to hemin and subsequent electron transfer reactions. The further assembly of the His-rich peptides into the hemin complexes may introduce a potential acid−base catalyst (His) to the distal side of hemin. The proposed configuration and function of such an active site are analogous to the natural catalytic Arg/His/His triad and may result in the enhancement of the activity.

hydrogen bonds (up to five in different directions). Such exquisite function of the Arg residue is supposed to be evolved by nature from the assembled simple groups in the primitive catalytic species. The neighboring carboxamide groups with similar orientation may behave similarly to Arg regarding the mobility and number of the hydrogen bonds. We are inspired to arrange the carboxamide groups of amino acid residues to mimic the function of arginine via the assembly of elaborate βsheet peptides. Specifically designed DNA strands have shown potential in catalysis upon binding to metals to form enzyme-mimicking DNAzyme,13−15 which holds promise in the design of enzymelike active sites. In this work, a heme-containing peroxidasemimicking system, containing a Gln-containing peptide (Q peptide), a guanine-rich DNA (G-DNA), and hemin, was designed and assembled. The specially designed DNA strands are folded into a high-order G-quadruplex consisting of four base-stacked quartets and can effectively interact with hemin via π−π stacking and an axial coordination.16,17 Moreover, the G-quadruplex can serve as a robust molecular scaffold18 to stabilize and orient the hemin cofactor. The Q peptide was designed based on the Q11 sequence, which self-assembles into a β-sheet-rich fibrillar nanostructure in saline-containing aqueous environments.19,20 Therefore, the Gln residues may be arranged to distribute around the G-quadruplex via intermolecular self-assembly. The results reveal that Q peptide and DNA had a remarkable synergistic effect on the activities of hemin, and these effects were significantly dependent on the βforming capability of the Q peptides. The introduction of a His-rich peptide further enhanced the activities, indicating its effective cooperation with G-DNA and Q peptide promoted

the electron transfer between hemin and the substrates. The length of each peptide component does not exceed 11 residues, so that this catalytic system may resemble a primitive framework of peroxidase heme pocket assembled from short polymers. It is proposed that the assembly of both peptides with G-quadruplex DNA and hemin may constitute a threedimensional active site that mimics the enzyme, as shown in Scheme 1. Furthermore, the capping of the G-quadruplex/ hemin DNAzyme with Q peptide-forming fibrils creates a chiral environment for the selective oxidization of DOPA enantiomers. Circular dichroism demonstrated a dominant presence of βsheet conformation for the Gln-containing peptide, which produced a negative peak near 220 nm. The G-quadruplex conformation of DNA in the DNA/Q11 hybrid was partially extended, indicating the assembly of DNA with Q11 peptide (Figure 1A). TEM images show that the Q11 peptides selfassembled into fibril nanostructures (Figure 1B). Using molecular dynamics simulation, Q11 strands (spacing 0.474 nm) were estimated to align along the β-sheet to form micrometers-long fibrils, and the width of the individual fiber (ca. 3.5−5.0 nm) was attributed to the length of β-strands (ca. 3.47−3.60 nm) or β-sheet stacks (spacing ca. 1.0 nm; Figure S1 and Figure 1C). Incorporation of DNA did not alter the morphological change of the fibers (Figure 1B). The rigid molecular docking was performed to show that the assembled β-sheet peptides can approach the G-quadruplex from multiple directions via hydrogen bonding and electrostatic interactions with the same possibilities (Figure 1D and Figure S2). This illustrates that in buffer solutions, each DNA G-quadruplex may interact with multiple peptide fibrils. 7017

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of the carboxamide (−CONH2) groups of Gln residues (illustrated by Figure S5A−C), which may provide multiple hydrogen bonds similar to the role of distal Arg in the formation of compound I in the active site of natural peroxidase.26 It is noted that Glu bearing a carboxylate group acts as a potential distal acid−base group.27,28 However, Figure S5D showed that the interaction of Glu residues, lying between the stacked β-sheets, with H2O2 can be severely hindered by the enriched carboxamides that could form hydrogen bond networks with H2O2. Furthermore, the negatively charged Glu can be repelled by phosphate backbones of DNA and may have difficulty in approaching G-DNA-supported hemin. Therefore, the possibility of Glu to accelerate the catalytic process can be ruled out due to the steric hindrance. It can be rationally proposed that the assembly of Q11 with G-DNA rigidifies the spatial location of the catalytic Gln residues and the nucleotide ligand, which distribute to either side of hemin. Such a configuration of the active site allows the successive electron transfer between hemin and the substrates in the natural peroxidase.12,26 These features underscore the synergistic effect of DNA and peptide components on the peroxidase-mimicking catalysis. The catalytic rate of hemin in various environments was studied by oxidation of ABTS2− (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as a model reaction. The reaction was monitored at 414 nm, the absorption maximum for ABTS•+. In the presence of Q11 peptide or G-DNA, hemin exhibited higher peroxidative activity than free hemin (Figure S6). Within the Q11/G-DNA self-assembly, hemin exhibited a significantly higher catalytic velocity than that within DNA or peptide alone, indicating a cooperation of the peptide and DNA (Figure 2A). As the molar ratio between DNA and peptide components varied (at constant DNA concentration), an optimal activity of hemin was observed (Figure 2B), while the Q11−hemin complex exhibited saturation in the activity at a higher Q11 concentration (Figure S7). Similar dependence

Figure 1. (A) CD spectra of Q11, G-DNA, and Q11/G-DNA hybrids. (B) TEM images of self-assembled Q11 and Q11/G-DNA fibrils. (C) Stimulated stacked β-sheet structures of Q11 peptides. Some carboxamide groups (N atom, blue; O atom, orange; C atom, gray; H atom, white) that may cooperatively interact with H2O2 are shown. (D) Molecular docking structure of G-DNA (blue backbone) with Q11 β-sheet (gray backbone) at lowest energy (−6.59 kcal/mol). (E) UV−vis spectra of hemin encapsulated by G-DNA, Q11, or Q11/GDNA hybrids. [Q11], 40 μM; [G-DNA], 1.0 μM; [hemin], 0.5 μM.

The coordination state of hemin in the self-assembled hybrids was investigated using UV−vis spectra (Figure 1C). Hemin localized in the Q11 fibers displayed a Soret peak at 390 nm, a shoulder at 365 nm, and a low-intensity band at 610 nm, which are similar to the spectra features of free hemin.21,22 In the presence of G-DNA, hemin showed a Soret band at 403 nm and charge-transfer transitions at 626 and 504 nm along with a weak absorbance at 536 nm, which are characteristic spectra features of high-spin hexa-coordinated species.23−25 In the self-assembled Q11/G-DNA hybrid, hemin showed similar spectra features to those complexed with G-DNA, indicating coordination of hemin with DNA nucleotides. Flexible docking results reveal that hemin stacked on either end of the G-quartet forms stable structures with similar free energy changes. The exocyclic amine of the G9 base (GGGTAGGGCGGGTTGGG, in lowest-energy structure; Figure S4A) or the deoxyribose oxygen of the G3 base (GGGTAGGGCGGGTTGGG; Figure S4B) lies close to and axial to the hemin iron and may act as a proximal ligand. Figure S2 shows that Q11 peptides interacted with G-DNA via groove binding and stayed close to where hemin stacked. In the chemical structure of the Q11 peptide (Figure S3), the residue Lys with a protonated side group (−NH3+) at neutral pH and Phe with a hydrophobic phenyl group were incapable of promoting the activation of hemin by H2O2. The intermolecular assembly of the Q11 peptide that adopted β-sheet secondary structure allows spatial arrangement and orientation

Figure 2. (a) Time-dependent absorbance changes for the oxidization of ABTS2− catalyzed by hemin, indicating synergies between Q11 and G-DNA. [Q11], 20 μM. (b) Effect of Q-peptide sequence on the initial catalytic velocity (Vi) of the peptide/G-DNA/hemin hybrids. The table lists the peptide sequences. [G-DNA], 0.25 μM; [hemin], 0.1 μM. 7018

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treatment of the catalytic complex with D2O may alter the hydrogen bonds between the peroxide substrate and the peptide residues. The increase in D2O concentration to 80 vol % further reduced the catalytic activity of the Q11/G-DNA/ hemin complex, while the activity of G-DNA/hemin was also decreased. The change in hydrogen bonding interactions in the active site and G-DNA-hemin interactions may both contribute to the D2O-induced deactivation of hemin. The affinity of hemin to H2O2 was further investigated by treatment of hemin by H2O2 in the absence of the reducing substrates, since the excessive H2O2 can result in the transformation of hemin into compound I and subsequently compound II,32 as observed by the decrease in the absorbance of Soret bands and blue shift of the Soret maximum.33 Figure S12 shows that when subjected to H2O2 (1 mM), the Soret band of hemin within the Q11/G-DNA hybrid was decreased faster than that within G-DNA, which indicated higher affinity of H2O2 to the Q11/G-DNA/hemin complex. However, in the buffer solutions containing 50 vol % D2O, the kinetics of the Soret band changes was almost the same for Q11/G-DNA/hemin and G-DNA/hemin complexes (Figure S13). Under these conditions, the hydrogen-bonding network between Gln residues and peroxide was supposed to be changed. These results indicate that the assembly of Q11 with G-DNA enhanced the affinity of H2O2 to hemin, which facilitated the catalytic reactions. In natural peroxidase, the activation of hemin was assisted by a distal Arg, as well as a distal His that functioned as the acid− base catalyst.10,26 We thus introduced a third activator peptide composed of 10 His residues (H10) to the Q11/G-DNA/ hemin system to investigate the cooperation among H10, Q11 peptides and G-DNA. The morphologies of Q11/G-DNA fibers were not altered significantly by assembly with the H10 peptide (Figure 3A), while Q11/G-DNA/H10 fibrils show slightly larger width (ca. 6.2 nm∼ 7.0 nm) than the binary components hybrids (Q11/G-DNA or Q11/H10). The modification of H10, Q11, or G-DNA with FAM, TAMRA, and Cy5 dye allows us to image the self-assembled fibrils following the fluorescence of the dyes upon excitation at 488, 549, and 635 nm, respectively, under confocal microscopy. The confocal images reveal the colocalization of the three dyes, indicating the successful assembly of G-DNA with Q11 and H10 peptides (Figure 3B and Figure S14). Irradiation treatment of TAMRA dyes resulted in the enhancement in FAM fluorescence, which is attributed to an inhibited fluorescence energy transfer (FRET) from FAM to TAMRA. It indicates FAM was spatially close to TAMRA in the fibrils (Figure S15). Fluorescence spectra confirmed a FRET process in Q11/H10/G-DNA hybrids and any of the binarycomponents hybrids (Q11/G-DNA, H10/G-DNA, or Q11/ H10; for the figures and discussion, see Figure S16). Remarkable synergistic activities were observed for hemin encapsulated within G-DNA/H10 hybrids. Moreover, the hemin encapsulated within the triple-components hybrid exhibited significantly higher activity than that within the binary-components hybrid, reflecting G-DNA, Q11, and H10 performed different and complementary roles in the catalysis (Figure 3C). Optimal initial catalytic velocity was observed at a specific H10/Q11 ratio (20 μM/20 μM). The His-rich peptides also protected hemin against H2O2-induced degradation,16,34 which was confirmed by the constantly enhanced substrate conversion efficiency (CABTS) for the Q11/H10/GDNA/hemin complex as H10 concentration increased (Figure

of the catalytic activity on DNA concentration was also observed (Figure S8). To investigate the effect of β-sheet formation and peptide self-assembly on the catalysis, we replaced Gln with Glu at position 1 (at N terminus) and position 11 (at C terminus) to yield Q11-E and Q11-EE peptides, and an obvious decrease of the initial catalytic activity of hemin (Vi) was observed (Figure 2B). The introduction of Glu groups transformed the β-sheet of Q11 to random coils, due to the electrostatic repulsion between the carboxylate groups. The significantly reduced aggregation of the peptides may result in inefficient enrichment of the Gln residues. To test this hypothesis, we assembled Q7, a fragment of Q11 peptide containing two alternating Gln residues, with G-DNA and hemin, and the complex revealed similar catalytic activities to those containing Q11-E and Q11EE. The incapability of forming a β-sheet for Q7 (Figure S9A) resulted in ineffective activation of hemin. Then, we replaced Gln at position 1 with Gly residue, and the activities of the hemin were not decreased, while the CD intensity corresponding to the β-sheet was even stronger. However, the replacement of Gln at position 1 and 11 by Gly residue resulted in the significantly lowered activity of the hemin complex, as well as weakened CD intensity of the peptide. These findings again indicate that the efficient formation of βsheet secondary structures was important to enhance the activity of the peptide/G-DNA-complexed hemin, due to the local enrichment of Gln residues. It is noted that the peptide that has a strong β-forming tendency can alter the CD intensity of the G-quadruplex more evidently, particularly at 300 nm, which reflected the significance of the β-formation in the interactions and cooperation of the peptide with DNA components (Figure S9B). We also replaced Gln residues in the Q11 peptide with Asn residues, which also bear a sidechain carboxamide, to form the N11 peptide. N11 could form a β-sheet with a stronger CD response than Q11 and effectively interacted with G-DNA, while the catalytic activity of hemin within the N11/G-DNA hybrid was lower. The shorter side chain in the Asn residue, compared to that in the Gln residue, may reduce the contribution of the carboxamide to the activation of hemin due to the constrained group mobility.29 The chemical structures of the peptides and CD spectra can be found in Figure S3 and Figure S9. In the peroxidase catalysis, the conversion of hemin-H2O2 to the reactive intermediate (e.g., compound I) is usually the ratelimiting step.12 The potential hydrogen-bonding interactions between the neighboring Gln residues and H2O2 may be a dominant factor to enhance the catalytic activity of the GDNA/hemin complex via promoting the adsorption of H2O2 to hemin. To validate the role of the hydrogen bonding network, we introduced D2O into the buffer solutions to exchange hydrogens in H2O2 and the side chains of Gln residues into deuteriums. The solvent kinetic isotope effects (KIEs) have been widely used to study the mechanism of enzyme-catalyzed reactions,30,31 in particular in the hydrogenbond-involved substrate binding and conversion, since a hydrogen bond with deuterium is slightly stronger. Figure S10 shows that when the volume concentration of D2O in the range of 20−50 vol %, the catalytic activity of the Q11/GDNA/hemin complex was significantly reduced, while the activity of G-DNA/hemin remained unchanged. CD spectra (Figure S11) showed that D2O did not affect the secondary structures of the Q11 peptides and G-DNA, and thus the spatial arrangement of Gln residues. This indicated the 7019

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with H10 peptide did not improve the activities, which revealed significant roles of G-DNA as the scaffold and the ligand donor. TEM images show the fibril shapes of Q11containing hemin complexes (Figure S20), indicating the βsheet association of Q11 and the contribution of carboxamide groups of Gln to the catalysis. Under saturated ABTS2− conditions (Figure S21 and Figure S22), the apparent kinetic parameters for H2O2 reduction (kcat (H2O2), Km (H2O2), kcat/Km (H2O2)) were calculated (Table 1) based on the construction of Lineweaver−Burk plots of the Table 1. Michaelis−Menten Parameters with Respect to Catalyzing Reduction of H2O2 with ABTS2− as the Reducing Substratea

Q11/hemin G-DNA/hemin Q11/G-DNA/hemin G-DNA/H10/hemin Q11/H10/hemin Q11/H10/G-DNA/ hemin

kcat (H2O2) (s−1)

Km (H2O2) (mM)

kcat/Km (H2O2) (s−1 mM−1)

0.137 0.109 0.493 0.425 0.138 0.712

1.998 2.45 2.183 2.02 1.79 1.25

0.0685 0.0444 0.225 0.210 0.077 0.567

a [Q11], 40 μM; [H10], 20 μM; [G-DNA], 0.25 μM; [hemin], 0.1 μM; [ABTS2−], 2 mM.

Figure 3. (A) TEM images of Q11/H10 and Q11/H10/G-DNA fibrils. (B) Confocal microscopy images of TAMRA-modified Q11/ FAM-modified H10/Cy5-modified G-DNA nanomaterials on the channels of 488, 559, and 635 nm, respectively. [Q11], 40 μM; [H10], 20 μM; [G-DNA], 1.0 μM. Scale bar: 5 μm. (C) Effect of H10 concentration on the initial catalytic velocity of peptide/G-DNA/ hemin hybrids. [Q11], 40 μM; [G-DNA], 0.25 μM; [hemin], 0.1 μM. (D) UV−vis spectra of hemin encapsulated by varials hemin complexes. [Q11], 40 μM; [H10], 20 μM; [G-DNA], 1.0 μM; [hemin], 0.5 μM.

initial catalytic velocity versus H2O2 concentration in the presence of 100 nM hemin (Figure S23).5,37 The kcat (H2O2) value, which indicates an enzyme’s turnover number of H2O2, and kcat/Km (H2O2), which reflects the catalytic efficiency for the reduction of H2O2, are both consistent with a synergistic effect of Q11, H10, and G-DNA components in the activation of hemin. In particular, the catalytic efficiency (kcat/Km (H2O2), 0.567 s−1 mM−1) of the Q11/H10/G-DNA/hemin complex was 10-fold higher than that of G-DNA/hemin (0.0444 s−1 mM−1) exceeds that of myoglobin (0.540 M−1 s−1).38 It is also noted that the Km value for the Q11/G-DNA/ hemin complex (Km (H2O2): 2.183 mM) was lower than that of G-DNA/hemin (Km (H2O2): 2.45 mM), which indicated higher affinity of H2O2 to hemin bound to Q11/G-DNA hybrid. This is consistent with the previous conclusions from kinetic isotopic effects (Figure S10) and H2O2-induced degradation of hemin (Figure S12 and Figure S13). The dependence of the catalytic activities on H2O2 concentration was also investigated by incorporation of glucose oxidase (GOx) into the system, considering the degradation effect of H2O2 on hemin. GOx catalyzed O2 oxidization of glucose to yield H2O2, which is the substrate of the artificial hemincontaining enzymes. As the reaction proceeded, the catalytic velocity of all the complexes was accelerated, which was attributed to the accumulation of the H2O2 in the solution. Remarkable synergies between Q11, H10, and DNA in the hemin catalysis were exhibited (Figure S24). In addition to ABTS2− as the electron donor substrate, the assembly of Q11 peptide with G-DNA/hemin also exhibited synergistic effects on the catalytic oxidization of 3,3′,5,5′tetramethylbenzidine (TMB), nicotinamide adenine dinucleotide (NADH), or pyrogallol (Pyr) with H2O2 as the oxidant (Figure S25). For the NADH peroxidase-mimicking catalysis, the initial catalytic velocity of the tricomponent complex was one order higher than that of either of the one-component complex. With respect to oxidization of pyrogallol, it is found

S17; for the time-dependence absorbance changes, see Figure S18; for detailed discussion, see SI). To validate the importance of the designed peptide sequences on the catalytic activity of the complex and to compare Gln residues with Arg, we investigated the effect of free arginine (Arg), histidine (His), and His-Arg dipeptide on G-DNA/hemin activity. As shown in Figure S19, even when His or Arg concentration reached up to 750 μM, the enhancement in the catalytic activity of G-DNA/hemin was quite limited, and the synergy between G-DNA and the monomeric residues was much less efficient than that between G-DNA and peptides (Figure 3B). Similarly, the enhancement in the activity of G-DNA/hemin by His-Arg dipeptide (e.g., 130 μM) was also much lower than that by Q11/H10 peptides containing 120 μM Gln (from 20 μM Q11) and 100 μM His (from 10 μM H10). The more effective synergy between the peptides and G-DNA was attributed to their multiple weak interactions. UV−vis spectra (Figure 3D) show the H10/hemin complex exhibited a Soret band at 414 nm and a pronounced band at 530 nm and a subtle shoulder at 570 nm. The spectral features are attributed to the imidazole → iron charge-transfer transition and the low-spin six-coordinated species35,36 In Q11/H10 hybrids, hemin had similar spectra features to that in H10, indicating the coordination of hemin to H10. Hemin within Q11/G-DNA, H10/G-DNA, and H10/Q11/G-DNA hybrids shows similar spectra features to that in G-DNA, in particular in the range of 500−700 nm. Preferential complexation of hemin with G-DNA in the G-DNA-containing hybrids can thus be concluded. It is noted that the assembly of Q11 7020

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Figure 4. QM models for the contribution of Gln and His residues to the formation of compound I (state iii) based on DFT/B3LYP calculations. One H atom of each CH3 group and the N atoms of the porphyrin ring were fixed in the geometry optimization process. The results showed the cooperative effect of the two Gln, one His, and the G9 coordination group on the adsorption of H2O2 to hemin iron and cleavage of O−O bonds. (N atom, blue; O atom, orange; C atom, gray; H atom, white; Fe atom, red).

that the kcat (Pyr) value for hemin within Q11/H10/G-DNA hybrid (1.51 s−1) was significantly higher than that for hemin conjugated with cyclodextrin (0.08 s−1),39encapsulated in supramolecular hydrogel which contained Phe and His residues (0.828 s−1)39 and closer to natural HRP (29.1 s−1;40 Figures S26 and S27, Table S2). To understand the mechanism of synergistic contribution of DNA and peptide components, a molecular model was constructed for the active site to simulate the formation of the reactive intermediate compound I, which is a rate-limiting step in natural horseradish peroxidase.12,41 Herein, the lowest energy mechanism of H2O2 activation of the peroxidase26 was studied using the B3LYP42,43 functional of density functional theory (DFT).44−47 One coordinating nucleotide (guanine) and three activating distal residues (one His and two neighboring Gln residues) were arranged around hemin (Figure 4). H2O2 (Hα−Oα−Oβ−Hβ) was held by the His and Glnα via an Fe−Oα bond and double hydrogen bonds, N(His)···Hβ-Oβ and N−H (Glnα)···Oβ (state i). This step was followed by the abstraction of a distal proton by His from H2O2 and a flip of the Fe−Oα−Hα−Oβ moiety to the Fe−Oβ− Oα−Hα complex, which was stabilized by hydrogen bonding between His(H+)/Glnα and Hβ−Oα−Oβ groups (state ii, compound 0). Then, the Hβ−Oα group on the distal side of Fe was reprotonated by His(H+), and the heterolytic cleavage of the Oα−Oβ bond led to the formation of the compound I analogue (state iii). The bond lengths were in line with effective coordination and the hydrogen bonds. In this model, His, Gln residues, and guanine nucleotide acted as the acid− base catalyst, hydrogen-bond donors, and the proximal ligand, respectively. They functioned in the H2O2 activation of hemin similarly to the catalytic His−Arg−His triad in the heme pocket of the natural peroxidase.10,26 In the absence of His residues, H2O2 adsorbed to hemin iron to form Fe−H2O2, followed by the migration of the proton bonded to Oα to Oβ, and a water molecule split off to form compound I directly (Figure 5). The heterolytic cleavage initiated from Fe−H2O2 had a much higher energy barrier than that nascent from Cpd0, since the 1,2-proton shift path is usually a “forbidden” process.48 On the other hand, the hydrogen bonds network provided by the neighboring carboxamide groups of Gln residues (as shown in Figure S4) promoted the adsorption of H2O2 to hemin iron, as well as the subsequent formation of compound I, which accounted for the synergy between Qpeptide and G-DNA. It is noteworthy that the mobility of the

Figure 5. QM models for the activation of hemin by H2O2 for Q11/ G-DNA/hemin complex.

carbon spacer between the carboxamide and the backbones enabled the effective contribution of hydrogen bonds by Gln. We also investigated the kinetic parameters of the various complexes with respect to the oxidization of ABTS2− (kcat (ABTS2−), Km (ABTS2−), and kcat/Km (ABTS2−)) (Table S1), under saturated H2O2 conditions (Figure S22). The hemin complexes containing Q11/DNA hybrids revealed improved catalytic kinetics toward ABTS2− oxidization (kcat (ABTS2−): 0.122 s−1 and kcat/Km (ABTS2−): 0.1025 s−1 μM−1) compared to those containing either Q11 (kcat (ABTS2−): 0.08 s−1 and kcat/Km (ABTS2−): 0.0284 s−1 μM−1) or DNA (kcat (ABTS2−): 0.088 s−1 and kcat/Km (ABTS2−): 0.0237 s−1 μM −1). Particularly, the inclusion of H10 enhanced the turnover rate (kcat (ABTS2−): 0.512 s−1) more significantly. The electronwithdrawing effect of the polar imidazole groups49,50 adjacent to the porphyrin ring may facilitate the electron transfer from the reducing aromatic substrate to compound I, while the π−π interactions of the imidazoles with the aromatic substrate may hinder the diffusion of the substrate to hemin. The helical conformations of DNA molecules promise their potential as the chiral scaffold in enantioselective catalysis. The Li group employed G-quartet-Cu2+ complexes for the enantioselective catalysis of the Friedel−Crafts and Diels− Alder reactions, in which Cu2+ was probably localized within the G-quartet pocket.15,51 However, in the DNA-assembled peroxidase mimics, hemin with a relatively larger size than Cu2+ stacks upon the G-quartet. The reducing substrate 7021

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amyloid-β peptide/heme complex-triggered oxidative stress and neuropathologies in Alzheimer’s disease brains.53,54

donates electrons to compound I by approaching the catalytic center iron (Fe(IV))O) from the distal side of the porphyrin ring (away from the G-quadruplex), while the lack of a structured environment leads to the lack of enantioselectivity of the G-quadruplex/hemin complexes. This is confirmed by the identical activities of G-DNA/hemin toward the oxidization of enantiomeric L- and D-DOPA (3,4-dihydroxyphenylalanine).14 However, upon self-assembly of G-DNA with Q11 peptide, L-DOPA was oxidized faster than its Denantiomer according to the turnover rate (kcat (DOPA)) and catalytic efficiency values (kcat/Km (DOPA); Table 2 and

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.8b00896. Materials and methods, details of computational simulations, Figures S1−S28, Tables S1 and S2 (PDF)

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L-DOPA D-DOPA

Km (DOPA) (mM)

kcat/Km (DOPA) (s−1 mM−1)

1.11 0.778

70 105.81

0.015 0.00735

AUTHOR INFORMATION

Corresponding Authors

Table 2. Apparent Kinetic Parameters for Catalyzed Oxidization of DOPA Enantiomers by Q11/G-DNA/Hemin Hybrida kcat (DOPA) (s−1)

ASSOCIATED CONTENT

S Supporting Information *

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Baoquan Ding: 0000-0003-1095-8872 Author Contributions #

These authors contributed equally.

[Q11], 40 μM; [G-DNA], 0.25 μM; [hemin], 0.1 μM; [H2O2], 10 mM. a

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Figure S26). A lower Km (DOPA) value for L-DOPA was observed, implying a stronger affinity of the complexed hemin to L-DOPA than D-DOPA. The results indicate that the selfassembled Q11 molecules acted as the chiral corona around hemin to allow the faster catalyzed oxidization of L-DOPA. As a control, the chiral Q11 encapsulating hemin exhibited no chiroselectivity over the enantiomeric DOPA, which indicated the prerequisite role of G-DNA as the scaffold. These results suggest a modular-assembly strategy for enantioselective discrimination between L-DOPA, an effective drug to treat Parkinson’s disease, and D-DOPA, which is inactive and may even cause side effects.52 However, G-DNA/H10/hemin and G-DNA/Q11/H10/hemin complexes did not exhibit chiroselectivity over DOPA enantiomers, probably because H10 was incapable of forming ordered structures like Q11 and may interfere with DNA−Q11 interactions (as concluded from Figure S16B). In summary, the present study introduced a novel concept to mimic the active site of natural peroxidase by controlled assembly of the designed Gln-containing peptides with Gquadruplex DNA/hemin DNAzyme. Theoretical simulation revealed that intermolecularly associated Gln peptides allowed the proper arrangement of Gln residues, which may provide a hydrogen-bond network to facilitate the adsorption of H2O2 to hemin iron. G-DNA can provide an axial ligand and a supramolecular scaffold for supporting and orienting hemin. Remarkable synergies between the Gln peptides and DNA in the activation of hemin were observed. The chiroselective catalysis was also achieved by the Gln peptide/DNA selfassembly that created a chiral catalytic environment. Furthermore, His-containing peptides were incorporated into the Gln peptides/DNAzyme system, resulting in significant enhancement of the catalytic activities. According to the experimental and theoretical results, it is proposed that the spatial configuration and the function of the assembled Gln/ His/Guanine groups may be similar to those of natural catalytic Arg/His/His triad, which was also theoretically exhibited. Our results may have implications for the potential biological roles of some in vivo-occurring peptide aggregates in catalytically triggering the cell function or dysfunctions, such as

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support from National Science Foundation China (21573051, 51761145044, 11422215, 11272327, and 11672079), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (21721002), National Basic Research Programs of China (2016YFA0201601, 2018YFA0208900), Beijing Municipal Science & Technology Commission (No. Z161100000116036), Key Research Program of Frontier Sciences, CAS (Grant No. QYZDB-SSWSLH029), CAS Interdisciplinary Innovation Team, K. C. Wong Education Foundation, Youth Innovation Promotion Association CAS, and the National Science Foundation of Beijing (2184130). We thank Prof. Di Li from East China Normal University for discussions on KIE.

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