Peptide–Gold Nanoparticle Conjugates as Artiﬁcial Carbonic Anhydrase Mimics

: We herein describe the design and synthesis of a catalytically active peptide–gold nanoparticle conjugate (Pep-Au-NP) that binds Zn(II) within its peptide monolayer and develops carbonic anhydrase activity. Speciﬁcally, a modiﬁed variant of the β -sheet forming IHIHIQI-peptide (IHQ), which forms an interstrand 3-His Zn(II)-binding site, was used as a ligand for spherical gold nanoparticles (Au-NPs). The resulting immobilized peptide maintains its ability to form β -sheets, as determined by circular dichroism (CD)-spectroscopy and, thus, maintains its ability to form Zn(II)-binding sites. The addition of Zn(II)-ions to the peptide–gold nanoparticle conjugates (Au@IHQ-NP) resulted in signiﬁcant improvements in rates of ester hydrolysis of 4-nitrophenyl acetate (4-NPA) and the hydration of CO 2 compared to the unconjugated peptide variants. Recycling of the catalyst revealed that Au@IHQ-NP remains intact with at least 94% of its initial activity after ﬁve rounds of CO 2 hydration. The herein reported results reveal that Pep-Au-NPs are able to perform reactions catalyzed by natural metalloenzymes and open up new possibilities for the implementation of these conjugates.


Introduction
The development of nanostructures that mimic the remarkable functions and catalytic properties of natural enzymes, i.e., to create nanozymes [1, 2], has been an exciting topic in the field of nanotechnology for the last two decades [3]. Among several approaches used to create nanozymes [4,5], the self-assembly of thiolated ligands onto the surface of gold nanoparticles (Au-NPs) to obtain monolayer-protected gold clusters (Au-MPCs) has emerged as a powerful strategy [6]. In particular, the conjugation of peptides on to the surface of gold nanoparticles to yield peptide-gold nanoparticle conjugates (Pep-Au-NPs) expanded the possibilities for the design of artificial enzymes [7][8][9][10]. Peptides are available at low cost by solid-phase peptide synthesis, and their modular design paired with the available repertoire of natural and non-natural amino acids allows for facile diversification of functional groups, and thus the resulting catalytic activities and substrate specificities of the peptide shell. The conjunction of peptides and Au-NPs leads to dense peptide monolayers in which new hydrogen bond and/or charge interactions between individual peptides are established. This enables known enzyme-like effects, such as the perturbation of pK a values of functional groups and cooperativity in substrate binding [9,11], and can even lead to new catalytic mechanisms [7,8]. Altogether, these effects can greatly improve the catalytic performance of a conjugated versus an unconjugated peptide.
To date, the design and application of Pep-Au-NPs have been limited to organic esterase chemistry, in which catalysis is mediated by a His-imidazole [7][8][9][10][11]. Considering the fact that zinc metalloenzymes perform numerous other types of reactions in nature, including phosphodiester and amide hydrolysis as well as the (de)hydrogenation of alcohols [12,13], it is of great interest to reconstitute such transformations in vitro by developing appropriate robust synthetic systems. For example, carbonic anhydrase (CA) is a naturally occurring zinc metalloenzyme that efficiently and reversibly catalyzes the hydration of CO 2 into hydrogen carbonate (HCO 3 − ). Its high activity originates from its zinc (Zn(II)) containing active site, in which three histidine imidazole moieties coordinate a Zn(II)-ion leaving a vacant space for the association of a water molecule. Zn(II) lowers the pK a of the bound water and forms a highly nucleophilic hydroxide species which reacts with CO 2 , bound in close proximity, to yield HCO 3 − [14]. CA isoforms have been extensively studied due to their clinical importance as drug targets and artificial lungs [15], and their great potential as catalysts in carbon capture systems [16][17][18]. Especially the latter application drove our interest, as the emission of CO 2 and the associated anthropogenic climate change steadily proceeds and new approaches for the absorption of CO 2 become increasingly urgent. In order to further increase our understanding of the design of Pep-Au-NPs, and to present a new approach for the absorption CO 2 , we herein describe the design and synthesis of the first Pep-Au-NP metalloenzyme mimicking carbonic anhydrase activity.

Peptide Design
The chosen peptide was designed according to the minimalistic β-sheet-forming heptapeptide IHIHIQI (IHQ) consisting of alternating hydrophobic residues and His ligands in positions i and i + 2 as reported by Rufo et al. [19]. The peptide assembles into extended parallel amyloid β-structures in which three His ligands form an interstrand 3-His Zn(II)-binding site (Figure 1, green) that functions as a hydrophobic primary coordination sphere, analogous to the active site of CA [20].
Grafting of IHQ or its cysteine-containing variant onto Au-NPs leads to irreversible aggregation and precipitation of the resulting Pep-Au-NPs. Thus, IHQ was modified as follows to obtain colloidal stable Pep-Au-NPs. The N-terminus was elongated by a spacer sequence of 9 amino acids comprised of Cys, Gly, Ala, and chromogenic Tyr (NH 2 -CAGYGAGAG-) to prevent aggregation of the Pep-Au-NPs by steric hindrance between the extended peptide chains and to enable conjugation to the gold-surface by stable S-Au-bonds. The C-terminus was elongated by a peptide sequence comprising the polar amino acids Thr, Gln, and Lys (-GTQK), which are residues found in the secondary coordination sphere of CA, for two reasons: first, positively charged Lys residues increase the electrostatic stabilization of the peptide monolayer by Coulomb repulsion between individual peptide chains; second, this sequence introduces a polar region that potentially assists in the catalytic process by engaging in stabilizing interactions with the transition state, or by providing a region which facilitates the release of product and/or solvent exchange [20,21]. The C-terminus was amidated to prevent possible aggregation of Pep-Au-NP after the addition of Zn(II).
The fully modified peptide sequence, referred to as IHQ-NP (Figure 1), was directly applied as a stabilizing agent in the reduction reaction of HAuCl 4 by NaBH 4 . The obtained bench stable Pep-Au-NPs are referred to as Au@IHQ-NP. The synthesized spherical Au@IHQ-NP possess a mean diameter of 10.8 ± 2.8 nm, which was determined by transmission electron microscopy (TEM, Supplementary Materials). Successful conjugation of IHQ-NP to the Au-NPs was verified by FT-IR-spectroscopy (Supplementary Materials). FT-IR stretching frequencies of characteristic bands of the peptide, including the Amide A, Amide I, and Amide II band, are present within the IR-spectrum of Au@IHQ-NP, confirming the presence of bound IHQ-NP. Characterization by UV/vis-spectroscopy (Supplementary Materials) shows a single surface plasmon resonance maximum at 525 nm, which is characteristic for spherical Au-NP. By applying supernatant analysis, the mean peptide loading per nanoparticle was determined to be 859 peptides/Au-NP. This corresponds to an average peptide density of 2.26 pep/nm 2 , which is consistent with literature reported peptide densities in the range of 1.41-2.55 pep/nm 2 , depending on the peptide length [9,11,22,23]. As the formation of β-sheets is a prerequisite for the generation of the 3-His Zn(II)-binding site, and thus the successful coordination of Zn(II) [19], circular dichroism (CD) spectroscopy was performed in order to assess the secondary structure of (un)conjugated IHQ-NP and its Zn(II)dependency.
The CD-spectra of IHQ-NP and Au@IHQ-NP show zinc-independent single ellipticity minima around 215 nm, respectively, which is characteristic for β-sheet structures ( Figure 2). Hence, IHQ-NP and conjugated IHQ-NP maintain the ability to form β-sheet structures even when immobilized at the surface of Au-NPs, indicating the presence of functional Zn(II)-binding domains and potential catalytic sites.  As the formation of β-sheets is a prerequisite for the generation of the 3-His Zn(II)-binding site, and thus the successful coordination of Zn(II) [19], circular dichroism (CD) spectroscopy was performed in order to assess the secondary structure of (un)conjugated IHQ-NP and its Zn(II)-dependency.
The CD-spectra of IHQ-NP and Au@IHQ-NP show zinc-independent single ellipticity minima around 215 nm, respectively, which is characteristic for β-sheet structures ( Figure 2). Hence, IHQ-NP and conjugated IHQ-NP maintain the ability to form β-sheet structures even when immobilized at the surface of Au-NPs, indicating the presence of functional Zn(II)-binding domains and potential catalytic sites. As the formation of β-sheets is a prerequisite for the generation of the 3-His Zn(II)-binding site, and thus the successful coordination of Zn(II) [19], circular dichroism (CD) spectroscopy was performed in order to assess the secondary structure of (un)conjugated IHQ-NP and its Zn(II)dependency.
The CD-spectra of IHQ-NP and Au@IHQ-NP show zinc-independent single ellipticity minima around 215 nm, respectively, which is characteristic for β-sheet structures ( Figure 2). Hence, IHQ-NP and conjugated IHQ-NP maintain the ability to form β-sheet structures even when immobilized at the surface of Au-NPs, indicating the presence of functional Zn(II)-binding domains and potential catalytic sites.

Esterase Activity
Although the natural reaction catalyzed by CA is the reversible hydration of CO 2 to HCO 3 − , a frequently used benchmark reaction to study the successful coordination of Zn(II) and function of the active sites is the hydrolysis of model substrate 4-nitrophenyl acetate (4-NPA) [24]. Both reactions are initiated by a similar mechanism, that is, the nucleophilic attack of the Zn(II)-bound hydroxide to the substrate (Scheme 1) [25,26]. The hydrolysis of 4-NPA can readily be traced by measuring the UV/vis-absorbance of the 4-nitrophenolate (4-NP) product at 405 nm.

Esterase Activity
Although the natural reaction catalyzed by CA is the reversible hydration of CO2 to HCO3 − , a frequently used benchmark reaction to study the successful coordination of Zn(II) and function of the active sites is the hydrolysis of model substrate 4-nitrophenyl acetate (4-NPA) [24]. Both reactions are initiated by a similar mechanism, that is, the nucleophilic attack of the Zn(II)-bound hydroxide to the substrate (Scheme 1) [25,26]. The hydrolysis of 4-NPA can readily be traced by measuring the UV/vis-absorbance of the 4-nitrophenolate (4-NP) product at 405 nm. To verify the Zn(II)-binding and function of the catalytic Zn(II)-center, IHQ-NP and Au@IHQ-NP were studied in the context of their ability to cleave 4-NPA in absence or presence of Zn(II). In the absence of Zn(II) nucleophilic and/or general acid/base catalysis performed by the His imidazole is expected, while in presence of Zn(II), hydrolysis is executed by the more rapid Zn(II)-bound hydroxide mechanism [19].
To ensure proper metal coordination [19], 10 equivalents of Zn(II) were added to the catalyst mixture and the resulting Zn(II)-containing peptide(-gold nanoparticle conjugate) are referred to as Zn(II)-IHQ-NP and Zn(II)-Au@IHQ-NP. Figure 3a and b show the blank-corrected saturation curves of IHQ-NP and Au@IHQ-NP in the presence or absence of Zn(II). While the dependence of unconjugated Zn(II)-/IHQ-NP-catalyzed 4-NPA hydrolysis on 4-NPA concentration displays hyperbolic behavior (Figure 3a), which is in accordance with the Michaelis-Menten theory, sigmoidal behavior was observed for Zn(II)-/Au@IHQ-NP ( Figure 3b). A sigmoidal profile indicates a positive cooperative effect for the binding of substrate molecules among individual active sites and has been previously observed to be an effect evoked by the conjugation and the resulting high peptide density within the peptide monolayer [9,11]. Positive cooperativity in substrate binding is classified as a regulatory mechanism also found in nature. Instead of a linearly increasing behavior of the catalytic response curve at low substrate concentrations (0-0.1 mM), the ascent of the catalytic response curve starts shallow, as above a certain concentration, small changes in the substrate concentration lead to greater catalytic responses. The Hill-coefficient n is frequently used to express the magnitude of cooperativity or slope of the sigmoidal curve. We found positive cooperativity in Zn(II)-Au@IHQ-NP-catalyzed 4-NPA hydrolysis (n = 2.5) to be 0.6 units higher compared to Au@IHQ-NP (n = 1.9), indicating that Zn(II)binding alters substrate interactions and the associated catalytic process.  To verify the Zn(II)-binding and function of the catalytic Zn(II)-center, IHQ-NP and Au@IHQ-NP were studied in the context of their ability to cleave 4-NPA in absence or presence of Zn(II). In the absence of Zn(II) nucleophilic and/or general acid/base catalysis performed by the His imidazole is expected, while in presence of Zn(II), hydrolysis is executed by the more rapid Zn(II)-bound hydroxide mechanism [19].
To ensure proper metal coordination [19], 10 equivalents of Zn(II) were added to the catalyst mixture and the resulting Zn(II)-containing peptide(-gold nanoparticle conjugate) are referred to as Zn(II)-IHQ-NP and Zn(II)-Au@IHQ-NP. Figure 3a,b show the blank-corrected saturation curves of IHQ-NP and Au@IHQ-NP in the presence or absence of Zn(II). While the dependence of unconjugated Zn(II)-/IHQ-NP-catalyzed 4-NPA hydrolysis on 4-NPA concentration displays hyperbolic behavior (Figure 3a), which is in accordance with the Michaelis-Menten theory, sigmoidal behavior was observed for Zn(II)-/Au@IHQ-NP (Figure 3b). A sigmoidal profile indicates a positive cooperative effect for the binding of substrate molecules among individual active sites and has been previously observed to be an effect evoked by the conjugation and the resulting high peptide density within the peptide monolayer [9,11]. Positive cooperativity in substrate binding is classified as a regulatory mechanism also found in nature. Instead of a linearly increasing behavior of the catalytic response curve at low substrate concentrations (0-0.1 mM), the ascent of the catalytic response curve starts shallow, as above a certain concentration, small changes in the substrate concentration lead to greater catalytic responses. The Hill-coefficient n is frequently used to express the magnitude of cooperativity or slope of the sigmoidal curve. We found positive cooperativity in Zn(II)-Au@IHQ-NP-catalyzed 4-NPA hydrolysis (n = 2.5) to be 0.6 units higher compared to Au@IHQ-NP (n = 1.9), indicating that Zn(II)-binding alters substrate interactions and the associated catalytic process.  Table 1 summarizes the catalytic parameters kcat, KM, and kcat/KM of IHQ-NP, Au@IHQ-NP in the absence or presence of Zn(II). The immobilization of IHQ-NP onto the surface of Au-NPs (Au@IHQ-NP) led to an overall increase in catalytic efficiency (kcat/KM) by almost 2-fold. Interestingly, the increase in kcat/KM originates exclusively from a decrease in KM, indicating a higher affinity for the peptide monolayer. We assume that the high peptide density leads to the formation of a secondary coordination sphere with sophisticated substrate interactions and possibly the formation of transient binding pockets within the peptide monolayer that lead to improved binding. Riccardi et al. previously verified the formation of such pockets for the binding of small molecules using even less complex Au-MPCs consisting of oligo(ethylene glycol)-based thiols [27]. Thus, it may be expected that Pep-Au-NPs composed of peptides with a more diverse set of functionalities are able to form similar binding cavities. The addition of Zn(II) to the peptide catalysts further improved the catalytic efficiency for the Zn(II)-IHQ-NP-and Zn(II)-Au@IHQ-NP-catalyzed ester hydrolysis by more than 3-fold, compared to the respective peptides in the absence of Zn(II). This time, the improvement in catalytic efficiency of Zn(II)-IHQ-NP and Zn(II)-Au@IHQ-NP results from both a decrease in KM and an increase in turnover number kcat by 2-and 3-fold, respectively. The increase in catalytic activity upon addition of Zn(II) is in accordance with the rate enhancements reported for the IHQ peptide when the catalytic Zn(II)-center is developed [19]. Hence, IHQ-NP and Au@IHQ-NP are able to bind Zn(II) and execute 4-NPA hydrolysis by the Zn(II)-bound hydroxide mechanism rather than nucleophilic and/or general acid/base catalysis performed by the His imidazole.  Table 1 summarizes the catalytic parameters k cat , K M , and k cat /K M of IHQ-NP, Au@IHQ-NP in the absence or presence of Zn(II). The immobilization of IHQ-NP onto the surface of Au-NPs (Au@IHQ-NP) led to an overall increase in catalytic efficiency (k cat /K M ) by almost 2-fold. Interestingly, the increase in k cat /K M originates exclusively from a decrease in K M , indicating a higher affinity for the peptide monolayer. We assume that the high peptide density leads to the formation of a secondary coordination sphere with sophisticated substrate interactions and possibly the formation of transient binding pockets within the peptide monolayer that lead to improved binding. Riccardi et al. previously verified the formation of such pockets for the binding of small molecules using even less complex Au-MPCs consisting of oligo(ethylene glycol)-based thiols [27]. Thus, it may be expected that Pep-Au-NPs composed of peptides with a more diverse set of functionalities are able to form similar binding cavities. Table 1. Determined kinetic parameters k cat , K M , and k cat /K M of IHQ-NP-catalyzed 4-NPA hydrolysis in absence and presence of Zn(II). Reactions were performed in Tris/HCl buffer pH 8 with 5% acetonitrile at 25 • C 10 µM (Immobilized) IHQ-NP was used and 100 µM ZnCl 2 was added for reactions performed in presence of Zn(II).  The addition of Zn(II) to the peptide catalysts further improved the catalytic efficiency for the Zn(II)-IHQ-NP-and Zn(II)-Au@IHQ-NP-catalyzed ester hydrolysis by more than 3-fold, compared to the respective peptides in the absence of Zn(II). This time, the improvement in catalytic efficiency of Zn(II)-IHQ-NP and Zn(II)-Au@IHQ-NP results from both a decrease in K M and an increase in turnover number k cat by 2-and 3-fold, respectively. The increase in catalytic activity upon addition of Zn(II) is in accordance with the rate enhancements reported for the IHQ peptide when the catalytic Zn(II)-center is developed [19]. Hence, IHQ-NP and Au@IHQ-NP are able to bind Zn(II) and execute 4-NPA hydrolysis by the Zn(II)-bound hydroxide mechanism rather than nucleophilic and/or general acid/base catalysis performed by the His imidazole.
Compared to other literature on known Pep-Au-NPs by Koksch and co-workers [9][10][11] or Pengo et al. [7,8], (Zn(II)-)Au@IHQ-NP shows only moderate catalytic efficiency. However, both particle size and peptide loading deviate from the reported conjugates and most probably influence the catalytic properties. Thus, comparisons of catalytic efficiency should be done carefully. Interestingly, independent of the conjugate, all show a similar K M of approximately 0.5 mM [9][10][11], indicating a similar affinity of 4-NPA for the respective peptide monolayers.

Carbonic Anhydrase Activity
We were particularly interested in establishing Au@IHQ-NP as a catalyst for the hydration of CO 2 to HCO 3 − . Since H + is generated during the catalytic process, the chemical absorption of CO 2 can readily be monitored by following the change in pH over time, which is the basis for the frequently used delta-pH method [28][29][30]. Here, the delta-pH method serves as a qualitative tool to investigate the kinetics of CO 2 -hydration and proof-of-concept for Au@IHQ-NP functioning as a CA mimic. Kinetic experiments were conducted in Tris/HCl buffer pH 8.9 in order to sufficiently cover the drop in pH over 2 units of pH, in the absence or presence of Zn(II). Figure 4a shows the time-dependent change in pH during the hydration of CO 2 in presence of IHQ-NP and Au@IHQ-NP, compared to the buffer control. Compared to other literature on known Pep-Au-NPs by Koksch and co-workers [9][10][11] or Pengo et al. [7,8], (Zn(II)-)Au@IHQ-NP shows only moderate catalytic efficiency. However, both particle size and peptide loading deviate from the reported conjugates and most probably influence the catalytic properties. Thus, comparisons of catalytic efficiency should be done carefully. Interestingly, independent of the conjugate, all show a similar KM of approximately 0.5 mM [9][10][11], indicating a similar affinity of 4-NPA for the respective peptide monolayers.

Carbonic Anhydrase Activity
We were particularly interested in establishing Au@IHQ-NP as a catalyst for the hydration of CO2 to HCO3 -. Since H + is generated during the catalytic process, the chemical absorption of CO2 can readily be monitored by following the change in pH over time, which is the basis for the frequently used delta-pH method [28][29][30]. Here, the delta-pH method serves as a qualitative tool to investigate the kinetics of CO2-hydration and proof-of-concept for Au@IHQ-NP functioning as a CA mimic.
Kinetic experiments were conducted in Tris/HCl buffer pH 8.9 in order to sufficiently cover the drop in pH over 2 units of pH, in the absence or presence of Zn(II). Figure 4a shows the timedependent change in pH during the hydration of CO2 in presence of IHQ-NP and Au@IHQ-NP, compared to the buffer control.  Both, IHQ-NP and Au@IHQ-NP show a marginal drop in pH over the buffer control, thus marginal increases in CO2 hydration in the absence of Zn(II) by ~3% and 6%, respectively. The small rate increase of IHQ-NP and Au@IHQ-NP can be attributed to the His imidazole which is reported to promote CO2 hydration [31][32][33].
More strikingly, the pH drops significantly faster for Zn(II)-IHQ-NP and Zn(II)-Au@IHQ-NP, compared to the buffer control (Figure 4b). CO2-hydration increases by 21% and 55%, respectively. The significantly higher activity in the presence of Zn(II) indicates that the catalytic process proceeds via the catalytic Zn(II)-center. Interestingly, Zn(II)-Au@IHQ-NP shows a significantly greater rate acceleration compared to the unconjugated Zn(II)-IHQ-NP. We assume that the peptide monolayer again acts as a more elaborate secondary coordination sphere in which CO2 interactions and/or transition state stabilization take place. To date, increased rates of reaction, evoked solely by conjugation of a peptide to the surface of Au-NPs, has only been observed in ester hydrolysis reactions performed by a His imidazole [7][8][9][10][11]. The herein described CO2 hydration indicates that the cooperative or synergistic effects that lead to the rate improvements are also applicable to other reactions executed by catalytic (metal) centers. Moreover, we hypothesize that the introduced polar region at the solvent exposed end of the peptide monolayer facilitates water/H + -shuttling as is the Both, IHQ-NP and Au@IHQ-NP show a marginal drop in pH over the buffer control, thus marginal increases in CO 2 hydration in the absence of Zn(II) by~3% and 6%, respectively. The small rate increase of IHQ-NP and Au@IHQ-NP can be attributed to the His imidazole which is reported to promote CO 2 hydration [31][32][33].
More strikingly, the pH drops significantly faster for Zn(II)-IHQ-NP and Zn(II)-Au@IHQ-NP, compared to the buffer control (Figure 4b). CO 2 -hydration increases by 21% and 55%, respectively. The significantly higher activity in the presence of Zn(II) indicates that the catalytic process proceeds via the catalytic Zn(II)-center. Interestingly, Zn(II)-Au@IHQ-NP shows a significantly greater rate acceleration compared to the unconjugated Zn(II)-IHQ-NP. We assume that the peptide monolayer again acts as a more elaborate secondary coordination sphere in which CO 2 interactions and/or transition state stabilization take place. To date, increased rates of reaction, evoked solely by conjugation of a peptide to the surface of Au-NPs, has only been observed in ester hydrolysis reactions performed by a His imidazole [7][8][9][10][11]. The herein described CO 2 hydration indicates that the cooperative or synergistic effects that lead to the rate improvements are also applicable to other reactions executed by catalytic (metal) centers. Moreover, we hypothesize that the introduced polar region at the solvent exposed end of the peptide monolayer facilitates water/H + -shuttling as is the case in native CA. However, detailed mechanistic studies are necessary in order to elucidate the complex process that leads to the observed rate enhancements of Zn(II)-Au@IHQ-NP.
Since conjugation of peptides onto Au-NPs represents a transition from homogenous to heterogeneous catalysis, recycling of Zn(II)-Au@IHQ-NP is feasible. Subsequent to CO 2 hydration, we released the absorbed CO 2 by elevating the temperature of the reaction mixture to 60 • C and bubbling a stream of N 2 -gas through the solution, until the initial pH of 8.9 was reached. We observed a slight change in the color of the Pep-Au-NP dispersion from red to violet, indicating some degree of instability of the Au-NPs. However, to our surprise, the treatment of the reaction mixture and subsequent color change did not affect the catalytic activity as Zn(II)-Au@IHQ-NP maintained almost 94% of its activity over five cycles of CO 2 hydration (Figure 5a,b).
Catalysts 2019, 9, x FOR PEER REVIEW 7 of 11 case in native CA. However, detailed mechanistic studies are necessary in order to elucidate the complex process that leads to the observed rate enhancements of Zn(II)-Au@IHQ-NP. Since conjugation of peptides onto Au-NPs represents a transition from homogenous to heterogeneous catalysis, recycling of Zn(II)-Au@IHQ-NP is feasible. Subsequent to CO2 hydration, we released the absorbed CO2 by elevating the temperature of the reaction mixture to 60° C and bubbling a stream of N2-gas through the solution, until the initial pH of 8.9 was reached. We observed a slight change in the color of the Pep-Au-NP dispersion from red to violet, indicating some degree of instability of the Au-NPs. However, to our surprise, the treatment of the reaction mixture and subsequent color change did not affect the catalytic activity as Zn(II)-Au@IHQ-NP maintained almost 94% of its activity over five cycles of CO2 hydration (Figure 5a and b).

Peptide Stock Solution
Amyloidogenic IHQ-NP was handled according to Rufo et al. [19]. Briefly, IHQ-NP stock solution was prepared by dissolving lyophilized IHQ-NP in 10 mM HCl. Concentration of stock solution was determined by using a Varian Cary 50 UV-Vis Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) and by applying an experimentally determined extinction coefficient (ε 280 ) of 1263 L mol −1 cm −1 . Working solutions at elevated pH >8 were freshly prepared and the pH verified before each individual measurement.

Synthesis of Au@IHQ-NP
Pep-Au-NP synthesis was performed according to our previously established procedure [10]. Briefly, an aqueous solution of HAuCl 4 (0.8 mL, 2.49 × 10 −2 M; Sigma-Aldrich ® , Merck KGaA, Darmstadt, Germany) was added to enzyme-free water (EF-H 2 O; Water for Molecular Biology, Merck KGaA, Darmstadt, Germany) containing IHQ-NP (1.5 mL of 0.6 mM Solution) while carefully adjusting the pH to a value of 6 (total reaction volume 10 mL). Slightly acidic reaction conditions prevented premature aggregation of the peptide. An aqueous solution of freshly prepared NaBH 4 (0.3 mL, 0.01 M) was added after 5 min of stirring. Following an additional 1 h of vigorous stirring, the formed red dispersion was centrifuged using centrifugal filters (Amicon Ultra-0.5 NMWL 30 kDa; Sigma-Aldrich ® , Merck KGaA, Darmstadt, Germany) to recover Au@IHQ-NP as a dark red pellet. The pellet was redispersed in EF-H 2 O and again centrifuged to remove excess NaBH 4 . The resulting pellet was redispersed in EF-H 2 O and stored at room temperature. Nanoparticle concentration was calculated according to equations provided by Liu et al. [34]. The average peptide loading was determined by supernatants as previously described [9,10]. Briefly, after centrifugation, the supernatant was analyzed and the amount of unbound peptide determined by UV/vis-spectroscopy (see Section 3.2). The amount of unbound peptide was subtracted from the amount of peptide added during the reaction, giving the amount of bound peptide on the Au-NPs.

Esterase Activity
The rates of hydrolysis of 4-NPA were monitored in 50 mM Tris/HCl pH 8 at a constant temperature of 25 • C by measuring the UV/vis-absorbance at 405 nm of the formed product 4-nitrophenolate (4-NP). Using a literature-reported extinction coefficient of 16,600 L mol −1 cm −1 [19], initial rates of reaction (v 0 ) of uncatalyzed (blank) and catalyzed reactions were determined over a range of substrate concentrations until saturation conditions were observed. Initial rates of reaction of the catalysts were obtained by subtracting v 0 of the blank reactions from v 0 of the corresponding catalyzed reaction. Kinetic experiments were performed at least three times to determine standard deviations. The Kinetic parameters v max , K M , and the Hill-coefficient n were obtained by fitting the measured data points to the Hill or Michaelis-Menten equation by performing a nonlinear regression using OriginLab (OriginPro, Version 2019 Academic; OriginLab Corporation, Northampton, MA, USA.) Calculation of errors and error propagation was carried out according to Gaussian equations for normally distributed errors and the derived error propagation.
Ester hydrolysis was performed in quartz cuvettes (QS, 1 cm path length 3.5 mL, Hellma Analytics) using Varian Cary 50 UV-Vis Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) thermostatted at 25 • C (Lauda RKS). Ester hydrolysis was initiated by addition of a defined volume of 4-NPA stock solution in acetonitrile. In total, 5% acetonitrile was present in each reaction mixture.
Kinetic studies were conducted using a calculated (immobilized) peptide concentration of 10 µM, while 100 µM ZnCl 2 was added when the reaction mixture contained Zn(II). Initial 4-NPA concentration was gradually increased so 0.2, 0.4, 0.6, and 0.8 mM were applied in 4-NPA hydrolysis.

Carbonic Anhydrase Activity
The delta-pH method was used to determine the feasibility of peptide/Pep-Au-NP-catalyzed CO 2 hydration. A setup reported by Kellsey et al. was modified and used for all hydration reactions [29]. Briefly, a pH probe and glass tubing were immersed into the two necks of a 2-necked 100 mL round-bottom flask containing 10 µM of the respective (immobilized) IHQ-NP in the absence and presence of 100 µM ZnCl 2 in Tris/HCl-buffer pH 8.9 (total volume 40 mL) at 22 • C. CO 2 gas (Carbon dioxide N35, Air Liquide) was streamed through the gas tubing into the reaction mixture at a constant flow of 0.1 L/min. The changes in pH were recorded automatically using a pHenomenal ® pH 1100L pH-meter (VWR International GmbH, Darmstadt, Germany). Rates of hydration were determined by the slope of the plot of time-dependent pH changes.

Conclusions
In summary, the catalytically active Zn(II)-binding peptide IHQ-NP was conjugated onto Au-NPs leading to the formation of a peptide monolayer that is able to bind Zn(II) and develop a catalytic Zn(II)-center. Peptide variants IHQ-NP and Au@IHQ-NP were tested for their ability to serve as esterase and carbonic anhydrase mimics. While IHQ-NP and Au@IHQ-NP both noticeably catalyze ester hydrolysis and marginally catalyze CO 2 hydration in the absence of Zn(II), significant improvements in catalytic efficiency and rate acceleration were observed in the presence of Zn(II) for both reactions. The Zn(II)-Au@IHQ-NP showed even greater rate improvements for ester hydrolysis and CO 2 hydration compared to the unconjugated Zn(II)-IHQ-NP variant. This is in accordance with previously reported results showing that Pep-Au-NPs can generate secondary coordination spheres in which synergistic effects and cooperativity in substrate binding and the associated improvements in catalysis over the unconjugated peptide are observed. Recycle experiments further revealed Zn(II)-Au@IHQ-NP to maintain at least 94% of its catalytic activity over 5 cycles of CO 2 hydration. Altogether, the herein reported results validate the proof-of-concept that Pep-Au-NPs can be designed to bind metal ions within their peptide monolayer and develop metalloenzymatic activities. The presented results provide a design principle based on a well-defined peptide secondary structure and demonstrate the exponentially increasing possibilities, due to the combination of Au-NPs and catalytically active peptide sequences, as their catalytic toolbox can be further expanded based on nature's examples.