Vesicular Mixed Gemini−SDS−Hemin−Imidazole Complex as a

ARTICLE pubs.acs.org/JPCB

Vesicular Mixed Gemini
SDS
Hemin
Imidazole Complex as a Peroxidase-Like Nano Artificial Enzyme Hussein Gharibi,*,† Zainab Moosavi-Movahedi,† Sohaeila Javadian,† Khodadad Nazari,^ and Ali A. Moosavi-Movahedi‡ †

Department of Physical Chemistry, Faculty of Science, Tarbiat Modares University, Tehran, Iran Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran ^ Research Institute of Petroleum Industry, N.I.O.C., Tehran, Iran ‡

ABSTRACT: A biomimetic was designed for the construction of a new efficient peroxidase-like nano artificial enzyme with a heme
imidazole component complexed with gemini 12-2-12/ SDS supramolecules. The presence of a simple surfactant mixture (SDS/gemini 12-2-12 at a particular concentration) provided an apoprotein-like hydrophobic pocket for the heme
imidazole moiety, which produced a peroxidase active site containing positive and negative charges distributed on the colloidal surface. Vesicular structures that stabilized the heme
imidazole complexes formed multienzyme advanced colloids. The enzymatic activation parameters indicated that the catalytic efficiency of the novel nano artificial enzyme was 27% as efficient as the native horseradish peroxidase (HRP). The imidazole moiety, which functionally corresponded to the histidine ligand in the native HRP, increased the reactivity and catalytic efficiency of the artificial enzyme. The nano biocatalyst did not exhibit suicide inactivation until high concentrations of hydrogen peroxide, indicating that the vesicle hydrophobic pocket effectively shielded the active site, thereby controlling the concentration of hydrogen peroxide at the heme moiety and enabling high rates of enzymatic turnover.

1. INTRODUCTION Biomimetic chemistry is concerned with designing novel chemical strategies inspired by the chemistry of living systems1 and attempts to improve the performance of chemical reactions and catalysts by imitating enzymatic processes.2 Robust biocatalysts may be formed by encapsulating metalloporphyrins in micelles or vesicles that mimic the polypeptide envelope protecting the catalytic center of the natural enzymes.3 With the goal of replicating and modeling the physicochemical roles of enzymes, artificial enzymes are biomimetically constructed from synthetic materials to simulate the catalytic functions exhibited by natural enzymes.4 Molecular assemblies can provide specific microenvironments for substrate binding and catalysis in aqueous media, similar to apo-enzymes. Such assemblies may be designed with a hydrophobic interior or core composed of surfactant hydrocarbon tails covered with a hydrophilic, usually ionic, surface layer made from surfactant head groups. These assemblies are similar to the structural organizations of globular proteins and biological membranes. The successful demonstration of biomimetic catalysis in supramolecular assemblies would constitute a significant advance in the field.3,5 The design of biomimetic protein pockets similar to those present in hemoproteins and heme-based enzymes has maintained a high level of interest, despite several challenges. The electronic properties and biochemical functions of heme prosthetic groups in hemoproteins depend strongly on the nature of r 2011 American Chemical Society

the protein environment surrounding the heme group.6
8 The porphyrin macrocycle is equatorially coordinated to the metal center, leaving two axial coordination sites, L1 and L2, for occupation by amino acid ligand donors from the protein, solvent, or substrate. One of these axial sites is, in many cases, the site at which catalysis occurs.9
11 The nature of the axial amino acid ligand determines the biochemical function.9 Several hemoproteins, including the peroxidases (L1 = His) and catalase (L1 = Tyr), use H2O2 to form an oxy-ferryl intermediate [Pþ•Fe4þL1O] or [PFe4þL1O, R•, where R• is a radical located on a protein amino acid residue that oxidizes the substrate.12 Previous studies have shown that porphyrins may be solubilized by detergents, such as sodium dodecyl sulfate (SDS). Strong hydrophobic interactions between the detergent and the porphyrin overcome the porphyrin
porphyrin forces to solubilize the porphyrin at detergent concentrations corresponding to the critical micelle concentration (CMC).13,14 The positioning of the porphyrin molecules within the micelle and the average distance between the micellar carbon and the iron center of the porphyrin complex has been determined.6,15 Micelles of different sizes may mimic the heme environments in different proteins. Moreover, the effect of electrostatic interactions, as well Received: December 19, 2010 Revised: February 5, 2011 Published: April 05, 2011 4671

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The Journal of Physical Chemistry B as hydrophobic effects, can be studied using heme complexes incorporated into micellar solutions with different surface charges.6 Histidine has been shown to improve the activity of artificial peroxidase models.3,16,17 In many peroxidases, the catalytically active iron center is coordinated to the four pyrrole nitrogen atoms of the porphyrin ligand in addition to an axial imidazole donor.18 The axial donor (usually an imidazole or pyridine or derivatives thereof) is believed to facilitate heterolytic cleavage of the O
O bond.19,20 In such cases, a fifth axial donor, preferably an imidazole group, covalently attaches to the heme complex. Mimics of this structure have provided efficient artificial minienzymes capable of reproducing peroxidase reactions with good biocatalytic activity. Hydrogen peroxide binds to the vacant sixth (axial) coordination site and is activated, as in native heme peroxidases. Hemoproteins have been the subject of extensive mechanistic,21
28 kinetic,29
32 and thermodynamic33
36 studies.37,38 At high hydrogen peroxide concentrations, all hemoproteins, including peroxidases, are inactivated21,39
41 through a pathway referred to as “suicide-peroxide inactivation”. The kinetic model for suicide-peroxide inactivation of catalase (as a monosubstrate hemoenzyme)42 and horseradish peroxidase (as a bisubstrate hemoenzyme) has been described previously.40 The inactivation process for horseradish peroxidase was monitored by measuring the time course (progress curve) of the oxidation reaction of an aromatic hydrogen donor substrate.40 These results were used to construct a kinetic model governing the process. The phase behavior and microstructures have been investigated for aqueous mixtures of cationic gemini surfactants, such as 12-3-12, 2Br
as a kind of m-s-m geminies, and the anionic surfactant sodium dodecyl sulfate (SDS). The phase diagram includes several regions characterized by different microstructures. Most of the regions occupied by vesicles coexist with micelles in dilute solutions.43
47 The dynamics of the micelle
vesicle transitions in aqueous anionic and cationic surfactant mixtures have been modeled previously.48,49 In this work, we designed a nano artificial peroxidase enzyme by embedding a heme complex inside the hydrophobic environment of mixed SDS
Gemini (12-2-12, 2Br
) supramolecules containing imidazole ligands. We obtained reasonable enzymatic efficiencies and evaluated the suicide inactivation parameters. The presence of both negative and positive charges in the hydrophobic pocket of this new nano artificial multienzyme, which mimicked the native apoprotein environment of the heme moiety, significantly improved the enzyme parameters (activation and suicide inactivation).

2. MATERIALS AND METHODS Materials. Bovine hemin, sodium dodecyl sulfate, and imidazole were obtained from Sigma. Gemini (12-2-12, 2Br
) was synthesized and purified. Other chemicals were purchased from Merck, were of analytical grade, and were used without further purification. Doubly distilled water was used throughout. Hemin, hydrogen peroxide, and guaiacol solutions were prepared daily. Absorption spectra were collected using a model Shimadzu3100 spectrophotometer with 1 cm path length cells equipped with a thermostatted holder. The steady-state kinetics of guaiacol oxidation by hydrogen peroxide, catalyzed by the model catalyst, were monitored at 470 nm (colored product of the reaction)50 in 5 mM phosphate buffer solution, pH = 7 (PBS).

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Methods. Steady-State Kinetics. The steady-state kinetics of guaiacol (as a hydrogen donor) oxidation by hydrogen peroxide, catalyzed by the artificial peroxidase enzyme, were obtained at 470 nm (colored product of the reaction)3 in PBS. Progress curves for the reactions were obtained at various guaiacol concentrations, and the initial rates were used to construct Michaelis
Menten curves. The concentration of H2O2 was kept high (1.2 mM) and constant with respect to the hydrogen donor during the course of the reaction to ensure pseudofirst-order kinetics. Steady-state conditions were reached after a lag time of 7 s. The concentration of hydrogen peroxide was determined from the absorbance measurements at 240 nm, with ε240 = 43.6 cm
1 M
1.3 The dilute solutions were freshly prepared. Suicide-Peroxide Inactivation. Suicide-peroxide inactivation of the peroxidase-like artificial enzyme was monitored at 470 nm (λmax for the product of the catalytic reaction) by difference spectrophotometry. The details of the procedures for measuring the product concentration, rate of reaction, and progress curves have been described previously.40 Progress curves were determined by following the absorbance of the reaction mixture at 470 nm during a time course of 10 min. After the reaction had been initiated, the first 30 s were considered to be dead time required for the reaction to reach the stationary state. The unreacted hydrogen donor or reductant
substrate (AH) concentration was calculated from the absorbance measurements and suitable relations.40 “End-point procedure” experiments were conducted to determine the maximum increase in absorbance (A¥) and the value of the intact activity (Ri). The reaction mixture was incubated for an extended period of time (2 h) according to the “end-point procedure” described in detail elsewhere.21,40 In the end-point method, an excess of fresh HRP solution was added to the incubated mixture. A¥ was used to calculate the concentration of unreacted AH ([AH]¥), and the intact activity of the enzyme (Ri) was estimated. The intact activity (Ri) was defined as the enzyme activity before it was exposed or reacted with the substrate. Dynamic Light Scattering. The hydrodynamic radii and zeta potentials of several biocatalyst mixtures, including our new artificial enzyme, were determined using a Zetasizer IIC dynamic light scattering and electrophoretic light scattering instrument (Malvern Instruments, UK). Transmission Electron Microscopy. Transmission electron micrographs were recorded on a ZEISS electron microscope (EM-10C) operated at 100 kV. Nano Calorimetric Measurements. Isothermal titration calorimetric (ITC) experiments were carried out using a VP-ITC ultrasensitive titration calorimeter (NanoCal, LLC, Northampton, MA). The nanocalorimeter consisted of a reference cell and a sample cell 1.8 mL in volume, with both cells insulated by an adiabatic shield. All solutions were thoroughly degassed before use by stirring under vacuum. The sample cell was loaded with an SDS/gemini 12-2-12 vesicle solution (90 mM/0.8 mM), and the reference cell contained buffer solution. The solution in the cell was stirred at 307 rpm using a syringe (equipped with a micropropeller) filled with heme/imidazole solution (175 μM/ 43.75 mM) to ensure rapid mixing. Injections were started after the baseline stability was achieved. The titration of SDS/gemini 12-2-12 vesicles with the heme/ imidazole solution involved 30 consecutive injections of the ligand solution, with a first injection of 5 μL and the remaining injections of 10 μL. Each injection took 6 s, and they were introduced at 3 min intervals. The thermal effects due to 4672

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Scheme 1. Schematic Diagram Showing the Micelle Heme/Imidazole and Vesicle Heme/Imidazole Artificial Peroxidase-Like Models

heme/imidazole dilution were corrected based on control experiments in which aliquots of the heme/imidazole solution were injected into buffer solutions with or without the SDS/gemini 12-2-12 vesicles. The enthalpy changes associated with processes occurring at a constant temperature were measured in ITC experiments. The measurements were performed at a constant temperature of 27.0 C, and the temperature was controlled using a Poly-Science water bath. Surface Tension. Surface tension measurements were performed at 27 C on a Kr€uss K100 tensiometer using the Wilhelmy plate method at atmospheric pressure. The platinum plate was thoroughly cleaned and flame-dried prior to each measurement. Measurements of the surface tension of pure water were performed to calibrate the tensiometer and check the cleanliness of the glassware. Gaussian Program. The volume of each gemini 12-2-12 molecule was calculated by the B3LYP/6-31G* method in the Gaussian 98 program.

3. RESULTS AND DISCUSSION The heme group plays a fundamental role in the activation of hemoproteins and hemoenzymes, such as horseradish peroxidase (HRP). Heme moieties in the absence of protein environments also play a catalytic role, but direct exposure to the solvent and substrate can reduce heme groups to inactive oxo complexes. An alternative approach to the preparation of robust biocatalysts involves the encapsulation of metalloporphyrins into micelles or vesicles that mimic the polypeptide envelope that protects the catalytic centers of natural enzymes.3 The apo-proteins that surround heme groups have negative and positive charges on their surfaces. Mixed positive and negative surfactants, micelles, or supramolecular hydrophobic pockets can mimic this property of the protein scaffold. As discussed in Section 3.1, mixtures containing SDS and a type

Table 1. DLS Parameters for the Various Catalysts compounds SDS (90 mM) SDS (90 mM) þ heme

diameter (nm)

zeta-potential (mV)

1.65 2.23


74.51
59.60

45.59


65.47

47.32


66.89

(10 μM) þ imidazole (3 mM) SDS (90 mM) þ gemini 12-2-12 (0.8 mM) SDS (90 mM) þ gemini 12-2-12 (0.8 mM) þ heme (10 μM) þ imidazole (3 mM)

of m-s-m gemini surfactant, 12-2-12, can produce stable vesicles. This supramolecule may be able to provide a proper and convenient hydrophobic pocket for heme
imidazole complexes as a mimic of peroxidase active sites in aqueous solutions (Scheme 1). 3.1. Structural Approach. To achieve a better understanding of heme-based biocatalysts, a structural study was performed. Quantitative insights into the micelle-to-vesicle transition from pure SDS (90 mM) to a mixed SDS (90 mM)/gemini 12-2-12 (0.8 mM) with inclusion of the heme
imidazole group in both systems were gained by performing dynamic light scattering measurements. As shown in Table 1, the addition of heme
imidazole complexes to SDS micelles increased the hydrodynamic radii of the micelles due to the proximity of the heme to the charged micelle surface and due to deep embedding of the heme in the hydrophobic micelle core. Mixing gemini 12-2-12 with SDS produced a large shift in the particle hydrodynamic radii, indicating creation of vesicles. This transition was accompanied by a decrease in the zeta-potential, from
74.51 to
65.47, indicating the presence of positively charged gemini 12-2-12 surfactants adjacent to the negatively charged SDS in the vesicles. Furthermore, addition of the heme
imidazole complex 4673

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Figure 3. Activity plot for titration of imidazole in the 90 mM SDS þ 0.8 mM gemini þ 10 μM heme solution, [H2O2] = 5.9 mM and [guaiacol] = 0.3 mM.

Figure 1. TEM image of the sample: SDS (90 mM) þ gemini 12-2-12 (0.8 mM) þ heme (10 μM) þ imidazole (3 mM).

Figure 2. UV
vis absorption spectra of solutions containing 12 μM heme ((), 90 mM SDS þ 12 μM heme (2), 90 mM SDS þ 3 mM imidazole þ 12 μM heme (b), 90 mM SDS þ 0.8 mM gemini þ 12 μM heme (/), 90 mM SDS þ 0.8 mM gemini þ 12 μM heme þ 3 mM imidazole (), and native HRP 12 μM (—).

to the SDS/gemini 12-2-12 vesicles induced swelling in the vesicles as well as incorporation of the heme
imidazole into the micelles. This experiment confirmed that the heme
imidazole group bound to vesicles in the interior or near the charged surface. To better observe the structures, a sample was analyzed by TEM. Figure 1 shows TEM images, which indicate the appearance of vesicle structures in mixed SDS/gemini 12-2-12 surfactant solutions upon addition of the heme
imidazole complex, in good agreement with DLS vesicle size measurements. Figure 2 shows the absorption of the Soret bands in the heme group for different heme
catalyst systems. The Soret band of the uncomplexed heme is present at 389.5 nm. Insertion of the heme group into a hydrophobic environment causes it to shift hyperchromicitically and increased the absorption intensity, indicating formation of a monomeric heme structure in the hydrophobic pockets.13 As Figure 2 shows, the hydrophobic

Scheme 2. Schematic Representation of the Vesicle Radius and 2LSDS, Determined by DLS

pockets of the SDS (90 mM) micelle and the mixed SDS (90 mM)/gemini (0.8 mM) as vesicles provided similar hydrophobic environments for the heme, as indicated by the presence of identical Soret bands (400 nm) of similar intensity, most likely due to identical SDS and gemini 12-2-12 tails. Moreover, it should be noted that the Soret band appearing in the UV
vis spectra for the heme (12 μM) þ SDS (90 mM) was nearly identical to that of the native HRP. The addition of imidazole (3 mM) to the heme (12 μM)/SDS (90 mM) and heme (12 μM)/SDS (90 mM)/gemini (0.8 mM) solutions produced a more pronounced blue shift in the Soret band (413 nm) and a large increase in the absorption intensity. The spectral intensity approached that of the Soret band of the native HRP (∼12 μM). The enzymatic activity varied as a function of imidazole concentration for each biocatalyst. The optimum imidazole concentration was 3 mM for the representative nano biocatalyst heme (10 μM)/SDS (90 mM)/gemini (0.8 mM)/imidazole (Figure 3). 3.1.1. Molecular Weights of the Vesicles. The hydrodynamic radius of a bilayer vesicle (Scheme 2) determined by DLS 4674

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(RDLS), along with the SDS tail length (LSDS = 1.8 nm),51 permits estimation of the volume of a vesicle bilayer (Vbilayer) using eq 1 Vbilayer ¼

4 πðRDLS 3
ðRDLS
2LSDS Þ3 Þ 3

ð1Þ

Vbilayer, obtained from eq 1, could be used to compute the aggregation number (Nagg) of the vesicles. Assuming a spherical vesicle, the aggregation number is computed using the following relation52
54 Nagg ¼

Vbilayer X SDS V SDS þ X GEM V GEM

ð2Þ

Table 2 tabulates the values of molecular volumes for SDS and 12-2-12 gemini monomers. Figure 4 shows the surface tension versus concentration of the three components, SDS, gemini 122-12, and the mixed SDS/gemini 12-2-12 (R = 90 mM/0.8 mM) solutions. The critical aggregation concentrations (CAC) of the three components (in 5 mM phosphate buffer pH = 7 and 27 C) are summarized in Table 3. The mole fractions of SDS (XSDS) and gemini surfactant (XGEM) in vesicles can be obtained using a regular solution model with the three determined CACs.55 Table 3 summarizes the calculated mole fractions, the

β interaction parameters, aggregation number in each vesicle, and the molecular weight of each vesicle (=8.52  106 g/mol). 3.1.2. Calorimetric Measurements and Heme Binding to the Vesicles. Figure 5 shows the calorimetric curves that indicate the interactions between heme
imidazole complexes and the SDS/ gemini vesicles (R = 90 mM/0.8 mM). As shown in the figure, the curve saturated at a molar ratio of ([heme
imidazol]/ [vesicle]) = 6. The curve indicating the heat of each injection as a function of the heme
imidazole complex/vesicle molar ratio could be fit according to two binding set programs. The fits yielded the calorimetric parameters summarized in Table 4. As can be seen in this table the ΔH of the first binding set is
2.01  105 cal/mol, and the ΔH of the second binding set is
8.17  105 cal/mol. Previous studies indicated that the hydrophobic interactions made endothermic contributions to the enthalpy of mixing, whereas the electrostatic interactions made exothermic

Table 2. Volume of the Compound Used for Equation 2 V (nm3)

compound SDS monomer

0.362b

12-2-12 Gemini monomer

2.01a 0.03b

H2O a

b

Calculated using the B3LYP/6-31G* method. Ref 58.

Figure 4. Surface tension versus concentration for pure 12-2-12 gemini ((), pure SDS (9), and mixed SDS/12-2-12 gemini (R = 90 mM/ 0.8 mM) (b).

Figure 5. Calorimetric curves obtained from measurements of the heat of inserting heme
imidazole (175 μM/43.75 mM) complexes into a 5 mM phosphate buffer, pH = 7, subtracted from the heat of inserting heme
imidazole (175 μM/43.75 mM) complexes into SDS/gemini 122-12 (90 mM/0.8 mM) vesicles. The resulting heat was subtracted from the heat of inserting a 5 mM phosphate buffer into SDS/gemini 12-2-12 (90 mM/0.8 mM) vesicles. (A) The heat/s (heat power) of each injection as a function of time. (B) Heat of each injection as a function of the molar ratio ([heme
im]/[vesicle]) (9) experimental, (—) fitted curves.

Table 3. Critical Micelle Concentration of Each Compound in 5 mM Phosphate Buffer (pH = 7), Mole Fraction of the Monomers in Vesicles, Aggregation Number of the Vesicles, and Molecular Weight of the Monomers and Vesicles compound

CAC mM

β

X in vesicle

Nagg

molecular weight (g/mol)

SDS

4.05

0.6439

-

-

288.38

12-2-12 Gemini

0.97

0.3561

-

-

614

SDS/gemini vesicle (bilayer)

0.80

-


9.39 4675

21066.59

8.52  106

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Table 4. Calorimetric Parameters of the Fit Curves at 27C N

Kb (M
1)

first binding set

3.21 ( 0.19

1.03  106 ( 2.30  105


2.01  105 ( 2.46  104


642

second binding set

1.11 ( 0.03

2.60  10 ( 5.90  10


8.17  105 ( 3.36  104


2.69  103

binding statues

7

ΔH (cal/mol) 6

ΔS (cal/mol deg)

Table 5. Kinetic Parameters for the Designed Catalysts Km (μM)

biocatalyst heme alonea heme (10 μM) þ imidazole (3 mM)

7.39 32.06

catalytic efficiency (μM
1 s
1)

0.0065 0.077

0.88 2.39

heme (10 μM) þ SDS micelle (90 mM)a

4.33

0.00442

1.02

heme (10 μM) þ SDS micelle (90 mM) þ gemini 12-2-12 (0.8 mM)

1.17

0.010

8.57

geme (10 μM) þ SDS micelle (90 mM) þ gemini 12-2-12 (0.8 mM) þ imidazole (3 mM)

1.52

0.030

heme (10 μM) þ SDS micelle (90 mM) þ imidazole (3 mM)

4.69

0.036

native HRPa a

kcat (s
1)

5800

420

19.8 7.67 72.4

Ref 3.

Scheme 3. Catalytic Reaction Cycle and Inactivation Pathway for Peroxidase-Like Enzymes

contributions to the enthalpy.56 According to Table 4, both binding sets were associated with an exothermic net ΔH, indicating that the electrostatic interactions between the charged regions of the heme
imidazole complexes (especially iron(III)) and the ionic walls of the vesicles were strong. However, the heat of the first binding set was much less than that of the second one. As mentioned, the spectra of the heme complexes indicated incorporation into the hydrophobic region. The heats of each binding set comprised the sum of both hydrophobic and electrostatic interactions. Therefore, the first binding set, corresponding to the lower exothermic ΔH (see Table 4), was associated with the more hydrophobic interactions involved in accommodating the heme
imidazole complexes deep in the hydrophobic core of the vesicles (see Scheme 1). The maximum number of heme
imidazole complexes that could be incorporated into each vesicle (N) in the first binding set was three, so that multiple active sites were present in the hydrophobic pockets to yield multienzymes. The second binding set, associated with a more exothermic enthalpy, corresponded to the electrostatic interactions of the heme
imidazole complex near the charged ionic walls of the vesicle, with an N of one. 3.2. Activity Measurements. Table 5 shows the kinetic parameters associated with the Michaelis
Menten constants (Km, eq 3) as a function of guaiacol concentration for the various peroxidase-like catalyst biomimetic models. rate ¼

Vmax ½S K m þ ½S

ð3Þ

The turnover number (catalytic rate constant, kcat), which indicates the maximum moles of a substrate converted to product

per mole catalyst per unit time, and the catalytic efficiency (kcat/ Km) of the artificial enzymes, measured by the Lineweaver
Burk linearized Michaelis
Menten plot (eq 4), are reported in Table 5.57 1 Km 1 ¼ þ rate Vmax ½S Vmax

ð4Þ

The biocatalysts consisting of heme
gemini 12-2-12/SDS vesicles demonstrated that the cationic gemini 12
2
12 improved the catalytic efficiency by a factor of 8 relative to the heme
SDS micelles. This indicates that the presence of both negative and positive charges at the active site, mimicking the native peroxidase, increased the catalytic efficiency toward that of the native HRP. However, the optimal peroxidatic reaction productivity occurred in the presence of a biocatalyst containing the heme/gemini 12-2-12/SDS vesicles in the presence of imidazole, yielding a 19.8 μM
1 s
1 catalytic efficiency that was about 27% that of the native HRP. The imidazole moiety, which played the role that histidine plays in the native HRP, increased the reactivity and catalytic efficiency of this artificial enzyme. The imidazole group covalently binds to the iron center of the heme group as a donor in peroxidases. This proximal donor facilitates the heterolytic cleavage of O
O bonds. To provide an efficient biomimetic enzyme, an imidazole group was introduced as a basic component into the artificial peroxidaselike enzyme. As indicated in Section 3.1, vesicles may enhance the activation of the artificial nano multienzymes. The high observed enzymatic efficiency was due to the presence of both positive and negative charge distributions on the supramolecular surfaces (similar to 4676

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the charges on protein or enzyme surfaces) and to complexion of the imidazole agent to the heme, which imitated binding of the histidine to the iron center in the native HRP. 3.3. Suicide Inactivation. Scheme 3 shows the peroxidase-like process including the activation cycle and the suicide inactivation for artificial enzyme. AEa, AEi, AH, P, and the parameter ki denote, respectively, the whole forms of active artificial enzyme, inactivated artificial enzyme, hydrogen donor (reductant
substrate), the catalytic reaction product (tetraguaiacol), and the apparent rate constant of inactivation (Scheme 3).21 In the catalytic reaction cycle, the active enzyme species are free active artificial enzymes (AEa), compound I (C
I), and compound II (C
II).21 In principle, monitoring the AH concentration during the catalytic reaction cycle permits estimation of the extent to which the levels of the active enzymatic forms decrease due to the suicide inactivation process. In fact, the concentration of the collection of all active enzyme species, R (in the form of C
I), defines the first-order rate law at each time (R = Re(
kit)). Instead of the activity measurements, R may be obtained by fitting the experimental data to the overall integrated kinetic equation (eq 5). Indeed, the rate of consumption of the reductant substrate (AH) and the overall rate of conversion of the active artificial enzyme (AEa) to the inactivated form (AEi) enable us to obtain the differential kinetic equation as well as the integrated kinetic relation (according to our previous model)21,40,42
ki t

½AHt ¼ ½AH  eR =ki ðe


1Þ

ð5Þ

Figure 6. Progress curves for suicide inactivation of the catalyst: 90 mM SDS þ 0.8 mM gemini þ 3 μM heme þ 3 mM imidazole with 3.4 mM hydrogen peroxide, as a function of guaiacol concentration over the reaction time. Data were fit to eq 1. Experimental ((); calculated (—) curves.

where [AH] is the molar concentration of AH at t = 0 and R is the whole active enzyme species at t = 0. Equation 5 describes the relationship between the concentration of the remaining aromatic substrate over time, taking into account the decrease in enzyme activity during the concurrent inactivation reaction. Equation 5 can be used to determine R and ki using a nonlinear regression fit of the experimental data to the equation. Common computer software, such as the Excel Solver, was used for this purpose. Figure 6 illustrates the accuracy of the kinetic model, and eq 5 describes the suicide-peroxide inactivation of the biocatalyst containing hemin
gemini 12-2-12/SDS vesicles in the presence of imidazole. The excellent agreement between experimental data and the curve fit (calculated) based on eq 5, shown in Figure 6, indicates the similarity between the suicide mechanisms of the biocatalysts and HRP. However, this process has been observed for all HRP-like catalysts, even for the bare heme group. The kinetic parameters for suicide inactivation of the biocatalysts, obtained by fitting the experimental curves to eq 5, are summarized in Table 6. As can be seen, ki for the hemin
imidazole
gemini 12-2-12/SDS vesicle catalyst was less than ki for native HRP42 in 3 mM hydrogen peroxide solutions; the ki for the hemin
imidazole
SDS micelle catalyst was much higher than that of the native HRP; and R for HRP was higher than for either catalyst. These results indicate excellent protection of the heme
imidazole group by the vesicle structure. As shown in Table 6, although catalysis by the heme in the SDS/gemini vesicles (without imidazole) had a lower inactivation constant (ki), R, which indicates the presence of active enzyme, was very low in the absence of imidazole. The high values of ki for the heme alone indicate the importance of a hydrophobic environment, such as that presented by micelles, vesicles, or an apoenzyme pocket, to the shielding of the heme moiety catalytic center. A plot of R/R as a function of time at various concentrations of hydrogen peroxide for different HRP-like biocatalysts (Figure 7) showed that the heme
imidazole in SDS/gemini vesicles gave the most active enzyme during an 8 min reaction period, even with respect to the native HRP in 3 mM hydrogen peroxide. A plot of R/R for the heme
imidazole in SDS micelles was lower than that of the native HRP under almost all conditions. Table 6 and Figure 7 show that although the nano multienzyme had a lower catalytic activity than the native HRP it was reactive over a longer period of time without considerable inactivation. As mentioned, the vesicle structure of the mixed SDS/gemini facilitated the catalytic activity and reduced the suicide rate. Figure 8 plots the values for ki, for the heme, heme
imidazole in gemini 12-2-12/SDS vesicles, heme
imidazole in SDS micelles, and native HRP as a function of hydrogen peroxide concentration. As shown in Figure 8, the presence of SDS

Table 6. ki and r Values for the Designed Catalysts Obtained during a Time Course of 8 Min, From Fits to the Progress Curves biocatalyst

a

[H2O2] (mM)

ki (min
1)

R (min
1)

hemin 3 μM þ imidazole 3 mM þ SDS 90 mM

3.4

0.091

0.085

hemin 3 μM þ imidazole 3 mM þ SDS 90 mM þ gemini 0.8 mM

3.4

0.039

0.070

hemin 3 μM þ SDS 90 mM

3.4

≈0

0.005

hemin 3 μM þ SDS 90 mM þ gemini 0.8 mM

3.4

≈0

0.012

hemin 3 μM HRP 20 nMa

3.4 3

0.902 0.044

0.057 0.18

Ref 42. 4677

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(Scheme 3). As shown in Figure 8, the heme
imidazole in gemini 12-2-12/SDS vesicles yielded a value of ki that was low compared to that of the heme
imidazole
SDS micelles. However, ki was still better than that of HRP, which was 4 mM hydrogen peroxide. Therefore, the heme
imidazole
gemini 12-2-12/SDS biocatalyst was better shielded than the native HRP below 4 mM hydrogen peroxide. Diffusion of the free heme group in the apo-artificial enzyme into and out of the hydrophobic environment, which contrasted with the behavior of the bound heme group in the native peroxidase, may have been responsible for the differences between HRP and heme
imidazole
gemini 12-2-12/SDS biocatalyst activity described in Figure 8. Therefore, the more stable structure of the active site (heme
imidazole or heme
histidine) in the hydrophobic pocket provides a next step for biomimetic strategies. Figure 7. Dependence of R/R on time in different concentrations of hydrogen peroxide. [Guiacol] = 0.22 mM for different biocatalysts, pH = 7.0, phosphate buffer 5.0 mM, and temperature of 27 C. A solution containing 90 mM SDS þ 0.8 mM gemini þ 3 mM imidazole þ 3 μM heme, [H2O2] = 2.53 mM (() and [H2O2] = 3.4 mM (2), 90 mM SDS þ 3 μM heme þ 3 mM imidazole, [H2O2] = 2.95 mM (9), native HRP (20 nM), [H2O2] = 3 mM (ref 42) (b).

4. CONCLUSION The four-component new peroxidase-like nano biocatalyst system containing heme
SDS
gemini 12-2-12 imidazole was found to have a (1) high catalytic efficiency and (2) good protection of the catalyst center at higher concentrations of hydrogen peroxide, with respect to the native HRP shielding. These properties resulted from the presence of both negative and positive charges on the hydrophobic pocket, which mimicked the native apoprotein heme binding pocket. The structural features of the mixed SDS/gemini 12-2-12 indicate that the vesicles were surrounded by positive and negative charges to provide suitable environments for multiple heme active sites, yielding multienzymes. The imidazole moiety, mimicking the histidine in the native HRP, was found to be an important component. This biocatalyst provided a catalytic efficiency that was 27% that of the native HRP. Such a high catalytic efficiency indicates that these peroxidase mimics provide an efficient nanoartificial peroxidase enzymatic model in aqueous solutions (see Scheme 1). ’ AUTHOR INFORMATION Corresponding Author

Figure 8. Dependence of ki on the concentration of suicide substrate, [H2O2]. [Guaiacol] = 0.22 mM for different biocatalysts, pH = 7.0, phosphate buffer 5.0 mM, and at a temperature of 27 C. A solution containing 90 mM SDS þ 0.8 mM gemini þ 3 μM heme þ 3 mM imidazole (9), heme alone 3 μM (2), 90 mM SDS þ 3 mM imidazole þ 3 μM heme ((), and the native HRP 20 nM (ref 42) (b).

micelles near the heme moiety significantly decreased the rate of inactivation (suicide parameters) and increased the initial hydrogen peroxide suicide concentration relative to that of the hemeonly solution. These effects indicate that the hydrophobic pocket provided good shielding and effectively controlled the hydrogen peroxide concentration at the heme
imidazole active site complex, thereby protecting the catalyst center. The presence of imidazole near the heme group in the micelles and vesicles considerably increased the concentration of suicide onset. As seen in Figure 8, no suicide was observed for these two catalysts until [H2O2] ≈ 2.5 mM. In contrast, low initial hydrogen peroxide concentrations induced HRP suicide (extrapolated line in Figure 8). This result highlights the importance of axial ligand occupation by the hydrogen peroxide and the imidazole in compound I (C
I). Up to [H2O2] ≈ 2.5 mM, the imidazole moiety was dominant and prevented suicide inactivation

*E-mail: [email protected]. Tel.: þ9821-82884401. Fax: þ9821-82884401.

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