Chaperonin-Nanocaged Hemin as an Artificial Metalloenzyme for

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A chaperonin-nanocaged hemin as an artificial metalloenzyme for oxidation catalysis Xiaoqiang Wang, Chao Wang, Meihong Pan, Junting Wei, Fuping Jiang, Rongsheng Lu, Xuan Liu, Yihui Huang, and Fang Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08963 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

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ACS Applied Materials & Interfaces

A chaperonin-nanocaged hemin as an artificial metalloenzyme for oxidation catalysis Xiaoqiang Wang†, Chao Wang†, Meihong Pan, Junting Wei, Fuping Jiang, Rongsheng Lu, Xuan Liu, Yihui Huang, Fang Huang*

State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580, P. R. China

†

These authors contributed equally

*

To whom correspondence may be addressed: [email protected] Tel: 0086-532-86981560, FAX: 0086-532-86981560

ACS Paragon Plus Environment


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ABSTRACT Taking inspiration from biology’s effectiveness in functionalizing protein-based nanocages for chemical processes, we describe here a rational design of an artificial metalloenzyme for oxidations with the bacterial chaperonin GroEL, a nanocage for protein folding in nature, by supramolecular anchoring of catalytically active hemin in its hydrophobic central cavity. The promiscuity of the chaperonin cavity is an essential element of this design, which can mimic the hydrophobic binding pocket in natural metalloenzymes to accept cofactor and substrate without requiring specific ligand-protein interactions. The success of this approach is manifested in the efficient loading of multiple monomeric hemin cofactors to the GroEL cavity by detergent dialysis, and the good catalytic oxidation properties of the resulting biohybrid in tandem with the clean oxidant of H2O2. Investigation of the mechanism of hemin-GroEL-catalyzed oxidation of two model substrates reveals that the kinetic behavior of the complex follows a ping-pong mechanism in both cases. Through the comparison with horseradish peroxidase (HRP), the oxidative activity and stability of hemin-GroEL were observed to be similar to those found in natural peroxidases. ATP-regulated partial dissociation of the biohybrid, as assessed by the reduction of its catalytic activity with the addition of the nucleotide, raises the prospect that ATP may be used to recycle the chaperonin scaffold. Moreover, hemin-GroEL can be applied to the chromogenic detection of H2O2, which (or peroxides in general) are commonly contained

in

industrial

wastes.

Considering

the

rich

chemistry

of

free

metalloporphyrins and the ease of production of GroEL and its supramolecular complex with hemin, this work should seed the creation of many new artificial metalloenzymes with diverse reactivity. KEYWORDS ： chaperonin, protein nanocage, hemin, artificial metalloenzyme, oxidation, catalytic material


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INTRODUCTION Biology has evolved to make protein nanocages to localize and constrain chemical processes.1-3 These biological compartments assembled from multiple subunits can encapsulate catalytically active components and may exert unique effects on the confined reaction. For example, reaction rates can be increased by holding substrate and active site near to each other in a vessel with dimensions on the nanoscale, while side reactions can be restrained by controlling the access of reactive species.4-5 This makes protein nanocages good scaffolds for biomimetic design. In nature there are diverse reactions that protein cages catalyze. Ferritin is a protein cage of 12nm diameter, which catalyzes the formation of hydrated iron oxide and stores it for living organisms.6 The chaperonin, a critical group of molecular chaperones that ensure protein homeostasis, forms a large cylindrical complex of 800-1000kDa and provides a protective chamber for protein folding to occur in isolation, unimpaired by aggregation.7-8 Another example is lumazine synthase, an assembly of 60 subunits arranged in a capsid that catalyzes the penultimate step of the riboflavin biosynthesis in the hyperthermophilic bacterium Aquifex aeolicus.9 Thus the ability to mimic or redesign these natural nanocompartments for the purpose of chemical synthesis presents a great opportunity for the elaboration of catalytic materials. Here, we report a new design of protein nanocage-based catalyst with the chaperonin cage of bacterial GroEL, which exploits the extensive promiscuity of the central hydrophobic cavity of the chaperonin as a scaffold for the catalytically active hemin (iron(III)-porphyrin) to catalyze oxidation reactions. Such reactions are biologically catalyzed by metalloenzymes such as hemoproteins under conditions fitting the current concept of green chemistry (i.e., aqueous solution, room temperature and atmospheric pressure). The use of ecologically friendly iron as an active site also makes these reactions very clean.10-11 Artificial metalloenzymes that address the question of clean oxidation were previously obtained by the association of synthetic iron-porphyrins with monoclonal antibodies.12-14 However, the development of new biomimetic metalloenzymes through supramolecular anchoring of the



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iron-containing cofactors in a cavity of a non-relevant protein constitutes a growing field of interest in sustainable catalysis. For example, ferritin and virus capsids have been well described as attractive platforms in this regard,15-20 and even porous non-protein materials have been explored.21-22 The bacterial GroEL is a tetradecameric protein complex composed of two chemically identical heptameric rings stacked back-to-back.23-24 In the open, substrate-receptive state, GroEL takes on a cylindrical structure with a diameter of ~13.7nm and a length of ~14.6nm.7-8, 23-24 Two ~4.5nm wide and deep cavities form at the two ends of the cylinder, one in each heptameric ring, which are separated from each other by the confluence of the C-terminal segments of the seven subunits.7-8, 23-24 Hence each GroEL complex has two separate cavities, which work alternately in vivo like a two-stroke engine. This is different from most natural protein-based nanocompartments. Moreover, an advantage of GroEL compared with other protein cages is that the chaperonin possesses multiple hydrophobic grooves facing into the openings of its central cavity, which capture a diverse range of polypeptides with accessible hydrophobic residues.25-26 Thus, GroEL should be an ideal cage-like protein complex for constructing novel metalloenzymes by introducing hemin into its central cavity as a mimic of the hydrophobic pocket that contains a hemin-like prosthetic group in natural hemoproteins such as horseradish peroxidase (HRP) and cytochrome P450 enzyme.10-11, 27-28 In this work GroEL-nanocaged hemin was prepared by detergent dialysis (Scheme 1). We examined the dispersity and amount of hemin encapsulated within the GroEL cage. The catalytic properties and stability of the hemin-GroEL complex were illustrated by the oxidation of model substrates with hydrogen peroxide (H2O2) used as the oxidant, which were also compared to those of the natural peroxidase HRP. Some basic mechanistic insights into hemin-GroEL-catalyzed oxidation were also obtained through the investigation of its activity over a range of substrate concentrations.25-26 The following advantages also make the hybrid metalloenzyme a useful tool for a broad range of applications such as the chromogenic detection of H2O2 shown here. These are (i) ease of production of GroEL in bacteria as well as its


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supramolecular complex with hemin, and (ii) the possibility of recycling of the protein cage via the ATP-regulated dissociation as for GroEL-peptide complex in nature. To our knowledge, this is the first GroEL-nanocage-based biocatalyst reported for oxidation reactions.

Scheme 1. Schematic Illustration of the Formation of the Hemin-GroEL Supramolecular System and Catalytic Process. The cross-section of the barrel-shaped chaperonin placed in side-on orientation is shown (middle), with the hydrophobic amino acid residues facing the upper passageway of the protein cavity highlighted in yellow. The diagrams of hemin and GroEL are rendered at different scales.

MATERIALS AND METHODS 1. Materials. Hemin of 98% purity was purchased from J&K Chemical Company (Beijing,

China).

Orange

II

(98%)

was

purchased

from

Sigma-Aldrich.

3,3′,5,5′-tetramethylbenzidine (98%) and horseradish peroxidase (300units/mg) were purchased from Shanghai Aladdin Bio-Chem Technology Company (China). All aqueous solutions were prepared with Millipore water (resistivity >18MΩ·cm). 2. Protein expression, purification and characterization. The bacterial chaperonin GroEL was prepared to ~95% purity as described previously.23, 29 Briefly, GroEL was overexpressed in E. coli strain BL21 from plasmid pTric 99 carrying the GroEL

gene.

The

bacterial

cells

were

induced

with

1mM

isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 37°C when the OD600 reached 0.4-0.6. After 4h, cells were harvested and then broken in a French pressure cell press. The lysate was clarified by centrifugation at 10,000×g for 30min at 4oC, and the



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supernatant was collected into a fresh tube. Saturated ammonium sulfate was then added to a final concentration of 50% and incubated on ice for 1h with gentle agitation. The sample was subsequently centrifuged (10,000×g) for 30min at 4°C. The pellet was collected and resuspended gently. The sample was then loaded onto a HiTrap Q Sepharose FF column (GE Healthcare), and the column was eluted with a linear gradient of 0-1M NaCl. The GroEL-containing fraction was concentrated in Amicon Ultra-15 Centrifugal Filter Units (Millipore). GroEL concentration was determined by the Bradford method. The morphology of GroEL was characterized by transmission electron microscopy (TEM) with a JEOL JEM 1400Plus electron microscope operated at 120 kV. 3. Construction of hemin-chaperonin by dialysis. A stock solution of hemin (1mM) was prepared in NaOH solution (20mM) containing detergent Triton X-100 with a micellar concentration of ~1.5mM. 1ml of diluted hemin solution (250µM, unless otherwise stated, in 10mM phosphate buffer) with a pH value adjusted to 7.0 was mixed with the same volume of GroEL solution (2.5µM in 10mM phosphate buffer with a pH value of 7.0). This was followed by dialysis against 10mM phosphate buffer for 48h to prepare hemin-chaperonin complex and/or to adjust the pH to desired values. As controls, dialysis on the mixture of the micellar solution of hemin with the same volume of phosphate buffer lacking GroEL was also performed. Triton X-100 was employed for two purposes, (i) to enhance the dispersion and stabilization of monomeric hemin at alkaline pH or the subsequently adjusted neutral pH, and (ii) to facilitate the hemin-GroEL preparation via dialysis at neutral pH, as opposed to alkaline condition, and thus ensure that GroEL structure would not be altered or even denatured. UV-Vis absorption spectra of hemin samples after dialysis in the absence or presence of GroEL were recorded on a Shimadzu UV-1700 UV-Vis spectrophotometer. The amount of hemin encapsulated by GroEL was calculated with Lambert-Beer law using an extinction coefficient of 5.2 × 104 M-1·cm-1 around 396nm, which was determined by making a standard curve at varied hemin concentrations (see Figure S1 in the Supporting Information). The hydrodynamic diameter distribution of the


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samples was also measured with dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS spectrometer. Preliminary UV-Vis experiments with stock or diluted micellar solutions of hemin gave a spectrum characteristic of monomeric hemin, indicating that hemin was monodisperse in the presence of Triton X-100. Presumably the hydrophobic interaction between the detergent micelle and hemin overcomes the hemin-hemin interactions leading to monomer formation. 4. Catalytic experiments. The catalytic properties of the hemin-chaperonin complex were evaluated by studying the oxidation of two different substrates by H2O2, namely, Orange II and 3, 3′, 5, 5′-tetramethylbenzidine (TMB). When Orange II was used as the substrate, its solution (0.125mM in 10mM phosphate buffer) was mixed with the same volume of H2O2 solution (10mM in 10mM phosphate buffer) and hemin-chaperonin solution (the concentration of hemin was set to 10µM unless otherwise stated) at specific pH values and temperatures. The reactions were monitored in time course mode at 484nm, which is around the maximum absorbance of Orange II, using a Shimadzu UV-1700 UV-Vis spectrophotometer. With respect to TMB, its concentration was set to 1mM prior to the mixing, and the reactions were monitored at 644nm, which is around the maximum absorbance of the oxidation product. For both substrates, the color change developed after the mixing was recorded with a camera, and the changes in the characteristic absorption peaks were detected with time by UV-Vis spectroscopy. The initial velocity (ν) of the oxidation of Orange II or TMB was measured by following a linear change in the intensity of the characteristic absorption within the first 60 seconds, and was then calculated with Lambert-Beer law. In this process, an average molar extinction coefficient determined of 1.8×104M-1·cm-1 at 484nm was used for Orange II (Table S1), and a value of 3.9×104M-1·cm-1 at 644nm was used for TMB-derived oxidation product.30 The two important parameters in enzymatic kinetics, maximum initial velocity (Vmax) and Michaelis-Menten constant (Km), were obtained based on the Michaelis-Menten equation ν=Vmax×[S]/(Km+[S]), where [S] is the substrate concentration. To investigate the mechanism of oxidation catalysis of hemin-chaperonin, experiments were performed under standard reaction conditions as



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described above (pH7.0, 20oC) by varying the concentration of Orange II or TMB at a fixed concentration of H2O2 or vice versa. Catalytic experiments in which HRP was used as a catalyst were also carried out for comparison with the hemin-GroEL complex prepared in this study. The sensitivity of the hemin-GroEL complex to ATP binding was tested by measuring its oxidative activity before and after addition of ATP to a final concentration of 5mM. 5. Hemin-GroEL-based detection of H2O2. Hemin-GroEL (with the concentration of hemin fixed at 10µM) was first mixed with the same volume of TMB (3mM) and H2O2 with different concentrations at 20°C. H2SO4 (20%, v/v) was then added to the reaction mixture 30min after the initiation of the reaction to a final concentration of 0.4% to stop the color reaction. The absorbance of the resultant mixture at 450nm was measured for quantification of TMB-derived product due to H2O2 oxidation.31 The absorbance at 450nm was plotted against the concentration of H2O2 added in the reaction mixture, based on which the detection limit was evaluated. RESULTS 1. Rational design of the supramolecular hemin-GroEL system. The construction of the supramolecular hemin-chaperonin hybrid for oxidation catalysis is illustrated in Scheme 1. The chaperonin GroEL used in this study was expressed in E. coli. Typical yields of purified protein were 50-60mg·L-1 culture and thus adequate amounts of sample can be prepared through scale-up. Figure 1A and Figure S2 show TEM images of the purified GroEL sample, which revealed not only the homogeneity of the sample but that the tetradecameric GroEL complex were all intact. As shown in Figure 1B and 1C, the doughnut-like end-on view and the rectangular side view of GroEL are clearly observed; their overall shape, height, cavity size and wall thickness are also comparable to those of the X-ray crystal structure.7-8, 23-24


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Figure 1. The bacterial chaperonin GroEL as a nanocage for inclusion of hemin complex. (A) TEM micrographs of intact GroEL tetradecameric particles. (B) The typical top view show a stain filled central chamber that appears as a doughnut shaped end-on view; a top view of GroEL crystal structure (right) (Protein Data Bank (PDB) 5DA8). (C) The side view is visible as rectangular particle with four stripes which correspond to thicker protein regions on cross-section; a side view of GroEL crystal structure (right) (PDB 5DA8). (D) UV-Vis spectra of GroEL-hemin complex and free hemin obtained after Triton X-100 dialysis in phosphate buffer (pH7.0). (E) Hydrodynamic diameter distribution of the hemin-GroEL complex compared with free hemin in phosphate buffer (pH7.0); hemin concentration was fixed at 10µM prior to dialysis.

The striking ability of the hydrophobic central cavity of GroEL to capture a diverse range of non-native substrate proteins via the hydrophobic interactions represents one of the most fascinating molecular recognition events in protein chemistry. In this work the Triton X-100-solubilized hemin, which was in monomeric form as assessed by spectroscopic measurement (Figure S3), was loaded into the hydrophobic GroEL cavity by detergent dialysis. Figure 1D shows the UV-Vis spectrtrum of GroEL-hemin



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complex compared with free hemin that was prepared by dialyzing Triton X-100-solubilized hemin against a buffer solution in absence of the chaperonin. GroEL-hemin displays a Soret peak at 396 nm with a band at 272 nm and a very weak band at 550 nm. The Soret peak of the hemin complexed with the chaperonin is similar to that of monomeric hemin (around 400nm).32 The absorbance band at 272nm indicates the presence of protein, and the weak band at 550nm may be ascribed to charge transfer transition.33-34 In contrast, hemin in pH7.0 buffer solution displays a Soret peak at 360nm, suggesting the aggregation of hemin.35 Based on DLS measurements (Figure 1E), the average hydrodynamic diameter of hemin in the buffer is approximately 7.0nm, which is much larger than the monomeric form (no more than 1.5nm), while for hemin-GroEL this value changed to be about 15.0nm (in agreement with the size of GroEL) indicative of an efficient encapsulation of hemin into the chaperonin cavity after dialysis.36 Overall, the hydrophobic patches on the inner wall of GroEL can effectively reduce the aggregation of hemin through supramolecular interactions to localize monomeric hemin within the cavity. The encapsulation of hemin by barrel-shaped GroEL was measured by UV-Vis spectroscopy with respect to the initial equilibrium concentration of hemin (Figure 2). The encapsulation proceeded efficiently with the equilibrium concentration of hemin and hit a constant level when the concentration of hemin reached 120µM, indicating that the supramolecular interaction reached an equilibrium state. One GroEL complex is thus estimated to accommodate up to ~70 hemin molecules, that is, ~35 in each of the two cavities of GroEL which are chemically identical. This is reasonable because the volume of each GroEL chamber is measured in the crystal structure to be ~85,000Å3, while each hemin is only about 15 Å across.36-37 For the subsequent experiments, the ratio of catalytically active hemin to GroEL was set to be ~50 in the constructed metalloenzyme (or hemoprotein), with their absolute concentrations determined by UV-Vis spectroscopy and the classical Bradford method respectively, to eliminate the possible effect of steric hindrance by overloaded active units on the catalytic process, as indicated by our preliminary experiments.


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Figure 2. Encapsulation of hemin by the hydrophobic central cavity of GroEL was spectrophotometrically measured with respect to the equilibrium concentration of hemin. The equilibrium concentration of GroEL was fixed at 1µM. The line is drawn to guide the eyes.

2. Oxidation of Orange II catalyzed by hemin-GroEL. The catalytic properties of the chaperonin-based artificial metalloenzyme were evaluated by using the oxidation of Orange II in the presence of H2O2 as a model reaction. As shown in Figure 3A, the maximum absorbance of Orange II (at 484nm) decreased with an increase in reaction time, which originates from the oxidative degradation of the substrate.38 In the absence of H2O2 or hemin-GroEL, the spectra displayed a negligible change (data not shown), indicating that no oxidation reaction occurred. Thus hemin-GroEL exhibited peroxidase-like ability to activate H2O2 for the catalytic oxidation of the model compound. Moreover, this hybrid biocatalyst was observed to show evidently higher catalytic performance than the monomeric hemin solubilized in Triton X-100 solution when the concentrations of hemin were set at the same level in the reaction (Figure S4).



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Figure 3. The oxidative activity of hemin-GroEL is concentration, temperature and pH dependent, which is compared with HRP. (A) The change in the maximum absorbance at 484nm for Orange II with the time of oxidation catalyzed by hemin-GroEL complex. The relative activity on the vertical axis in (B-D) stands for the specific initial velocity of the oxidation divided by the maximum value, and the maximum point was set as 100%. (B) The concentration of hemin-GroEL or HRP was varied (represented by the concentration of the catalytic center of hemin), with temperature and pH fixed at 20oC and 7.0. (C) Temperature was varied, with hemin concentration and pH fixed at 10µM and 7.0. (D) The pH was varied, with hemin concentration and temperature fixed at 10µM and 20oC. The dashed lines in (B-D) are simple guides for the eye.

The influence of the concentration of hemin-GroEL as well as temperature and pH on the catalytic activity of the artificial metalloenzyme was next evaluated and compared with HRP. Specifically, we measured the activity of hemin-GroEL and HRP by determining the initial velocity while varying the catalyst concentration from 5µM to 30µM, the temperature from 20oC to 70oC, and the pH from 5 to 11. As can be seen in Figure 3B, the activity of hemin-GroEL was dependent on its concentration, and the higher the concentration, the higher the activity, as was the case for HRP. The activity


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of hemin-GroEL was less sensitive to the temperature change than that of HRP over the whole range of temperature (Figure 3C). However, like non-enzymatic catalysts, the catalytic activity in both cases increases on raising the temperature. It is important to note that the denaturation temperatures of intact GroEL and HRP are both around 70oC,39-40 close to the upper limit of the temperature range tested. With respect to the effect of pH change, while the optimal pH was found to be about pH 9.0 for HRP, the activity of hemin-GroEL increased with pH over the whole range (Figure 3C). Moreover, the artificial biohybrid showed an abrupt elevation in activity from pH 9.0 to 11.0 similar to the behavior observed for HRP from pH 7.0 to 9.0. Taken together, the oxidative activity of the supramolecular hemin-GroEL system is, like HRP, dependent on the catalyst concentration, temperature and pH. 3. Mechanistic insights into the oxidative activity of hemin-GroEL. The apparent steady-state kinetic parameters for the oxidative activity of hemin-GroEL were determined to gain mechanistic insights into the reactivity. In this process, we changed the concentration of Orange II or H2O2 while keeping the other constant, and determined the initial reaction velocities at different substrate concentrations based on the change with time of the maximum absorbance of Orange II (at 484nm with an average molar extinction coefficient determined to be 1.8×104M-1·cm-1 (Table S1)). The data (initial velocity vs. substrate concentration) were fitted to the Michaelis– Menten model to obtain the parameters shown in Table 1, which are compared with those previously reported for HRP.38, 41 The apparent Km value of hemin-GroEL with Orange II as the substrate was much lower than that for HRP. Since Km is inversely related to the affinity between the substrate and the enzyme, i.e. the smaller the Km value the stronger the affinity and vice versa, our results indicate that hemin-GroEL has a higher affinity for Orange II than HRP. By contrast, the Km value of hemin-GroEL with H2O2 as the substrate was higher than that for HRP, suggesting that the artificial biocatalyst has a lower affinity for H2O2 than HRP. Moreover, the result that the Km value of hemin-GroEL with H2O2 as the substrate was significantly higher than that with Orange II as the substrate is consistent with the observation that a higher concentration of H2O2 was required to



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Page 14 of 25

observe maximal activity for hemin-GroEL. Table 1. Apparent Michaelis-Menten Constant (Km) and Maximum Velocity (Vmax) of Hemin-GroEL and HRP for Catalyzing the Oxidation of Orang II by H2O2 Catalyst

Substrate

Km (mM)

Vmax (×10-8M·s-1)

Hemin-GroEL

OrangeII

0.02

16.58

H2O2

10.63

39.12

Orange II

2.93a

232.67a

H2O2

3.70a

8.71a

HRP

a

Data from ref 35 & 38.

To further investigate the catalytic mechanism of hemin-GroEL in the system, we measured its activity over a range of Orange and H2O2 concentrations. The double reciprocal plots of the initial velocities against the concentrations of one substrate were obtained over a series of concentrations of the second substrate (Figure 4). The lines in each case were approximately parallel, which is characteristic of a ping-pong mechanism, as was previously observed for HRP.42 The results then indicate that, as for HRP, the hemin-GroEL complex binds and reacts with the first substrate, then releases the first product before reacting with the second substrate.41-43

Figure 4. Catalytic mechanism of hemin-GroEL. (A) Double-reciprocal plots of activity of hemin-GroEL with the concentration of H2O2 fixed and that of Orange II varied, or (B) vice versa.

4. Effect of temperature, pH and ATP on hemin-GroEL stability. In addition to activity, enzyme stability is another important aspect to be considered for enzyme assays. In this work we tested the stability of hemin-GroEL by incubating the artificial enzyme at a series of varied temperatures or pH for 2h, followed by the measurement


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of its activity under standard conditions (pH 7.0, 20oC), which was also compared with HRP (Table 2). Although the denaturation temperatures of intact GroEL and HRP are both high (~70oC) as described above, the optimum temperatures for maintaining their activity are both around 37oC. With respect to the effect of pH, while HRP showed a higher stability in an alkaline environment, hemin-GroEL was shown to be relatively stable at about pH 7.0. ATP binding by GroEL has been shown to cause an expansion of the opening to the chaperonin cavity, resulting in a reduced affinity of the chaperonin for unfolded hydrophobic protein substrates.8 To test whether the hemin-GroEL complex was sensitive to ATP binding, 5mM ATP was added to preformed hemin-GroEL complex. This caused an immediate reduction of the catalytic activity by ~1/4 (Table 2), indicating that ATP may have a direct effect on the stability of hemin-GroEL complex probably causing the partial dissociation of the complex by lowering the affinity of the chaperonin for hydrophobic hemin. This result also raises the prospect that ATP may be used to disrupt the hemin-GroEL complex and recycle the chaperonin scaffold. Table 2. Relative stability of hemin-GroEL (%) at different temperatures and pH compared with HRP and the effect of ATP Temperature (oC)

Catalyst

pH

ATP

20

37

50

60

70

5

7

9

11

(-)

(+)

Hemin-GroEL

99.4

100

73.2

73.2

65.1

90.9

100

90.9

85.5

100

75.1

HRP

98.2

100

98.7

94.1

67.9

73.6

100

106.9

118.9

N/A

N/A

N/A—Not applicable

5. Application of hemin-GroEL in chromogenic detection of H2O2. Clean oxidations under mild conditions are needed in many areas such as the conversion of relatively cheap alkanes into more valuable oxidized products, detection of industrial wastes and clinical diagnosis10. Besides the assessment of catalytic properties of the hemin-GroEL artificial metalloenzyme, its potential applications in the chromogenic detection of H2O2 were also tested with 3,3′,5,5′-tetramethylbenzidine (TMB) used as a substrate, to address the practical need of the detection and removal of peroxide from



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materials such as food stuffs and industrial wastes44. Figure 5A shows the color change of TMB solution mixed with H2O2 following the addition of hemin-GroEL. The colorless TMB-H2O2 mixture turned blue within 1min and the color became darker with time, suggesting the oxidation of TMB catalyzed by the chaperonin-based enzyme in the presence of H2O2. The UV-Vis spectroscopic measurements revealed a dramatic absorbance increase of the reaction mixture at 644nm, ascribable to the TMB-derived oxidation products (Figure 5B).30

Figure 5. Hemin-GroEL catalyzes the oxidation of TMB in the presence of H2O2 to produce a

color change. (A) Images of the color change with reaction time; (B) the change in the UV-Vis absorbance of the reaction mixture; (C) the catalytic mechanism of hemin-GroEL in the oxidation reaction; (D) A dose–response curve for H2O2 detection with hemin-GroEL.

Investigation of the mechanism of hemin-GroEL-catalyzed oxidation of TMB by H2O2 reveals that the catalytic reaction also follows ping-pong kinetics (Figure 5C). This suggests that, as for HRP and for the Orange II-H2O2 system, the hemin-GroEL complex binds and reacts with the TMB and H2O2 substrates only one at a time. The addition of ATP to preformed hemin-GroEL complex also led to a clear decrease of its


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catalytic activity in the TMB oxidation reaction, confirming the sensitivity of the hybrid biocatalyst to ATP binding to the chaperonin, as of GroEL-peptide complex in vivo (data not shown).8 We next studied the sensitivity of hemin-GroEL-based detection for H2O2 combined with the use of the chromogenic substrate of TMB. The absorbance change at 450nm of the reaction mixture quenched by adding H2SO4 was plotted against the concentrations of H2O2 added in the system. This change is directly related to the amount of TMB-derived oxidation product.31 As can be seen in Figure 5D, the absorbance increased with the concentration of H2O2, and a good linear relationship between the two variables can be observed in the concentration range of 0-300µM of H2O2. The limit of H2O2 detection was estimated to be 10µM, which is comparable to those values reported previously.45 DISCUSSION Over the past two decades, the issue of clean oxidation has been addressed by developing various original strategies to construct new biocatalysts dedicated to process oxidation efficiently using clean oxidants such as O2 or H2O2 under mild conditions.10-11 Here we have shown that catalytic oxidation can be achieved by incorporating hemin cofactor into the bacterial chaperonin GroEL. No exploration of GroEL’s effectiveness in non-covalent encapsulation of metal cofactors for the purpose of catalysis has been reported previously, despite the fact that this protein complex, along with its cofactor GroES, is a well-studied paradigmatic molecular machine of protein folding.7-8 An advantage of GroEL compared to other protein cages is that the chaperonin is evolved to capture polypeptides with its central cavity through sequence-independent hydrophobic interactions, a feature similar to natural metalloenzymes accommodating the catalytically active site and the substrate, and thus constitutes a promising scaffold for artificial metalloenzymes.10-11, 24-25 Moreover, besides the openings at the end of GroEL cavity, the natural pores on the wall are also large enough to facilitate general substrate or product chemicals to pass through.46 A red shift in the Soret band of hemin was evident after detergent dialysis in the presence of GroEL, as compared to the case without the chaperonin, suggesting that a heme-GroEL complex was formed. The main purpose of the use of detergent Triton



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X-100 was to prevent hemin aggregation, with its micelles serving as a favorable microenvironment to monomeric hemin.34 This is achieved as judged from the preliminary experiments and the resultant efficient introduction of hemin to GroEL in its monodisperse form, as indicated by UV-Vis spectroscopic and DLS measurements. The observation that one GroEL can bind multiple guest hemin cofactors (up to ~35 in each cavity with a total molecular weight of ~23kDa) is far from exceeding the capacity of each GroEL cavity, which can accommodate a polypeptide of up to 60 kDa in assisted folding, comparable with the estimation based on the volume of each GroEL chamber (~85,000Å3) and the molecular size of hemin (~15Å across). 36-37, 47 It is also conceivable that a much larger volume of GroEL chamber compared with the hydrophobic binding pocket of natural metalloenzymes (ranging from~400Å3 to over 2000Å3) may endow the chaperonin-based hybrid biocatalyst with unique properties of a nanoreactor such as the enhancement of reaction rate and the suppression of side reaction.4-5, 48 However, the distribution and orientation of hemin cofactors in GroEL cage and how they interact with other components during catalysis are important aspects of the catalytic reaction that deserve further examination. The success of creating a GroEL-based metalloenzyme by supramolecular anchoring of hemin to the chaperonin central cavity is manifested in the good catalytic properties of the hemin-GroEL complex. The artificial biocatalyst was observed to have the ability to activate H2O2 for the catalytic oxidation of the model substrates of both Orange II and TMB, similar to that of natural hemoprotein HRP. It is well-known that enzymatic activity are influenced by various parameters, among which temperature, pH and enzyme concentration are commonly considered. Our results demonstrate that the activity of hemin-GroEL is directly proportional to its concentration, especially in the lower concentration range where the number of this artificial biohybrid is significantly lower than that of the model substrate. Meanwhile, the catalytic activity is shown to be highly dependent on temperature, as for HRP,38 and in essence high temperatures encouraged the oxidation reactions in the temperature range tested, probably due to increased molecular motion and collision. Nevertheless, the GroEL-based became unstable and lost some of its activity quickly


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(i.e., within 2h) at the higher temperatures. Although GroEL itself was reported to denature at about 70oC,40 higher temperature may alter to some extent the delicate supramolecuar structure of the artificial hybrid biocatalyst, leading to a decreased stability. With respect to the effect of pH value, while hemin-GroEL showed higher activity in an alkaline solution, the optimal pH for the artificial biocatalyst to keep relatively stable was found to be about pH 7.0. Hence the reactivity and stability are two important facets for evaluating the catalytic performance of artificial metalloenzyme hemin-GroEL and should be balanced in practical application, reminiscent of the research on natural biocatalyst or man-made inorganic catalyst.49-51 Investigation of the mechanism of hemin-GroEL-catalyzed oxidation of both Orange II and TMB reveals that, like HRP,42 the catalysis follows a ping-pong mechanism, which, also known as double-displacement reaction, is characterized by the change of an enzyme from its intermediate state to its standard state just like a ping-pong ball (Scheme 2). Another important feature of the mechanism is that one substrate is converted to the product and dissociates before the second one binds. In addition to hemin-GroEL complex, the kinetic behavior of several other hemin-functionalized nanomaterials in oxidation reaction was also consistent with this mechanism,43, 52 suggesting that the catalytically active hemin rather than the scaffold of HRP or the mimetic catalysts plays a decisive role in their general kinetic behavior. Moreover, it is generally accepted that in the catalytic cycle of natural hemoprotein such as cytochrome P450, the interaction with oxidants induces the change of iron(III) in its heme cofactor into a highly reactive iron(V)-oxo species, which are responsible for the oxidation of the second substrate.10-11 Similar species or oxidizing intermediate may also form in the oxidation of Orange II and TMB catalyzed by hemin-GroEL in the presence of H2O2. Thus, as shown in Scheme 2, it is possible that H2O2 first binds to hemin-GroEL and gives rise to the formation of the iron(V)-oxo intermediate, which, after the first product of H2O leaves, bounces back to its standard state with iron(III) by binding and oxidizing Orange II or TMB, followed by the dissociation of the second product and another reaction cycle.



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Scheme 2. Schematic illustration of the ping-pong mechanism, where S1 and S2 represent two different substrates, P1 and P2 the corresponding products, and E and E(*) denote enzyme in standard and intermediate state, respectively.

The hemin-GroEL is shown in tandem with TMB to be a good tool for H2O2 detection. Basically, this artificial metalloenzyme is useful in all potentially applicable fields of HRP, including but not limited to decolorization of synthetic dyes, removal of phenolic contaminants and related compounds, organic and polymer synthesis, biosensors and enzyme immunoassays.44 Moreover, the ATP-regulated dissociation of GroEL-nanocaged polypeptides, nanoparticles, or even hemin demonstrated here by decreased reactivity in the oxidation of either Orange II or TMB, makes it possible to recycle the chaperionin cage.25, 53 Considering the ease of production of the bacterial chaperonin GroEL and its supramolecular complex with hemin, together with the rich chemistry of free metalloporphyrins, this work should seed the creation of many new artificial metalloenzymes with diverse reactivity, or even selectivity. CONCLUSIONS The work presented here demonstrates a novel design of an artificial metalloenzyme, created by supramolecular anchoring of catalytically active hemin complex in the hydrophobic central cavity of the bacterial chaperonin GroEL. To our knowledge, this is the first reported chaperonin nanocaged-hemin biocatalyst for oxidations. The success of the present design is manifested by the efficient loading of multiple hemin cofactors to one GroEL molecule in their monomeric form, and the good catalytic oxidation properties of the resulting artificial hemoprotein. Investigation of the mechanism of hemin-GroEL-catalyzed oxidation reveals that the kinetic behavior of the complex follows a ping-pong mechanism. Moreover, through comparison with HRP, the results of this study show that the oxidative activity of hemin-GroEL is similar to that found in natural peroxidases. HRP has been used for diverse applications; hemin-GroEL is envisioned to be adapted readily for these applications


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in tandem with the clean oxidant of H2O2, as assessed by the chromogenic detection described here. It is notable that the present design by utilizing the extensive promiscuity of the central hydrophobic cavity of GroEL is highly flexible for binding other metalloporphyrins and catalysis for many other reactions. Supporting Information Figure S1, showing the absorbance curve of GroEL encapsulating different amounts of hemin; Figure S2, showing an additional TEM picture of purified GroEL; Figure S3, showing UV-Vis spectrum of hemin solubilized with Triton X-100; Figure S4, showing time course of the degradation of Orange II catalyzed by hemin-GroEL or hemin solubilized with Triton X-100; Table S1, showing molecular extinction coefficients of Orange II at 484nm determined at different temperatures. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21503278), China Postdoctoral Science Foundation (2014M560588, 2015T80756), the Fundamental Research Funds for the Central Universities. REFERENCES 1.

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Chaperonin-Nanocaged Hemin as an Artificial Metalloenzyme for

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