The Fe (III)/Fe(II) redox couple as a probe of immobilized tobacco peroxidase: Effect of the immobilization protocol

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Electrochimica Acta on Olloqui‐Sariego, J. L., Zakharova, G.S., Poloznikov, А. А., Calvente, J. J., Hushpulian, D. M., Gorton, L., & Andreu, R. (2019). The FE (III)/FE(II) redox couple as a probe of immobilized tobacco peroxidase: Effect of the immobilization protocol. Electrochimica Acta, 299, 55-61., available at: https://doi.org/10.1016/j.electacta.2018.12.153” 1 The Fe (III) / Fe(II) Redox Couple as a Probe of Immobilized Tobacco Peroxidase: Effect of the Immobilization Protocol José Luis Olloqui-Sariego,a Galina S. Zakharova,b Andrey A. Poloznikov,b Juan José Calvente,a Dmitry M. Hushpulian,b Lo Gorton,c Rafael Andreu a,* a Department of Physical Chemistry. University of Sevilla. Profesor García González 1. 41012 Sevilla. Spain. b D. Rogachev center of Pediatric Hematology, Oncology and Immunology, 1 Samory Mashela str., Moscow 117997, Russia. c Department of Biochemistry and Structural Biology. Lund University, P. O. Box 124, 221 00, Lund, Sweden. *Corresponding author’s e-mail: [email protected] 2 Abstract Non-turnover voltammetry is a sensitive tool to characterize the electrochemical properties of redox proteins. However, the catalytically competent oxidation states of most peroxidases do not display the required electrochemical reversibility. In this report, we circumvent this limitation and exploit the voltammetric response associated with the Fe(III)/Fe(II) redox couple of tobacco peroxidase to probe the energetics and electronic connectivity of the heme pocket. We have applied this approach to rationalize the previously reported influence of the immobilization protocol on the electrocatalytic activity of tobacco peroxidase. To decouple proton and electron transfer steps, measurements have been carried out over the 3 ≤ pH ≤ 9 range and a 1e-/2H+ ladder scheme has been adopted for their analysis. At each pH, thermodynamic and kinetic parameters associated with the Fe(III)/Fe(II) redox conversion were determined as a function of temperature in the 0-30ºC range. Reduction entropies and reorganization energies displayed different values for covalently immobilized and physisorbed enzymes, pointing to a larger involvement of the solvent in the last case. These findings, together with a larger electronic coupling between the prosthetic group and the electrode, are indicative of a partial denaturation of the physisorbed enzymes as the origin of their lower electrocatalytic activity. Keywords: Tobacco Peroxidase; Proton Coupled Electron Transfer; Fe(III)/Fe(II) Couple; Immobilization Protocol; Thermodynamics and Kinetics 3 1. Introduction Peroxidases are heme enzymes that catalyze the oxidation of a wide variety of organic and inorganic substrates by peroxides. Their catalytic function involves the oxidation of the heme iron to Compound I, which is then reduced by the substrate to Compound II and Fe(III) in two consecutive electron transfer steps [1]. Anionic tobacco peroxidase (TOP) is a class III plant peroxidase, which has been recognized as a promising building block for biosensors due to its high stability and substrate specificity [2,3]. When peroxidases are integrated in mediatorless electrocatalytic devices, such as third generation biosensors, an electrode replaces the substrate as the source of electrons to reduce Compound I and Compound II [4,5]. To generate a reliable response, this configuration requires that electroactive enzymes preserve their tertiary structure around the catalytic pocket after immobilization, and that redox cofactors are adequately wired to the electrode surface [6]. A number of studies have described both direct and mediated electrocatalytic responses of tobacco peroxidase adsorbed on a variety of electrode surfaces [716], but little is known on the impact of the enzyme immobilization protocol on the redox properties of the heme cofactor. Recently, we have shown that the covalent immobilization of crosslinked TOP oligomers (ci-olig-TOP) on a graphite electrode results in a significant improvement of the recorded electrocatalytic currents, as compared to those produced by covalently immobilized (ci-mon-TOP) and physically adsorbed (pa-mon-TOP) TOP monomers [14]. To explore the origin of this behavior, non-turnover voltammetry provides a simple and effective method to probe the electronic wiring and energetics of the redox center of immobilized enzymes [17]. However, this type of analysis can only be applied to the Fe(III)/Fe(II) redox couple of the heme center since the higher oxidation states involved in the catalytic cycle of 4 peroxidases do not provide a reversible voltammetric response under non-turnover conditions, except in the case of cytochrome c peroxidase [18,19]. Our previous voltammetric study of immobilized TOP was performed at pH 7 and 0ºC, where the three aforementioned immobilization protocols produced similar nonturnover voltammograms, in stark contrast with their distinct electrocatalytic responses [14]. In this work we have carried out a systematic analysis of the influence of pH and temperature on the Fe(III)/Fe(II) redox conversion of immobilized TOP enzymes. Though only c.a. one fifth of the electroactive heme groups of covalently immobilized TOP enzymes retain their ability to reduce hydrogen peroxide via the Compound I/Compound II/Fe(III) catalytic cycle [15], a single and characteristic Fe(III)/Fe(II) voltammetric response is observed for each immobilization protocol. To decouple the contributions from proton and electron transfer steps we have adopted a 1e-/2H+ ladder scheme, where proton steps are assumed to remain in equilibrium. Proton coupling is shown to lead to a pH dependent contribution to the reduction entropy of the enzyme, which may help to explain the broad range of reduction entropy values reported in literature for peroxidases [20]. The effect of temperature on the thermodynamic and kinetic charge transfer parameters is observed to be different for physisorbed and chemisorbed enzymes, and delineates a physical scenario where physisorbed enzymes undergo conformational changes that may explain their loss of electrocatalytic activity. 2. Material and methods 2.1. Enzyme production Wild-type recombinant TOP (hydrogen peroxide oxidoreductase, EC 1.11.1.7) was expressed in the form of inclusion bodies in E. coli BL21(DE3) CodonPlus cells as was 5 described earlier [21]. The construction of the expression vector and the procedure of TOP expression, refolding and purification are covered in detail elsewhere [21]. Briefly, the biomass from 600 mL of culture medium was disrupted, and the precipitate of inclusion bodies was washed, solubilized in 6 M urea and added drop by drop to the refolding medium, containing 1.8 M urea, 0.1 mM DTT, 0.5 mM oxidized glutathione, 3 mM СаСl2, 5 µM hemin and 5% glycerol in 50 mM Tris-HCl pH 9.5. After in vitro reactivation, r-TOP was concentrated and purified on a Toyopearl HW-55 column. The concentration of peroxidase was measured by the absorbance at 403 nm, using the experimentally determined extinction coefficient value of 108.0 ± 0.5 mM-1cm-1 for the protein monomer [22]. The resulting preparation was homogeneous as judged by SDSPAGE. The activity of purified TOP towards ABTS was about 4100 U/mg and the RZ value was 3.0. 2.2. Reagents 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy sulfosuccinimide sodium salt (NHS) were from Sigma-Aldrich. Sodium phosphate buffer solutions (SPB) of fixed ionic strength were prepared from phosphoric acid, and mono and dihydrogen sodium phosphate from Fluka, and sodium perchlorate and sodium hydroxide from Sigma-Aldrich. All reagents were of high purity, and were used as received. Water was purified with a Millipore Milli-Q system (resistivity 18 M cm). 2.3. Enzyme immobilization Pyrolytic graphite electrodes were constructed by fitting a rod of highly oriented pyrolytic graphite from Mineral Technologies into a PEEK casing, so that it exposed the edge of the graphite planes with a circular geometric area of 0.07 cm2. Prior to enzyme 6 immobilization, graphite surfaces were polished with abrasive P2400 sandpaper, rinsed with Millipore water and dried with an argon stream. To physisorb TOP, a 17 µL drop of 0.11 mg mL-1 TOP in 0.01 M SPB pH 6 was deposited on the graphite surface, and physisorption from solution was allowed to proceed for 20 h at 4ºC under wet conditions. For covalent coupling of TOP monomers, the electrode was incubated for 2 h at 4ºC in a solution formed by mixing a 4.5 µL drop of 20 mM NHS and a 5.5 µL drop of 40 mM EDC, both solutions being also 0.01 M SPB pH 6. After activation of the oxidized species at the graphite surface, the electrode was thoroughly rinsed with water and dried with an argon stream. Then, in a second incubation step that lasted 20 h at 4ºC, the electrode was capped with a 17 µL drop of a 0.11 mg mL-1 TOP solution in 0.01 M SPB pH 6. For covalent trapping of TOP oligomers the electrode surface was exposed to a mixture of three droplets: a 4.5 µL drop of 20 mM NHS, a 5.5 µL drop of 40 mM EDC and 7 µL drop of a 0.26 mg mL-1 TOP solution, that were also 0.01 M SPB pH 6, for 20 h at 4ºC. Irrespective of the immobilization protocol, the enzyme concentration in the deposition solution was 3 M, and the electrodes were thoroughly rinsed, first with water and then with the working buffer solution, after enzyme incubation. 2.4. Electrochemical measurements All electrochemical measurements were performed with an AUTOLAB PGSTAT 30, from Eco Chemie B.V, in a conventional three electrode undivided glass cell, equipped with a gas inlet and thermostated with a water jacket. The counter and reference 7 electrodes were a Pt bar and a AgAgClNaCl saturated electrode, respectively. The reference electrode was connected to the cell solution via a salt bridge, and kept at room temperature (23 ± 2ºC) in a non-isothermal configuration. Reported potential values have been referred to the SHE potential scale by adding +192 mV to the experimental potential values. All measurements were carried out under argon atmosphere. Working solutions contained 0.02 M sodium phosphate buffer at the desired pH plus sodium perchlorate to adjust the ionic strength to 0.12 M. Positive feedback for ohmic drop compensation was applied whenever the potential scan rate was higher than 1 V s-1. 3. Results and discussion 3. 1. Impact of Solution pH on the Fe(III) / Fe(II) redox conversion of TOP Figure 1 illustrates some voltammetric characteristics of immobilized TOP under non-turnover conditions at 25ºC. A couple of surface voltammetric waves, associated with the Fe(III)/Fe(II) redox conversion, can be observed at potentials close to 0 V at pH 3. As the solution is made more basic, the waves shift towards more negative potentials (see Figure 1a), indicating that the redox process is accompanied by proton exchange with solution. The surface concentration of electroactive heme groups, as determined by integration of the baseline corrected voltammograms, was found to lie within 80  20 pmol cm-2 for ci-olig-TOP and pa-mon-TOP, and within 40  10 pmol cm-2 for ci-mon-TOP. The full width at half maximum of the peaks at low scan rate (typically between 150 mV and 200 mV) is larger than the value expected for a population of identical non-interacting redox centers (90.6 mV at 25ºC), suggesting the presence of a variety of environments around the heme groups, including the possibility of conformational changes that may affect the catalytic activity of the enzymes. The 8 peak potential separation p E  remains insensitive to the potential scan rate v up to 10 V s-1, which is indicative of a fast electron exchange between protein and electrode [23]. Irrespective of the immobilization protocol, the midpoint potential 12/ E (determined from the average of the anodic and cathodic peak potentials) varies linearly with pH in basic solutions, it reaches a plateau for pH values close to 5, and it increases again as the solution is made more acidic (see Figure 1b). Previously reported 12/ E values for physisorbed TOP at pH 5 [13] and 7 [14] are in good agreement with those displayed in Figure 1b. The shape of these 12/ E vs pH curves is analogous to that of the potentiometric titration curves of isoenzymes C and A of horseradish peroxidase (HRP), though they are shifted towards more acidic pHs and more positive potentials in relation to those of HRP-C [24] and HRP-A [25]. The simplest reaction scheme that can describe the observed variation of the midpoint potential with pH is a six membered ladderscheme (see Scheme 1), involving two acid/base groups A1 and A2 whose acid dissociation equilibrium constants are dependent on the oxidation state of the heme group, and are denoted as 1ox, K , 2ox, K , 1red , K and 2red , K [26,27]. Moreover, the three protonation states of the oxidized heme are related to their corresponding reduced forms through three electron transfer steps, each one being characterized by its formal potential f i E and standard rate constant of electron transfer s,i k (i = 1, 2, 3). According to this reaction scheme, three plateaus corresponding to 1 2 1f / EE , 1 2 2f / EE and 1 2 3f / EE are to be expected in a 12/ E vs pH plot when the electron transfer takes place sequentially through each of the three parallel electron pathways, as the solution is made more basic, according to:         12 1 2 2 12 1 ln 1 red, red , fHH / ox, ox, HH c / K K / c RT EEFc / K K / c      (1) 15 where P and xi stand for pressure and solution composition, respectively. The slope of the 12/ E vs T plots turns out to be the sum of two contributions: the   2i f P,x E / T term, which is pH independent, and a second pH dependent term, whose value evolves from positive to negative as the solution becomes more basic, and it accounts for the change of slope observed in Figure 3. The   2i f P,x E / T term in eq. (5) is directly related to the reduction entropy 2f,rc S  corresponding to the electron transfer pathway 2 in Scheme 1, which can be expressed in the non-isothermal cell configuration as [3335]: 2 2 i f f,rc P,x E S nF T       (6) where n = 1 and F has its usual meaning. Analogously, the reduction enthalpy 2f,rc H  (referred to the normal hydrogen electrode at room temperature) is given by:       2 2 NHE 1 i f f,rc P,x E / T H nF /T        (7) Over the limited temperature range considered here, both 2f E vs T and 2f E / T vs 1/T plots are linear (see Figures 4a and b), and the corresponding 2f,rc S  and 2f,rc H  values have been collected in Table 2. These values lay well within the ranges reported for peroxidases in solution at pH 7, namely, 210 J mol-1 K-1 ≤ f rc S  ≤ -120 J mol-1 K-1 and 91 kJ mol-1 K-1 ≤ f rc H  ≤ -18 kJ mol-1 K-1 [20]. It is interesting to note that the 2f,rc S  and 2f,rc H  values obtained for ci-olig-TOP and ci-mon-TOP are similar and negative, in stark contrast with the positive values determined for pa-mon-TOP. In the case of electron transfer proteins with positively charged redox centers, more positive reduction entropies are generally associated with an increase of the solvent accessibility to the 16 redox center [36]. Therefore, the more positive 2f,rc S  value of physisorbed TOP enzymes suggests that they acquire a more open and solvent accessible polypeptide structure upon adsorption on graphite. On the other hand, crosslinking with the graphite surface and with other TOP molecules helps to preserve the native and catalytically active structure of ci-mon-TOP and ci-olig-TOP, but it also restricts the solvent access to the redox center. Extensive enthalpy-entropy compensation, as observed in Table 2 for the three immobilization protocols, points also to a substantial contribution of the solvent exchange between protein and solution to the energetics of TOP reduction [37]. 17 Figure 4. (a) Formal potential 2f E vs absolute temperature T and (b) 2f E / T vs 1 T plots for the three indicated immobilization protocols. Solid lines in plots (a) and (b) are linear leastsquares fits. Other experimental conditions: 0.02 M SPB (I = 0.12 M). Table 2. Thermodynamic and Kinetic Parameters Describing the Influence of Temperature on the Fe(III) / Fe(II) Redox Conversion of Immobilized TOP. pa-mon-TOP ci-mon-TOP ci-olig-TOP ∆𝑆2,𝑟𝑐 𝑓 / J K-1 mol-1 13 ± 7 –37 ± 7 –24 ± 8 ∆𝐻2,𝑟𝑐 𝑓 / kJ mol-1 2 ± 1 –8 ± 2 –4 ± 1 10−4 ∙ 𝐴2 / s-1 23 ± 8 3.0 ± 0.9 1.7 ± 0.7 ∆𝐻2 ≠ / kJ mol-1 10 ± 1 5.8 ± 0.5 6.2 ± 0.5 Within the temperature range of interest, the ap s k vs pH profiles were similar to those already discussed at 25ºC (see Figure 5), and were also fitted to eq. (3) to obtain sets of optimized 1 Q , 2s, k and 3 Q values at each temperature. Both 1 Q and 3 Q displayed a stronger variation with temperature (not shown) than that of 2s, k , but we have limited our analysis to the 2s, k values due to the impossibility of decoupling the contributions of rate and equilibrium constants to the observed variation of 1 Q and 3 Q with temperature. 18 Figure 5. pH and temperature dependence of the standard electron transfer rate constant ap s k for the three indicated immobilization protocols. Solid lines are theoretical fits computed from equation (3). Other experimental conditions: 0.02 M SPB (I = 0.12 M). The three immobilization protocols lead to linear Arrhenius plots displayed in Figure 6. The intercept and slope of these plots provide the pre-exponential factor ( 2 A ) and activation enthalpy ( 2 ΔH ) for electron transfer, according to: 2 22 Δ ln ln s, H kA RT   (8) 2 A and 2 ΔH values are collected in Table 2, and both may be seen to decrease as the extent of TOP crosslinking increases, so that their combined values tend to mitigate the influence of the immobilization protocol on the observed rate of electron transfer. A more detailed interpretation of the 2 A and 2 ΔH values requires the adoption of a specific electron transfer mechanism. The rate of electron exchange between TOP and graphite is quite fast, in spite of the low density of electronic states of graphite (compare 0.0022 eV-1 for graphite [38,39] with 0.28 eV-1 for a typical metal like gold [40]), which reduces the probability of electron transfer between enzyme and electrode, and reinforces the possibility that this electron exchange takes place through a nonadiabatic mechanism [40]. The adequacy of 19 this mechanism is also supported by the observed insensitivity of the measured ap s k values to the replacement of water by a highly viscous solution as solvent (specifically, a 2.8 M glucose solution with  = 4.9 cP at 22ºC [41]). Then, within the framework of a nonadiabatic electron transfer regime [41]: 2 2 AB AH (9a) 2 2 λ Δ4 H (9b) where AB H and 2  are the electronic coupling and reorganization energies, respectively. The extent of electronic coupling varies with the thickness and electronic conductivity of the intervening medium between electrode and redox center [42]. Since the immobilization protocol is not expected to induce dramatic changes in the electronic conductivity of the polypeptide matrix, the sequence of 2 A values in Table 2 is likely to reflect differences in the distance of closest approach of the heme group to the electrode, which would be then smaller for pa-mon-TOP than for ci-mon-TOP and ciolig-TOP. According to this interpretation, physisorption of TOP enzymes would be accompanied by conformational changes that bring the heme group closer to the electrode surface. In the case of chemisorbed TOP enzymes, the formation of inter- and intramolecular amide bonds would help to fix the native protein structure and prevent a closer approach of the heme group to the electrode. 20 Figure 6. Arrhenius plot for the standard electron transfer rate constant 2s, k , for the three indicated immobilization protocols. Solid lines are linear least-squares fits. Other experimental conditions: 0.02 M SPB (I = 0.12 M). The complex molecular scaffold of proteins restricts the mobility of the various dipoles (such as peptide groups or solvent molecules) that may reorient when the charge of a redox cofactor is changed in an electron transfer event, and thereby it helps to lower its reorganization energy in comparison to similar reactions involving simple reactants in solution [43]. Moreover, the immobilization of a redox protein on a surface is expected to reduce further the contribution of the surrounding medium to the reorganization energy [44,45]. Thus, the reorganization energy of cytochrome c has been reported to decrease from 0.6 eV in solution down to 0.3 eV in the adsorbed state [46]. This last value is close to the reorganization energies derived from the 2 H   values collected in Table 2, i. e.: 0.23 eV for ci-mon-TOP, 0.25 eV for ci-olig-TOP and 0.41 eV for physisorbed TOP. The higher value obtained for physisorbed TOP is again consistent with an easier access of the solvent to the redox center and its involvement in 21 the activation process [47,48], whereas the lower values determined for covalently immobilized proteins can be considered as a direct consequence of the more rigid and tight molecular envelope that crosslinking generates around the redox center [43]. 3. Conclusions In this work we have shown how the Fe(III)/Fe(II) redox couple of tobacco peroxidase can be used to probe the energetics and electronic connectivity of the heme pocket. Analysis of the combined effects of pH and temperature on the voltammetric response leads to a detailed interpretation of the influence of the enzyme immobilization protocol on its electrochemical behavior. As compared with covalently immobilized TOP, physisorbed enzymes are characterized by a more positive reduction entropy and a higher reorganization energy, which are indicative of a more extensive solvent involvement in the electron transfer process. These observations, together with a larger electronic coupling between heme and electrode point to a partial denaturation of the physorbed TOP enzymes as the origin of their low electrocatalytic activity. Thermodynamic parameter values are similar for covalently immobilized monomers and oligomers, while a larger electronic coupling is responsible for the somewhat higher electron transfer rate constants determined for the monomers. However, intermolecular crosslinking results in a higher population of electroactive oligomers and, overall, in a more active electrocatalytic film. Acknowledgements J. L. O., J. J. C. and R. A. acknowledge financial support from the Spanish Ministry of Economy and Competitiveness and the European Union FEDER (grants CTQ2014- 22 52641-P and CTQ2015-71955-REDT (ELECTROBIONET)) and L. G. from the Swedish Research Council (grant 2014-5908). References [1] H. B. Dunford, Heme Peroxidases, John Wiley, Chichester, 1999. [2] I. G Gazaryan, L. M. Lagrimini, Purification and Unusual Kinetic Properties of a Tobacco Anionic Peroxidase, Phytochem. 41 (1996) 1029-1034. [3] I. G. Gazaryan, L. Gorton, T. Ruzgas, E. Csöregi, W. Schuhmann, L. M Lagrimini, D. M. 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