Solvent tunes the peroxidase activity of cytochrome c ...

Applied Clay Science 118 (2015) 316–324

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Research paper

Solvent tunes the peroxidase activity of cytochrome c immobilized on kaolinite Elena Castellini a, Fabrizio Bernini a, Marcello Berto b, Marco Borsari a, Marco Sola b,c, Antonio Ranieri b,⁎ a b c

Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy CNR-NANO Institute of Nanoscience, Via G. Campi 213/A, 41125 Modena, Italy

a r t i c l e

i n f o

Article history: Received 5 June 2015 Received in revised form 27 August 2015 Accepted 9 October 2015 Available online 18 October 2015 Keywords: Cytochrome c Ethanol Kaolinite Adsorption Heterogeneous catalysis

a b s t r a c t The adsorption process and the peroxidase activity of yeast cytochrome c (ycc) immobilized on kaolinite (Kaol) were investigated in mixed ethanol/water solutions. The protein strongly adsorbs on the surface of the clay mineral and the thermodynamic adsorption constant increases with increasing ethanol concentration. The adsorption parameters suggest that in ycc a conformational transition from molten globule to helical state occurs in solution for ethanol concentration above 20%. The peroxidase activity of ycc immobilized on Kaol increases from 0% to 20% ethanol (v/v), then it progressively decreases and almost vanishes in pure ethanol. The catalytic properties of adsorbed ycc were studied in 20 and 40% ethanol solutions which correspond to the molten globule and to the helical state, respectively. In both cases, catalysis adheres to the Michaelis–Menten model. The molten globule state, which binds more weakly to kaolinite than the helical state, was found to be more catalytically active. This study is meant to identify the physicochemical factors that modulate the catalytic activity of this kaolinite-based interface of broad applicability. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Cytochrome c (cytc) is an ubiquitous metalloprotein involved in several physiological functions (Scott and Mauk, 1996). This protein possesses a robust structure and physico-chemical properties modulated by the pH and solvent composition (Battistuzzi et al., 1995, 1997, 1998; Moore and Pettigrew, 1990). In the last two decades, several studies have been carried out on the possibility of replacing enzymes, which are rather delicate molecules, with cytochromes c – either native or engineered – able to carry out the same catalytic function also in a hostile environment (Vazquez-Duhalt, 1999). It is known that the native six-coordinated (Met, His)-ligated form of cytc in solution does not show peroxidase activity due to the absence of a free coordination position for H2O2 binding to the heme iron to yield the ferryl group (Diederix et al., 2002). Breaking or weakening of the axial methionine bond to the heme iron is therefore needed for induction of a peroxidase-like activity in cytc (Diederix et al., 2002; Ying et al., 2009). For the native protein, these effects can be easily obtained by lowering the pH or using strong unfolding agents like urea at high concentration (Fedurco et al., 2004). Moreover, by means of sitedirected mutagenesis, recombinant five-coordinated heme containing cytc can be obtained, as the M80A variant. Also adsorption on some surfaces has been found to induce an unfolding effect on cytc involving ⁎ Corresponding author. E-mail address: [email protected] (A. Ranieri).

http://dx.doi.org/10.1016/j.clay.2015.10.012 0169-1317/© 2015 Elsevier B.V. All rights reserved.

such change in axial heme iron coordination (Castellini et al., 2009; Ranieri et al., 2011). A big effort is being devoted to exploit these catalytic properties also in heterogeneous catalysis, which would facilitate the separation of the products and recovering of the catalyst from the reaction environment. Within this perspective, adsorption of cytc onto a proper supporting material may play the double function of immobilizing cytc onto a solid material and inducing or enhancing the catalytic properties thanks to an adsorption-driven conformational change of the protein. Yeast cytochromes c (ycc) adsorb onto various surfaces such modified metal electrodes and clay minerals (Castellini et al., 2009; Ranieri et al., 2011). In particular, ycc are strongly adsorbed by kaolinite (Kaol hereafter) and, under these conditions (Kaol-ycc), display peroxidase activity towards the oxidation of guaiacol in a wider pH range compared to the protein in solution. Indeed, Kaol-ycc catalyst is active also at pH values above 8.2, while in solution at the same pH values ycc is inactivated by the alkaline transition (Ranieri et al., 2011). The peroxidase activity of Kaol-ycc at pH 7 is ascribed to the presence of a non-native lowspin (Met, His) species, imparted with catalytic activity (Ranieri et al., 2011). The catalytic efficiency of Kaol-ycc dramatically increases (20 fold) in the presence of urea at high concentration (Castellini et al., 2013). Urea causes a progressive unfolding of the polypeptide chain leading to the detachment of the methionine residue from the heme iron (Monari et al., 2010, 2011; Ranieri et al., 2012a). In this study the adsorption process of ycc on Kaol and the peroxidase activity of Kaol-ycc catalyst in mixed ethanol/water solutions was

E. Castellini et al. / Applied Clay Science 118 (2015) 316–324

investigated. The aim is two-fold. First, to assess conditions in which the organic solvent improves the catalytic properties of Kaol-ycc and to gain information on the underlying protein conformational changes. Second, to evaluate the solvent effects on the thermodynamics of the adsorption process onto kaolinite. Ethanol was selected due to its weak unfolding properties. This allows the behavior of ycc to be studied in experimental conditions which are intermediate between the strong unfolding conditions induced by highly concentrated urea and those of the unperturbed protein. In this way, the contribution given to the peroxidase activity of a kaolinite-based catalyst by mild unfolding effects – likely involving the breaking of H bonds of the tertiary structure – with no change in the axial ligand set of the iron, will be assessed. 2. Experimental 2.1. Materials High purity and fully characterized kaolinite, signed as KGa-1b, was purchased from the Clay Minerals Society (The Clay Minerals Society, Source Clays Repository, University of Missouri, Columbia, MO). Yeast cytochrome c from Saccharomyces cerevisiae (ycc) was purchased from Sigma-Aldrich (St. Louis, MO) and further purified by ionic exchange chromatography with Sephadex G-15 resin. Guaiacol (2-methoxyphenol), ethanol and H2O2 were purchased from Carlo Erba Reagenti (Rodano, Italy) and used without further purification. Buffer solutions (acetate buffer at pH 3.5 and phosphate buffer at pH 7) were from J. T. Backer (Deventer, The Netherlands). All chemicals were of reagent grade. Water was purified through a MilliQ Plus Ultrapure Water System coupled with an Elix-5 Kit (Millipore). The water resistivity was over 18 MΩ cm. 2.2. Adsorption measurements The adsorption of ycc solubilized in ethanol/water mixtures with percentage ethanol content (by volume), Φeth, of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50% on Kaol has been investigated at 293 K and at pH 3.5. Higher Φeth caused ycc precipitation. The choice of pH 3.5 was made considering the key role played by the pH on the properties of kaolinite and on the protein–mineral interaction. In fact, kaolinite shows a pronounced pH-dependence of the surface charge (Schroth and Sposito, 1997; Diz and Rand, 1989; Tombacz and Szekeres, 2006) and ycc is strongly adsorbed on kaolinite in a wide pH range, from pH 2 to 10 (Ranieri et al., 2011). At pH 7, the interactions between ycc and kaolinite have been proposed to be mostly electrostatic in nature (Sallez et al., 2000; Castellini et al., 2009; Ranieri et al., 2011). At pH 3.5 both electrostatic and hydrogen bonding interactions are most likely responsible for the formation and stabilization of the adsorbed film of the protein. At this pH, the charge of the edges and octahedral alumina sheets of the Kaol crystals is positive while that of the tetrahedral silica faces is negative and almost pH-independent (Gupta and Miller, 2010). The experiments were carried out following the procedure described previously (Castellini et al., 2009): dispersions were prepared by mixing 2 mg of Kaol with 1 ml of ycc solubilized in ethanol/water mixtures at pH 3.5 (for Φeth = 0%, 10 mM acetate buffer was employed; for Φeth values in the range 5–50%, the pH of the acetate buffer was set at 3.5 by calibrating the pH-meter with two buffer solutions for each Φeth value (Bates, 1973; Battistuzzi Gavioli et al., 1988; Perrin and Dempsey, 1979). The starting concentrations of the freshly prepared ycc solutions were in the range 0.0005–0.06 mM and were checked spectrophotometrically. The dispersions were continuously shaken at 250 rpm in an orbital incubator (Stuard Scientific Orbital Incubator mod. SI50) at 20 °C for 2 h. Thereafter, the dispersions were allowed to rest in the thermostated incubator to achieve the solid–liquid separation and then the separated supernatant was centrifuged for 2 min at 13,000 rpm (Spectrafuge mod. 24D) and subjected to UV–Vis measurement (Jasco mod. V-570 spectrophotometer). Invariably, the UV–Vis

317

spectrum of ycc solubilized in ethanol/water mixtures showed that ycc was in the oxidized form. As previously stated, the absorption spectrum showed a Soret band at 407 nm which was selected for the quantitative analysis (Battistuzzi et al., 2012). The experimental data were fitted using Sigma Plot v. 11.0 software (Dundas Software LTD, Germany) to select the suitable adsorption isotherm and determine the related parameters. 2.3. Preparation of the Kaol-ycc solid catalyst A batch of dispersions was prepared by adsorbing ycc onto Kaol from an aqueous solution buffered at pH 3.5 at a concentration corresponding to the plateau in the adsorption isotherm. UV–Vis spectra were acquired on the clarified supernatants to determine the number of adsorbed protein moles per kilogram of Kaol. The obtained mean value was 0.61 ± 0.02 mmol/kg. The separated solid phase was washed at least three times with 10 mM acetate buffer at pH 3.5 to eliminate the excess protein. These samples constitute the Kaol-ycc solid catalyst. 2.4. Initial reaction rate and peroxidase activity measurements Measurement of the peroxidase activity (P.A.) of the Kaol-ycc solid catalyst in different ethanol/water mixtures at 20 °C was carried out with the conventional procedure which exploits the oxidation of guaiacol (Gc) to tetraguaiacol (tetraGc) in the presence of H2O2, according to the reaction: 4Gc þ 4H2 O2 →tetraGc þ 8 H2 O

ð1Þ

by measuring spectrophotometrically the intensity of the tetraguaiacol signal at 480 nm (extinction coefficient, ε tetraGc,480 = 26,600 M− 1 cm− 1) (Mandelman et al., 1998). In this case, the medium in which the reaction takes place is a mixed ethanol/water solution and for this reason the Gc is diluted in proper ethanol/ water solutions. A 1.46 \cdot 10− 4 M Gc solution at pH 7 (phosphate buffer) was used; proper buffers were used for the pH measurements in mixed ethanol/water solutions (Bates, 1973; Battistuzzi Gavioli et al., 1988; Perrin and Dempsey, 1979). The solid catalyst was mechanically dispersed in 2 ml of the Gc solution and, as soon as an homogeneous dispersion was obtained, 50 μl of H2O2 0.5 M ([H2O2]0 = 12.2 mM) were rapidly added to the dispersion. After 15 s from H2O2 injection, the spectrum of the products was instantaneously recorded on the filtered liquids (∅ = 0.45 μm). The same procedure was carried out using Kaol lacking the adsorbed ycc (control sample). A H2O2 concentration of 12.2 mM was selected as this concentration is far from the values at which the reaction rate is independent of substrate concentration (catalyst saturation). From the absorbance of the tetraGc product at 480 nm (A480 nm) and the measured time of contact between the adsorbed ycc and H2O2 (Δt), the initial reaction rate (V0) was calculated. The initial reaction rate is defined as the concentration of reagent Gc, expressed in nanomolarity, which was consumed per second and per micromole of adsorbed ycc (μmolycc), namely: V0 ¼ ½Gc= Δt μmolycc

ð2Þ

½V0 ¼ nMGc = s μmolycc :

ð3Þ

Thus V0 is obtained from the experimental data as follows: V0 ¼ 4 109 ΔAλ¼480nm = Δt εtetraGc;480 d μmolycc

ð4Þ

where ΔAλ = 480 nm is the difference between the Aλ = 480 nm value recorded at t = Δt and at t = 0; εtetraGc,480 is the molar extinction coefficient of the tetraGc at λ = 480 nm (εtetraGc,480 = 26,600 M−1 cm−1);

318


d is the optic pathway of the UV–Vis cell (1 cm); the coefficient 4 · 109 is needed to transform tetraGc molarity into Gc nanomolarity; μmolycc is the number of adsorbed ycc micromoles. The average time of contact (15 s) is within the linear dependence of Aλ = 480 nm vs. time. The P.A. of the adsorbed cytochromes is defined as the concentration of Gc, expressed in nanomolarity, which is consumed per second, per micromole of adsorbed ycc, and per unit of starting concentration of H2O2, expressed in millimolarity, in reaction (1):

3. Results 3.1. Adsorption measurements

P:A: ¼ ½Gc= Δt μmolycc ½H2 O2 0

ð5Þ

½P:A: ¼ nMGc = s μmolycc mMH2O2

ð6Þ

therefore: P:A: ¼ V0 =½H2 O2 0 :

20 min, the dispersions were centrifuged, the supernatants separated and the soaked solids used for the measurements. DR UV–Vis spectra were recorded on a V-570 Jasco instrument equipped with an integrating sphere attachment (Jasco model ISN-470) in the 220–900 nm range. Kaolinite was used as standard instead of BaSO4.

ð7Þ

Ycc invariably adsorbs onto Kaol at all the Φeth investigated at 293 K. The plots representing the adsorbed ycc moles for mass unit of Kaol, q, as a function of the ycc equilibrium concentrations, c, follow a typical Langmuir or Frumkin adsorption isotherm for all the Φeth values. The limit value, qmax, is independent of Φeth (Fig. 1A and B). A Langmuir-type adsorption isotherm neglects the lateral interactions occurring among the adsorbed molecules. It is expressed by the following equation: θ ð1−θÞc

2.5. Peroxidase activity measurements of Kaol-ycc catalyst as a function of Φeth

K ads ¼

Experimental conditions were as follows: 2 mg of Kaol-ycc; 1.46 · 10−4 M Gc solutions (2 ml) prepared at pH 7 in mixed ethanol/ water solutions with Φeth values of 10, 15, 20, 25, 30, 40, 60, 80, and 100%; [H2O2]0 = 12.2 mM, T = 20 °C. These measurements served to recognize the ethanol/water ratios at which Kaol-ycc displays a peculiar catalytic behavior (vide infra). On the basis of the results, the Φeth values of 20 and 40% were selected to investigate the initial reaction rate, V0, as a function of substrate concentration.

where Kads is the adsorption constant, c the adsorbate equilibrium concentration, and θ the coverage. In the present case, θ can be defined as the q/qmax ratio. The Frumkin isotherm, instead, accounts for the

2.6. Determination of the initial rate as a function of the substrate concentration (H2O2) at Φeth 20 and 40% for Kaol-ycc, Kaol-E20-ycc and Kaol-E40-ycc catalysts The V0 values for reaction (1) catalyzed by Kaol-ycc were determined as a function of H2O2 concentration in mixed ethanol/water media with Φeth values of 20 and 40%, following the procedure described above. The standard experimental conditions were as follows: 2 mg of Kaol-ycc; 1.46 · 10− 4 M Gc solutions (2 ml) buffered at pH 7 at Φeth values of 20 or 40%; [H2O2]0 in the range 3–500 mM. To investigate how adsorption process affects the role of ethanol on the peroxidase activity of cytochrome c, the determination of the initial rate V0 as a function of H2O2 concentration was performed on Kaol-ycc prepared adsorbing cytochrome c solubilized in mixed ethanol/water solutions. In fact, it is well known that the effect of unfolding agents on adsorbed cytochrome c is significantly different from that on the protein in solution (Monari et al., 2010, 2011; Ranieri et al., 2015a). The other solid catalysts were prepared adsorbing onto Kaol-ycc from mixed ethanol/water solutions (Φeth 20 and 40%) buffered at pH 3.5; in this case, the adsorbing solutions has the same ethanol content of the reaction media. These catalysts are named Kaol-E20-ycc and Kaol-E40-ycc, respectively. Measurements of V0 for reaction (1) catalyzed by Kaol-E20-ycc and Kaol-E40-ycc were also performed in the same conditions as for Kaol-ycc. 2.7. Diffuse-Reflectance UV–Vis measurements In order to detect possible changes in the heme environment of ycc induced by the mixed solvent, Diffuse-Reflectance (DR) UV–Vis measurements have been performed on Kaol-ycc treated with solution at different Φeth. In fact, the Soret and α,β bands are sensitive to changes in the coordinative sphere of the metal center (Ranieri et al., 2011). DR UV–Vis measurements were taken for three samples in which Kaol-ycc was dispersed in buffer solution at pH 7 and in ethanol/water solvents with Φeth of 20 and 80% at pH 7. After a contact time of

Fig. 1. Adsorbed ycc mmoles for mass unit of Kaol, q, as a function of the ycc equilibrium concentrations, c, for Φeth = 0, 5, 10, 15, and 20% (A) and for Φeth = 25, 30, 35, 40, 45, and 50% (B). T = 293 K.


319

interactions between the adsorbed molecules according to the equation: K ads ¼

θ e2aθ ð1−θÞc

where a is the Frumkin interaction parameter and the other terms have the usual meaning. A positive (negative) Frumkin interaction parameter is indicative of repulsive (attractive) interactions between the adsorbed molecules. For a = 0 no interactions between molecules occur and the Frumkin isotherm becomes Langmuir's. Therefore, the Langmuir isotherm can be considered as a particular case of the Frumkin isotherm. To understand if the adsorption process follows a Langmuir or Frumkin model at the various Φeth values, the slope of the plot of ln[θ/c(1 − θ)] vs. θ (Langmuir test, Fig. S1) was evaluated. For a Langmuir-like adsorption process the slope should be zero. As slopes invariably differ from zero, adsorption of ycc on Kaol at all Φeth values is better described by the Frumkin isotherm. Therefore, the q vs. c data were fitted with the Frumkin model to calculate qmax, Kads and a values (Table 1). The sign of a, which is positive, accounts for the existence of repulsive interactions between the adsorbed ycc molecules. The qmax values allow the coverage value, θ (θ = q/qmax), to be calculated for each equilibrium concentration value. Kads increases with increasing Φeth. The standard Gibbs energy, ΔG°′ads, can be calculated at all the Φeth values at 20 °C by the equation ΔG°′ads = − RTlnKads (Table 1). All values of ΔG°′ads are negative suggesting that the adsorption process is thermodynamically favored.

3.2. Peroxidase activity of Kaol-ycc catalyst as a function of Φeth The P.A. of Kaol-ycc increases from Φeth 0% to 20%, then it progressively decreases and almost vanishes in pure ethanol solution (Fig. 2). The Φeth values of 20 and 40% (at which the peroxidase activity of Kaol-ycc is, respectively, maximum and average) were selected as the two significant solvent compositions at which the catalytic behavior of Kaol-ycc in mixed ethanol/water medium is worth studying. No ycc was detected in the reaction solution at pH 7 (from the absorbance spectrum), indicating that no desorption of ycc from Kaol occurs during the measurements. This testifies the good quality of the catalyst and is likely the result of the pH-independent charge of the adsorption sites on Kaol and the high adsorption constant of ycc onto Kaol at pH 3.5 (Table 1).

Table 1 Frumkin interaction parameter a, adsorption constant Kads, and limit adsorbed amount qmax for the Frumkin model applied to the adsorption of ycc from solutions at different Φeth on Kaol, as obtained from best fitting (Sigma Plot v.11.0) of the q vs. c data, along with the corresponding ΔG°′ads values. T = 293 K. Φeth

aa

Kadsa 10−7/mol−1 L

qmaxa 104/mol kg−1

ΔG°′adsa/kJ mol−1

0 5 10 15 20 25 30 35 40 45 50

1.05 1.10 1.11 1.08 1.24 1.35 2.05 1.97 1.94 1.94 1.96

0.31 0.40 0.47 0.60 0.76 1.23 2.49 4.77 10.90 28.97 72.48

6.1 6.1 6.1 6.1 6.1 6.0 6.0 6.0 6.0 6.0 6.0

−36.4 −37.0 −37.4 −38.0 −38.6 −39.8 −41.5 −43.1 −45.0 −47.4 −49.7

a Errors associated to a, Kads and q max, are ± 0.04, ± 0.02 · 10 7 mol − 1 L, ± 0.2 · 10 − 4 mol kg − 1 and ± 0.1 kJ mol− 1 , respectively.

Fig. 2. Peroxidase activity (P.A.) of Kaol-ycc catalyst as a function of Φeth at T = 293 K and pH 7.

3.3. Determination of the initial rate as a function of substrate concentration (H2O2) at Φeth 20 and 40% for Kaol-ycc, Kaol-E20-ycc and Kaol-E40-ycc catalysts Independently of the catalyst and the Φeth conditions, the V0 vs. [H2O2]0 curves show a monotonic increase in V0 which tends to level off to a constant value at high [H2O2]0 (Fig. 3A and B). The curves are similar to those predicted by the Michaelis–Menten model, so the corresponding equation was selected to fit the data. Although the Michaelis– Menten model was developed to describe homogeneous catalysis, its application in heterogeneous catalysis is allowed because the hypotheses of this model are independent of whether the catalyst and substrate are in the same or in a different physical phase. Substrate saturation is achieved under all the experimental conditions employed (Fig. 3A and B). V0 values are strongly affected by the ethanol content of the reaction medium. In particular, V0 values at Φeth = 20% (Fig. 3A) are larger than those at Φeth = 40% (Fig. 3B), in line with the peroxidase activity results (Fig. 1). Data were fitted using Sigma Plot v. 11.0 (Dundas Software, Germany) for the calculation of the Michaelis–Menten parameters Vmax and KM (Table 2). As for the P.A. measurements, ycc was found not to desorb from kaolinite.

3.4. Diffuse-Reflectance (DR) UV–Vis measurements The DR UV–Vis spectra for Kaol-ycc catalysts soaked at pH 7 in aqueous buffer solution and in mixed ethanol/water solutions with Φeth = 20 and 80% are shown in Fig. 4. Φeth = 20% was selected to mimic the experimental conditions of the V0 measurements, while Φeth = 80% was selected to investigate limit conditions in terms of alcohol content. The DR UV–Vis spectra show the Soret band at 409 nm and the Q-band at 531 nm (with a shoulder at about 555 nm), while the charge transfer band at 695 nm is undetectable (Ranieri et al., 2011). These bands are characteristic of a low-spin ferric cytochrome c. The weak shoulder at about 630 nm, however, can be attributed to a minor form of ycc characterized by a high-spin heme (Fedurco et al., 2004; Ranieri et al., 2011). This species has been already observed, but its contribution to the catalytic activity of ycc adsorbed on kaolinite was found negligible (Ranieri et al., 2011). Interestingly, the spectral features in mixed ethanol/water solutions are almost unchanged with respect to those in aqueous buffer (Fedurco et al., 2004; Ranieri et al., 2011). This means that the iron coordination sphere remains unchanged at increasing ethanol content in the solution from 0 to 80%. These results, therefore, suggest that the ethanol content-related changes in the peroxidase activity of ycc are the

320


Fig. 4. DR UV–Vis spectra of Kaol-ycc soaked at pH 7 in aqueous buffer and in mixed ethanol/water solutions. T = 293 K.

4. Discussion 4.1. Adsorption

Fig. 3. (A) Plots of V0 of reaction (1) at 293 K, pH 7 and Φeth = 20% as a function of H2O2 starting concentration for the Kaol-ycc catalyst (●) and for the Kaol-E20-ycc catalyst (○). (B) Plots of V0 of reaction (1) at 293 K, pH 7 and Φeth = 40% as a function of H2O2 starting concentration for the Kaol-ycc catalyst (▲) and for Kaol-E40-ycc catalyst (Δ). Best fitting curves obtained with Sigma Plot v.11.0 (Dundas Software LTD, Germany).

result of alterations of the polypeptide conformation around the metal center which do not affect heme iron ligation. The spectra do not change with time (data not shown) suggesting that ycc does not degrade on kaolinite during turnover. The peculiar stability of this biocatalytic interface is most likely related to the strong affinity of ycc for Kaol (Castellini et al., 2009). However, as previously observed, ycc is quickly degraded by H2O2 in the absence of Gc substrate (Ranieri et al., 2012a, 2012b; Shakir et al., 2010).

Table 2 Vmax e KM values as obtained by the best fitting of the V0 vs. [H2O2]0 data for reaction (1) catalyzed at 293 K and pH 7 by the different catalysts at Φeth = 20 and 40%. Catalyst

V0 measurement conditions

Vmax 10−4/nMGc s−1 μmol−1a cyt

KM/mMa

Kaol-ycc Kaol-ycc Kaol-E20-ycc Kaol-ycc Kaol-E40-ycc

Φeth = 0 Φeth = 20% Φeth = 20% Φeth = 40% Φeth = 40%

4.13b 13.0 8.4 3.1 3.3

27.3b 34 82 23 27

a b

Errors on Vmax and KM values are of ±5% and ±7%, respectively. Ranieri et al. (2011).

4.1.1. Coverage The value of qmax (0.00061 mol/kg, Table 1) is independent of ethanol concentration, indicating that the coverage is controlled only by the number of available adsorption sites on the Kaol surface. As ycc features a molecular charge of + 6 at pH 3.5, the adsorbed cytochromes under adsorption saturation conditions carry 0.00366 equivalents of charge per kilogram of Kaol, an amount higher than the CEC value of Kaol at the same pH (0.003 equivalents/kg (Zhou and Gunter, 1992)). This fact suggests that the adsorption driving force is not only electrostatic, but probably also involves hydrogen bonding interactions at the octahedral alumina face and at the edges of the kaolinite crystal. Consequently, adsorption of ycc likely occurs on all the surface of the kaolinite crystal. From the qmax values and the specific surface area (SBET = 11.62 m2/g, Castellini et al., 2005) of Kaol, the area of Kaol surface potentially available for each ycc molecule can be estimated about 3200 Å2. This area is much higher than that calculated by considering ycc as a rigid sphere of 34 Å diameter (910 Å2) and that determined for a full-densely packed layer of cytochromes c adsorbed onto anionic self assembled monolayers (880–950 Å2) (Casalini et al., 2008; Monari et al., 2008; Tarlov and Bowden, 1991). 4.1.2. Frumkin interaction parameter The Frumkin interaction parameter a is positive for all the Φeth investigated. This means, as expected, that repulsive forces exist among the ycc molecules adsorbed on kaolinite surface. The a value for same system in which ycc adsorption is performed at pH 7 in aqueous buffer is higher than that at pH 3.5 (1.94 (Castellini et al., 2009) vs. 1.05, see Table 1), in line with the higher coverage found at pH 7 with respect to pH 3.5 (qmax = 0.00232 vs. 0.00061 mol/kg, respectively). The behavior of a as a function of Φeth is worthy of note (Fig. 5). a increases with increasing Φeth up to Φeth = 20%, then it undergoes a sudden increase and levels off for Φeth N 30%. Such increase of a as a function of Φeth can be ascribed to the lowering of the dielectric constant of the medium caused by ethanol in water. In fact, the dielectric constant calculated for mixed ethanol/water solutions shows a linear decrease as a function of Φeth (Timmermans, 1960). However, this effect does not justify the steep increase in a above Φeth 20%. It is then conceivable that under these conditions the adsorbed protein undergoes a structural change and/or modifies the adsorption geometry in a fashion that amplifies the repulsive interactions. That would be also consistent with the near invariance of a above 30% Φeth,


321

new term has been introduced (Castellini et al., 2009), namely the apparent adsorption constant, Kappads, which describes the affinity of an adsorbate for and adsorbent at a fixed θ value, defined as follows: K appads ¼

Fig. 5. Frumkin interaction parameter, a, as a function of ethanol volume fraction Φeth for the adsorption of ycc onto Kaol at 293 K.

despite the lowering of the dielectric constant. It is known that in the presence of short-chain alcohols, several proteins, including cytochromes c, are subjected to a collapse of the internal hydrogen bond network without changes in the secondary structure (Bychkova et al., 1996). The protein maintains its globular structure while the intramolecular hydrogen bond network changes resulting in an increase in protein flexibility and accessibility to solvent. This structure, called “molten-globule”, is known to occur for cytochrome c subject to heating or in the presence of mild unfolding agents, like alcohols, or phospholipids (Bychkova et al., 1996). At high alcohol concentrations, the molten-globule structure changes to form a more unfolded structure called “helical state”, due to the large amount of solvent-exposed helical regions. Therefore, it can be hypothesized that upon increasing ethanol volume fraction from 0 to about 20% native ycc switches to the moltenglobule state and converts to the helical state for larger ethanol contents. These conformational transitions, namely native to molten globule and molten globule to helical state, would respectively account for the increase in the Frumkin parameter a from 1.05 to 1.24 and from 1.24 to the final value of about 2. a = 2 is invariant at higher ethanol volume fractions, and can therefore be taken as distinctive of the helical state of ycc. 4.1.3. Adsorption constant The adsorption constants at 293 K for different ethanol volume fractions are listed in Table 1. In the absence of ethanol, the value of Kads at pH 3.5 (0.31 · 107) is remarkably lower than that obtained at pH 7 (6.63 · 107) (Castellini et al., 2009) as the negative surface charge of kaolinite increases with increasing pH. Kads increases with increasing the ethanol volume fraction, in a fashion similar to the Frumkin interaction parameter. In particular, for Φeth ≥ 20%, Kads increases steeply, most probably due to the transition of ycc from molten globule to helical state and the ethanol-induced lowering of the dielectric constant of the medium. Although the lowering of the dielectric constant of the medium amplifies both the attractive forces between kaolinite and cytochromes and the repulsive forces among adsorbed cytochromes (as observed for the a values), the former effect prevails on the latter. In fact, the thermodynamic constant describes an adsorption process occurring at infinite dilution, so the Kaol surface strongly attracts ycc (and vice versa), but in a condition under which there are no adsorbed cytochromes repelling one another on the surface, because in principle the surface is almost empty (θ tends to 0). The relationship between the coverage (θ) and the adsorbate–adsorbent affinity is worth studying. In fact, if the adsorbed molecules experience repulsion, the adsorbate– adsorbent affinity would decrease with increasing the coverage and vice versa. For this reason, Kads being independent of the coverage, a

θ : ð1−θÞc

This term depends on the coverage and, at a fixed coverage, allows determination of the equilibrium concentration of the adsorbing species, c, which is inversely proportional to adsorbate–adsorbent affinity. Once verified the Frumkin adsorption isotherm, Kappads can be calculated from Kadse−2aθ and represents a sort of adsorbate–adsorbent affinity index. The behavior of Kappads calculated for different coverages (θ = 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9) as a function of Φeth at 293 K is illustrated in Fig. 6. The value of Kappads for θ = 0 is the thermodynamic constant Kads. Like Kads, the Kappads values increase with Φeth, meaning that not only the thermodynamic stability, but also the affinity of ycc towards Kaol increases with increasing the ethanol content of the medium. Although the shape of the curves obtained for different θ values is similar, the Kappads values progressively diminish with increasing coverage. Likely, the increasing repulsion among the adsorbed ycc molecules progressively offsets attractive adsorbate–adsorbent forces. However, even for a saturated Kaol surface (θ = 0.95), the Kappads values increase with increasing Φeth and invariably soar above 30% Φeth. This indicates that the affinity of ycc towards the surface is greater for the “helical state” than that for the molten globule state. Possibly, an increased flexibility of the protein would facilitate a favorable positioning of the charged groups of the polypeptide matrix for interaction with Kaol. The steep rise in Kappads above 30% Φeth, which is remarkable for low coverages, is attenuated at high coverages, making the affinity of the moltenglobule state comparable to that of the helical state towards Kaol. 4.2. Catalytic activity The catalytic activity of cytc at pH 7 is known to be the result of unfolding-induced conformational changes of the heme pocket and, in particular, of axial heme iron ligation (Castellini et al., 2013; Diederix et al., 2002; Fedurco et al., 2004; Monari et al., 2010, 2011; Ranieri et al., 2011, 2012a, 2012b; Tavagnacco et al., 2011). For cytc immobilized on a self-assembled monolayer, the peroxidase activity has been put in relation with the formation of a (His, His) or (His, OH−/H2O) axial iron coordination (Bortolotti et al., 2012; Casalini et al., 2008, 2010; Ranieri et al., 2012a). The same activity shown at pH 7 by ycc adsorbed on Kaol in the presence of unfolding agents has

Fig. 6. Kappads (=Kadse−2aθ) values calculated for different coverages θ of ycc on Kaol as a function of ethanol volume fraction. T = 293 K.

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been ascribed to the weakening of the Fe–S methionine bond (Castellini et al., 2013). In the present case, partial unfolding needed for peroxidase activity is due to the contemporary effects of the surface and ethanol. In fact, ycc adsorbed on Kaol shows catalytic properties also in the absence of alcohol (Castellini et al., 2009; Ranieri et al., 2011), but peroxidase activity undergoes a 3-fold increase in the presence of 20% ethanol (Table 2). Here, by tuning the concentration of a weak unfolding agent such as ethanol, it is possible to distinguish the peroxidase activity of the “molten-globule” form of ycc at pH 7, in which the ligand coordination set of the iron center remains almost unchanged, from that of a “helical form”. Previous data indicated that ycc and its Lys to Ala mutants interact with Kaol probably through electrostatic and hydrogen bonding interactions (Castellini et al., 2009). The biphasic behavior of the dependence of P.A. vs. ethanol content indicates formation of two distinct ethanolinduced forms of ycc, one at low concentrations and another at larger concentrations which is much less active. These results parallel those obtained for cytc in mixed methanol/water solutions (Bychkova et al., 1996). In fact, at low methanol concentrations, fluorescence and circular dichroism spectra indicated the presence of a compact, but flexible, form (molten-globule state) that maintains the secondary and, at least in part, also the tertiary structure. At high methanol concentration, however, the molten globule is transformed into a more helical state. It can therefore be concluded that the behavior of P.A. of ycc as a function of ethanol concentration can be ascribed to these two forms. To gain more information on the ethanol-induced conformational changes, the starting reaction rates (V0) were measured as a function of [H2O2]0 at Φeth = 20 and 40%. As shown above, at these medium compositions the prevailing form of ycc is molten-globule and helical state, respectively. Ycc was adsorbed on Kaol at pH 3.5 from both an aqueous solution and from mixed ethanol/water solutions (the same used then as working solutions). The data obtained were analyzed using the Michaelis–Menten model which fits the data very well (Fersht, 1998) (Fig. 3A and B, Table 2). For an enzyme in solution, Vmax depends on the starting concentration (Vmax = kcat[E]0, being kcat the turnover number, i.e. the maximum number of substrate moles converted into product per second and volume unit by the catalytic site of an enzyme (or a catalyst) for a unit enzyme concentration). In the present case, ycc is adsorbed and the value of Vmax is calculated per the number of protein moles adsorbed on kaolinite, which numerically coincides also with the turnover number. Thus, as the native folded ycc lacks any

peroxidase activity, Vmax depends on the fraction of unfolded ycc adsorbed on Kaol (Diederix et al., 2002). The catalytic mechanism of peroxidases also accounts for the reactivity of cytochrome c towards H2O2 (Scheme 1). The main steps of the process include the H2O2 entrance in the catalytic pocket to form compound I (cytc•Fe+ 4=O), through a reversible addition of H2O2 to the metal center (to form an hydroperoxo complex, compound 0) and the subsequent oxidation of the substrate (Gc) by compounds I (cytc•Fe+ 4=O) and II (cytcFe+4 = O) (Dunford, 2002; Ranieri et al., 2011). In such a way, the Michaelis constant KM (an index of the kinetic instability of the enzyme-substrate adduct) accounts for the affinity of H2O2 for the heme center (compound 0), while the maximum rate achieved by the system, Vmax, is a measure of the reactivity of the ferryl compounds (I or II) towards the Gc substrate. It is worth noting that the involvement of ferryl heme group in the peroxidase activity of cytochrome c has been recently demonstrated through spectroscopic and kinetics studies (Bischin et al., 2011; Lawrence et al., 2003). In the peroxidase enzymes, the formation of the hydroperoxo complex (compound 0) is favored by the onset of a hydrogen bond between H2O2 and a histidine or arginine residue of the protein (Jones and Dunford, 2005; Rodriguez-Lopez et al., 1996, 2001). Also in the present case a hydrogen bond involving the hydroperoxo group and an arginine in position 13 could occur and be taken as responsible for the stabilization of Compound 0. In 20% ethanol, the KM and Vmax values obtained by adsorbing ycc from aqueous and 20% ethanol solutions are quite different, while in 40% ethanol these values are nearly independent of the composition of the solution used to immobilize the protein (Table 2). This suggests that in 40% ethanol ycc is characterized by the same conformation both when it is immobilized on kaolinite from water and from mixed ethanol/water solution at Φeth = 40%. This is not the case for measurements in 20% ethanol. In this case the V0 vs. [H2O2] plots strongly differ (Fig. 3a) and the corresponding Michelis–Menten fits show that both KM and Vmax are very different (Table 2). The two curves strongly differ and the corresponding Michelis–Menten fits show that both KM and Vmax are very different. This indicates the presence of two different forms of ycc on the kaolinite surface. Conceivably, ycc in the conformation stable in 20% mixed ethanol solution remains almost unchanged after adsorption and subsequent contact with the same solution. Conversely, the protein adsorbed from the aqueous solution likely modifies to some extent its conformation when placed in contact with the mixed ethanol solution.

Scheme 1. Essential catalytic mechanism of peroxidases (Dunford, 2002). For the sake of clarity, axial ligation of the iron center has been omitted.


In this case, however, the structural constrains due to the interaction with the kaolinite surface force the structure evolution towards a conformation different from that obtained in the former case. The nature of these structural differences is as yet unknown. The DR-UV–Vis spectra indicate that the heme coordination does not change, therefore other changes in the catalytic pocket must occur. KM value for Kaol-ycc at 20% ethanol (34 mM) is higher than at 40% ethanol (23 mM) (Table 2). As at 20% ethanol the prevailing form of ycc is molten-globule, while it is prevalently helical at 40% ethanol (Bychkova et al., 1996), these results suggest that the catalytic pocket shows higher affinity for H2O2 when the protein is in the helical state. This form differs from the molten-globule because of a larger surface exposed to the solvent including the catalytic pocket. The decrease in KM in the helical state can therefore be tentatively related to an increased accessibility of the heme center to solvent (and to H2O2). Alternatively, differences in H-bond-induced stability of compound 0, due to different conformations of the catalytic pocket, can account for the observed differences in the KM values. Unfortunately, specific structural information concerning the molten-globule and helical forms (in particular in adsorbed state) are lacking. The Vmax values confirm the existence of different catalytically active species of ycc on kaolinite depending on the composition of the working and adsorption solutions (Table 2). In 20% ethanol the Vmax values for Kaol-ycc and Kaol-E20-ycc are much higher than those obtained in 40% ethanol for Kaol-ycc and Kaol-E40-ycc. This can tentatively be interpreted as follows. The accessibility of the metal center of ferryl compounds to guaiacol increases from water to 20% ethanol, for decreasing at higher ethanol concentrations. In the molten-globule structure, in fact, the catalytic pocket probably widens but still maintains a strong hydrophobic character that warrants a high accessibility and affinity for the guaiacol substrate. In the helical form, the extended opening of the catalytic pocket could increase solvent access, thereby reducing the affinity for the organic substrates (as Gc). This would induce a decrease in the reaction rate between Gc and the ferryl compounds, and consequently a decrease in Vmax. In 40% ethanol, Vmax is nearly the same for Kaol-ycc and Kaol-E40ycc (31,000 and 33,000 nMGc s−1 μmol−1 cyt ) and is also similar to Vmax obtained in water for Kaol-ycc (41,300 nMGc s−1 μmol−1 cyt ). This further suggests that the helical and the native states of ycc share a similar degree of hydrophilicity that decreases their affinity towards Gc with respect to the molten globule state. At the extreme ethanol concentrations (N 80%), the formation of oligomeric structures among ycc molecules (Hirota et al., 2010, 2012) could account for the loss of the peroxidase activity (Fig. 2). The results obtained by adsorbing directly the molten globule and helical forms, namely those for Kaol-E20-ycc and Kaol-E40-ycc, are noteworthy. In fact, while the helical form has almost the same catalytic properties when formed before and after adsorption, the molten globule shows remarkable differences (Table 2). The molten globule form obtained after adsorption shows a lower KM value (34 with respect to 82 mM), therefore a higher affinity of the heme center for hydrogen peroxide, and also a higher Vmax, (13,000 with respect to 8400 nMGc s−1 μmol−1 cyt ), thereby showing a greater affinity of the ferryl compounds for the organic substrate, Gc. The reasons for these differences are not clear. In both cases a molten globule form is obtained, but apparently the conformation and/ or adsorption geometry of the two forms is different. The geometry of the protein on a charged adsorbing surface is driven by the distribution of ionized residues and by the overall protein conformation. The adsorption process, therefore, reasonably exerts motional restrictions on the unfolding processes of ycc (Castellini et al., 2013). In this view, the immobilizing surface plays a key role. Consistently, it is known that that the adsorption properties of cytc changes considerably for kaolinite and montmorillonite (Castellini et al., 2009; Hristova and Zhivkov, 2015). Surfaces of phospholipid vesicles are known to drive cytochrome c into the molten globule state (Bychkova and Ptitsyn, 1993). An

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interesting but still open problem is the role of this form in the physiology of mitochondria. The condition experienced by cytochrome c at or in proximity of the surface of the inner mitochondrial membrane can be mimicked in simple artificial systems such as the one studied here, in which the protein is immobilized on the surface of kaolinite in a mixed water/alcohol solvents. The systems described in this work simulate both the denaturing actions suggested for membrane surfaces, one owing to the local increase in the electric field due to the membrane charge and the other related to the local decrease in the dielectric constant. Therefore, the fact that motionally-restricted cytc is imparted with a peroxidase activity modulated by both the electric field of the immobilizing surface and the solvent-induced conformational change of cytochrome c, is a relevant finding. This peroxidase activity is known to activate cellular apoptosis through oxidation of cardiolipin in cytochrome c/cardiolipin adduct (Basova et al., 2007; Kagan et al., 2005, 2009; Santucci et al., 2010; Ranieri et al., 2015b). 5. Conclusions Ethanol induces the formation of two forms of ycc adsorbed on kaolinite, which are imparted with different properties, in particular the peroxidase activity. These forms likely correspond to the molten globule and helical state, at low and high ethanol concentration, respectively. The results show that the catalytic activity is inversely proportional to the strength of adsorption. In fact, the molten globule state, which is adsorbed less firmly onto kaolinite with respect to the helical state (probably due to a less favorable positioning of the charged groups on the protein surface for the electrostatic interaction with the kaolinite surface), is more active in catalyzing the oxidation of guaiacol. The remarkably higher Vmax value shown by this form in the Michaelis– Menten model could be put in relation with an easier accessibility of the catalytic pocket to the guaiacol substrate. Indeed, dimensions, exposure to solvent and hydrophobicity of the catalytic pocket are found to be critical points for the catalytic properties of yeast cytochrome c immobilized on kaolinite. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clay.2015.10.012. Acknowledgments This work was performed with the financial support of the University of Modena and Reggio Emilia (Fondo di Ateneo per la Ricerca, FAR2010). References Basova, L.V., Kurnikov, I.V., Wang, L., Ritov, V.B., Belikova, N.A., Vlasova, I.I., Pacheco, A.A., Winnica, D.E., Peterson, J., Bayir, H., Waldeck, D.H., Kagan, V.E., 2007. Cardiolipin switch in mitochondria: Shutting off the reduction of cytochrome c and turning on the peroxidase activity. Biochemistry 46, 3423–3434. Bates, R.G., 1973. Determination of pH, Theory and Practice. Wiley, New York. Battistuzzi Gavioli, G., Borsari, M., Pellacani, G.C., Menabue, L., Sola, M., Bonamartini Corradi, A., 1988. Effectiveness of the cadmium(II) ion in promoting sulfonamide nitrogen deprotonation. 113Cd NMR, polarographic, and pH-metric investigations on the cadmium(II)-N-tosylglycinate and cadmium(II)-N-dansylglycinate systems in aqueous and methanolic solutions. Inorg. Chem. 27, 1587–1592. Battistuzzi, G., Borsari, M., Ferretti, S., Sola, M., Soliani, E., 1995. Cyclic voltammetry and 1 H-NMR of Rhodopseudomonas palustris cytochrome c2 pH-dependent conformational states. Eur. J. Biochem. 232, 206–213. Battistuzzi, G., Borsari, M., Rossi, G., Sola, M., 1998. Effects of solvent on the redox properties of cytochrome c: Cyclic voltammetry and 1H NMR experiments in mixed waterdimethylsulfoxide solutions. Inorg. Chim. Acta 272, 168–175. Battistuzzi, G., Borsari, M., Sola, M., Francia, F., 1997. Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c. Biochemistry 36, 16247–16258. Battistuzzi, G., Bortolotti, C.A., Bellei, M., Di Rocco, G., Salewski, J., Hildebrandt, P., Sola, M., 2012. Role of Met80 and Tyr67 in the low-pH conformational equilibria of cytochrome c. Biochemistry 51, 5967–5978. Bischin, C., Deac, F., Silaghi-Dumitrescu, R., Worrall, J.A.R., Rajagopal, B.S., Damian, G., Cooper, C.E., 2011. Ascorbate peroxidase activity of cytochrome c. Free Radic. Res. 45, 439–444.

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