His ligation switch.

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Effect of motional restriction on the unfolding properties of a cytochrome c featuring a His/Met–His/His ligation switch† Antonio Ranieri,a Carlo A. Bortolotti,ab Gianantonio Battistuzzi,c Marco Borsari,c Licia Paltrinieri,c Giulia Di Roccoa and Marco Sola*ab The K72A/K73H/K79A variant of cytochrome c undergoes a reversible change from a His/Met to a His/His axial heme ligation upon urea-induced unfolding slightly below neutral pH. The unfolded form displays a dramatically lower reduction potential than the folded species along with a pseudo-peroxidase activity. We have studied electrochemically the effects of urea-induced unfolding on the protein electrostatically immobilized on an electrode surface functionalized by means of a negatively charged molecular spacer. The latter mimics the electrostatic interaction with the inner mitochondrial membrane. This behavior has

Received 16th October 2013, Accepted 17th December 2013

been compared with the unfolding of the same species in solution. This system constitutes a model to

DOI: 10.1039/c3mt00311f

pH upon interaction with the membrane component phospholipid cardiolipin in the early stages of the

decipher the role of the above electrostatic interaction in the unfolding of cytochrome c at physiological apoptosis cascade. We found that immobilization obstacles protein unfolding due to structural constraints

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at the interface imposed by protein–SAM interaction.

Introduction The comprehension of the molecular events and the physical chemistry of biological electron transport (ET) owes much to the research on cytochrome c (cytc hereafter), a single centered, single chain heme protein.1–3 This small protein (10–14 kDa), thanks to the ease of purification and engineering, and stability under a variety of conditions, has long constituted one of the paradigms for the investigation of the functioning of respiratory chains in cells.4–7 Moreover, cytc has been the model protein for studying the coordinative and electronic properties of metalloporphyrins in biological matrixes,8–10 folding–unfolding processes,11–13 protein–protein interactions,14–16 as well as being the benchmark for the application of a variety of physico-chemical techniques to metalloproteins.17–21 More recently, cytc has proved to be a valuable constituent of functional interfaces for nanobiotechnological applications.22–25 In recent years, research on this protein has received further and new stimulus from its involvement in apoptosis as a consequence of the interaction a

Department of Life Sciences, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy. E-mail: [email protected]; Fax: +39-059373543; Tel: +39-0592055037 b CNR-NANO Institute of Nanoscience, via Campi 213/A, I-41125 Modena, Italy c Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy † Electronic supplementary information (ESI) is available. See DOI: 10.1039/ c3mt00311f

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with the negatively charged phospholipid cardiolipin (CL) of the inner mitochondrial membrane (IMM) and the induced peroxidase activity.26–35 The latter is the result of a perturbed protein folding involving rupture of the Met80–Fe(III) bond and ligand swapping by a Lys or His residue.31,36–42 The understanding of this unfolding event must include assessment of the role of molecular recognition and binding of cytc to CL in terms of involved residues and structural changes. Some important advancements have been made recently in this respect.26,32–41,43–45 However, full physico-chemical comprehension of the process should include the role played by the membrane in terms of electrostatic interaction and cytochrome c anchoring. This, in fact, suppresses/modifies the protein translational, rotational and vibrational degrees of freedom of the protein. It follows that spatial constraints are imposed to the conformational changes which influence the thermodynamic and kinetic parameters of the unfolding process and the resulting peroxidase activity. It has been shown that binding of cytochrome c to reconstituted membranes composed of negatively charged lipid depresses the rotational mobility of the lipid chains relative to that in fluid lipid membranes.46 Here, we study the mutual effect on the bound protein in terms of unfolding propensity. Disentangling the role of the interaction with the membrane in the unfolding event is not obvious and requires a tailored protein model system. With this in mind, here we have studied electrochemically the unfolding induced by a chemical denaturant (urea) on a variant of yeast iso-1-cytochrome c resulting in axial

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a Milli-Q Plus Ultrapure Water System coupled with an Elix-5 Kit (Millipore). The water resistivity was over 18 MO cm.


Electrochemical measurements

Fig. 1 Yeast iso-1-cytc showing the side chain of His73 (K73H mutation) as stick model and the approximate positions of Ala79 (K79A mutation) and Ala72 (K72A mutation). The heme cofactor is shown in magenta along with the Met80 and His18 heme axial ligands. The substructures of cytochrome c as defined in ref. 84 are shown from least to most stable in the colors gray, red, yellow, green, and blue.

iron ligand swapping (as for cytc interacting with CL) electrostatically immobilized on an electrode surface functionalized by means of a negatively charged molecular spacer. The latter would mimic the electrostatic interaction with IMM. This behavior was then compared with the urea-unfolding of the same variant in solution. In particular, we have focused on the K72A/K73H/K79A variant of yeast cytc (Fig. 1).22 This variant was chosen because urea-induced unfolding causes replacement of the axial Met iron ligand by a His residue (somewhat below neutral pH, a condition resembling that of the mitochondrial intermembrane space where cytc is located and in which protons are pumped from the mitochondrial matrix) which results in the appearance of a new voltammetric peak. Therefore this variant can be exploited as a redox probe for the comparative study of the unfolding event of cytc under diffusing and immobilized conditions. Further, this variant features only one substituting ligand (His73) in a large range of urea concentration (beyond which other His residues can be involved, vide infra)22,47–54 because the lysines which could serve as substituting heme iron ligands,31,33,34,43 are mutated to Ala. Therefore, the absence of competing ligands eliminates a potential interfering factor in the comparison between the unfolding properties in solution and in the immobilized state. All these properties make this triple variant a useful model system to understand if and to what extent an electrostatic interaction of cytochrome c with a molecular layer, inducing a restriction to protein mobility, affects the thermodynamics of unfolding and axial Met substitution.

Experimental Materials The K72A/K73H/K79A variant of yeast iso-1-cytochrome c was produced and purified following the procedure reported elsewhere.55 All chemicals were reagent grade. 11-Mercapto-1undecanoic acid (MUA) and 11-mercapto-1-undecanol (MU) were purchased from Sigma-Aldrich. Water was purified through


A potentiostat/galvanostat model 273A (EG&G PAR, Oak Ridge, USA) was used to perform cyclic voltammetry (CV) measurements. Experiments under both diffusive and nondiffusive (electrode-immobilized protein) regimes were carried out at different scan rates (0.02–5 V s1) using a cell for small volume samples (0.5 mL) under argon. A polycrystalline gold wire, a platinum sheet, and a saturated calomel electrode (SCE) were used as the working, counter, and reference electrodes, respectively. The electric contact between the SCE and the working solution was achieved with a Vycors (from PAR) set. Reduction potentials were calibrated against the MV2+/MV+ (MV = methyl viologen) and ferrocene/ferrocenium couples under all experimental conditions employed in this work to make sure that the effects of liquid junction potentials were negligible. All the reduction potentials reported here are referred to the standard hydrogen electrode (SHE). The working gold electrode was cleaned as reported elsewhere.56 Measurements in diffusive conditions were conducted using a gold electrode functionalized with 4-mercapto-pyridine. Electrode functionalization is reported elsewhere.57 Electrode coating with the MUA-MU SAM for electrostatic protein immobilization was obtained by dipping the polished gold electrode into a 1 mM ethanolic solution of both MUA and MU for 12 h and then rinsing it with water. The functionalized electrodes were subjected to 10 voltammetric cycles from +0.2 V to 0.4 V in a 0.1 M sodium perchlorate solution (outgassed with argon) to align the SAM. The resulting CV was taken as the background and checked for the absence of spurious signals. Protein solutions were freshly prepared before use in 5 mM phosphate buffer at pH 7 and their concentration was carefully checked spectrophotometrically (with a Jasco model V-570 spectrophotometer). Protein adsorption on the SAM-coated Au electrodes was achieved by dipping the functionalized electrode into a 0.2 mM protein solution made up in 10 mM sodium perchlorate and 5 mM phosphate buffer at pH 7, at 4 1C for 5 h. The CV experiments were carried out using a working solution containing 10 mM and 50 mM sodium perchlorate (for the immobilized and solution protein, respectively) as base electrolyte, plus 5 mM acetate and phosphate buffer at pH 5 and 7.4, respectively. Urea concentration was varied between 0 and 9 M. Since ions in solution influence the redox thermodynamics and kinetics of cytochrome c also in the adsorbed state and the strength of anion binding can be redoxstate dependent,58 measurements have been carried out under the same conditions in terms of buffers and ionic composition, as specified above. For the protein under both diffusive and electrode-immobilized regimes, the formal potentials Eo 0 were calculated from the average of the anodic and cathodic peak potentials and were found to be almost independent of scan rate in the range 0.02–5 V s1. The experiments were performed at least twice and the Eo 0 values were found to be reproducible within 0.002 V. The current intensities for the immobilized protein are linearly dependent on the scan rate, as expected for

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a diffusionless electroactive species (see for example Fig. S1, ESI†). The surface coverage G0 for the immobilized protein was calculated from the overall charge Qtot exchanged by the protein (determined upon integration of the baseline-corrected cathodic peaks) and the area A of the gold electrode by applying the relationship: Ð i(V) dV = n(nFAG0) = nQtot (1) where n is the sweep rate (in V s1), n (= 1) is the number of electrons exchanged in the redox center half reaction, and F is the Faraday constant. The area of the electrode was determined electrochemically by applying the Randles–Sevçik relationship to the CV signal obtained for aqueous solutions of ferrocenium tetrafluoroborate of known concentration in a diffusion-controlled regime, in which the bare electrode was dipped at exactly the same depth as for the measurements with the adsorbed protein. A coverage of 18.5 0.8 pmol cm2 was determined,59 which was almost constant with increasing urea concentration. Cyclic voltammograms at variable scan rate were recorded to determine the rate constant ks for the interfacial electrochemical ET process for the adsorbed protein, according to Laviron.60 Effects of uncompensated cell resistance were minimized using the positive-feedback iR compensation function of the potentiostat, set at a value slightly below that at which current oscillations emerge.61 The ks values were averaged over five measurements and found to be reproducible within 6%, which was taken as the associate error. The separation between the anodic and the cathodic peak increases with increasing scan rate as is expected for systems adsorbed in both oxidation states, while the Eo 0 values remain unchanged. The ks values were also measured at 5, 10, 15, 20, 25, 35 1C to determine the activation enthalpies (DH #) using the Arrhenius equation, namely from the slope of the plot of ln ks versus 1/T. The CV experiments at different temperatures for both diffusive and electrode-immobilized regimes were carried out with a cell in a ‘‘non-isothermal’’ setting, namely in which the reference electrode was kept at constant temperature (21 0.1 1C) whereas the half-cell containing the working electrode and the Vycors junction to the reference electrode was under thermostatic control with a water bath.62–64 The temperature was varied from 5 to 50 1C. With this experimental configuration, the standard entropy change for Fe(III) to Fe(II) cytochrome c o0 is given by:62–64 reduction DSrc o0 dE o0 o0 o0 (2) DSrc ¼ Sred Sox ¼ nF dT 0

o was determined from the slope of the plot of Eo0 versus thus, DSrc temperature which turns out to be linear under the assumption 0

o is constant over the limited temperature range investithat DSrc 0 gated. With the same assumption, the enthalpy change DHrco was obtained from the Gibbs–Helmholtz equation, namely as the negative slope of the Eo0 /T versus 1/T plot. Repeated cycling does not affect the voltammograms from 5 to 35 1C, indicating that the protein monolayer is stable. The nonisothermal behavior of o0 o0 the cell was carefully checked by determining the DHrc and DSrc 63,64 values of the ferricyanide/ferrocyanide couple.

876 | Metallomics, 2014, 6, 874--884

Paper

Results The voltammetric responses and the temperature dependence of E o 0 for the Fe3+/Fe2+ couple of the triple K72A/K73H/K79A variant of yeast iso-1 cytc (‘protein’ hereafter) immobilized on a polycrystalline gold electrode coated with a MUA/MU SAM and in freely diffusing conditions were studied as a function of urea concentration in the working solution at pH 5 and 7.4. Urea does not affect the properties of the MUA/MU SAM under the conditions employed. This is shown by the capacitance curves collected for the SAM-functionalized electrode in the presence of urea over the whole concentration range investigated (0–8 M), which are identical. The CVs for the immobilized protein at pH 5 and 7.4 in the absence of denaturant consist of a single voltammetric peak typical of an electrochemically quasi-reversible process with an E o0 value of +0.240 (HPpH5 signal) and 0.208 V (LPpH7 signal), respectively (Fig. 2A and B, Table 1) (despite removal of three Lys residues, immobilization of the present triple variant is ensured by other Lys residues on the cytc surface; Lys 13, 27, 86, 87 and 88 for the oxidized protein plus Lys22 and 25 for the reduced protein have been shown to be involved in the electrostatic binding of cytc to carboxyalkanethiol SAMs).65–67 It has been shown previously that the above signals correspond to the heme iron featuring a His/Met and a His/His axial ligation, respectively, the latter due to the replacement of Met80 by His73 as the ‘sixth’ ligand with a pKa value of 5.7 (immobilized) and 6.2 (diffusing).22,59 For both signals, the cathodic and anodic peak currents are linearly dependent of the scan rate (Fig. S1, ESI†) and their ianodic/icathodic ratio is about one for all temperatures and scan rates investigated, as expected for an electrochemical response originating from a protein in the adsorbed state. At pH 5, addition of urea at concentrations above 1 M causes the appearance of a new cathodic peak at negative potentials (LPpH5 signal) (Fig. 2C and 3, Table 1). This signal shows the anodic counterpart only at sweep rates (n) higher than 0.2 V s1. At n = 0.5 V s1, the anodic and cathodic currents become comparable. No new signals appear for the protein at pH 7.4 upon urea addition up to 9 M. Only the LPpH7 signal undergoes a limited shift to more negative values (Table 1, Fig. 3A). The LPpH5 signal, arising from an adsorbed species as well (Fig. S1, ESI†), shows E o 0 values and reduction thermodynamics (vide infra) almost coincident with those of the LPpH7 signal at the same urea concentration (Table 1, Fig. 3A). Therefore these two signals must correspond to the same protein form. Moreover, the current intensity of the LPpH5 signal increases with increasing urea concentration to the detriment of that of the initial HPpH5 signal which disappears at 9 M urea, while the sum of the two currents remains nearly constant (Fig. 3B). As a result, it is apparent that urea-induced protein unfolding causes axial heme ligand swapping from Met to His. This occurs at a lower pH than in the absence of denaturant. In particular, as currents of the HPpH5 and LPpH5 match at 5.2 M urea (Fig. 3B), the apparent pKa for His deprotonation and heme Fe(III) binding at this urea concentration is 5 (i.e. the same as the pH of the working solution). Compared to 0 M urea, the pKa decreases by about 0.7 pH units.22 The E o0 value of the HPpH5 signal undergoes a cathodic shift upon increasing


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dependent of urea concentration (Fig. S2, ESI†); the resulting o0 thermodynamic parameters for protein reduction (DSrc and 0


o ) are listed in Table 1. DHrc The cathodic peak current intensity (i) profiles for the HPpH5 and LPpH5 signals (Fig. 3B) due to the urea-induced conversion of the His/Met ligated form to a His/His form at pH 5 allow the free energy of unfolding in the presence (DGou) and absence (DGuo H2 O ) of denaturant, to be determined for the protein under both regimes. As shown elsewhere,47 for a simple two-state denaturation process these two terms are related by the equation:

DGou ¼ DGuo H2 O m½urea

(3)

where m is a parameter proportional to the increase in the solvent-exposed surface area of the denatured state compared to the native state of the protein. Therefore, smaller m values correspond to a larger degree of residual structure in the denatured state.47,68,69 DGou in turn can be calculated from the equilibrium constant of the denaturation process at a given urea concentration, Ku: DGou = RT ln Ku

(4)

where Ku is given by: Ku = aprotein

unfolded/aprotein folded

E [protein unfolded]/

[protein folded] = i LPpH5/i HPpH5

Fig. 2 Cyclic voltammograms for the K72A/K73H/K79A variant of yeast iso-1-cytochrome c adsorbed on a polycrystalline gold electrode coated with a SAM of MUA/MU in the presence of varying urea concentrations. (A) 0 M urea, pH 7.4; (B) 0 M urea, pH 5; (C) 8 M urea, pH 5. Working solution: 10 mM phosphate (pH 7.4) or acetate (pH 5) buffer, plus 10 mM sodium perchlorate. Sweep rate: 0.5 V s1, T = 20 1C.

urea concentration (about 50 mV), while the LPpH5 signal (measured at n = 0.5 V s1) is much less affected (16 mV) (Table 1, Fig. 3A). The latter shows the same E o0 values and thermodynamics of reduction (vide infra) as the HPpH7 signal (Table 1, Fig. 3A). The CV response of the protein under freely diffusing conditions is qualitatively very similar to that of the immobilized protein in terms of pH-dependent urea effects. The ureadependent cathodic current intensity for the LPpH5 and HPpH5 signals match at 3.1 M urea (Fig. 3B). The slopes of the Eo 0 vs. T plots for both signals at pH 5 and the signal at pH 7.4 for the protein under both diffusive and immobilized conditions are


(5)

The plots of DGou versus urea concentration for the immobilized and freely diffusing protein along with wt cytc and the K72A/K73A/K79A variant in the same conditions (calculated using data from ref. 70) are shown in Fig. S3 (ESI†). According to eqn (3), DGuo H2 O and m are obtained from the intercept and the slope of the least-square linear fit of the data points, respectively. Values are listed in Table 2. The rate constants for the heterogeneous electron transfer process are comparable with those for wt cytc and their Lys to Ala variants immobilized on carboxyl-terminated SAMs,25,71–75 and decrease with increasing urea concentration (Table 3). The activation enthalpy (DH#) and the pre-exponential factor values were determined according to the Arrhenius equation by measuring the temperature dependence of the ks values from 5 to 35 1C with a non-isothermal cell at different urea concentrations (Fig. 4, Table 3). The pseudoperoxidase activity of the immobilized His/Hisligated form at pH 7.4 in the presence of 8 M urea has been measured following the electrocatalytic waves generated upon addition of increasing concentrations of hydrogen peroxide, as reported previously22,59 and analyzed within a classical Michaelis– Menten scheme (Fig. 5, Table 4). jcat = jmax[H2O2]/KM + [H2O2]

(6)

where jcat is the electrocatalytic current density (taken as the ratio of the difference between the cathodic peak current in the presence and in the absence of a given concentration of hydrogen peroxide and the electrochemically determined area of the electrode), jmax is the maximum current density at substrate saturation and KM is the Michaelis constant.

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Table 1 Thermodynamic parameters for the electron-transfer process for the K72A/K73H/K79A variant of yeast iso-1-cytochrome c adsorbed on a polycrystalline gold electrode coated with a SAM of MUA/MU and in freely diffusing conditions in the presence of varying urea concentrationsa

HPpH5 signal

LPpH5 signal


o0 c

o 0 b,c

(V)

o0 c

LPpH7 signal o0 c

DHrc (kJ mol1)

DSrc (J mol1 K1)

E

o 0 b,c

(V)

o0 c

0

DHrc (kJ mol1)

DSrc (J mol1 K1)

E

o 0 b,c

(V)

0

oc DHrc (kJ mol1)

oc DSrc (J mol1 K1)

[Urea]

E

Adsorbed 0 1 2 3 4 5 6 7 8 9

+0.240 +0.237 +0.234 +0.230 +0.218 +0.198 +0.192 +0.190 — —

34.9 34.9 35.2 36.3 38.6 40.2 41.4 42.4 — —

40 41 43 48 60 72 78 82 — —

— 0.216 0.217 0.217 0.218 0.218 0.220 0.227 0.232 0.233

— 24.1 24.1 24.2 24.0 24.0 29.1 36.0 42.6 43.0

— 11 11 11 10 10 27 48 69 70

0.208 0.210 0.213 0.215 0.216 0.216 0.218 0.224 0.236 0.237

35.0 31.3 27.3 25.0 24.0 24.2 28.2 33.5 42.8 43.1

50 38 23 15 11 11 24 41 68 69

Solution 0 1 2 3 4 5 6 7 8 9

+0.257 +0.255 +0.245 +0.228 +0.215 +0.205 +0.198 +0.195 — —

44.1 44.7 46.2 47.6 48.8 50.7 51.6 52.4 — —

66 68 77 88 96 106 111 115 — —

— 0.189 0.191 0.194 0.196 0.201 0.204 0.207 0.208 0.209

— 25.4 25.2 25.6 25.9 28.1 31.1 40.3 44.1 44.8

— 24 23 23 24 30 40 69 82 84

0.186 0.187 0.189 0.191 0.193 0.196 0.196 0.203 0.208 0.209

35.8 35.7 35.0 34.0 33.2 33.1 33.3 39.0 44.1 44.8

61 60 57 53 50 48 49 66 82 84

a The HPpH5, LPpH5 and LPpH7 signals refer to the His/Met form at pH 5 and the His/His form at pH 5 and 7.4, respectively. The protein-coated electrode was dipped in a working solution made up in 5 mM acetate (at pH 5) or phosphate (at pH 7.4) buffer plus 10 mM sodium perchlorate. b o0 o0 T = 20 1C. c Average errors on Eo 0 , DSrc , DHrc are 0.002 V, 2 J K1 mol1, 0.3 kJ mol1, respectively.

Discussion o

Unfolding-induced E changes for cytochrome c Cytochrome c can be unfolded by denaturing agents in solution (acid pH,76 urea,70 GdCl47) or upon interaction with (mainly hydrophobic) surfaces.72,77 In all cases, the main effect on the metal center is the loss of Met 80 resulting in an Eo 0 decrease as the new axial heme iron ligand stabilizes the oxidized state. In fact, a His residue serves as the replacing ligand upon urea70 and GdCl47 unfolding, while this role is played by an exogenous OH ion or H2O molecule or an endogenous histidine or lysine residue for cytochrome c bound to PGE77 or hydrophobic SAMs.72 The differences in Eo 0 for unfolded immobilized cytc in these cases are due to a different axial ligation and/or a different heme exposure to solvent, (the conformational change is driven by the nature of the unfolding agent) along with a different protein adsorption geometry. In fact the different surfaces impose peculiar constraints to the adsorbed protein in terms of orientation and nature of the binding interaction. Changes in the thermodynamics of reduction for K72A/K73H/ K79A cytc due to urea-induced unfolding At pH 5, the urea-induced substitution of the HPpH5 signal with the LPpH5 signal for both solution and immobilized K72A/ K73H/K79A cytc parallels that observed previously for wt cytc at pH 7.70 Therefore, this is most likely the result of the opening of the 71–85 O loop (Fig. 1) and substitution of the axial methionine ligand to Fe(III) with a histidine residue.70 Such change in heme

878 | Metallomics, 2014, 6, 874--884

coordination due to unfolding was studied previously by Bowler for the single K73H variant by spectroscopic means.47 The engineered His73 was proposed to bind at low denaturant concentration yielding a native-like intermediate, whereas His26, 33 and 39 likely replace it at larger denaturant concentrations.47 The anodic counterpart for the LPpH5 signal is observed only at relatively large potential scan rates. This is because ligand replacement is disfavored for the reduced form owing to the larger affinity of the Met thioether sulfur for Fe(II) compared to the imidazole nitrogen of His. Consequently, the reduced His/His heme converts with time to the reduced His/Met form. The kinetics of His to Met ligand switching have been studied using the Laviron model for an electrochemical reaction followed by a first order chemical reaction (see ESI,† p. 4).78 A rate constant value, k, of 0.28 0.01 s1 has been obtained, which is remarkably lower than that for the Lys to Met switching measured for the reduced alkaline form of cytc from various sources (0.98–1.05 s1 for bacterial cytc,79 60 s1 for yeast cytc and 30 s1 for horse cytc80). The Eo 0 values for the His/Met and His/His forms for the immobilized protein are shifted cathodically by a few mV compared with those for the diffusing protein under the same conditions (Table 1). This is due to stabilization of the more positively charged oxidized form by the negatively charged SAM, as is largely documented in the literature.71,73 The high-potential HPpH5 signal corresponding to the His/Met ligated heme center shows negative reduction enthalpy and entropy values because of the enthalpic stabilization of the reduced heme by the sulfur atom of the axial Met ligand and the hydrophobic environment


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Table 2 Thermodynamic parameters of urea-induced protein unfolding for wt and the Lys to Ala/His variants of yeast iso-1-cytochrome c adsorbed on a polycrystalline gold electrode coated with a SAM of MUA/ MU and in freely diffusing conditionsa

Protein

DGuoH2 O b,c (kJ mol1)

mb,c (kJ mol1 M1)

Adsorbed wtd K72A/K73H/K79A K72A/K73A/K79Ad

13.4 7.3 11.3

3.2 1.6 2.9

Solution wte K72A/K73H/K79A K73He

20.8 12.7 15.2

6.9 4.2 5.1

a

The protein-coated electrode was dipped in a working solution made up in 5 mM acetate buffer at pH 5 plus 10 mM sodium perchlorate. b T = 5 1C. c Average errors on DGuo H2 O and m are 0.8 kJ mol1 and 10% (relative error), respectively. d From ref. 70. e From ref. 47. Data were collected in 5 mM Tris/10 mM NaCl buffer at pH 7.4. Errors on DGuo H2 O for wt and K73H are 1.7 and 0.6 kJ mol1, respectively.

Table 3 Kinetic parameters for the electron-transfer process for the K72A/K73H/K79A variant of yeast iso-1-cytochrome c adsorbed on a polycrystalline gold electrode coated with a SAM of MUA/MU in the presence of varying urea concentrationsa

HPpH5 signal

LPpH5 signal

LPpH7 signal

ksb,c ksb,c ksb,c DH# c DH# c DH# c 1 c 1 1 c 1 1 c 1 [Urea] (s ) ln A (kJ mol ) (s ) ln A (kJ mol ) (s ) ln A (kJ mol )

Fig. 3 (A) Eo 0 values for the His/Met ligated form at pH 5 (HPpH5 signal) (K,J), His/His ligated form at pH 5 (LPpH5 signal) (’,&) and His/His ligated form at pH 7.4 (LPpH7 signal) (m,n) for the K72A/K73H/K79A variant of yeast iso-1-cytochrome c adsorbed (open symbols) on a polycrystalline gold electrode coated with a SAM of MUA/MU and freely diffusing (closed symbols) in the presence of varying urea concentrations. (B) Relative current intensity (cathodic) for the HPpH5 signal (K,J) and LPpH5 signal (’,&) for the K72A/K73H/K79A variant of yeast iso-1-cytochrome c adsorbed (open symbols) on a polycrystalline gold electrode coated with a SAM of MUA/MU, and freely diffusing (closed symbol), at pH 5, as a function of urea concentration. Lines are simply drawn through the points. Working solution: 10 mM phosphate (pH 7.4) or acetate (pH 5) buffer, plus 10 mM sodium perchlorate. Sweep rate: 0.5 V s1, T = 20 1C.

and because of reduction-induced solvent reorganization effects, respectively.79,81 The Eo 0 value of this form decreases by about 50–60 mV upon increasing urea concentration from 0 to 8 M under both regimes (Table 1). Invariably, the corresponding 0

0

o changes in DHrco and DSrc are in part compensative (indeed, both terms become more negative, see Table 1). Reduction-induced solvent reorganization effects are known to be perfectly compensative.82,83 Therefore, the resulting decrease in Eo 0 is likely to be due to an enthalpic effect related to the increased polarity of the heme environment. This is consistent with the increased exposure of the heme center to solvent due to the urea-induced breaking of the H-bonding network that stabilizes the ‘yellow’ O foldon responsible for the hydrophobicity of the heme crevice,84 as shown previously for wt cytc.70 This is prodromal to axial ligand


0 2 4 7 8 9

8.75 2.95 1.87 0.59 — —

5.54 4.61 4.32 4.28 — —

8.2 8.6 9.0 11.7 — —

— 0.96 0.95 0.95 0.94 0.92

— 4.50 4.51 4.53 4.50 4.52

— 10.9 10.9 11.0 11.1 11.2

1.75 0.88 0.89 0.91 0.90 0.90

4.11 5.23 6.57 5.65 4.49 4.48

8.6 13.2 16.4 14.0 11.2 11.2

a The HPpH5, LPpH5 and LPpH7 signals refer to the His/Met form at pH 5 and the His/His form at pH 5 and 7.4, respectively. The protein-coated electrode was dipped in a working solution made up in 5 mM acetate (at pH 5) or phosphate (at pH 7.4) buffer plus 10 mM sodium perchlorate. b T = 20 1C. c Average errors on ks, ln A and, DH # are 6% (relative error), 0.3 and 0.3 kJ mol1, respectively.

swapping. We note that the urea-induced Eo 0 change for the HPpH5 signal for the protein in solution is somewhat larger and occurs at lower urea concentration than the immobilized protein (Fig. 3A). This indicates that immobilization obstacles urea-induced protein unfolding, in agreement with the more quantitative data gained from the peak currents discussed below. The LPpH5 and LPpH7 signals corresponding to the same His/ His ligated species that appear at pH 5 above 1 M urea and at pH 7.4, respectively, show a lower urea-induced change in Eo 0 compared to the His/Met form under both regimes (Table 1, Fig. 3A). However, as above most of the Eo 0 change is shifted toward larger urea concentration (between 5 to 8 M) compared to the solution species. Moreover, the Eo 0 /[urea] profile is different (Fig. 3A) suggesting that a peculiar unfolding pathway occurs for the immobilized His/His protein. The negative Eo 0 value for the LPpH5 and LPpH7 signals is the result of positive reduction enthalpy and entropy values. The enthalpic term is mostly due to the stabilization of the ferric heme by the axial

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Fig. 5 Plot of the catalytic current vs. hydrogen peroxide concentration for the K72A/K73H/K79A variant of yeast iso-1-cytochrome c adsorbed on a polycrystalline gold electrode coated with a SAM of MUA/MU, in the presence of urea. (J) pH 5, 8 M urea; (&) pH 7.4, 8 M urea; (’) pH 7.4, 4 M urea. Working solution: 10 mM phosphate (pH 7.4) or acetate (pH 5) buffer, plus 10 mM sodium perchlorate. Sweep rate: 0.5 V s1, T = 20 1C. Solid lines are the best fit curves to the Michaelis–Menten equation.

Table 4 Pseudo-peroxidase activity for the folded and unfolded K72A/ K73H/K79A variant of yeast iso-1-cytochrome c adsorbed on a polycrystalline gold electrode coated with a SAM of MUA/MUa,b

Protein

KMc (mM)

jmaxc,d (mA cm2)

jmax/KM (mA mM1 cm2)

Folded (0 M urea), pH 7.4 Unfolded (4 M urea), pH 7.4 Unfolded (8 M urea), pH 7.4 Unfolded (8 M urea), pH 5

0.95e 0.96 0.60 0.59

0.5e 4.0 2.7 2.8

0.5 4.2 4.5 4.7

0.1 0.3 0.5 0.5

a

The protein-coated electrode was dipped in a working solution made up in 5 mM acetate or phosphate buffer plus 10 mM sodium perchlorate at pH 5 and 7.4, respectively. b T = 20 1C. c Average errors on KM and jmax are 0.04 mM1 and 0.1 mA cm2, respectively. d jmax is the maximum catalytic current density. e From ref. 22.

Fig. 4 Arrhenius plots for the K72A/K73H/K79A variant of yeast iso-1cytochrome c adsorbed on a polycrystalline gold electrode coated with a SAM of MUA/MU, in the presence of varying urea concentrations: 0 M (J), 2 M (K), 4 M (&) 7 M (’), 8 M (n), 9 M (m). (A) HPpH5 signal, (B) LPpH5 signal; (C) LPpH7 signal. Solid lines are least-squares fits to the data points.

His which has replaced the Met ligand, and the solvent accessibility of the heme center. The entropy gain upon reduction is indeed typical of heme centers exposed to solvent, due to the reduction-induced decrease of solvent ordering following the charge change of the heme center from +1 to 0.79,81–83 The Eo 0 value is almost invariant up to 5 M urea (Table 1, Fig. 3A), once again as a result of a solvation-related compensatory change (decrease) of the reduction thermodynamics, which in this case is almost perfect, indicating that the changes in reduction thermodynamics upon increasing urea concentration are dominated by changes in solvent reorganization effects, as expected for an increasingly unfolded structure with the heme center

880 | Metallomics, 2014, 6, 874--884

more exposed to solvent. The conformational changes must affect mostly the oxidized form, as the reduced protein does not undergo significant unfolding up to 6 M urea.79 Above 5 M urea, the reduction thermodynamics increase, opposite to above, and induces a somewhat larger Eo0 decrease (Table 1, Fig. 3A). This behavior, and particularly reduction of the solvent-related H/S compensation, indicates that above 5 M urea the protein conformational change is larger both in solution and in the immobilized state. This observation is consistent with the substitution of His73 by another His residue as an axial iron ligand at a high denaturant concentration proposed previously.47 Effect of electrostatic protein–SAM interaction on the thermodynamics of urea-induced protein unfolding The changes in Eo 0 values due to urea-induced unfolding for both the His/Met and His/His forms are very similar under immobilized and diffusing regimes. Therefore, the structural changes in the vicinity of the heme center are independent of whether the protein is free or restricted on a surface. However, it is worthy of note that signal swapping due to Met for His


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substitution occurs at larger urea concentration for the immobilized protein compared to the solution state. In fact, the urea-dependent cathodic currents (Fig. 3B) show that the populations of the oxidized His/Met and His/His forms for the immobilized protein match at about 5.2 M urea, against 3.1 M urea for the protein in solution. Analogous behaviors were found previously for wt cytc at pH 7 (3.9 M vs. 3.5 M) and for the triple K72A/K73A/K79A variant at pH 5 (3.5 M vs. 3.2 M).70 Therefore, protein immobilization on MUA/MU inhibits the unfolding effect of urea due to structural constraints at the interface imposed by the electrostatic protein–SAM interaction (which by itself has no major consequences on cytc conformation, particularly in the heme environment).85–88 The much larger increase in denaturant concentration needed for unfolding for the present variant with respect to the other two species also tells us that this effect is dependent of protein surface features. Based on these results, it can be hypothesized that the binding mode of cytochrome c to IMM is optimized to yield the needed combined effect of structural and dynamic changes due to specific binding to CL and resistance to unfolding due to membrane binding.89 From the above, it follows that the thermodynamics of protein unfolding (Table 2) cannot be interpreted easily. In fact, the DGuo H2 O values for the immobilized proteins are smaller compared to those for the diffusing species indicating that immobilization would result in a lower intrinsic thermodynamic stability of cytc and a larger propensity to unfolding. One possible explanation for this conflicting result is that the mechanism of unfolding for the immobilized proteins changes compared to the solution state and the DGuo H2 O values are not directly comparable. Indeed, evidence has been gained that the confinement of the protein along with its hydration sphere on a surface results in unfolded state(s) different from those in solution.90 In particular, the confined solvent affects the unfolding thermodynamics allowing the folded and unfolded states of the immobilized protein to experience energy levels different from those in solution. The main determinants of the free energy change are solvent reorganization effects at the protein–solvent interface that tend to maximize solvent entropy.90 This is not surprising if we consider that the protein is motionally constrained and subjected to an electric field (at the electrode as well as at IMM). This is consistent with the abovementioned different profile of the ureainduced Eo0 changes for both immobilized His/Met and His/His forms compared to the solution species (Fig. 3A). Influence of urea-unfolding on the kinetics of heterogeneous ET and pseudoperoxidase activity for immobilized K72A/K73H/K79A cytc The changes in the kinetics of heterogeneous cytc-electrode ET between the His/Met and His/His forms help recognizing differences between these two species. The rates are indeed sensitive to protein conformation and solvent accessibility of the heme center.70,91 As shown by Bowden et al., the ks values obtained with the Laviron method can be assumed to correspond to the kinetic constant for ET at zero driving force.73,92,93 Therefore, the Marcus equation for heterogeneous ET:94 kET = n0 exp[b(r r0)] exp[(FZ + l)2/(4lRT)]


(7)

(where Z is the applied overpotential, the other parameters have the usual meaning and the signs + and refer to the reduction and oxidation reaction respectively), in conditions of DGo 0 = FZ = 0, assumes the form: ks = n0 exp[b(r r0)] exp[l/(4RT)]

(8)

ks = n0 exp[b(r r0)] exp[DG#/(RT)]

(9)

DG# = l/4

(10)

Since:

we obtain:

For folded cytochromes c the activation entropy is considered negligible,73,95 and therefore DG# is assumed to correspond to DH#. However, unfolding of immobilized cytochrome c induces removal of structural constraints, an increased access of solvent to the prosthetic center and the presence of water molecules at the protein/SAM interface.96 All this will likely lead to a significant DS# value (water molecules at the protein/SAM interface are known to play a dominant role as determinants of DS#).97 Therefore, the above assumption is no longer justified. It follows that the reorganization energy l for the immobilized unfolded protein and the electron donor–acceptor distance r cannot be calculated by such an approach. The Marcus equation could be rewritten as: ks = n0 exp[b(r r0)] exp(DS#/R) exp[DH#/(RT)]

(11)

From above, here we must restrict the discussion to the urea-induced unfolding effects on ks, the pre-exponential factor A (= n0 exp[b(r r0)] exp(DS#/R)) and DH# (Table 3). For the His/Met ligated form at pH 5, the ks values decrease upon increasing urea concentration, due to unfavorable changes of both ln A and DH#. The main determinant here is the increase in activation enthalpy that may be put in relation to the increase in outer-sphere reorganization energy due to the increased solvent exposure of the heme center to solvent following unfolding. In contrast, the ks values for the His/His ligated form at pH 7.4, after an initial decrease from 0 to 2 M urea, are independent of urea concentration. This is the result of opposite changes in ln A and DH# which both increase up to 4 M urea and then decrease at larger urea concentrations (Table 3). This behavior parallels the urea-dependence of the reduction thermodynamics for this form (Table 1). Therefore the increase in activation enthalpy up to 4 M urea can be due to the opening of the heme center to solvent, whereas above 5 M urea it could tentatively be ascribed to a protein conformational change including substitution of His73 heme iron ligand by another His residue that decreases solvent exposure and/or yields a more favorable tunneling pathway. With the data at hand it is not possible to assess the influence of the entropic terms and of changes in the tunneling factor b on the concomitant changes in ln A. We finally note that the kinetic parameters for the His/His forms at pH 5 and 7.4 coincide (Table 3) thereby confirming that these forms represent the same species, as indicated by the reduction thermodynamics.

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Hydrogen peroxide turnover (pseudo-peroxidase activity) of the immobilized His/His ligated forms (Table 4) will likely include the following steps:59 cytFe(III)–His + e - cytFe(II)–His

(12)

cytFe(II)–His + H2O2 - cytFe(IV)QO(His)uncoord + H2O (13) cytFe(IV)QO(His)uncoord + e + 2H - cytFe(III)–His + H2O (14) Published on 19 December 2013. Downloaded by York University on 30/06/2014 10:40:37.

+

The catalytic efficiency for the unfolded species above 4 M urea is the same independent of the pH (as are the reduction thermodynamics and the rate of heterogeneous ET) and urea concentration, and much larger than that of the folded protein at pH 7.4. While the latter effect is clearly related to the larger accessibility of the heme to solvent due to unfolding, the invariance of the jmax/KM values from 4 to 8 M urea is intriguing. This effect is the result of the perfect balance between a decrease in KM and a decrease in jmax. In particular, at pH 7.4 the kinetic affinity of hydrogen peroxide for the reduced heme center is nearly the same from 0 to 4 M urea probably because the bound His which dissociates is the same. jmax increases remarkably because of a faster reduction of the ferryl group and/or reattachment of the His. At 8 M urea, the decrease in KM is consistent with a change in the His residue coordinated to the heme iron, as suggested above by the urea dependence of Eo 0 and DH#. This residue could bind the heme center more weakly, which therefore would feature a larger affinity for the substrate. While jmax could decrease because of a slower His re-association caused by a larger solvent exposure of the site. The latter could also cause a slower ET rate and therefore a slower reduction of the ferryl group. Also, a slower proton transfer may be involved.

Conclusions The K72A/K73H/K79A variant of yeast iso-1 cytochrome c undergoes an unfolding-induced change in axial heme ligation from His/Met to His/His and a resulting pseudo-peroxidase activity at pH values near neutrality. It does so both under freely diffusing conditions and electrostatically immobilized on a gold electrode coated with a negatively charged SAM. These properties make it a suitable model for studying the role of membrane bindinginduced restriction of protein motional freedom on the unfolding process of cytochrome c that occurs upon interaction with IMM where it specifically binds CL in one of the initial events of apoptosis. Here we found that protein unfolding at the surface is inhibited to some extent by the electrostatic interaction with the SAM-coated electrode. Such an immobilization-induced effect is modulated by the charge properties of the cytochrome c surface. Therefore we may expect that in vivo the specific effect of the electrostatic interaction with the negatively charged membrane is to strengthen to some extent the folded conformation, in a surface- and therefore orientation-dependent manner. It follows that the membrane probably plays an active role in influencing

882 | Metallomics, 2014, 6, 874--884

the cytc propensity to unfolding needed for the progress of the apoptosis cascade.

Acknowledgements This work was supported by a grant from the Ministero ` e della Ricerca (MIUR) of Italy (Programmi di dell’Universita Ricerca Scientifica di Rilevante Interesse Nazionale 2009 prot. n. 20098Z4M5E_002 (MB)).

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In-Cell Enzymology To Probe His-Heme Ligation in Heme Oxygenase Catalysis.

Rate related block in the canine His-Purkinje system after ligation of the anterior septal artery--observation by His bundle electrocardiography.

"His turn".

Second degree His-Purkinje block during his bundle pacing.

Adler and His Psychology: Seen through His Early Memories.

His-bundle bigeminy evidenced by short his-ventricular interval.

Re: Bach and His.

His name is Alton.

His father's Oldsmobile.

Allison-And His Parents.

Draffin and his rods.

Man and his genes.

Thomas Bewick--his mark.

Bach and His.

Heimlich defends his maneuver.

His bundle electrocardiography.

Bigger than his bite.

Engelmann and his laddergram.

Atrio-His bundle tracts.

It is in his eyes.

Louis Joblot and his microscopes.

A psychiatrist moves his office.

Harvey's account of his "discovery".

Otto Ullrich and his syndromes.