Article pubs.acs.org/Langmuir
pH and Solvent H/D Isotope Effects on the Thermodynamics and Kinetics of Electron Transfer for Electrode-Immobilized Native and Urea-Unfolded Stellacyanin Antonio Ranieri,‡ Gianantonio Battistuzzi,† Marco Borsari,*,† Carlo Augusto Bortolotti,‡,§ Giulia Di Rocco,‡ and Marco Sola*,‡,§ †
Department of Chemical and Geological Sciences, and ‡Department of Life Sciences, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy § CNR-NANO Institute of Nanoscience, Via Campi 213/A, I-41125 Modena, Italy S Supporting Information *
ABSTRACT: The thermodynamics of Cu(II) to Cu(I) reduction and the kinetics of the electron transfer (ET) process for Rhus vernicifera stellacyanin (STC) immobilized on a decane-1-thiol coated gold electrode have been measured through cyclic voltammetry at varying pH and temperature, in the presence of urea and in D2O. Immobilized STC undergoes a limited conformational change that mainly results in an enhanced exposure of one or both copper binding histidines to solvent which slightly stabilizes the cupric state and increases histidine basicity. The large immobilization-induced increase in the pKa for the acid transition (from 4.5 to 6.3) makes this electrode−SAM−protein construct an attractive candidate as a biomolecular ET switch operating near neutral pH in molecular electronics. Such a potential interest is increased by the robustness of this interface against chemical unfolding as it undergoes only moderate changes in the reduction thermodynamics and in the ET rate in the presence of up to 8 M urea. The sensitivity of these parameters to solvent H/D isotope effects testifies to the role of protein solvation as effector of the thermodynamics and kinetics of ET.
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INTRODUCTION The study of the redox behavior of an electron shuttling metalloprotein immobilized on a solid electrode by means of a molecular spacer that ensures electrical contact and mimics molecular recognition with the physiological partner is a valuable approach for understanding the mechanism of biological ET.1−11 This approach would be particularly informative for those proteins which accomplish the electron exchange not as a diffusible shuttles but as components of a protein complex embedded into the cell membrane or attached to the cell surface. The latter is the case of the plant cupredoxin stellacyanin (STC) which exchanges electrons while covalently bound through a glycosyl-phosphatidyl-inositol anchor to the cell wall. Rhus vernicifera stellacyanin is a cupredoxin belonging to the subclass of the phytocyanins.12−17 Its physiological role is presently unknown, although hypotheses on its involvement in redox processes during primary defense and or lignin formation and cell to cell signaling during morphogenesis have been put forward.12,18−20 STC displays peculiar properties within the cupredoxin family and among ET metalloproteins in general. In particular, it contains a type-1 copper site with His2Cys coordination plus a strong fourth glutamine ligand, instead of the classical Met ligand, creating a distorted tetrahedral geometry.12,15,21 The site is accessible to solvent to a significant extent, contrary to most cupredoxins, as both binding histidines (His46 and His94) are solvent exposed.12,15,21 Moreover, © 2012 American Chemical Society
glycosylation of Asn60 shields the metal site making it accessible only to small-sized, likely nonprotein, physiological redox partners.12,21 The solvent exposed binding histidines are most likely involved in the ET pathway and the presence of the carbohydrate chain reasonably affect the kinetics of the ET process. Moreover, STC shows a remarkably lower E°′ value (+0.187 V vs NHE) than other blue copper proteins.12,22,23 In this work, we have studied the thermodynamics and kinetics of electron transfer for STC immobilized on a solid electrode coated with a hydrophobic self-assembled monolayer (SAM). To the best of our knowledge, this is the first electrochemical investigation of electrode-immobilized STC. The choice of a hydrophobic surface was dictated by the observation that STC binds more strongly to neutral than charged surfaces, yielding a high protein coverage. The study was also made at varying pH values. In fact, STC, like most cupredoxins, undergoes the so-called “acid transition”, in which one solvent-exposed metal bound histidine protonates and detaches from the Cu(I) ion at low pH. This protonation triggers a conformational change that leads to the establishment of a trigonal-planar metal coordination geometry and a dramatic increase in reduction potential that may suppress Received: August 3, 2012 Revised: September 23, 2012 Published: September 25, 2012 15087
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redox activity.24−28 The acid transition, which is fully reversible, may thus in principle act as a physiological redox switch in response to environmental changes in proton concentration. Here, we have studied the thermodynamics of the acid transition for immobilized STC, which is found to occur at much higher pH values compared to the solution protein as a result of a favored balance of bond breaking/formation processes. Moreover, the influence of urea-unfolding and the solvent H/D isotope effect on the E°′ value and heterogeneous ET rate have been investigated. A discussion in terms of molecular changes that influence the free energy of the oxidized and reduced states of the copper center and the kinetic parameters within the framework of the Marcus theory, respectively, is offered. Besides being attractive for basic research, investigation of immobilized cupredoxins and ET proteins in general would also have technological applications, especially for the development of protein-based molecular electronic devices.5,6,29
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dipped at exactly the same depth as for the measurements with adsorbed STC. CVs at variable scan rates (from 0.020 to 10 V s−1) were recorded to determine the heterogeneous ET rate constant (ks) for the adsorbed protein using the Laviron method.33 Effects of uncompensated cell resistance were minimized using the positivefeedback iR compensation function of the potentiostat, set at a value slightly below that at which current oscillations emerge.34 The ks values were averaged over five measurements and found to be reproducible within 6% which was taken as the error on these measurements. The reaction thermodynamics of Cu(II) to Cu(I) reduction for STC can be determined from variable-temperature E°′ measurements carried out using a “non-isothermal” electrochemical cell, in which the reference electrode is kept at constant temperature (21.0 ± 0.1 °C) whereas the half-cell containing the working electrode and the Vycor junction is under thermostatic control.35−37 With this experimental configuration, the reaction entropy for reduction of the oxidized protein (ΔS°′rc) is given by35−37
° ′ − Sox ° ′ = nF ΔSrc° ′ = Sred
dE°′ dT
(1)
thus, ΔS°′rc was determined from the slope of the plot of E°′ versus temperature which is linear under the assumption that ΔS°′rc is constant over the temperature range investigated. With the same assumption, the enthalpy change (ΔH°′rc) was obtained from the Gibbs−Helmholtz equation, namely as the negative slope of the E°′/T versus 1/T plot. The nonisothermal behavior of the cell was carefully checked by determining the ΔH°′rc and ΔS°′rc values of the ferricyanide/ferrocyanide couple.36,37
EXPERIMENTAL SECTION
Materials. Stellacyanin (STC) was isolated from R. vernicifera acetone powder (Saito, Osaka, Japan) according to a literature method.30,31 Decane-1-thiol (DT) (Sigma-Aldrich) was used without further purification. Buffers and sodium perchlorate were reagent grade (Sigma-Aldrich). Ultrapure water (MILLIQ) was used throughout. Electrochemical Measurements. Cyclic voltammetry experiments were performed with a Potentiostat/Galvanostat PAR mod. 273A, at different scan rates (0.02−10 V s−1) using a three-electrode cell for small volume samples (0.5 mL) under argon. A 1 mm-diameter polycrystalline gold wire was used as working electrode, and a Pt sheet and a saturated calomel electrode (SCE) were used as counter and reference electrodes, respectively. A Vycor (PAR) set ensured the electric contact between the SCE and the working solution. Reduction potentials (E°′) were calibrated against the MV2+/MV+ (MV = methylviologen) and ferrocene/ferrocenium couples under all experimental conditions employed in this work (see below) to make sure that the effects of liquid junction potentials were negligible. All values are referenced to the SHE. Prior to use the working electrode was treated in concentrated nitric acid for 10 min, flamed under oxidizing conditions, heated in concentrated sulfuric acid for 2 h and then in 2.5 M KOH for 4 h, and, after rinsing in water, soaked in concentrated sulfuric acid for 12 h. To minimize residual adsorbed impurities, the electrode was subjected to 15 voltammetric cycles between +1.5 and −0.3 V (vs SCE) at 0.1 V s−1 in 1 M H2SO4. Electrode coating with the decane-1-thiol (DT) SAM was done by dipping the cleaned electrode into a 1 mM ethanolic DT solution for 12 h at 4 °C. After rinsing with MILLIQ water, the SAM-coated electrode was subjected to 10 voltammetric cycles from +0.2 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. STC was adsorbed on the alkanethiol-coated gold electrodes by dipping the functionalized electrode into a 0.2 mM protein solution (checked spectrophotometrically, using an extinction coefficient value of 4200 M−1 cm−1 at 605 nm)32 in 5 mM Hepes pH 7 at 4 °C for 4 h. The CV experiments were carried out using the washed functionalized electrode in solutions at different pH values using sodium perchlorate (10 mM), plus 5 mM acetate (from pH 4 to 6.3), 5 mM HEPES (from pH 6.3 to 7.5), or 5 mM Tris (Tris-(hydroxymethyl)-aminomethane) (from pH 7.5 to 11) as base electrolytes. The experiments were performed at least in duplicate, and the E°′ values were found to be reproducible within ±0.002 V. The surface coverage was calculated from the overall charge exchanged by the protein (determined upon integration of the baseline-corrected anodic or cathodic peaks) and the area of the gold electrode. The latter was determined electrochemically by recording the CV of a standard aqueous solution of ferrocenium tetrafluoborate in a diffusion-controlled regime, in which the bare electrode was
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RESULTS STC was adsorbed on a polycrystalline gold electrode coated with a SAM of decane-1-thiol (DT) taking advantage of the hydrophobic interaction between the SAM and a nonpolar region of the protein. The CV response consists of a single well-defined signal arising from the quasi-reversible oneelectron reduction/oxidation process of the Cu(II)/Cu(I) couple (Figure 1). This signal persists in the presence of up
Figure 1. Cyclic voltammograms for R. vernicifera stellacyanin immobilized on a polycrystalline gold electrode coated with a SAM of decane-1-thiol in 5 mM Hepes buffer, 10 mM sodium perchlorate in H2O at pH 7, in the presence of varying urea concentration. Sweep rate: 0.05 V s−1, T = 20 °C. Solid line: 0 M urea; dotted line: 2 M urea; dashed line: 8 M urea.
to 8 M urea (Figure 1). The apparent E°′ values (determined as the average of the cathodic and anodic peak potentials) (Table 1) are independent of the potential scan rate, v, over the entire range studied (from 0.02 to 10 V s−1). Urea induces a concentration-dependent E°′ shift of approximately 0.010− 0.012 V (Figure 2). In particular, a cathodic shift is observed up 15088
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Table 1. Thermodynamic and Kinetic Parameters for the Electron-Transfer Process of R. vernicifera stellacyanin Adsorbed on a Gold Electrode Coated with a 1-Decanethiol SAM in the Presence of Varying Concentrations of Urea, and in D2Oa ks (s−1) urea (M)
E°′ (V)
0
0.143 0.152b 0.187c 0.140 0.135 0.134 0.131
2 4 6 8
ΔS°′rc (J K
−1
−54 −74b −22c −102 −115 −86 −62
−1
mol )
−1
ΔH# (kJ mol−1)
ln A
ΔH°′rc (kJ mol )
H2O
D2O
H2O
D2O
H2O
D2O
−29.6 −36.5b −25.0c −43.6 −46.8 −37.9 −30.8
33.2
15.8
13.7
14.8
9.1
8.8
33.9
n.d.
13.5
n.d.
9.1
n.d.
46.3
n.d.
18.2
n.d..
11.3
n.d
a
The stellacyanin-coated electrode was dipped in a working solution made up in 5 mM Hepes buffer plus 10 mM sodium perchlorate at pH 7.0. T = 20 °C. Average errors on E°′, ΔS°′rc, ΔH°′rc, ks, ΔH#, and ln A are ±0.002 V, ±2 J K−1 mol−1, ±0.3 kJ mol−1, ±6%, ±0.3 kJ mol−1, and ±0.1, respectively. bValues measured for immobilized stellacyanin in the same conditions but in D2O. cValues for freely diffusing stellacyanin in the same working solution in water (from ref 23).
Figure 2. Plot of E°′ for R. vernicifera stellacyanin immobilized on a polycrystalline gold electrode coated with a SAM of decane-1-thiol in 5 mM Hepes buffer, 10 mM sodium perchlorate in H2O at pH 7, versus urea concentration.
Figure 3. Plot of E°′ versus temperature for R. vernicifera stellacyanin immobilized on a polycrystalline gold electrode coated with a SAM of decane-1-thiol in 5 mM Hepes buffer, 10 mM sodium perchlorate in H2O (●) and D2O (○) at pH 7.
to 4 M urea, whereas no further E°′ changes occur at larger urea concentration (up to 8M). Urea also causes some current decrease due to the decrease in protein surface coverages (which were 1.1 × 10−11, 7 × 10−12, and 4 × 10−12 mol cm−2 at 0, 2, and >4 M urea, respectively). The current intensity is linearly dependent on the scan rate, as expected for a diffusionless electroactive species (Figure S1). Repeated cycling does not affect the voltammograms from 5 to 40 °C, indicating that the protein monolayer is stable. Above 40 °C, the currents decrease and signal distortion occurs due to protein desorption and/or unfolding, possibly accompanied by metal loss. The enthalpy and entropy changes of Cu(II) to Cu(I) reduction (ΔH°′rc and ΔS°′rc, where the subscript rc stands for “redox couple”) for immobilized STC, determined from variable temperature E°′ measurements22,35−37 in the presence of varying concentrations of urea, and in D2O (Figure 3), are listed in Table 1. The difference in E°′ for STC measured in H2O and D2O is small but larger than the experimental error (Table 1). Moreover, the E°′ values in D2O show a much larger temperature dependence than in H2O. The rate constants for heterogeneous ET (ks) between the electrode and immobilized STC have been determined from the scan rate dependence of the anodic and cathodic peak potentials, according to Laviron (Figure S2).33 The ks values increase with increasing urea concentration and decrease remarkably in D2O (Table 1). The activation enthalpy (ΔH#) values were determined according to the Arrhenius equation by measuring the temperature dependence of the rate constants from 5 to 40 °C with a nonisothermal cell (Figure 4). The ΔH#
Figure 4. Arrhenius plot for R. vernicifera stellacyanin immobilized on a polycrystalline gold electrode coated with a SAM of decane-1-thiol in 5 mM Hepes buffer, 10 mM sodium perchlorate at pH 7 in aqueous and D2O solution and in the presence of varying urea concentration. Solid lines are least-squares fits to the data points.
values increase in D2O and in the presence of urea (Table 1). The pH dependence of E°′ for immobilized STC is shown in Figure 5. Two acid−base equilibria are observed, one below pH 6 and the other above pH 9, which correspond to the so-called acid and alkaline transition of cupredoxins, respectively, due to redox-state dependent ionizations involving the copper binding histidines.24,31,32,38−41 Measurements could not be performed below pH 4 and above pH 11 because of the deterioration of the CV signal. The E°′/pH profile was fit to the following twoproton equilibrium equation (vide infra): 15089
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pK a =
ΔH °′AcT 1 ΔS°′AcT − 2.3R T 2.3R
(3)
Since for the acid transition it is generally accepted to refer to the protonation reaction,24,31,39,41 we here report the transition enthalpies and entropies ΔH°′AcT, and ΔS°′AcT with the sign change. Protonation of the copper binding histidine turns out to be exothermic and to occur with an entropy gain (Table 2). Evaluation of the same parameters for the alkaline transition was hampered by the worsening of the voltammetric responses at high pH upon increasing temperature.
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DISCUSSION Effects of Electrode Immobilization on the Reduction Thermodynamics of STC. The firm STC binding to the nonpolar electrode surface used here indicates the presence of a hydrophobic patch on the protein surface. As an efficient heterogeneous protein-electrode ET occurs (vide infra), this region likely surrounds the solvent-exposed Cu binding His46 and His94.21 Such an exposure would indeed facilitate reversible and fast ET between the electrode and the copper center which is located near the protein surface. The positive E°′ value for freely diffusing STC originates from the selective enthalpic stabilization of the cuprous state (ΔH°′rc = −25.0 kJ mol−1) which prevails on the entropic term (TΔS°′rc = −6.6 kJ mol−1) that would induce an opposite effect (Table 1). However, among cupredoxins, STC shows one of the lowest E°′ values due to the presence of the axial amide oxygen donor atom of a glutamine residue which favors Cu(II) and to the pronounced accessibility of the two Cu-binding His46 and His94site to solvent.21,22,42 The negative ΔS°′rc value has been proposed to originate mostly from the strengthening of the van der Waals and H-bonding interactions among the water molecules within the hydration shell of STC due to the increase of the negative protein charge following Cu(II) to Cu(I) reduction.22 The E°′ values for immobilized STC are about 40 mV lower compared to the freely diffusing protein under the same conditions (Table 1). Conceivably, the adsorption process (involving hydrophobic interactions) selectively stabilizes oxidized STC probably due to conformational, charge and solvent reorganization effects. In particular, the adsorption-induced partially compensative reduction enthalpy and entropy changes (Table 1) is the signature of the alteration of the reduction-induced changes in the network of hydrogen bonds at the protein−solvent interface.23,38,43 These in fact are known to induce exactly compensative ΔH°′rc and ΔS°′rc changes. As discussed previously for other electrode adsorbed metalloproteins and peptides,10,44−47 the resulting change in reduction free energy leading to the E°′ decrease is determined by the enthalpy-based stabilization of the oxidized protein. In this case, the adsorption-induced changes in the hydrogen bonding network surrounding the exposed copper binding His residues could be the result of an enhanced accessibility of the copper binding His to solvent which would favor Cu(II) and/or affect the extensive structural changes in the ligand environment of the metal center associated with copper reduction.12 Influence of Urea-Unfolding and Solvent-Isotope (HD) Effect on the Reduction Thermodynamics for Immobilized STC. The small E°′ decrease for STC upon increasing urea concentration up to 4 M (Figure 2) must be the result of moderate protein unfolding events that influence the free energy of the oxidized and reduced states of the copper
Figure 5. pH dependence of E°′ for R. vernicifera stellacyanin immobilized on a polycrystalline gold electrode coated with a SAM of decane-1-thiol in 10 mM sodium perchlorate plus 5 mM acetate (from pH 4 to 6.3), 5 mM HEPES (from pH 6.3 to 7.5), and 5 mM Tris (Tris-(hydroxymethyl)-aminomethane) (from pH 7.5 to 11) in H2O. Solid line is the best fit curve to eq 2).
E°′(pH) = E°′pH7 + −
RT ⎛ [H +] ⎞ ln⎜⎜1 + red ⎟⎟ F ⎝ K a1 ⎠
K ox ⎞ RT ⎛ ln⎜1 + a2+ ⎟ F ⎝ [H ] ⎠
(2)
in which E°′pH7 is the E°′ value in the plateau region (at pH 7), Kred a1 is the apparent (being measured at finite ionic strength) proton dissociation constant of the solvent exposed binding histidine of reduced STC, which is responsible for the low-pH ox equilibrium, and Ka1 is the apparent proton dissociation constant of the acid−base residue of oxidized STC responsible for the high-pH equilibrium (vide infra). The pKa values determined at 278 K for immobilized STC are listed in Table 2 Table 2. Thermodynamic Parameters for the Acid Transition for Immobilized and Solution R. vernicifera stellacyanin and Other Blue Copper Proteins pKred a1 immobilized R. vernicifera stellacyanina freely diffusing R. vernicifera stellacyanin P. versutus amicyanin cucumber plastocyanin spinach plastocyanin umecyanin CBP (cucumber basic protein)
pKox a2
ΔH°′AcT (kJ mol−1)
ΔS°′AcT (J K−1 mol−1)
6.2
10.4
−14.9
+67
4.5b
10.0c,d
−11e
+49e
−24e −41e −47e
+42e −54e −77e
+34e
+197e
6.4b 4.4e 4.2b 4.2b
9.5d 10.0d
a Values were obtained in 5 mM buffer solution (see the Experimental Section) plus 10 mM sodium perchlorate. T = 20 °C. Average errors on ΔH°′AcT and ΔS°′AcT values are ±0.6 kJ mol−1 and ±5 J mol−1 K−1, respectively. bFrom refs 24 and 39. cFrom ref 62. dFrom ref 31. eFrom ref 24.
along with the values determined previously for the same protein in solution and for other cupredoxins. The pH dependence of E°′ was measured at different temperatures in the range 5−40 °C. The pKa values for the acid transition yielded a linear van’t Hoff plot (Figure S3) from which the transition thermodynamics (ΔH°′AcT and ΔS°′AcT) were evaluated using the integrated equation 15090
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k 0 = ν0 exp[−ΔG # /RT ] = ν0 exp[−λ /4RT ]
center. Pseudomonas aeruginosa azurin (Az) and the purple CuA domain from Thermus thermophilus cytochrome oxidase in solution were previously found to undergo an increase in E°′ upon unfolding, attributed to a change in the coordination geometry of the copper center.48 In the immobilized state, however, Az showed a negligible E°′ change, implying that urea, also at high concentration, does not affect the coordinative properties of the metal.49 This must also be the case for STC under the present conditions. Therefore, the moderate stabilization of Cu(II) relative to Cu(I) up to 4 M urea is likely the result of changes in the H-bonding network and/or a larger exposure of the copper-binding His residues to solvent. The concomitant decrease in protein surface coverage up to 4 M urea indicates that these unfolding events involve residues at the protein/SAM interface. Indeed, from 0 to 4 M urea, both reduction enthalpy and entropy for STC become more negative (Table 1) and these compensatory effects are typical of changes in the reduction-induced solvent reorganization effects within the hydration sphere of the protein.23,38,43,50,51 We may speculate that an increased solvent exposure of the copper ligand His residues would enhance the ordering effect of the reduction-induced increase of the (negative) protein charge on the surrounding water molecules. This effect would be compensated by a larger enthalpy loss due the strengthened H-bonding interactions.22 At urea concentrations above 4 M, E°′ remains constant as a result of a fully compensatory increase in ΔH°′rc and ΔS°′rc, in a fashion opposite to that observed at lower denaturant concentration (Table 1). As also protein surface coverage is unchanged, it can be hypothesized that further protein unfolding occurs in regions not involved in the interactions with the SAM influencing reduction-induced solvent reorganization effects. The sensitivity of the ΔH°′rc and ΔS°′rc values to solvent H/ D isotope effects (Table 1) parallels that observed for azurin52 and cytochrome c53 and testifies the role of the reductioninduced changes in protein solvation as determinants of the reduction thermodynamics. The effects on the ΔS°′rc values may be due to small differences in thermal expansion of the protein in the two solvents and in the amplitude and frequency of collective normal modes of vibration of the protein and/or to a different sensitivity of the equilibrium configuration around the metal center to the changes of the electric field associated with the change in the oxidation state of the metal center.52 Influence of Urea-Unfolding and Solvent-Isotope (HD) Effect on the Kinetics of Heterogeneous ET for Immobilized STC. STC exhibits a very high electron transfer rate with small molecular weight species.12 However, the ks value of 33.2 s−1determined here for immobilized STC (Table 1) is distinctly lower than that determined previously for Az adsorbed on the same DT-coated Au electrode under the same conditions (ks = 510 s−1).11,54 This difference is due to the larger activation enthalpy for STC compared to Az (ΔH# = 13.7 and 9.0 kJ mol−1 for STC and Az, respectively). Possibly, the carbohydrate chain linked to Asn6021 which most likely lies at the protein/SAM interface allows for the presence of more water molecules, thus increasing the reorganization energy of the ET process and therefore ΔH#. According to the model proposed by Fujita et al.,55 the Marcus equation for STC immobilized on a hydrophobic surface is
(5)
In eq 4, rSTC is the intramolecular ET distance from the copper center to the protein surface facing the SAM; rint is the intermolecular distance between the protein surface and the SAM headgroup; (n + 3) corresponds to the number of bonds through the alkanethiolate H3C(CH2)nS- SAM chain (n = 9 for decane-1-thiol); βSTC, βint, and βSAM represent the tunneling factors for intramolecular ET through the protein, for the intermolecular ET through the interface, and for tunneling through the SAM, respectively. Moreover, ΔG# = λ/4 (λ is the reorganization energy) and ν0 is the frequency factor (kT/h = 6 × 1012 s−1). In case the activation entropy (ΔS#) is negligible,31,54,56 ΔH# would correspond to ΔG#. In this way, λ could be easily calculated as 4ΔH#. Under this hypothesis, a λ value of 0.57 eV would be obtained for immobilized STC in water solution at pH 7. However, this approach holds for adsorbed folded metalloproteins and organic molecules.54,56−58 In our case it cannot be applied as is owing to the presence of the glycosylate chain which could induce the presence of several water molecules at the protein/SAM interface. A waterrich interface can be responsible for ΔS# values different from zero. Nor it can be applied for the urea-unfolded protein. Therefore, here we restrict the discussion to the effect of urea and D2O on ks, ΔH#, and A (the pre-exponential factor corresponding to ν0 exp[−(βSTCrSTC + βintrint)] exp[−βSAM(n + 3)] exp[ΔS#/R]). All of these parameters are almost unchanged up to 4 M urea but increase remarkably beyond this limit (Table 1) in that paralleling the behavior of the reduction thermodynamics. Therefore, above 4 M urea, a specific conformational change in STC must occur that strongly influences the thermodynamics and kinetics of the protein− STC ET process. The urea-induced larger A values, which are responsible for the increase in the ET rate, cannot be explained safely. A is related to ΔS#, β, and r, and all of these parameters may in principle be affected by urea-induced unfolding. The increase in ΔH# could be related to the larger exposure of the copper binding histidines to solvent which may increase the reorganization energy λ of the ET process owing to a more pronounced oxidation-state dependent change in the Hbonding network connecting the water molecules in proximity of the metal site.50,51 The ks values sensibly decrease in D2O (Table 1) due to an increase in ΔH# (Table 1). This is most likely the result of a larger reorganization energy due to the change in the H(D)bonding network associated with ET, owing to the increased strength (by 2 kJ mol−1) of the hydrogen bond involving deuterium compared to protium.52,59 Acid−Base Equilibria for Immobilized STC. The low-pH equilibrium in the E°′/pH profile for immobilized STC corresponds to the well-known “acid transition” of cupredoxins in which Cu(II) reduction is coupled to a proton uptake. The E°′ values increase linearly upon lowering the pH (in this case below 6), with a slope of approximately 60 mV/pH (Figure 5). The residue responsible for the proton uptake is a solvent exposed metal binding histidine (either His46 or His94) which detaches from the Cu(I) ion triggering a conformational change leading to a trigonal planar Cu(I) metal coordination geometry.24−28 At intermediate pH values (from 6.5 to 9.5) no ionizable residues affect STC in both redox states. In the alkaline region (above pH 9.5), an additional proton loss occurs at experimentally accessible pH values only for the oxidized protein, as the pKa for the reduced form is much higher. It
ks = k 0 exp[−(βSTCrSTC + βint rint)] exp[ − βSAM (n + 3)] (4)
where 15091
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of urea do not result in relevant conformational changes, but only in an enhanced exposure of one or both copper binding His responsible for the moderate E°′ decrease and for the shift of the acid transition toward higher pH values. The latter is one of the most interesting outcomes of this work. In fact, this electrode-SAM-protein construct represents a robust hybrid interface able to switch ET on and off (by changing E°′ and therefore the driving force for ET) through a pH change near neutral pH, of interest for utilization in protein-based molecular electronic devices.63−67
follows that the deprotonated oxidized form would protonate again upon reduction, originating the high-pH equilibrium known as the “alkaline transition” of cupredoxins.31,32,60−62 Within this model, the pH dependence of E°′ has been fit to eq 2 which includes two proton uptakes, at low and high pH, coupled with the reduction of the oxidized protein. The agreement between experimental and calculated E°′ values is very good (Figure 5). It is worthy of note that the pKred a1 value for the copper binding His in immobilized reduced STC responsible for the acid transition is higher by almost two pH units compared to the corresponding pKa value for the freely diffusing protein (Table 2). Therefore, proton affinity of this residue increases upon interaction with the nonpolar SAM. This effect is likely to be put in relation with the fact that both binding His are probably located in proximity to the hydrophobic patch with which STC interacts with the SAM. Both transition enthalpy and entropy become more favorable for immobilized STC (Table 2). However, as noted elsewhere24,39,41 and analogously to the above discussion for immobilization-induced E°′ changes, the free energy of transition is ultimately controlled only by the features of the coordination of the copper ion and its immediate environment, which determine the protein-based enthalpy change. Immobilization does not change copper coordination (see above) but protein−SAM interaction could induce a localized conformational change in reduced STC that increases His basicity/solvent exposure and/or remove structural constraints to the transition of copper coordination from tetrahedral to trigonal planar upon reduction and His protonation. There is no clear evidence about the nature of the residue(s) responsible for the alkaline transition in cupredoxins. Studies of protein variants subjected to mutation of the axial copper ligand suggested a binding histidine as the deprotonated residue.31 Therefore, in oxidized STC, Cu(II) could induce a decrease in the pKa of one of the binding His determining the transition to a histidinate. Reduction to Cu(I) would result in an increase in pKa of the binding His and therefore in residue protonation. The immobilization-induced pKa increase, although modest (Table 1), would be consistent with the involvement of one of the copper binding histidines in the equilibrium as these are likely located in proximity of the protein-SAM interaction domain.
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ASSOCIATED CONTENT
S Supporting Information *
Plot of the current intensity as a function of the scan rate and van’t Hoff plot for the acid transition for immobilized R. vernicifera stellacyanin. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected];
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a grant from the Ministero dell’Università e della Ricerca (MIUR) of Italy (Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale 2009 Prot. No. 20098Z4M5E_002 (M.B.)).
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CONCLUSIONS R. vernicifera stellacyanin adsorbs firmly to a decane-1-thiol coated gold electrode and yields an efficient heterogeneous electron transfer. Immobilization occurs through hydrophobic protein−SAM interactions which must involve a nonpolar patch on the protein surface. The good voltammetric response (even though the rate of ET is much lower than other cupredoxins immobilized under the same conditions, such as azurin) indicates that this domain must be in proximity of the solvent-exposed binding copper binding histidines, which in general mediate ET in these systems. The presence of such domain is only inferred, as the three-dimensional structure of the protein is still unavailable. The carbohydrate chain of STC (which hampers protein crystallization and X-ray analysis) is most likely placed at the protein/SAM interface (as the site of attachment, Asn60, is near the exposed binding histidines) and probably slows down the rate of ET contributing to increase the hydration of the interface. Several lines of evidence indicate that immobilization on the electrode and subsequent addition 15092
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