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Factors Affecting the Electron Transfer Properties of an Immobilized Cupredoxin Stefano Monari,† Gianantonio Battistuzzi,† Christopher Dennison,*,‡ Marco Borsari,† Antonio Ranieri,† Michal Jan Siwek,† and Marco Sola*,†,§ Department of Chemistry, UniVersity of Modena and Reggio Emilia, Via Campi 183, 41125 Modena, Italy, CNR-INFM National Center nanoStructures and bioSystems at Surfaces - S3, Via Campi 213/A, I-41125 Modena, Italy, and Institute for Cell and Molecular Biosciences, Medical School, Newcastle UniVersity, NE2 4HH Newcastle upon Tyne, United Kingdom ReceiVed: October 21, 2010
°′ °′ The ionic strength (I) dependence of the reduction thermodynamics (E°′, ∆Hrc , and ∆Src ) and the kinetics of electron transfer (ET) for Pseudomonas aeruginosa azurin (AZ) adsorbed on a gold electrode coated with alkanethiolate SAMs has been investigated between pH 4.5 and 10.5 by cyclic voltammetry. The change in the reduction thermodynamics with I (sodium perchlorate) adheres to the Debye-Hu¨ckel model and allows the charges of the two redox states of AZ to be determined at different pH values. From pH 4 to 8 the protein charges are in agreement with those calculated considering the protonation states of the noncoordinating His35 and His83 residues and highlight that a single phosphate ion binds to both redox states of AZ, most likely at Lys122. A composite, Lys-based, equilibrium occurs at higher pH values, involving the loss of five protons at pH 10.5. The reduction thermodynamics extrapolated to zero I shows that the largely buried His35 dominates the electrostatic effects on E°′ for the equilibrium at around pH 7, whereas the residues involved in the high pH effect are more solvent exposed. At pH 10.5, the ET rate constants for AZ on all investigated SAMs are lower than the corresponding values at pH 4.5, probably due to a decrease in the tunneling efficiency at the AZ-SAM interface in terms of electronic coupling. It is suggested that Lys122 plays a distinctive role in this effect.
Introduction The investigation of electron transfer (ET) between a solid electrode and an immobilized redox metalloprotein can be highly informative.1,2 Such systems may, to some extent, mimic the physiological conditions in which the protein operates (e.g in proximity or embedded in a membrane) and can imitate the interaction with a redox partner. Insight can therefore be gained into the thermodynamics and kinetics of ET and the coupling with proton exchange processes or protein conformational changes, that occur in vivo and upon redox catalysis.1-7 Furthermore, the rational design and fabrication of hybrid interfaces can be exploited in nanostructured bioelectronic devices, from FET-like transistors to biosensors.8-10 In this study we have focused on the redox properties of the blue-copper protein azurin (AZ) adsorbed via hydrophobic interactions on a gold electrode coated with alkanethiol self-assembled monolayers (SAMs). Previous studies show that such an assembly gives rise to fast protein-electrode ET.11-19 In AZ, the copper is strongly coordinated by two histidines and a cysteine, in an approximately trigonal arrangement, with weak axial interactions from a methionine and a peptide carbonyl oxygen.20,21 The interaction of this biological redox shuttle with physiological partners occurs through a hydrophobic patch close to the type 1 (T1) copper site (Figure 1) through which the partially solvent-exposed His117 ligand protrudes. This His residue is also the most likely conduit for effectively * Corresponding authors. (M.S.) Tel: +39 059 2055037. Fax: +39 059 373543. E-mail:
[email protected]. (C.D.) Tel: +44 191 222 7127. Fax: +44 191 222 7424. E-mail:
[email protected]. † University of Modena and Reggio Emilia. ‡ Newcastle University. § CNR-INFM National Center - S3.
Figure 1. Space-filling representations of the two opposite faces of Pseudomonas aeruginosa AZ.20 The side containing the copper binding loop (Cys112-Met121) and that opposite are shown in (A) and (B), respectively. Acidic residues are colored red, while basic residues are blue, polar residues are light brown, nonpolar residues are white, the copper-binding loop (Cys112-Met121) is yellow, His117 is green, and Lys122 is purple.
channeling electrons between an electrode and the metal center.22 The electrochemistry and mechanism of long-range interfacial ET of AZ on SAM-modified gold have been thoroughly investigated. Particular focus has been put on investigating the effects exerted on E°′ and the ET kinetics by the chain length of the SAM molecule,14,17-19 pH,13,16,18 I,12,16 the nature of the SAM headgroup,17 and urea-induced unfolding.18 Insight into the mechanism of SAM-mediated protein-electrode ET has been gained, which was exploited as a model system to experimentally and theoretically analyze the dependence of the ET regime on donor-acceptor distance.19
10.1021/jp110096a 2010 American Chemical Society Published on Web 11/29/2010
Reduction Thermodynamics and Kinetics of Azurin In this work we have investigated how the redox and ET properties of immobilized AZ respond to changes in ionic composition, pH, and temperature. In particular, we have performed a detailed investigation of the effects exerted by the shielding of the protein surface charge by the ionic environment and by specific protein-ion interactions on the thermodynamics (measured here for the first time for immobilized AZ) and kinetics of heterogeneous ET at different pH values. This approach, based on Debye-Hu¨ckel theory,23 and applied from pH 4 to 10.5, allows the protein charges (related to the oxidation and protonation states) to be determined across this wide range of pH. Novel information has been extracted about the pHdependent conformers of the immobilized protein, their ionbinding properties and their ET efficiencies. We have also investigated how the charge change at pH 10.5 affects the activation parameters and the mechanism of ET. This has been achieved by measuring the kinetics of ET at different donor-acceptor distances, using SAMs of alkanethiols of variable chain length,16,17,19 and comparing the data with those determined at pH 4.5.19 Experimental Methods Materials. Recombinant wild type Pseudomonas aeruginosa azurin (AZ) was isolated and purified as described elsewhere.24 Pentane-1-thiol (PT), decane-1-thiol (DT), and hexadecane-1thiol (HDT) (Sigma-Aldrich) were used without further purification. Buffers and sodium perchlorate were reagent grade (SigmaAldrich). Ultrapure water (MILLIQ) was used throughout. Electrochemical Measurements. Cyclic voltammetry (CV) experiments were carried out with a Potentiostat/Galvanostat PAR mod. 273 A at different scan rates using a cell for small volume samples (0.5 mL) under an argon atmosphere. A 1 mmdiameter polycrystalline gold wire was used as the working electrode and a Pt ring and a saturated calomel electrode (SCE) as counter and reference electrodes, respectively. Electrical contact between the SCE and the working solution was maintained with a Vycor (PAR) set which was washed in an ultrasonic pool for about 5 min prior to use. Reduction potentials [E°′ values calculated as E°′ ) (Epa + Epc)/2] were calibrated against the MV2+/MV+ couple (MV ) methylviologen),25 and all values reported here are referenced to the standard hydrogen electrode (SHE). Prior to use the working electrode was treated in concentrated nitric acid for 10 min, flamed under oxidizing conditions and heated 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. The SAMs of PT, DT, and HDT were obtained by dipping the polished electrode into a 1 mM ethanolic solution of the alkanethiol for 12 h. After rinsing with MILLIQ water, the SAM-coated electrode was subjected to 10 voltammetric cycles from +0.2 to -0.4 V (vs SCE) in 100 mM sodium perchlorate to align the SAM, set the background and check for the absence of signals. AZ solutions, freshly prepared before use, were made up in 5 mM N-[2hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (Hepes) buffer at pH 7 and their concentration was checked spectrophotometrically (ε ) 5100 M-1 cm-1 at 628 nm).26 AZ was adsorbed on the alkanethiol-coated gold electrodes by dipping the functionalized electrode into a 0.2 mM protein solution in 5 mM Hepes pH 7 for 5 h at 4 °C. The CV experiments were carried out immersing the washed functionalized electrode in solutions at different pH values and ionic strength (I), varied using sodium perchlorate (from 5 to 150 mM) in the presence of I ) 5 mM
J. Phys. Chem. C, Vol. 114, No. 50, 2010 22323 acetate (from pH 4 to 6.3), I ) 5 mM tris(hydroxymethyl)aminomethane (Tris) (from pH 6.3 to 10.5), and I ) 15 mM phosphate buffer (at pH 7.7 and 8.7) (vide infra). The experiments were performed at least in duplicate and the E°′ values were found to be reproducible within (2 mV. The surface coverage was calculated from the overall charge exchanged by the protein (determined upon integration of the baselinecorrected 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 dipped at exactly the same depth as for the measurements with adsorbed AZ. CVs at variable scan rates (from 0.020 to 60 V s-1) were recorded to determine the heterogeneous ET rate constant (ks) for the adsorbed protein using the Laviron method.27 The ks values were averaged over five measurements and found to be reproducible ((6%) which was taken as the error on these measurements. The CV experiments at different temperatures were carried out with a “non-isothermal” cell in which the reference electrode was kept at a constant temperature (21.0 ( 0.1 °C) whereas the half-cell containing the working electrode and the Vycor junction to the reference electrode was kept under thermostatic control with a water bath. The temperature was varied from 5 to 45 °C. With this experimental configuration, the reaction entropy for reduc°′ ) is given by28-30 tion of the oxidized protein (∆Src ◦′ ◦′ ∆S◦′ rc ) Sred - Sox ) nF
dE°′ dT
(1)
°′ thus, ∆Src was determined from the slope of the plot of E°′ versus temperature which is linear under the assumption that °′ is constant over the temperature range investigated. With ∆Src °′ ) was obtained the same assumption, the enthalpy change (∆Hrc from the Gibbs-Helmholtz equation, namely as the negative slope of the E°′/T versus 1/T plot. The nonisothermal behavior °′ and of the cell was carefully checked by determining the ∆Hrc °′ 29,30 ∆Src values of the ferricyanide/ferrocyanide couple.
Results The CV response of adsorbed AZ has been measured at different temperatures, I values, scan rates and pH values on a DT-coated gold electrode. Moreover, at pH 10.5 measurements were extended to AZ immobilized on PT and HDT SAMs. Typical CVs of AZ adsorbed on DT SAM are shown in Figure 2. The signals consist of single well-defined peaks arising from a remarkably efficient quasi-reversible one-electron reduction/ oxidation process from the T1 copper center of the adsorbed protein. The peak-to-peak separation varies from 5 to 120 mV over the scan rates investigated (at 20 °C) and is also influenced by temperature and I (vide infra). Peak currents increase linearly with increasing scan rate, as expected for an adsorbed species (not shown), and are approximately constant from 5 to 30 °C. At higher temperatures, decreases in the observed currents, most likely due to protein desorption, were observed. Between 5 and 30 °C, the CV responses of AZ under varying solution conditions are reproducible and persist for several cycles. The E°′ values were independent of the scan rate and the surface coverage was 11.1 ( 0.9 pmol cm-2, and was almost unaffected by temperature and the ionic composition of the medium. This value is comparable to that determined previously under analogous conditions (10 ( 5 pmol cm-2).11 At pH 10.5, the CV signals of AZ immobilized on PT, HDT, and DT SAMs
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Figure 2. Cyclic voltammograms (scan rate 0.05 V s-1) of AZ adsorbed on a gold electrode coated with a DT SAM at 20 °C. Data obtained in a I ) 65 mM solution at pH 6.2 (made up of I ) 5 mM acetate and I ) 60 mM sodium perchlorate; solid line) and in a I ) 10 mM solution at pH 8.6 (made up of I ) 5 mM Tris and I ) 5 mM sodium perchlorate; dotted line).
Monari et al.
Figure 4. E°′ versus T plots for AZ adsorbed on a gold electrode coated with a decane-1-thiol SAM at I ) 15 mM (I ) 10 mM sodium perchlorate plus I ) 5 mM buffer) at pH 5 (acetate buffer) (b), pH 7.7 (Tris) (O), pH 8.7 (Tris) (4), pH 10.5 (Tris) (9), and pH 7.7 (I ) 15 mM, phosphate buffer only) (1). Solid lines are least-squares fits to the data points and error bars are smaller than the symbols.
TABLE 1: Reduction Thermodynamics for AZ Freely Diffusing and Immobilized on a Gold Electrode Coated with DT at Different pH Values pH immobilized azurinb
freely diffusing azurinc
5.0 6.4 7.7 8.7 10.5 5 7 8
°′ a °′ a E°′ (V)a ∆Src ∆Hrc (vs SHE) (J mol-1 K-1) (kJ mol-1)
0.332d 0.311d 0.272d 0.232d 0.201d 0.338e 0.307e 0.286e
-8 -10 -14 -23 -25 -65 -65 -65
-34.3 -33.1 -30.4 -29.1 -26.7 -52 -49 -47
Figure 3. pH dependence (20 °C) of E°′ for AZ adsorbed on a gold electrode coated with a decane-1-thiol SAM. CV measurements were carried out in a I ) 15 mM solution (made up of I ) 10 mM sodium perchlorate plus I ) 5 mM acetate or Tris, see Experimental Section). Error bars are smaller than the symbols.
a °′ °′ Errors on E°′, ∆Src , and ∆Hrc are (0.002 V, (2 J mol-1 K-1, and (0.3 kJ mol-1, respectively. b Working solution: I ) 15 mM (I ) 10 mM NaClO4 + I ) 5 mM Tris or acetate). c I ) 0.1 M phosphate (from ref 32). d Experiments performed at 20 °C. e Experiments performed at 25 °C.
are alike, although the shortest SAM yielded larger currents and smaller peak-to-peak separations. The pH dependence of E°′ for adsorbed AZ between pH 4.0 and 10.5 (Figure 3) is similar to previously reported data for the freely diffusing and immobilized protein.13,16,18,24,31 AZ has been used for many years to study the effect of protonation of the surface His35 and His83 residues.32,33 The sigmoidal titration pattern centered at around pH 7 is typical of an acid-base equilibrium affecting both oxidation states of the protein. The pKa values obtained from a fit of these data to a conventional one-proton equilibrium equation (pKa values of 6.3 ( 0.1 and 7.4 ( 0.1 for oxidized and reduced, AZ, respectively, fit not shown) match very closely those obtained from electrochemical studies of freely diffusing AZ31 and values determined by NMR for His35 in the two oxidation states of the protein.34 We therefore assign this effect primarily to His35. His83, which is situated further from the copper site, will have some influence with NMR-determined pKa values of 7.5 and 7.7 for oxidized and reduced AZ, respectively.34 For immobilized AZ pKa values of 7.0-7.3 and 7.7-7.9 have been determined for His83 in the Cu(II) and Cu(I) proteins, respectively.13 The extent of the influence of His83 protonation on E°′ in our experiments is unclear but the agreement between experimental and calculated charges at pH 7.7, in which we have assumed deprotonation of both surface His residues, is very good (vide infra). A second titration step is observed above pH 8, which reaches a plateau at approximately pH 10. At these pH values previous reports show a somewhat less pronounced decrease in E°′.16,18 This E°′
vs pH change is likely due to ET being coupled to the acid-base equilibria of Lys residues in both redox states of the protein. As noted earlier,13 above pH 8 the heterogeneous ET of adsorbed AZ is coupled to additional acid-base equilibria. This is supported by the determination of the protein charges obtained from the dependence of E°′ on I, as described below. The E°′ versus T profiles for adsorbed AZ at pH 5.0, 7.7, 8.7, and 10.5 obtained at I ) 15 mM are shown in Figure 4. These pH values were chosen for the determination of the reduction thermodynamics and of the protein charges (from the dependence of E°′ on I, as described below) because under these experimental conditions they correspond to distinct AZ protonation states. In particular, at pH 5.0 His35 and His83 are protonated in both the oxidized and reduced proteins.34 pH 7.7 corresponds to the inflection point between the low- and highpH equilibria (Figure 3). Therefore this represents the pH value in which reduced AZ features the highest population of deprotonated surface His residues (vide supra) and is still insensitive to the high-pH equilibrium. Finally, pH 10.5 is the highest pH at which AZ, that experiences further residue deprotonation(s) (vide infra), yields a good electrochemical signal. The E°′ values invariably show a monotonic linear decrease with increasing temperature in the 5-30 °C range. Table 1 lists the reduction thermodynamics of adsorbed AZ at different pH values at a fixed I, along those from experiments for freely diffusing AZ under comparable conditions. Table 2 reports the
Reduction Thermodynamics and Kinetics of Azurin
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TABLE 2: Reduction Thermodynamics for AZ Immobilized on a Gold Electrode Coated with DT at Different pH and I (Obtained with Sodium Perchlorate) Values pH 5.0 (acetate)b
7.7 (Tris)b
8.7 (Tris)b
10.5 (Tris)b
[NaClO4] (mM)
I (mM)
E°′ (V)a,c (vs SHE)
°′ a ∆Src (J mol-1 K-1)
°′ a ∆Hrc (kJ mol-1)
5 10 30 60 150 5 10 30 60 150 5 10 30 60 150 5 10 30 60 150
10 15 35 65 155 10 15 35 65 155 10 15 35 65 155 10 15 35 65 155
0.332 0.332 0.331 0.312 0.298 0.272 0.272 0.273 0.274 0.275 0.233 0.232 0.232 0.233 0.233 0.198 0.201 0.204 0.210 0.213
-3 -8 -12 -15 -16 -6 -14 -15 -17 -18 -17 -25 -26 -28 -29 -16 -25 -25 -28 -30
-33.0 -34.3 -35.5 -34.5 -33.7 -28.1 -30.4 -30.6 -31.2 -31.7 -27.4 -29.9 -30.1 -30.6 -30.9 -23.8 -26.9 -27.0 -28.5 -29.3
°′ °′ Errors on E°′, ∆Src , and ∆Hrc are (0.002 V, (2 J mol-1 K-1, -1 and (0.3 kJ mol , respectively. b Ibuffer ) 5 mM, taking into account the protonation state at this pH. c Experiments performed at 20 °C. a
TABLE 3: Reduction Thermodynamics for AZ Immobilized on a Gold Electrode Coated with DT at Different I Values (Obtained with Sodium Perchlorate) in Phosphate Buffer (I ) 15 mM), pH 7.7 [NaClO4] (mM)
I (mM)
E°′ (V)a,b (vs SHE)
°′ a ∆Src (J mol-1 K-1)
°′ a ∆Hrc (kJ mol-1)
0 20 50 140
15 35 65 155
0.251 0.254 0.256 0.258
-25 -26 -28 -30
-31.6 -32.1 -32.8 -33.5
Errors on E°’, ∆S°’rc and ∆H°’rc are (0.002 V, ( 2 J mol-1 -1 K and (0.3 kJ mol-1, respectively. b Experiments performed at 20 °C.
reduction thermodynamics measured at different perchlorate concentrations in Tris and acetate buffer at pH 5.0, 7.7, 8.7, and 10.5. In Table 3 the thermodynamic parameters measured at different perchlorate concentrations in phosphate buffer at pH 7.7 are listed. °′ °′ , and ∆Hrc values for freely diffusing and The E°′, ∆Src electrode immobilized metalloproteins have been found to be influenced by the nature and concentration of the electrolyte(s) in solution.23,35-39 The data in Table 2 demonstrate that this is also the case for immobilized AZ, whose reduction thermodynamics are affected by the ionic strength of the solution in contact with the electrode. This can be attributed to the surface charge of the two redox states of the protein being shielded to a different extent by the “ionic atmosphere”. In particular, the redox state of the protein bearing the larger charge is selectively stabilized with a consequent change in E°′. Applying the extended Debye-Hu¨ckel equations for the activity coefficient of the two protein redox states in the Nernst equation, the following E°′ versus I relationship is obtained23,40,41 0.5√I RT [z 2 - zox2] nF red 1 + 0.33a√I
protein, and a is the “ion-size parameter”, defined as the mean distance of closest approach between the protein and an ion of opposite charge belonging to the ionic atmosphere (taken simply as the sum of the ionic radii).41 By defining a function f(I) as
f(I) ) (0.5√I)/(1 + 0.33a√I)
(2)
°′ where EI)0 is the redox potential extrapolated to zero I, zred and zox are the charges on the reduced and oxidized forms of the
(3)
eq 2 can be written as: ◦ E◦ ) EI)0 + 2.303
a
◦ + 2.303 E°′ ) EI)0
Figure 5. Reduction potential (20 °C) of AZ adsorbed on a gold electrode coated with a DT SAM as a function of f(I). f(I) was changed by altering the concentration of sodium perchlorate. pH 5.0 (I ) 5 mM acetate buffer) (b), pH 7.7 (I ) 5 mM Tris) (O), pH 8.7 (I ) 5 mM Tris) (4), pH 10.5 (I ) 5 mM Tris) (9), pH 7.7 (I ) 15 mM, phosphate buffer only) (1). Solid lines are least-squares fits to the data points and error bars are smaller than the symbols.
RT (z 2 - zox2)f(I) nF red
(4)
For a negatively (positively) charged species, eq 4 predicts a linear increase (decrease) in E°′ with increasing f(I). This equation, which would imply that the protein behaves like a low-dielectric rigid cavity with the charge uniformly distributed on the surface, embedded in an ionic atmosphere of equal and opposite net charge,41,42 has proved to be surprisingly effective in describing the dependence of E°′ on I for freely diffusing and adsorbed cytochrome c23,39,43 and plastocyanin.38 Here, we observe that adsorbed AZ also adheres to this model. In fact, E°′ changes linearly with increasing f(I), in a pH-dependent fashion (an a value for AZ of 22.5 Å was used for calculating f(I)). Figure 5 shows the E°′ versus f(I) plots (obtained by varying the sodium perchlorate concentration) at pH 5.0, 7.7, 8.7, and 10.5 (chosen as representative of different protonation states of AZ, as described above) in acetate or Tris buffer, along with the same plot in the presence of phosphate buffer at pH 7.7. At pH 5.0, the linearity of the plot is lost at higher perchlorate concentration (above 0.03 M). It is conceivable that at this pH value, as observed previously for cytochrome c,23,43 the perchlorate ions interact specifically with the protein inducing charge and conformational changes which would cause deviations from the model. From the slope of the E°′ versus f(I) plots, the charges of oxidized and reduced AZ at the different pH values investigated can be determined, assuming zox - zred ) 1. These are listed in Table 4 along with the theoretical values. At pH 8.7 the slope of the plot is zero; therefore, zox ) zred. This represents a peculiar case and can only be the result of the selective deprotonation of the oxidized state (see below). °′ ) From the E°′ versus f(I) plots, the E°′ values at zero I (EI)0 can be extrapolated at each temperature (eq 4). This allows the °′ °′ °′ °′ and ∆Hrc values (∆Src,I)0 corresponding ∆Src , ∆Hrc,I)0 ) to be
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TABLE 4: Calculated and Experimental Protein Charge Values for Oxidized (zox) and Reduced (zred) AZ Immobilized on a Gold Electrode Coated with DT at Different pH Values in Various Buffers calculateda
TABLE 6: pH-Induced Changes in Reduction Thermodynamics at Zero and Finite I for AZ Immobilized on a Gold Electrode Coated with DT His35(83) equilibriuma High-pH equilibriumb
experimentalb
I)0
pH
buffer
zox
zred
zox
zred
5.0 7.7 7.7 8.7 10.5
acetate Tris phosphate Tris Tris
+1 -1 -1 n.d.c n.d.c
0 -2 -2 n.d.c n.d.c
+1.3 -0.8 -2.8 -2 -5.6
+0.3 -1.8 -3.8 -2 -6.6
-0.067 ∆E°′ (V) °′ ∆∆Src (J mol-1 K-1)d -8 °′ ∆∆Hrc (kJ mol-1)d +3.8 c,d
a From the protein sequence, considering the protonation state of the various residues of the immobilized protein at the pH values shown; in particular, using the calculated pKa values in the 6-8 range, which are presumed to be dominated by His35, and considering His83 to be deprotonated when calculating the charges at pH 7.7 and above. b From the E°’ vs f(I) plots (see text). c Not determined at this pH as protein charges are affected by multiple residue deprotonation(s) whose pKa values are not known.
I ) 15 mM
I)0
I ) 15 mM
-0.060 -6 +3.9
-0.090 -5 +7.2
-0.071 -11 +3.5
a Values obtained from subtraction of the data at pH 5.0 from those at pH 7.7. b Values obtained from subtraction of the data at pH 7.7 from those at pH 10.5. c Experiments performed at 20 °C. d °′ °′ Errors on ∆E°′, ∆∆Src , ∆∆Hrc are (0.006 V, (6 J mol-1 K-1, and (0.9 kJ mol-1, respectively.
TABLE 7: Kinetic Parameters of Heterogeneous ET for AZ Immobilized on a Gold Electrode Coated with Different Alkane-1-thiols at Various pH Valuesa pH
ks (s-1)b
∆H# (kJ mol-1)
∆Hsr# (kJ mol-1)
10.5 10.5 9.2 10.5 4.5 4.5 4.5
755 195 570 0.96 1098 171 1.42
10.6 ( 0.3 7.6 ( 0.3 5.8 ( 0.3 6.3 ( 0.3 15 ( 2 11.7 ( 1.3 7.6 ( 0.6
4.3 ( 0.6 1.3 ( 0.6 n.a.c ≈0 8(3 4.1 ( 1.5 ≈0
SAM PT DT HDT PTd UDTd,e HDTd
a I ) 15 mM (I ) 5 mM Tris plus I ) 10 mM sodium perchlorate). b At T ) 20 °C, estimated relative error on ks is 6%. c Not available because the measurements at pH 9.2 were made only on this SAM (see text). d From ref 19. e Undecane-1-thiol.
°′ Figure 6. EI)0 versus T plots for AZ adsorbed on a gold electrode °′ coated with a DT SAM at different pH values. The EI)0 values were extrapolated from the data set at pH 5.0 (acetate) (b), pH 7.7 (Tris) (O), pH 8.7 (Tris) (4), pH 10.5 (Tris) (9), and pH 7.7 (phosphate) (1). Solid lines are least-squares fits to the data points and error bars are smaller than the symbols.
TABLE 5: Reduction Thermodynamics at Zero I for AZ Immobilized on a Gold Electrode Coated with DT at Different pH Values and Buffers acetate Tris phosphate Tris Tris pH 5.0 pH 7.7 pH 7.7 pH 8.7 pH 10.5 °′ EI)0 (V)a,b (vs SHE) °′ a (J mol-1 K-1) ∆Src,I)0 °′ a ∆Hrc,I)0 (kJ mol-1)
0.335 +11 -28.8
0.268 +3 -25.0
0.239 -17 -28.1
0.232 0 -22.4
0.178 -2 -17.8
a °′ °′ °′ Errors on EI)0 , ∆Src,I)0 , ∆Hrc,I)0 are (0.004 V, (4 J mol-1 K-1, and (0.6 kJ mol-1, respectively. b Experiments performed at 20 °C.
calculated, which do not contain the influence of the “ionic atmosphere” of the protein (Figure 6). These data at different pH values, and for different buffers, are shown in Table 5. The pH-induced changes in reduction thermodynamics at zero and finite I between pH 5.0 and 7.7 and pH 7.7 and 10.5 (corresponding to the two pH titration steps in Figure 3) are listed in Table 6. The ks for AZ adsorbed on PT, DT, and HDT were determined at pH 10.5 in the presence of 10 mM sodium perchlorate, and at pH 9.2 on the DT SAM only, as a function of temperature. Table 7 lists the activation enthalpies (∆H#) under these conditions calculated using the Arrhenius equation
(
ks ) A′ exp
-∆H# RT
)
(5)
Figure 7. Arrhenius plot (ln ks versus 1/T) for AZ adsorbed on a gold electrode coated with a SAM of DT at pH 9.2 (b), PT at pH 10.5 (O), DT at pH 10.5 (1), and HDT at pH 10.5 (4). I ) 15 mM (I ) 5 mM Tris buffer plus I ) 10 mM sodium perchlorate). Solid lines are leastsquares fits to the data points and are smaller than the symbols.
namely from the slope of the plot of ln ks versus 1/T (Figure 7). Discussion Effects of Protein Immobilization on E°′. The E°′ values for adsorbed AZ in the pH range 5-8 are very similar to those for the protein in solution (Table 1). This means that the free energy of adsorption of the protein on the DT SAM, which most likely involves the hydrophobic patch of AZ, is comparable for the reduced and oxidized states, and that the properties of the metal site are scarcely affected by the protein-SAM interaction. °′ °′ The compensatory differences in ∆Hrc and ∆Src between adsorbed and freely diffusing AZ (Table 1) are most likely due to differences in reduction-induced changes of the hydrogenbonding network of water molecules within the hydration sphere of AZ between the unconstrained conditions in solution and the more confined state for the immobilized protein.44-46
Reduction Thermodynamics and Kinetics of Azurin Ionic Strength Effects. Reduction enthalpy in metalloproteins is controlled by the first-coordination sphere and electrostatics at the metal site, while reduction entropy is dictated by the effects of solvent reorganization and changes in conformational degrees of freedom.44 For immobilized AZ under the conditions used in this study, both of these thermodynamic terms become more negative with increasing I at all pH values (Table 2). As these changes are compensatory, the corresponding variations in the E°′ values are modest. This is likely the result of changes in reduction-induced reorganization of the hydrogen-bonding network of water molecules within the hydration shell of the molecule (which are known to induce compensatory enthalpy-entropy changes)47,48 induced by non specific proteinion interactions, as noted previously for c-type cytochromes.44,45 The same effect is observed with phosphate which also specifically binds to both redox states of the protein (see below). The charges of the two oxidation states of AZ immobilized on DT obtained from the slope of the E°′ versus f(I) plots at each pH value are invariably in good agreement with the calculated protein charges (Table 4 and Figure 5). This is noticeable as the Debye-Hu¨ckel theory assumes that the protein is embedded in an ionic atmosphere of equal and opposite charge (as in a bulk medium) and that the charge is distributed with spherical symmetry on the molecule. Therefore for an adsorbed molecule embedded into an ideally planar diffuse layer generated by an electrified surface the Gouy-Chapman theory49 would appear to be more appropriate. However, if this was the case, adsorbed AZ would experience an ionic cloud also influenced by the charge density on the metal surface. As a consequence, application of eq 4 would not result in a linear plot and, mostly, the protein charge values obtained from it would not correspond to those calculated. Therefore, our data indicate that adsorbed AZ adheres to the Debye-Hu¨ckel model. Therefore at the I values and applied potentials investigated, AZ protrudes outside the diffuse layer so as to experience the ionic atmosphere. This is consistent with the majority of the charged residues on AZ being located on the side of the protein oriented toward the solution opposite the nonpolar patch facing the hydrophobic SAM (Figure 1). In particular, it is worth noting that the charges determined at pH 5 coincide with those calculated from the sequence of the protein. A pH increase from 5.0 to 7.7 results in the loss of two positive charges from both oxidation states of AZ, consistent with the deprotonation of both His35 and His83, which is probably not fully complete as discussed earlier. The AZ charges at pH 5.0 and 7.7 also indicate that acetate and Tris do not specifically interact with the protein. This is not the case for phosphate at pH 7.7, as the protein charges are more negative than those calculated indicating the binding of one HPO42- ion to both redox states (see below). The data at pH 10.5 (corresponding to the alkaline limit of E°′ in the second titration event, see Figure 3) indicate an increase in the protein negative charge of both AZ redox states by approximately 5 units (Table 4). This is likely due to the deprotonation of surface Lys residues (11 Lys residues are found on the surface of AZ). The AZ charges determined at pH 8.7 show that the effects of residue deprotonation(s) are less pronounced, as expected, and involve, overall, the loss of one proton from the oxidized protein. °′ °′ °′ , ∆Src,I)0 , and ∆Hrc,I)0 (Table 5) The pH-dependence of EI)0 allows the electrostatic effect on the reduction thermodynamics due to His35 and His83 deprotonation, unsuppressed by the ionic environment effect, to be determined. In particular, we note that the ∆E°′I)0, ∆∆H°′rc,I)0, and ∆∆S°′rc,I)0 values, obtained by subtract-
J. Phys. Chem. C, Vol. 114, No. 50, 2010 22327 ing the values at pH 5.0 from those at pH 7.7 are very similar to those at finite I (15 mM, Table 6). This indicates that the charge effect accompanying proton loss from the two noncoordinating His residues in AZ is sensed by the copper center via a mechanism that is hardly affected by shielding of the surface charge of the protein by the ions in the medium, consistent with the protonation state of the largely buried His35 exerting a larger influence on the E°′ of AZ.13,16,18,31 The pHinduced changes in the reduction thermodynamics at zero I for the equilibrium at alkaline pH values can also be estimated from the differences between the values at pH 10.5 and 7.7 (Table 6). This ∆E°′I)0 value is larger indicating an increased electrostatic effect of residue deprotonations on the E°′ of the copper center at high pH, unsuppressed by I effects. This finding is consistent with the larger change in protein charge due to the high-pH equilibrium compared to the His35(83) equilibrium (Table 4), thus providing confirmation of this model. Moreover, we infer that these residues are, on average, more exposed to solvent than His35 because the difference in ∆E°′ at zero and finite ionic strength is greater than that for the His equilibrium (Table 6). Kinetics of ET. As shown by Jeuken et al.,13 the ks values for AZ adsorbed on an alkanethiolate-modified gold electrode cannot be determined from pH 6.0 to 8.5 as ET is coupled to slow proton transfer associated with the acid-base equilibrium of His35. Here, we have investigated the distance dependence of the ET rate and the activation parameters of AZ immobilized on alkanethiols of varying lengths on which AZ experiences different ET regimes,14,15,17,19 at pH 10.5 (Table 7 and Figure 7). The comparison of these data with those obtained previously by Khoshtariya et al.19 allows information to be gained on how residue deprotonations occurring at this high pH limit influence the ET mechanism. It has been proposed that long- and shortrange ET for AZ occurs through nonadiabatic (tunneling) and ‘frictional’ mechanisms, respectively.19 Others suggested that the short-range ET process is gated by AZ reorientation on the SAM surface which precedes ET,12,14 as well as for adsorbed cytochrome c.50 As for AZ adsorbed on a hydrophobic layer the activation entropy is negligible,19 the ∆H# values can be taken as the ∆G# values from which, under the tunneling regime (long-range ET), the reorganization energy (λ) can be calculated as ∆H#/4. For short-range ET, irrespective of whatever the rate limiting process is, either a “protein friction” mechanism in which the relaxation component is thermally activated, or a gating conformational rearrangement, an additional term ∆Hsr# , (where sr stands for short-range) adds to the activation enthalpy # # . Therefore: ∆H# ) ∆Hsr# + ∆Htun . for electron tunneling, ∆Htun # # # For thick SAMs ∆Hsr is negligible, thus ∆H ) ∆Htun. The temperature dependent kinetic measurements made on # ()∆H#) AZ immobilized on HDT at pH 10.5 yields a ∆Htun value which is smaller than the corresponding value determined # , the ∆Hsr# previously for AZ at pH 4.5 (Table 7).19 Using ∆Htun # values can be obtained from the ∆H values for AZ on PT and DT (Table 7). Again, these values are lower than the shortrange enthalpic terms reported at pH 4.5 (undecane-1-thiol was used at this pH).19 Such a decrease in activation enthalpies is possibly a consequence of changes in either the relaxation properties or reorientation process of AZ induced by the increased protein negative charge at high pH due to the deprotonation of Lys residues. Nonetheless, the ET rate constants for AZ on the investigated SAMs at pH 10.5 are invariably lower than the corresponding values at pH 4.5. It follows that the tunneling efficiency at the AZ-SAM interface [in terms of electronic coupling factor (β) and/or donor-acceptor
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distance (r)] is lowered at the higher pH value and that this effect prevails on the decrease in the activation enthalpies. Remarkably, at pH 9.2, namely within the high-pH titration step, AZ immobilized on DT features a higher ks and a lower ∆H# value with respect to both pH 10.5 and 4.5. Interpretation of this behavior is not obvious. One possible explanation could be that not all the residues involved in the composite, likely Lys-based, high-pH deprotonation equilibrium affect the ET parameters in the same fashion. An increase in the ET rate constant with increasing pH for AZ immobilized on a partially hydrophilic SAM was previously attributed to the enhanced coupling of the SAM to Asn47 caused by the deprotonation of nearby residues.16,17 In our system this could be the case for Lys122 which occupies a distinctive position close to the copper center and is on the periphery of the protein’s hydrophobic patch, and therefore is likely to experience a lower pKa than other surface Lys residues (see below). This deprotonation could decrease the polarizability of the SAM-protein interface and affect the tunneling factors. Deprotonation of the remaining Lys residues, which are mainly located much further from the copper site, opposite to the hydrophobic patch (Figure 1), could conceivably have a different influence on the tunneling efficiency resulting in an increase in the ∆H# value. Phosphate Ion-Azurin Interaction. The HPO42- ion can bind specifically to positively charged protein surface patches, such as those found in cytochrome c.23 Moreover, phosphate has been implicated in His ligand protonation in amicyanin,51 and similar effects have been found for cupredoxin loop mutants,24 fern plastocyanin42 and pseudoazurin.53 The interaction between amicyanin and phosphate has been investigated by computational studies54 and Arg100, adjacent to Met99 (corresponding to Lys122 in AZ) has been implicated in binding along with His96. The charges of AZ obtained from the E°′ vs f(I) plot at pH 7.7 in phosphate buffer (Table 4) are lower by approximately two units compared to those determined in Tris at this pH value. This is indicative of the specific binding of one HPO42- ion to both redox states of AZ. Surface lysine residues are most often involved in phosphate binding sites.23 Inspection of the surface features of AZ (Figure 1) shows that all Lys residues, with the exception of Lys122, are close to negatively charged side chains and are largely exposed to the solvent (values range from 40 to 76%)55 and thus electrostatic interactions will be shielded by solvent and I (Figure 1). Lys122 is only 35% solvent-exposed and is found in a nonpolar environment close to the copper binding Cys112 to Met121 loop. These features should favor the formation of a specific Lys122HPO42- couple. Specific phosphate binding induces a decrease in E°′, which is consistent with the electrostatic stabilization of the oxidized state due to the bound negative charge. The E°′ change °′ °′ ) EI)0 (pH 7.7, phosphate) determined at zero I (∆EI)0 °′ (pH 7.7, Tris)) amounts to -0.029 V (Table 5). As noted EI)0 above, this value does not contain the shielding effects of the electrostatic interaction by I. The components -∆∆H°′rc,I)0/F () °′ 0.032 V) and T∆∆Src,I)0 /F () -0.061 V, at T ) 293 K) are partly compensatory, indicative of the presence of solvent reorganization effects.44 Under these conditions, it has been shown that the change in E°′ coincides with a change in reduction enthalpy,44 which is therefore indicative of a shortdistance electrostatic interaction, fully consistent with Lys122 as the binding site. At pH 8.7 and 10.5 the protein charges calculated from the E°′ vs f(I) plots in the absence and presence of phosphate are similar indicative that under these conditions no specific phosphate binding occurs. Therefore, Lys122 is likely
Monari et al. to be one of the positively charged residues which deprotonate in this pH range with the lowest pKa values, consistent with its rather low exposure to solvent which would favor proton loss to the uncharged state. This would support the above-mentioned role of Lys122 in the pH-dependent ET kinetics. We also observe that at pH 7.7 a specific AZ-phosphate interaction increases the peak-to-peak separation in the AZ CV signal by approximately 30 mV. Although, as mentioned above, the ks values cannot be measured at this pH value, this observation indicates that phosphate binding depresses the ET rate. This effect is consistent with the phosphate binding site being close to the metal center and could result from an increase in polarizability and solvent exposure of the metal environment following formation of the Lys122-HPO42- ionic couple. Conclusions The thermodynamics of ET of AZ noncovalently immobilized on a SAM-modified gold electrode measured at varying I, pH and temperature are found to follow a Debye-Hu¨ckel model from which the charges of the two protein redox states can be determined. These in turn allow charge effects due to residue deprotonations over a wide pH range to be obtained and detection of a specific phosphate ion-protein interaction (which likely occurs at Lys122), which elucidate the pH-dependent redox behavior of AZ. Although the mechanisms of short- and long-range ET (obtained using alkane-thiolate SAMs of different length) is apparently independent of pH, the rate of ET above pH 9 is affected by multiple (predominantly Lys-based) residue deprotonations. These influence the properties of the SAMprotein interface and the tunneling factors in a complex way, yet invariably decrease the activation enthalpies. Lys122 is suggested to play a distinctive role as an effector of the ET rate in this pH range. Acknowledgment. This work was supported in part by a grant from the Fondazione Cassa di Risparmio di Modena (Project No. 1297.08.8C) [M.S.] and by the Marie Curie Research Training Network “EdRox” project funded the Commission of the European Communities (Contract No. MRTNCT-2006-035649) [M.S. and C.D.]. References and Notes (1) Heering, H. A.; Weiner, J. H.; Armstrong, F. A. J. Am. Chem. Soc. 1997, 119, 11628–11638. (2) Hirst, J.; Armstrong, F. A. Anal. Chem. 1998, 70, 5062–5071. (3) Nahir, T. M.; Bowden, E. F. J. Electroanal. Chem. 1996, 410, 9– 13. (4) Chen, K. S.; Hirst, J.; Camba, R.; Bonagura, C. A.; Stout, C. D.; Burgess, B. K.; Armstrong, F. A. Nature 2000, 407, 814–817. (5) Jeuken, L. J. C.; van Vliet, P.; Verbeet, M.; Camba, R.; McEvoy, J. P.; Armstrong, F. A.; Canters, G. W. J. Am. Chem. Soc. 2000, 122, 12186– 12194. (6) Pershad, H. R.; Duff, J. L. C.; Heering, H. A.; Duin, E. C.; Albracht, S. P. J.; Armstrong, F. A. Biochemistry 1999, 38, 8992–8999. (7) Zu, X. L.; Lu, Z. Q.; Zhang, Z.; Schlenkman, J. B.; Rusling, J. F. Langmuir 1999, 15, 7372–7377. (8) Willner, I.; Basnar, B.; Willner, B. FEBS J. 2007, 274, 302–309. (9) Murphy, L. Curr. Opin. Chem. Biol. 2006, 10, 177–184. (10) Rinaldi, R.; Biasco, A.; Maruccio, G.; Arima, V.; Visconti, P.; Cingolani, R.; Facci, P.; De Rienzo, F.; Di Felice, R.; Molinari, E.; Veerbeet, M. Ph.; Canters, G. W. Appl. Phys. Lett. 2003, 82, 472–474. (11) Jeuken, L. J. C.; Armstrong, F. A. J. Phys. Chem. B 2001, 105, 5271–5282. (12) Jeuken, L. J. C.; McEvoy, J. P.; Armstrong, F. A. J. Phys. Chem. B 2002, 106, 2304–2313. (13) Jeuken, L. J. C.; Wisson, L.-J.; Armstrong, F. A. Inorg. Chim. Acta 2002, 331, 216–223. (14) Chi, Q.; Zhang, J.; Andersen, J. E. T.; Ulstrup, J. J. Phys. Chem. B 2001, 105, 4669–4679.
Reduction Thermodynamics and Kinetics of Azurin (15) Chi, Q.; Farver, O.; Ulstrup, J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16203–16208. (16) Yokoyama, K.; Leigh, B. S.; Sheng, Y.; Niki, K.; Nakamura, N.; Ohno, H.; Winkler, J. R.; Gray, H. B.; Richards, J. H. Inorg. Chim. Acta 2008, 361, 1095–1099. (17) Fujita, K.; Nakamura, N.; Ohno, H.; Leigh, B. S.; Niki, K.; Gray, H. B.; Richards, J. H. J. Am. Chem. Soc. 2004, 126, 13954–13961. (18) Guo, Y.; Zhao, J.; Yin, X.; Gao, X.; Tian, Y. J. Phys. Chem. C 2008, 112, 6013–6021. (19) Khoshtariya, D. E.; Dolidze, T. D.; Shushanyan, M.; Davis, K. L.; Waldeck, D. H.; van Eldik, R. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 2757–2762. (20) Nar, H.; Messerschmidt, A.; Huber, R.; van de Kamp, M.; Canters, G. W. J. Mol. Biol. 1991, 221, 765–772. (21) Lowery, M. D.; Solomon, E. I. Inorg. Chim. Acta 1992, 200, 233– 243. (22) Van Pouderoyen, G.; Mazumdar, S.; Hunt, N. I.; Hill, H. A. O.; Canters, G. W. Eur. J. Biochem. 1994, 222, 583–588. (23) Battistuzzi, G.; Borsari, M.; Sola, M. Eur. J. Inorg. Chem. 2001, 2989–3004. (24) Yanagisawa, S.; Dennison, C. J. Am. Chem. Soc. 2004, 126, 15711– 15719. (25) Battistuzzi, G.; Borsari, M.; Ranieri, A.; Sola, M. Arch. Biochem. Biophys. 2002, 404, 227–233. (26) Li, C.; Yanagisawa, S.; Martins, B. M.; Messerschmidt, A.; Banfield, M. J.; Dennison, C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7258– 7263. (27) Laviron, E. J. Electroanal. Chem. 1979, 101, 19–28. (28) Yee, E. L.; Cave, R. J.; Guyer, K. L.; Tyma, P. D.; Weaver, M. J. J. Am. Chem. Soc. 1979, 101, 1131–1137. (29) Yee, E. L.; Weaver, M. J. Inorg. Chem. 1980, 19, 1077–1079. (30) Taniguchi, V. T.; Sailasuta-Scott, N.; Anson, F. C.; Gray, H. B. Pure Appl. Chem. 1980, 52, 2275–2281. (31) St. Clair, C. S.; Ellis, W. R., Jr.; Gray, H. B. Inorg. Chim. Acta 1992, 191, 149–155. (32) Hill, H. A. O.; Leer, J. C.; Smith, B. E.; Storm, C. B.; Ambler, R. P. Biochem. Biophys. Res. Commun. 1976, 70, 331–338. (33) Hill, H. A. O.; Smith, B. E. J. Inorg. Biochem. 1979, 11, 79–93. (34) Van de Kamp, M.; Canters, G. W.; Andrew, C. R.; Sanders-Loehr, J.; Bender, C. J.; Peisach, J. Eur. J. Biochem. 1993, 218, 229–238.
J. Phys. Chem. C, Vol. 114, No. 50, 2010 22329 (35) Battistuzzi, G.; Borsari, M.; Loschi, L.; Righi, F.; Sola, M. J. Am. Chem. Soc. 1999, 121, 501–506. (36) Battistuzzi, G.; Bortolotti, C. A.; Borsari, M.; Di Rocco, G.; Ranieri, A.; Sola, M. J. Phys. Chem. B 2007, 111, 10281–10287. (37) Petrovic, J.; Clark, R. A.; Yue, H.; Waldeck, D. H.; Bowden, E. F. Langmuir 2005, 21, 6308–6316. (38) Ranieri, A.; Battistuzzi, G.; Borsari, M.; Casalini, S.; Fontanesi, C.; Monari, S.; Siwek, M. J.; Sola, M. J. Electroanal. Chem. 2009, 626, 123–129. (39) Battistuzzi, G.; Loschi, L.; Borsari, M.; Sola, M. J. Biol. Inorg. Chem. 1999, 4, 601–607. (40) Goldkorn, T.; Schejter, A. Arch. Biochem. Biophys. 1976, 177, 39– 45. (41) Margalit, R.; Schejter, A. Eur. J. Biochem. 1973, 32, 492–499. (42) Tanford, C. J. Am. Chem. Soc. 1957, 79, 5340–5347. (43) Battistuzzi, G.; Borsari, M.; Sola, M. Eur. J. Inorg. Chem. 2001, 2989–3004. (44) Battistuzzi, G.; Borsari, M.; Sola, M. Chemtracts - Inorg. Chem. 2005, 18, 73–86. (45) Battistuzzi, G.; Borsari, M.; Di Rocco, G.; Ranieri, A.; Sola, M. J. Biol. Inorg. Chem. 2004, 9, 23–26. (46) Bortolotti, C. A.; Battistuzzi, G.; Borsari, M.; Facci, P.; Ranieri, A.; Sola, M. J. Am. Chem. Soc. 2006, 128, 5444–5451. (47) Grunwald, E.; Steel, C. J. Am. Chem. Soc. 1995, 117, 5687–5692. (48) Liu, L.; Guo, Q.-X. Chem. ReV. 2001, 101, 673–695. (49) Bockris, J. O’M., Reddy, A. K. N., Eds.; Modern Electrochemistry Eds.; Plenum Press, New York, 1977; pp 722-724. (50) Avarez-Paggi, D.; Martin, D. F.; DeBiase, P. M.; Hildebrandt, P.; Martı´, M. A.; Murgida, D. H. J. Am. Chem. Soc. 2010, 132, 5769–5778. (51) Lommen, A.; Canters, G. W. J. Biol. Chem. 1990, 265, 2768–2774. (52) Dennison, C.; Lawler, A. T.; Kohzuma, T. Biochemistry 2002, 41, 552–560. (53) Yanagisawa, S.; Crowley, P. B.; Firbank, S. J.; Lawler, A. T.; Hunter, D. M.; McFarlane, W.; Li, C.; Kohzuma, T.; Banfield, M. J.; Dennison, C. J. Am. Chem. Soc. 2008, 130, 15420–15428. (54) Buning, C.; Comba, P. Eur. J. Inorg. Chem. 2000, 1267–1273. (55) Chen, M. E.; Cang, H. X.; Nymeyer, H. NOC Viewer Vers. 3.01 [computer software], Florida State University, 2007.
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