Orientation-Dependent Kinetics of Heterogeneous Electron Transfer

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J. Phys. Chem. C 2007, 111, 12100-12105

Orientation-Dependent Kinetics of Heterogeneous Electron Transfer for Cytochrome c Immobilized on Gold: Electrochemical Determination and Theoretical Prediction Carlo Augusto Bortolotti,† Marco Borsari,‡ Marco Sola,‡ Rita Chertkova,| Dmitry Dolgikh,| Alexander Kotlyar,§ and Paolo Facci*,† Contribution from the CNR-INFM National Center nanoStructures and bioSystems at Surfaces S3, Modena, Italy, Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel AViV UniVersity, Ramat AViV, Israel, the Department of Chemistry, UniVersity of Modena and Reggio Emilia, Modena, Italy, and Shemyakin-OVchinnikoV Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia ReceiVed: April 11, 2007; In Final Form: June 13, 2007

A systematic comparison between electron-transfer rate constants measured electrochemically for different cysteine-bearing mutants of cytochrome c chemisorbed on gold surfaces in different orientations has been performed. Experimental data have been correlated with electronic coupling theoretical estimates obtained from two different empirical models for the kinetics of protein electron transfer, the tunneling pathway model and the average packing density model. The results indicate that both models also hold in the case of immobilized redox proteins, outlining their role in the rational design of optimized electron-transfer-based bioinorganic interfaces.

Introduction

kET )

Almost any approach aiming at exploiting the peculiar properties of redox metalloproteins in sensors and/or electronic devices requires immobilization of the biomolecules in a stable and function-preserving way either on metal electrodes or on insulating surfaces (e.g., SiO2).1,2 In the case of immobilization of biomolecules onto electrode surfaces, it is essential to develop strategies for controlling the electrical communication between the adsorbed species and the substrate.3-8 The elucidation of the parameters playing a key role in the electron-transfer (ET) process involving immobilized biomolecules is therefore fundamental to the rational design of nanoelectronic hybrid devices. Within these parameters, protein orientation toward electrodes is believed to seriously affect the interfacial ET process and in particular the rate at which the electron-transfer reaction occurs.9-13 Many efforts have been made in the last few years to understand the role of the protein structure in mediating the electron coupling between donor and acceptor and to gain new insight into biological electron transfer.14-16 Most biological ET reactions take place between redox cofactors that are separated by 10-15 Å or even more.15 According to theory, the rate constant, kET, for ET between a distant donor (D) and an acceptor (A) can be expressed as the product of the square of an electronic-coupling matrix element (HDA) and a nuclear (or Franck-Condon) factor (FC):17 * Corresponding author. Paolo Facci National Center for nanoStructures and bioSystems at Surfaces (S3) of INFM-CNR, via Campi 213/ A, 41100, Modena, Italy. Phone: +39-059-2055654. Fax: +39-059-374794. E-mail: [email protected]. † CNR-INFM National Center - S3. ‡ University of Modena and Reggio Emilia. § Tel Aviv University. | Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS.

2π |HDA|2(FC) p

(1)

Several measurements concerning intramolecular ET reactions involving modified proteins in solution proved that the intervening medium between donor and acceptor influences the electroniccoupling matrix element (HDA) and, consequently, the ET rate constant, kET.15,18,19 Moreover, different theoretical approaches meant to elucidate the ET process in proteins were developed. These computational tools allow prediction of how the composition and three-dimensional structure of a biomolecule affect its electron-transfer efficiency.20 In particular, much progress has been made in computing the electronic coupling matrix element between the electronic states of donor and acceptor, HDA, which is also called transfer integral.21 In this field, exhaustive ab initio methods are usually avoided because proteins are still too large for these calculations, making them computationally too demanding.22 Recently, computation of transfer integrals within a density-functional theory (DFT) scheme was proposed.22 Given their simplicity, empirical models for estimating HDA remain in wide use.18 Among the most widely used are the tunneling pathway23-25 and the average packing26,27 models, both yielding predictions of relative or absolute ET rates. These methods are usually applied to estimate ET rates for modified proteins in solution aiming at shedding new light onto the influence of the structure of the intervening medium on electron transfer for freely diffusing proteins.15,23,28 Concerning adsorbed proteins, the effects of molecular orientation and edge-to-edge distance between donor and acceptor on the interfacial electrontransfer still lack further theoretical and experimental investigation. Some recent studies suggest that adsorption orientation can affect the electron-transfer rate constant for both electrostatically9,29 and covalently30 adsorbed cytochrome c. This behavior is probably due to the fact that adsorbing the biomolecule through a patch or a residue that is linked to the heme by an efficient electron-tunneling pathway could lead to a faster interfacial ET reaction.

10.1021/jp072813g CCC: $37.00 © 2007 American Chemical Society Published on Web 07/21/2007

Heterogeneous ET for Immobilized Cytochrome c In this work, we investigated the redox behavior of a set of cytochrome c cysteine-containing mutants that were chemisorbed on gold via S-Au covalent binding. All of the mutants were engineered using site-directed mutagenesis so that each one contained a single exposed cysteine residue in a different position, thus enabling investigation of the ET process over a wide range of protein orientations toward the electrode. Following the same procedure described previously for native and mutated yeast cytochrome c,30 the proteins were chemisorbed on polycrystalline bare gold. Using the model proposed by Laviron,31 we obtained and discussed the ET rate constant, ks, for each variant. Moreover, the experimental results were compared with calculations performed using both the tunneling pathway model and the average packing density approach. Our work aims at verifying whether these empirical models, that were shown to be reliable for determining the relative efficiencies of different ET pathways in solution, can also be exploited when the goal is tuning the rate of interfacial ET between an adsorbed protein and the surface by changing the orientation of the biomolecule toward the surface. Experimental Methods Materials. Iso-1-cytochrome c from the yeast Saccharomyces cereVisiae was purchased from Sigma. The samples featured an Rz value (Rz ) A410/A280) greater than 4.5 and were used without further purification.32-34 Expression, purification, and site-directed mutagenesis of the N62C mutant of Saccharomyces cereVisiae iso-1-cytochrome c were performed as described previously.30 All chemicals were reagent-grade. Nanopure water was used throughout. Preparation of Mutated Horse-Heart Cytochromes. The horse-heart cytochrome c genes with singular substitutions V11C, G45C, and G56C were engineered using site-specific mutagenesis. The QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used for the experiments. The mutagenesis procedure included synthesis of the full-length single-stranded plasmid DNA by means of thermostable DNA polymerase Pfu, using two complementary primers that contained the necessary substitutions. Each primer was composed of 30-35 nt. The substitutions were localized in the middle part of the sequence, being flanked by approximately 15 nt. All of the mutant genes were sequenced to confirm their primary structure. Then the mutated genes were cloned into expression system pBP(CYC1)35 for yeast cytochrome c and modified for horse cytochrome c and its mutants.36 The system includes coexpression of the cytochrome c gene and the yeast heme lyase gene; the cytochrome c gene expression efficiency reaches up to 20 mg of the heme-containing cytochrome c per liter of culture. The mutant cytochrome c genes were expressed in E. coli strain JM 109 for 20-22 h at 37 °C in the rich SB media. The mutant proteins were purified from the supernatant, obtained after cell disruption by French Press and centrifugation (100 000 g for 20 min at 4 °C) using modified protocol.37 The protocol includes two steps of liquid chromatography-cation exchange on a Mono-S column and adsorption on a hydroxyapatite column. Final purity of the obtained cytochrome c mutants was >95% according to SDS-PAGE electrophoresis. Electrochemistry. Adsorption of cyt c horse-heart mutants on polycrystalline bare gold was carried out following the procedure described previously for yeast cytochrome c.30 Cyclic voltammetry experiments were carried out with a potentiostat/ galvanostat PAR model 273A in Ar atmosphere. A polycrystalline gold wire was used as a working electrode, and a Pt wire and a saturated calomel electrode (SCE) were used as counter

J. Phys. Chem. C, Vol. 111, No. 32, 2007 12101 and reference electrode, respectively. The electrical contact between the SCE and the working solution was obtained with a Vycor set. Potentials were calibrated against the MV2+/MV+ couple (MV ) methylviologen).39 All of the redox potentials reported here are referred to SHE. All of the electrochemical measurements involving adsorbed cytochrome c species were carried out in 0.01 M phosphate buffer in the presence of 0.2 M sodium chloride. The values for the electron-transfer rate constant, ks, for the adsorbed proteins were determined by recording cyclic voltammograms at variable scan speed and extrapolating the rate constant values using the model proposed by Laviron.31 ks values were averaged over five measurements and found to be reproducible within 10%, which was taken as the associate error. All of the experiments were performed at least five times, and the reduction potentials were found to be reproducible within (2 mV. Computational Estimates. The application of both average packing density and tunneling pathway models was carried out through the HARLEM program40 loaded with wild-type horseheart cytochrome c and yeast iso-1-cytochrome c PDB structure files (1HRC and 1YCC, respectively); hydrogen bonds were identified as implemented in the HARLEM program. To run these simulations, one has to define which atoms of the 3D structure are being considered as belonging to the “donor” and which belong to the “acceptor”. We chose to consider the CR atom belonging to the backbone of the mutated residue as the “acceptor” atom; that is, the metal surface was not theoretically modeled. Consequently, we performed simple predictions of intramolecular electronic pathways between the heme cofactor and the mutated residue used for immobilization. The question of which heme atoms should be taken into account when defining the “donor” unit was also addressed. Calculations were performed by repeatedly changing the set of atoms belonging to the “donor” including (i) the iron atom only, (ii) the Fe atom and all directly bound atoms, (iii) the Fe atom, all directly bound atoms, and atoms belonging to the porphyrin ring, and (iV) all heme atoms, including propionates and porphyrin ring substituents. We found that the results were largely unaffected by the choice of the donor atom set. Therefore, we chose to define the cofactor (i.e., the “donor” unit in the HARLEM modeling) as the iron atom plus all atoms directly bound to it, including also the aromatic rings of the porphyrin macrocycle, according to the choice of Moser and Dutton.27 Results and Discussion Electrochemical Determination of ks. Figure 2 shows a typical cyclic voltammogram obtained for chemisorbed V11C horse-heart cytochrome c mutant covalently immobilized on a polycrystalline bare gold electrode. Similar waves were recorded for G45C and G56C variants. The quasi-reversible cyclic voltammogram originates from the one-electron reduction/oxidation of the adsorbed cytochrome. The E°′ values at pH 7 and 20 °C for the mutants (Table 1) show small differences. It is worth noting that the E°′ values for the adsorbed species are significantly more positive than those for the freely diffusing wild-type protein (256 mV vs SHE),41,42 likely as a consequence of the electrostatic repulsion among the closely packed proteins forming the dense monolayer, leading to stabilization of the reduced form of cytochrome c.30 Also for immobilized horse-heart cytochrome c, we obtained a high surface coverage, which amounts to approximately 80% of that expected for a full densely packed monolayer, (19 pmol/ cm2), as estimated from the crystallographic dimensions of the protein.13

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Figure 3. Dependence of logarithm of experimental ET rates vs edgeto-edge distance between iron atom and CR of each cysteine residue used as a tethering point for protein immobilization. Distances were calculated on the basis of yeast and horse-heart cytochrome c structures (PDB codes: 1YCC and 1HRC, respectively).

TABLE 1: Redox Potentials for Cytochrome c Species Covalently Bound to a Polycrystalline Gold Electrode through a (Cys)S-Au Bond; Calculated Euclidean Distances between the Heme Iron and Cr of the Cysteine Residue Used for Surface Immobilization on Gold; Electrochemically Determined Rate Constants for Electron Transfer between the Heme and the Electrode for the Immobilized Species

Figure 1. 3D representations of the structures of yeast iso-1cytochrome c (top) and horse-heart (bottom) cytochrome c. The labeled residues are those that were mutated into cysteines (except for native Cys 102 in yeast cyt c) for surface immobilization. Heme is represented in red. These images were generated using VMD (Visual Molecular Dynamics program).38

Figure 2. Cyclic voltammogram for V11C mutant of horse-heart cytochrome c immobilized on bare polycrystalline gold electrode through the (Cys)S-Au bond in 0.01 M phosphate buffer, 0.2 M NaCl, pH 7, sweep rate 20 mVs-1, and T ) 20 °C.

Analysis of the three-dimensional structure of both wild-type horse-heart and yeast cytochrome c allowed calculation of the edge-to-edge distance between the iron atom of the heme and the CR of each residue that was mutated into a cysteine. It is

species

E°′ a-c (mV)

Fe-CysCR distanced (Å)

ks e,f (s-1)

horse-heart V11C horse-heart G45C horse-heart G56C yeast native yeast N62C

+381 +394 +388 +370g +378g

12.5 15.4 16.7 13.9 16.1

8.34 2.22 0.63 0.20g 0.46g

a At 20 °C. b 0.01 M phosphate buffer, 0.2 M NaCl, pH 7. c All of the electrochemical experiments were performed at least five times, and the reduction potentials were found to be reproducible within (2 mV. d Calculated from three-dimensional structures (PDB codes 1YCC and 1HRC, whose resolution is 1.23 Å and 1.90 Å, respectively) e Measured from the scan-rate dependence of peak potentials using the model proposed by Laviron for diffusionless electrochemical systems.31 f At 20 °C; the error affecting the ks values is (10%. g From ref 30.

worth noting that, for the three mutants of horse-heart cyt c considered here, ks decays with increasing distance. This is in good agreement with theoretical predictions15,17,18 and experimental results27 because the coupling matrix element is known to exponentially decay with increasing distance between donor and acceptor. It should be mentioned that it is quite unlikely that the edge-to-edge distances resulting from analysis of the 3D structure are unaltered by direct protein binding to a bare surface. However, it appears that the relationship between ks and the calculated ET distance is well conserved within the series, as if adsorption had similar effects on V11C, G45C, and G56C mutants. The finding that the ET rate constant is higher for the V11C mutant is in agreement with electrochemical experiments carried out previously on cytochrome c mutants electrostatically bound to carboxylate-terminated alkanethiol SAMs, which revealed highly anisotropic electronic coupling across the protein/ monolayer interface. The coupling between the heme and Lys13, which lies very close to Val11, was found to be much higher than that for other lysine residues surrounding the cyt c heme

Heterogeneous ET for Immobilized Cytochrome c

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Figure 4. Best pathways between heme cofactor and CR of (A) yeast cyt c, Cys 102, (B) yeast cyt c, Asn 62; (C) horse-heart cyt c, Val 11; and (D) horse-heart cyt c, Gly 56. Calculation of the best pathway between donor and acceptor and their visual representation were performed with the HARLEM program.40

crevice.9,29 This result proved that Lys13 is likely to be connected to heme by more efficient electron tunneling pathways than other residues, thus suggesting that immobilization via Lys13 or close residues could lead to efficient communication between the conducting substrate and the adsorbed protein. Moreover, the cytochrome c patch surrounding Lys13 was found to be involved in the binding of cytochrome c to the cytochrome c1 subunit of its physiological partner cytochrome bc1 complex,43 thus strengthening the belief that interfacial ET for cytochrome c adsorbed via surface-exposed residues positioned close to Lys13 can be more efficient than that with different protein orientations. Nevertheless, the Euclidean distance itself is unlikely to be the only factor affecting the ET rate. In fact, the N62C variant of yeast cyt c featured a higher ks value than the native protein,

despite the smaller edge-to-edge distance between heme and the CR of the cysteine residue.19 This is the result of the fact that, besides distance, other factors affect the rate of the ET process between the heme and the surface, such as the intervening medium and the orientation of the protein toward the surface. Computational Predictions. The HARLEM software40 was used to calculate the electronic coupling matrix element, HDA, between the heme and the CR carbon of the residue that was then mutated into a cysteine to anchor the protein to the gold substrate. It should be noted that, in contrast to the ET process taking place between redox centers in solution, the ET reaction involving an electrode surface and an immobilized protein is heterogeneous because in the latter a continuum of electrode states from the surface acts as donor/acceptor. This is likely to

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Figure 5. Logarithmic graphs of experimental ks for yeast and horse-heart cyt c mutants (covalently immobilized on polycrystalline bare gold electrodes) vs square of tunneling pathway (A) and average packing density (B) calculated HDA.

TABLE 2: HDA Values Calculated with the Tunneling Pathway and Average Packing Density Models

acceptor

tunneling pathway coupling value (arbitrary units)

average packing density HDA (arbitrary units)

horse-heart Val11 horse-heart Gly45 horse-heart Gly56 yeast Cys102 yeast Asn62

1.31 10-3 1.83 10-4 1.67 10-5 1.12 10-4 8.10 10-5

2.02 10-3 3.03 10-4 4.67 10-5 2.80 10-4 5.84 10-5

make the electron-tunneling probability at electrodes different from that between two localized molecular states.9 This means that, neglecting the presence of the surface in our calculations and restricting the calculation to the protein matrix only, predictions of absolute heterogeneous ET rate constants become impossible. Nevertheless, these calculations can still lead to estimates of relatiVe ks values, predicting the sites that are most strongly coupled to the heme center, thus helping to identify the best tethering sites for fast ET. We calculated the electronic coupling HDA value by using both tunneling pathway23-25 and average packing density26,27 approaches. The key idea on which both approaches are based is that the electronic coupling between donor and acceptor decays across a number of discrete steps and that electronic interactions decay more rapidly through space than through bond. Notably, the ratio of the through-bond and through-space decay parameters in the two models is different. Moreover, the pathway-coupling estimate provides as a result the single strongest donor-acceptor route, whereas, in the average density approach, the overall packing density (F) of protein atoms in the volume between redox centers, rather than the pathway connectivity, defines the coupling. F is defined as the fraction of the volume between redox cofactors that is within the van der Waals radius of intervening atoms.27 Given their simplicity and their mathematical isomorphism,20 the average packing density and tunneling pathway models are usually in qualitative agreement.20 The HARLEM package allows prediction of the best path between the CR atom of each mutated residue and the heme cofactor, along with its HDA value, as well as a visual representation of it. Some of these pathways calculated for the present species are shown in Figure 4, and the HAD values are listed in Table 2. Because the pathway model accounts for a large difference in through-bond and through-space separation and is based on

the assumption that electronic interactions decay much more slowly through bond than through space, pathways featuring nonbonded or hydrogen-bonded steps will lead to a weaker coupling than purely covalently bridged ones. This fact justifies the calculated HDA values for the yeast cytochrome c species. The coupling between heme and CR of Asn62 is higher than that for Cys102, although the edge-to-edge distance between the native Cys102 and the heme is smaller. This effect is due the fact that the most probable calculated pathway between the heme and the CR of Cys102 features two nonbonded jumps, whereas the heme-Asn62 only one. The HARLEM package also allows calculations of the electronic coupling matrix element, HDA, with the average packing density model.40 Table 2 shows that, despite the difference in parametrization, predictions made with the two models are comparable. As stated before, the two different models lead to similar predicted values when dealing with donor and acceptor pairs separated by mostly covalent linkages, that is, rather high packing density values. Comparison between Experimental Results and Computational Predictions. One of the main goals of this work is to assess whether the use of empirical models for predicting intraand intermolecular donor-acceptor couplings can be extended to immobilized proteins. This would greatly help the rational design of hybrid edifices with highly efficient communication because one could predict in advance the protein orientation toward the substrate leading to the fastest electron transfer. To consider the tunneling pathway model and/or the average packing density one as reliable tools for predicting relative efficiencies of different electron-tunneling pathways in immobilized biomolecules, one should observe a satisfactory fitting between experimental and theoretical values. Figure 5 shows the experimental rates versus the predicted electronic coupling values. A roughly linear correlation is expected on the basis of eq 1, assuming that the nuclear factor, FC, is approximately constant for all mutants. This assumption is based on the consideration that cytochrome c is an electrontransfer biomolecule featuring a quite rigid structure and that, subsequently, low reorganization energy differences for each mutant are expected. A linear correlation between ks and HDA2 indeed emerges. For both models, theoretical predictions seem to be in good agreement with experimental results, except for native yeast cytochrome c. It is worth noting that structural alterations in chemisorbed molecules could occur with respect to X-ray

Heterogeneous ET for Immobilized Cytochrome c structures because of the interaction of the molecules with highnergy inorganic surfaces. The residue used for chemisorption of native yeast cyt c on a gold substrate, Cys102, is known to be scarcely exposed off the external protein surface because it occupies a predominantly hydrophobic pocket.44 Adsorption via the Cys102 thiol group can therefore lead to a slight structural rearrangement that could result in altered Euclidean distance between the heme iron and the sulfur atom. This fact could provide a possible explanation for the discrepancies concerning the expected and experimental relative values for the electrontransfer rate constant for the yeast cytochrome c species we examined. The satisfactory agreement between the calculations and the experimental data concerning the other mutant species suggests that the two computational models examined here can be employed for analysis of heterogeneous electron transfer between a redox cofactor buried in the protein matrix and the electrode. This analysis enables us to select areas on the protein surface for their efficient electronic coupling to the electrode, providing a useful tool for designing bioelectronic interfaces and biosensors. Conclusions A systematic analysis of the electron-transfer rate constants for cytochrome c mutants bearing a single exposed cysteine and chemisorbed on gold has been carried out by correlating the measured experimental data with the electronic coupling values estimated by two computational models. The results provide a qualitative agreement between experimental data and theoretical estimates confirming the use of these models for designing protein mutants with optimized electronic coupling to solid electrodes. This finding would facilitate the rational design of bio-inorganic interfaces for biosensors and bioelectronic devices. Abbreviations E°′ CV SCE SHE cyt c

standard reduction potential cyclic voltammetry saturated calomel electrode standard hydrogen electrode cytochrome c.

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