Characterization of the Electron Transfer of a Ferrocene Redox Probe

May 6, 2009 - This outcome was different from the result obtained during SERR experiments coupled to electrochemistry in which a direct electrochemica...
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Characterization of the Electron Transfer of a Ferrocene Redox Probe and a Histidine-Tagged Hemoprotein Specifically Bound to a Nitrilotriacetic-Terminated Self-Assembled Monolayer Veronique Balland,*,† Sophie Lecomte,‡ and Benoıˆ t Limoges*,† † Laboratoire d’Electrochimie Mol eculaire, Universit e Paris Diderot, UMR CNRS 7591, 15 rue Jean-Antoine de Baı¨f, 75205 Paris Cedex 13, France, and ‡Chimie Biologie des Membranes et Nano-objets, Universit e Bordeaux 1, UMR CNRS 5248, 2 rue Robert d’Escarpit, 33607 Pessac, France

Received January 7, 2009. Revised Manuscript Received February 26, 2009 We report the selective, controlled binding of a model redox probe, 1,10 -bis(N-imidazolylmethyl)ferrocene (Fc-Im2), and a small redox hemoprotein, histidine-tagged recombinant human neuroglobin (hNb), at the surface of metal electrodes (gold and SER-active silver) modified by a self-assembled monolayer (SAM) of a nitrilotriacetic(NTA)terminated thiol. The resulting SAMs were characterized by cyclic voltammetry and surface-enhanced resonance Raman (SERR) spectroscopy coupled to electrochemistry. Once specifically bounded to the Ni(II)-NTA-modified gold electrode, nearly ideal cyclic voltammetric behavior with relatively fast electron-transfer (ET) communication through the SAM was determined for the Fc-Im2 redox probe. However, no direct electron transfer could be evidenced for the hNb redox protein under the same conditions. This outcome was different from the result obtained during SERR experiments coupled to electrochemistry in which a direct electrochemical conversion of hNb immobilized on a Ni(II)-NTA-modified SER-active Ag electrode was observed. The SERR spectra of the immobilized hNb was the same as the resonance Raman spectra of the protein in homogeneous solution, allowing us to conclude that the native structure of hNb was retained upon immobilization and that the direct ET was not the result of some partial or complete protein denaturation. The long-range ET rate constant (kET) through the SAM was determined by time-resolved SERR spectroscopy. A value of kET = 0.12 s-1 was obtained, which is within the predicted range of a fully nonadiabatic ET through a SAM thickness of ∼26 A˚ and close to the values previously determined for analogous small redox proteins at similar long-range ET distances. A SERR spectroelectrochemical titration of the immobilized hNb was also carried out, showing both an apparent standard potential (E00 ) negatively shifted by 100 mV compared with hNb in solution and a gentle slope in the titration curve. These results suggest a range of chemical environments in the surroundings of the redox protein and a variety of interactions with the NTA-terminated SAM. The influence of protein immobilization on E00 is discussed together with the long-range ET rate constant and molecular orientation of the surface-immobilized hNb.

Introduction Electrochemical investigation of the electron-transfer (ET) kinetics of chemical or biological redox molecules immobilized as a monolayer on conductive surfaces has been recognized to be a powerful approach for better understanding long-range electron transfer, which is one of the fundamental processes in nature.1,2 This subject is also of interest for potential applications, for example, in biomolecular electronics where biological molecules are integrated on the surface of electronic transducers to provide new strategies for biosensing.3 The electrochemistry of redox biomolecules immobilized on an electrode surface affords several advantages relative to ET kinetic studies performed in other formats. In particular, the electrode potential control affords the experimenter a variable driving force that facilitates the measurement of valuable thermodynamic and kinetic parameters including, for example, the formal potential, reorganization energy, and ET rate constants. Furthermore, the absence of diffusive and conductive transport leads to simplified kinetic *Corresponding authors. E-mail: veronique.balland@univ-paris-diderot. fr, [email protected]. (1) Finklea, H. O. Electrochemistry of Organized Monolayers of Thiols and Related Molecules on Electrodes; Marcel Dekker: New York, 1996; Vol. 19. (2) Leger, C.; Bertrand, P. Chem. Rev. 2008, 108, 2379–2438. (3) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1180–1218. (4) Finklea, H. O.; Hanshew, D. D. J. Am. Chem. Soc. 1992, 114, 3173–3181.

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analysis.4 In many instances, however, the redox center of the biomolecule of interest is not easily accessible to the electrode. This is particularly true for redox proteins in which the redoxactive site is embedded deeply within the 3D structure of the polypeptide matrix and is accessible only through a specific path in the surrounding of the protein. Accordingly, a number of strategies have been developed to promote efficient electron exchange between an electrode and an immobilized redox-active protein. This includes the use of free to diffuse5,6 or coimmobilized redox mediators,7,8 natural or artificial electron relays,9-12 conducting molecular wires,13 and specifically designed electrode (5) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667–671. (6) Limoges, B.; Marchal, D.; Mavre, F.; Saveant, J.-M. J. Am. Chem. Soc. 2006, 128, 2084–2092. (7) Maidan, R.; Heller, A. Anal. Chem. 1992, 64, 2889–2896. (8) Anicet, N.; Anne, A.; Moiroux, J.; Saveant, J.-M. J. Am. Chem. Soc. 1998, 120, 7115–7116. (9) Haas, A. S.; Pilloud, D. L.; Reddy, K. S.; Babcock, G. T.; Moser, C. C.; Blasie, J. K.; Dutton, P. L. J. Phys. Chem. B 2001, 105, 11351–11362. (10) Heering, H. A.; Wiertz, F. G. M.; Dekker, C.; de Vries, S. J. Am. Chem. Soc. 2004, 126, 11103–11112. (11) Leger, C.; Elliott, S. J.; Hoke, K. R.; Jeuken, L. J. C.; Jones, A. K.; Armstrong, F. A. Biochemistry 2003, 42, 8653–8662. (12) Willner, I.; Heleg-Shabtai, V.; Blonder, R.; Katz, E.; Tao, G.; Buckmann, A. F.; Heller, A. J. Am. Chem. Soc. 1996, 118, 10321–10322. (13) Hess, C. R.; Juda, G. A.; Dooley, D. M.; Amii, R. N.; Hill, M. G.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 2003, 125, 7156–7157.

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coatings that facilitate direct electron-transfer communication by means of a favorable spatial connection and/or electronic coupling between the electrode and the immobilized redox protein.14,15 One important example of electrode coatings is alkanethiolate-based self-assembled monolayers (SAMs) on metal surfaces, which provides a highly ordered interfacial environment to promote both the binding of redox proteins and direct electronic coupling to the electrode. In this way, direct, fast electron transfer was achieved for cytochrome c (cyt c) electrostatically adsorbed on carboxylic-terminated SAMs14,16,17 or coordinatively bound to pyridine-terminated SAMs,18 a strategy that was subsequently extended to other small redox proteins such as cytochrome c0 ,19 copper-containing nitrite reductase on a SAM of cysteamine,20 and azurin in hydrophobic interactions with a H3C-terminated SAM.21,22 This approach also has the specific advantage to allow a high degree of distance control between the redox centers and the electrode by simply varying the chain length of the SAM linker, thus offering a way to characterize long-range ET kinetics. However, in many cases, it is not clear whether the redox protein has retained its native 3D structure or if there has been some change in conformation to bring the protein redox center close enough to the electrode to allow significant direct electron transfer. This uncertainty is largely due to the lack of structural information for most of the immobilized redox proteins that have been investigated so far. Among the rare surface analytical methods available for probing structural changes in immobilized proteins, surfaceenhanced resonance Raman (SERR) spectroscopy and surfaceenhanced infrared absorption (SEIRA) spectroscopy are certainly the most powerful. The effectiveness of the SERR and SEIRA methods for identifying structural changes was clearly demonstrated in the case of cyt c electrostatically adsorbed on a Ag electrode coated with different lengths of carboxylic-terminated SAMs. For long SAMs, the SERR and SEIRA spectra were essentially identical to those measured for cyt c in homogeneous solution, indicating that the native structure of cyt c was essentially unaffected by the electrostatic adsorption.23-25 However, this was not the case for shorter SAMs, where a significant structural alteration of cyt c was revealed in the SERR spectra, a result that was attributed to the loss of axial ligand Met-80 in the heme pocket. Analogous structural changes accompanied by a significant downshift of the redox potential were also observed upon hydrophobic binding of cyt c at electrodes covered with H3C-terminated SAMs26 or after adsorption at a bare pyrolytic (14) Kasmi, A. E.; Wallace, J. M.; Bowden, E. F.; Binet, S. M.; Linderman, R. J. J. Am. Chem. Soc. 1998, 120, 225–226. :: (15) Rudiger, O.; Abad, J. M.; Hatchikian, E. C.; Fernandez, V. M.; De Lacey, A. L. J. Am. Chem. Soc. 2005, 127, 16008–16009. (16) Niki, K.; Hardy, W. R.; Hill, M. G.; Li, H.; Sprinkle, J. R.; Margoliash, E.; Fujita, K.; Tanimura, R.; Nakamura, N.; Ohno, H.; Richards, J. H.; Gray, H. B. J. Phys. Chem. B 2003, 107, 9947–9949. (17) Kranich, A.; Ly, H. K.; Hildebrandt, P.; Murgida, D. H. J. Am. Chem. Soc. 2008, 130, 9844–9848. (18) Khoshtariya, D. E.; Wei, J.; Liu, H.; Yue, H.; Waldeck, D. H. J. Am. Chem. Soc. 2003, 125, 7704–7714. (19) de Groot, M. T.; Evers, T. H.; Merkx, M.; Koper, M. T. M. Langmuir 2007, 23, 729–736. (20) Zhang, J.; Welinder, A. C.; Hansen, A. G.; Christensen, H. E. M.; Ulstrup, J. J. Phys. Chem. B 2003, 107, 12480–12484. (21) Chi, Q.; Zhang, J.; Andersen, J. E. T.; Ulstrup, J. J. Phys. Chem. B 2001, 105, 4669–4679. (22) 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. (23) Murgida, D. H.; Hildebrandt, P. J. Phys. Chem. B 2001, 105, 1578–1586. (24) Ataka, K.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 9445–9457. (25) Murgida, D. H.; Hildebrandt, P. Phys. Chem. Chem. Phys. 2005, 7, 3773– 3784. (26) Rivas, L.; Murgida, D. H.; Hildebrandt, P. J. Phys. Chem. B 2002, 106, 4823–4830.

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graphite electrode.27 These few examples advised that great care has to be taken to ensure the integrity of the immobilized protein before examining the redox processes of proteins or enzymes at electrochemical interfaces. Another key parameter that has to be considered is the spatial distribution and/or orientation of redox proteins on the surface, which has been predicted to play a significant role in the electrochemistry of the redox protein monolayer because the redoxactive site is generally not located at the center of the protein.28-32 As a result, a distribution of molecular orientations should generate a distribution of redox site-electrode separation distances and hence a distribution of ET rate constants and/or redox potentials. Such a hypothesis has been invoked to explain the nonideal voltammetry of cyt c immobilized on SAMs.31,33 On the basis of this previous work, it appears that the key issue in properly investigating the direct electrochemistry of a monolayer of redox-active proteins is to design a biocompatible interface that is able to (i) bind the protein of interest strongly enough to give a stable protein layer, (ii) preserve the immobilized protein from structural and functional changes, (iii) allow precise control over the distance between the protein metallic center and the electrode surface, (iv) permit homogeneous repartition over the surface, and finally (v) provide molecular-level control of the protein orientation in such a way as to promote efficient electronic coupling with the electrode. Among the few protein-immobilization strategies able to meet these multiple criteria, the method based on the nitrilotriacetic acid (NTA)/histidine-tag (his-tag) technology34 is certainly the most suitable for the following reasons: (i) it leads to the mild modification of proteins (i.e., without a significant alteration of the protein functionality), (ii) it is site-specific and thus allows us to control the protein orientation and distance to the electrode surface, (iii) it is very flexible because it can be adapted to any kind of genetically engineered protein, and (iv) it can be easily implemented in SAMs. Such immobilization was advantageously exploited to study redox proteins or enzymes by surface plasmon resonance,35 surface-enhanced vibrationnal spectroscopy,36-38 and cyclic voltammetry at gold39-41 and carbon surfaces.42 To achieve the immobilization of his-tagged proteins or enzymes on an electrode, (27) Ye, T.; Kaur, R.; Senguen, F. T.; Michel, L. V.; Bren, K. L.; Elliott, S. J. J. Am. Chem. Soc. 2008, 130, 6682–6683. (28) Dick, L. A.; Haes, A. J.; Van Duyne, R. P. J. Phys. Chem. B 2000, 104, 11752–11762. (29) Runge, A. F.; Mendes, S. B.; Saavedra, S. S. J. Phys. Chem. B 2006, 110, 6732–6739. (30) Nahir, T. M.; Bowden, E. F. J. Electroanal. Chem. 1996, 410, 9–13. (31) Yue, H.; Waldeck, D. H.; Petrovic, J.; Clark, R. A. J. Phys. Chem. B 2006, 110, 5062–5072. (32) Araci, Z. O.; Runge, A. F.; Doherty, W. J.; Saavedra, S. S. J. Am. Chem. Soc. 2008, 130, 1572–1573. (33) Clark, R. A.; Bowden, E. F. Langmuir 1997, 13, 559–565. (34) Crowe, J.; Dobeli, H.; Gentz, R.; Hochuli, E.; Stiiber, D.; Henco, K. In Protocols for Gene Analysis; Harwood, A. J., Ed.; Humana Press: Totowa, NJ, 1994; Vol. 31, pp 371-387. (35) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490–7. (36) Friedrich, M. G.; Giess, F.; Naumann, R.; Knoll, W.; Ataka, K.; Heberle, J.; Hrabakova, J.; Murgida, D. H.; Hildebrandt, P. Chem. Commun. 2004, 2376– 2377. (37) Ataka, K.; Giess, F.; Knoll, W.; Naumann, R.; Haber-Pohlmeier, S.; Richter, B.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 16199–16206. (38) Hrabakova, J.; Ataka, K.; Heberle, J.; Hildebrandt, P.; Murgida, D. H. Phys. Chem. Chem. Phys. 2006, 8, 759–766. (39) Madoz-Gurpide, J.; Abad, J. M.; Fernandez-Recio, J.; Velez, M.; Vazquez, L.; Gomez-Moreno, C.; Fernandez, V. M. J. Am. Chem. Soc. 2000, 122, 9808– 9817. (40) Johnson, D. L.; Martin, L. L. J. Am. Chem. Soc. 2005, 127, 2018–2019. (41) Balland, V.; Hureau, C.; Cusano, A. M.; Liu, Y.; Tron, T.; Limoges, B. Chem.;Eur. J. 2008, 14, 7186–7192. (42) Blankespoor, R.; Limoges, B.; Schoellhorn, B.; Syssa-Magale, J.-L.; Yazidi, D. Langmuir 2005, 21, 3362–3375.

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a conductive surface functionalized either by a SAM of NTAterminated alkanethiols on gold35 or by the electrochemical grafting of NTA-terminated phenyl diazonium salt on carbon,42 followed by metalation of the NTA ligand by NiII or CuII ions, was proposed. Accordingly, the mediated electrochemistry of his-tagged ferredoxin/NADP+ reductase,39 horseradish peroxidase,42 and laccase,41 specifically bound to NTA-modified electrodes, was reported. From the quantitative analysis of the catalytic current recorded at these enzyme electrodes corroborated by an independent measurement of the active-enzyme surface concentrations, it was shown that the activities of the immobilized enzymes were all fully preserved. These results indicate that NTA coatings provide a biocompatible environment for preserving the functionality of bounded proteins. Nevertheless, whatever the redox enzymes investigated, no enzymatic activity could be observed in the absence of a soluble mediator. The lack of direct electron transfer between the electrode and the active site of these enzymes involved electron-transfer distances that were too long. Similar results were obtained for the cytochrome c oxidase (CcO), a membrane enzyme that was tethered to an NTA-thiol-functionalized gold electrode via a histidine tag on the C termini of subunits I or II and embedded into a reconstituted phospholipid bilayer. In the absence of natural electron mediator cyt c, the authors were unable to observe in cyclic voltammetry a catalytic current in the presence of the O2 substrate, signifying that CcO was not electrically coupled to the electrode.37 However, it was shown by SERR spectroscopy coupled to electrochemistry that the oxidized or reduced states of CcO could be fully transformed by direct electron transfer (i.e., without added cyt c).36 Using time-resolved SERR spectroscopy, the same authors were able to measure an ET rate constant of 0.002 s-1, which is rather fast in view of the considerable electrontransfer distance between the electrode surface and the CcO redox centers (>40 A˚).38 More recently, the same CcO assembly on gold was investigated by cyclic voltammetry. In contrast to the SERR result previously obtained, an impressively fast direct ET rate ranging from ca. 10 to 4000 s-1 was deduced from a reversible wave in the cyclic voltammogram, which was attributed to direct electrical communication between the electrode and CcO.43 It is worth mentioning that E 00 of the reversible wave was shifted 450 mV from the standard potential determined for the electron acceptor center in the isolated CcO, thus raising some doubts about the structural integrity of the immobilized protein. Another facilitated direct electron transfer between a gold electrode and his-tagged redox proteins was proposed at an analogue NTA ligand covalently attached to a short-length SAM on gold.40 From the distinct reversible waves recorded in cyclic voltammetry for several his-tagged redox proteins, the authors concluded that there was rapid electronic communication between the protein redox sites and the underlying electrode. However, the apparent standard potentials of the immobilized proteins were not corroborated with those obtained in homogeneous solution, and the possible structural changes and/or protein denaturation on such a very short length SAM were not examined, preventing one from commenting on the protein integrity and on the origin of the direct electron transfer. In view of these efforts directed toward improved control of the direct ET between a redox protein and an electrode through a sitespecific binding orientation of the protein and on account of the several questions that remain open, we have chosen to examine the long-range ET between a metal electrode and a small (43) Friedrich, M. G.; Robertson, J. W. F.; Walz, D.; Knoll, W.; Naumann, R. L. C. Biophys. J. 2008, 94, 3698–3705.

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his-tagged hemoprotein bound on a moderately short NTAterminated SAM. The NTA-terminated SAM was the same as previously used by us for the immobilization of a his-tagged laccase (i.e., based on N-(5-([1,2-dithiolane-3-pentanoylamino]1-carboxypentyl)iminodiacetic acid).41 As a small hemoprotein, we have selected recombinant histidine-tagged human neuroglobin (hNb), which has a size (17 kDa) comparable to that of cyt c. The physiological role of hNb is not yet clearly understood,44 but its 3D structure has been determined by crystallography.45 The hemoprotein presents an accessible heme pocket and specific heme iron bis-histidine hexacoordination in its native state with a characteristic resonance Raman spectrum.46 It is therefore a good candidate for both investigating its direct electron transfer at an NTA-modified gold electrode and characterizing its structure by SERR spectroscopy. The his-tagged hNb on NTAmodified electrodes was thus investigated by cyclic voltammetry, potential-dependent SERR spectroscopy, and time-resolved SERR spectroscopy. We have also examined the binding and long-range electron transfer of the 1,10 -bis(N-imidazolylmethyl) ferrocene (Fc-Im2). This redox probe was used as a standard for evaluating the binding properties of the NTA-terminated SAM coating and for calibrating the long-range ET through the SAM. Another important objective of the work was to characterize quantitatively all of the steps of the immobilization procedure and to determine the extent to which his-tagged hNb is preserved from conformational changes. The influence of protein immobilization on E 00 is finally discussed together with the long-range ET rate constant and molecular orientation of the surfaceimmobilized hNb.

Results and Discussion Electrochemical Characterization of the Functionalized Electrodes. NTA-thiol-modified gold electrodes were prepared, as previously described, in a single step from the selfassembly of N-(5-([1,2-dithiolane-3-pentanoylamino]-1-carboxypentyl)iminodiacetic acid.41 The surface concentration of the chemisorbed NTA-thiol molecules was deduced from the voltammetric oxidative desorption of the SAM in a 0.5 M KOH solution.47 After correcting for oxidation of the gold surface and assuming a three-electron oxidation peak, the surface concentration of sulfur atoms was found to be (7 ( 0.5)  10-10 mol 3 cm-2 . This value is close to the (8 ( 0.5)  10-10 mol 3 cm-2 value found under the same conditions for a SAM of thioctic acid and is consistent with the theoretical value of a close-packed monolayer. Assuming two sulfur atoms grafted per molecule, the surface concentration of the NTAthiol linker was finally estimated to be (3.50 ( 0.25)  10-10 mol 3 cm-2. Metalation of the NTA-modified electrodes was achieved by soaking them in a 1 mM NiCl2 solution (phosphate buffer 100 mM, pH 7.4) for 30 min. The Ni(II)-NTA complex that formed on the electrode surface was not electroactive within the potential window of the aqueous buffer. Therefore, with the aim of determining the amount of Ni(II)-NTA on the electrode, we have used an indirect method based on the specific binding of (44) Hankeln, T.; Ebner, B.; Fuchs, C.; Gerlach, F.; Haberkamp, M.; Laufs, T. L.; Roesner, A.; Schmidt, M.; Weich, B.; Wystub, S.; Saaler-Reinhardt, S.; Reuss, S.; Bolognesi, M.; De Sanctis, D.; Marden, M. C.; Kiger, L.; Moens, L.; Dewilde, S.; Nevo, E.; Avivi, A.; Weber, R. E.; Fago, A.; Burmester, T. J. Inorg. Biochem. 2005, 99, 110–119. (45) Pesce, A.; Dewilde, S.; Nardini, M.; Moens, L.; Ascenzi, P.; Hankeln, T.; Burmester, T.; Bolognesi, M. Structure 2003, 11, 1087–1095. (46) Uno, T.; Ryu, D.; Tsutsumi, H.; Tomisugi, Y.; Ishikawa, Y.; Wilkinson, A. J.; Sato, H.; Hayashi, T. J. Biol. Chem. 2004, 279, 5886–5893. (47) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335–59.

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Scheme 1. (A) Fc(CH2)16SH-modified electrode. (B) Fc-Im2/ Ni(II)-NTA-thiol-modified electrode

Figure 1. (A) Cyclic voltammograms of Fc-Im2/Ni(II)-NTA-

the Fc-Im2 redox probe to the free coordination site of the Ni(II)-NTA complex (Scheme 1). For such a purpose, the Ni(II)-NTA-modified electrode was immersed in a 5 μM FcIm2 aqueous solution (pH 7.4) and scanned by cyclic voltammetry. Under such dilute conditions, the voltammetric contribution of the ferrocene probe free to diffuse in solution is negligible compared with that of Fc-Im2 chelated to the Ni(II)-NTA complex on gold. The binding kinetics of Fc-Im2 was followed by measuring the reversible wave of ferrocene as a function of immersion time into the Fc-Im2 solution. A maximal faradaic current response was obtained after 10 min. Once the equilibrium binding was reached, the reversible wave of Fc-Im2 chelated to the Ni(II)-NTA SAM was stable for more than 2 h and does not show any significant variation in peak height or shape over repetitive scans. Typical cyclic voltammograms recorded at the equilibrium binding and different scan rates (v) are shown in Figure 1A. At a low potential scan rate of 0.2 V 3 s-1, a remarkably well defined symmetrical pair of redox peaks, characteristic of the Fc-Im2/Fc-Im+ 2 couple, is observed at E 00 = 0.39 V versus SCE. An analysis of the anodic and cathodic peak currents as a function of the scan rate shows a linear dependence, as expected for a surface-confined electroactive center (Figure 1B). At 0.2 V 3 s-1, the peak-to-peak potential separation (ΔEp) differs by less than 10 mV, and the fwhm for each peak was 110 ( 5 mV, which is relatively close to that expected theoretically for an ideal Nernstian redox species adsorbed on an electrode surface (i.e., ΔEp = 0 mV and a fwhm value of 89 mV at 20 °C).48 Such nearly ideal behavior suggests few interactions between the immobilized ferrocene/ferricinium molecules and a highly homogeneous environment around the redox centers, a result that reflects somewhat the high degree of self-organization afforded by the selected immobilization strategy. This is a rather uncommon result compared with electroactive SAMs made of covalently attached ferrocene for which important peak width broadening and even peak dedoubling are commonly observed in aqueous buffer at similar ferrocene surface concentrations.49 An increase in the peak width broadening was nonetheless observed for the highest ferrocene coverages (Figure 2A), which is compatible with an increase in the interactions as the redox probe surface concentration is raised.50 From the integration of the anodic or cathodic peak current in Figure 1A, an equilibrium Fc-Im2 coverage (48) Laviron, E. J. Electroanal. Chem. 1979, 101, 19–28. (49) Lee, L. Y. S.; Sutherland, T. C.; Rucareanu, S.; Lennox, R. B. Langmuir 2006, 22, 4438–4444 and references therein. (50) Laviron, E.; Roullier, L. J. Electroanal. Chem. 1980, 115, 65–74.

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modified gold electrodes recorded at 0.2, 0.5, 1, and 2 V/s in the presence of 5 μM Fc-Im2 in phosphate buffer (0.1 M NaPi, pH 7.4, T = 20 °C). Dashed red lines are the numerical simulations of cyclic voltammograms based on Laviron’s model involving interactions between the immobilized molecules: ΓFc, eq = 1.05  10-10 mol/cm2, E 00 = 0.39 V, S = 0.0246 cm2, R = 0.5, kET = 80 s-1, β = -0.3, γ = 0.5, λ = 0, μ = 0. (B) Variation of the anodic (9) and cathodic (O) peak intensities as a function of the scan rate. The solid red line shows the linear regression fitting.

Figure 2. (A) Cyclic voltammograms of Fc-Im2/Ni(II)-NTA-

modified gold electrodes recorded at 0.1 V 3 s-1 in phosphate buffer (0.1 M NaPi, pH 7.4, T = 20 °C) containing (black line) 5 or (red line) 50 μM Fc-Im2. Contributions from diffusing ferrocene were subtracted. (B) Binding isotherm of Fc-Im2 at a Ni(II)-NTA SAM on a gold electrode. Surface concentrations of Fc-Im2 were obtained from the integration of the voltammetric anodic and cathodic waves recorded at 0.1 V 3 s-1, and the resulting values were corrected from the electrode roughness factor. The solid red line is the fit to a Langmuir isotherm.

of 1.05  10-10 mol 3 cm-2 could be estimated (corrected from a gold surface roughness factor of 2). This value corresponds to ca. 23% of the theoretical maximal coverage that can be calculated for hexagonal close-packing of 6.6-A˚-diameter ferrocene spheres (i.e., 4.5  10-10 mol 3 cm-2).51 The apparent binding constant (Kapp) of Fc-Im2 for the Ni(II)-NTA-terminated SAM monolayer and the maximum Fc-Im2 surface coverage (Γsat Fc ) were determined from the plot of the binding isotherm measured at pH 7.4 and 20 °C (Figure 2B). The variation in the surface coverage of Fc-Im2 with its concentration in solution follows a simple Langmuir (51) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. J. Am. Chem. Soc. 1990, 112, 4301–6.

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isotherm according to eq 1. Γeq, Fc ¼

Γsat Fc Kapp ½Fc-Im2  1 þ Kapp ½Fc -Im2 

ð1Þ

where Γeq,Fc is the equilibrium surface concentration of Fc-Im2. -10 mol 3 cm-2 and Kapp = The best fit gave Γsat Fc = 1.63  10 sat 5 -1 3.7  10 M . The value of ΓFc thus corresponds to ∼36% of the theoretical maximal coverage calculated for a close-packed monolayer of ferrocene heads and to ∼45% of the overall NTA-thiol molecules attached to the gold surface, assuming 1:1 stoichiometry between Fc-Im2 and the Ni(II)-NTA complex. It also means that up to nearly half of the NTA ligand of the SAM can be loaded with NiII, an amount that is slightly lower than the value of 70% previously measured by us for CuII at the same NTA-modified gold electrode.42 Such a loading difference between the two metal ions is in accordance with the lower affinity of NiII for the NTA ligand. At unmodified gold electrodes, the standard potential of FcIm2 in homogeneous solution was E 0 = 0.37 V versus SCE. Once specifically immobilized onto the modified electrode, the redox potential of Fc-Im2 was slightly shifted in the anodic direction (+20 mV, E 00 = 0.39 V), suggesting the stabilization of the reduced state relative to the oxidized state. Such an effect may arise from two opposite influences. First, because the metalation of the NTA monolayer is partial, the SAM exhibits many uncomplexed negatively charged carboxylate ions that stabilize the neighboring ferrocenium ions. Second and most likely predominant, the binding of Fc-Im2 to the Ni(II)-NTA complex is achieved through coordination of the imidazole substituents of Fc-Im2 (Scheme 1), which may decrease the electronic density and, in turn, destabilize the oxidized state of ferrocene. At high scan rates (v > 0.5 V 3 s-1), the peak potential difference between the anodic and cathodic waves increases, reflecting progressive kinetic control by the rate of electron transfer of the ferrocene units (Figure 3). From the shift of the anodic and cathodic peak potentials as a function of the scan rate, the ET rate constant kET was then estimated using the Laviron method.48 Positions of the peak potentials as a function of the scan rate are represented in Figure 3. Because the electrochemical behavior of the ferrocene probe was close to ideal, the data were first fitted to theoretical peak potential values calculated using the classical Butler-Volmer equations for a surface-confined redox reaction (dashed lines). Using a ferrocene charge-transfer coefficient R of 0.5, the best fit gave a kET value of 80 s-1. However, the fitting was not perfect on the cathodic branch, a result that discloses the occurrence of interactions between the immobilized redox probes. To take into account these slight interactions, we have simulated the voltammograms using the general expression developed by Laviron for a surface-confined redox reaction with interaction between the immobilized molecules.50 In the simulation of the voltammetric peaks, we have assumed the same value of kET (i.e., 80 s-1) and have adjusted the four interaction parameters β, γ, λ, and μ so as to give the best-fit curves (Figure 1A). The peak potentials of the simulated voltammograms were reported in Figure 3, showing improved fitting on the cathodic branch (solid lines). The question that was addressed with the measured value of kET for the Fc-Im2 probe through the Ni(II)-NTA SAM was how it compares with other long-range ET through SAM, assuming that the metal Ni center within the linker does not significantly influence the electron-transfer tunnelling.52 Among the diverse redox probes that have been used to study the (52) The Ni(II) center is not redox-active within the potential window of the solvent and so cannot be involved in the redox hopping mechanism.

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Figure 3. Positions of the anodic (9) and cathodic (O) peak potential as a function of the scan rate. Dashed blue lines: theoretical curves calculated from the Buttler-Volmer equations for a surface-confined redox reaction.48 Solid red lines: theoretical curves calculated from the Laviron’s model involving interactions between the immobilized molecules50 (ΓFc,eq = 1.05  10-10 mol/ cm2, E 00 = 0.39 V, S = 0.0246 cm2, R = 0.5, kET = 80 s-1, β = -0.3, γ = 0.5, λ = 0, μ = 0). The overall data were collected from cyclic voltammograms recorded at several Ni(II)-NTAmodified gold electrodes immersed in a buffer solution (0.1 M NaPi, pH 7.4, T = 20 °C) containing 5 μM Fc-Im2.

long-range ET kinetics through SAMs of alkanethiol, the ferrocene molecule was undoubtedly the most investigated.19,53-58 Using this ideal outer sphere one-electron transfer probe covalently attached to the extremity of alkanethiols SAMs, it was established that the ET rate constant through a SAM exhibits an exponential decay with the increase of the SAM thickness (L). A number of studies have demonstrated an exponential decay constant ( β = -(d ln[kET])/dL) of ∼1 A˚-1 through SAMs of alkanethiols.54,55,58 On account of the numerous kET values reported in the literature for different lengths and natures of SAMs, the ferrocene probe can then be used as a standard to evaluate the long-range ET distance through our SAM coating. By reporting the kET value determined for Fc-Im2 (i.e., 80 s-1) on the linear plot of Figure 4 in ref 54 (representing ln kET vs the bridge length l59), an electron-transfer distance of ∼19 A˚ was deduced. This distance is consistent with the theoretical molecular length of the Fc-Im2/Ni(II)-NTA-terminated thiol molecule (i.e., ∼23 A˚) used in this work (Scheme 1). It is also comparable to the length of a SAM made of HS-(CH2)16Fc (20 A˚) for which a kET value of 28 s-1 was determined by cyclic voltammetry.60

(53) Kiani, A.; Alpuche-Aviles, M. A.; Eggers, P. K.; Jones, M.; Gooding, J. J.; Paddon-Row, M. N.; Bard, A. J. Langmuir 2008, 24, 2841–2849. (54) Chidsey, C. E. D. Science 1991, 251, 919–22. (55) Smalley, J. F.; Finklea, H. O.; Chidsey, C. E. D.; Linford, M. R.; Creager, S. E.; Ferraris, J. P.; Chalfant, K.; Zawodzinsk, T.; Feldberg, S. W.; Newton, M. D. J. Am. Chem. Soc. 2003, 125, 2004–2013. (56) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Chem. Soc., Faraday Trans. 1997, 93, 1367–1370. (57) Murgida, D. H.; Hildebrandt, P. J. Am. Chem. Soc. 2001, 123, 4062–4068. (58) Liu; Paddon-Row, M. N.; Gooding, J. J. J. Phys. Chem. B 2004, 108, 8460– 8466. (59) The bridge length l is the shortest distance between the carbon attached to the sulfur and the linked carbon of the cyclopentadiene ring of the ferrocene. For the aliphatics, it is assumed that L = 2.1 + l cos(30°)A˚.55 (60) Weber, K.; Creager, S. E. Anal. Chem. 1994, 66, 3164–72.

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Figure 4. RR spectra of the oxidized (A) and reduced (E) forms of his-tagged hNb in homogeneous solution (0.1 M NaPi, pH 7.4) measured under Soret excitation at λexc = 406.7 nm. SERR spectra (λexc = 406.7 nm) of his-tagged hNb immobilized on different SERactive Ag electrodes recorded in a phosphate buffer (0.01 M NaPi, 0.05 M KCl, pH 7.4) and under diverse electrode polarization (Eappl): (B) Ni(II)-NTA-SAM-coated Ag electrode under Eappl = 0.1 V; (C) bare Ag electrode under Eappl = 0 V; (D) NTA-SAM-coated Ag electrode in the absence Ni2+; (F) Ni(II)-NTA-SAM-coated Ag electrode under Eappl = -0.8 V.

After having demonstrated the good binding and ET properties of the Fc-Im2 redox probe at the NTA-SAM-coated gold electrode, we examined those of his-tagged hNb. For such a purpose, the Ni(II)-NTA-modified gold electrodes were soaked in a 1 μM hNb solution for 1 h and subsequently scanned by cyclic voltammetry in a deaerated protein-free buffer solution. We have selected this protein concentration on the basis of our previous work with Ni(II)-NTA-modified electrodes41,42 wherein saturated monolayer coverage was reached for histagged protein concentrations higher than 0.5 μM. Electrode saturation can be achieved at such a low protein concentration because affinity binding constants toward his-tagged proteins (107-108 M-1)41,42 are much higher than those of the Fc-Im2 redox probe (3.7  105 M-1). Despite repetitive voltammetric experiments at different modified electrodes and different scan rates (i.e., ranging from 0.01 to 1 V 3 s-1), we were unable to distinguish any oxidation and/or reduction wave in the accessible potential window of the modified electrode (i.e., between -0.8 and +0.6 V vs SCE), and only the capacitive current of the dielectric layer was finally measured.61 Even in the presence of O2, which is known to bind and oxidize the reduced prosthetic group of hNb, there was no discernible catalytic response upon scanning the electrode in the cathodic direction. Several hypotheses can be advanced to explain the absence of faradaic current in cyclic voltammetry. First, protein binding does not occur or is so inefficient that the surface concentration of the redox protein (61) The capacitive current of the gold electrodes is strongly decreased upon SAM assembly. The capacitance of the Ni(II)-NTA-SAM-modified gold electrode is ca. 4 μM 3 cm-2, whereas it is 30 μM 3 cm-2 for a bare gold electrode.

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Article

is too low to be electrodetectable. Second, the immobilized protein is not or is poorly electronically coupled to the electrode so that the ET rate is too low for appreciable electrochemistry to be observed. Third, during immobilization, the redox protein is denaturated and hence its redox properties are lost. With the aim of demonstrating that an effective binding of his-tagged hNb occurs at the NTA-modified electrode and that the native structure of the immobilized protein is preserved, we have characterized the hNb/Ni(II)-NTA SAM by SERR spectroscopy. For such a purpose, roughened SER-active silver electrodes were used and functionalized by the Ni(II)-NTA-SAM using the same procedure as for gold electrodes. Spectroscopic Characterization of Immobilized hNb. The reference resonance Raman (RR) spectra of the oxidized FeIII and dithionite-reduced FeII forms of his-tagged hNb were first recorded in homogeneous solution. The spectra shown in Figure 4 (spectra A and E) are identical to those previously reported in the literature, showing characteristic in-plane vibrational modes of the heme.46 The preparation of SER-active Ag electrodes and their modification by a self-assembled monolayer of N-(5-([1,2dithiolane-3-pentanoylamino]-1-carboxypentyl)iminodiacetic acid are described in the Experimental Section. After Ni(II) complexation, the immobilization of his-tagged hNb was achieved by soaking the electrodes in a 1 μM protein solution for 1 h. The Raman experiments were subsequently performed in a protein-free buffer solution. To be sure that the immobilization strategy lead to a sufficiently stable protein monolayer during Raman experiments, the binding kinetics of his-tagged hNb (0.2 μM in solution) at a Ni(II)-NTA-modified Ag electrode was monitored by SERR spectroscopy (Experimental Section). A maximal, stable SERR intensity was reached after 45 min and, once equilibrium was established, the signal was stable for at least 2 h. A typical SERR spectrum recorded at a hNb/Ni(II)-NTAmodified Ag electrode, under 0.1 V of electrode polarization, is presented in Figure 4 (spectrum B). The spectrum shows characteristic bands of heme, signifying the presence of hNb on the electrode surface. In spite of the use of a moderately long NTA-thiol (i.e., equivalent to a C16 alkanethiol SAM), spectra with high intensity and good quality were systematically recorded. This is in contrast to the low surface enhancement of RR signals reported for cyt c adsorbed on a carboxylic-terminated SAM containing 16 methylene groups.23 This observation suggests that the selected binding strategy leads to high protein coverages on the SER-active NTA-modified Ag electrode. The SERR spectrum in Figure 4B is also very analogous to the RR spectrum of native hNb in Figure 4A. In the high-frequency region, the SERR spectrum contains several bands that are particularly diagnostic of the redox state, spin, and coordination pattern of the heme iron. The coordination/spin state-sensitive ν2 and ν3 bands and the redox state ν4 marker band observed at 1578, 1504, and 1373 cm-1, respectively, are typical of heme iron in an oxidized six-coordinated (6c) low-spin (LS) state.62 All of these observations show that essentially no major structural change of the heme pocket occurs upon immobilization of histidine-tagged hNb and that the bis-imidazole structure of the FeIII ion is maintained. Upon application of a reducing potential at -0.68 V, the ν2, ν3, and ν4 bands were shifted to 1581, 1491, and 1362 cm-1, respectively (spectrum F in Figure 4). These frequencies are (62) Spiro, T. G.; Li, X.-Y. Biological Applications of Raman Spectroscopy; Wiley Interscience: New York, 1988; Vol. III.

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characteristic of a fully reduced 6-coordinated low-spin iron heme and are very similar to those recorded in solution for the reduced FeII form of hNb (Figure 4, spectrum E). This result strongly supports direct ET between the immobilized protein and the underlying SER-active Ag electrode. As in the case of the oxidized form of the protein, the great similarity between SERR and RR spectra of the reduced form of hNb indicates that no major structural modification of the heme pocket occurs upon reduction of the immobilized hNb and that the bis-imidazole coordination around the FeII ion is maintained. Moreover, the sharp ν4 band at 1362 cm-1 shows that no protein remains in the oxidized state after a few seconds of cathodic polarization, suggesting that all of the immobilized proteins are capable of direct electronic communication with the electrode. The specificity of the immobilization process was controlled and found to be excellent because no characteristic SERR band of hNb could be observed when no Ni(II) ions were coordinated to the NTA SAM (Figure 4, spectrum D). This was not the case at the bare Ag electrode because a SERR spectrum with characteristic heme bands was recorded, signifying that histagged hNb could adsorb nonspecifically onto the metallic Ag electrode. However, the SERR spectrum of the nonspecifically adsorbed protein (spectrum C in Figure 4) strongly differs from the RR spectrum of hNb in homogeneous solution (Figure 4, spectrum A). The bands attributed to the oxidation (ν4), spin, and coordination states (ν3, ν2) are located at 1371, 1491, and 1572 cm-1, respectively, which are characteristic of a fivecoordinated (5c) high-spin (HS) FeIII ion and analogous to that reported for immobilized microperoxidase 8.63 This result revealed that, in spite of the well-known high stability of hNb in homogeneous solution (especially toward elevated temperatures64 or high urea concentrations), its direct adsorption on a rough metallic surface leads to important structural changes in the heme iron(III) environment, most likely induced by partial or complete denaturation of the protein as previously reported for several other heme proteins adsorbed on similar surfaces.65 Redox Titration of Immobilized Neuroglobin. Spectroelectrochemical titrations of hNb were performed both for the protein in solution and for the protein immobilized on a SERactive NTA-modified Ag electrode. Figure 5A shows the UV-visible spectroelectrochemical titration of his-tagged hNb in homogeneous solution and in the presence of a one-electron mediator. Fully reversible oxidation-reduction without hysteresis was observed in the UV-visible absorption spectra, with a well-defined isosbestic point at 416 nm. The Nernst equation (eq 22) was fitted to the experimental plot shown in Figure 5B (curve O). ½red 1 ¼ ½ox þ ½red 1 þ enF=RTðE -E°0 Þ

ð2Þ

The excellent fit to the experimental data gives E 00 = -0.340 ( 0.010 V and n = 1.05. The latter is in agreement with the one electron involved in the FeIII/FeII heme couple. As observed for the protein in homogeneous solution, the direct electrochemical transformation of the immobilized hNb by varying the applied potential from 0.1 to -0.8 V was a fully reversible process that could be performed several times without degrading the protein-modified SAM electrode. It was thus (63) Lecomte, S.; Ricoux, R.; Mahy, J. P.; Korri-Youssoufi, H. J. Biol. Inorg. Chem. 2004, 9, 850–858. (64) Hamdane, D.; Kiger, L.; Dewilde, S.; Uzan, J.; Burmester, T.; Hankein, T.; Moens, L.; Marden, M. C. FEBS J. 2005, 272, 2076–2084. (65) Smulevich, G.; Spiro, T. G. J. Phys. Chem. 1985, 89, 5168–73.

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Figure 5. (A) Oxidative titration of hNb in solution monitored by UV-visible spectroelectrochemistry. (B) Fraction of ferrous form as a function of the potential applied during (O) spectroelectrochemical titration of hNb in solution and (9) SERR spectroscopic titration (λexc = 406.7 nm) of hNb immobilized on a Ni(II)-NTASAM Ag electrode (the data values were obtained from three independent measurements with a standard error of less than 10 mV). Dashed black lines: fits to the Nernst equatio (eq 22). Red line: fit to eq 33, assuming a square distribution of E 00 . (C) Oxidative titration of hNb immobilized on a Ni(II)-NTA-SAM Ag electrode monitored by SERR spectroelectrochemistry. The Soret excitation is λexc = 406.7 nm and Eappl is (a) 0.1, (b) -0.4, (c) -0.5, and (d) -0.7 V. All experiments were carried out at T = 20 °C.

possible to perform a spectroelectrochemical SERR titration of hNb using the same hNb/Ni(II)-NTA-modified Ag electrode (Figure 5C). A typical experimental plot obtained from the deconvolution of the ν4 band is shown in Figure 5B (curve 9). From fitting to the Nernst equation (eq 22), reproducible E 00 (-0.440 ( 0.010 V) and n (0.45) values were obtained. These values were independent of the anodic or cathodic direction of the titration, indicating that for each potential value the laser exposure had no influence on the ratio of reduced over oxidized forms of the protein. However, the number n significantly deviates from its theoretical value of 1. Similar behavior was reported for microperoxidase 8 or cyt c adsorbed on carboxylicterminated SAM Ag electrodes, and it was ascribed to a dispersion of protein orientations that leads to a redox potential distribution.63,66 In the present case, binding of the protein via the his-tag may constrain a specific protein orientation toward the SAM. Therefore, the most likely hypothesis is to consider different interactions between the protein and the SAM due to the nonhomogeneous SAM chemical composition. As depicted in Scheme 2, the NTA ligand exhibits at least three different chemical environments in the SAM: (i) uncomplexed, (ii) complexed by the NiII ion and most likely water molecules to complete the metal coordination sphere, and (iii) complexed by the NiII ion and the histidine-tag of hNb. This statement is based on the respective surface concentrations of NTA-thiol molecules (350 pmol 3 cm-2), NiII ions (165-250 pmol 3 cm-2), and his-tagged hNb (theoretically 16 pmol 3 cm-2 for a saturated monolayer assuming for Nb a close packing of 3.5-nm-diameter spheres). The chemical heterogeneity of the SAM can thus be at the origin of the heterogeneous environment of the bound protein and therefore of the distribution of the apparent redox potentials of the heme. The simplest way to introduce a potential distribution into the Nernst equation is to consider a square distribution centered on an average E 00 value with a width of (ΔE. For such a distribution, the relative concentration of reduced proteins as a function of the applied potential E is (66) Murgida, D. H.; Hildebrandt, P. J. Mol. Struct. 2001, 565-566, 97–100.

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Scheme 2. Model for immobilized Fc-Im2 and hNb (PDB entry 1OJ6) on a Ni(II)-NTA-SAM electrode. The chemical heterogeneity of the SAM is depicted by assuming three different environments of the NTA ligand. The positively charged residues are presumed to interact with the negatively charged NTA monolayer extremity, as highlighted by the space-filling model

given by eq 3: ½red 1 ¼ ½ox þ ½red 2ΔE

Z

0

E 0 þΔE

E 00-ΔE

dE i 1 þ e þnF=RTðE -E i Þ

¼ 00

RT 1 þ e -nF=RTðE -E -ΔEÞ ln ð3Þ 0 2ΔEnF 1 þ e -nF=RTðE -E 0 þΔEÞ Equation 3 was then used to fit the SERR spectroelectrochemical titration data of Figure 5B, assuming that n = 1. The best fit gave an average apparent redox potential of E 00 = -0.440 V and a ΔE value of 0.135 V. Whatever the method used to extract E 00 , the apparent standard potential of immobilized hNb is significantly downshifted by 100 mV compared with that measured in homogeneous solution. Negative E 00 shifts of similar magnitude have been reported for cyt c binding on anionic surfaces and are attributed to a stronger interaction of the oxidized protein with the SAM than the reduced form.67 In a recent study on tetraheme cytochrome c3, it was also shown that the impact of the SAM charge on the heme redox potential is strongly dependent on the heme-SAM distance.68 By analogy, we propose that the negative shift of the redox potential observed for the immobilized hNb pinpoints a specific protein orientation, in which the heme pocket is brought close to the SAM surface (Scheme 2). This hypothesis is consistent with the large dispersion of E 00 and supports favorable electrostatic and hydrogen-bonding interactions between the heme pocket and the heterogeneous chemical environment at NTA surface. These (67) Petrovic, J.; Clark, R. A.; Yue, H.; Waldeck, D. H.; Bowden, E. F. Langmuir 2005, 21, 6308–6316. (68) Rivas, L.; Soares, C. M.; Baptista, A. M.; Simaan, J.; Di Paolo, R. E.; Murgida, D. H.; Hildebrandt, P. Biophys. J. 2005, 88, 4188–4199.

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interactions may also involve the heme propionates, for which the environment is known to influence the heme redox potential strongly.69,70 It has been proposed that the potential drop across the SAM may be responsible for the potential shift of the redox potential of immobilized cyt c,23 but this hypothesis is unlikely because the potential drop can affect only the electron-transfer kinetics but not the apparent standard potential measured, here under thermodynamic equilibrium conditions. This is analogous to the effect of the potential drop in a double layer (Frumkin effect).71 Determination of the Heterogeneous Electron-Transfer Rate. After having characterized the thermodynamics of immobilized hNb and established that direct electron transfer occurs between the protein and the electrode, we have next determined the rate of heterogeneous electron transfer. For such a purpose, we have used time-resolved surface-enhanced resonance Raman spectroscopy (TR-SERRS, Experimental Section). Figure 6 shows a selection of the TR-SERR spectra in the region of the ν4 mode, which is a sensitive marker for the oxidation state of the heme iron. Spectrum A corresponds to the stationary SERR spectrum recorded at an hNb/Ni(II)-NTAcoated Ag electrode polarized at 0 V (the hNb is in its fully oxidized state). Immediately after stepping the electrode potential from 0 to -0.68 V, we observed with increasing delay time δ a decrease in the prominent peak located at 1373 cm-1 (characteristic of the oxidized protein) at the expense of the appearance of a new one located at 1361 cm-1 (characteristic of the (69) Voigt, P.; Knapp, E.-W. J. Biol. Chem. 2003, 278, 51993–52001. (70) Rivera, M.; Seetharaman, R.; Girdhar, D.; Wirtz, M.; Zhang, X.; Wang, X.; White, S. Biochemistry 1998, 37, 1485–1494. (71) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; 2nd ed.; John Wiley & Sons: Hoboken, NJ, 2001.

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Figure 6. (A) Stationary and time-resolved SERR spectra of hNb

immobilized on a Ni(II)-NTA-SAM Ag electrode using λexc = 406.7 nm. The stationary SERR spectrum was measured at an initial potential of Ei = 0 V, and the time-resolved SERR spectra were acquired after a potential step from Ei = 0 V to Ef = -0.68 V and delay times δ of 30, 120, and 500 ms (T = 20 °C; 0.01 M NaPi, 0.05 M KCl, pH 7.4). (B) Fraction of oxidized hNb as a function of delay time δ. The data were fitted to an exponential decay (Experimental Section).

reduced protein). The ν4 bands recorded at different delay times were normalized and deconvoluted from the complete component spectra of the various species as determined in stationary experiments. This leads to the plot in Figure 6 representing the ratio of oxidized protein as a function of time. The resulting kinetic curve was fitted to an exponential decay corresponding to a one-step relaxation process, allowing us to deduce an apparent rate constant of τ = (70 ( 10)  10-3 s. The kET value was finally obtained from the Butler-Volmer equation (eq 4) after correction from the applied overpotential. 0 1 nF ¼ kET ½e -Rξ þ eð1 -RÞξ  with ξ ¼ ðE -E 0 Þ τ RT

ð4Þ

Because of the implication of a distribution of redox potentials centered on an average E 00 , the rate constant derived is an 00 average apparent rate constant of kapp ET . Assuming an average E of -0.44 V, a theoretical n value of 1, and R = 0.5, we finally -1 obtained an apparent ET rate constant of kapp ET = 0.12 s . On the basis of the electron-transfer rate constants determined for both the immobilized Fc-Im2 and hNb and assuming in each case that the one-electron transfer proceeded in a fully nonadiabatic regime and through an outer-sphere mechanism with equivalent reorganization free energies, we can roughly estimate the heme-electrode distance in the neuroglobin assemblage. Considering an exponential decay factor of 1.0 A˚-1, the 660-fold decrease of kET may be caused by an extra electrontransfer distance of ∼7 A˚. This value is quite small considering the crystallographic dimensions of neuroglobin (ellipsoidal protein of 25  35  50 A˚3), but in agreement with the closest approach of the heme iron center to the surface of the neuroglobin, which is 8 A˚.45 Thus, an extra distance of 7 A˚ between Fc-Im2 and hNb most likely indicates that the hemoprotein is lying flat on the monolayer rather than stretched normal to the SAM interface (Scheme 2), as also reported for cyt c bound to anionic SAM.72,73 This remark provides further support for the hypothesis that the negative shift of E 00 observed in the SERR (72) Yu, Q.; Golden, G. Langmuir 2007, 23, 8659–8662. (73) Nakano, K.; Yoshitake, T.; Yamashita, Y.; Bowden, E. F. Langmuir 2007, 23, 6270–6275.

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titration curve should result from close interaction of the protein with the negatively charged NTA monolayer. Moreover, the kapp ET value determined for immobilized hNb is within the range of values determined by cyclic voltammetry or TR-SERR spectroscopy for analogous small redox proteins (e.g., cyt c) adsorbed to carboxylic-terminated alkanethiol SAMs of similar length (Table 1).19,57 Considering the proposed orientation of hNb, one may notice that several positively charged amino acids (Arg47, 66, and 94 and Lys95) located near the heme pocket point to the solvent and may therefore interact with the negatively charged NTA SAM (Scheme 2). Assuming this orientation, the heme propionates would be located near the NTA extremities and should therefore be very sensitive to their local environment. Small variability of the propionates’ orientation toward the SAM surface together with variability of the local charge of the surface may be responsible for the large distribution of E 00 observed by spectroelectrochemistry. On the basis of the results obtained in this work, an important question remains as to why no voltammetric response of hNb could be observed at the NTA-modified gold electrode whereas direct electron transfer was clearly demonstrated at the SERactive NTA-modified Ag electrode. This question has pushed us to examine the SER-active NTA-modified Ag electrode in cyclic voltammetry.74-76 However, no significant faradaic current could be discernible at this electrode. Such a result is unexpected because the apparent ET rate constant of 0.12 s-1 determined by time-resolved SERR spectroscopy is not especially slow and not very different from that of other small redox proteins immobilized on SAMs of similar thickness and for which a distinct reversible wave was observed in cyclic voltammetry (Table 1).19 A low protein surface concentration could explain such a result, but the very intense SERR spectra that have been obtained do not favor this hypothesis. Moreover, we have previously shown using similar immobilization conditions that a saturated his-tagged protein monolayer could be reached at NTA-modified electrodes.41,42 For example, a saturation coverage of Γsat = 5 pmol cm-2 was obtained for his-tagged laccase (60-90 kDa) immobilized at the same NTA SAM coating as that used in this work. At such a relatively high protein coverage and assuming direct electron coupling with all immobilized protein, a distinct reversible or quasi-reversible wave of hNb would thus be observed in cyclic voltammetry recorded at a sufficiently low scan rate. One hypothesis that may explain the undetectable faradaic response in cyclic voltammetry is the large dispersion of E 00 , as revealed in the SERR titration curve. The large potential distribution should result in considerable voltammetric wave broadening with a concomitant faradaic current decrease. Such a phenomenon was previously reported for cyt c immobilized on carboxylic-terminated alkanethiol SAM.33 For example, it showed a 40% decrease in current density for a 40 mV Gaussian width distribution of the redox potential. In our case, the total width of ∼270 mV is far larger and might be the cause for a strong extinction of the voltammetric signal. (74) Leopold, M. C.; Bowden, E. F. Langmuir 2002, 18, 2239–2245. (75) Brolo, A. G.; Irish, D. E.; Szymanski, G.; Lipkowski, J. Langmuir 1998, 14, 517–527. (76) With the aim of being sure that the electrode surface topography cannot influence the ET rate constant as already shown for cyt c through carboxylicterminated alkanethiol SAMs,74 we have repeated the experiments with highroughness gold electrodes. The latter were either polished on 1200 grit sandpaper or treated by repetitive scans in a KCl solution according to a typical roughening procedure used for the preparation of the SER-active surface of silver or gold.75 In all cases, the redox potential of the immobilized Fc-Im2 probe and the kET values were found to be independent of the electrode roughness factors (>2 for all electrodes).

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Table 1. Long-Range ET Rate Constants Determined by Cyclic Voltammetry (CV) and TR-SERR Spectroscopy for Different Ferrocenes and Hemoproteins Immobilized on Metallic Electrodes through SAM MW (kDa)

SAM composition

L (A˚)a

kET (s-1)

pH

technique

ref

Fc Fc-Im2 cyt c

-S-(CH2)16-Fc 20 28 CV 60 0.35 -S-C5-CO-LysNTA ∼19 80 7.4 CV this work 24 + 5b 0.073 7.0 TR-SERRS 57 12.3 -S-(CH2)16-COOH 24 + 5b 0.42 4.8 CV 19 -S-(CH2)16-COOH ∼26 0.12 7.4 TR-SERRS this work hNb 17 -S-C5-CO-LysNTA >40 0.002 8.0 TR-SERRS 38 CcO 68 -S-C2-CO-LysNTA a L corresponds to the distance between the electrode and the metal redox center. b 5 A˚ accounts for the distance between the protein surface and the heme iron center.73

Conclusion In this study, we have addressed the characterization of metallic electrodes modified with an NTA-thiol monolayer for the specific immobilization of histidine-tagged proteins under mild conditions. The Ni(II)-NTA-thiol modified electrodes were first characterized by using an immobilized ferrocene probe. High surface coverage was reached without significant interaction between the ferrocene molecules. Determination of the heterogeneous electron rate constant (kET = 80 s-1) allowed us to indirectly estimate a SAM thickness of ca. 19 A˚. The NTA-thiol modified electrodes were also used to perform immobilization of his-tagged neuroglobin. The resulting electrodes are stable enough to be studied for a few hours in a protein-free buffer at neutral pH. The absence of spectral change in the SERR spectrum of hNb immobilized on the Ni(II)-NTA SAM tends to confirm that the native structure of the immobilized protein is retained on such a modified surface probably because the immobilized protein is maintained on a sufficiently highly polar and hydrophilic environment at a distance far enough from the hydrophobic underlying metallic surface. Direct electronic communication between the electrode and the immobilized protein was evidenced by SERR spectroscopy but not by cyclic voltammetry. From SERR experiments, all of the immobilized proteins remain electroactive and nondenaturated in both redox states. Careful analysis of the spectroelectrochemical titration of the immobilized protein showed a large distribution of the heme redox potential, together with a global negative shift of ca. -100 mV compared to the value reported for the protein in solution. Both observations are consistent with an immobilized protein orientation where the heme pocket faces the SAM surface, allowing direct electron transfer within the heme redox center and the electrode. The obtained kET = 0.12 s-1 value accounts for an extra distance of ca. 7 A˚ in the case of hNb as compared to the ferrocene probe. Experimental Section Reagents. All reagents were purchased from Sigma-Aldrich. The phosphate buffer (0.1 M NaPi, pH 7.4) was prepared using a Milli-Q water purification system from Millipore. 1,10 -Bis(Nimidazolylmethyl)ferrocene (Fc-Im2),42 [OsII(bpy)pyCl](PF6,)77 and N-(5-([1,2-dithiolane-3-pentanoylamino]-1-carboxypentyl) iminodiacetic acid (NTA-thiol)41 were prepared as previously described. Electrode Preparation and Apparatus. The roughened SER-active Ag electrodes (ring electrodes) were prepared as described elsewhere.63 Prior to chemisorption of thiols, the polycrystalline gold electrodes (disk of 1 mm diameter) were polished to a mirrorlike finish with 3, 1, and 0.1 μm alumina slurry on microcloth pads. After the removal of trace alumina from the surface by rinsing with water and brief cleaning in an ultrasonic bath with ethanol and then water, the gold electrodes (77) Kober, E. M.; Caspar, J. V.; Sullivan, B. P.; Meyer, T. J. Inorg. Chem. 1988, 27, 4587–98.

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were electrochemically cleaned in 0.5 M H2SO4 by cycling the electrode potential between 0.2 and 1.8 V vs SCE until a reproducible voltammogram was obtained. An evaluation of the effective area of the gold electrode was carried out as previously reported,78 and a rugosity factor of ca. 2 could be reproducibly found. The general procedure used for electrode modification is as follows: the electrodes were placed in a solution containing 1 mM NTA-thiol in ethanol (filtered through a 0.45 μm Sartorius membrane) for one night at 4 °C. Nickel ion complexation was carried out at room temperature by immersing the NTA-modified electrodes in 1 mM NiCl2-buffered solution (0.1 M NaPi, pH 7.4) for 30 min. The electrodes were then rinsed with copious amount of buffer solution and directly used. Cyclic voltammetry was carried out with a PST 20 Autolab potentiostat (Eco-Chemie) interfaced with a PC. A saturated calomel electrode was employed as a reference electrode. The counter electrode was a platinum wire. Experiments were performed in a water-jacketed electrochemical cell maintained at 20 °C with a circulating water bath. All potentials in the text are quoted relative to the saturated calomel electrode (+0.24 V vs NHE). Protein Immobilization Procedures. hNb solutions were freshly prepared daily by purifying a stock solution with ionexclusion column chromatography (Amersham, PD10 desalting column). The hNb concentration was determined by UV-visible spectroscopy using a Soret extinction coefficient of ε412 = 120 mM-1 cm-1. For the binding of hNb, the Ni(II)-NTAmodified electrodes were immersed in a buffer solution (0.05 M NaPi, pH 7.4) containing 1 μM hNb for 1 h at room temperature.

UV-Visible Spectroelectrochemical Titration of hNb.

The redox-mediated spectroelectrochemical titration of hNb was performed in solution using a homemade two-compartment bulk electrolysis cell.41 The working electrode was a fine wire mesh gold minigrid (Goodfellow). A DRIREF-2 Ag/AgCl/KCl 3 M (World Precision Instruments) was used as the reference electrode (E0 = 0.21 V vs NHE, T = 20 °C). A platinum wire (1 mm diameter), separated from the bulk of the solution by a Vycor frit and Teflon heat-shrink tubing (Princeton Applied Research) filled with the buffer solution, was used as an auxiliary electrode. The three electrodes were inserted into a 3 mL quartz cell (1 cm path length) through a silicon cap that hermetically closes the cell. An additional tygon tube for degassing was introduced. The spectroelectrochemical cell was purged with argon during the entire experiment. An hNb concentration of 8 μM in a total volume of 1.4 mL of phosphate buffer solution (0.05 M NaPi, 0.05 M KCl, pH 7.4) was used. The titration was performed in the presence of a pool of redox mediators: Ru (NH3)6Cl3 (5 μM), indigotrisulfonate (5 μM), anthraquinone2,6-disulfonate (10 μM), and phenosafranin (5 μM). Electrolysis at controlled potential was carried out with a homemade potentiostat and under magnetic stirring. UV-visible spectral changes of the electrolysis solution were simultaneously monitored with an 8452 A diode array spectrophotometer (HewlettPackard). After each applied potential, the solution was left to (78) Anne, A.; Demaille, C.; Moiroux, J. Macromolecules 2002, 35, 5578–5586.

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equilibrate until two identical UV-visible spectra were recorded. The absorption spectrum at 0 V was identical to that of the starting ferric hNb, exhibiting a Soret band maximum at 412 nm. A potential of -0.6 V was applied for 20 min before oxidative titration from -0.5 to 0 V. After the oxidative titration, the potential was swept back to rereduce the protein (reductive titration). Resonance Raman Experiments. RR spectra of hNb in solution were measured under Soret excitation at λexc = 406.7 nm with a Kr+ laser. A laser power of 5 mW at the sample was systematically used. The spectra were recorded at ambient temperature with a Dilor XY spectrograph equipped with a liquidnitrogen-cooled CCD camera. The spectral resolution was 2.5 cm-1, and the wavenumber increment per data point was 0.5 cm-1. Solution experiments were performed in 50 μL containing 7 μM hNb. The reduced form of hNb was prepared anaerobically by adding dithionite (5 μL of a 1 mM solution) to the protein sample. Reported spectra were the result of the averaging of 12 single spectra recorded with a CCD exposure time of 60 s. SERR Experiments. SERR spectra were measured under Soret excitation at λexc = 406.7 nm. The excitation was carried out with a Kr+ laser and by applying a low laser power of 1 mW at the sample. The working electrode used for SERR experiments was a homemade rotating Ag cylinder electrode, constructed according to a previously published method.79 Platinum and saturated calomel electrodes were used as counter and reference electrodes, respectively. The spectroelectrochemical cell was filled with 25 mL of a phosphate buffer (0.01 M NaPi, 0.05 M KCl, pH 7.4) and was flushed with argon prior to and during the entire experiment. For the oxidized and reduced states of the protein, reported spectra were the result of the averaging of 12 single spectra recorded with a CCD exposure time of 30 s. Localization of the laser on the SERR electrode was modified for each spectrum because the spectral intensity decreased rapidly upon long laser exposure of the electrode. During the recording of the SERR spectra of the fully oxidized redox proteins, a continuous anodic potential and a low laser power were applied to avoid partial photoreduction of the protein in the laser beam. The SERR redox titration of immobilized neuroglobin was performed by using an EG&G 273A potentiostat (Princeton Applied Research). A potential step of (79) Hildebrandt, P.; Macor, K. A.; Czernuszewicz, R. S. J. Raman Spectrosc. 1988, 19, 65–9.

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0.1 or 0.05 V was applied, and the solution was left to equilibrate for 30 s before spectrum acquisition (30 s of CCD exposure time). The background was manually subtracted, and the ν4 band of the spectrum was deconvoluted from the spectral contribution of the oxidized and reduced forms of hNb by subtraction, giving the relative contribution of each form to the spectrum. Time-resolved SERR experiments were obtained by applying a rapid potential step (,100 μs) from an initial potential Ei to a final potential Ef and then by acquiring the SERR spectra after (variable) delay times δ (ranging within a time interval Δt of 10 or 20 ms). During the experiment, laser power excitation of 7.2 mW at the sample (λexc = 406.7 nm) was used. Then, the potential was set back to Ei to restore the initial equilibrium. The sequence was repeated 100 times so that the effective total accumulation time NΔt was for the total duration of 1 to 2 s, as required for a sufficient signal-to-noise ratio. The time resolution was controlled by gated excitation rather than gate signal detection. A Pockel cell, synchronized with the potential jump trigger, achieved the gating of the laser beam. The background was manually subtracted, and the ν4 band of each spectrum was completely removed by subtraction of the spectra of the oxidized and reduced forms of the protein, giving the relative contribution of each species to the spectrum. The data were analyzed by assuming a first-order rate reaction using the following equation: fraction FeIII = A exp(-t/τ) + B (where τ is the time constant, and A and B are the FeIII fractions at E = 0 and -0.68 V, respectively). Determination of the Protein Kinetic Binding. A SERactive electrode was functionalized with the Ni(II)-NTA SAM and then immersed in the SERR spectroscopic cell filled with 25 mL of phosphate buffer (0.025 M NaPi, 0.05 M KCl, pH 7.4) containing 0.2 μM hNb. A potential at 0 V was continuously applied to maintain the protein under its ferric form and to avoid photoreduction in the laser beam. The 1 min acquisition procedure was performed every 5 min, and the intensity of the ν4 band at 1373 cm-1 was selected as the analytical response for estimating the protein coverage kinetics. According to the heterogeneity of the SER electrode surface, we assume 10% error in the band intensity.

Acknowledgment. We thank Dr. Laurent Kiger (INSERM U779) for the generous gift of the N-terminal histidine-tagged human neuroglobin.

Langmuir 2009, 25(11), 6532–6542