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In situ STM Imaging and Direct Electrochemistry of Pyrococcus furiosus Ferredoxin Assembled on Thiolate-Modified Au(111) Surfaces Jingdong Zhang, Hans E. M. Christensen, Bee Lean Ooi, and Jens Ulstrup* Department of Chemistry, Building 207, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark Received May 10, 2004. In Final Form: August 10, 2004 We have addressed here electron transfer (ET) of Pyrococcus furiosus ferredoxin (PfFd, 7.5 kDa) in both homogeneous solution using edge plane graphite (EPG) electrodes and in the adsorbed state by electrochemistry on surface-modified single-crystal Au(111) electrodes, This has been supported by surface microscopic structures of PfFd monolayers, as revealed by scanning tunneling microscopy under potential control (in situ STM). Direct ET between PfFd in phosphate buffer solution, pH 7.9, and EPG electrodes is observed in the presence of promoters. Neomycin gives rise to a pair of redox peaks with a formal potential of ca -430 mV (vs SCE), corresponding to [3Fe-4S]1+/0. The presence of an additional promoter, which can be propionic acid, alanine, or cysteine, induces a second pair of redox peaks at ∼-900 mV (vs SCE) arising from [3Fe-4S]0/1-. A robust neomycin-PfFd complex was detected by mass spectrometry. The results clearly favor an ET mechanism in which the promoting effect of small organic molecules is through formation of promoter-protein complexes. The interaction of PfFd with small organic molecules in homogeneous solution offers clues to confine the protein on the electrode surface modified by the same functional group monolayer and to address diffusionless direct electrochemistry, as well as surface microstructures of the protein monolayer. PfFd molecules were found to assemble on either mercaptopropionic acid (MPA) or cysteine-modified Au(111) surfaces in stable monolayers or submonolayers. Highly ordered (2x3 × 5)R30° cluster structures with six MPA molecules in each cluster were found by in situ STM. Individual PfFd molecules on the MPA layer are well resolved by in situ STM. Under Ar protection reversible cyclic voltammograms were obtained on PfFd-MPA/Au(111) and PfFd-cysteine/Au(111) electrodes with redox potentials of -220 and -201 mV (vs SCE), respectively, corresponding to the [Fe3S4]1+/0 couple. These values are shifted positively by 200 mV relative to homogeneous solution due to interactions between the promoting layers and the protein molecules. Possible mechanisms for such interactions and their ET patterns are discussed.

1. Introduction Ferredoxins constitute a large family of redox proteins with iron-sulfur clusters as the redox center and with important biological roles in electron transfer (ET) and catalysis.1,2 ET patterns of ferredoxins (Fd) are, however, not straightforward due to their multiple-step nature. Electrochemistry has offered effective means to ET features of Fd’s. Direct electrochemistry of iron-sulfur proteins has been extensively studied either in homogeneous solutions or in the adsorbed state,3,4 particularly by Armstrong and associates.4-8 In the present work, we address one of the Pyrococcus furiosus Ferredoxins (PfFd), a small protein (MW ca. 7.5 kDa) with a single [3Fe-4S] redox center, i.e., an incomplete iron-sulfur cluster with Fe and S at alternative corners. Despite the small size, the X-ray crystallographic structure of this protein has * Author to whom correspondence should be addressed. E-mail address: [email protected]. Fax: +45 4588 3136. (1) Johnson, M. K. In Encyclopaedia of Inorganic Chemistry; King R. B. Ed.; Wiley: Chichester, 1994; Vol. 4, pp 1896-1915. (2) Spiro, T. G., Ed. Iron Sulfur Proteins; Wiley: New York, 1982. (3) Landrum, H. L.; Salmon, R. T.; Hawkridge, F. M. J. Am. Chem. Soc. 1977, 99, 3154-3158. (4) Armstrong, F. A. Adv. Inorg. Chem. 1992, 38, 117-163. (5) Duff, J. L. C.; Breton, J. L. J.; Butt, J. N.; Armstrong, F. A.; Thomson, A. J. J. Am. Chem. Soc. 1996, 118, 8593-8603. (6) Hirst, J.; Jameson, G. N. L.; Allen, W. A.; Armstrong, F. A. J. Am. Chem. Soc. 1998, 120, 11994-11999. (7) Fawcett, S. E. J.; Davis, D.; Breton, J. L.; Thomson, A. J.; Armstrong, F. A. Biochem. J. 1998, 335, 357-368. (8) Jung, Y.-S.; Bonagura, C. A.; Tilley, G. J.; Gao-Sheridan, H. S.; Armstrong, F. A.; Stout, C. D.; Burgess, B. K. J. Biol. Chem. 2000, 275, 36974-36983.

long been elusive. Christensen et al. have, however, recently developed a new method for expression of recombinant PfFd with high yield and improved preparations of PfFd single crystals. The X-ray crystallographic structure of this protein could be determined to 1.5 Å resolution,9 Figure 1A. The diameter of PfFd is noted to be 3.1 nm. As for other Fd’s, the protein surface is strongly negatively charged in neutral solution (Figure 1B). This detailed structural information is crucial in further studies of interfacial ET. We have studied surface microscopic structures and ET properties of redox protein and enzyme monolayers over the past years. Immobilization of redox proteins on metallic substrate surfaces has led to detailed characterization of the adsorbed state and diffusionless interfacial ET of proteins. In some cases, proteins containing natural or engineered surface linker groups, such as thiol or disulfide groups, directly assemble on the gold surface in monolayers or submonolayers, which retain ET or catalytic function. This applies to the blue copper protein Pseudomonas aeruginosa azurin,10-12 yeast cytochrome c,13 an artificial protein,14 and the redox enzyme copper (9) Nielsen, M. S.; Harris, P.; Ooi, B. L.; Christensen, H. E. M. Biochemistry 2004, 43, 5188-5194. (10) Friis, E. P.; Andersen, J. E. T.; Kharkats, Yu. I.; Kuznetsov, A. M.; Nichols, R. J.; Zhang, J.; Ulstrup, J. Proc. Natl. Acad. Sci. USA 1999, 96, 1379-1384. (11) Chi, Q.; Zhang, J.; Nielsen, J. U.; Friis, E. P.; Chorkendorff, I.; Canters, G. W.; Andersen, J. E. T.; Ulstrup, J. J. Am. Chem. Soc. 2000, 122, 4047-4055. (12) Chi, Q.; Zhang, J.; Friis, E. P.; Andersen, J. E. T.; Ulstrup, J. Electrochem. Commun. 1999, 1, 91-96.

10.1021/la048853i CCC: $27.50 © 2004 American Chemical Society Published on Web 10/01/2004

Imaging and Electrochemistry of P. furiosus Ferredoxin

Figure 1. (A) Three-dimensional structure and (B) a model for the protein surface charge distribution of PfFd [3Fe-4S]. Red and blue colors denote negatively and positively charged residues, respectively. For details, see ref 9.

nitrite reductase.15 Direct adsorption of protein on the metallic surface is ineffective in many cases due to protein denaturation and no detectable ET signal. Self-assembled monolayers (SAMs) of small thiolate-based molecules on the metallic surface, particularly on gold surfaces, have instead been shown to be efficient. Furthermore, molecular orientations of the protein on the surface could be controlled by tailoring the terminal group of the thiolate compounds. This strategy was first employed for the heme protein horse heart cytochrome c16-23 using carboxylgroup-terminated alkanethiol monolayers and later for azurin.24 Pioneering studies in the 1990s were based on polycrystalline Au (PolyAu) electrodes. The surface nature of PolyAu did not allow observation of protein monolayer structures at the molecular level. Introduction of singlecrystal electrodes into protein electrochemistry reduced experimental background currents and increases drastically the signal-to-noise ratio, providing in this way high voltammetric resolution. A combination of single-crystal electrochemistry and in situ scanning tunneling microscopy (STM) have here offered novel effective approaches to surface microscopic structures and ET properties of monolayers of redox proteins and enzymes.15,25-29 (13) Hansen, A. G.; Boisen, A.; Nielsen, J. U.; Wackerbarth, H.; Chorkendorff, I.; Andersen, J. E. T.; Zhang, J.; Ulstrup, J. Langmuir 2003, 19, 3419-3427. (14) Brask, J.; Wackerbarth, H.; Jensen, K. J.; Zhang, J.; Chorkendorff, I.; Ulstrup, J. J. Am. Chem. Soc. 2003, 125, 94-104. (15) Zhang, J.; Welinder, A. C.; Hansen, A. G.; Christensen, H. E. M.; Ulstrup, J. J. Phys. Chem. B 2003, 107, 12480-12484. (16) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 18471849. (17) Collinson, M.; Bowden, E. F.; Tarlov, M. J. Langmuir 1992, 8, 1247-1250. (18) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (19) Kasmi, A. El.; Wallace, J. M.; Bowden, E. F.; Binet, S. M.; Linderman, R. J. J. Am. Chem. Soc. 1998, 120, 225-226. (20) Feng, Z.-Q.; Sagara, T.; Niki, K. Anal. Chem. 1995, 67, 35643569. (21) Feng, Z.-Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Chem. Soc., Faraday Trans. 1997, 93, 1367-1371. (22) Avila, A.; Gregory, B. W.; Niki, K.; Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759-2766. (23) Niki, K. Electrochemistry 2002, 70, 82-90. (24) Gaigalas, A. K.; Niaura, G. J. Colloid Interface Sci. 1997, 193, 60-70. (25) Chi, Q.; Zhang, J.; Andersen, J. E. T.; Ulstrup, J. J. Phys. Chem. B 2001, 105, 4669-4679. (26) (a) Friis, E. P.; Andersen, J. E. T.; Madsen, L. L.; Møller, P.; Ulkstrup, J. J. Electroanal. Chem. 1997, 431, 35-38. (b) Zhang, J.; Chi, Q.; Kuznetsov, A. M.; Hansen, A. G.; Wackerbarth, H.; Christensen, H. E. M.; Andersen, J. E. T.; Ulstrup, J. J. Phys. Chem. B 2002, 106, 11311152. (27) Davis, J. J.; Hill, H. A. O. Chem. Commun. 2002, 393-394. (28) Andolfi, L.; Cannistraro, S.; Canters, G. W.; Facci, P.; Ficca, A. G.; van Amsterdam, I. M. C.; Verbeet, M. P. Arch. Biochem. Biophys. 2002, 399, 81-88.

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In the present work, we address an iron-sulfur protein along these lines. We first investigated the electrochemical behavior of PfFd in homogeneous solution using small organic promoter molecules. It is shown by mass spectrometry and in other ways that promotion is induced by stable promoter-protein complex formation. These data serve as a clue to appropriate organic thiolate compounds as molecular linkers and ET bridges in PfFd monolayer studies. PfFd was found to assemble in stable monolayers on single-crystal Au(111) electrodes modified by highly ordered adlayers of mercaptopropionic acid or cysteine. High-resolution in situ STM under electrochemical potential control has further enabled direct observation of individual protein molecules on the surface, while monolayer voltammetry characterizes the diffusionless direct ET. These results represent the first case for this type of protein (ferredoxins) in which in situ STM imaging at molecular resolution and direct monolayer ET have been achieved. 2. Experimental Section 2.1. Cloning, Expression, and Purification of P. furiosus [Fe3S4]-Ferredoxin. The gene encoding P. furiosus ferredoxin (PfFd) [Fe3S4] was expressed in Escherichia coli and the protein purified according to procedures to be published elsewhere.30 In brief, the PfFd gene was amplified from P. furiosus cells (DSM No. 3638) by the PCR method. The gene was then cloned into the pET3a expression vector and the construct transformed into BL21(DE3) cells (Novagen) for expression.31 PfFd was purified to homogeneity in a form suitable for crystallization.30 The PfFd concentration was determined by UV-vis spectroscopy at 408 nm using a molar absorption coefficient of 18 000 M-1cm-1.32 2.2. Electrochemical Measurements and In Situ STM Imaging. 2.2.1. Reagents. L-cysteine (Cys, >98%, Sigma), L-alanine (Ala, Puriss Fluka AG), propionic acid (PA, >99%, Acros Organics), 3-mercaptopropionic acid (MPA, >98%, Merck), 3-mercaptopropanol (MPL, >95%, Aldrich), 4,4′-dithiodipyridine (DTPy, Sigma), 1-octanethiol (>98.5%, Aldrich), cysteamine (>98%, Fluka), and neomycin sulfate (BDH Biochemical) were used without further purification. Phosphate buffer (PB) was prepared by mixing KH2PO4 (suprapur, 99.995%, Merck) and K2HPO4 (suprapur, 99.99%, Merck). Millipore (Milli-Q Housing, 18.2 MΩ) water was used throughout. 2.2.2. Electrochemical Measurements. Cyclic voltammetry was performed on an Autolab potentiostat (Eco Chemie, The Netherlands) controlled by the general-purpose electrochemical system software. The electrochemical cell (homemade) and electrodes were confined in a Faraday cage to minimize electrical noise. A three-electrode system was used. The reference electrode was a freshly prepared reversible hydrogen electrode (RHE) checked against a saturated calomel electrode (SCE) after each measurement. All potentials are reported vs SCE. A clean Pt wire was the counter electrode. Edge plane graphite (EPG) (homemade) or Au(111) (homemade) were used as working electrodes. Before each measurement, the EPG was polished with 1.0, 0.1, and 0.05 µm Al2O3 slurry, followed by supersonication twice in Millipore water. Clean EPG was dried by Ar flow before assembly in the electrochemical cell. Au(111) electrodes for electrochemistry and STM were prepared as before11,12 and checked by the method of Clavilier and Hamelin.33 Prior to use, Au(111) electrodes were annealed in a hydrogen flame and quenched in Millipore water saturated with hydrogen gas. The electrodes were transferred to freshly prepared thiolate-contain(29) Bonanni, B.; Alliatra, D.; Bizarri, A. R.; Cannistraro, S. ChemPhysChem 2003, 4, 1183-1188. (30) Barat-Jankovics, H.; Ooi, B. L.; Christophersen, S.; Nielsen, M. S.; Siu, Y. S.; Christensen, H. E. M. submitted for publication. (31) Studier, F. W.; Rosenberg, A. H.; Dunn, J. J.; Dubendorff, J. W. Methods Enzymol. 1990, 185, 60-89. (32) Conover, R. C.; Kowal, A. T.; Fu, W. G.; Park, J. B.; Aono, S.; Adams, M. W.; Johnson, M. K. J. Biol. Chem. 1990, 265, 8533-8541. (33) Hamelin, A. J. Electroanal. Chem. 1996, 401, 1-16. (34) Clavilier, J.; Faure, R.; Guinet, G.; Durand, R. J. Electroanal. Chem. 1980, 107, 205-209.

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ing solutions. Self-assembled monolayers were formed by soaking Au(111) in 0.5-1.0 mM thiol solution for 2-5 h at room temperature. After rinsing with Millipore water, the electrodes were transferred to 1 mg mL-1 Pf Fd in 5 mM PB (pH 7.9) solution for 3-5 h to prepare the protein adlayers. The resulting electrodes were rinsed with Millipore water. Mercaptopropionic acid and cysteine-covered electrodes are denoted as MPA/Au(111) and Cys/ Au(111), respectively, and protein-containing electrodes are denoted as PfFd-MPA/Au(111) and PfFd-Cys/Au(111). Since Pf Fd is extremely sensitive to oxygen, electrolyte solutions were deoxygenated for several hours by Ar (5N), purified by Chrompack (oxygen < 50 ppb) before measurements. All systems were blanketed with Ar atmosphere during measurement. 2.2.3. STM Imaging. A Pico SPM instrument (Molecular Imaging Co., USA) was used for potential-controlled STM. STM tips were prepared from Pt/Ir (80/20, 0.25 mm diameter) wire by electrochemical etching and coated by Apiezon wax. In-housebuilt STM cells (2.5 mL volume) were made of Teflon. All glassware and STM cells were cleaned prior to use as previously described.12,26 Prior to measurement, the STM scanner was calibrated against the reconstruction lines on the Au(111) surface to obtain accurate XY dimensions from STM images. The STM cell and tip were enclosed in an Ar-filled chamber (5N). An Ar environment was found to be crucial for high quality STM images of protein molecules. All STM images are in the constant current mode. 2.3. Preparation and Mass Spectrometry of PromoterPfFd Complexes. The promoter-protein complex was prepared by incubating 36 µM PfFd and 5 mM promoter (e.g., neomycin) in 5 mM PB (pH 7.9) at room temperature for 2-3 h. Excess promoter was removed by ultrafiltration (four times) in an Amicon cell (YM3, 3000 MW) with 5 mM PB (pH 7.9). Samples were stored at 4 °C. Prior to mass spectrometry, the samples were desalted using a 5 mL HiTrap desalting column (Amersham Biosciences) and diluted to an approximate concentration of 10 µM. The mass spectra were acquired on a Nanoflow Electrospray Ionization (ESI) quadrupole-time-of-flight instrument (Micromass, UK). A typical needle voltage of 850 V and a cone voltage of 25 V were applied. Au/Pd-coated borosilicate glass capillary tips (cat. no. ES380, MDS Proteomics, Denmark) were used throughout. The instrument was calibrated by NaI. The mass spectra were calculated from the m/z-spectra using the transform algorithm provided with the MassLynx v3.4 software (Micromass).

3. Results and Discussion 3.1. Direct Eectrochemistry of PfFd in Homogeneous Solution. No redox signal was detected from PfFd at the EPG electrode in the absence of promoter. The presence of neomycin gives rise to a pair of well-defined redox peaks from PfFd with a formal potential (E°′) of ca. -430 mV (vs SCE) (Figure 2A). A linear relation between peak currents and the square root of the scan rate in the range 2-200 mV s-1 is indicative of diffusion control. Peak positions and other characteristics are similar to the data of Armstrong et al.7 The peaks originate from the one-ET [3Fe-4S]1+/0 redox couple. The apparent rate constant is 7.8 × 10-5 cm s-1 from the peak separation (∆Ep1),34,35 suggesting a slow electrode process with neomycin as the sole promoter. We did not observe the second pair of redox peaks as in ref 7 with neomycin alone as promoter, reported to appear only a very slow scan rates.7 This could result from different experimental conditions including buffer, pH, ionic strength, and temperature, although exact reasons are not clear. The conditions of ref 7 were 20 mM Hepes (pH 7.0) in 0.1 M NaCl and 0.1 mM EGTA at 0 °C,7 those of the present data are 5 mM phosphate buffer (pH 7.9) at room temperature (22 ( 1 °C). These conditions are the same as in situ STM and PfFd monolayer electrochemistry on Au(111), cf. below. (35) Nicholson, R. S. Anal. Chem. 1965, 37, 1351-1355.

Figure 2. Cyclic voltammograms of PfFd in 5 mM phosphate buffer (pH 7.9) at EPG electrodes in the presence of various promoters: (A) 5 mM neomycin, (B) 5 mM neomycin + 5 mM proponic acid, (C) 5 mM neomycin + 5 mM alanine, and (D) 5 mM neomycin + 5 mM L-cysteine. Concentration of PfFd: (A) 36 µM, (B) 38 µM, (C) 30 µM, (D) 35 µM. Scan rate: (A) and (B) 2 mV s-1, (C) and (D) 5 mV s-1. “I” and “II” indicate positions of the first and second pair of redox peaks, respectively.

A second pair of redox peaks around -900 mV (vs SCE), along with the first couple, however, appears on addition of a second promoter. This can be propionic acid (PA), alanine (Ala) or L-cysteine (Cys) (Figure 2B-D). In the previous report, the second pair has been assigned to superreduced [3Fe-4S]0/2- (i.e., two-electron transfer).7 The peak features under the present conditions, particularly

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Table 1. Redox Parameters of Pf Ferredoxin (36 µM) in Homogeneous Solutions Containing Small Organic Molecules as Promotersa promotersb

E1°′ (mV vs SCE)

∆Ep1c (mV)

10-3 ko′ (cm s-1)

E2°′ (mV vs SCE)

∆Ep2c (mV)

10-3 ko′ (cm s-1)

neomycin neomycin + propionic acid neomycin + alanine neomycin + cysteine

-430 ( 10 -408 ( 2 -427 ( 3 -425 ( 3

100 ( 15 58 ( 2 68 ( 7 55 ( 2

0.078 2.4 0.36 2.4

no peak -880 ( 5 -925 ( 2 -910 ( 5

94 ( 5 100 ( 3 105 ( 5

0.084 0.078 0.061

a Data obtained from CVs in 5 mM PB (pH 7.9) on the basis of three or more measurements. b The concentration of all promoters is 5 mM. c Peak separations estimated from CVs with a scan rate of 5 mV s-1.

the peak half-width, however, suggest a one-ET couple (i.e., [3Fe-4S]0/1-). There are only a few reports on superreduced [3Fe-4S] protein clusters, but synthetic [3Fe4S] clusters36 commonly display two reversible one-ET steps corresponding to [3Fe-4S]1+/0 and [3Fe-4S]0/1-. The common structural feature among PA, Ala, and Cys is the carboxyl group. As a comparison, electrochemical measurements using small organic molecules without the -COOH group, such as 1-propanol and 1-aminopropane as the second promoter, did not induce the second redox couple. The -COOH group thus seems to be crucial for the second redox step. Table 1 lists the electrochemical parameters. Rate constants are estimated from peak separations. The following suggestions emerge from the data: First, formal potentials (both E1°′ and E2°′, i.e., the midpoint potential of the anodic and cathodic peaks) vary slightly with the second promoter. Second, the second promoter not only induces the second redox couple but also accelerates the first ET step. The rate constant thus increases by more than 1 order of magnitude. Third, the second ET step is much slower than the first step. Adsorption of positively charged promoters, such as neomycin on the graphite surface, with neutralization of negative electrode surface groups has been suggested as the promotion mechanism. However, propionic acid, alanine, and L-cysteine are all negatively charged under the present conditions (pH 7.9), and the carboxyl group is present in deprotonated form (-COO-).37 The neomycin charge state is determined by the protonation state of six amino groups and is more complex.38 Neomycin is positively charged at pH 7.9 but with no more than three net charges.39 This implies that a strong positive charge is not essential in direct electrochemistry of PfFd. Instead, structural interactions between PfFd and promoter molecules could be important. This was elucidated by further electrochemical measurements and mass spectroscopy. 3.2. Formation and Mass Spectrometry of the PfFd-Promoter Complexes. The 5 mM phosphate buffer (pH 7.9) containing 5 mM neomycin and 36 µM PfFd was incubated at room temperature. After 2-3 h, excess promoter was removed by ultrafiltration (four times) and exchanged in an Amicon cell (YM3, 3000 MW) at 4 °C with 5 mM PB (pH 7.9). No free promoter is present in the final solution. Such samples were used for voltammetry and mass spectroscopy (MS). Cyclic voltammograms in promoter-free buffer (Supporting Information, Figure S1A) were indistinguishable from that in Figure 2A. This clearly indicates that free promoter in the electrolyte solution does not induce direct ET between PfFd and the graphite electrode. Instead, PfFd and the (36) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, John Wiley and Sons: New York, 1980; pp 213-248. (37) Zhou, J.; Hu, Z.; Mu¨nck, E.; Holm, R. H. J. Am. Chem. Soc. 1996, 118, 1966-1980. (38) The pKa of propionic acid is 4.87, and the PIs of alanine and L-cysteine are, respectively, 6.00 and 5.07.39 (39) The pKa’s of six -NH3+ at neomycin are, respectively, about 5.7, 7.6, 7.6, 8.1, 8.6, and 8.8.39

Figure 3. Nanoflow electrospray ionization time-of-flight mass spectra: (A) PfFd alone and (B) the neomycin-PfFd complex.

promoter interact to form an adduct. This is clearly supported by mass spectrometry (Figure 3). Figure 3A shows a MS for pure PfFd. The main peak at 7461 agrees with the calculated molecular mass of 7460.9 for oxidized PfFd with an intact disulfide bridge. Figure 3B shows the MS of protein treated with neomycin. Besides the 7461 peak, two other peaks at 8076 and 8690 were observed. These correspond exactly to the masses for adducts with 1:1 and 1:2 ratios of PfFd to neomycin, respectively. These peaks also show that neomycin binds to the protein in neutral rather than positively charged form (the molecular masses of neomycin are 615 for the neutral and 621 Da for the fully protonated state of six amino groups). The relative abundance of the 8076 vs 8690 peaks suggests dominance of the 1:1 PfFd adduct. Together, these data prove that the promoting effect of neomycin in PfFd voltammetry is dominated by promoter-protein complex formation in solution. It is not surprising that neomycin binds to PfFd. Neomycin and its derivatives are extensively used as antibiotics by their interaction with proteins and RNAs.39 Other, MS data were recorded for PfFd samples treated with both neomycin and L-cysteine. No mass peak corresponding to a neomycin-PfFd-cysteine complex was

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Figure 4. A proposed scheme for the ET mechanism of PfFd in homogeneous solution. Neo and Cys denote neomycin and cysteine, respectively. Protonation and deprotonation are disregarded for simplicity.

detected, although two pairs of redox peaks similar to Figure 2D were observed in cyclic voltammetry (Supporting Information, Figure S1B). This could be due to labile cysteine binding to PfFd, which does not survive the conditions used in MS. The electrochemical and MS data suggest that PfFd and the promoters first form an intermediate complex in solution. The interactions result in changes in the protein structure which open favorable (tunneling) ET pathways and facile access to the electrode surface. A schematic view illustrating such a mechanism is proposed in Figure 4. Details of the interactions between PfFd and promoters remain a challenge, where, e.g., NMR spectroscopy and X-ray crystallography of the complex structures are required. This is, however, beyond the objectives of the present work. 3.3. STM Imaging of MPA and PfFd Monolayers. The interaction between promoter and PfFd in homogen-

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eous solution suggest that the dominating promoting effects are caused by the carboxyl group. This was exploited to confine PfFd on the electrode surface and address diffusionless ET. PfFd was found to be adsorbed on carboxyl-terminal organic molecules on Au(111), in stable self-assembled monolayers, and direct ET between adsorbed protein and the electrode was observed. The most effective adlayers are MPA and Cys SAMs. Formation of the protein monolayer was evidenced by potential-dependent capacitances (Supporting Information, Figure S2), and by STM. We address first the surface microstructures of MPA and PfFd monolayers by in situ STM with molecular resolution. Interfacial ET is addressed in the next section. Figure 5A shows an in situ STM image of the MPAmodified Au(111) in phosphate buffer (pH 7.9). A dense monolayer with numerous pits (diameter ) 3-7 nm) is clearly seen. Pit formation is a common surface feature of thiol-containing SAMs. High-resolution images disclose detailed microstructures of the MPA monolayer (Figure 5B and C), with a highly ordered cluster-like, periodic organization. Each cluster contains six bright spots, aligned in two rows. Analysis of the domains and the atomic directions on the Au(111) substrate shows that the distance between neighboring clusters along the [112h ] direction is 1.01 ((0.03) nm, and the distance in the perpendicular direction is 1.43 (( 0.04) nm. The rectangular cluster unit can therefore be described as (2x3 × 5)R30° with a coverage of 1.15 × 10-10 mol cm-2. If each bright spot represents one MPA molecule, the molecular MPA monolayer density is 6.9 × 10-10 mol cm-2. This agrees well with the surface coverage of 6.8 × 10-10 mol cm-2 obtained from reductive desorption by Sawaguchi et al.40 Other surface structures of MPA/Au(111) have been observed.40-42 Sawaguchi et al. concluded that an ordered

Figure 5. (A)-(C) In situ STM images of MPA monolayer self-assembled on the Au(111) surface in 5 mM phosphate buffer (pH 7.9) under potential control in Ar atmosphere. It ) 0.25 nA, Vbias ) -0.10 V, and Ew ) -0.15 V (vs SCE). Scan area: (A) 200 × 200 nm2, (B) 12.5 × 12.5 nm2, (C) 6.5 × 6.5 nm2, and (D) Schematic model for orientation of MPA molecules on Au(111).

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Figure 6. In situ STM images of PfFd molecules immobilized on the MPA-modified Au(111) surface obtained in 5 mM phosphate buffer (pH 7.9) under potential control in Ar atmosphere. It ) 0.10 nA, Vbias ) -0.35 V, and Ew ) -0.12 V (vs SCE). Scan area: (A) 160 × 160 nm2 and (B) 80 × 80 nm2.

rhombic (3 × 3) structure was found on MPA/Au(111) in 50 mM HClO4 with three MPA molecules in triangular positions kept close together by intermolecular hydrogen bonding.40,41 Tao et al. reported on incommensurate (p × x3) structure as the dominant phase with (5 × x3), (6 × x3), (8 × x3), and (10 × x3) commensurate phases for MPA/Au(111) in 25 mM phosphate buffer (pH 7).42 It is not surprising that surface microstructures of the MPA adlayer vary with buffer, pH, and electrolyte concentration. The protonation state of the MPA monolayer depends on these parameters, and the cluster formation is likely to be controlled by hydrogen-bonding among carboxylic groups. A network-like cluster structure was also observed for L-cysteine monolayers on Au(111).43 Each cluster holds six cysteine molecules. The unit cell is (3x3 × 6)R30°, i.e., larger than MPA ((2x3 × 5)R30°). This can be assigned to structural differences of the two molecules. Cysteine has an additional amine group and a larger area per molecule. Further analysis based on in situ STM image recording of both bare Au(111) and the MPA/Au(111) layer under potential control reveals that the six MPA molecules in a cluster are aligned with an angle of 18° vs the [1h 10] direction. Since each MPA molecule contains two flexible C-C bonds, different MPA molecular conformations may coexist. The overall information is summarized in the model in Figure 5D with emphasis on the (2x3 × 5)R30° unit cell and six-molecule-based clusters. In situ STM of the PfFd monolayer in 5 mM phosphate buffer (pH 7.9) was also recorded. The protein is structurally and functionally stable in this medium. PfFd is much more sensitive to dioxygen than azurin or yeast cytochrome c. An argon atmosphere was found to be crucial for highquality STM imaging. Figure 6 shows two representative in situ STM images with different scan areas. No periodic lattice structure is observed, but the distribution of protein molecules is uniform. The lateral diameter for most of the protein molecules is 3.0-3.5 nm, close to the crystallographic dimensions. A small portion (ca. 10%) of larger spots (4.5-5.0 nm) most likely originates from PfFd dimers. PfFd molecules tend to form dimers even in the crystalline phase.9 The coverage of PfFd calculated from the density of white spots in the in situ STM images is 7 (( 1) × 10-12 mol cm-2. This value is comparable with but lower than the value of 1.2 × 10-11 mol cm-2 from cyclic voltammetry, cf. below. This discrepancy is most likely caused by tip influence since the interaction between PfFd and the MPA layer is noncovalent and not as strong as the Au-S bond in the case of azurin/Au(111).11 PfFd gives overall strong STM contrasts, likely to be caused by significant electronic contributions from the Fe-S cluster. The uniform size and image contrast indicate that PfFd molecules are adsorbed in a narrow orientation distribution. In conclusion, use of STM for mapping the PfFd

Figure 7. Monolayer cyclic voltammograms of Cysteine/Au(111) (dashed lines) and PfFd-Cysteine/Au(111) electrode (solid lines). 5 mM phosphate buffer (pH 7.9). Scan rate 5 mV s-1.

adlayer structure and organization on the MPA monolayer, directly in the aqueous buffer environment, has been successful. 3.4. Direct Electron Transfer of PfFd Monolayers. Different thiolate SAMs with neutral, positively, and negatively charged terminal groups as supporting adlayers for immobilization and ET of PfFd were compared. No voltammetric signal from PfFd could be detected for 3-mercaptopropanol (-OH), cysteamine (-NH2 or -NH3+), 1-octanethiol (-CH3), and 4,4′-thioldipyridine. In contrast, carboxyl-group-containing MPA and L-cysteine act efficiently as linker molecules and ET promoters. These results follow those for homogeneous solution, cf. above. Figure 7 shows cyclic voltammograms of cysteine/ Au(111) and PfFd-cysteine/Au(111) electrodes. Cysteine/ Au(111) does not itself give any redox signal but a pair of well-defined redox peaks from absorbed PfFd appear at ca -200 mV vs SCE. Similar observations are obtained for PfFd-MPA/Au(111) electrodes (Figure 8A), except for a slightly more negative formal potential (-220 mV). Both cathodic and anodic peak currents follow a linear dependence on the scan rate (Figure 8B), characteristic of diffusionless electrode processes. Other characteristics (e.g., half-peak width, etc.) are that the reaction involves a one-electron step, [3Fe-4S]1+/0 . The second redox couple observed for PfFd in homogeneous solution in the presence of two promoters could not be observed due to the limited potential window available for the thiolate-modified Au(111). Extension of the potential window in the negative direction caused reductive desorption of the MPA or L-cysteine monolayer, which in turn results in collapse of the protein adlayer. The redox potential of adsorbed PfFd is shifted positively by about 200 mV compared to the homogeneous solution (the first redox couple). This is likely to reflect the different environment of the redox reaction in solution and in the adsorbed state. The difference is unlikely to be caused by protein denaturation on adsorption, as there is no evidence for PfFd unfolding or for loss of the iron-sulfur cluster. This protein is extremely thermostable with a transition temperature well above (40) See, for recent examples: (a) Hoch, I.; Berens, E.; Westhof, E.; Schroeder, R. J. Mol. Biol. 1998, 282, 557-569. (b) Fourmy, D.; Recht, M. I.; Puglisi, J. D. J. Mol. Biol. 1998, 277, 347-362. (c) Ma, C.; Baker, N. A.; Joseph, S.; McCammon, J. A. J. Am. Chem. Soc. 2002, 124, 14381442. (d) Borda, E. J.; Sigurdsson, S. Th. Bioorg. Med Chem. 2004, 12, 1023-1028. (41) Sawaguchi, T.; Sato, Y.; Mizutani, F. J. Electroanal. Chem. 2001, 507, 256-262. (42) Sawaguchi, T.; Sato, Y.; Mizutani, F. Phys. Chem. Chem. Phys. 2001, 3, 3399-3404. (43) Giz, M. J.; Duong, B.; Tao, N. J. J. Electroanal. Chem. 1999, 465, 72-79.

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Figure 9. Schematic representation of the orientation of PfFd protein molecules on the organic thiolate-modified Au(111) surface.

Figure 8. (A) Monolayer cyclic voltammograms of the MPA/ Au(111) (dashed lines) and PfFd-MPA/Au(111) electrode (solid lines). 5 mM phosphate buffer (pH 7.9). Scan rate 5 mV‚s-1. (B) Plots of anodic and cathodic peak currents against scan rates obtained from the PfFd-MPA/Au(111) electrode in 5 mM phosphate buffer (pH 7.9). Table 2. Redox Parameters of Pf Ferrdoxin Immobilized on the Thiolate Monolayers with Various Terminal Groupsa

electrodes PfFd-Cys/Au(111) PfFd-MPA/Au(111) PfFd-MPA/Au(111)b PfFd-(MPA+MPL)/ Au(111)

E°′ (mV)

∆Ep (mV)

k°′ (s-1)

-201 ( 5 106 ( 8 0.040 ( 0.005 -220 ( 3 37 ( 3 0.20 ( 0.02 -255 ( 3 40 ( 3 0.18 ( 0.02 -198 ( 2 32 ( 2 0.22 ( 0.02

surface coverage (× 10-12 mol cm-2) 7.5 ( 0.5 12 ( 3 4.2 ( 0.5 5.1 ( 0.5

a Data obtained from CVs in 5 mM PB (pH 7.9) solution, scan rate 5 mV s-1. b The buffer solution containing 5 mM neomycin.

100 °C.9 Similar potential shifts for other redox proteins have been reported. Rusling et al. found negative shifts of 150 and 240 mV, respectively, for myoglobin and hemoglobin confined in clay films.44 Riveral et al. reported a positive reduction potential shift of cytochrome b5 at a β-mercaptopropionate-modified gold electrode,45 attributed to polylysine-cytochrome b5 complex formation on the electrode surface.45 A similar explanation with complex formation between PfFd and surface carboxyl groups may apply in the present case. Table 2 summarizes other PfFd surface chemical ET properties. The surface coverage was estimated from the Faradic charge, and rate constants (ko′) from the peak separations according to Laviron’s method.46 An average (44) Zhang, J.; Chi, Q.; Nielsen, J. U.; Friis, E. P.; Andersen, J. E. T.; Ulstrup, J. Langmuir 2000, 16, 7229-7237. (45) Zhou, Y.; Hu, N.; Zeng, Y.; Rusling, J. F. Langmuir 2002, 18, 211-219.

value of about 1.2 × 10-11 mol cm-2 for the PfFd surface coverage of at the MPA monolayer was found, comparable to theoretical expectations (1.5 × 10-11 mol cm-2 ) for an ideal closed-packed PfFd monolayer. Second, direct ET is faster with MPA as supporting layer than with L-cysteine (rate constants 0.2 and 0.04 s-1, respectively), an indication that MPA is the more efficient promoter, possibly due to unfavorable influence from the amino group of cysteine. Third, the presence of neomycin (5 mM) in the buffer solution does not affect the ET kinetics of adsorbed PfFd, but the surface population of PfFd dramatically decreases to less than 40% (0.42 × 10-11 vs 1.2 × 10-11 mol cm-2). This is a strong indication that neomycin complex formation has occurred and removed absorbed protein molecules from the surface. Finally, the ET kinetics based on a mixed monolayer of 3-mercaptopropanol (MPL) and MPA as supporting layer, is almost unchanged, but the PfFd surface coverage is less than half the values at the pure MPA monolayer (0.51 × 10-11 vs 1.2 × 10-11 mol cm-2). This indicates that either the -OH group cannot confine protein molecules or that MPL cannot promote ET. This further supports the essential role of the carboxyl group in both immobilization and ET promotion. The other observation is, finally, suggestive as to possible mechanisms for interaction between PfFd and carboxyl groups in the MPA monolayer. The surface pKa of MPA/Au was reported to be in the range of 5.8-8.0, estimated by various methods.47-51 This is obviously higher than in bulk solution (pKa1 4.16 for -COOH and pKa2 10.1 for -SH). In contrast to bulk solution, the surface pKa also depends strongly on solution ionic strength and the surface potential.47-51 The MPA layer would tend to be significantly deprotonated and therefore negatively charged at the present conditions. Pure electrostatic attraction might thus not be solely responsible for immobilization of the strongly negatively charged PfFd (at (46) Rivera, M.; Seetharaman, R.; Girdhar, D.; Wirtz, M.; Zhang, X.; Wang, X.; White, S. Biochemistry 1998, 37, 1485-1494. (47) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28. (48) Hu, K.; Bard, A. J. Langmuir 1997, 13, 5114-5119. (49) Sugihara, K.; Shimazu, K.; Uosaki, K. Langmuir 2000, 16, 71017105. (50) Kim, K.; Kwak, J. J. Electroanal. Chem. 2001, 512, 83-91. (51) Kakiuchi, T.; Iida, M.; Imabayashi, S.; Niki, K. Langmuir 2000, 16, 5397-5401. (52) Smalley, J. F.; Chalfant, K.; Feldberg, S. W.; Nahir, T. M.; Bowden, E. F. J. Phys. Chem. B 1999, 103, 1676-1685.

Imaging and Electrochemistry of P. furiosus Ferredoxin

least 10 net negative charge on the protein surface at pH 7.0, Figure 1B). Instead, the -COO- group could approach the iron-sulfur cluster as a surface ligand. A summarizing schematic model for PfFd immobilized on the MPA layer is shown in Figure 9.

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In situ STM images to molecular resolution directly reveal that adsorbed protein molecules keep their expected lateral dimensions with uniform distribution of protein molecules over the surface. Direct and diffusionless ET was characterized by cyclic voltammetry. The ET kinetics are slow compared to azurin or cytochrome c.

4. Concluding Summary We have investigated comprehensively interfacial ET of PfFd both in homogeneous solution and in the adsorbed state by electrochemistry, electrochemical STM, and mass spectrometry. Results using various techniques support each other. Direct electrochemistry of PfFd in homogeneous solution was detected in the presence of promoters. The broadly used promoter for ferredoxins, neomycin, only promotes the first ET step. Together with neomycin, a second promoter containing the carboxyl group (propionic acid, alanine, or L-cysteine) is required to observe the second couple at significantly lower potential. A combination of electrochemical and MS data has enabled us to propose a mechanism in which the promoting effects are through formation of a robust PfFd-promoter complex. PfFd can further assemble in stable functional monolayers or submonolayers on carboxyl-group-containing SAMs.

Acknowledgment. We greatly appreciate the assistance of Dr. Ole Nørregaard Jensen with the mass spectrometry analysis, performed at the Danish Mass Spectrometry Instrument Center, University of Southern Denmark, Odense, Denmark. We acknowledge assistance and many helpful discussions from Dr. Qijin Chi, and financial support from The Danish Technical Science Research Council. Supporting Information Available: Cyclic voltammograms of the neomycin-PfFd complex and the neomycinPfFd-cysteine complex in phosphate buffer and potentialdependent capacitance curves of the Au(111) electrodes. This material is available free of charge via the Internet at http://pubs.acs.org. LA048853I