Microemulsion-Controlled Reaction Sites in ... - ACS Publications

Sep 21, 2006 - Pharmacology, UniVersity of Connecticut Health Center, Farmington, Connecticut 06032. ReceiVed April 26, 2006. In Final Form: July 20, ...
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10788

Langmuir 2006, 22, 10788-10795

Microemulsion-Controlled Reaction Sites in Biocatalytic Films for Electrochemical Reduction of Vicinal Dibromides† Abhay Vaze‡ and James F. Rusling*,‡,§ Department of Chemistry, UniVersity of Connecticut, Storrs, Connecticut 06269-3060, and Department of Pharmacology, UniVersity of Connecticut Health Center, Farmington, Connecticut 06032 ReceiVed April 26, 2006. In Final Form: July 20, 2006 We report herein the electrochemical dehalogenation of vicinal dibromides in microemulsions using cross-linked films of the redox protein myoglobin (Mb) and poly-L-lysine (PLL) covalently bonded to carbon electrodes. Catalytic reduction of the dibromides to olefins was more efficient in an SDS microemulsion than in a CTAB microemulsion. SDS shifts the Mb redox potential more negative, but a comparison to Mb-SDS films suggests that the activation free energy of the reduction is controlled by an inner-sphere mechanism. SDS also enters the positively charged Mb-PLL films and preconcentrates the dibromide reactants, enhancing catalytic efficiency in SDS microemulsions. Shifts in formal potential and Soret absorbance bands for Mb-PLL films suggested binding of trans-1,2-dibromocyclohexane in the iron heme distal pocket with little catalysis. Results are consistent with active catalytic reduction sites for reactant bound on the protein surface and less-reactive sites in the distal heme pocket. Preconcentration into catalytic PLL films using SDS incorporated from microemulsions may be a general way to improve catalytic efficiency for nonpolar reactants in microemulsions.

Introduction Biocatalysts that provide regio- and stereoselectivity are expected to play a major role in the future synthesis of drugs and specialty chemicals.1 Microemulsions are clear, stable, nanoheterogeneous mixtures of oil, water, surfactant, and cosurfactant that offer advantages over traditional organic solvents as reaction media, including lower toxicity and cost and the ability to solubilize polar and nonpolar reactants.2 Manipulating the composition of these fluids can provide unique pathway control in electrochemical syntheses.3-5 Recent efforts in our laboratory seek to combine stable, reusable biocatalytic films of inexpensive redox proteins with the pathway control and reactant solubilization afforded by microemulsions. For the above reasons, we developed covalently linked films of redox proteins and poly-L-lysine (PLL) on electrodes that have very good stability in microemulsions under a wide range of reaction conditions, including small millimolar quantities of hydrogen peroxide used to activate the protein for oxidations6 and at elevated temperatures.7 Covalent linkage of PLL to the electrode and of myoglobin (Mb) to PLL was critical to obtain the necessary stability in microemulsions, with their high dissolving power for polyions.6 Films of Mb and PLL show Soret band and UV circular dichroism (CD) spectra consistent with near-native Mb conformation residing in a water-rich †

Part of the Electrochemistry special issue. * Corresponding author. E-mail: [email protected]. ‡ Department of Chemistry, University of Connecticut. § Department of Pharmacology, University of Connecticut Health Center. (1) (a) Schoemaker, H. E.; Mink, D.; Wubbolts, M. G. Science 2003, 299, 1694-1697. (b) Koeller, K. M.; Wong, C.-H. Nature, 2001, 409, 232-240. (2) Bourrel, M.; Schechter, R. S. Microemulsions and Related Systems; Marcel Dekker: New York, 1988. (3) Rusling, J. F. In Reactions and Synthesis in Surfactant Systems; Texter, J., Ed.; Marcel Dekker: New York, 2001; pp 323-335. (4) Rusling, J. F. In Interfacial Kinetics and Mass Transport; Calvo, E. Ed.; Encyclopedia of Electrochemistry; Wiley-VCH: Weinheim, Germany, 2003; Vol. 2, pp 418-439. (5) Rusling, J. F.; Campbell, C. J. In Encyclopedia of Surface and Colloid Science; Marcel Dekker: New York, 2002; pp 1754-1770. (6) Vaze, A. S.; Parizo, M.; Rusling, J. F. Langmuir 2004 20, 10943-10948. (7) Guto, P. M.; Kumar, C. V.; Rusling, J. F. Angew. Chem., Int. Ed., submitted for publication.

environment within the films when they are immersed in neutral buffers or microemulsions.6,8 We have used Mb-PLL films for the electrochemical catalytic oxidation of styrene in the presence of oxygen and found 40fold-larger turnover rates in bicontinuous microemulsions compared to those in oil-in-water (o/w) microemulsions correlated mainly with faster mass transport of reactants in the oil phases of the bicontinuous fluids.6 However, residence sites of nonpolar reactants within the Mb-PLL films as monitored by the fluorescent probe pyrene were more polar in CTAB than in SDS microemulsions, which was a secondary factor in determining reactivity.9 We found that the reduction of tert-butylhydroperoxide by Mb-PLL films with simultaneous oxidative activation of the iron heme protein follows Michaelis-Menten kinetics.8 A unique property of iron heme proteins such as Mb is that they can transfer electrons relatively efficiently from MbFeII to vicinal dibromides.10 However, inner-sphere reductants FeI, Fe0, NiI, and CoI porphyrins,11 the CoI corrin vitamin B12s,12 and NiI macrocyles13 utilize the lower metal oxidation states to effect these reactions efficiently. We previously reported electrochemical vicinal dibromide dehalogenations catalyzed by the FeIII/FeII redox couple of Mb in films of surfactant in buffer14 and used these reactions to evaluate reactivity control in microemulsions.15 Possible pathways for electrochemical vicinal dihalide (RX2) reduction catalyzed by Mb films are shown in eqs 1-3. Electron injection into the film from the electrode gives MbFeII (eq 1), which can attack the carbon-halogen bond to form a carbon (8) Guto, P. M.; Rusling, J. F. J. Phys. Chem. B 2005, 109, 24457-24464. (9) Vaze, A. S.; Rusling, J. F. Faraday Discuss. 2005, 129, 265-274. (10) (a) Wade, R. S.; Castro, C. E. J. Am. Chem. Soc. 1973, 95, 231-234. (b) Bartnicki, E. W.; Belser, N. O.; Castro, C. E. Biochemistry 1978, 17, 5582-5586. (11) (a) Lexa, D.; Saveant, J.-M.; Su, K. B.; Wang, D. L. J. Am. Chem. Soc. 1987, 109, 6464 (b) Lexa, D.; Saveant, J.-M.; Schafer, H. J.; Su, K.-B.; Vering, B.; Wang, D. L. J. Am. Chem. Soc. 1990, 112, 6162-6177. (12) Zhou, D.-L.; Gao, J.; Rusling, J. F. J. Am. Chem. Soc. 1995, 117, 11271134. (13) Campbell, C. J.; Rusling; J. F.; Bru¨ckner, C. J. Am. Chem. Soc. 2000, 122, 6679-6685. (14) Nassar, A.-E.; Bobbitt, J. M.; Stuart, J. D.; Rusling; J. F. J. Am. Chem. Soc. 1995, 117, 10986-10993. (15) Kamau, G. N.; Guto, M. P.; Munge, B.; Panchagnula, V.; Rusling, J. F. Langmuir 2003, 19, 6976-6981.

10.1021/la061138j CCC: $33.50 © 2006 American Chemical Society Published on Web 09/21/2006

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radical RX• intermediate, which can in turn be reduced by another MbFeII to give the olefin (eq 3). Alternatively, concerted E2 elimination can follow eq 1 to give the olefin directly, a pathway followed by FeI, Fe0, and CoI porphyrins.11a

dihalides reside almost entirely in the oil microphases of microemulsions and thus may also reside in hydrophobic regions that might exist in Mb-PLL films in contact with microemulsions. In this article, we examine the influence of bicontinuous microemulsions having a cationic and an anionic surfactant on the reduction of three vicinal dibromides with the aim of elucidating how reaction sites within the Mb-PLL films influence the catalytic efficiency of Mb-PLL films. Results suggest that the incorporation of SDS into the PLL films preconcentrates the reactant and enhances reactivity, and that the Mb reaction may involve reactant binding to amino acid residues on the Mb surface.

MbFeIII + e- T MbFeII (at electrode)

(1)

Experimental Procedures

RX2 + MbFeII f RX• + MbFeIII + X-

(2)

RX• + MbFeII f olefin + MbFeIII + X-

(3)

Materials and Solutions. Horse heart myoglobin (Mb) from Sigma was dissolved in 10 mM acetate buffer, pH 5.5 and filtered through a YM30 filter (Amicon, 30 000 MW cutoff).22 Organic halides were from Aldrich. Cetyltrimethylammonium bromide (CTAB), dodecane, pentanol, sodium dodecyl sulfate (SDS) and tetradecane were obtained from Acros, and poly(L-lysine) (MW 150 000-300 000) was obtained from Sigma. Water was purified to a specific resistance >15 MΩ cm-2. Previously characterized bicontinuous microemulsions were made by mixing CTAB/water/ pentanol/tetradecane (17.5/35/35/12.5 wt %) and SDS/0.1 M NaCl/ pentanol/tetradecane (13.3/52/26.7/8 wt %).6,8 Basal plane pyrolytic carbon (PG) disk electrodes (A ) 0.16 cm2) were prepared as described previously. 23 Film Preparation and Characterization. Mb-PLL films were bonded to oxidized pyrolytic graphite (PG, 0.16 cm2) disk electrodes by carbodiimide-assisted amide bond formation as reported previously6,8 in the sequence PLL/Mb/PLL/Mb/PLL. These films contained ∼0.4 nmol cm-2 Mb. Mb-surfactant films were made by adsorption onto rough PG from microemulsions containing Mb.15 The distal histidine of Mb in PLL films was modified to make the tetrazolyl imidazole form of Mb by reaction with cyanogen bromide and sodium azide by a published procedure.24 Mb-PLL films for optical studies were made on fused silica slides.6 Film assembly and surfactant accumulation were monitored with a quartz crystal microbalance (QCM, USI Japan) using 9 MHz QCM resonators (AT-cut, International Crystal Mfg.). The gold-coated (0.16 ( 0.01 cm2) quartz resonators were first reacted with 0.3 mM 3-mercaptopropionic acid in ethanol.25 Films were assembled on resonators as for PG electrodes. Resonators were dried in a stream of nitrogen before measuring the frequency change. Cyclic voltammetry on MbPLL films was done in oxygen-free microemulsions as described previously.6

Scheme 1. Illustration of Conformational Equilibrium of Vicinal Dihalides for DBCH

The anti-periplanar conformer of vicinal dihalides gave faster reduction rates.11 Examples of structural rearrangement prior to electron transfer have been demonstrated at low temperatures where two different conformations react at an electrode at distinctly different potentials and the equilibrium between them is slow.16 trans-1,2-Dibromocyclohexane (DBCH) illustrates the two conformers, axial-axial (DBCHaa) and equatorial-equatorial (DBCHee), as shown in Scheme 1. Lexa et al.11 estimated the standard redox potential of β-bromoalkyl radical formation for (DBCHaa) at -0.87 V versus SCE and at -1.23 V (DBCHee). Evans et al. studied the electrochemical reduction of two interconverting conformers of vicinal dihalides using voltammetry at low temperatures.16 Conformational equilibria were solventdependent with (DBCHaa) favored in nonpolar solvents and roughly equal populations of (DBCHaa) and (DBCHee) favored in polar solvents.17,18 Conformational interconversion needs to be considered for a full mechanistic picture, but at ambient temperature, the rapid interconversion of conformers of vicinal dihalides used in the present article is a reasonable assumption for fluid phases. MbFeII may reduce vicinal dihalides faster than most nonprotein divalent metal macrocycles because substrate binding to the reaction site lowers the activation free energy via an inner-sphere pathway.19 Such binding can involve the iron heme or specific amino acid sequences on the protein surface. Substrates for heme enzymes such as cytochrome P450s are thought to bind in the distal pocket above the iron heme prior to reaction.20 We previously used vicinal dihalide reductions to probe the influence of microemulsions on reaction efficiency for catalysts dissolved in water phases or in films.12,15,21 Hydrophobic organic (16) (a) Nelsen, S. F.; Echegoyan, L; Evans, D. H. J. Am. Chem. Soc. 1975, 97, 3530-3532. (b) Klein, A. J.; Evans, D. H. J. Am. Chem. Soc. 1979, 101, 757-758. (17) Abraham, R. J.; Rossetti, Z. L. J. Chem. Soc., Perkin Trans. 1973, 2, 582-587. (18) Reeves, L. W.; Stromme, K. O. Trans. Faraday Soc. 1961, 57, 390-398. (19) (a) Save´ant, J.-M. AdV. Phys. Org. Chem. 1990, 26, 1-130. (b) Andrieux, C. P.; Save´ant, J. M. In Molecular Design of Electrode Surfaces; Murray, R. W. Ed.; Techniques of Chemistry Series; Wiley-Interscience: New York, 1992; Vol. 22, pp 207-270. (20) (a) Schenkman, J. B., Greim, H., Eds. Cytochrome P450; SpringerVerlag: Berlin, 1993. (b) Ortiz de Montellano, P. R. Ed. Cytochrome P450, 2nd ed.; Plenum: New York, 1995. (c) Montellano, P. R.; Catalano, C. E. J. Biol. Chem. 1985 260, 9265-9271. (d) Ortiz de Montellano, P. R.; Fruetel, J. A.; Collins, J. R.; Camper, D. L.; Loew, G. H. J. Am. Chem. Soc. 1991 113, 31953196. (e) Fruetel, J. A.; Collins, J. R.; Camper, D. L.; Loew, G. H.; Ortiz Montellano, P. R. J. Am. Chem. Soc. 1992 114, 6987-6993. (f) Loida, P. J.; Sligar, A. G. Prot. Eng. 1993, 6, 207-212. (g) Sibbesen, O.; Zhang, Z.; Ortiz de Montellano, P. R. Arch. Biochem. Biophys. 1998, 353, 285-296. (21) (a) Owlia, A.; Wang, Z.; Rusling, J. F. J. Am. Chem. Soc. 1989, 111, 5091-5098. (b) Zhou, D.-L.; Njue, C. K.; Rusling, J. F. J. Am. Chem. Soc. 1999, 121, 2909-2914.

Results Catalytic Reduction of DBCH. Mb-PLL films showed reversible voltammetry for the redox protein in SDS and CTAB microemulsions. Formal potentials as midpoints between reduction-oxidation peak pairs of the heme FeIII/FeII redox couple were -0.33 V versus SCE in the CTAB microemulsion and -0.41 V versus SCE in the SDS microemulsion. As reported previously for Mb-PLL films in microemulsions,6 all CV characteristics were consistent with nonideal thin film protein electrochemistry. Catalytic voltammetry of Mb-PLL films with DBCH present showed very different CVs in the two microemulsions. Films in the SDS microemulsion showed classic evidence of catalytic (22) Nassar, A. E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 2386-2392 (23) Njue, C. K.; Rusling, J. F. J. Am. Chem. Soc. 2000, 122, 6459-6463. (24) (a) Shiro, Y.; Morishima, I. Biochemistry 1984, 23, 4879-4884. (b) Taniguchi, I.; Sonoda, K.; Mie, Y. J. Electroanal. Chem. 1999, 468, 9-18. (25) Zhou, L.; Yang, J.; Estavillo, C.; Stuart, J. D.; Schenkman, J. B.; Rusling J. F. J. Am. Chem. Soc. 2003, 125, 1431-1436.

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Figure 2. Cyclic voltammograms of Mb-PLL films in an SDS microemulsion with (a) 0 mM DBCH, 15 mV s-1; (b) 400 mM DBCH, 15 mV s-1; and (c) 400 mM DBCH, 200 mV s-1.

Figure 1. Cyclic voltammograms of Mb-PLL films at 15 mV s-1 (A) in an SDS microemulsion with (a) 0 mM DBCH or (b) 160 mM DBCH and (B) in a CTAB microemulsion with (a) 0 mM DBCH and (b) 160 mM DBCH.

reduction26 (Figure 1A, wave b) with an increase in the reduction current at low scan rates and nearly complete disappearance of the oxidation peak characteristic of the ferrous form of Mb since it reacted with the dibromide. However, in the CTAB microemulsion Mb-PLL films retained their reversibility in the presence of DBCH at the same low scan rates but showed an unusual positive shift in redox potential to -0.20 V versus SCE (Figure 1B, wave b) for DBCH concentrations of about 160 mM and above. Mb-PLL films in SDS microemulsions showed increased catalytic current with increasing DBCH concentration at 15 mV s-1. However, Mb-PLL films in CTAB microemulsions showed almost no catalytic current even at very large DBCH concentrations and then only at scan rates e5 mV s-1. At higher scan rates, above 160 mM DBCH, no catalysis was found, and the Mb-PLL redox potential in the CTAB microemulsions remained at -0.20 V versus SCE. These results show that the catalytic reduction of DBCH by Mb-PLL films is much faster in the SDS microemulsion than in the CTAB microemulsions. When the Mb-PLL films exposed to DBCH in a CTAB microemulsion were washed with a dibromide-free microemulsion, the peak pair returned to -0.37 V versus SCE (Figure S1, Supporting Information), close to the midpoint potential of the original peaks. This shows that the peak shift caused by DBCH in the CTAB microemulsion can be reversed by removing DBCH. Voltammetry of Mb-PLL films in the SDS microemulsion at very high concentrations of DBCH (e.g., 400 mM) also caused a positive shift in both the reversible and catalytic peaks (Figure 2). This can be seen in catalytic wave b at 15 mV s-1 and more clearly in the reversible peaks (Figure 2, wave c) at 200 mV s-1, where the catalytic reaction does not have enough time to occur at this scan rate. The midpoint potential of this reversible voltammogram is -0.16 V versus SCE. Ferric myoglobin (metmyoglobin) is six coordinate with histidine as the proximal axial ligand and water hydrogen bonded (26) (a) Rusling J. F.; Zhang, Z. In Handbook of Surfaces and Interfaces of Materials; Nalwa, H. S., Ed.; Academic Press: San Diego, CA, 2001; Vol. 5, pp 33-71. (b) Rusling, J. F.; Zhang, Z. In Biomolecular Films; Rusling, J. F, Ed.; Marcel Dekker: New York, 2003; pp 1-64.

to another histidine as the distal ligand. Upon reduction to ferrous Mb, water dissociates from the heme iron but remains hydrogen bonded to the histidine.27 This distal water is part of a hydrogenbonding network that extends from the distal cavity to the exterior water. Armstrong and co-workers28 showed that myoglobin reduction is associated with heme geometry changes that are easier in the absence of the sixth water ligand because of lower solvent reorganization energy. Thus, the absence of the water ligand in the distal cavity reduces the energy barrier for electron transfer, lowering the reduction potential. For this reason, electrontransfer kinetics and the redox potential of Mb depend on the spin state and coordination number of the heme iron.24b,28,29 In light of the above discussion, we considered that the midpoint potential shifts of Mb in PLL films in the presence of DBCH could be caused by the entry of DBCH into the heme pocket, forcing out water. Thus, we investigated DBCH catalysis at PLLMb films featuring ligands that replace the axial water in metmyoglobin and bind to the heme iron as the sixth ligand. Imidazole remains associated in both ferric and ferrous forms of myoglobin,24 and presumably blocks the binding of DBCH in the heme pocket. We wished to know if Mb with imidazole as the sixth ligand can catalyze vicinal dibromide reduction, so we used voltammetry with 35 mM imidazole added to the microemulsion. Cyclic voltammetry in SDS and CTAB microemulsions showed comparable catalysis for the reduction of DBCH by the resulting imidazole-Mb formed in the film (Figure 3), with only small shifts in redox potentials from Mb-PLL films. This can be ascertained by observing the ratio of catalytic current with DBCH added to that in the absence of DBCH (i.e., the so-called catalytic efficiency of the reaction11,26). These ratios for imidazole-Mb (Figure 3) are similar for similar conditions in the two microemulsions. For the CTAB microemulsion, catalysis by imidazole-Mb was observed under CV conditions for which it does not occur with metmyoglobin (cf. Figure 1B). We also modified the distal histidine to make the tetrazolyl imidazole form of Mb (Mb-TzImd).24 The distal water ligand is replaced by tetrazolyl imidazole, and the heme iron is coordinated to the nitrogens of six ligands. Here there is little chance for the substrate to enter the distal pocket and bind above the heme. Films of Mb-TzImd-PLL gave CVs with midpoint potentials that were similar to those for Mb-PLL films but provided characteristic evidence of the catalytic reduction of DBCH (Figure 3C) for conditions under which there was no catalysis with Mb(27) King, B. C.; Hawkridge, F. M.; Hoffman, B. M. J. Am. Chem. Soc. 1992, 114, 10603-10608. (28) Van Dyke, B. R.; Saltman, P.; Armstrong, F. A. J. Am. Chem. Soc. 1996, 118, 3490-3492. (29) Cohen, D. J.; King, B. C.; Hawkridge, F. M. J. Electroanal Chem. 1998, 447, 53-62.

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Figure 3. Cyclic voltammograms of films at 15 mV s-1 (A) in an SDS microemulsion for (a) Mb-PLL, 0 mM DBCH; (b) Mb-Imidazole-PLL, 0 mM DBCH; (c) Mb-Imidazole-PLL, 160 mM DBCH; and (d) Mb-Imidazole-PLL, 240 mM DBCH; (B) in a CTAB microemulsion for (a) Mb-PLL, 0 mM DBCH; (b) Mb-Imidazole-PLL, 0 mM DBCH; (c) Mb-Imidazole-PLL, 160 mM DBCH; and (d) Mb-Imidazole-PLL, 240 mM DBCH; and (C) in a CTAB microemulsion for Mb-TzImd-PLL films for (a) 0 mM DBCH, (b) 80 mM DBCH, and (c) 160 mM DBCH.

PLL films. Thus, results in Figure 3 with forms of myoglobin in which water cannot be replaced above the heme by DBCH suggest that the most efficient catalytic binding site of DBCH is probably not the heme pocket but may involve sites on the exterior protein surface. To help assess the influence of the film matrix on catalytic efficiency, we also investigated the catalysis of DBCH reduction using Mb-surfactant films adsorbed from microemulsions onto PG electrodes without PLL. In these films, surfactant is coadsorbed along with myoglobin onto the electrode surface.15 As reported previously, Mb-surfactant films showed reversible reduction-oxidation peak pairs at about -0.5 V versus SCE in the SDS microemulsion and at about -0.3 V in the CTAB microemulsion. Mb-surfactant films showed catalytic currents indicating the reduction of DBCH in both microemulsions at 15 mV s-1(Figure 4). Mb-surfactant films in the SDS microemulsion showed catalysis with no reverse peak in the range of 15 to 100 mV s-1, whereas in the CTAB microemulsion, the MbFeII oxidation peak began to appear at a scan rate of 55 mV/s. At a scan rate of 100 mV s-1 in the CTAB microemulsion, Mbsurfactant films showed a reversible peak. The catalytic peak current increased with increasing DBCH concentration. Catalytic Voltammetry with 1,2-Dibromobromide (DBB) and meso-1,2-Dibromo-1,2-diphenylethane (DBDPE). Catalytic reduction of these dibromides was found in the SDS microemulsion with significantly larger catalytic currents at the same scan rate and in the same concentration range as for the CTAB microemulsion (Figure 5). In the CTAB microemulsions, much smaller shifts in the Mb-PLL redox potentials were found than for DBCH. DBB gave a relatively small catalytic current with Mb-PLL films in CTAB microemulsion at 15 mV s-1 and 160 mM DBB. Mb-surfactant films showed some catalysis by CV in both microemulsions at the same scan rate and and in the same concentration range for DBB. DBDPE showed no catalysis in the CTAB microemulsion at Mb-PLL and Mb-surfactant films; however, the concentration of this reactant was limited by poor solubility in the microemulsions. For all of the dibromides and films (Table 1), better catalysis was observed in the SDS microemulsion than in the CTAB microemulsion. Direct Voltammetry of Vicinal Dihalides. Carbon electrodes reduce vicinal dihalides via outer-sphere electron transfer.11 The influence of surfactant and PLL on the direct voltammetry of the

Figure 4. Cyclic voltammetry at 15 mV s-1 of Mb-surfactant films adsorbed on PG from microemulsions: (A) in an SDS microemulsion for (a) 0 mM DBCH and (b) 80 mM DBCH and (B) in a CTAB microemulsion for (a) 0 mM DBCH and (b) 80 mM DBCH.

vicinal dibromides was evaluated in microemulsions at bare PG and PG-PLL electrodes. In SDS microemulsions, direct irreversible reduction of DBCH occurred at -1.65 V versus SCE at a bare PG electrode and at -1.6 V versus SCE at PG-PLL (Figure 6A). However, about twice the peak current was found at PGPLL. In CTAB microemuslions, peak potentials were about -1.5 V for PG and -1.6 V for PLL-PG, but the peak currents were similar. Although CVs for the direct reduction of DBB did not give peaks that were as well defined as those for DBCH in SDS microemulsions, results were generally similar in that the current was larger for PG-PLL electrodes in SDS microemulsions than for PG but similar on the two electrodes for CTAB microemulsions. Results are consistent with preconcentration of the vicinal

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Figure 5. Cyclic voltammetry of Mb-PLL films at 15 mV s-1: (A) in an SDS microemulsion with DBB at (a) 0, (b) 80, and (c) 160 mM and (B) in a CTAB microemulsion with DBB at (a) 0, (b) 40, (c) 80, and (d) 160 mM DBCH.

Figure 6. Cyclic voltammograms at 5 mV s-1 for direct reductions at (a) PG and (b) PG-PLL electrodes in (A) an SDS microemulsion containing 16 mM DBCH and (B) a CTAB microemulsion containing 4 mM DBCH.

Table 1. Characteristic Potentials and Catalytic Efficiencies (Icat/Id)a of Mb-PLL and Mb-Surfactant Films in CTAB and SDS Microemulsions at 22 °C for the Reduction of Vicinal Dihalides by CV

reactant none DBCH DBCH DBB DBDPE none DBCH DBB DBDPE

concn, mM

80 160 80 1

80 90 1

CTAB microemulsion E1/2b Emb Icat/Id Mb-PLL Films -0.33 -0.30 1.0 -0.20 1.0 -0.30 1.2 -0.29 1.0

SDS microemulsion E1/2b Emb Icat/Id -0.41 -0.36 -0.36 -0.34

Mb-Surfactant Films -0.27 -0.22 2.5 -0.46 -0.27 2.1 -0.38 -0.27 1.0 -0.44

-0.40

2.0 3.4 2.2 1.2

-0.48 2.6 3.9 1.8

a At 15 mV s-1, Icat is the peak or plateau current in the presence of vicinal dibromide, and Id is the peak current in its absence. E1/2 is reported for the catalytic waves; midpoint potential Em is reported for reversible peak pairs. b V vs SCE.

dihalides in the PLL films in SDS microemulsions but not in CTAB microemulsions. Soret Band Spectra. Soret band spectra provide information about the distal environment, spin state, and coordination number of the heme iron in Mb.30 When dissolved in a medium-pH buffer, Mb shows a Soret band absorption maxima at 409 nm when water is the distal ligand. When the distal environment is nonpolar, the absorption maxima shifts to ∼390-402 nm.30,31 We recently showed that intact Mb in PLL films has Soret bands at 411-413 nm in neutral buffer and neutral CTAB or SDS microemulsions and that the CD spectra of these films are (30) (a) Morikis, D.; Champion, P. M.; Springer, B. A.; Egeberg, K. D.; Sligar, S. G. J. Biol. Chem. 1990, 265, 12143-12145. (b) Cao, W.; Christian, J. F.; Champion, P. M.; Rosca, F.; Sage, J. T. Biochemistry 2001, 40, 5728-5737. (c) Li, Q. C.; Mabrouk, P. A. J. Biol. Inorg. Chem. 2003, 8, 83-94.

Figure 7. Absorbance spectra of Mb-PLL films on fused silica slides upon exposure to a microemulsion containing DBCH and then washing with water: (A) CTAB microemulsion with DBCH at (a) 0 and (b) 40 mM and (c) rinsed with water and (B) SDS microemulsion with DBCH at (a) 0 and (b) 40 mM and (c) rinsed with water. Vertical lines represent 409 nm.

consistent with the native secondary structure of a polypeptide backbone.6,8 In the present work, Mb-PLL films showed shifts in the Soret band maxima to ∼390-400 nm when exposed to a CTAB microemulsion containing DBCH (Figure 7A). In SDS microemulsions containing DBCH, absorption maxima shifted to ∼405 nm (Figure 7B). (Absorbance in the region toward 300 nm is caused by DBCH.) Upon rinsing with water and returning to microemulsions with no DBCH, the Soret band moved back to its original position. The decrease in absorbance upon washing

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cm-2 that is consistent with the entry of SDS into the films. The absence of a significant change in ∆F for films exposed to the CTAB microemulsion indicated a negligible change in mass (Figure 8).

Discussion

Figure 8. QCM frequency changes for two Mb-PLL films after each layering step of fabrication and drying. The last points show measurements after exposure of the films to the CTAB (4) or SDS (b) microemulsion.

is related to the relative instability of the films on fused silica, to which they are not covalently bound as they are to electrode surfaces. Visible spectroscopy of the Mb-PLL film in microemulsions containing DBB or DBDPE showed no significant shifts in the Soret bands (Figures S2 and S3, Supporting Information) nor were Soret band shifts observed for Mb dissolved in either microemulsion when DBCH was present. Quartz Crystal Microbalance Studies. SDS entering into the cationic PLL films was revealed previously from quartz crystal microbalance (QCM) studies of PLL films containing cobalt corrin catalysts23 and by preliminary studies of Mb-PLL films.9 Here we provide more detailed QCM results for Mb-PLL films made on gold-coated quartz QCM resonators and then immersed in the CTAB and SDS microemulsions. In QCM, the change in frequency (-∆F) for dry films is proportional to mass per unit area (M/A) in the absence of viscoelasticity changes.32 For 9 MHz quartz resonators, the relation for dry films is

-∆F (Hz) M (g cm-2) ) A 1.83 × 108

(4)

The nominal thickness (d) of dry films can be estimated from ∆F with an expression confirmed by high-resolution electron microscopy:33,34

d (nm) ≈ (-0.016 ( 0.002)∆F (Hz)

(5)

Figure 8 shows the results of QCM monitoring of film construction and microemulsion exposure. There was a reproducible decrease in ∆F with each layer of PLL and Mb added onto the gold resonator. ∆F for Mb layers was much smaller than for PLL layers, indicating a larger amount of protein in the film than PLL. This behavior is consistent with reproducible layer formation at each step of film construction. By using eqs 4 and 5, we extract an average thickness for the films in Figure 8 of 415 nm and an average protein content of 6.5 nmol cm-2. Comparing to the value of 0.4 nmol cm-2 of electroactive Mb6 suggests that ∼10% of the protein is electroactive. Mb-PLL films showed a further decrease in ∆F upon exposure to SDS microemulsions, indicating a mass increase of 27 µg (31) Manchester, J. I.; Paulsen, M. D.; Rein, R.; Ornstein, R. L. Chem. Phys. 1996, 204, 223-231. (32) Buttry, D. A.; Ward, M. D. Chem. ReV. 1992, 92, 1355-1379. (33) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T., J. Am. Chem. Soc. 1995, 117, 6117-6123. (34) Lvov, Y. In Handbook of Surfaces and Interfaces of Materials; Nalwa, R. W., Ed.; Academic Press: San Diego, CA, 2001; Vol. 3, pp 170-189.

To set the stage for interpreting the data, we first review the general nature of the reaction site and the reactant residence site in Mb-PLL films. Mb in polyion films resides in a water-rich environment as supported by spectra of Mb/PLL films suggesting near-native conformation in films bathed in neutral microemulsions. In particular, Soret absorbance band positions and CD spectra of Mb-PLL films in a range of neutral surfactant-based fluids were nearly identical to those in neutral pH buffers.6,8 Fluorescence probe studies with pyrene in Mb-PLL films exposed to microemulsions showed that the pyrene residence site in CTAB microemulsions is somewhat more polar than the residence site in films that are exposed to SDS microemulsions.9 Pyrene sites in both systems were more polar than a hydrocarbon environment but much less polar than water. Because the vicinal dibromides in the present study are relatively nonpolar, we assume that they reside in the films in regions similar to the pyrene residence sites. Therefore, reactions of vicinal dibromides with reduced Mb (eqs 1-3) in these film take on a biphasic nature, with the catalyst in a water-rich phase and the reactant beginning in a relatively less polar phase and perhaps reacting at a biphasic interface. The situation is further complicated in the SDS microemulsions in which QCM data (Figure 8) suggest that SDS enters the cationic films and most likely forms micelles as suggested23 for PLL films with attached metal complex catalysts. In this scenario, the SDS micelles within the Mb-PLL film can preconcentrate the nonpolar reactant and improve the bimolecular reaction rate (eq 2). Such preconcentration is suggested by direct voltammetry of the dibromides at PG-PLL electrodes in SDS microemulsions but not in CTAB microemulsions (Figure 6). QCM results also suggest that CTAB does not enter the films A general experimental finding in this article is that the catalytic efficiency of Mb-PLL films for vicinal dibromide reduction is always better in the SDS microemulsion than in the CTAB microemulsion (Table 1 and Figures 1 and 5). The formal potential of Mb-PLL in the SDS microemulsions is 80 mV more negative than in the CTAB microemulsion (Table 1), whereas the value in pH 6.5 buffer is -0.30 V.8 The difference in formal potential is possibly caused by specific electrostatic interactions of DSwith the protein in the films, which would be stronger for MbFeIII than for MbFeII,4 whereas there are no such interactions with cationic CTA+ because it is repelled by the similarly charged PLL film. For the surfactant-Mb films (Table 1, Figure 4), the formal potential in CTAB microemulsions is 210 mV positive of that in SDS microemulsions. However, catalytic activity for the reduction of DBCH and DBB is now found for these films in the CTAB microemulsion, although it is a bit less than for the SDS microemulsion. This shift in formal potential while improving catalysis compared to that for the Mb-PLL films suggests that the catalyst redox potential does not control the catalytic efficiency and is consistent with an inner-sphere mechanism.11,19 Voltammetric and spectroscopic results provide clues to the catalytic reaction sites. First, at higher concentrations of DBCH (Figure 1B, Table 1) in CTAB microemulsions, the formal potential of Mb shifts positively by 130 mV with little catalysis. This shift does not occur with hemin-PLL films (Supporting

10794 Langmuir, Vol. 22, No. 25, 2006

Information, Figure S5), supporting the view that it is a function of intact Mb in the film. Second, the Soret band spectrum of Mb-PLL shifts to about 385 nm in the presence of >40 mM DBCH in CTAB microemulsions (Figure 7A), but the shift is reversed when DBCH is removed. Only a very small reversible blue Soret band shift was found in SDS microemulsions (Figure 7B), and shifts were negligible for the other vicinal dibromides in Mb-PLL films exposed to microemulsions. In addition, very high DBCH concentrations in the SDS microemulsion shifted the reversible formal potential of Mb-PLL positively by 250 mV (Figure 2c). The small potential shifts found under catalytic conditions (Table 1) are those expected from the usual influence of a chemical reaction step following electron transfer.35 No shifts in reversible formal potentials (Table 1) or Soret bands were found for the surfactant-Mb films. As mentioned previously, the shifts in formal potentials and Soret bands for Mb-PLL films in the presence of DBCH in may be due to the replacement of water by DBCH in the distal pocket of Mb above the heme. If this is true, then why does it occur only with DBCH and not with the other vicinal dibromides? The volume of the distal pocket of Mb has been estimated to be 0.13 to 0.15 nm3 with the water removed and 0.10 nm3 with distal water present.36 The van der Waals volume of DBCH is 0.141 nm3, which is within the range of the volume of the water-free pocket. Thus, DBCH can be envisioned as fitting into the distal pocket and displacing water. However, van der Waals volumes are 0.117 nm3 for DBB, 0.218 nm3 for DBDPE, and 0.130 nm3 for trans-1,2-dichlorocyclohexane, none of which gave Soret band or formal potential shifts with the Mb-PLL films in microemulsions. These data suggest that the smaller DBB may enter the heme pocket but is too small to affect heme coordination and that DBDPE is too large to enter the pocket. Our results also suggest that the Mb distal heme pocket has remarkable highresolution size selectivity and that DBCH is a relatively unique probe for this binding site. The conventional pathway proposed for cytochrome P450 iron heme enzymes involves the binding of the substrate in the heme pocket with the displacement of the distal water causing a positive shift of the redox potential and a blue shift of the Soret band.37 Similar binding for other iron heme proteins has been suggested.38 However, with Mb-PLL films, it seems that if DBCH is bound in the distal pocket it cannot be easily reduced when MbFeIII is electrochemicaly converted to the MbFeII form. This explains the shift in redox potential for Mb-PLL in CTAB microemulsions with a concomitant lack of catalysis in the DBCH reduction (Figure 1B and Table 1). Mb-imidazole and Mb-tetrazole-imidazole have the nonlabile imidazole ligand distal to the iron, precluding binding of DBCH in the distal pocket. With these biocatalysts, catalytic reduction was observed by CV under conditions for which it was not found for Mb-PLL (Figure 3). In addition, catalytic efficiency was only slightly smaller for these species in CTAB microemulsions compared to that in SDS microemulsions. Finally, comparisons of Mb-PLL, PLL-Mb-Imd, and PLL-Mb-TzImd films in CTAB microemulsions (Figure 3) reveal that the catalytic efficiency (35) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed; Wiley: New York, 2001; pp 501-505. (36) (a) Gibson, Q. H.; Regan, R.; Elber, R.; Olson, J. S.; Carver, T. E. J. Biol. Chem. 1992, 267, 22022-22034. (b) Carver, T. E.; Brentley R. E.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, J. N.; Olson, J. S. J. Biol. Chem. 1992, 267, 14443-14450. (37) Ortiz de Montelllano, P. R.; DeVoss, J. J. In Cytochrome P450, 3rd ed; Ortiz de Montelllano, P. R., Ed.; Kluwer Academic/Plenum Publishers: New York, 2005; pp 183-230. (38) (a) Wishnia, A. Biochemistry 1969, 8, 5064-5069. (b) Hershberg, R. D.; Chance, B. Biochemistry 1975, 14, 3885-3891.

Vaze and Rusling

Figure 9. Space-filling myoglobin model (Protein Data Bank, http:// www.rscb.org/pdb): green: heme iron; blue: positively charged nitrogens; red: negatively charged carboxyl oxygens.

does not depend on the redox potential. In general, results with Mb-PLL films with imidazole, tetrazolyl-imidazole, or water as the axial ligand suggest that the most active site of reduction of the vicinal dihalides may be on the outer surface of the protein rather than in the distal pocket. Because SDS clearly enters the Mb-PLL films and probably forms micelles, this may have an influence on catalytic efficiency. Surfactant structures such as micelles can preconcentrate nonpolar reactants,39 which is confirmed by the direct reduction data showing a larger peak current for PG-PLL electrodes than for PG electrodes in SDS microemulsions (Figure 6A). In the catalytic reactions, such preconcentration in the film increases the local concentration of reactant near the catalytic sites, which can enhance the catalytic efficiency.39 In addition, SDS aggregates in the film may restrict the access of DBCH to the distal pocket either by sequestering it in micelles or somehow blocking access to the distal pocket. However, it seems that distal pocket access is not totally blocked in SDS microemulsions because a large positive shift in the formal potential of Mb-PLL was observed at very large DBCH concentration (Figure 2c), implying competitive binding. SDS could interact with positively charged amino acid residues on the surface of Mb near the heme group (Figure 9). As speculated for proteins in solution, small micellelike clusters could form at these sites on the protein surface.40 These protein-surface micelles could trap DBCH and inhibit access to the heme pocket. However, there are only few positive charges near the heme group (Figure 9), and spectral changes in the Soret band (Figure 7) and in protein secondary structure as monitored by CD spectra6,8 that would be characteristic of strong Mb-surfactant interactions40 are absent. Thus, competition for DBCH between the heme pocket and SDS micelles in the films, probably strongly associated with protonated amine groups of PLL, seems a likely scenario. However, localized micelles located on positive charges near the heme edge (Figure 9) could still play a role in catalysis by increasing the local concentration of DBCH near reaction sites. Gray and co-workers41 estimated possible electron-transfer pathways in myoglobin and identified three cationic amino acid residues on Mb connected to the central heme iron via throughbond or through-space networks. These surface residues may be (39) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369. (40) Tofani, L.; Feis, A.; Snoke, R. E.; Berti, D.; Baglioni, P.; Smulevich, G. Biophys J. 2004, 87, 1186-1195. (41) Onuchic, J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B. Ann. ReV. Biophys. Biomol. Struct. 1992, 21, 349-377.

Microemulsion-Controlled Reaction Sites

involved in electron transport from heme iron to vicinal dibromides in micelles on the Mb surface. A remaining issue that is very difficult to address is conformational equilibria (eqs 1-3) of vicinal dibromides within PLL films. This is relevant because one conformer is more easily reduced than the other. Can conformations be frozen in PLL films at ambient temperature and influence the observed catalytic efficiency? We have so far been unsuccessful in obtaining NMR spectra of vicinal dibromides in PLL films or other evidence to support or disprove this hypothesis, so we are actively pursuing other experimental approaches. However, bromine-nitrogen association bonds (i.e., R-Br‚‚‚NH2-R) have an interaction enthalpy of ∼4.8 kcal/mol,42 which is similar to that of hydrogen bonds, and could increase the energy barrier to the conformational conversion of the dibromide bound in PLL films. In summary, inner-sphere catalytic reduction of vicinal dibromides with Mb-PLL films in an SDS microemulsion was more efficient than in a CTAB microemulsion. SDS enters the positively charged Mb-PLL films and preconcentrates the (42) Metrangolo, P.; Neukirch, H.; Pilati, T.; Resnati, G. Acc. Chem. Res. 2005, 38, 386-395.

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dibromide reactants, enhancing catalytic efficiency in the SDS micelles. Shifts in formal potentials and Soret bands of Mb-PLL films suggesting binding of DBCH in the iron heme distal pocket with little catalysis are consistent with the most efficient catalytic reduction sites involving the protein surface. The influence of conformational equilibria in PLL films is currently unknown but may also play a role. Preconcentration of nonpolar reactants into catalytic PLL films using SDS incorporated from microemulsions may be a general approach to improved electrochemical catalytic efficiency in microemulsions. Acknowledgment. This work was supported financially by the National Science Foundation (grant no. CTS-0335345). Supporting Information Available: Reversibility of CV peak shifts due to DBCH, visible absorbance spectra of Mb-PLL in microemulsions containing DBB and DBDPE, absorbance spectrum of tetrazolyl imidazole-Mb, and CVs of control hemin-PLL films. This material is available free of charge via the Internet at http://pubs.acs.org. LA061138J