Enhanced Rates of Electrolytic Styrene Epoxidation Catalyzed by

in microemulsions feature the protein in a water-rich film environment with reactant in an oil-rich environment. .... Bruno Bühler , Katja Bühle...
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Langmuir 2004, 20, 10943-10948

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Enhanced Rates of Electrolytic Styrene Epoxidation Catalyzed by Cross-Linked Myoglobin-Poly(L-lysine) Films in Bicontinuous Microemulsions Abhay Vaze,† Michael Parizo,†,§ and James. F. Rusling*,†,‡ Contribution from Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269-3060, and Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032 Received May 25, 2004. In Final Form: September 10, 2004 Redox proteins attached to surfaces designed for biocatalysis hold promise for future clean synthetic routes. It is advantageous for these biocatalysts to operate in low-toxicity fluids with a high capacity to dissolve reactants. Here we report cross-linked films of myoglobin (Mb) and poly(L-lysine) (PLL) chemically attached to oxidized carbon cloth cathodes that in microemulsions feature the protein in a water-rich film environment with reactant in an oil-rich environment. These cross-linked Mb/PLL films were the most stable in microemuslions and had the largest turnover rates for epoxidation of styrene compared to lightly cross-linked or uncross-linked Mb/poly(styrene sulfonate) films. Up to 40-fold larger turnover rates were found in bicontinuous microemulsions compared to oil-in-water microemulsions and micelles. Enhanced turnover rates are correlated with up to 10-fold faster mass transport of solutes in the oil phases of the bicontinuous fluids.

Introduction Biocatalysis promises to be a major facilitator of future clean synthetic routes.1 Microbiological or enzymecatalyzed processes2-5 are often regio- and stereoselective and efficient. Current research efforts continue to discover or genetically engineer enzymes for stereoselective synthesis,6 boding well for development of valuable future biosynthetic processes. Other advantages of biocatalysts include mild operating conditions and high turnover rates. Synthesis of semisynthetic β-lactam antibiotics catalyzed by penicillin amidase7 and the production of high fructose corn syrup catalyzed by a three-enzyme process involving R-amylase, glucoamylase, and glucose isomerase are current examples of viable commercial processes.8 Oxidoreductases can catalyze redox reactions with the aid of coenzyme NADH to prepare chiral alcohols, hydroxy acids, and aldehydes from carbonyl compounds.9 Reductases in nature transfer electrons from donors to cytochrome P450 enzymes in aerobic media to drive oxidation of styrene,10 alkylbenzenes,11 and many other * Author to whom correspondence should be addressed. E-mail: [email protected]. † Department of Chemistry, University of Connecticut. ‡ Department of Pharmacology, University of Connecticut Health Center. § Present address: Pfizer Pharmaceuticals, Eastern Point Rd, Groton, CT. (1) Schoemaker, H. E.; Mink, D.; Wubbolts, M. G. Science 2003, 299, 1694-1697. (2) For examples of biocatalysis with bacteria, see: (a) Waterman, M. R.; Jenkins, C. M.; Pikuleva, I. Toxicol. Lett. 1995, 82/83, 807-813. (b) Dror, Y.; Freeman, A. Appl. Environ. Microbiol. 1995, 61, 855-859. (3) Joo, H.; Lin, Z.; Arnold, F. H. Nature 1999, 399, 670-673. (4) (a) Lehman, J. P.; Ferrin, L.; Fenselau, C.; Yost, G. S. Drug. Metab. Dispos. 1981, 9, 15-18. (b) Hara, M. Iazvovskaia, S.; Ohkawa, H.; Hideo, A.; Miyake, J. J. Biosci. Bioeng. 1999, 87, 793-797. (5) Fernandez-Salquero, P.; Bunch, A. W.; Gutierrez-Merina, C. Prog. Membr. Biotechnol. 1991, 291-305. (6) Rouhi, A. M. Chem. Eng. News 2001, Feb. 18, 86-87. (7) Matsumoto, K. In Industrial Applications Of Immobilized Biocatalysts; Dekker: New York, 1993; pp 67-88. (8) Pederen, S. In Industrial Applications Of Immobilized Biocatalysts; Dekker: New York, 1993; pp 185-208. (9) Hummel, W. TIBTECH 1999 17, 487-492.

unsaturated molecules.12 Myoglobin (Mb) is an iron heme protein that stores and transports oxygen in mammals, but it can also serve as an oxidation or reduction catalyst.13 We demonstrated that Mb, cytochrome P450s, and other metalloproteins immobilized on electrode surfaces in ordered surfactant films14 or in multilayered polyionprotein films15 facilitate direct electrode exchange with electrons involving the protein in its native state. This approach bypasses the requirement for electron donors and reductases. We have used Mb and cyt P450 in films on electrodes to catalyze dehalogenation16 and epoxidation17 in aqueous buffers. However, many organic substrates have low solubility in water, and this can bottleneck reactant supply and catalyst turnover. Microemulsions are thermodynamically stable, macroscopically isotropic mixtures of oil, water and surfactants with dynamic internal nanostructures.18 They are capable of solubilizing polar, nonpolar, and ionic reactants in relatively large amounts. Furthermore, mediated electrolysis in microemulsions provides an attractive approach (10) (a) Ortiz de Montellano, P. R.; Fruetel, J. A.; Collins, J. R.; Camper, Loew, G. H. J. Am. Chem. Soc. 1991, 113, 3195-3196. (b) Fruetel, J. A.; Collins, J. R.; Camper, D. L.; Loew, G. H.; Ortiz Montellano, P. R. J. Am. Chem. Soc. 1992, 114, 6987-6993. (11) (a) Loida, P. J.; Sligar, A. G. Protein Eng. 1993, 6, 207-212. (b) Sibbesen, O.; Zhang, Z.; Ortiz de Montellano, P. R. Arch. Biochem. Biophys. 1998, 353, 285-296. (12) (a) Schenkman, J. B., Greim, H., Eds. Cytochrome P450; SpringerVerlag: Berlin, 1993. (b) Ortiz de Montellano, P. R., Ed. Cytochrome P450; Plenum: New York, 1995. (13) (a) Montellano, P. R.; Catalano, C. E. J. Biol. Chem. 1985, 260, 9265-9271 (b) Wade, R. S.; Castro, C. E. J. Am. Chem. Soc. 1973, 95, 231. (14) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369. (15) Lvov, Y. M.; Lu, Z.;. Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. (16) Nassar, A. E. F.; Bobbitt, J. M.; Stuart, J. D.; Rusling, J. F. J. Am. Chem. Soc. 1995, 117, 10986-10993. (17) (a) Zu, X.; Lu, Z.; Zhang, Z.; Schenkman, J. B.; Rusling, J. F. Langmuir 1999, 15, 7372-7377. (b) Munge, B.; Estavillo, C.; Schenkman, J. B.; Rusling, J. F. ChemBiochem 2003, 4, 82-89. (18) (a) Bourrel, M.; Schechter, R. S. Microemulsions and Related Systems; Marcel Dekker: New York, 1988. (b) Rusling, J. F. In Modern Aspects of Electrochemistry; Conway, B. E., Bockris, J. O’M., Eds.; Plenum Press: New York, 1994; Vol. 26, pp 49-104.

10.1021/la048712g CCC: $27.50 © 2004 American Chemical Society Published on Web 11/04/2004

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Table 1. Composition, Specific Conductance (K), and Viscosity (η) of Microheterogeneous Fluids Used fluid type

materials

composition

κ, MΩ-1

η, cP

bicontinuous µΕ bicontinuous µΕ O/w µΕ O/w µΕ micellar micellar

CTAB/pentanol/tetradecane/H2O SDS/pentanol/ tetradecane/H2O CTAB/butanol/ hexadecane/H2O SDS/pentanol/dodecane/0.1M NaCl CTAB/buffer + 50 mM NaCl SDS/ buffer + 50 mM NaCl

17.5/35/12.5/35 (wt) 13.3/26.7/8/52 (wt) 5/5.4/1/88.6 (wt) 3.35/6.65/1.0/89 (wt) 27 mM CTAB 34 mM SDS

1.6 6.0 4.2 10.5

14.8 12.5 5.4 4.1

to environmentally benign organic synthesis,19 avoiding toxic, expensive organic solvents, and often providing unique kinetic and pathway control.20,21 Advantages of microemulsions apply to synthesis of small molecules and polymers.20b,22 We previously used dissolved Mb in microemulsions to demonstrate a 50-fold increase in turnover rate for styrene epoxidation compared to aqueous buffer.23 This impressive rate increase was attributed to improved availability of the styrene reactant, which is much more soluble in microemulsions than in water, where the reactant mixture was a water-styrene emulsion. Efficient enzyme-catalytic electrodes optimized for synthetic applications in microemulsions could have high technological value, especially if relatively cheap catalytic proteins could be employed as “enzymes”. Such electrodes might provide reusable biocatalytic surfaces specifically designed for efficient production of fine chemicals in lowtoxicity microemulsions using cheap electricity. Recently, we developed cross-linked Mb-polyion films that were catalytically active in aqueous buffers from pH 1 to 12 in which Mb retained a near-native secondary structure at pH of the contact buffer as low as 2.24 In this paper, we describe cross-linked protein-polyion films developed specifically for operational stability in microemulsions. We evaluated two types of layered myoglobin-polyion films: (a) films of poly(styrenesulfonate) (PSS) and Mb assembled layer-by-layer that were subsequently cross-linked, and (b) layered films in which myoglobin was cross-linked at each step with poly(L-lysine) (PLL), the first layer of which was covalently bound to a carbon electrode. The Mb/PLL films catalyzed the epoxidation of styrene with up to 40-fold higher turnover rates in bicontinuous microemulsions compared to oil-in-water (o/w) microemulsions or micellar solutions. Efficient reactant supply achievable in microemulsions is a major factor in rate enhancement. Experimental Section Materials and Solutions. Sodium poly(styrenesulfonate) (PSS, MW 70 000, Aldrich) was used at 3 mg mL-1 in 0.5 M NaCl, and poly(dimethyldiallylammonium chloride) (PDDA, Aldrich) at 2 mg mL-1 in water. Horse heart myoglobin (Mb) from Sigma was dissolved in acetate buffer, pH 5.45 containing 10 mM NaCl, and filtered through a YM30 filter (Amicon, 30 000 MW cutoff),25 giving 2.9 mg mL-1 Mb. 1-Butanol, hexadecane, dodecane, pentanol, 1-[3-(dimethylamino)propyl]-3-ethyl carbo(19) Rusling, J. F. Pure Appl. Chem. 2002, 73, 1895-1905. (20) (a) Rusling, J. F. In Reactions and Synthesis in Surfactant Systems; Texter, J., Ed.; Marcel Dekker: New York, 2001; pp 323-335. (b) Rusling, J. F.; Campbell, C. J. In Encyclopedia of Surface and Colloid Science; Hubbard, A., Ed.; Marcel Dekker: New York, 2002; pp 17541770. (21) (a) Gao, J.; Njue, C.; Mbindyo, J. K. N.; Rusling, J. F. J. Electroanal. Chem. 1999, 464, 31-38 (b) Zhou, D.-L.; Carrero, H.; Rusling, J. F. Langmuir 1996, 12, 3067-3074. (22) Co, C. C.; De Vries, R.; Kaler, E. W. In Reactions and Synthesis in Surfactant Systems; Texter, J., Ed.; Marcel Dekker: New York, 2001; pp 455-470. (23) Onuoha, A. C.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1997, 119, 3979-3986. (24) Panchagnula, V.; Kumar, C. V.; Rusling, J. F. J. Am. Chem. Soc. 2002, 124, 12515-12521.

Table 2. Mb Films Assembled for This Work short notation Mb/PSS MbX/PSS MbX/PLL

film as assembled on carbon cloth (PSS/PDDA)3/(PSS/Mb)2/PSS (PSS/PDDA)3/(PSS/Mb)2/PSS, cross-linked afterward (PLL/Mb)2/PLL cross-linked at each layer addition

diimide (EDC), and cetyltrimethylammonium bromide (CTAB) were from Aldrich. Sodium dodecyl sulfate (SDS) was from Kodak. Tetradecane was from Acros, and poly(L-lysine) (MW 150 000300 000) was from Sigma. Water was purified by a Hydro Nanopure system to specific resistance >15 MΩ cm-2. The microemulsions used (Table 1) were characterized previously by a collection of methods as having bicontinuous or o/w structures.21a,26, Microemulsions were made by mixing appropriate weight ratios of components and stirring. Micellar solutions of CTAB and SDS were made in 50 mM tris buffer, pH 7.4 containing 50 mM NaCl. Apparatus and Procedures. Film Assembly. Films of PSS and myoglobin were constructed layer-by-layer on carbon cloth (Zoltek Corp.) electrodes by repeated alternate adsorption for 15 min from aqueous solutions of polyanion PSS, positive PDDA and Mb.15,17,24,27 Films were washed with water between adsorption steps. Typical Mb/PSS films comprised three PSS/PDDA precursor layers24 to improve stability and coverage, two Mb/ PSS layers, and a final PSS layer (Table 2). These films were used as is, or cross-linked after assembly by immersing filmcoated carbon cloth electrodes in freshly prepared, aqueous 24 mM EDC for 5 min to promote cross-linking of Mb,24 followed by rinsing with water. Using poly(L-lysine) (PLL) instead of PSS in the films allowed cross-linking between PLL and Mb, as well as self-cross-linking of Mb. Carbon cloth electrodes were first oxidized electrochemically as reported previously28 to form carboxylate groups on the surface. The oxidized electrode was immersed into 10 mL of 24 mM EDC for 20 min, which was subsequently replaced with aqueous PLL 4 mM in lysines. This step forms amide linkages between carboxylates on carbon cloth to amino groups on PLL.29 After 4 h, the carbon cloth was drained and rinsed with water and then covered with 10 mL of Mb solution and 10 mL of 48 mM EDC and let stand overnight. Additional layers of PLL and Mb were added in a similar manner to make two bilayers of PLL/Mb followed by a final layer of PLL (Table 2). For voltammetry, films were made on pyrolytic graphite (PG) or small carbon cloth electrodes as described above. For optical studies, PLL-Mb films were made on fused silica slides.24 UV-vis absorption was done with an HP 8453 UV-vis (25) Nassar, A.-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 2386-2392. (26) (a) Georges, J.; Chen, J. W. Colloid Polym. Sci. 1986, 264, 896902. (b) Mackay, R. A.; Myers, S. A.; Brajter-Toth, A. Electroanalysis 1996, 8, 759-764. (27) (a) Lvov, Y., In Protein Architecure: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mo¨hwald, H., Eds.; Marcel Dekker: New York, 2000; pp 125-167. (b) Lvov, Y., In Handbook Of Surfaces And Interfaces Of Materials, Vol. 3. Nanostructured Materials, Micelles and Colloids; Nalwa, R. W., Ed.; Academic Press: San Diego, 2001; pp 170-189. (c) Rusling, J. F.; Zhang Z., In Handbook Of Surfaces And Interfaces Of Materials, Vol. 5. Biomolecules, Biointerfaces, and Applications; Nalwa, R. W., Ed.; Academic Press: San Diego, 2001; pp 33-71. (d) Rusling, J. F.; Zhang, Z. In Biomolecular Films; Rusling, J. F., Ed.; Marcel Dekker: New York, 2003; pp 1-64. (28) Vaze, A. Rusling, J. F. J. Electrochem. Soc. 2002, 149, D193D197. (29) Zhou, D.-L.; Njue, C. K.; Rusling, J. F. J. Am. Chem. Soc. 1999, 121, 2909-2914.

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Figure 1. Cyclic voltammograms at 0.3 V s-1 of cross-linked Mb-polyion films in the bicontinuous CTAB microemulsion (Table 1) for (a) a cross-linked [PSS/PDDA]3[PSS/Mb]5/PSS film on PG successively after (1) 1 min in µE; (2) 373 min in µE; (3) 790 min total in µE, 420 min after adding 1 mM H2O2; and (4) 940 min total in µE for film used for scan 3 after adding 5 mM H2O2 for 145 min exposure; and (5) 1320 min total in µE, with final 180 min after adding 10 mM H2O2. (b) Cross-linked [PLL/Mb]2/PLL on oxidized PG successively after (1) 1 min in µE; (2) 210 min in µE with 1 mM H2O2; (3) 285 min total in µE for film used for scan 2 after adding 5 mM H2O2 for 70 min exposure; and (4) 560 min total in µE for film used for scan 3 after adding 10 mM H2O2 for 270 min exposure. diode array spectrophotometer. A JASCO 710 spectropolarimeter was used for circular dichroism. Voltammetry. A three-electrode thermosatted cell was used at 22 °C with a BAS-100B/W electrochemical analyzer. The working electrode was protein-coated carbon cloth or pyrolytic graphite (PG), the counter electrode was a Pt wire, and the reference electrode was saturated calomel (SCE). Surface coverage of active catalyst was measured by integrating cyclic voltammograms (CVs) at low scan rates. Polished glassy carbon electrodes were used with a previously reported method30 to obtain apparent diffusion coefficients of ferrocene in the various fluids. Electrolysis. Electrolysis was done in a stirred batch H-cell with anode and cathode compartments separated by a salt bridge (1 g agar + 7 g KCl + 23 mL water)31 behind a medium-porosity glass frit. The working electrode was a 12 cm2 geometric area carbon cloth coated with a Mb film, and the counter electrode was a spectroscopic graphite rod. Electrolyses were done in 10 mL of fluid containing 100 µL (0.86 mmol) of styrene in 10 mL of fluid with oxygen bubbled into the working electrode compartment for the first 20 min of reaction. Electrolysis was then continued under an oxygen atmosphere with magnetic stirring for a total reaction time of 1 h. The applied potential was -0.6 V vs SCE. After electrolysis, product mixtures were analyzed by gas chromatography as described previously.17

Results Voltammetry. Cyclic voltammograms of Mb/PSS and cross-linked MbX/PSS or MbX/PLL films (X denotes crosslinked) in microemulsions were reproducible on second and repeated scans on the time scale of 10 s to several minutes. All these films showed chemically reversible CV peaks (Figure 1) characteristic of the FeIII/FeII heme redox couple of myoglobin in polyion films, in the same potential range as in aqueous solutions.15,17 At 0.3 V s-1, crosslinked MbX/PSS film showed an initial reduction peak at -0.35 V and an oxidation peak at -0.20 V vs SCE (Figure 1a). Cross-linked MbX/PLL films showed reversible peak pairs at around -0.38 V vs SCE with a smaller reductionoxidation peak separation (Figure 1b). These films were tested for stability by CV in bicontinuous microemulsions and also at various hydrogen peroxide concentrations in a CTAB bicontinuous microemulsion. These media were designed to mimic conditions during electrochemical catalytic reduction of oxygen and styrene epoxidation, in which peroxide is generated and often reaches concentrations of ∼10 mM.15,17 The MbX/ (30) Rusling, J. F.; Shi, C.-N.; Kumosinski, T. F. Anal. Chem. 1988, 60, 1260-1267. (31) Meites, L. Polarographic Techniques, 2nd ed.; Wiley: New York, 1965; p 63.

Table 3. Surface Concentrations (Γ) of Mb in Polyion Films (X ) cross-linked) on Carbon Electrodes and D′-values for Ferrocene (Fc) in Different Fluids solvent CTAB bicon. µE SDS bicon. µE CTAB o/w µE SDS o/w µE CTAB micelles SDS micelles

106 × D′(Fc) MbX/PSS Γ (cm2 s-1) Mb/PSS (nmol cm-2) MbX/PLL 0.85 0.85 0.9 1 0.74 1

1.3 1.2 1 1.3 1.1 1.12

0.37 0.39 0.29 0.29 0.45 0.43

3.4 2.9 0.28 0.40 0.76 0.95

PSS films lost considerable peak current after storage in the microemulsion for 370 min and decreased further after addition of H2O2 (Figure 1a). On the other hand, while the Mb peak current was smaller, the covalently MbX/PLL films showed remarkable stability in microemulsions before and after addition of H2O2. CV peaks were virtually unchanged after hours of contact with microemulsions containing up to 10 mM H2O2 (Figure 1a). Uncross-linked Mb/PSS films showed well-defined, reversible CV peaks (not shown), but these films were less stable in microemulsions than MbX/PSS films. Thus, film stability in microemulsions deceased in the order MbX/PLL > MbX/ PSS > Mb/PSS. The CV reduction peaks of Mb in the films in microemuslions increased linearly with scan rate between 0.01 and 0.3 mV s-1, consistent with thin-film voltammetry.32 Integrations of CVs in the lower scan rate range of films that had not been aged were used to obtain the initial amount of electroactive Mb in the films in each fluid (Table 3). Values reflect the initial CV results as in Figure 1. The nature of the fluid did not have a large influence on the amount of electroactive Mb in the films. As reflected in Figure 1, MbX/PLL films had less electroactive Mb but were much more stable. CV was also used to measure the apparent diffusion coefficients (D′) in the oil phases or of micelles in the fluids in Table 1 by using bare glassy carbon electrodes with 2 or 3 mM dissolved ferrocene as a probe (Table 3). Since nearly all the ferrocene resides in the oil phase or the micellar interior of these fluids, these D′ values reflect the inherent mass transport rates of molecules in the oil phase or micelles in the bulk fluid.30,33 These data indicate up to 10-fold faster mass transport in the oil phases of (32) Rusling, J. F.; Zhang, Z. In Handbook Of Surfaces And Interfaces Of Materials, Vol. 5. Biomolecules, Biointerfaces, and Applications; Nalwa, R. W., Ed.; Academic Press: San Diego, 2001; pp 33-71.

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Figure 2. Voltammetry at 0.3 V s-1 of cross-linked [PLL/Mb]2/ PLL film in anaerobic bicontinuous CTAB microemulsion: (a) CV with no H2O2; (b) CV with 1 mM H2O2 present; and (c) rotating disk voltammetry at 1800 rpm with 1 mM H2O2 present.

bicontinuous microemulsions compared to o/w microemulsions and micellar fluids. Peroxidase activity of Mb in thin films can be detected by observing catalytic voltammetric peaks when H2O2 was added to generate active oxyferryl radicals.34,35 Similar experiments with added oxygen can be used to detect the catalytic reduction of oxygen by Mb. Figure 2 shows cyclic and rotating disk voltammetry for a cross-linked PLL/Mb film in the bicontinuous CTAB microemulsion with and without hydrogen peroxide. The new peak at -0.6 V vs SCE after adding H2O2 is due to catalytic reduction of H2O2 by Mb. The catalytic peak in RDV is larger due to the convective mass transport of H2O2 to the electrode surface. Similar results were obtained with other Mb films and other microemulsions. Furthermore, catalytic reduction peaks in the presence of oxygen (no H2O2) were also observed with Mb films in microemulsions. These experiments confirm that Mb retains its catalytic activity in the films. None of the catalytic peaks showed a large dependence on fluid type. Spectral Characterization of Mb. Native Mb has a strong iron heme Soret absorption band in neutral solutions at 410 nm.36 Vis spectra of cross-linked PLL/Mb films on quartz slides showed a well-defined Soret band of FeIII heme group of Mb at 411 nm. Figure 3 shows UVvis spectra of cross-linked PLL/Mb films on exposure to CTAB bicontinuous microemulsion up to 1 h. The Soret band remained at 411 nm, and the intensity decreased slightly because the films are slightly less stable on fused silica that lacks covalent bonds to the film. Figure 4 compares a circular dichroism (CD) spectrum of a cross-linked PLL/Mb film exposed to bicontinuous CTAB microemulsion with a spectrum of Mb in a neutral solution. CD is sensitive to the secondary structure of proteins. The double minima at 210 and 222 nm and the maximum at 195 nm are characteristic of the large R-helical content of Mb37 comprising 76% of the polypeptide backbone in the native conformation in neutral solution.38 Previous studies found similar CD spectra for cross-linked Mb/PSS films.24 The Vis and CD spectra suggest that Mb in the films in contact with microemulsions retains a nearnative secondary structure. (33) Rusling, J. F. In Modern Aspects of Electrochemistry, No. 26; Conway, B. E., Bockris, J. O’M., Eds.; Plenum Press: New York, 1994; pp 49-104. (34) Zhang, Z.; Chouchane, S.; Magliozzo, R. S.; Rusling, J. F. Anal. Chem. 2002, 74, 163-170. (35) Yu, X. Chattopadhyay, D. Galeska, I.; Papadimitrakopoulos, F. Rusling, J. F. Electrochem. Commun. 2003, 5, 408-411. (36) Goto, Y.; Fink, A. L. J. Mol. Biol. 1990, 214, 803-805. (37) Holzwarth, G.; Doty, P.; J. Am. Chem. Soc. 1965, 87, 218-228. (38) Rusling, J. F.; Kumosinski, T. F. Nonlinear Computer Modeling of Chemical and Biochemical Data; Academic Press: New York, 1996; pp 117-134.

Figure 3. Absorbance of Myoglobin in cross-linked PLL/Mb film on fused silica slide at different times of exposure to CTAB bicontinuous microemulsion. Curve baselines are offset for clarity.

Figure 4. Circular dichroism spectra of (a) 6 µΜ Mb in pH 7 buffer (b) cross-linked [PLL/Mb]2/PLL film on fused silica slide after dipping into CTAB microemulsion.

Epoxidation of Styrene. Catalytic electrochemical reduction of oxygen by Mb generates hydrogen peroxide, which activates Mb for styrene epoxidation.15,17 The three types of Mb films were used on carbon cloth cathodes to catalyze electrolytic epoxidation of styrene. The performance of these films were compared for 1 h epoxidations in different microemulsions and micellar solutions and in some cases at several temperatures (Table 4). The sole products of the reaction detected by gas chromatography were styrene oxide and benzaldehyde, but styrene oxide is the only Mb-catalyzed product. Benzaldehyde forms in relatively small amounts by a slow reaction between hydrogen peroxide and styrene.13,17 Turnover rates for Mb in Table 4 (nmol styrene oxide/nmol Mb/hr-1) were obtained by using the total moles of electroactive Mb on the electrode used for the synthesis assayed by slow scan voltammetry. Electrolysis results showed that turnover rates are the largest in bicontinuous SDS and CTAB microemulsions (Table 1), regardless of the types of films used. These rates are up to 40-fold faster in bicontinuous fluids compared to SDS micelles and SDS o/w microemulsions, and 4-fold larger than in CTAB micelles and CTAB o/w microemulsions. Addition of H2O2 to the CTAB bicontinuous microemulsion without electrolysis (Table 4, entry 3) gave turnover rates comparable to electrolysis, as found previously in aqueous media without surfactant added.17 In the cases documented, the turnover rate increased with temperature. The MbX/PLL films gave the best turnover

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Table 4. Styrene Oxide (SO) Yields for 1 h and Turnover Rates (TR) with Mb-Polyion Filmsa Mb/PSS fluid

T, °C

SO

1. CTAB bicon. µE 2. SDS bicon. µE 3.CTAB bicon. µE+10 mM H2O2 4. CTAB bicon. µE 5. CTAB bicon. µE 6. CTAB o/w µE 7. SDS o/w µE 8. CTAB micelles 9. CTAB micelles 10. SDS micelles

22 22 22 30 40 22 22 22 30 22

859 845 538 1290 1340 301 32 294 306 101

MbX/PSS TR,

hr-1

85 ( 16 84 53 ( 19 129 133 30 3.2 ( 0.4 29 34 ( 2 9.6 ( 2

SO 1230 845 854 1185 1245 347 20 208 182 136

TR,

MbX/PLL hr-1

79 ( 9 54 55 76 ( 18 80 22 1.3 ( 0.2 13 ( 0 13 8.85 ( 0.6

SO

TR, hr-1

953 920 948 1106 1230 236 70 175 648 84

375 380 391 457 507 ( 65 97 11 ( 1 73 ( 16 90 ( 14 14 ( 1

a All reactions run by electrolysis with oxygen present for 1 h at -0.6 V vs SCE except entry 3, CTAB bicon. µE driven by adding 10 mM H2O2. Most reactions were done in duplicate for SO yields ((15%), except that TR values given with (sd were run in triplicate. Symbols: X ) cross-linked film; SO ) nanomoles styrene oxide found; TR ) turnover rate (nmol styrene oxide/nmol Mb/hr-1).

Scheme 1. Pathway for Electrolytic and Hydrogen Peroxide Driven Epoxidation of Styrene

rates of the three film types in all fluids. Turnover rates in Mb/PSS and MbX/PSS films were comparable to one another. Discussion Explanation of the larger turnover rates in bicontinuous microemulsions (Table 4) rests on an understanding of the pathway of the Mb-mediated electrolytic epoxidation and the properties of the microemulsions. Onuoha et al.23 first investigated the electrochemical catalytic reaction pathway with Mb in aqueous solution and dissolved in microemulsions. Zu et al. later studied the reaction with Mb in films in homogeneous aqueous buffers.17a These studies revealed the pathway in Scheme 1. The applied voltage used in the present work is well negative of the CV peak for reduction of MbFeIII in our films (Figure 1), and thus, rapid reduction yields MbFeII. Dioxygen in solution reacts with MbFeII to give MbFeII-O2, a very fast reaction whose rate constant is 2 × 107 M-1 s-1 in neutral solution.39 Electrochemical reduction of MbFeII-O2 is fast at -0.6 V vs SCE. It yields hydrogen peroxide and recycles MbFeII for reaction with more oxygen.23 H2O2 formed by reduction of MbFeII-O2 can react with MbFeIII in the film to give the active ferryloxy radical oxidant •MbFeIVdO. The rate constant of this reaction39 in neutral solution at 25 °C is 440 M-1 s-1, and the lifetime of the ferryloxy radical is ∼30 s at room temperature at pH 7. This radical is the active oxidant that adds oxygen (39) Wazawa, T.; Matsuoka, A.; Tajima, G.; Sugawara, Y.; Nakamura, K.; Shikama, K. Biophys. J. 1992, 63, 544-550 and references therein.

to the olefinic bond of styrene.13 Epoxidation occurs by two pathways when oxygen is present, either by oxygen transfer from the ferryloxy radical, or by addition of dioxygen to a radical amino acid residue on •MbFeIVdO followed by epoxidation of styrene by the resulting peroxy radical oxyferryl Mb.13,17a Comparing the rapid electrochemical steps and the fast rates of reaction of oxygen with MbFeII (107 M-1 s-1) and of H2O2 with MbFeIII (440 M-1 s-1) with the much slower turnover rates in the range of several hundred per hour at best for electrolytic styrene epoxidation by the films (Table 4) suggests oxygen transfer to styrene as the most likely rate-limiting factor. Since the amount of protein catalyst in the films is limited, this reaction is likely to depend on mass transport of styrene to the electrode. D′ values of electroactive ferrocene, which track with mass transport in oil phases, were 10-fold larger in the bicontinuous microemulsions than in the o/w fluids (Table 3). Styrene has an o/w distribution coefficient of 6 and resides predominantly in the oil phase. Thus, mass transport of styrene should be much faster in the bicontinuous microemulsions than in o/w or micellar systems, facilitating turnover in these fluids. Furthermore, styrene is compartmentalized into droplets in the o/w and micellar fluids, while it is more freely distributed in dynamic oil tubules of the bicontinuous microemulsions. We observed a similar rate-limiting compartmentalization phenomenon previously in the electrochemical reduction of vicinal dihalides in the oil phase catalyzed by vitamin B12 in water droplets of a waterin-oil microemulsion.40 However, we believe that the present work is the first example of a compartmentalization effect in electrochemical catalysis using a mediatorcoated electrode. In contrast, large differences in turnover rates for electrochemical cobaltcorrin-PLL catalysis of dibromocyclohexane reduction between o/w and bicontinuous SDS microemulsions were not observed, possibly because of mechanistic differences.41 Interfacial dynamics near the reaction site in the film may be an additional factor controlling epoxidation rates. Studies in water-in-oil microemulsions suggested that increased amounts of cosurfactant can improve interfacial fluidity and may facilitate dynamics of solute transfer between phases.42-44 Table 1 shows that the bicontinuous fluids used have 35% and 27% cosurfactant for CTAB and SDS bicontinuous fluids, respectively, while the o/w (40) Owlia, A.; Wang, Z. Rusling, J. F. J. Am. Chem. Soc. 1989, 111, 5091-5098. (41) Njue, C. K.; Rusling, J. F. J. Am. Chem. Soc. 2000, 122, 64596463. (42) Garcia, E.; Song, S.; Oppenheimer, L. E.; Antalek, B.; Williams, A. J.; Texter, J. Langmuir 1993, 9, 2782-2785.

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microemulsions have 5-7% cosurfactant and the micelles have no cosurfactant. Thus, it cannot be ruled out that high interfacial fluidity in the bicontinuous microemulsions mediated by high cosurfactant content leads to efficient delivery of styrene from the its oil-phase microenvironment predominating in the bulk microemulsions to a water-rich microenvironment that probably surrounds Mb molecules in the films. The view that the Mb microenvironment is water-rich in the films is supported by VIS and CD spectra (Figures 3 and 4) that suggest a near-native conformation for Mb in the films. If the environment was oil-rich, some influence on the Mb secondary structure might be expected. More-detailed studies are underway to investigate the importance of this and other factors controlling reaction rates within the films in microemulsions. Increases in turnover rates for DBCH reduction by cobaltcorrin-PLL films in microemulsions were correlated with larger conductivities and lower bulk viscosity.41 Since, for our bicontinuous microemulsions, the conductivities are similar or smaller and the viscosities are larger than for the o/w fluids (Table 1), it would seem that these factors are not important in increasing turnover rates in the present case. MbX/PLL films have both the protein and lysine groups from PLL cross-linked, with the first layer of PLL chemically attached to the oxidized carbon electrode. The (43) Garcia, E.; Texter, J. J. Colloid Interface Sci. 1994, 162, 262264. (44) Antalek, B.; Williams, A. J.; Garcia, E.; Texter, J. Langmuir 1994, 10, 4459-4467.

Vaze et al

MbX/PSS films are only cross-linked once at the end of film assembly. Here, only the protein component is capable of being cross-linked to itself and there is less possibility of covalent attachment to the electrode. The Mb/PSS are not cross-linked at all. While the more-extensive crosslinking of MbX/PLL shifts the Mb FeIII/FeII midpoint potential about 100 mV negative of the value for the lightly cross-linked MbX/PSS films, the MbX/PLL films are much more stable in the presence of hydrogen peroxide. Even though the MbX/PLL films contain somewhat less electroactive Mb (Table 3), their improved stability during electrolysis leads to higher effective turnover rates. In summary, cross-linked MbX/PLL films on carbon cloth cathodes offered better stability and higher turnover rates for styrene epoxidation in microemulsions. The general method of film preparation should be useful for a wide variety of protein catalysts on carboxylated surfaces for use in microemulsions. Improved yields should result from reactor designs that maximize total catalyst while minimizing volume of the reaction solution and maximize efficiency of mass transport. In the simple stirred batch electrochemical reactors used here, up to 40-fold larger turnover rates in bicontinuous microemulsions compared to o/w microemulsions and micelles was correlated with ∼10-fold more efficient mass transport in the oil phase. Acknowledgment. This work was supported by Grant No. CTS-0335345 from NSF. M.P. is grateful for an undergraduate research fellowship from NSF. LA048712G