Conductive Polypyrrole via Enzyme Catalysis - The Journal of

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19278

J. Phys. Chem. B 2005, 109, 19278-19287

Conductive Polypyrrole via Enzyme Catalysis Hyun-Kon Song† and G. Tayhas R. Palmore*,†.‡ DiVision of Engineering and DiVision of Biology and Medicine, Brown UniVersity, ProVidence, Rhode Island 02912 ReceiVed: March 22, 2005; In Final Form: July 25, 2005

Laccase catalyzes the polymerization of pyrrole into a conducting polymer using dioxygen as the terminal oxidant. This finding is significant, because it identifies an enzymatic route, and thus an environmentally benign method, for preparing a technologically important polymer. In addition, the rate of oxidation of pyrrole increases when the redox molecule, ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate)], is included in a reaction medium that contains laccase. This increase in rate occurs because laccase catalyzes the oxidation of ABTS to ABTS•. In addition to laccase, the biocatalytically generated ABTS• oxidizes pyrrole to its corresponding radical cation to yield polypyrrole. Moreover, oxidation of pyrrole by ABTS• regenerates ABTS for subsequent biocatalytic turnover. Including ABTS in the reaction medium has two important consequences for the final product: (a) The reaction proceeds rapidly enough to form polymeric films instead of oligomeric precipitates, and (b) ABTS remains within the polymeric film as a redox-active dopant. The charge transport properties of the resulting polymers, both with and without ABTS as the counteranion, are compared to those of other conducting materials including polypyrrole prepared electrochemically or chemically.

Introduction The development of enzymes for manufacturing processes is of great commercial interest, both in the production of chemicals, food, paper, and textiles and in applications such as diagnostics, health care, and environmental remediation.1 This interest is driven by the need to develop processes that are environmentally benign, energy efficient, and selective toward specific molecular targets. Enzymes catalyze a wide range of chemical transformations under relatively mild conditions (e.g., aqueous conditions, room temperature, atmospheric pressure) and more recently have been found to function under more extreme conditions.2-4 Enzymes can be efficient catalysts, although the hallmarks of most enzymatic catalysts are their selectivity and specificity at converting substrate to product and their susceptibility to metabolic control. Most commercial enzymes to date are wild-type proteins isolated from either natural or genetically engineered hosts, which catalyze the transformation of substrates found in their natural environment. The development of enzymes for new commercial applications typically targets the modification of wild-type proteins in terms of their substrate selectivity and specificity via genetic engineering, or the discovery of enzymatic transformations of nonnatural substrates.5-10 Pursuing the latter approach, we observed that laccase catalyzes the oxidation and polymerization of pyrrole by dioxygen. Laccase belongs to a subclass of oxidoreductases that includes ascorbate oxidase, bilirubin oxidase, and ceruloplasmin. These enzymes are known to couple the oxidation of 4 equiv of substrate to the four-electron, four-proton reduction of dioxygen to water using a catalytic site that contains four Cu (II/I) ions.11-18 The natural substrate of laccase (EC 1.10.3.2) is monolignol, which upon oxidation polymerizes into lignin.19-21 * Corresponding author: [email protected]. † Division of Engineering. ‡ Division of Biology and Medicine.

In addition to monolignols, laccase catalyzes in vitro the polymerization of a number of other derivatives of phenol (e.g., syringic acid,22-24 2,6-dimethylphenol,25 1-naphthol,26 coniferyl alcohol27), derivatives of benzenediol (e.g., urushiols28), and acrylamide.29 Although a number of polymers can be prepared via enzymatic catalysis,30-34 the preparation of a conducting polymer via enzymatic catalysis is limited to only a few examples: polyaniline,35-37 poly(2,5-diaminobenzenesulfonate),38 and polybenzidine39 via horseradish peroxidase; or polyaniline via bilirubin oxidase.40 Thus, our observation that laccase catalyzes the oxidation and polymerization of pyrrole is significant, because it defines the first enzymatic route for preparing conductive polypyrrole. Conducting polymers have attracted a great deal of attention since doped polyacetylene was discovered to be conductive.41,42 After this discovery, numerous other conductive polymers were identified including polypyrrole, polythiophene, and polyaniline.43,44 Electrical conductivity in these materials is the result of delocalization of π-electrons in a conjugated system. Conducting polymers initially were prepared via chemical catalysis (e.g., Ziegler-Natta). Subsequent studies showed that pyrrole, aniline, and thiophene could be polymerized into their corresponding conductive polymers with strong chemical oxidants such as ammonium peroxydisulfate [(NH4)2S2O8], ferric ions (Fe3+), permanganate (MnO4-), dichromate (Cr2O72-), or hydrogen peroxide (H2O2).45 Electrocatalysis also produces conductive films of polypyrrole,46 polyaniline,47 and polythiophene.48 The results reported herein show that laccase catalyzes the oxidation and polymerization of pyrrole into a conducting polymer by dioxygen. Included is evidence that the addition of a redox mediator facilitates the biocatalytic reaction between laccase and pyrrole, which ultimately improves the physical properties of the resulting conducting polymer.

10.1021/jp0514978 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/28/2005

Polymerization of Pyrrole via an Oxidoreductase

Figure 1. The rate of reaction of pyrrole in the presence (solid circles) or absence (open circles) of laccase. The initial solution was 0.5 mM pyrrole, 0.5 µM laccase in 10 mM sodium acetate buffer, pH 4.0. Inset photo: black precipitate that forms upon mixing a more concentrated solution of pyrrole: 200 mM pyrrole, 5 µM laccase in 200 mM acetate buffer, pH 4.0. Acetate is the counteranion in the precipitate.

Results and Discussion A Black Precipitate Forms in Mixtures of Pyrrole and Laccase. The discovery that laccase catalyzes the oxidation and polymerization of pyrrole stems from the observation that a black precipitate slowly forms upon addition of laccase to a solution of pyrrole exposed to air. Shown in the inset of Figure 1 is a black precipitate typically obtained 24 h after mixing a 200 mM solution of pyrrole in 200 mM sodium acetate buffer, pH 4.0, with a 5 µM solution of laccase. If the solution is purged with argon to remove all dissolved oxygen prior to the addition of laccase, a significantly smaller amount of black precipitate forms. These results suggest that oxygen, although not required, is involved in the reaction that leads to the formation of the black precipitate. To determine if pyrrole is consumed during the formation of the black precipitate, the concentration of pyrrole was monitored by absorbance spectroscopy. Shown in Figure 1 is the concentration of pyrrole relative to its initial concentration plotted as a function of time. Data were obtained by measuring the absorbance at 205 nm of a solution containing 0.5 mM pyrrole in 10 mM acetate buffer, pH 4.0, with and without 0.5 µM laccase. A dilute solution of pyrrole was used to ensure that no precipitate formed during the course of the experiment. Absorbance was converted to concentration using the Beer-Lambert law and an experimentally measured extinction coefficient of 6000 M-1 cm-1 for pyrrole. Note that the ratio of pyrrole to laccase is 1000:1, and therefore, the biocatalytic reaction is at saturation kinetics. Thus, increasing the amount of laccase will result in a proportional increase in the rate at which pyrrole is oxidized. The data shown in Figure 1 reveals that the concentration of pyrrole decreases with time whether or not laccase is present, although at different rates. It is known that pyrrole is protonated at the R-position under acidic conditions, which subsequently leads to an acid-catalyzed polymerization of protonated pyrrole.49-52 The polypyrrole that forms via this mechanism, however, has poor conductivity, because saturated rings are present in its molecular structure.52 On the basis of the slope of the data within the first 10 h, the rate of polymerization of pyrrole was calculated to be 0.068 µM min-1 for an acidcatalyzed mechanism (open circles, -d[Py]/dt|acid) and 0.11 µM min-1 for a laccase-catalyzed mechanism (solid circles, -d[Py]/ dt|Lcc). It should be noted that the value for -d[Py]/dt|Lcc is corrected for the reaction of pyrrole via the acid-catalyzed

J. Phys. Chem. B, Vol. 109, No. 41, 2005 19279

Figure 2. Proposed mechanism for laccase-catalyzed oxidation of both monomers and multimers of pyrrole by dioxygen.

mechanism. These results indicate that laccase is involved not only in the reaction that leads to a decrease in the concentration of pyrrole but that it also increases the rate at which the reaction occurs. To obtain the activity of laccase with pyrrole, the rate at which laccase catalyzes the oxidation of pyrrole by dioxygen (-d[Py]/dt|Lcc ) 0.11 µM min-1) is divided by the concentration of laccase in the reaction mixture. Thus, the activity of laccase with pyrrole is 3.7 mU pyrrole/mg laccase where U ) µmol min-1. This value is considerably smaller than the activity of laccase with a commercially available substrate, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) or ABTS, which is >2000 mU ABTS/mg laccase. These two observations, (i) the increase in the amount of black precipitate that forms in the presence of oxygen and (ii) the increase in the rate at which pyrrole reacts in the presence of laccase, provide evidence that laccase catalyzes the oxidation of pyrrole by dioxygen. This conclusion is supported further by reports that laccase catalyzes the polymerization of small organic molecules19-29 and that pyrrole is oxidized by other Cu(II/I) catalysts such as CuCl2, CuBr2, or CuCl-AlCl3-O2.52-55 Proposed Mechanistic Pathway for Laccase-Catalyzed Oxidation of Pyrrole by Dioxygen. Shown in Figure 2 is a proposed mechanistic pathway for laccase-catalyzed oxidation of pyrrole by dioxygen and subsequent chemical reactions that lead to polypyrrole. First, pyrrole is oxidized to its corresponding radical cation by reducing one of the four Cu(II) ions to Cu(I) in the active site of laccase. After 4 equiv of pyrrole have been oxidized, four electrons in the form of Cu(I) ions are available to reduce 1 equiv of dioxygen to water.16 Reduction of dioxygen to water regenerates the active site of laccase to its fully oxidized state to complete one biocatalytic cycle. Subsequent chemical steps in the mechanistic pathway include the coupling of two radical cations of pyrrole to form a dimer and its release of two protons to restore aromaticity to the pyrrole rings.43 The basis for the chemical steps shown in Figure 2 are the known mechanisms for both electrochemically catalyzed and chemically catalyzed polymerization of pyrrole.43,52 It is likely that dimers of pyrrole as well as higher-order multimers enter the biocatalytic cycle and are oxidized to their corresponding radical cations at the active site of laccase. Coupling of these radicals increases the length of the polymer chain. Because the oxidation potential of a lengthening multimer shifts to values more negative than pyrrole, the thermodynamic driving force of the reaction between laccase and multimers of pyrrole becomes more favorable with increasing multimer lengths.43,56 The rates at which multimers are oxidized by

19280 J. Phys. Chem. B, Vol. 109, No. 41, 2005

Song and Palmore

Figure 3. (a) Molecular structure of ABTS includes a diazo chromophore and two sulfonate substituents. Sulfonate substituents afford ABTS the ability to function as an anionic dopant in the conductive form of polypyrrole. (b) Conversion of ABTS (colorless) to ABTS• (blue-green) is a reversible, one-electron oxidation that occurs at 685 mV vs NHE.

laccase, however, is limited by physical parameters such as the diffusion coefficient of a multimer and its solubility. The mechanistic pathway illustrated in Figure 2 can be summarized by the following balanced equations:

pyrrole oxidation: (n + 1)Py f [Py]n+1 + 2nH+ + 2ne(1) dioxygen reduction: n/2O2 + 2nH+ + 2ne- f nH2O (2) overall: (n + 1)Py + n/2O2 f [Py]n+1 + nH2O

(3)

where Py is monomeric pyrrole and [Py]n+1 is polypyrrole consisting of (n + 1) pyrrole units. From eq 1, it can be seen that protons are produced during the oxidation of pyrrole and its multimers, which leads to a decrease in the pH of the solution if the oxidation reactions are catalyzed electrochemically or chemically. In contrast, the reduction of dioxygen to water (eq 2) consumes protons liberated from a biocatalyzed oxidation of pyrrole, resulting in a constant pH. In fact, the pH of a solution containing pyrrole, dioxygen, and laccase remains unchanged even in the absence of buffer. This observation is consistent with the proposed overall mechanism for laccasecatalyzed oxidation of pyrrole by dioxygen. Laccase-Catalyzed Oxidation of Pyrrole by a Redox Mediator. The electroactive compound 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate), or ABTS, is useful in a number of applications that involve laccase, including (i) optical screening for biocatalytic activity and (ii) mediating electron transfer between laccase and the cathode of a biofuel cell.57,58 These types of applications take advantage of the diazo chromophore in the molecular structure of ABTS (Figure 3a) and the reversible, one-electron redox reaction that occurs at 685 mV vs normal hydrogen electrode (NHE) (Figure 3b). Laccase catalyzes the oxidation of ABTS (λmax ) 225 and 340 nm) by dioxygen to produce the radical cation, ABTS• (λmax ) 415 nm) and water. This radical is known to oxidize organic

compounds such as phenols, uric acid, ascorbic acid, and bilirubin.59 Our studies reveal that ABTS• also oxidizes pyrrole. For example, dark blue to black films form within 1 h at the air-solution interface of a mixture of 200 mM pyrrole, 200 mM sodium acetate buffer, and 5 µM laccase in the presence of 2.5 mM (Figure 4a) or 25 mM (Figure 4b and c) ABTS, respectively. Increasing the concentration of ABTS from 2.5 to 25 mM results in thicker, more robust films. Because the molecular structure of ABTS contains two sulfonate groups (cf., Figure 3a), ABTS is expected to function as an anionic dopant in the cationic or conductive form of polypyrrole. Elemental analysis indicates that the ratio of pyrrole to ABTS in these films remains constant at 10:1 (i.e., five pyrrole units per one negative charge) regardless of the concentration of ABTS in the reaction mixture. Typically, there are four pyrrole units per anionic charge in conductive polypyrrole prepared electrochemically or chemically.43,46 Regardless of the concentration of ABTS used, we conclude that biocatalytically generated ABTS• rapidly oxidizes pyrrole to form films of polypyrrole doped with ABTS. Shown below is spectroscopic evidence in support of this conclusion. Spectroscopic Analysis of the Reaction between Pyrrole and the ABTS Redox Couple. Absorption spectra of dilute solutions containing 0.1 mM pyrrole, 1.0 µM laccase, 10 mM sodium acetate buffer (pH 4.0), and 25 µM ABTS were obtained for two purposes: (1) to determine if ABTS or ABTS• chemically oxidize pyrrole and (2) to determine the effect of laccase on the kinetics of oxidation of pyrrole in the presence of ABTS. Dilute solutions of pyrrole were used to avoid the formation of precipitates, which would complicate the interpretation of the absorption spectra. Shown in Figure 5 are the end-point absorption spectra over a 20 h period of two control solutions (e.g., ABTS, pyrrole) and a solution containing both ABTS and pyrrole (e.g., ABTS + pyrrole) that provide evidence that ABTS alone does not oxidize pyrrole. The spectra of a solution containing only ABTS (closed circles) remains unchanged for the duration of the experiment, indicating that ABTS is not oxidized spontaneously to ABTS• by dioxygen within 20 h. The spectra of a solution containing only pyrrole (open and closed squares) decreases slightly at wavelengths lower than 225 nm, which correspond to the acidcatalyzed oxidation of pyrrole discussed previously. The spectra of a solution containing both ABTS and pyrrole (solid and dashed lines) also shows a slight decrease at wavelengths lower than 225 nm similar to the spectral changes observed for a solution containing only pyrrole. Spectral changes at longer wavelengths would be expected if ABTS oxidized pyrrole because of the concomitant conversion of ABTS (225 and 340 nm) to ABTS• (415 nm). Such spectral changes, however, were not observed, providing conclusive evidence that ABTS alone does not oxidize pyrrole. Shown in Figure 6a are time-dependent absorption spectra of a solution containing 0.1 mM pyrrole, 10 mM sodium acetate buffer (pH 4.0), and electrochemically generated ABTS•. For this experiment, ABTS• was generated electrochemically prior to its addition to the acetate solution containing pyrrole. On the basis of the absorption spectrum (not shown) obtained prior to the addition of pyrrole, the initial concentrations of ABTS• and ABTS were 20 µM and 5 µM ABTS, respectively. During the time period of the experiment, both the absorbance due to pyrrole (205 nm) and ABTS• (415 nm) decrease with a concomitant increase in the absorbance due to ABTS (340 nm). These spectral changes indicate that ABTS• oxidizes pyrrole to produce ABTS and the radical cation of pyrrole (Py•+).

Polymerization of Pyrrole via an Oxidoreductase

J. Phys. Chem. B, Vol. 109, No. 41, 2005 19281

Figure 4. Films of polypyrrole typically produced from solutions containing 200 mM pyrrole, 5 µM laccase, 200 mM sodium acetate buffer (pH 4.0) and (a) 2.5 mM ABTS or (b and c) 25 mM ABTS. Images in (a) and (b) are photographs, whereas the image in (c) is a scanning electron micrograph.

Figure 5. Absorption spectra of a solution containing 0.1 mM pyrrole, 25 µM ABTS, and 10 mM sodium acetate buffer (pH 4.0) at t ) 0 and 20 h. The absorption spectra of 25 µM ABTS in 10 mM sodium acetate buffer (pH 4.0) and 0.1 mM pyrrole in 10 mM sodium acetate (pH 4.0) are included for comparison.

Simultaneously, three new peaks appear at 220, 275, and 303 nm (indicated by the *). The isobestic point at 216.5 nm corresponds to the interconversion between pyrrole and the radical cation of pyrrole, whereas the isobestic point at 366.5 nm corresponds to the interconversion between ABTS• and ABTS. The appearance of three new peaks in the spectra suggests that Py•+ rapidly forms an unidentified intermediate complex that ultimately becomes polypyrrole doped with ABTS. It has been shown previously that a donor-acceptor complex between a transition metal ion and the π-system of pyrrole appears during the chemical synthesis of polypyrrole using metal ion oxidants.52 Thus, the unidentified intermediate complex observed in this study is likely to be an ion pair between monoanionic ABTS• or dianionic ABTS and the radical cation of pyrrole. This conclusion is further supported by the fact that the three new peaks related to the unidentified intermediate are not observed in spectra where ABTS is absent (cf., Figure 1). For comparison, shown in Figure 6b are time-dependent absorption spectra of a solution containing 0.1 mM pyrrole, 10 mM sodium acetate buffer (pH 4.0), and laccase-generated ABTS•. This experiment is identical to the experiment shown in Figure 6a except that laccase continuously regenerates the ABTS• consumed during the oxidation of pyrrole. Prior to the addition of pyrrole at t ) 0 min, the entire 25 µM solution of ABTS is oxidized to ABTS• via biocatalysis (i.e., the ABTS peak at 340 nm is absent). Upon addition of pyrrole, ABTS• (415 nm) is reduced to ABTS concurrently with the oxidation of pyrrole (205 nm) to its radical cation. By t ) 1.0 min, only 76% (19 µM) of ABTS• remains. Because laccase is present in the reaction mixture, however, any ABTS generated from this reaction is reoxidized immediately to ABTS• for further reaction with pyrrole. Consequently, unlike what is observed in the

Figure 6. Time-dependent absorption spectra of a solution containing 0.1 mM pyrrole, 10 mM sodium acetate buffer (pH 4.0), and 25 µM ABTS. ABTS• was generated electrochemically in (a) and via laccase in (b).

spectra shown in Figure 6a, an absorption peak corresponding to ABTS (340 nm) does not appear in the spectra shown in Figure 6b. The absorption peaks corresponding to the intermediate complex (218, 275, and 303 nm; indicated by *) however, are better resolved than in Figure 6a. All three peaks increase with isobestic points at 212 and 348 nm during the course of the reaction. Proposed Mechanism for the Oxidation of Pyrrole by Laccase-Generated ABTS•. Shown in Figure 7 is a schematic illustrating electron transfer steps between pyrrole (Py) and laccase-generated ABTS•. As described previously, laccase catalyzes the oxidation of ABTS by dioxygen to produce ABTS• and water. This reaction introduces an additional oxidant (ABTS•) to the reaction medium, which converts pyrrole to its radical cation, thereby regenerating ABTS for subsequent biocatalytic turnover. Included in the schematic are the potentials associated with each electron transfer step illustrating that all reactions are thermodynamically downhill and that the overall process is catalytic. The oxidation potential of pyrrole is 700

19282 J. Phys. Chem. B, Vol. 109, No. 41, 2005

Figure 7. Schematic illustrating the electron transfer steps involved in the oxidation of pyrrole (Py) to its radical cation (Py•+) and ultimately polypyrrole (pPy) via a biocatalytic cycle that includes ABTS. The potentials of each step relative to NHE are given.

Song and Palmore calculate fractional concentrations. Briefly, the intermediate complex that forms in the solution containing electrochemically generated ABTS• was assumed to be identical to the intermediate complex that forms in the solution containing biocatalytically generated ABTS•. This assumption ignores the fact that both ABTS and ABTS• can be involved in the formation of the intermediate complex in the solution containing electrochemically generated ABTS•, whereas only ABTS• is involved in the formation of the intermediate complex in the solution containing biocatalytically generated ABTS•. Nevertheless, this assumption appears to be valid, because the peaks related to the intermediate complex are at the same wavelengths in the spectra of both solutions. Despite the possible error caused by this assumption, it is possible to fit the experimental data using eq 19 (see Experimental Section) up to 125 min with a regression coefficient of >0.999 for wavelengths between 250 and 370 nm. Furthermore, the sum of the fractional concentrations is consistent for the entire time range of both experiments. The rates at which ABTS• decreases (-d[ABTS•]/dt) were estimated from the slope of the data within the first 2 min of reaction. The rate for the electrochemically generated ABTS• (Figure 8a) is given by

-d[ABTS•]/dt|ABTS ) 1.5 µM min-1 Figure 8. Fractional concentrations of ABTS (open circle), ABTS• (solid circle), and the intermediate complex (triangle) plotted as a function of time. ABTS• was generated electrochemically in (a) and biocatalytically in (b). The fractional concentration is defined as the concentration of each component divided by their corresponding bases: 25 µM ABTS, 25 µM ABTS•, and 25 µM ABTS• equivalent are used as the bases for ABTS, ABTS•, and the intermediate complex, respectively.

mV vs NHE. The measured potential of a solution containing equal concentrations of ABTS• and ABTS is 685 mV vs NHE. In the presence of laccase, however, excess ABTS• is generated, and the potential shifts to 900 mV vs NHE according to the Nernst equation. Thus, electron transfer from pyrrole to ABTS• is favored thermodynamically by about 200 mV. The potential of the O2/H2O couple is 1.0 V vs NHE at pH 4, and thus, in the presence of laccase, electron transfer occurs from ABTS to dioxygen to yield ABTS• and water. The laccase-generated ABTS• subsequently is available to oxidize another molecule of pyrrole. Shown in Figure 8 are the fractional concentrations of ABTS, ABTS•, and the intermediate complex plotted as a function of time for reactions that occur in acetate buffer between pyrrole and (a) electrochemically generated ABTS• or (b) biocatalytically generated ABTS•. The fractional concentrations were calculated from the time-dependent spectra shown in Figure 6 using the Beer-Lambert law (see Experimental Section). Both experiments used solutions containing 0.1 mM pyrrole, 10 mM sodium acetate buffer (pH 4.0), and 25 µM ABTS; only the second experiment (b) included 1.0 µM laccase. The fractional concentration of pyrrole is not shown, because one of the three absorption bands associated with the intermediate complex overlaps with the absorption band of pyrrole at 205 nm. Ideally, the sum of the fractional concentrations of ABTSrelated components in a solution should be equal to 1.0. Experimentally, the sum of ABTS• + ABTS + intermediate complex is 1.15 for the solution containing electrochemically generated ABTS• (Figure 8a) and 1.0 for the solution containing biocatalytically generated ABTS• (Figure 8b). The 15% excess observed for the solution containing electrochemically generated ABTS• (Figure 8a) is likely due to the basis spectra used to

(4)

and the rate for the biocatalytically generated ABTS• (Figure 8b) is given by

-d[ABTS•]/dt|ABTS+Lcc ) 5.0 µM min-1

(5)

Thus, the fractional concentration of ABTS• decreases more rapidly when ABTS• is generated biocatalytically despite the fact that ABTS• is produced continuously during the course of the experiment. The rate at which pyrrole is oxidized by electrochemically generated ABTS• (-d[Py]/dt|ABTS) can be estimated from the rate at which the concentration of ABTS increases (d[ABTS]/ dt|ABTS). If one assumes that ABTS does not participate in the formation of the intermediate complex, then the rate is given by

-d[Py]/dt|ABTS ≈ d[ABTS]/dt|ABTS ) 0.98 µM min-1

(6)

which is a value that is based on the slope of the ABTS data within the first 2 min of the reaction (Figure 8a). In contrast, if ABTS participates in the formation of the intermediate complex, then the rate is given by

-d[Py]/dt|ABTS > d[ABTS]/dt|ABTS ) 0.98 µM min-1. (7) The decrease in fractional concentration of electrochemically generated ABTS• is ascribed not only to the conversion of ABTS• to ABTS during the oxidation of pyrrole, but also to the inclusion of ABTS• in the intermediate complex. Consequently, only the minimum value of -d[Py]/dt|ABTS is obtained from d[ABTS]/dt|ABTS in the manner described above, because the value of -d[Py]/dt|ABTS cannot be calculated directly from -d[ABTS•]/dt|ABTS. In contrast, the decrease in fractional concentration of biocatalytically generated ABTS• is directly related to the increase in concentration of the intermediate complex. Recall that absorption peaks corresponding to ABTS are not observed in the spectrum of a solution containing biocatalytically generated ABTS• (Figure 6b). The absence of these peaks is evidence that laccase converts ABTS to ABTS• more rapidly than pyrrole converts ABTS• to ABTS. Therefore,

Polymerization of Pyrrole via an Oxidoreductase

J. Phys. Chem. B, Vol. 109, No. 41, 2005 19283

the rate at which pyrrole is oxidized by biocatalytically generated ABTS• (-d[Py]/dt|ABTS+Lcc) can be determined directly from the rate at which ABTS• decreases (-d[ABTS•]/dt|ABTS+Lcc). Because 1 equiv of ABTS combines with 10 monomeric units of pyrrole (equivalent to ∼2 cations), then

-d[Py]/dt|ABTS+Lcc ) 10 × -d[ABTS•]/dt|ABTS+Lcc ) 50 µM min-1 (8) In summary, the rate at which pyrrole is oxidized (-d[Py]/dt) varies according to reactants present as follows:

laccase (0.11 µM min-1) < ABTS• (>0.98 µM min-1) < ABTS• + laccase (50 µM min-1) (9) The rates of formation of the intermediate complex (d[complex]/dt) were calculated from its change in concentration in Figure 8. For electrochemically generated ABTS• (Figure 8a)

d[complex]/dt|ABTS+Lcc ) 5.0 µM equiv of ABTS• min-1 (11)

Figure 9. FTIR spectra of polypyrrole prepared in the presence of laccase and different anions: (a) pPy[Ac-]; (b) pPy[ClO4-]; (c) pPy[ABTS] from 8:1 Py to ABTS; (d) pPy[ABTS] from 80:1 Py to ABTS. FTIR spectra of polypyrrole prepared in the absence of laccase (i.e., electrochemically synthesized): (e) pPy[ABTS]. Peaks characteristic of the polypyrrole backbone are indicated by vertical dashed lines.60 Peaks characteristic to the counteranions in the polypyrrole films are indicated with asterisks (acetate: 2970; 1700 [CdO]), triangles (perchlorate: 2900, 1650, 1450, 1130), and dots (ABTS: 1480, 1125, 1090, 655 [ionic sulfonate]). Normalized spectra are offset on the ordinate.

Regardless of whether ABTS• is generated electrochemically or biocatalytically, the oxidation of pyrrole and the formation of the intermediate complex occur by the same reaction. Thus, the difference in the rate of formation of the intermediate complex in the two reactions is due to the amount of ABTS• available at any given time, which is higher when laccase is present because laccase oxidizes ABTS to ABTS• continuously. As a consequence, the presence of ABTS in the reaction mixture increases the rate of formation of polypyrrole via biocatalysis for two reasons. First, the activity of laccase with ABTS (>2,000 mU ABTS/mg laccase) is considerably larger than its activity with pyrrole (3.7 mU pyrrole/mg laccase). Second, ABTS• oxidizes pyrrole (>0.98 µM min-1) more rapidly than laccase alone (0.11 µM min-1). Infrared Spectroscopy of Polypyrrole Films. Shown in Figure 9 are FTIR spectra of polypyrrole prepared from a solution containing pyrrole and laccase in the absence (Figure 9a) or presence (Figure 9c,d) of ABTS. For comparison, FTIR spectra of electrochemically synthesized polypyrrole prepared from solutions containing ClO4- (b) or ABTS (e) are included. Despite the different counteranions present or the method of synthesis, peaks known to correspond to the backbone of polypyrrole60 are present in all five spectra. These peaks are located at 3400 [-NH], 1550, 1180, 1050, 880, and 800 cm-1 and are indicated by vertical dashed lines. The presence of these peaks in all five spectra confirms that mixing pyrrole with laccase and dioxygen produces polypyrrole both in the presence and the absence of ABTS. Moreover, the presence of these peaks in all five spectra supports the proposed mechanistic pathway for laccase-catalyzed oxidation of pyrrole by dioxygen both in the presence and the absence of ABTS. In addition to the peaks associated with the backbone of polypyrrole, peaks corresponding to the counteranion incorporated within the film also are observed. For example, peaks characteristic of the acetate anion are present in the spectrum of the black precipitate that results when a sodium acetate buffer solution of pyrrole is mixed with laccase in the presence of dioxygen (Figure 9a, indicated with asterisks). Peaks charac-

teristic of perchlorate are present in the spectrum of polypyrrole electrochemically synthesized from a solution containing 50 mM NaClO4 (Figure 9b, indicated with triangles). The spectra of the polypyrrole films made from solutions containing laccase and ABTS have characteristic peaks at 1480, 1125, 1090, and 655 cm-1 that correspond to stretching and bending modes of the sulfonate groups on ABTS (Figure 9c and d, indicated with dots). The intensity of these peaks is independent of the concentration of ABTS in the reaction mixture, confirming that ABTS is the limiting reagent that coprecipitates with polypyrrole during polymerization. These peaks also are present in the spectrum of pPy[ABTS] synthesized electrochemically (Figure 9e, indicated with dots). Shown in Figure 10a is a comparison of the charge transport properties of polypyrrole prepared via biocatalysis, both with and without ABTS as the counteranion, to those of other conductive materials including polypyrroles and poly-Nsubstituted pyrroles prepared electrochemically. The values of the conductivity of the other polypyrroles and poly-N-substituted pyrroles are taken from the literature.46,61-63 The conductivity of pPy[ABTS] (∼20-µm-thick film) prepared via laccase is approximately 0.2 S cm-1. This value is comparable to the conductivity of polypyrrole prepared electrochemically from an aqueous solution containing F- ions (0.14-0.16 S cm-1) or from an acetonitrile solution containing HSO4- ions (0.3 S cm-1). In contrast, the conductivity of pPy[Ac-] prepared from a sodium acetate solution containing laccase is considerably lower (∼10-3 S cm-1). Shown in Figure 10b is a comparison of the charge mobility (µ) of different materials as a function of carrier density (n). On the basis of Hall measurements, the charge carrier in pPy[ABTS] is positive or holelike, the carrier density is ∼2.2 × 1015 cm-3, and the charge mobility is ∼700 cm2 V-1 s-1 at room temperature (solid square with error bars). These values are comparable to that of silicon doped with arsenic, phosphorus, or boron. Conventional conducting polymers have charge mobilities on the order of 10-8 to 100 cm2 V-1 s-1. For example, the charge mobility (i.e., field effect mobility) of polypyrrole

d[complex]/dt|ABTS ) 0.58 µM equiv of ABTS• min-1 (10) and for biocatalytically generated ABTS• (Figure 8b)

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Song and Palmore

Figure 10. (a) Comparison of the conductivity of films of polypyrrole that contain different counteranions. The solvents used during synthesis (aq ) aqueous, ACN ) acetonitrile) and the associated counteranions are included for polypyrrole. The square symbols represent the conductivity of polypyrrole films prepared in this study (i.e., pPy[ABTS] and pPy[Ac-]). Both pPy[ABTS] or pPy[Ac-] were prepared from acetate buffer containing laccase. (b) Charge mobility of different materials plotted as a function of carrier density: pET2[ClO4-] ) perchlorate-doped poly(4,4′-dipentoxy2,2′-bithiophene);67,68 p(Si-Py)[I3-] ) iodine-doped poly[(6-N-pyrrolyhexyl)hexylsilane];69 1-D or 2-D pPy[Tos] ) one- or two-dimensional polypyrrole doped with tosylate.71

doped with p-toluenesulfonate is 1.77 cm2 V-1 s-1 at room temperature,64 or less than 0.1 cm2 V-1 s-1 (i.e., Hall mobility) and 40 cm2 V-1 s-1 (i.e., magnetoresistance mobility) at 270 K.65 Thus, the estimated charge mobility of pPy[ABTS] prepared in this study is significantly larger than that of conventional conducting polymers. Only a few organic polymers have such large values for charge mobility: µ > 103 cm2 V-1 s-1 for the highly oriented polyacetylene doped with iodine;66 µ ) ∼430 cm2 V-1 s-1 at n ) 3 × 1015 cm-3 for electrochemically synthesized poly(4,4′-dipentoxy-2,2′ bithiophene) doped with perchlorate (pET2[ClO4-], open diamonds in Figure 10b);67,68 µ ) 1.07 × 103 cm2 V-1 s-1 at n ) 1.03 × 1014 cm-3 for iodine-doped poly[(6-N-pyrrolyhexyl)hexylsilane] 69 (p(Si-Py)[I3-], open circles). Several explanations are given in the literature to explain the high mobilities of these organic polymers: a planar and straightened conformation of polyacetylene chains forced by delocalization of π-electrons; a bandlike conducting mechanism due to overlapping of cationic states followed by generation of preferential paths for the carrier conduction in pET2[ClO4-]; and σ-conjugation in p(Si-Py)[I3-]. The high charge mobility associated with pPy[ABTS] appears to be a consequence of the ABTS dopant, which has two sulfonate groups that ion-pair with cationic sites on the backbone of polypyrrole. This ion-pairing provides a structural bridge between two polypyrrole chains and, as such, may enforce structural ordering of the polypyrrole chains.70 In addition to the structural effects of the dopant, ABTS may function as a conjugated bridge that facilitates electron transport between polymer chains. Thus, electron transport in pPy[ABTS] may be similar to that of two-dimensional polypyrrole,71 which has a cross-linked structure via a large number of R-β linkages. In two-dimensional polypyrrole (solid triangle in Figure 10b; µ ) 20 cm2 V-1 s-1 at n ) 1019 cm-3), the conduction mechanism consists of transport in large metallic clusters (cross-linked by 75 monomeric units) and hopping between the clusters over a small tunneling dimension (a dimension of 2 monomeric units). In contrast, the mobility of one-dimensional polypyrrole (open triangle in Figure 10b; µ ) 0.02 cm2 V-1 s-1 at n ) 1021 cm-3) is limited by the length of the one-dimensional chain or by the barriers to electron hopping between neighboring chains.

Conclusions We have shown that laccase catalyzes the oxidation of pyrrole to its corresponding radical with concomitant reduction of dioxygen to water. The pyrrole radicals subsequently couple to form higher-order multimers, which, after aromatization and anionic doping, results in conductive polypyrrole. In addition, we have shown that the rate of oxidation of pyrrole increases when the redox molecule, ABTS {2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate)}, is included in the reaction medium. This increase in rate occurs because ABTS mediates electron transfer between pyrrole and the active site of laccase. The resulting polymeric films contain a redox-active dopant and show improved mechanical properties. This method of increasing the rate of polymerization is not limited to the reaction between laccase, ABTS, and pyrrole but could be applied to other reactions involving a combination of enzyme, mediator, and monomer. For the reaction between laccase, ABTS, and pyrrole, however, ABTS functions as both the mediator during the biocatalyzed polymerization of pyrrole and the dopant in the resulting conductive polymer. The conductivity of these polymers, both with and without ABTS as the counteranion, is similar to that of polypyrrole prepared electrochemically or chemically. The high charge mobility of pPy[ABTS], however, appears to be a consequence of the structural features of ABTS. The effect of ABTS dopant and similarly structured dopants on the electron transport properties of polypyrrole is the subject of a separate study and will be reported elsewhere.72 The merits to synthesizing polypyrrole via biocatalysis are twofold. First, the polymerization reaction is environmentally benign, because all reagents are consumed in the making of the polymer and the catalyst is biodegradable. Second, the pH of the solution does not decrease during the reaction, because protons generated during the polymerization of pyrrole are consumed by oxygen to form water. As a result, the poorly conductive polymer produced via acid catalysis is suppressed. Experimental Section Chemicals. Fungal laccase (benzenediol/oxygen oxidoreductase; EC 1.10.3.2) from Trametes Versicolor (2 units mg-1 protein using ABTS as substrate at pH 4) was a gift from Wacker Consortium fur Elektrochemische Industrie GmbH and used as received. The redox mediatior, 2,2′-azinobis(3-ethyl-

Polymerization of Pyrrole via an Oxidoreductase benzothiazoline-6-sulfonate) diammonium salt (ABTS), and other chemicals used to prepare buffered solutions were purchased from Aldrich and used without further purification. Infrared spectra were recorded on a Perkin-Elmer 1600 FTIR spectrometer. All samples were prepared as KBr pellets. Preparation of Polypyrrole. Pyrrole was oxidized by laccase in the presence of two different dopant anions, ABTS or acetate (Ac-). Both pPy[ABTS] and pPy[Ac-] were prepared in a Petri dish by mixing pyrrole (200 mM) and laccase (5 µM) in sodium acetate buffer (200 mM) to a final volume of 10 mL with or without ABTS (25 mM). The buffer was adjusted to pH 4.0, which is the pH at which this isozyme of laccase is the most active using ABTS as the electron donor (i.e., >2000 mU mg-1 at pH 4.0 vs 60 mU mg-1 at pH 7.0). Within 1 h, a black film of pPy[ABTS] forms at the air-liquid interface, which continues to develop overnight. In contrast, at least 1 day is required before a black solid of pPy[Ac-] precipitates from solution. The precipitate was collected for characterization by filtering the reaction mixture through a nylon membrane (47 mm diameter with 0.2 µm pores, Millipore). All samples of polypyrrole were rinsed with deionized water and dried at 60° C for 6 h prior to characterization. For comparison, pPy[ABTS], pPy[Ac-], and pPy[ClO4-] (using 50 mM NaClO4) were synthesized electrochemically from solutions identical to those used for the biocatalyzed polymerization of pyrrole, except that laccase was absent. The electrochemical cell was purged with nitrogen for 10 min prior to the electrochemical oxidation of pyrrole. The voltage of the working electrode was cycled between 0.2 and 1.2 V vs NHE for 30 cycles at 100 mV s-1. For pPy[ABTS] and pPy[ClO4-], the current corresponding to the oxidation of pyrrole at >0.7 V vs NHE increased with each cycle, indicating that the surface area of the conductive polymer was increasing. For pPy[Ac-], the current decreased with each cycle, indicating that a nonconductive coating formed on the surface of the electrode. UV-vis Spectroscopic Measurements. Absorption data were collected (190-1000 nm) on a dual-beam Cary-500 scanning spectrophotometer using matched quartz cuvettes (3 mL capacity, 1 cm pathway length) for reference and sample beams. The concentration of reactants was chosen for adherence to the Beer-Lambert law. Absorption spectra were scanned at 2000 nm min-1 with a reduced slit height. Time-dependent absorption spectra were obtained from solutions containing the following components dissolved in 10 mM sodium acetate buffer, pH 4.0: 1 ) 0.5 µM laccase/25 µM ABTS; 2 ) 0.5 mM pyrrole; 3 ) 0.5 mM pyrrole/0.5 µM laccase; 4 ) 0.1 mM pyrrole/25 µM ABTS; 5 ) 0.1 mM pyrrole/ABTS•; and 6 ) 0.1 mM pyrrole/1.0 µM laccase/25 µM ABTS. For solutions 1-4, time ) 0 when all components were mixed. Time ) 0 when pyrrole was added to a solution of ABTS• electrochemically generated from 25 µM ABTS at 1.0 V vs NHE for solution 5 or biocatalytically generated from a mixture of ABTS and laccase for solution 6. The electrochemically generated ABTS• is found to be stable at room temperature for at least 1 day. The corresponding reference solutions were equivalent to the sample solutions, except pyrrole, ABTS, ABTS•, and laccase were absent. The activity of laccase with ABTS as substrate was determined using solution 1 by measuring the change in the absorption at 340 nm (ABTS, max ) 3.45 × 104 M-1 cm-1). The change in concentration of pyrrole as a function of time was determined using solutions 2 and 3 by measuring the change in the absorption at 205 nm (pyrrole, max ) 6.00 × 103 M-1 cm-1). By plotting absorbance data vs time, the rate of the decrease in concentration of ABTS or pyrrole can be calculated

J. Phys. Chem. B, Vol. 109, No. 41, 2005 19285 from the slope of the line obtained within the first minute of data for ABTS or the first 10 h for pyrrole, respectively. The temporal changes in fractional concentration (x) of each component in ABTS•-containing solutions (5 and 6) were determined from their time-dependent spectra using the BeerLambert law. In time-dependent spectra, absorbance (A) is a function of wavelength (λ) and time (t), while the fractional concentration is only time dependent over all wavelengths

Ai ) Ai(λ, t) and xi ) xi(t)

(12)

The subscript i identifies the species included in a solution from which an absorbance value was obtained (e.g., a ) ABTS•, b ) ABTS, c ) pyrrole, and d ) intermediate complex). If the concentration is fixed, the absorbance is only wavelength dependent

Ai0 ) Ai0(λ)

(13)

where the superscript 0 indicates a fixed concentration that is used as a basis from which fractional concentrations are calculated. The basis concentration for all ABTS-related compounds was 25 µM, because the sum of the concentrations of all ABTS-related compounds (i.e., ABTS, ABTS•, and the intermediate complex) in solutions 5 and 6 should be 25 µM at all times. The basis spectra for ABTS•, ABTS, and the intermediate complex were measured experimentally as follows: (1) The basis spectrum for ABTS• (Aa0) was obtained from a solution containing 25 µM ABTS and 1.0 µM laccase in 10 mM sodium acetate buffer, pH 4. (2) The basis spectrum for ABTS (Ab0) was obtained from a solution containing 25 µM ABTS in the same buffer. A reference solution was used for (1) and (2), which was equivalent to the sample solution, except pyrrole, ABTS, and ABTS• were absent. The basis spectrum for the intermediate complex (Ad0) was the spectrum of solution 6 at time ) 125 min, because all ABTS• was consumed at this time. Thus, the concentration for Ad0 was defined as “25 µM ABTS• equivalent” because the intermediate complex forms via the consumption of 25 µM ABTS•. Since only ABTS• has an absorbance at λ ) 415 nm, where max ) 3.50 × 104 M-1 cm-1, the fractional concentration of ABTS•, xa(t), can be determined

Aabcd(415 nm, t) ) Aa(415 nm, t) ) xa(t) Aa0(415 nm) or xa(t) ) Aa(415 nm, t)/Aa0(415 nm) (14) where Aabcd is the spectrum of either solution 5 or 6, and Aa0(λ) is the basis spectrum for ABTS•. After xa(t) is determined, the spectra of the solution containing all components except ABTS• (Abcd) can be calculated

Abcd(λ, t) ) Aabcd(λ, t) - xa(t) Aa0(λ)

(15)

For the range of wavelengths from 250 to 370 nm in which there are no peaks related to pyrrole

Abd(λ, t) ) Abcd(λ, t) for λ ) 250-370 nm

(16)

Because all the ABTS in solution 6 is converted to ABTS• by laccase, Abcd(λ, t) is equivalent to the spectrum of the intermediate complex over the range from 250 to 370 nm

Ad(λ, t) ) Abcd(λ, t) for λ ) 250-370 nm and for solution 6 (17) Finally, the fractional concentration of the intermediate complex,

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Song and Palmore

xd(t), in solution 6 is obtained

Ad(λ, t) ) xd(t) Ad0(λ) or xd(t) ) Ad(λ, t)/Ad0(λ) for any wavelength or xd(t) ) Abcd(λ, t)/Ad0(λ) for λ ) 250-370 nm and for solution 6 (18)

of sample. Because sulfur is unique to ABTS, we used the ratios of N to S and C to S, in combination with the molecular formulas of pyrrole (C4H5N) and ABTS (C18H18N4O6S4), to determine the molar ratio of pyrrole to ABTS in pPy[ABTS]. Thus, a repeat unit in pPy[ABTS] that includes x mol of pyrrole and 1 mol of ABTS will contain (x + 4) mol nitrogen, (4x + 18) mol carbon, and 4 mol of sulfur. Therefore

N/S ) (x + 4)/4

(20)

C/S ) (4x + 18)/4

(21)

where Ad0(λ) is the basis spectrum for the intermediate complex of 25 µM ABTS• equivalent. The value for xd(t) was calculated at 303 nm (a distinct peak in the absorption spectra of solution 6); however, it can be calculated at any wavelength between 250 and 370 nm. The analysis of solution 5 is more complicated than that of solution 6, because an additional component (ABTS) is present. Equations 14-16 used in the analysis of solution 6 are applicable to the analysis of solution 5. Equations 14 and 15 were used to calculate, respectively, the values for xa(t) and Abcd(λ, t) in solution 5. Given that Abcd(λ, t) obtained from eq 15 leads to Abd(λ, t) for 250-370 nm according to eq 16, the following is true:

For samples of pPy[ABTS] prepared from a solution containing an 8:1 ratio of pyrrole to ABTS, the weight percents of N, C, and S were 15.09, 54.39, and 9.66, respectively (average value of the three nearest data points with one data point discarded). Thus, the mole percents of each element were N ) 1.077, C ) 4.529, and S ) 0.3012. These values can be used to determine the ratio of nitrogen to sulfur or carbon to sulfur (N/S ) 3.576; C/S ) 15.04) in the samples, which subsequently were used in following equations to solve for the ratio of pyrrole units to ABTS (x) in pPy[ABTS]:

Abd(λ, t) ) xb(t) Ab (λ) + xd(t) Ad (λ) for any wavelength or alternatively

Similarly, the molar ratio of pyrrole to ABTS can be calculated for samples of pPy[ABTS] prepared from a solution containing an 80:1 ratio of pyrrole to ABTS. In this case, the ratio of pyrrole units to ABTS was similar to that found for pPy[ABTS] prepared from a solution containing an 8:1 ratio of pyrrole to ABTS: x ) 10.70 from N/S ) 3.675, or x ) 10.48 from C/S ) 14.62. Measurement of Electrical Properties. Samples of pPy[Ac-] were prepared by filtering the precipitated polypyrrole through filter paper. Samples of pPy[ABTS] were prepared by transferring films of polypyrrole onto a glass surface. The conductivities of pPy[Ac-] and pPy[ABTS] were measured with a four-point probe. Voltage (V) between the two inner probes was measured while passing current (i) through the two outer probes. Conductivity (σ in S cm-1) is calculated using the following equation: σ ) F-1 and F ) tkgeo(V/i), where F (ohm cm) is the resistivity of the filtered precipitate or film, t (cm) is the thickness of the sample, and kgeo (dimensionless) is the correction factor for sheet resistance. For this configuration, where the ratio of sample size to spacing between probes was greater than 40, the value of kgeo was set to 4.53. The thicknesses of the samples were measured using a micrometer with friction stop or with scanning electron microscopy. Hall measurements were used to measure the carrier density and carrier mobility in samples of pPy[ABTS]. A film of pPy[ABTS] was fixed to a square cover glass with epoxy adhesive. Copper leads were attached to the four corners of the sample in a van der Pauw configuration. The carrier density was determined from the Hall voltage (VH) and the equation: n ) iB/(dqVH), where n is the carrier density in reciprocal cubic centimeters, i is the applied current in amperes, B is the applied magnetic field in gauss, d is the thickness of the sample in centimeters, and q is the elementary charge on an electron (1.6 × 10-19 C). Carrier mobility (µ, cm2 V-1 s-1) subsequently was calculated from the equation µ ) σ/(nq).

0

0

Abcd(λ, t) ) xb(t) Ab0(λ) + xd(t) Ad0(λ) for λ ) 250-370 nm Dividing by Ad0(λ) and substituting leads to

Y ) xb(t)X + xd(t) for λ ) 250-370 nm

(19)

where Y ) Abcd(λ, t)/Ad0(λ) and X ) Ab0(λ)/Ad0(λ). The values for xb(t) and xd(t) can be obtained by fitting (X, Y) data with eq 19 at each time t. A least-squares fitting with linear regression was performed on data between 250 and 370 nm, resulting in regression coefficients more than 0.999 for all fittings. Electrochemical Measurements. An EG&G potentiostat/ galvanostat, model 273A, was used to measure cyclic voltammograms of the polypyrrole films. A single-compartment cell was configured with a glassy carbon working electrode (3.14 × 10-2 cm2), a platinum gauze counter electrode, and a saturated silver/silver chloride reference electrode (0.197 V vs NHE). All potentials are reported vs NHE. The working electrode was polished prior to use, first with 1 µm R-Al2O3, followed by 0.05 µm γ-Al2O3 (Micropolish II, Buehler). Elemental Analysis. An elemental analyzer (NCS2100, CE instruments) was used to measure the weight percent of nitrogen, carbon, and sulfur in pPy[ABTS] prepared from a solution containing 2.5 mM ABTS and pPy[ABTS] prepared from a solution containing 25 mM ABTS. Four samples of pPy[ABTS] (∼5 mg) prepared from each of the two solutions were tested. The samples were vaporized (e.g., NOx, CO2, and SO2) via dynamic flash combustion, moved through an absorptive trap for water, transferred to a reactor to reduce NOx to N2, and swept through a gas chromatography (GC) column where the products separated and were subsequently detected by a thermal conductivity detector. The molar ratios of pyrrole to ABTS in the pPy[ABTS] samples were calculated as follows. First, the measured weight percent of each element was divided by its corresponding atomic weight (N ) 14.01, C ) 12.01, S ) 32.07 g mol-1) to obtain the number of moles of each element relative to the total mass

x ) [4(N/S)] - 4 ) 10.30

(22)

x ) [4(C/S) - 18]/4 ) 10.54

(23)

Acknowledgment. The authors thank the National Science Foundation, the Whitaker Foundation, and the Office of Naval Research for generous support. References and Notes (1) Layman, P. Chem. Eng. News 1990, 68, 17-18.

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