Direct Voltammetry and Catalysis with Mycobacterium tuberculosis

Amperometric responses for these films to H2O2 at 0 V are likely to contain ... Soft-Landed Protein Voltammetry: A Tool for Redox Protein Characteriza...
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Anal. Chem. 2002, 74, 163-170

Direct Voltammetry and Catalysis with Mycobacterium tuberculosis Catalase-Peroxidase, Peroxidases, and Catalase in Lipid Films Zhe Zhang,† Salem Chouchane,‡ Richard S. Magliozzo,‡ and James F. Rusling*,†

Department of Chemistry, University of Connecticut, Box U-60, 55 North Eagleville Road, Storrs, Connecticut 06269-3060, and Department of Chemistry, Brooklyn College, City University of New York, 2900 Bedford Avenue, Brooklyn, New York 11210-2889

Stable films of dimyristoylphosphatidylcholine and M. tuberculosis catalase-peroxidase (KatG), several peroxidases, myoglobin, and catalase showed reversible FeIII/ FeII voltammetry on pyrolytic graphite electrodes and catalytic current for hydrogen peroxide and oxygen. Amperometric responses for these films to H2O2 at 0 V are likely to contain significant contributions from catalytic reduction of oxygen produced during the catalytic cycles. Relative apparent turnover rates at pH 6 based on steadystate currents at 0 V versus SCE in the presence of H2O2 were in the order horseradish peroxidase > cytochrome c peroxidase (CcP) > soybean peroxidase > myoglobin > KatG > catalase. Lower currents for the very efficient peroxide scavengers KatG and catalase may be related to the instability of their compounds I in the presence of H2O2. KatG catalyzed the electrochemical reduction of oxygen more efficiently than catalase and CcP but less efficiently than the other peroxidases. DMPC films incorporating glucose oxidase and peroxidases gave good analytical responses to glucose, demonstrating the feasibility of dual enzyme-lipid films for biosensor fabrication. Biosensors featuring immobilized enzymes on electrodes can generate electrical signals selectively proportional to substrate concentration.1-3 Over the past decade, several types of enzyme films have been developed to achieve direct electron exchange with electrodes and avoid the complications of mediators.4,5 To this end, we developed films of metalloproteins in membrane-like surfactant and layered polyion matrixes on electrodes that facilitate direct reversible voltammetry.6,7 †

University of Connecticut. Brooklyn College. (1) Habermuller, K.; Mosbach, M.; Schuhmann, W. Fresenius’J. Anal. Chem. 2000, 366, 560-568. (2) Wang, J. Anal. Chem. 1999, 71, 328R-332R. (3) Nakabayashi, Y.; Omayu, A.; Morii, S.; Yagi, S. Sens. Actuators, B 2000, B66, 128-130. (4) Armstrong, F. A.; Wilson, F. S. Electrochim. Acta 2000, 45, 2623-2645. (5) Rusling, J. F.; Zhang, Z. In Handbook Of Surfaces And Interfaces Of Materials. Vol. 5. Biomolecules, Biointerfaces, and Applications; Nalwa, R. W., Ed.; Academic: San Diego, 2001; pp 33-71. (6) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369. (7) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. ‡

10.1021/ac010701u CCC: $22.00 Published on Web 11/30/2001

© 2002 American Chemical Society

Peroxidases are iron heme enzymes widely used in electrochemical biosensors,8 with applications that include DNA hybridization9-11 and electrochemical immunosensors.12,13 Reactions of cytochrome c peroxidase (CcP) and horseradish peroxidase (HRP) with H2O2 to form reactive oxidants are well documented.14 CcP forms compound ES featuring an oxoferryl iron heme [‚PFeIVdO, P ) protein] with a cation radical located on Trp191.15,16 HRP forms compound I with oxoferryl iron and the cation radical located on the heme porphyrin.17,18 In peroxidase catalysis, compound I can accept an electron from an organic substrate to form compound II [nonradical PFeIVdO], resulting in oxidation of the substrate. Compound II accepts a second electron to regenerate the resting enzyme. Direct electrochemical oxidations of CcP and HRP on specially treated electrodes have been reported.19-21 Like peroxidases, myoglobin (Mb), hemoglobin (Hb), and cytochrome P450s (cyt P450s) have catalytic cycles that can be activated by hydrogen peroxide, thought to produce reactive oxyferryl radical species similar to compound I in formal oxidation state.22,23 Direct electron transfer with Mb, Hb, and cyt P450s was (8) Gorton, L.; Bremle, G.; Csoeregi, E.; Joensson-Pettersson, G.; Persson, B. Anal. Chim. Acta 1991, 249, 43-54. (b) Ruzgas, T.; Csoregi, E.; Emneus, J.; Gorton, L.; Marko-Varga, G. Anal. Chim. Acta 1996, 330, 123-138. (9) Palacek, E.; Fojto, M. Anal. Chem., 2001, 73, 75A-83A. (10) Azek, F.; Grossiord, C.; Joannes, M.; Limoges, B.; Brossier, P. Anal. Biochem. 2000, 284, 107-113. (11) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-774. (12) Warsinke, A.; Benkert, A.; Scheller, F. W. Fresenius J. Anal. Chem. 2000, 366, 622-634. (13) Killard, A. J.; Micheli, L.; Grennan, K.; Franek, M.; Kolar, V.; Moscone, D.; Palchetti, I.; Smyth, M. R. Anal. Chim. Acta 2001, 427, 173-180. (14) Everse, J., Everse, K. E., Grisham, M. B., Eds. Peroxidases in Chemistry and Biology; CRC Press: Boca Raton, Fl, 1991; Vol. II. (15) Sivaraja, M.; Goodin, D. B.; Smith, M.; Hoffman, B. M. Science 1989, 245, 738-740. (16) Erman, J. E.; Vitello, L. B.; Mauro, J. M.; Kraut, J. Biochemistry 1989, 28, 8, 7992-7995. (17) Schulz, C. E., Rutter, R., Sage, J. T., Debrunner, P. G., Hager, L. P. Biochemistry 1984, 23, 4743-4754. (18) Roberts, J. E.; Hoffman, B. M.; Rutter, R.; Hager L. P. J. Biol. Chem. 1981, 256, 2118-2121. (19) Madhu, S.; Fuller H. A.; Armstrong, F. A. J. Am. Chem. Soc. 1996, 118, 263-264. (20) Chattopadhyay, K.; Mazumdar, S. New J. Chem. 1999, 23, 137-139. (21) Li, J.; Dong, S. J. Electroanal. Chem. 1997, 431, 19-22. (22) Ortiz de Montellano, P. R., Ed. Cytochrome P450; Plenum Press: New York, 1995. (b) Schenkman, J. B., Greim H., Eds. Cytochrome P450; SpringerVerlag: Berlin, 1993.

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achieved in lipid films on electrodes,24-26 which were used to epoxidize olefins.27 Mycobacterium tuberculosis KatG is an iron heme catalaseperoxidase of two 80-kDa subunits that oxidatively activates the prodrug isoniazid used to treat tuberculosis. This enzyme can be activated by peroxides to oxidize isoniazid.28 In a preliminary communication, we reported direct reversible FeIII/FeII voltammetry and catalytic reduction of H2O2 for M. tuberculosis KatG in lipid films on PG electrodes, suggesting participation of KatG compound I.29 In the present paper, we expand this study to comparisons with HRP, CcP, soybean peroxidase (SP), Mb, and catalase (Cat) in lipid films. Our goals in this work were to obtain additional insight into the catalytic electrochemical responses to hydrogen peroxide common to iron heme enzymes and to address analytical possibilities of peroxidases in lipid films. We show herein that stable films of all these enzymes give reversible FeIII/FeII voltammetry and catalyze the reduction of H2O2 and dioxygen. EXPERIMENTAL SECTION Chemicals and Enzymes. M. tuberculosis catalase-peroxidase was obtained from Escherichia coli strain UM262 expressing the M. tuberculosis KatG gene, as reported previously.29,30 Catalase (EC 1.11.1.6, 3260 units/mg, bovine liver), HRP (EC 1.11.1.7, type II, 240 units/mg from horseradish), SP (EC 1.11.1.7, 54 units/ mg from soybean), glucose oxidase (EC 1.1.3.4, type II-S, 18.8 units/mg from Aspergillus niger), and DMPC (99+%) were from Sigma. Cytochrome c peroxidase was obtained from E. coli BL21(DE3) expressing the CcP (MKT) gene as described previously31 and was a gift from Grant Mauk, University of British Columbia. Water had specific resistance of g18 MΩ‚cm. Other chemicals were reagent grade. Equipment and Procedures. A CHI430 electrochemical workstation (CH Instruments) was used for cyclic voltammetry (CV) and amperometry. The three-electrode cell employed a saturated calomel reference electrode (SCE), a Pt wire counter electrode, and a basal plane pyrolytic graphite disk (PG, Advanced Ceramics, A ) 0.2 cm2) working electrode. PG disks were abraded with 600-grit SiC paper prior to coating with enzyme-lipid films. Ohmic drop was compensated to 300 mV. Other enzyme-DMPC films showed similar 166 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002

amperometric behavior but with different sensitivities to H2O2. Relative apparent turnover rates based on the steady-state currents37 on rotating electrodes were in the order HRP > CcP > SP > Mb > KatG > Cat (Table 1). Peroxidases can be coupled with oxidases that produce H2O2 when oxidizing substrates, and the resulting signal from catalytic reduction of H2O2 can be measured.8 Glucose oxidase (GO) converts glucose to gluconic acid and produces H2O2. We immobilized GO and SP or HRP in DMPC films in a single casting step. These bienzyme films gave good responses to glucose, with SP giving better sensitivity (Figure 5). The signal derives from the peroxidase-catalyzed response to peroxide formed in the glucose oxidation. Films with a GO/SP ratio 3:1 gave better sensitivity to glucose than those with a 1:1 ratio. This suggests that the rate-controlling step is the oxidation of glucose by GO. All of the enzyme films also reduced oxygen in electrochemical catalytic cycles. In air-saturated buffers, enzyme-lipid films gave greatly increased FeIII reduction peaks accompanied by the disappearance of FeII oxidation peaks (Figure 6). This is consistent with catalytic reduction of oxygen to H2O2, as observed previously for Mb, Hb, and Cyt P450cam in lipid films.6 This catalytic reduction is ∼0.4 V more positive than direct reduction of oxygen on DMPC/PG electrodes for KatG, HRP, CcP, SP, and ∼0.3 V for catalase. It was also of interest to observe the development of catalytic peaks in the presence of limiting reactant. Comparison of CVs of KatG films in the presence of small amounts of O2 and H2O2 shows that the catalytic peaks are quite similar including potentials and shapes. In both cases, an increase in the limiting amount of reactant leads to the growth of catalytic peaks ∼100 mV more positive than the FeIII reduction peaks (Figure 7). This behavior is characteristic of rapid electrochemical catalysis.38 Since amperometric responses of enzyme-DMPC films to H2O2 were readily apparent at 0 V vs SCE, we investigated whether catalytic reduction of oxygen would produce responses under the same conditions. Figure 8 shows that all of the peroxidases and Mb gave significant responses to oxygen at 0 V. KatG enzyme (37) Armstrong, F. A. In Bioelectrochemistry of Biomacromolecules; Lenaz G., Milazzo G., Eds.; Birkhauser Verlag: Basel, Switzerland, 1997; pp 205255. (38) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980.

Figure 8. Amperometric responses in stirred solutions of various enzyme-DMPC films in pH 6.0 buffer purged with pure nitrogen, then with air beginning at t ) 200 s, and then with nitrogen again beginning at t ) 400 s. Control electrode was coated with DMPC alone. Figure 6. CVs at 0.1 V s-1 for enzyme-DMPC films in pH 6 buffer saturated with air (- - -) and without oxygen (s). (a) KatG shown with CV for control DMPC film without enzyme in air-saturated buffer (...), (b) Cat, (c) HRP, (d) CcP, and (e) SP. Curves for different enzymes are offset for clarity.

Figure 9. Spectrum of dry HRP-DMPC film on quartz, and spectra at specific times after wetting of the film with 200 µL of 2 mM H2O2.

treatment with H2O2, Soret absorbance bands of HRP (Figure 9), Mb, and SP, shifted to longer wavelengths characteristic of oxyferryl intermediates.39-41 After 1-3 min. in air, the bands returned to original Soret band wavelengths. KatG, CcP, and catalase bands in the films were not influenced by this H2O2 treatment. Similar results were found in solutions of these proteins, but return to the original Soret band took 7 h for HRP. Mb and SP bands decayed to nearly zero in this period. Red Soret band shifts were also found in CcP solutions containing lipid vesicles.

Figure 7. Comparison of CVs of KatG-DMPC films in the presence of H2O2 and oxygen at 0.1 V s-1 in pH 6 buffer initially purged with purified nitrogen for 20 min. CVs were run 30 s after adding H2O2 or air.

gave the smallest response of all peroxidases, and catalase gave a current increase only slightly larger than the control. In attempts to detect intermediates, protein-lipid films on quartz slides were wet with 2 mM H2O2. Immediately after

DISCUSSION Enzyme FeIII/FeII Electrochemistry. Several peroxidases, most notably HRP, have been immobilized on electrodes by adsorption, in polymer films or in carbon paste.8 Carbon electrodes with adsorbed HRP can provide direct electron transfer to detect H2O2.42-45 Electrochemical reduction of FeIII HRP on modified or unmodified graphite, Pt, and Au was reported to be very slow.8 An exception to this was the nearly reversible voltammetry (39) Onuoha, A. C.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1997, 119, 39793986. (40) Hayashi, Y.; Yamazaki, I. J. Biol. Chem. 1979, 254, 9101-9106. (41) Vitello, L. B.; Erman, J. E.; Miller, M. A.; Mauro, J. M.; Kraut, J. Biochemistry 1992, 31, 11524-11535.

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reported for a layer of HRP adsorbed from DMSO onto glassy carbon,45 for which peak separations (∆Ep) at 0.1 V s-1 were ∼80 mV and E°′ was -0.365 V versus Ag/AgCl (-0.32 V vs SCE). The problem with adsorption of enzymes onto electrodes for biosensor applications is the ever present possibility of desorption. The advantages of the lipid-protein films include their ease of preparation, good operational stability, and a biomembrane-like environment for the enzyme.6 These films feature ordered multiple bilayer structures that can incorporate a variety of metalloproteins.5,6,24-29 Typically, DMPC-protein films thin over several minutes to ∼0.5 µm during first exposure to buffer and then are stable under storage for up to several months.24 The main features of peroxidase and catalase voltammetry in DMPC films in anaerobic, peroxide-free solutions are the reversible peaks (Figure 1) for the heme FeIII/FeII redox couples. The peroxidase peaks appear at potentials similar to those for HRP adsorbed on glassy carbon from DMSO, and peak separations are slightly smaller than in that system, ranging from 66 mV for HRP to 86 mV for KatG (Table 1). Except for catalase, peak widths were in excess of the ideal 90 mV predicted for a one-electron surface reaction. This peak broadening can be interpreted in terms of dispersions of formal potentials and electrochemical rate constants, as previously documented for Mb in lipid films and for a wide variety of protein films on electrodes.5,6,25,36 Reversible changes of E°′ and Γ with pH show that the properties of enzymes in DMPC films are controlled by the acidity of the external solution, consistent with the large water content of these films.6 The change of E°′ with pH for all peroxidase enzymes between pH 6 to 11 is close to the -59 mV pH-1 expected at 25 °C for the reversible transfer of one proton and one electron.34,35 The intersections of linear portions of E°′ versus pH plots at about pH 5.8 for the peroxidases suggests that enzyme residues with a pKa of 5.8 participate in the protonation. This value is close to the pKa of 6 of histidylimidazole,46 which is the proximal iron heme ligand in HRP and CcP. The proximal ligand for KatG was identified as histidineimidazolate from resonance Raman spectra.47 Thus, the proximal histidine in these enzymes is a likely candidate for involvement in the redox coupled proton transfer. For Mb, the discontinuity in the E°′ versus pH plot at pH 4.7 has been correlated with similar effects of pKa 4.7 residues on electrochemical rate constants, surface concentrations, and secondary structure.36 Proximal and distal histidines are likely to be associated with this pKa. The discontinuity in the E°′-pH dependence of catalase suggests involvement of a residue with pKa 8.2. This enzyme has Tyr357as the fifth iron heme ligand,48 which may be the residue involved in a proton dissociation coupled with electron transfer. (42) Csoregi, E.; Joensson-Pettersson, G.; Gorton, L. J. Biotechnol. 1993, 30, 315-337. (43) Ho, W. O.; Athey, D.; McNeil, C. J.; Hager, H. J.; Evans, G. P.; Mullen, W. H. J. Electroanal. Chem. 1993, 351, 185-197. (44) Lindgren, A.; Munteanu, F.-D.; Gazaryan, I. G.; Ruzgas, T.; Gorton, L. J. Electroanal. Chem. 1998, 458, 113-120. (45) Guo, Y.; Guadalupe, A. R. Chem. Commun. 1997, 1437-1438. (46) Voet, D.; Voet J. F. Biochemistry, 2nd ed.; John Wiley & Sons: New York, 1995. (47) Lukat-Rodgers, G. S.; Wengenack, N. L.; Rusnek, F.; Rodgers, K. R. Biochemistry 2000, 39, 9984-9993. (48) Eventoff, W.; Tanaka, N.; Rossmann, M. G. J. Mol. Biol. 1976, 103, 799801. (b) Fita, I.; Rossmann, M. G. J. Mol. Biol. 1985, 185, 21-37. (c) Fita, I.; Silva, A.; Murthy, M. R. N.; Rossmann, M. G. Acta Crystallogr., Sect. B: Struct. Sci. 1986, B42, 497-515.

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Tyrosine has a pKa of 10.5,46 but the pKa of Tyr357 is probably lower than this. Tyr357 is tightly bonded to the heme Fe (Fe-phenolic oxygen distance 1.9 Å), and Arg353 may also promote ionization of Tyr357 by lowering the pKa of the tyrosine phenol (the two side chains are 3.5 Å apart). Absorption spectra showed that KatG catalase-peroxidase is stable under basic conditions. All the other hemoproteins had reversible spectral shifts between pH 7 and 11. The alkaline forms of Mb,49 HRP,50 and CcP51 contain heme FeIII-OH species, and their formation may involve conformational changes.52 However, formation of these species cannot be the sole source of the pH dependence of E°′ in the films, since the pH dependence begins at a much lower pH than the pK’s for FeIII-OH formation. Nevertheless, it is possible that protonation of these species may be involved in the reductions at the higher end of the pH range. Below pH 5, the smaller shifts in E°′ may involve conformational transitions driven by protonation. These are well documented for Mb53 and HRP.54 At pH 3, the spectra of KatG, HRP, Cat, and CcP suggest that the enzymes have partially unfolded, but unfolding is reversible for all but KatG. Data for SP suggest conformational change but not complete unfolding. KatG was irreversibly denatured at pH 3. The enzyme-lipid films typically showed increased Γ as pH decreased below 5, possibly also due to partial unfolding of the enzymes providing a more exposed heme to communicate with the electrode surface as documented for Mb.6,36 Catalytic Electrochemistry. Current responses for the reduction of H2O2 were detected for all enzyme-lipid films by CV and amperometry (Figures 3 and 4, Table 1). The peak potentials for catalytic reduction of H2O2 in these films were slightly positive of the formal potentials of the heme FeIII/FeII couple, consistent with fast electrochemical catalysis.38 Significant catalytic current can also be observed well positive of the peak values, but not as positive as the 700-750 mV versus SHE reported for CcP and HRP on other electrode surfaces and films.19-21 Relative enzyme turnover rates toward H2O2 in the lipid films based on amperometric currents at 0 V versus SCE were in the order HRP > CcP > SP > Mb > KatG > Cat (Table 1). However, different loadings were obtained in the DMPC films for different enzymes, so that the order of H2O2 sensitivity was CcP > Mb > SP > HRP > KatG > Cat. Catalytic H2O2 current for peroxidases8,19,55,56 has been attributed to the reduction of compound I generated by reaction of the enzymes with H2O2. The formal potential of compound I (or (49) Antonini, E.; Brunori, M. Hemoglobin and Myoglobin in their Reactions with Ligands; North-Holland: Amsterdam, 1971. (50) Araiso, T.; Yamazaki, I. Biochemistry 1978, 17, 942-946. (51) Dhaliwal, B. K.; Erman, J. E. Biochim. Biophys. Acta 1985, 827, 174-182. (52) Epstein, N.; Schejter, A. FEBS, 1972, 25, 46-48. (b) Iizuka, T.; Ogawa, S.; Inubushi, T.; Yonezawa, T.; Mishima, I. FEBS Lett. 1976, 64, 156-158. (53) Shen, L. L.; Hermans, J. Biochemistry 1972, 11, 1836-1841. (b) Yang, A.S.; Honig, B. J. Mol. Biol. 1994, 237, 602-614. (c) Goto, Y.; Fink, A. L. J. Mol. Biol. 1990, 214, 803-805. (d) Stigter, D.; Alonso, D. O. V.; Dill, K. A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4176-4180. (e) Friend, S. H.; Gurd, F. R. N. Biochemistry 1979, 18, 4612-4619; 4620-4630. (54) Smulevich, G.; Paoli, M.; Sanctis, G. D.; Mantini, A. R.; Ascoli, F.; Coletta, M. Biochemistry 1997, 36, 640-649. (55) Ferri, T.; Poscia, A.; Santucci, R. Bioelectrochem. Bioenerg. 1998, 45, 221226. (b) Chen, X.; Peng, X.; Kong, J.; Deng, J. J. Electronal. Chem. 2000, 480, 26-33. (56) Scott, D. L.; Paddock, R. M.; Bowden, E. F. J. Electroanal. Chem. 1992, 341, 307-321.

ES)/FeIII couple for CcP adsorbed on edge plane PG electrodes was 0.74 V versus SHE,19 and that of compound I/II for HRP on mercaptopropane sulfonate-Au was 0.53 V versus Ag/AgCl.21 However, we did not detect direct FeIII peroxidase oxidation peaks for any of the enzyme-lipid films. Nevertheless, CcP and SP gave catalytic responses to H2O2 extending as positive to 0.3 V versus SCE (∼0.55 V vs SHE). It is possible that catalytic activity in this potential region reflects a more negative formal potential for FeIII/ compound I (or ES) of these peroxidases in the films. Catalase is a well-known efficient catalyst,46 with rate constant as high as 4 × 108 M-1 s-1, close to the diffusion limit. The process occurs as follows: III

IV

•

CatFe + H2O2 f [CatFe dO] + H2O IV

•

III

(1)

retain small oxidation peaks corresponding to PFeII oxidized to PFeIII (Figure 7). In contrast, the oxidation peaks of FeII enzymes completely disappeared in the CVs of CcP, HRP, and SP at similar H2O2 levels. These results are consistent with very fast regeneration of H2O2 by catalase and KatG (eq 1) and very fast regeneration of the ferric enzymes (eq 2). The pathway in Scheme 1 shown for Mb was suggested for the catalytic reduction of oxygen by Mb and cyt P450 in films and in solution:6,7,25,27,39

Scheme 1 MbFeIII + e- f MbFeII

(at electrode)

MbFeII + O2 f MbFeIIsO2

(3) (4)

1

[CatFe dO] + H2O2 f CatFe + /2O2 + H2O (2)

MbFeIIsO2 + 2 e- + 2 H+ f MbFeII + H2O2 (at electrode) (5)

A similar reaction pathway was recently confirmed for myoglobin.57 Our results show that the peroxidase-catalase KatG, is the least efficient peroxidase in generating catalytic current for H2O2. However, KatG and catalase are highly efficient in reducing peroxide, so in these cases, it is possible that the current at 0 V is limited by rapid conversion of compound I to the ferric enzyme as in eq 2. Thus, considering the fast reactions of catalase and Kat G with H2O2 and slow electron transfer between electrodes and compound I, direct reduction of catalase and KatG compounds I may not be major contributors to the catalytic current from H2O2. Since the catalytic reduction peak of H2O2 is close to the formal potential of catalase FeIII/FeII, a possible reaction process involves the product O2 (eq 2) reacting with CatFeII to form CatFeII-O2 that can be directly reduced at the electrode. Ferrous iron in Mb and cytochrome P450 react rapidly with dioxygen to form such ferrous-dioxygen complexes. The dioxygen complex of Mb was shown by spectroelectrochemistry to be reduced at electrodes in solution,39 and dioxygen complexes are implicated in production of H2O2 in lipid films containing Mb and cyt P450s.24,25 Nearly identical reduction peak potentials and shapes in CVs of KatG in the presence of small amounts of O2 and H2O2 (Figure 7) support the view that KatG may be acting as a catalase to form O2 as in eqs 1 and 2. Furthermore, all of the enzymes studied here give a catalytic response to O2 at potentials as low as 0 V versus SCE (Figure 8). This raises the possibility that all the observed catalytic and amperometric responses to H2O2 may involve significant contributions from catalytic reduction of O2 formed by catalase-like activity. Films containing HRP, CcP, and SP all catalyzed the reduction of O2 and H2O2 and showed similar but not identical electrochemical behavior to catalase and KatG (Figures 3, 4, and 6-8 and Table 1). Spectra of films treated with H2O2 (Figure 9) provided clear evidence of oxyferryl species for HRP, Mb, and SP that convert in minutes to the ferric proteins. These results suggest that these enzymes may react with H2O2 in pathways similar to those of catalase. HRP can act as a catalase in the absence of proper substrates,58 and H2O2 can behave either as an oxidant or a reductant. In the presence of H2O2, CVs of catalase and KatG (57) Egawa, T.; Shimada, H.; Ishimura, Y. J. Biol. Chem. 2000, 275, 3485834866.

MbFeIII + H2O2 f •MbFeIVdO + H2O

(6)

•

MbFeIVdO + H2O2 f MbFeIII + 1/2O2 + H2O (7)

•

MbFeIVdO + SH f MbFeIVdO

(8)

An electron from the electrode first reduces metmyoglobin (eq 3) to MbFeII, which reacts rapidly with oxygen to give MbFeIIsO2 (eq 4). This reaction occurs with rate constant 2 × 107 M-1 s-1 at neutral pH.59H2O2 is produced by electrochemical reduction of the ferrous dioxygen complex (eq 5). Peroxide now reacts with metmyoglobin to give •MbFeIVdO, equivalent to peroxidase compound I (eq 6). This oxyferryl radical can react with peroxide to give dioxygen (eq 7),57,60 a process similar to the catalase reaction in eq 2, and can also decay by reaction with hydrogen atom donor SH to nonradical oxyferryl species.39,60 A similar pathway can be envisioned for the catalytic reduction of oxygen by the other heme enzymes. Thus, the catalytic responses of Cat, KatG, and peroxidases to H2O2 may involve significant contributions from the catalytic reduction of oxygen generated by reaction of H2O2 with compound I (eq 7), even at 0 V versus SCE, a common potential for analytical applications of peroxidases. Following addition of H2O2, a complex sequence of reactions equivalent to eqs 6 and 7, followed by eqs 3-5, may occur. This pathway could be particularly relevant for enzymes in lipid bilayer structures, in which oxygen is 10-fold more soluble than in water.61 Clearly, this pathway involves the generation of compound I (or ES), and direct reduction of compounds I, ES, and II cannot be ruled out. Relative contributions to the catalytic current for a given enzyme would depend on the relative rates of the coupled chemical and electrochemical processes and may also depend on the relative stability of the (58) Nakajima, R.; Yamazaki, I. J. Biol. Chem. 1987, 262, 2576-2581. (b) Arnao, M. B.; Acosta, M.; Del-Rio, J. A.; Varon, R.; Garcia-Canovas, F. Biochim. Biophys. Acta. 1990, 1041, 43-47. (59) Wazawa, T.; Matsuoka, A.; Tajima, G.; Sugawara, Y.; Nakamura, K.; Shikama, K. Biophys. J. 1992, 63, 544-550 and references therein. (60) King N. K.; Winfield, M. E. J. Biol. Chem 1963, 238, 1520-1528. (b) King N. K.; Winfield, M. E. Aust. J. Biol. Sci. 1966, 19, 211-217. (61) Kotyk, A.; Janacek K.; Koryta, J. Biophysical Chemistry of Membrane Function; Wiley: Chichester, U.K., 1988.

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oxyferryl iron radicals (cf. eqs 2, 7, and 8). Kat G and catalase compounds I are unstable and may contribute less to the catalytic current than similar species of the other peroxidases and Mb. This might explain why the amperometric turnover rates for H2O2 are lowest for KatG and catalase, which also show relatively low rates of reduction of O2. Amperometry showed that HRP has the highest relative turnover rate for reduction of H2O2 at 0 V versus SCE in the lipid films (Table 1). CcP films gave the best sensitivity, however, because of the larger amount of electroactive enzyme present. Interestingly, Mb, available in good purity at low cost, gave a H2O2 sensitivity 67% of that of CcP, suggesting possible biosensor applications for Mb based on peroxide detection. HRP also had the fastest relative rate of reduction of oxygen at 0 V versus SCE in the lipid films. The sequence is HRP > SP > Mb > KatG > CcP > Cat (Table 1), different from that of H2O2 reduction. This difference may reflect differences in the rates of chemical reactions with H2O2 (eqs 6 and 7) or in the relative rates of reduction of compounds I. The dual enzyme system GO/SP in DMPC films gave good amperometric responses to glucose (Figure 6). This experiment demonstrates that cast lipid films are viable for sequential enzyme systems and may find application to various biosensors. In summary, stable peroxidase-lipid films gave analytically useful electrochemical catalytic responses to hydrogen peroxide

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and oxygen. Electrochemical responses of these films to H2O2 at 0 V are likely to contain a significant contribution from catalytic reduction of oxygen produced during a complex catalytic cycle. ACKNOWLEDGMENT This work was supported by U.S. PHS Grant ES03154 from the National Institute of Environmental Health Sciences (NIEHS), NIH and Grant AI43582 from the National Institute of Allergy and Infectious Diseases, NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. The authors thank Grant Mauk, University of British Columbia, for the generous gift of cytochrome c peroxidase, and Naifei Hu of Beijing Normal University for suggestions about the catalytic mechanism of peroxide reduction. SUPPORTING INFORMATION AVAILABLE Table of enzyme Soret band data at various pH values and three figures showing pH dependence of Γ, E°′, and spectra of enzymelipid films.

Received for review June 22, 2001. Accepted October 23, 2001. AC010701U