Effects of Redox Potential and Hydroxide Inhibition on the pH Activity Profile of Fungal Laccases*

(Received for publication, May 21, 1996, and in revised form, October 10, 1996)

Feng Xu Dagger

From Novo Nordisk Biotech, Davis, California 95616

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSIONS
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The electronic absorption spectrum, susceptibility to fluoride inhibition, redox potential, and substrate turnover of several fungal laccases have been explored as a function of pH. The laccases showed a single spectrally detectable acid-base transition at pH 6-9 and a fluoride inhibition that diminished by increased pH (indicating a competition with hydroxide inhibition). Relatively small changes in the redox potentials (<= 0.1 V) of laccase were observed over the pH 2.7-11. Under the catalysis of laccase, the apparent oxidation rates (kcat and kcat/Km) of two nonphenolic substrates, potassium ferrocyanide and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid),decreased monotonically as the pH increased. In contrast, the apparent oxidation rates (kcat and kcat/Km) of three 2,6-dimethoxyphenols (whose pKa values range from 7.0 to 8.7) exhibited bell-shaped pH profiles whose maxima were distinct for each laccase but independent of the substrate. By correlating these pH dependences, it is proposed that the balance of two opposing effects, one generated by the redox potential difference between a reducing substrate and the type 1 copper of laccase (which correlates to the electron transfer rate and is favored for a phenolic substrate by higher pH) and another generated by the binding of a hydroxide anion to the type 2/type 3 coppers of laccase (which inhibits the activity at higher pH), contributes to the pH activity profile of the fungal laccases.


INTRODUCTION

Laccases (EC 1.10.3.2) are a family of multi-copper oxidases that catalyze the oxidation of a range of inorganic and aromatic substances (particularly phenols) with the concomitant reduction of O2 to water (for recent reviews see Refs. 1, 2, 3, 4, 5, 6, 7, 8). In the past decades, significant progress has been made in elucidating the structure of the copper sites, the catalysis sequence, and the mechanism that governs the catalytic reduction of O2 to H2O. Laccase is receiving increased attention as a model system for characterizing the structure-function relationship of copper-containing proteins because of its potential biotechnological application in the fields such as delignification, plant fiber derivatization, textile dye or stain bleaching, and contaminated water or soil detoxification (4).

One of the most important characteristics of laccase enzymology with phenolic substrates is the pH dependence. In general, the phenol oxidase activity of laccase has a bell-shaped (bi-phasic) pH profile whose optimal pH varies considerably among different laccases (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). Various structural and mechanistic factors from laccase, phenolic substrate, and O2 may contribute to the pH activity profile. Detailed studies have been carried out with respect to the effect of protic equilibrium (related to the laccase-bound O2 and type 2/type 3 (T2/T3)1 copper sites) on the rate of laccase-catalyzed O2 reduction (12, 13). However, insight on other potential factors, such as those related to the type 1 (T1) copper and (reducing) substrate, is still limited, and a comprehensive understanding on the mechanism that governs the bi-phasic pH dependence has not been fully established. To address this problem, I investigated several fungal laccases for pH-induced changes in their electronic absorption spectrum, fluoride inhibition, redox potential, and oxidation rate of phenolic and nonphenolic substrates. The study showed that the redox potential difference between a phenolic substrate and the T1 copper of laccase could result in an increased substrate oxidation rate at higher pH, whereas the hydroxide anion binding to the T2/T3 coppers could lead to an inhibition of laccase activity at higher pH. The balance of these two opposing effects might play an important role in determining the pH activity profile of laccase.


EXPERIMENTAL PROCEDURES

Materials

Chemicals used as buffers and substrates were purchased from Aldrich (except for methyl syringate, which was from Acros) with the highest available grade. Recombinant Polyporus pinsitus (or Trametes villosa) laccase isoform-1 (PpL), Rhizoctonia solani laccase isoform-4 (RsL), and Myceliophthora thermophila laccase (MtL) were purified as reported previously (11, 14, 15). Fe(2,2'-dipyridyl)2Cl3 and Fe(2,2'-dipyridyl)2Cl2 were made by mixing FeCl3 or FeCl2 with two molar equivalent 2,2'-dipyridyl, respectively. The Britton and Robinson (B&R) buffers were made by mixing 0.1 M boric acid, 0.1 M acetic acid, 0.1 M phosphoric acid with 0.5 M NaOH to the desired pH.

Spectrophotometrical Redox Titration of Laccase

The spectrum of laccase was recorded in B&R buffer, pH 2.7-11, on a Shimadzu UV160U spectrophotometer with an 1-cm quartz cuvette. The redox potential (Eo) of the T1 copper in laccase was measured as reported previously (11), except that B&R buffer was used. Briefly, the Eo(PpL) was measured with 21 µM PpL, 0.2 mM Fe(bipyridyl)2Cl2, and 0.1-0.4 mM Fe(bipyridyl)2Cl3 (Eo[Fe(2,2'-dipyridyl)2Cl3/Fe(2,2'-dipyridyl)2Cl2] = 0.780 V); the Eo(RsL) was measured with 17 µM RsL, 0.2 mM Fe(bipyridyl)2Cl2, and 0.05-0.2 mM Fe(bipyridyl)2Cl3; and the Eo(MtL) was measured with 0.14 mM MtL, 23 mM K3Fe(CN)6, and 0-200 mM K4Fe(CN)6 (Eo[K3Fe(CN)6/K4Fe(CN)6] = 0.433 V). Under various potentials of the solution poised by various concentration ratio of the redox titrant couples, the absorbance changes of laccase in the range of 550-800 nm were monitored, and the concentrations of the copper(II) and copper(I) states were calculated after the spectral change reached an equilibrium. Anaerobicity was achieved by repetitive evacuating and argon flushing of the reaction chamber at 4 °C.

The pKa value of the phenolic substrate studied was determined spectrally in B&R buffer. At alkaline pH, the phenolic substrates showed a deprotonation-induced spectral change. For syringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde), the absorption maximum at 302 nm was replaced by two maxima at 250 and 365 nm (isosbestic points: 238, 266, and 327 nm); for acetosyringone (4'-hydroxy-3',5'-dimethoxyacetophenone), the absorption maximum at 284 nm was replaced by two maxima at 250 and 359 nm (isosbestic points: 237, 263, and 321 nm); and for methyl syringate (methyl 4-hydroxy-3,5-dimethoxybenzoate), the absorption maximum at 273 nm was replaced by one shoulder around 236 nm and one maximum at 322 nm (isosbestic points: 231, 251, and 293 nm). The pH profiles of the absorption at 302 and 365 nm yielded a pKa of 7.0 ± 0.2 for syringaldehyde, a pKa of 7.8 ± 0.2 for acetosyringone, and a pKa of 8.7 ± 0.2 for methyl syringate. Under the same conditions, a pKa values of 8.2 and 2.2 were previously found for syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine) and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), respectively (11).

Laccase Activity Assays

The laccase-catalyzed oxidation of syringaldehyde, acetosyringone, or methyl syringate was monitored by O2 consumption as described in Ref. 16, and the oxidation of syringaldazine or ABTS was determined photometrically as described in Ref. 11. Briefly, the phenol oxidation was monitored by a Hansatech DW1/AD O2 electrode with 0.04-15 µM laccases in 0.3-0.5 ml of B&R buffer at 20 °C. After the voltage reading stabilized, laccase was added into the solution to initiate the reaction, and the initial output voltage changes were used to calculate the initial reaction rate (v). The oxidation of the nonphenolic substrates were photometrically monitored on either a Shimadzu UV160U spectrophotometer with a 1-cm quartz cuvette (for ABTS; Delta epsilon at 418 nm = 36 mM-1 cm-1) or on a Molecular Devices Thermomax microplate reader with a 96-well plate (Costar, tissue culture grade) (for K4Fe(CN)6; Delta epsilon at 405 nm = 0.90 mM-1 cm-1). The initial absorbance changes were used to calculate the initial reaction rate v. The apparent kinetic parameter Km was determined by fitting initial reaction rate (v) and substrate concentration to v = Vmax× [substrate]/(Km + [substrate]) with the Prizm program of GraphPad (San Diego, CA), and the apparent kcat was determined from kcat = Vmax/[laccase]. All the experiments were carried out in air-saturated solutions. Thus the initial [O2] was kept constant (and was assumed to be the same as in plain water (0.28 mM)), and the observed Km and kcat were "apparent" values ([O2] needs to be systematically varied in order to measure the true Km and kcat).

Fluoride Inhibition

The inhibition of laccase by NaF was assayed with laccase-catalyzed ABTS oxidation in B&R buffer. The assay solutions contained 2 mM ABTS and 0.6 µM MtL or 40 nM PpL. Being a complex "linear mixed type," the inhibition showed convex type correlations when the slope and the y intercept of the Lineweaver-Burk plots (1/rate versus 1/[substrate]) were plotted against [NaF], similar to that observed with the Rhus laccase (12). The inhibition was quantitated by the parameter I50, the NaF concentration at which only 50% of the initial laccase activity remained, because the complexity of the plots complicated the extraction of the inhibition constant Ki.


RESULTS

The pH-induced changes in the Eo and the wavelength corresponding to the maximal absorption of the blue band of laccase are shown in Fig. 1 (A and B). At the pH range of 2.7-11, one spectrally detected acid-base transition occurred around pH 6.5 for MtL, pH 8 for RsL, and pH 9 for PpL (Fig. 1B). For MtL, the Eo decreased ~90 mV from pH 2.7 to 5 but increased ~20 mV from pH 5 to 11; for RsL, the Eo decreased ~80 mV from pH 2.7 to 7 but increased ~30 mV from pH 7 to 11; and for PpL, the Eo did not change from pH 2.7 to 7 but increased ~30 mV from pH 7 to 11. Under the experimental conditions, no obvious correlation between the two types of pH profile in Fig. 1 (A and B) was observed.


Fig. 1. The pH profiles for the redox and spectral properties of the laccases. A, dependence of the Eo. B, dependence of the maximal absorption wavelength lambda. Laccase studied: MtL (open circle ), RsL (triangle ), and PpL (×).
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Fig. 2 (A-D) shows the pH dependence of the initial oxidation rate for four phenolic substrates and two nonphenolic substrates at a given concentration. With PpL, all four phenols, whose pKa range from 7.0 to 8.7, had bell-shaped pH profiles from pH 2.7 to 10 with the same pH optimum of 5. With MtL, all four phenols also had bell-shaped pH profiles from pH 4 to 10 with the same pH optimum of 7. In contrast, the two nonphenolic substrates, K4Fe(CN)6 and ABTS, only showed monotonic pH profiles (with both PpL and MtL) in which the rate decreased as the pH increased.


Fig. 2. The pH profiles for the (relative) initial rate of the phenolic and nonphenolic substrates. Laccase studied: A and C, PpL (40 nM); B and D, MtL (6 µM). A and B, oxidation of syringaldehyde (open circle ), acetosyringone (triangle ), methyl syringate (×), and syringaldazine (+). C and D, oxidation of ABTS (triangle , +) and K4Fe(CN)6 (open circle ). The activities of syringaldehyde (0.1 mM), acetosyringone (0.1 mM), and methyl syringate (0.1 mM) were monitored by the O2 consumption; the activity of syringaldazine (20 µM) was measured photometrically (at 530 nm); the activity of K4Fe(CN)6 (2.5 mM) was measured photometrically (at 405 nm); and the activity of ABTS (2 mM) was determined either photometrically at 405 nm (triangle ) or by O2 consumption (+).
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The pH profiles of the apparent Km and kcat for the nonphenolic substrates are shown in Fig. 3 (A-D). With both PpL and MtL, the profiles of the apparent kcat for ABTS and K4Fe(CN)6 had a monotonic declining nature when the pH changed from acidic range to alkaline range (Fig. 3, B and D). With PpL, K4Fe(CN)6 showed an apparent Km that did not change much when the pH changed from 2.7 to 7, whereas ABTS showed an apparent Km that did not change much from pH 2.7 to pH 7 but increased approximately 4-fold from pH 7 to pH 8 (Fig. 3A). With MtL, K4Fe(CN)6 showed an apparent Km that increased about 3-fold when the pH changed from 2.7 to 4 but did not change much from pH 4 to pH 7, whereas ABTS showed an apparent Km that increased 4-fold from pH 2.7 to pH 6 but 40-fold from pH 6 to pH 8 (Fig. 3C). The catalyzed oxidation of K4Fe(CN)6 or ABTS was so slow above pH 7 or pH 8 that no accurate Km and kcat could be obtained.


Fig. 3. The pH profiles of the apparent Km and kcat for the nonphenolic substrates. Laccase studied: A and B, PpL (40-100 nM); C and D, MtL (6-120 µM). Substrates studied: ABTS (triangle ) and K4Fe(CN)6 (open circle ).
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The pH profiles of the apparent Km and kcat for the phenolic substrates are presented in Fig. 4 (A-D). Being oxidized by PpL, all these substrates showed the highest apparent kcat at pH 5 (Fig. 4B). With PpL, the apparent Km for syringaldehyde and acetosyringone did not change much from pH 4 to 7 but increased about 10-fold from pH 4 to 2.7, whereas the apparent Km for methyl syringate did not change much from pH 2.7 to 6 but decreased about 10-fold from pH 6 to 7 (Fig. 4A). With MtL, the apparent Km for methyl syringate and acetosyringone showed minimal change at pH 2.7 to 9, but the apparent Km for syringaldehyde increased about 3-fold from pH 6 to 2.7 (Fig. 4C). Being oxidized by MtL, all the phenols showed the highest apparent kcat at pH 7, although an increase in the apparent kcat was also observed when the pH changed from 4 to 2.7 (Fig. 4D). The catalyzed oxidation of all three phenols was so slow above pH 7 with PpL or pH 9 with MtL that no accurate Km and kcat could be obtained. Similar result was previously observed with syringaldazine (11).


Fig. 4. The pH profiles of the apparent Km and kcat for the phenolic substrates. Laccase studied: A and B, PpL (40-220 nM); C and D, MtL (6-60 µM). Substrate studied: syringaldehyde (open circle ), acetosyringone (triangle ), methyl syringate (×).
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The addition of NaF resulted in an immediate laccase inhibition with I50 <=  0.1 mM at acidic pH. Because HF has a pKa of 3.5, the laccase inhibition shown in Fig. 5 was most likely caused by F-. As the pH increased, the I50 became larger, indicating a weaker F- inhibition at higher pH (Fig. 5). This increase of I50 at higher pH did not correspond to an increase in laccase activity, however, because in the absence of NaF, the activity of laccase diminished as pH increased (Fig. 2, C and D).


Fig. 5. Inhibition of the laccases by NaF as the function of pH. The unit of I50 is M. The activity was based on the initial absorbance change rate at 405 nm with 2 mM ABTS and 0.6 µM MtL (open circle ) or 40 nM PpL (triangle ).
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DISCUSSIONS

Based on a wide range of physical and chemical characterizations, it is generally accepted that the catalysis of fungal laccase involves (a) the binding of a reducing substrate to the T1 pocket and subsequent reduction of the T1-Cu(II) to Cu(I), (b) the internal electron transfer from the T1 to the T2/T3 center, and (c) the binding and subsequent reduction of an O2 to H2O at the T2/T3 center (1, 2, 3, 4, 5, 6, 7, 8). Potentially, any pH-induced structural or mechanistic changes in either the reducing substrate, O2, or laccase (particularly on its T1 and T2/T3 centers) could contribute to the observed pH activity profiles.

The oxidation of phenol by laccase depends on the redox potential difference between the phenol and the T1 copper (16). Due to the oxidative proton release, the Eo of a phenol decreases when pH increases. At a rate of Delta E/Delta pH = 0.059 V at 25 °C, a pH change from 2.7 to 11 would result in an Eo(phenol) decrease of 0.49 V. However, over the same pH range, the Eo changes for the laccases studied were much smaller (<= 0.1 V), similar to the case of the Rhus laccase (17). Such different pH dependences of the Eo for phenolic substrate and laccase would then result in a larger difference in redox potential [Delta Eo = Eo(laccase, T1) - Eo(substrate, single electron)] or driving force (for the electron transfer from phenol to T1 copper) at higher pH (Fig. 6, A and C). Given the correlation of log(rate) = 7.1 × Delta Eo + 7.0 observed (at pH 5) for a wide variety of substrates and laccases (16), this Delta Eo effect should lead to a pH dependence in which the activity increases as the pH increases, thus contributing to the ascending part of the bell-shaped pH activity profile for phenols shown in Figs. 2, 4, and 6. The increase in kcat from pH 4 to 2.7 for MtL-catalyzed phenol oxidation (Fig. 4D) could also be related to the Delta Eo effect, becasue, as shown in Fig. 1A, the Eo(MtL) decreased 80 mV when pH changed from 2.7 to 4, thus reducing the oxidation potency of MtL.


Fig. 6. Contributions to the pH activity profile from the Delta Eo and the OH- inhibition. Laccase studied: A and B, PpL; C and D, MtL. A and C, dependence of Delta Eo = Eo(laccase, T1) - Eo(phenol, single electron) on pH. The values for the Eo(laccase, T1) are from Fig. 1A, and the values for the Eo(phenol, single electron) are from Ref. 18. B and D, dependence of Delta log(rate) on pH. For syringaldehyde (open circle ), acetosyringone (triangle ), and methyl syringate (×), the log(rate) = log(kcat/Km) values are obtained from Fig. 4 (A-D) and presented relative to the value at pH 5. The contribution of the Delta Eo to the pH activity profile is represented by the dashed line alpha , which is derived from the correlation log(rate) = 7.1 × Delta Eo + 7.0 (16) with the Delta Eo values from A and C and is presented relative to the value at pH 5. The contribution of the OH- inhibition to the pH activity profile is represented by the dotted line beta , which is derived from the apparent kcat and Km of ABTS (Fig. 3, A-D) and presented relative to the value at pH 8 (B) or 9 (D). (For clarity purpose, the lines for syringaldehyde and acetosyringone, which are superposable to line alpha , and the line for K4Fe(CN)6, which is superposable to line beta , are omitted in B and D.)
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The loss of F- inhibition at high pH did not result in recovery or increase of laccase activity. Likely the observed pH dependence of the F- inhibition was mainly due to an OH- competition (with F-) for inhibiting MtL and PpL, similar to the cases of other laccases in which OH- and F- are shown to competitively bind to the T2/T3 center and inhibit activity (1, 3, 19, 20, 21, 22). Such OH- inhibition interrupts the internal electron transfer from the T1 to the T2/T3 centers in laccase and, together with other rate-diminishing deprotonations (to be discussed later), could contribute to the descending part of the pH activity profile of phenols shown in Figs. 2, 4, and 6.

For phenol substrates, a bell-shaped pH activity profile with an optimal pH dependent on laccase (not substrate) is consistent with the mechanism in which the opposing effects of the OH- inhibition and Delta Eo contribute, respectively, at alkaline and acidic pH mainly. The oxidation of ABTS (to the stable, preferred cation radical) or K4Fe(CN)6 (to K3Fe(CN)6) does not involve protons, and thus possesses an Eo independent of pH (17). This would make any pH effect of Delta Eo minimal for these two substrates. The contribution of the OH- inhibition would then result in a monotonic pH activity profile consistent with the data presented in Figs. 2, C and D, and 3, C and D. As shown in Fig. 6, B and D, the descending part of the pH profile for the phenols (at neutral alkaline pH range) is similar to the profile for the nonphenolic substrates (ABTS and K4Fe(CN)6), suggesting the contribution of a common mechanism involving the OH- inhibition of laccase.

The laccases studied showed different, spectrally detected acid-base transitions (on their T1 center) with a pKa ranging from 6.5 to 9. The pH-induced change in the blue absorption band around 600 nm was probably caused by an energy perturbation (on the T1 Cu(II)) related to protic equilibria of nearby amino acid residue(s). The three phenol substrates studied also have different acid-base transitions because of their different pKa (ranging from 7 to 8.7). These protic equilibria related to the reducing substrate (or its oxidized intermediate product) and the T1 pocket could affect substrate docking (e. g. making Km larger or smaller) or activation (e. g. transforming phenol to phenoxide or vice versa). However, such protic events seem to be small in this study because the optimal pH appeared to be independent of either the pKa or the apparent Km of the laccases and substrates. As shown in Figs. 3, 4, and 6, the effects of Delta Eo and OH- were most significant on the apparent kcat or kcat/Km, indicating that it is the electron transfer kinetics rather than the substrate binding (as reflected by Km) that plays a more important role in determining the pH activity profile. Protic equilibria related to O2 (or its reduced intermediate product) and the T2/T3 pocket could diminish the overall catalytic rate at high pH (8, 12, 13). However, such an effect also seems to be small in this study, because a dominant effect of this type would result in a pH profile with a maximum independent of the reducing substrate.

The dependence of O2 reduction potential on pH could also impact the pH activity profile. At 20 °C, the Eo(O2/H2O) can drop from 1.23 V at pH 0 to 0.82 V at pH 7 or 0.58 V at pH 11 and thus become comparable with the Eo(T1) in PpL and MtL, respectively. Because the Eo(T2/T3) would generally be equal to or higher than the Eo(T1) in laccase (23, 24), this pH-induced change of Eo(O2/H2O) would lead to a decrease in reaction rate at high pH (due to unfavorable thermodynamics). The descending part of the observed pH profiles might be the result of the combination of the effect and that of the OH- inhibition.

The difference between the monotonic pH activity profile of a nonphenolic substrate (such as ABTS, K4Fe(CN)6, or Fe(EDTA)2-) and the bi-phasic (or bell-shaped) pH activity profile of a phenolic substrate (such as syringaldehyde) reported for other laccases (11, 25, 26, 27, 28) could also be interpreted by the Delta Eo and OH- effects discussed above. Another observation that could be attributed to these effects involves the different pH dependence of activity observed under steady-state and single-turnover (or anaerobic) conditions. It was reported that under steady-state conditions, the rate of the Rhus laccase-catalyzed hydroquinone oxidation has a bell-shaped pH dependence (29), whereas under single-turnover conditions, the rate only increases when pH increases (12, 19, 20). Like PpL and MtL, the Rhus laccase has an Eo quite insensitive to pH change (<= 0.1 V over pH 3-10) (17) and is inhibited by OH- at alkaline pH (1, 3, 19, 20, 21, 22). Thus the bell-shaped pH activity profile of the Rhus laccase under steady-state conditions could be attributed (at least partly) to the opposing effects of Delta Eo and OH- inhibition discussed above. Under single-turnover conditions, the effect of the OH- inhibition (excised at the T2/T3 center) would become insignificant because both the reduction of O2 (at the T2/T3 center), and the internal electron transfer (from the T1 to the T2/T3 center) in laccase would contribute little to the oxidation of hydroquinone (at the T1 center). In consequence, the predominant effect of Delta Eo should result in a higher substrate oxidation rate at more alkaline pH.

In summary, this study demonstrated that both the OH- inhibition at the T2/T3 center and the redox potential difference between a reducing substrate and the T1 center could affect the pH activity profile of a laccase. For a reducing substrate (such as K4Fe(CN)6) whose oxidation does not involve protons and has a minimal Eo dependence on pH, the activity of a laccase could decline monotonically when the pH increases, as the result of the possible involvement of an OH- inhibition on the T2/T3 center. For a reducing substrate (such as syringaldehyde), whose oxidation involves protons and has a significant Eo dependence on pH, the pH activity profile of a laccase could be bi-phasic, reflecting possible combinatory contribution from the opposing effects of the pH-induced redox potential change (on both the T1 center and substrate) and the OH- inhibition. It should be pointed out that laccase is a two-substrate enzyme and to obtain the true kinetic parameters, both substrates should be subjected to concentration variation. The Km and kcat reported above were observed with various reducing substrate concentrations in air-saturated solutions only and hence are apparent values. Thus the hypothesis proposed above should be further tested by a full kinetic analysis based on experiments in which [O2] is also systematically varied.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Novo Nordisk Biotech, 1445 Drew Ave., Davis, CA 95616. Tel.: 916-757-8100; Fax: 916-758-0317; E-mail: fengxu{at}nnbt.com.
1    The abbreviations used are: T2/T3, type 2/type 3 trinuclear copper cluster; T1, type 1 copper; PpL, recombinant P. pinsitus (or T. villosa) laccase isozyme-1; RsL, recombinant R. solani laccase isozyme-4; MtL, recombinant M. thermophila laccase; B&R, Britton and Robinson buffer; ABTS, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid); Eo, single-electron redox potential (referenced to the Normal Hydrogen Electrode); Delta Eo, single-electron redox potential difference between laccase (T1 copper) and reducing substrate.

Acknowledgments

I thank Drs. Alan V. Klotz, Alexander M. Blinkovsky, Mark S. Madden, Stephen H. Brown, and Kim M. Brown of Novo Nordisk Biotech for critical reading and helpful suggestions.


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