(Received for publication, October 8, 1996, and in revised form, January 16, 1997)
From the Department of Biochemistry and Molecular Biology and Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park, Pennsylvania 16802
The mechanism of veratryl alcohol-mediated oxidation of 4-methoxymandelic acid by lignin peroxidase was studied by kinetic methods. For monomethoxylated substrates not directly oxidized by lignin peroxidase, veratryl alcohol has been proposed to act as a redox mediator. Our previous study showed that stimulation of anisyl alcohol oxidation by veratryl alcohol was not due to mediation but rather due to the requirement of veratryl alcohol to complete the catalytic cycle. Anisyl alcohol can react with compound I but not with compound II. In contrast, veratryl alcohol readily reduces compound II. We demonstrate in the present report that the oxidation of 4-methoxy mandelic acid is mediated by veratryl alcohol. Increasing veratryl alcohol concentration in the presence of 2 mM 4-methoxymandelic acid resulted in increased oxidation of 4-methoxymandelic acid yielding anisaldehyde. This is in contrast to results obtained with anisyl alcohol where increased concentrations of veratryl alcohol caused a decrease in product formation. ESR spectroscopy demonstrated that 4-methoxymandelic acid caused a decrease in the enzyme-bound veratryl alcohol cation radical signal, which is consistent with its reaction at the active site of the enzyme.
To degrade the aromatic polymer lignin, the white-rot fungus Phanerochaete chrysosporium secretes H2O2 (1), and two families of H2O2-utilizing enzymes, the lignin peroxidase (LP)1 and manganese peroxidases (MnP). The MnPs catalyze the oxidation of Mn2+ to Mn3+ (2) whereas the LPs catalyze the oxidation of phenolic and nonphenolic methoxylated aromatic substrates (3). The catalytic cycle of both peroxidases is similar to that of other peroxidases. The enzyme is first oxidized by H2O2 to form a two-electron-oxidized intermediate, compound I (4). Compound I then returns to the resting ferric enzyme by two sequential one-electron reduction steps producing two, one-electron-oxidized products. The one-electron oxidized species of the enzyme formed during turnover is referred to as compound II. Unique to these fungal peroxidases is the recalcitrant nature of their substrate (5). Both act on substrates that other peroxidases are not capable of oxidizing.
The role and mechanism by which the LP and MnP interact with lignin is yet to be elucidated. For both enzymes, the role of mediators has been proposed. With MnP, the mediation phenomenon is well characterized and widely accepted. Complexed Mn2+ (6, 7) is oxidized by MnP and diffuses away from the active site of the enzyme to oxidize lignin (8). In the case of LP, the role of mediators and the mechanism by which lignin is depolymerized by LP is still unknown. A key component of LP catalysis is veratryl alcohol, a secondary metabolite also produced by ligninolytic cultures of P. chrysosporium (9). Harvey et al. (10) proposed that veratryl alcohol is the mediator for LP as Mn2+ is the mediator for MnP. They proposed that the initial one-electron oxidation of veratryl alcohol forms the cation radical which then diffuses away from the active site and oxidizes other substrates such as anisyl alcohol and 4-methoxymandelic acid (4-MMA). The mediation hypothesis explains the ability of LP to oxidize substrates such as anisyl alcohol and 4-MMA only in the presence of veratryl alcohol (10). The mediation mechanism is also attractive as it accounts for how a large bulky enzyme can interact with large bulky insoluble substrates.
Harvey's hypothesis was supported by the work of Khindaria et al. (11). They were able to generate the radical chemically and also enzymatically. Both forms were detected by ESR spectroscopy; the half-life was much longer for the enzyme-bound form (12). Candeias and Harvey (13) also generated the veratryl cation radical by chemical means and characterized it by pulse radiolysis. They determined the lifetime of the radical and calculated that it was capable of diffusing up to 7 µm. However, the work of Candeias and Harvey (13) also showed that the veratryl alcohol cation radical did not oxidize anisyl alcohol or 4-MMA. They postulated that the enzyme-bound radical could be more reactive or that it would have a longer half-life, thus allowing the radical to oxidize these monomethoxylated substrates.
Our previous work demonstrated that the veratryl alcohol "mediation" phenomenon with anisyl alcohol was not due to redox mediation (14). The enhancement was due to the requirement of veratryl alcohol to complete the catalytic cycle. Whereas most substrates can be oxidized by both compound I and II of LP, anisyl alcohol can only be oxidized by compound I (thus being stuck at compound II). Inclusion of veratryl alcohol results in its oxidation by compound II allowing the enzyme to complete the catalytic cycle. In the present study, we demonstrate, to our surprise, that the oxidation of the other monomethoxylated substrate that Harvey and co-workers (13) characterized, 4-MMA, is mediated by veratryl alcohol. Steady-state methods show that 4-MMA is oxidized by the enzyme-generated veratryl alcohol cation radical. We also provide ESR spectroscopy data consistent with this where the ESR signal of the veratryl alcohol cation radical is quenched by 4-MMA.
LP isozyme H1 (pI 4.7) was isolated
from P. chrysosporium strain PSBL-1 as described previously
(15). The H1 fraction from the Mono Q column was further purified
by preparative isoelectric focusing. For stopped-flow experiments, the
enzyme was dialyzed against distilled-deionized water. The
concentration of LP was determined at 409 nm using an extinction
coefficient of 169 mM1 cm
1
(16).
Hydrogen peroxide solutions were prepared daily
and the concentration was determined using 240 nm = 39.4 M
1 cm
1(17). Veratryl
alcohol, veratraldehyde, 4-MMA, and anisaldehyde were purchased from
Aldrich. Veratryl alcohol was vacuum-distilled before use; all other
chemicals were used without further purification.
Reaction mixtures were sampled (100 µl) and added to 400 µl of methanol. A 10-µl aliquot of the resultant solution was injected onto a reverse phase C18 (VYDACTM) column eluted at 0.5 ml/min with a 5-100% linear methanol gradient in water. Products were monitored at 280 nm and identified by comparison to retention times of authentic standards.
Steady-state KineticsThe rate of veratraldehyde and
anisaldehyde formation was determined spectrophotometrically. The
absorption spectra of the alcohols and their corresponding aldehydes
are shown in Fig. 1. Veratraldehyde formation was
monitored at 330 nm ( = 1.9 mM
1
cm
1) rather than at 310 nm as in previous studies since
anisaldehyde is transparent at this wavelength. Anisaldehyde formation
was determined by the absorbance increase at 300 nm (
= 4.5 mM
1 cm
1). The veratraldehyde
contribution to the absorbance increase at 300 nm (
= 7.4 mM
1 cm
1) was determined by its
absorbance at 330 nm. The substrates have no spectral contribution at
300 or 330 nm. The reaction mixtures contained 0.4 mM
H2O2, 0.05 µM LP, and as
specified in the figure legends, either 4 mM 4-MMA and
varying concentrations (0-6 mM) of veratryl alcohol or
varying concentrations (0-4 mM) of 4-MMA and 2 mM veratryl alcohol in 50 mM sodium tartrate
buffer at pH 3.5. Reactions were at 28 °C and initiated by addition
of H2O2.
Presteady-state Kinetics
The KinTek three-syringe stopped-flow apparatus was used as described previously (7). The reactions of LP compound I were monitored at 426 nm, the isosbestic wavelength of native enzyme and compound I. Rate constants for compound II were determined from experiments where compound II was formed from the reaction of compound I with the reductant of choice. The reactions were at 28 °C under pseudo-first order conditions. The double mixing system contained 3 µM LP in water which was mixed initially with 3 µM H2O2. The resultant compound I was then mixed with the contents of the third syringe which contained the reductant (4-MMA) in 150 mM sodium tartrate, pH 3.5.
ESR Quantitation of Cation RadicalThe veratryl alcohol
cation radical formed from LP was quantitated by acid quench with a
three-syringe fast-flow system as described previously (11). The
contents of the first two syringes, one containing LP and veratryl
alcohol, the other containing H2O2, were mixed
for 0.5 s. The contents were then quenched with 10% HNO3. Radical concentration was determined by double
integration of the first derivative spectrum. Tempol was used as a
standard. Tempol was quantitated at 240 nm using an extinction
coefficient of 1,440 M1 cm
1
(18).
Computer simulations of steady-state data using rate constants obtained from presteady-state studies were performed with the program KIMSIM (19), provided by Carl Frieden and Bruce Barshop (Washington University, St. Louis, MO).
The effect of veratryl alcohol
concentration on the rate of anisaldehyde formation from 4 mM 4-MMA was determined spectrophotometrically (Fig.
2). Formation of veratraldehyde and anisaldehyde in
these steady-state experiments was confirmed by high pressure liquid chromatography analyses (data not shown). Little or no anisaldehyde is
formed in the absence of veratryl alcohol. Increasing veratryl alcohol
concentration resulted in an increase in the rate of anisaldehyde formation. The hyperbolic curve fits Michaelis-Menton kinetics with an
apparent Km of 240 µM for veratryl
alcohol. This Km is within experimental error for
the Km value of 172 µM determined for
veratryl alcohol in the absence of 4-MMA (14). Due to the high 4-MMA
concentration used in these experiments and the little or no change in
the apparent Km for veratryl alcohol, this would
indicate that 4-MMA minimally competes with veratryl alcohol at the
active site. The effect of pH on 4-MMA oxidation is similar to that
obtained with just veratryl alcohol oxidation (data not shown). The
Vmax increases as the pH decreases.
The effect of 4-MMA concentration on veratraldehyde and anisaldehyde
formation at a constant veratryl alcohol concentration is shown in Fig.
3. With a constant veratryl alcohol concentration of 2 mM, increasing 4-MMA caused a rapid decrease in the rate of
veratraldehyde formation. Complete inhibition was observed at a
concentration of less than 1 mM. Concomitant with the
decrease in veratraldehyde formation is the increase in anisaldehyde
formation. The rate reached a peak at 150 µM 4-MMA. This
was followed by a slow and gradual decrease in rate. The decrease in
rate can be accounted for by competition of 4-MMA with veratryl alcohol at the active site. Based on the shape of the curve, the
Ki is predicted to be in excess of 4 mM.
This is consistent with the results shown in Fig. 2 where the
Km for veratryl alcohol determined in the absence of
4-MMA approximates the value determined in the presence of 4 mM 4-MMA.
Kinetic traces monitoring veratraldehyde formation at 330 nm showed lag
phase kinetics at very low 4-MMA concentrations (Fig. 4). This is reminiscent of the effect of guaiacol on
veratryl alcohol oxidation (14). The length of the lag period was
roughly proportional to the 4-MMA concentration, suggesting that
veratryl alcohol oxidation did not proceed until most if not all of the 4-MMA was first oxidized.
Presteady-state Kinetics
The reactivity of 4-MMA with
compound I and II of LP was studied with a three-syringe stopped-flow
technique under pseudo-first order reaction conditions. Compound I was
generated in the stopped-flow by mixing one equivalent of LP with
H2O2 and aged for 4 s before mixing with
4-MMA. Similar to results obtained with anisyl alcohol (14), 4-MMA
reacts with compound I but much more slowly than veratryl alcohol (Fig.
5). A rate constant of 4.3 × 103
M1 s
1 was calculated from the
slope for the reaction of compound I with 4-MMA. Reactions of compound
II with other reductants always exhibit a hyperbolic concentration
dependence. We were not able to observe this with 4-MMA at higher
concentrations (data not shown). This suggests that 4-MMA has little or
no reactivity with compound II. When extremely high concentrations are
required to observe a reaction, a low level contaminant could account
for the reaction; this we have observed with anisyl alcohol (14).
ESR Spectroscopy
The ESR spectrum of the veratryl alcohol
cation radical is shown in Fig. 6. The radical was
generated by enzymatic incubations quenched with HNO3.
Stoichiometric amounts of 25 µM enzyme was incubated with
H2O2 in the presence of 2 mM
veratryl alcohol and varying concentrations of 4-MMA. The ESR signal
intensity was determined by double integration and calculated as
described previously (12). Spectrum A is an incubation without 4-MMA; a
value of 7.3 µM was calculated for the cation radical.
Addition of 0.5 mM 4-MMA decreased the signal intensity by
53%. Addition of 1 mM 4-MMA resulted in an 85% decrease
in signal intensity.
Veratryl alcohol was first detected in ligninolytic cultures of P. chrysosporium by Lundquist and Kirk in 1978 (9), long before the discovery of LP (20, 21). It was not until the discovery of LP that a role for veratryl alcohol in lignin degradation was first proposed. Veratryl alcohol not only protects the enzyme from H2O2-dependent inactivation (22) but also serves as a convenient assay substrate for the enzyme (23). The most debated role is that of a redox mediator between LP and polymeric lignin. This role was initially formulated from data showing the ability of veratryl alcohol to enhance the oxidation of recalcitrant compounds that are not directly oxidized by the enzyme. Lignin (24), anisyl alcohol (10), 4-MMA (10), chloropromazine (25), and guaiacol (26) are only oxidized by LP if veratryl alcohol is included in the reaction mixture. Although the mediation mechanism provided a suitable explanation for the phenomenon and provided a model for how a large bulky enzyme could degrade an insoluble large bulky polymer, there were chemical arguments against the model.
Despite its proposed existence as a stable mediator able to participate in oxidation reactions at a distance, there was no direct proof of the radical for many years. Khindaria et al. (11) were the first to detect the veratryl alcohol cation radical by ESR spectroscopy. They first generated and characterized the species by oxidation with Ce(IV) in 10% HNO3. After establishing the ESR parameters, these workers were then able to detect the enzyme-bound radical during steady-state turnover of LP at pH 3.5. At this pH, the radical was not detected when generated by Ce(IV) oxidation indicating that the radical is not stable free in solution. Khindaria et al. (12) proposed that the enzyme extends the half-life of the radical.
Candeias and Harvey (13) also characterized the veratryl alcohol cation radical. They generated the species with thallium (II) and studied it by pulse radiolysis. The spectral and kinetic properties of the radical determined by Candeias and Harvey (13) did not totally agree with those of Khindaria et al. (12). Nevertheless, these workers determined that the half-life of the radical was approximately 60 ms in solution thus allowing it to diffuse a distance of 7 µm. This would allow for it to act as a diffusible oxidant. Khindaria et al. (12) determined a half-life of 0.57 ms resulting in a much lower diffusion radius. Regardless, Candeias and Harvey (13) demonstrated that the radical was capable of diffusing far enough to react with the polymeric dye Poly R-478. Surprisingly, no reaction was observed with either anisyl alcohol or 4-MMA. Candeias and Harvey (13) rationalized that the enzyme could serve to increase the reactivity of the radical (with positively charged residues) thus allowing the veratryl alcohol cation radical to oxidize both anisyl alcohol and 4-MMA. Alternatively, they indicated that if the enzyme extended the half-life of the cation radical, the probability of reaction would be increased.
Our studies with anisyl alcohol clearly showed that its oxidation by LP is not mediated by veratryl alcohol (14). We found that anisyl alcohol is not oxidized by compound II. Compound I, in contrast, is able to readily oxidize anisyl alcohol. Thus with only anisyl alcohol, the enzyme would be stuck at the compound II intermediate. The addition of veratryl alcohol, which is readily oxidized by compound II would allow the enzyme to complete the catalytic cycle. We showed that this mechanism would predict stimulation of anisyl alcohol oxidation at low veratryl alcohol concentrations. This is because reactions with compound II to complete the catalytic cycle (enhancement) would offset the effect of veratryl alcohol reacting with compound I (inhibitory). At higher concentrations of veratryl alcohol, the rate of anisyl alcohol oxidation should decrease because it would more effectively compete with anisyl alcohol at compound I. Consistent with the proposed mechanism, our data show that an optimal rate of anisyl alcohol oxidation is observed at 0.1 mM veratryl alcohol (14).
Two recent studies have shown that veratryl alcohol enhanced oxidation
of a second substrate is due to mediation. Goodwin et al.
(25) demonstrated veratryl alcohol-mediated oxidation of
chloropromazine; Koduri and Tien (26) showed that the oxidation of
guaiacol was mediated by veratryl alcohol. The proposed mechanism for
mediation of these substrates is shown in Fig. 7.
Kinetic simulation of mediation reveals two unique aspects which are
born out by the data: (i) veratryl alcohol oxidation does not occur until the secondary substrate is depleted (lag phase kinetic traces) and (ii) the effect of veratryl alcohol on the oxidation of the secondary substrate exhibits saturation kinetics. Both of these properties are observed with chloropromazine (25) and guaiacol (26).
These properties differ from those observed with anisyl alcohol where
no lag phases are observed and an optimal rate is observed at low
veratryl alcohol concentrations.
Results obtained here with 4-MMA oxidation by LP resemble those
obtained with guaiacol and chloropromazine oxidation. The addition of
4-MMA caused lag phase kinetics on veratryl alcohol oxidation (Fig. 4)
and saturation kinetics were observed when the veratryl alcohol
concentration was varied (Fig. 2). These two results clearly point to
mediation of 4-MMA oxidation by veratryl alcohol. Mediation is also
consistent with the ESR results. The veratryl alcohol cation radical
generated by compound I decreased in concentration in incubations
containing 4-MMA. The decrease in signal intensity can be attributed to
either competitive inhibition at the active site or to the reaction of
4-MMA with the cation radical. If the inhibition were due to
competition, the decrease in signal intensity can be predicted based on
the relative rate constants of compound I with veratryl alcohol and
4-MMA (1.5 × 105 M1
s
1 versus 4.3 × 103
M
1 s
1). These predicted values
are 0.7% inhibition at 0.5 mM 4-MMA and 1.4% inhibition
at 1 mM 4-MMA. The data indicates 53 and 85% inhibition;
this is consistent with 4-MMA reacting with the cation radical thus
causing the decrease in the ESR signal intensity.
Our results with anisyl alcohol would predict that 4-MMA oxidation is
not mediated by veratryl alcohol. Like anisyl alcohol, 4-MMA is a
monomethoxylated substrate whose reduction potential would be predicted
to be higher than that of veratryl alcohol. Examination of the 4-MMA
structure reveals how it, despite being a monomethoxylated substrate,
could be oxidized by the veratryl alcohol cation radical. The
elimination of the -carboxyl group as CO2 would drive
the reaction forward (Fig. 7B). Candeias and Harvey (13)
suggested that the lack of reactivity between the veratryl alcohol
cation radical and 4-MMA in solution can be explained by the short
half-life of the cation radical. Our kinetic simulations, described
below agree with this conclusion. We simulated the effect of 4-MMA on
veratraldehyde formation (similar to data shown in Fig. 4) with a
short-lived and a long-lived veratryl alcohol cation radical. The
mechanism used for this simulation is shown in Fig. 8A. Khindaria et al. (12) measured
the half-life of the cation radical in solution and enzyme-bound to be
0.57 and 370 ms, respectively. Candeias and Harvey (13) estimated the
rate constant between the veratryl alcohol cation radical with 4-MMA to
be 5 × 104 M
1
s
1 (we used 9 × 104
M
1 s
1). With a
kcat of 17 s
1 for LP and a
half-life of 370 ms for the enzyme bound cation radical, the
simulations shown in Fig. 8B were obtained which resemble
the real data shown in Fig. 4. When the half-life of the cation radical
is decreased to 0.57 from 370 ms, no inhibition of veratraldehyde
formation is observed (Fig. 8B), indicating that its
half-life was too short for reactions with 4-MMA.
In conclusion, we have shown that 4-MMA oxidation is mediated by veratryl alcohol. This work supports the hypothesis put forth by Candeias and Harvey (13) suggesting that extending the half-life of the radical can increase the reaction yield. However, our results still do not support the ability of veratryl alcohol to act as a diffusible oxidant. The cation radical is too short-lived when free in solution to oxidize other substrates. The results of Khindaria et al. (12) and ours clearly show that it must be stabilized to participate in electron transfer reactions with other aromatic substrates. Nevertheless, the list of substrates for which veratryl alcohol serves as an electron-transfer agent continues to grow. It therefore, would not be surprising to find that it serves such a role with lignin. Furthermore, as suggested by Shoemaker et al. (27), other mechanisms may be operating in fungal cultures to stabilize the cation radical and allow it to act as a diffusible oxidant.
We are indebted to Aditaya Khindaria and Steven D. Aust of the Utah State University for performing ESR experiments.