(Received for publication, January 18, 1995; and in revised form, July 3, 1995)
From the
We have investigated the lignin peroxidase-catalyzed oxidation
of guaiacol and the role of veratryl alcohol in this reaction by
steady-state and pre-steady-state methods. Pre-steady-state kinetic
analyses demonstrated that guaiacol is a good substrate for both
compounds I and II, the two- and one-electron oxidized enzyme
intermediates, respectively, of lignin peroxidase. The rate constant
for the reaction with compound I is 1.2 10
M
s
. The reaction
of guaiacol with compound II exhibits a K
of 64 µM and a first-order rate constant of 17
s
. Oxidation of guaiacol leads to tetraguaiacol
formation. This reaction exhibits classical Michaelis-Menten kinetics
with a K
of 160 µM and a k
of 7.7 s
. Veratryl alcohol,
a secondary metabolite of ligninolytic fungi, is capable of mediating
the oxidation of guaiacol. This was shown by steady-state inhibition
studies. Guaiacol completely inhibited the oxidation of veratryl
alcohol, whereas veratryl alcohol had no corresponding inhibitory
effect on guaiacol oxidation. In fact, at low guaiacol concentrations,
veratryl alcohol stimulated the rate of guaiacol oxidation. These
results collectively demonstrate that veratryl alcohol can serve as a
mediator for phenolic substrates in the lignin peroxidase reaction.
This study investigates the ability of 3,4-dimethoxybenzyl
(veratryl) alcohol to mediate the lignin peroxidase-catalyzed oxidation
of guaiacol. Lignin peroxidases are hemeproteins secreted by the white
rot fungus Phanerochaete chrysosporium during secondary
metabolism(1, 2) . These isozymes catalyze the
oxidation of lignin and a large number of phenolic and non-phenolic
substrates(3, 4) . The catalytic cycle of lignin
peroxidase is similar to that of other peroxidases (5, 6) where ferric enzyme is first oxidized by
HO
to generate the two-electron oxidized
intermediate, compound I(7) . Compound I is then reduced by one
electron donated by a substrate molecule, yielding the 1-electron
oxidized enzyme intermediate, compound II, and a free radical product.
The catalytic cycle is completed by the one-electron reduction of
compound II by a second substrate molecule.
In the absence of a
reducing substrate, the enzyme can undergo a series of reactions with
HO
to form compound III,
oxyperoxidase(6, 8) . It is also well documented that
prolonged incubation of enzyme with H
O
in the
absence of a reducing substrate such as veratryl alcohol can cause
irreversible inactivation of the enzyme(9) . In the presence of
veratryl alcohol, however, lignin peroxidase undergoes multiple
turnovers without any detectable inactivation. Because veratryl alcohol
is normally produced by ligninolytic cultures of P.
chrysosporium(10) , workers have proposed that its
physiological function is to protect the enzyme from
H
O
-dependent inactivation (11) .
An
alternate role for veratryl alcohol in lignin biodegradation has been
proposed by Harvey et al.(12) . These workers observed
that substrates that are not oxidized by lignin peroxidase such as
anisyl alcohol and 4-methoxymandelic acid are oxidized in the presence
of veratryl alcohol(12) . They proposed that the one-electron
oxidized product of veratryl alcohol, the aryl cation radical, is able
to mediate the oxidation of substrates typically not oxidized by the
enzyme. They further proposed that the aryl cation radical is a
diffusible species, capable of acting at a distance. In contrast to
Harvey et al.(12) , Valli et al.(13) proposed that the stimulation of 4-methoxymandelic
acid and anisyl alcohol oxidation is due solely to the ability of
veratryl alcohol to prevent inactivation of lignin peroxidase. They
claimed that enzyme in the presence of anisyl alcohol and excess
HO
leads to the formation of inactive compound
III*(13) . Veratryl alcohol is then capable of converting
inactive compound III* back to the native state(14) . The
existence of compound III* has been subsequently
questioned(8) .
In this study, we reinvestigated the ability
of veratryl alcohol to mediate the oxidation of guaiacol. We find that
unlike anisyl alcohol (15) , guaiacol is a substrate for
compounds I and II of lignin peroxidase. Nevertheless, our results
support the findings of Harvey et al.(12) and show
that veratryl alcohol can mediate the oxidation of guaiacol. We also
show that guaiacol, like veratryl alcohol, is capable of converting the
enzyme from the compound III state back to the resting ferric state in
the presence of HO
.
Figure 1:
Effect of guaiacol concentration on the
rate of tetraguaiacol formation. Reaction mixtures contained 0.1
µM lignin peroxidase, 0.3 mM HO
(saturating), and varying concentrations of guaiacol in 25
mM sodium tartrate (pH 3.5).
Steady-state analyses with
HO
as the variable substrate indicated that
H
O
inhibits the enzyme at high concentration (Fig. 2A). This has also been observed for veratryl
alcohol oxidation(5) . The rate of tetraguaiacol formation
increased as the H
O
concentration increased to
125 µM. A further increase in the H
O
concentration resulted in a decrease in the rate of tetraguaiacol
formation.
Figure 2:
Effect of HO
on
the rate and yield of guaiacol oxidation. A, effect of
H
O
on the rate of tetraguaiacol formation.
Incubations contained 2 mM guaiacol. Initial velocities were
monitored. Experimental conditions were identical to those described in
the legend of Fig. 1. B, effect of H
O
concentration on the final yield of tetraguaiacol formation. The
H
O
concentration was varied in the presence of
2 mM (closedcircles) and 10 mM (triangles) guaiacol. The superoxide scavenger
tetranitromethane (1 mM) was added to certain incubations
containing 2 mM guaiacol (opencircles).
Incubations were monitored, and the maximal tetraguaiacol concentration
was recorded. Incubations were identical to those described in the
legend of Fig. 1.
The observed decrease in the rate of tetraguaiacol
formation at high HO
concentrations was
reflected by the decreased yield of tetraguaiacol (Fig. 2B). In these experiments, the tetraguaiacol
concentration was monitored by absorbance at 470 nm, and the maximal
absorbance value was recorded. This maximal value was reached anywhere
from 10 to 50 min. In incubations containing 2 mM guaiacol and
low concentrations of H
O
, the yield of
tetraguaiacol was slightly less than the 4:1 ratio of
H
O
to tetraguaiacol, similar to that reported
for horseradish peroxidase(22) . At H
O
concentrations above 300 µM, the amount of
tetraguaiacol decreased.
Harvey and Palmer (23) proposed
that compound III was formed during steady-state oxidation of guaiacol.
Formation of compound III would account for the decreased rate and
yield of tetraguaiacol. To further investigate this possibility, we
added tetranitromethane to the incubations. Tetranitromethane scavenges
superoxide and also reacts directly with compound III, yielding ferric
enzyme(8) . This addition dramatically increased the yield of
tetraguaiacol formation (Fig. 2B). This observation is
consistent with the proposal that compound III formation accounts for
the decreased yield at high HO
concentrations.
The decrease in the yield of tetraguaiacol at higher
H
O
concentrations could be partially reversed
by increasing the guaiacol concentration (Fig. 2B).
Figure 3:
Effect of veratryl alcohol on the rate of
tetraguaiacol formation. A, experiments in which guaiacol
concentration was held constant at 3 mM with varying veratryl
alcohol concentrations; B, similar experiments except that the
veratryl alcohol concentration was held constant at 2 mM and
the guaiacol concentration was varied. Reaction conditions were similar
to those described in the legend of Fig. 1with 0.3 mM
HO
.
The effect of veratryl alcohol
on the rate of guaiacol oxidation was also studied when the
concentration of veratryl alcohol was held constant (2 mM) and
the guaiacol concentration was varied (Fig. 3B). In
contrast to the results shown in Fig. 1, a hyperbolic
relationship was not observed. Surprisingly, at low guaiacol
concentrations, the rate of its oxidation was even higher than the
calculated k of 7.7 s
. At
these low concentrations of guaiacol (with 2 mM veratryl
alcohol), the rate approached that of the k
for
veratryl alcohol (Fig. 3B). These data suggest that 2
mM veratryl alcohol saturates the enzyme (thus, the velocity
should approach the k
for veratryl alcohol),
resulting in the formation of the veratryl alcohol cation radical
intermediate. This radical, in turn, mediates the oxidation of
guaiacol. At higher guaiacol concentrations, competition by veratryl
alcohol at the active site is minimized; thus, the rate approaches the k
for guaiacol. At these low guaiacol
concentrations, no detectable veratraldehyde is detected until the
guaiacol is depleted (see below).
Figure 4: Oxidation of veratryl alcohol in the presence (open circles) and absence (closed circles) of 0.1 mM guaiacol. Reaction conditions were as described in the legend of Fig. 1.
Figure 5:
Effect of varying guaiacol concentration
on the time course of veratryl alcohol oxidation. Reaction mixtures
contained 2 mM veratryl alcohol and 0.3 mM HO
as described in the legend of Fig. 1. Increasing guaiacol concentrations from 0, 10, 20, 30,
40, and 50 µM caused a proportionate increase in the lag
period.
Figure 6: Reaction of lignin peroxidase compounds I and II with guaiacol. A, results from the reaction of compound I with varying concentrations of guaiacol; B, similar results with compound II and guaiacol. Generation of compounds I and II is as described under ``Materials and Methods.''
where LP II is lignin peroxidase compound II and Gu is guaiacol. If rapid equilibrium is assumed for the first step, the rate of guaiacol oxidation can be given by .
The line drawn in Fig. 6B is a best fit
according to with K = 64
µM and k = 17 s
. This
is in contrast to 280 µM and 16 s
for K
and k, respectively, for veratryl
alcohol(15) .
Figure 7:
Conversion of lignin peroxidase compound
III to ferric enzyme (solid line). Compound III was generated
by adding 40 eq of HO
to ferric enzyme (5.1
µM) in 25 mM sodium tartrate (pH 3.5). The
spectrum of compound III (scan 2) was recorded 2 min after the
addition of H
O
to ferric enzyme (scan
1). Guaiacol (50 eq) was added, and the resultant spectrum (scan 3) was similar to that of ferric
enzyme.
Veratryl alcohol has been shown to stimulate the lignin peroxidase-catalyzed oxidation of monomethoxy substrates such as anisyl alcohol and 4-methoxymandelic acid(12) . Harvey et al.(12) suggested that these substrates, which are not oxidized directly by the enzyme, could be oxidized by veratryl alcohol cation radicals generated by lignin peroxidase catalysis. This mediation phenomenon by veratryl alcohol, a secondary metabolite also produced by the fungus(10) , led to the proposal by Harvey et al.(12) that the physiological role of veratryl alcohol is to mediate the oxidation of lignin. Our previous studies have provided a different interpretation for the stimulation of anisyl alcohol and 4-methoxymandelic acid oxidation by veratryl alcohol(15) . Transient state kinetic studies showed that anisyl alcohol can be oxidized by compound I (but not by compound II) of lignin peroxidase. Therefore, inclusion of veratryl alcohol or another substrate that reacts with compound II is essential for completion of the catalytic cycle. This completion of the catalytic cycle explains the observed stimulation of anisyl alcohol oxidation by veratryl alcohol.
In
contrast to anisyl alcohol, guaiacol is a very good substrate for
lignin peroxidase. As summarized in the scheme shown in Fig. 8A, guaiacol is a good substrate for both
compounds I and II. With guaiacol, a second substrate like veratryl
alcohol would not be needed to complete the catalytic cycle. The rate
constant for compound I reacting with guaiacol is actually greater than
that with veratryl alcohol. The reactivity of compound II with these
two substrates is comparable with the exception that guaiacol has a
much lower K. These results indicate that the
stimulation of guaiacol oxidation by veratryl alcohol cannot be
attributed to the need of veratryl alcohol to complete the catalytic
cycle.
Figure 8:
Reaction scheme of lignin peroxidase (LP) with guaiacol (Gu) and HO
in the absence (A) and presence (B) of veratryl
alcohol (VA). Rate constants and references are as follows: k
= 5.8
10
M
s
(5) , k
= 1.5
10
M
s
(15) , k
= 1.2
10
M
s
(this work), k
= 16 s
(15) ,
and k
= 17 s
(this
work). The K
values for veratryl alcohol
and guaiacol are 280 µM(14) and 64 µM (this work), respectively.
Although our previous studies discounted a mediation role for
veratryl alcohol in the oxidation of anisyl alcohol(15) , this
study clearly shows that veratryl alcohol can mediate the oxidation of
a phenolic substrate such as guaiacol. This evidence comes from
steady-state kinetic studies. Fig. 8B shows the
proposed mechanism of guaiacol oxidation in the presence of veratryl
alcohol. Both guaiacol and veratryl alcohol are substrates for lignin
peroxidase. As shown in Fig. 8B, they are both oxidized
by compounds I and II. If the interaction of guaiacol and veratryl
alcohol was only at compounds I and II, the inhibition of the oxidation
of one substrate by the other would be expected to be
competitive(25) . However, veratryl alcohol has no inhibitory
effect on guaiacol oxidation. In fact, at low guaiacol concentrations
(below K), saturating concentrations of veratryl
alcohol (2 mM) stimulated guaiacol oxidation to rates higher
than its k
of 7.7 s
(Fig. 3B). This can be explained by veratryl
alcohol saturating the enzyme (K
= 168
µM) at 2 mM (thus resulting in its maximal
velocity) and the subsequent reaction of the veratryl alcohol cation
radical with guaiacol. In contrast, guaiacol was able to completely
inhibit veratryl alcohol oxidation at concentrations much lower than
its K
. Veratraldehyde production was not observed
until all of the guaiacol was depleted from the reaction mixture. This
indicates that the mode of inhibition is more than just competing at
the active site and can only be explained by guaiacol very effectively
reducing the veratryl alcohol cation radical, resulting in its own
oxidation (Fig. 8B). The rate constant for this
reaction of the veratryl alcohol cation radical and guaiacol must be
near the diffusion limit. The results from the inhibition studies can
only be explained by mediation of guaiacol oxidation by veratryl
alcohol.
There is still the question of why guaiacol oxidation does
not proceed in a linear fashion and that compound III accumulates
during turnover. Harvey and Palmer (23) were the first to
observe such a phenomenon. They attributed the inhibition to the
inability of guaiacol to convert compound III to ferric enzyme. Our
results are in agreement, in part. Although we showed that guaiacol can
partly convert compound III to resting enzyme, guaiacol is not as
effective as veratryl alcohol in this process(8) . The
oxidation of veratryl alcohol very efficiently results in the
conversion of compound III to resting enzyme (26, 27) by a mechanism that is yet to be defined.
Formation of compound III during turnover would explain the decreased
rate of guaiacol oxidation at high HO
concentrations. Compound III is readily formed with lignin
peroxidase at high H
O
concentrations(10) . However, this does not completely
explain the decrease in the yield of tetraguaiacol at high
H
O
concentrations. One would predict that the
yield would remain constant even with decreased rates. This decreased
yield appears to involve superoxide. The addition of tetranitromethane,
a superoxide scavenger and an agent capable of converting compound III
to ferric enzyme(8) , also increased the yield of
tetraguaiacol. Superoxide has been shown to be produced during lignin
peroxidase turnover(28) . The product tetraguaiacol is not
extremely stable(29) , and thus, the observed decrease in yield
might be due to the possible involvement of superoxide in the
decomposition of tetraguaiacol.
Many roles have been proposed for
veratryl alcohol in lignin biodegradation. Veratryl alcohol has been
shown to protect lignin peroxidase from
HO
-dependent
inactivation(11, 30) ; it has been shown to prevent
compound III accumulation(9) ; and it has been proposed to act
as a redox mediator in lignin depolymerization(12) . Strong
arguments can be made for the physiological significance of the first
two roles, whereas the latter role as a redox mediator has been
contested. Although our studies here demonstrated the ability of
veratryl alcohol to mediate the oxidation of a phenol, we doubt that
this is physiologically significant. No veratryl alcohol is bound to
lignin peroxidase upon purification from fungal cultures. Therefore,
veratryl alcohol cannot be viewed as a tightly bound prosthetic group.
In addition, the veratryl alcohol concentration in fungal cultures is
below its K
for lignin peroxidase. This indicates
that under physiological conditions, the enzyme is not saturated with
veratryl alcohol and that phenolic substrates would be just as readily
oxidized directly by the enzyme as they would be by the veratryl
alcohol cation radical. Furthermore, there are also no data indicating
that the recently detected veratryl alcohol cation radical is stable
and diffusible(31) . Thus, it does not seem likely that
veratryl alcohol can be acting at a distance as a diffusible oxidant.
Consequently, the mediation of phenol oxidation would have to occur
near or at the enzyme active site. Our results here are more consistent
with a physiological role of veratryl alcohol protecting the enzyme
from H
O
-dependent inactivation and serving as a
substrate for compound II, where more recalcitrant substrates, such as
those found in lignin, may not be able to serve such a
role(15) .