From the
The formation and decay of veratryl alcohol radical cation upon
oxidation of veratryl alcohol by thallium (II) ions was studied by
pulse radiolysis with spectrophotometric and conductometric detection.
In aqueous solution at pH 3 the radical cation decays by a first order
process, assigned to the deprotonation from the
Lignin peroxidase (EC 1.11.1.7), an extracellular heme
peroxidase produced by white rot, has been shown to catalyze the
depolymerization of lignin (1) as well as the oxidation of a
range of compounds of relatively high redox potential (2) (for
recent reviews see Refs. 3-5). Aromatic compounds, in particular
the fungal secondary metabolite veratryl alcohol (VA)
Further progress in this field has been
hampered by the lack of information on the lifetime and reactivity of
the veratryl alcohol radical cation. NMR experiments (11) suggest its formation during the oxidation of VA by lignin
peroxidase, but its detection by EPR has not been possible(12) .
The radical cations of aromatic compounds can be conveniently studied
by radiation chemistry methods, especially pulse
radiolysis(13) . In this technique(14) , a short pulse of
accelerated electrons, typically of a few nanoseconds, is used to
generate a quantifiable amount of free radicals in solution, usually in
the micromolar range, in less than a microsecond. The reactivity of
these radicals is usually monitored spectrophotometrically, by
conductometry or other detection techniques.
In the present study,
using radiation chemistry techniques we were able to measure the
veratryl alcohol radical cation absorption spectrum as well as its
lifetime and reactivity toward two model compounds, a polymeric dye
(Poly R-478) and 4-methoxymandelic acid.
Commercial chemicals from Sigma or Aldrich of the highest
purity available were used in this study. Solutions prepared with water
purified with a Millipore Milli-Q system were degassed for about 30 min
before irradiation with oxygen free nitrous oxide (N
Pulse radiolysis with spectrophotometric detection
was performed with a 4-MeV van de Graaff accelerator as described
previously(15) . The doses per pulse were calibrated with
N
Analysis of solutions used a HPLC equipped
with a Hichrom Hypersil 50DS column and a Waters 490 multiwavelength
detector, operating at 280 nm. Elution was achieved with a flow of 2 ml
min
The unstable ion Tl
On-line formulae not verified for accuracy
REACTION 1
In the present study solutions of Tl
We have applied the
reaction of radiolytically generated Tl
In longer time scales, the
absorption at 430 nm was observed to decay slowly, and the conductance
returned to the prepulse value. We conclude therefore that VA decays
with release of a proton, i.e. into an uncharged species that
does not absorb at 430 nm. Through experiments with optical detection,
we found that the rate of decay was independent both of the VA
concentration (20-80 µmol dm
Chromatographic analysis of identical solutions irradiated under
steady-state conditions showed that veratryl aldehyde is the main
stable product formed on oxidation of veratryl alcohol by
Tl
It can therefore be concluded that VA reacts
with Poly R to regenerate veratryl alcohol and bleach the polymer,
presumably involving a radical cation of the latter as intermediate.
On-line formulae not verified for accuracy
REACTION 4
In a previous study, 4-methoxymandelic acid (MMA;
hydroxy(4-methoxyphenyl)acetic acid) was used as a model recalcitrant
substrate that could only be oxidized by lignin peroxidase if veratryl
alcohol was present(6) . The one-electron oxidation of MMA
yields a zwitterion that undergoes rapid elimination of carbon dioxide,
and in the presence of oxygen the resulting radical is completely
converted into anisaldehyde.
In agreement with this mechanism, the oxidation of MMA by
Tl
Using the pulse radiolysis technique, we have been able for
the first time to observe directly the formation and decay of the
veratryl alcohol radical cation, to record its absorption spectrum (Fig. 1), and to measure its lifetime (59 ± 8 ms).
Previous attempts to detect this species by EPR were
unsuccessful(12) , and the lifetime reported here confirms that
VA may be too short lived to be detected by that technique.
One of
the reasons for suggesting an involvement of the veratryl alcohol
radical cation in the lignin peroxidase-catalyzed degradation of lignin
was the impossibility of a spatial interaction between the heme and the
polymer. It was therefore proposed that veratryl alcohol ``redox
cycles,'' i.e. the radical cation serves as an electron
carrier between the polymer and the enzyme. However, the short lifetime
attributed to VA until now seemed incompatible with this hypothesis. In
conjunction with the Einstein-Smoluschowski equation of diffusion, the
lifetime of 59 ms implies that VA can migrate about 7 µm in an
aqueous environment. Obviously, this distance is sufficient to allow
the oxidation of a polymer at a considerable distance from the
peroxidase.
The ability of veratryl alcohol to redox cycle is well
illustrated by the results with Poly R. In the chemical system used,
the veratryl alcohol radical cation acted as an electron carrier
between the oxidant (Tl
Different
results were obtained with 4-methoxymandelic acid; no electron transfer
between this compound and the veratryl alcohol radical cation could be
observed. This is in agreement with the prediction that
4-methoxymandelic acid, with a single methoxy substituent, has a higher
redox potential than veratryl alcohol. However, in the event of
reversible electron transfer, equilibrium 6 would be shifted to the
right by the short lifetime of the MMA radical cation.
On-line formulae not verified for accuracy
REACTION 6
Therefore, the failure to observe the oxidation of MMA by veratryl
alcohol radical cation may reflect not only a thermodynamic but also a
kinetic barrier to . In other words, the finite lifetime
of VA may be too short for the establishment of equilibrium 6.
With
respect to the oxidation of 4-methoxymandelic acid, the results of
radiation chemical oxidation contrast with the previous observations in
the enzyme system(6) . When equimolar mixtures of
4-methoxymandelic acid and veratryl alcohol were incubated with lignin
peroxidase (in the presence of hydrogen peroxide), MMA and not VA was
oxidized, although MMA alone was not a substrate for the enzyme. It
appears that veratryl alcohol can oxidize 4-methoxymandelic acid in the
enzyme system but not when generated in aqueous solution by a chemical
oxidant. In order to solve this apparent discrepancy, we suggest that
the veratryl alcohol radical cation may exist as an enzyme-bound
species and that the binding may overcome either the thermodynamic or
the kinetic barrier to the oxidation of methoxymandelic acid. Binding
of several peroxidases to their substrates has been demonstrated,
although the results obtained with veratryl alcohol and lignin
peroxidase are ambiguous (20). We hypothesize that veratryl alcohol
binds to the enzyme and that after electron transfer to the heme, the
veratryl alcohol radical cation may react with a second substrate prior
to its release.
It is conceivable that the suggested binding would
prolong the lifetime of the veratryl alcohol radical cation to the
extent of allowing to proceed. Cases are known in which
radicals that are unstable in aqueous solution become stabilized in an
enzyme environment; for example, the active ribonucleotide reductase
contains a long-lived tyrosine radical(21) . Alternatively, the
reduction potential of the bound veratryl alcohol radical cation may be
higher than when it exists free in solution and therefore make the free
energy variation associated with less positive. This
hypothesis is substantiated by recent calculations showing the
existence of a positively charged region around the active site of
lignin peroxidase (22), which would be expected to increase the
reduction potential of a radical cation immersed in it.
An
alternative role for the veratryl alcohol radical cation in the
catalytic cycle of lignin peroxidase is the conversion of an inactive
form (compound III) back into active enzyme(8) . With the
1,2,4,5-tetramethoxybenzene radical cation this reaction was directly
observed(8) , but in the case of veratryl alcohol only indirect
evidence can be obtained. Clearly, the possibility of a bound veratryl
alcohol radical cation is not contrary to the reactivation of compound
III. The lifetime of VA implies that its steady-state concentration
during the enzyme cycle will be of the order of micromolar
(steady-state concentration = rate of turnover/17
s
In summary, the lifetime of the veratryl alcohol
radical cation (59 ± 8 ms) is sufficient to enable it to migrate
over large distances (about 7 µm in aqueous solution) and possibly
act as a redox mediator. The oxidation of Poly R-478 shows that, at
least with this polymer, VA can act as a redox mediator. However,
4-methoxymandelic acid is not oxidized by VA in aqueous solution, in
disagreement with the conclusions from studies with lignin peroxidase.
We therefore suggest that VA can exist as an enzyme-bound species and
that in this state it has either a longer lifetime or a higher
reduction potential than in bulk solution.
We are grateful to Profs. P. Jones, J. Palmer, and P.
Wardman for helpful discussions, to L. K. Folkes for assistance, and to
Dr. B. Vojnovic and R. Locke for the development of the pulse
radiolysis equipment, in particular the conductance detection.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-carbon. On the
basis of its lifetime (59 ± 8 ms) and of its ability to oxidize
a polymeric dye (Poly R-478) we estimate that the radical cation can
diffuse about 7 µm in an aqueous environment to act as a mediator
of oxidations over long distances. However, 4-methoxymandelic acid is
not oxidized by the veratryl alcohol radical cation in homogeneous
solution, and the comparison with previous studies on lignin peroxidase
catalysis suggests a second role for veratryl alcohol radical cation in
the enzyme action: it may exist as an enzyme-bound species that has
either a longer lifetime or a higher reduction potential than the free
radical cation in bulk solution.
(
)(3,4-dimethoxybenzyl alcohol), have been shown to enhance
the enzyme action, but their function has not yet been satisfactorily
elucidated. It has been suggested (6) that veratryl alcohol is
oxidized by the peroxidase to a radical cation and that this species
plays a key role in the catalytic cycle by acting as a redox mediator
between the enzyme and its substrates(3, 6) , or by
converting compound III, an inactive enzyme intermediate formed by
reaction of compound II with hydrogen peroxide, to the native (ferric)
state(7, 8) . It has also ben proposed that VA
(unoxidized) protects against formation of compound III by supplying a
reducing equivalent that prevents accumulation of compound
II(9, 10) .
O) or
with a mixture of N
O and oxygen (5%, v/v) (from British
Oxygen Company).
O-saturated solutions of KSCN solutions (10 mmol
dm
), assuming the product of the radiation chemical
yield and extinction coefficient of (SCN) equal to 5.1
10
Gy
cm
(16).
Conductance detection used conventional conductance cells with platinum
electrodes (cell constant = 0.2 cm
) to which
a polarizing voltage 10 V/10 MHz AC was applied. Steady-state
irradiation used a
Co
-source with an activity of
about 35 TBq. Irradiations were performed at a dose rate of 7.9 Gy
min
, as determined by a Fricke
dosimeter(17) .
of a mixture of 15 mmol dm
potassium acetate at pH 3.8 and a gradient (10-50% in 5
min) of 75% acetonitrile.
is a strong oxidant
that reacts with many methoxylated aromatic compounds to yield the
respective radical cations(13) . It can be conveniently
generated by radiolysis of aqueous solutions of Tl
saturated with nitrous oxide. Under these conditions, the
hydroxyl radical is the main reactive species resulting from
irradiation, and it reacts with Tl
according to (18) .
(1 mol
dm
) in 1 mmol dm
HClO
were used. This concentration of Tl
is adjusted
to give the maximum yield of Tl
, whereas the
HClO
sets the pH close to that used in previous studies
with lignin peroxidase. Under these conditions, the radiation-chemical
yield of Tl
was 0.46 µmol J
,
determined by monitoring the oxidation of ABTS
(2,2`-azinobis(3-ethylbenzothiazoline 6-sulfonate)) to the stable
radical ABTS (
= 417 nm,
= 34,700
dm
mol
cm
(19) .
with VA to
produce the respective radical cation and study its spectroscopic
properties and reactivity. The transient absorption spectrum recorded
by pulse radiolysis on reaction of Tl
with VA is
shown in Fig. 1. The two absorption bands at 300 and 420 nm are
characteristic of the absorption spectra of the radical cations of
methoxylated aromatics(13) , suggesting that the veratryl
alcohol radical cation was formed. The increase of absorption at 430 nm
was exponential and had a rate constant proportional to the VA
concentration (in the range 10-50 µmol
dm
), from which the second order rate constant of Fig. R2was determined (k = (5.4 ± 0.1)
10
dm
mol
s
).
Figure 1:
Absorption spectrum and
decay kinetics of the veratryl alcohol radical cation. The radical
cation was generated by pulse radiolysis of a NO-saturated
solution of veratryl alcohol (70 µmol dm
) and
Tl
(1 mmol dm
) in 1 mmol
dm
HClO
. The spectrum was built from
measurements of the absorbance change at discrete wavelengths 100
µs after pulses of about 4 Gy. Inset a, time-dependence of
the absorbance at 430 nm. Inset b, and of the conductance of
the solution.
Figure R2:
Reaction 2.
Similar experiments were performed with conductometric detection.
Following the radiation pulse, a decrease of conductance was observed,
attributable to the formation of OH in and consequent neutralization of one equivalent of
H
. However, the decreased conductance persisted longer
than the completion of Fig. R2, which shows that the latter
reaction does not generate or deplete further H
, in
support of the formation of VA.
) and of the
initial concentration of radicals in the range
0.5 to
5
µmol dm
(i.e. of the radiation dose in
the range
1 to
10 Gy). In view of these observations, we
conclude that the radical cation decays by a first order process,
probably by deprotonation form the
-carbon. The rate of this decay
was determined as k = 17 ± 2
s
, i.e. the lifetime of the veratryl
alcohol radical cation, defined as the reciprocal of the rate constant,
is 59 ± 8 ms.
. From the dependence of the concentration of
veratryl aldehyde on the radiation dose (see Fig. 3), it can be
estimated that this product accounts for about 93% of the
Tl
generated by irradiation. The oxidation of the
carbon-centerd radical formed in Fig. R3leads to veratryl
aldehyde, and the high yield observed suggests that the solutions used
in the experiments described contain a substance that is able to carry
out this oxidation, probably Tl
.
Figure 3:
Formation of veratryl aldehyde and the
bleaching of Poly R. Veratryl aldehyde formation on oxidation of
veratryl alcohol (100 µmol dm) by Tl
generated radiolytically in the absence (opensquares) or in the presence (solidsquares) of Poly R-478 (25 mg dm
). The
bleaching of Poly-R is shown by the solidcircles.
Figure R3:
Reaction 3.
In order to
evaluate the possibility that the veratryl alcohol radical cation may
transfer its charge to polymers, we studied its reaction with Poly
R-478 (hitherto referred to simply as Poly R). In a pulse radiolysis
experiment, VA was generated by reaction with Tl in
the presence of Poly R (up to 25 mg dm
). Under these
conditions the decay of VA, monitored by the absorption at 430 nm (Fig. 2a), was fast and exhibited a rate proportional to
the concentration of the dye. Simultaneously, a bleaching of the dye
could be observed at 548 nm (Fig. 2b) which had the same
rate as the decay at 430 nm, a good indication that a reaction between
the veratryl alcohol radical cation and the polymer was taking place.
The second order rate constant of this reaction, determined from the
linear dependence of the rate of decay at 430 or 548 nm on the Poly R
concentration, is (5.1 ± 0.7)
10
dm
(mol of monomer)
s
.
Figure 2:
Reaction of the veratryl radical cation
with Poly R. Transient absorption at 430 nm showing the decay of the
veratryl alcohol radical cation (a) and at 548 nm showing the
simultaneous bleaching of Poly R (b). Traces were recorded on
pulse radiolysis of a NO-saturated solution of veratryl
alcohol (100 µmol dm
), Poly R (5 g
dm
), and Tl
and HClO
as
in Fig. 1.
In
addition, the same reaction was investigated by steady-state radiolysis
experiments with similar solutions containing Poly R (25 mg
dm). The bleaching of the dye was measured by the
decrease of absorption at 548 nm, and the formation of veratryl
aldehyde was monitored by HPLC. Pulse radiolysis experiments showed
that under these conditions VA is formed initially in 85% yield
relative to the Tl
. However, in the steady-state
experiments bleaching of the dye was observed, and no consumption of
veratryl alcohol could be measured. Moreover, the yield of veratryl
aldehyde was decreased to
7% of that measured in the absence of
Poly R (Fig. 3).
(
)
in a solution saturated with a mixture of nitrous
oxide and oxygen (20% v/v) yielded anisaldehyde in quantitative yield.
A similar experiment was performed with a solution that contained, in
addition, veratryl alcohol (1 mol dm
). Under these
conditions, most of the Tl
formed (about 78%) reacts
with veratryl alcohol, and accordingly the yield of anisaldehyde was
greatly reduced (Fig. 4). In fact, the anisaldehyde formed under
these conditions can be essentially accounted for by the fraction of
Tl
reacting with 4-methoxymandelic acid, suggesting
that the veratryl alcohol radical cation did not oxidize MMA
significantly. On the basis of our results, we estimate the rate
constant of this reaction to be
5
10
dm
mol
s
.
Figure 4:
Inhibition of the oxidation of
4-methoxymandelic acid by veratryl alcohol. MMA (squares) is
quantitatively oxidized to anisaldehyde (circles) in a
solution saturated with NO + O
(5%, v/v) (opensymbols), but in the presence of 1 mmol
dm
veratryl alcohol (1 mmol dm
)
the reaction is inhibited (solidsymbols). The
solutions contained Tl
and HClO
(as in
Fig. 1) and were irradiated and analyzed by
HPLC.
) and the polymeric dye.
Presumably, the veratryl alcohol radical cations generated by lignin
peroxidase will behave similarly, at least toward Poly R. However, due
to the difficulty of measuring degradation of an insoluble polymer, the
reaction of VA with lignin has not been directly observed.
). Therefore, the reaction with compound III at a
rate sufficient to prevent inactivation would require a very high
bimolecular rate constant. However, a veratryl alcohol radical cation
bound to the enzyme active site is readily available to react with
compound III.
)); HPLC, high pressure liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.