Lignin peroxidase catalysed oxidation of 4-methoxymandelic acid. The role of mediator structure

Article abstract of DOI:10.1016/S0040-4020(02)00997-3

A large number of substances have been tested as redox mediators in the LiP-catalysed oxidation of 4-methoxymandelic acid (4-MMA) to anisaldehyde. In some cases (i.e. thioanisole), the mediation efficiency is almost equal to the maximum value displayed by the natural mediator veratryl alcohol. The mediation efficiency is a function of the redox potential of the mediator and also appears to depend on the kinetic effectiveness with which the mediator is oxidised by the enzyme. In contrast, the lifetime of the mediator radical cation seems not to play any significant role, which would support the idea that the redox mediation is actually accomplished by a complex between the mediator radical cation and the enzyme.

Full text of DOI:10.1016/S0040-4020(02)00997-3

Tetrahedron 58 (2002) 8087–8093
Lignin peroxidase catalysed oxidation of 4-methoxymandelic acid.
The role of mediator structure
*
Enrico Baciocchi, Maria Francesca Gerini, Osvaldo Lanzalunga and Simona Mancinelli
Dipartimento di Chimica, Universit a` “La Sapienza”, P.le A. Moro, 5, I-00185 Rome, Italy
Received 27 March 2002; revised 19 July 2002; accepted 8 August 2002
Abstract—A large number of substances have been tested as redox mediators in the LiP-catalysed oxidation of 4-methoxymandelic acid
(
4-MMA) to anisaldehyde. In some cases (i.e. thioanisole), the mediation efficiency is almost equal to the maximum value displayed by the
natural mediator veratryl alcohol. The mediation efficiency is a function of the redox potential of the mediator and also appears to depend on
the kinetic effectiveness with which the mediator is oxidised by the enzyme. In contrast, the lifetime of the mediator radical cation seems not
to play any significant role, which would support the idea that the redox mediation is actually accomplished by a complex between the
mediator radical cation and the enzyme. q 2002 Elsevier Science Ltd. All rights reserved.
1
. Introduction
2
P–FeðIVÞvO þ H O ! P–FeðIIIÞO
þ H O
ð4Þ
2
2
2
2
Compound III ðLip IIIÞ
Lignin peroxidase (LiP, EC 1.11.1.7), an extracellular heme
enzyme isolated from ligninolytic cultures of the white-rot
basidiomycetous fungus Phanerochaete chrysosporium
has attracted great attention mainly because of its capacity
to degrade lignin, a process which is of fundamental
importance both in the carbon earth cycle and in the paper
and pulp industry. Interestingly, LiP is also able to promote
the H O -dependent oxidation of several compounds
It seems that LiP alone can catalyse lignin degradation;
however, there is evidence that such a process is
significantly accelerated by the presence of veratryl alcohol,
3,4-dimethoxybenzyl alcohol (VA), a secondary metabolite
1
2
,3
of the fungus P. chrysosporium, which acts as a cofac-
18–20
4
tor.
This observation has led to a lively discussion
2
2
about the actual role played by VA and three hypotheses
have been presented in order to explain the beneficial effect
of VA. The first hypothesis suggests that VA protects LiP
against H O inactivation preventing the formation of the
inactive form LiP III. The second is that VA reduces
LiP II back to the native enzyme, thus completing the
5
–16
characterised by redox potential ,1.4 V.
The LiP catalytic cycle involves initial reaction of the ferric
heme (the native state) with H O to yield Compound I (LiP
I), characterised as having an oxy-ferryl heme with
2
2
2
1,22
2
2
þz2
23–25
porphyrin p-cation radical (P Fe(IV)vO) (Eq. (1)).
catalytic cycle.
The third hypothesis, which presently
Thereafter, two steps of single electron oxidation of
substrates restore the enzyme to the native state, via the
intermediate Compound II (LiP II), which retains the oxy-
ferryl heme (P–Fe(IV)vO) (Eqs. (2) and (3)). LiP II can
also react with H O to yield the catalytically inactive
seems to receive the major credit, is that VA acts as a one-
electron redox mediator transferring oxidising equivalents
from the enzyme to the large, insoluble and hydrophobic
lignin polymer, as illustrated in Scheme 1. The oxidation of
VA would involve the transfer of one electron to LiP I,
leading to the formation of VA which would then
exchange one electron with one of the aromatic rings of
11,19,26,27
the macromolecule, thus initiating its degradation.
2
2
2
17
þz
Compound III (LiP III) (P–Fe(III)O ) (Eq. (4)).
2
þz2
P–FeðIIIÞ þ H O ! P FeðIVÞvO þ H O
ð1Þ
ð2Þ
2
2
2
LiP
Compound I ðLiP IÞ
Very interestingly, a beneficial effect of VA has also
been observed in the oxidation of simple organic substrates
which are ‘recalcitrant’ towards the oxidation by LiP.
Accordingly, LiP alone is practically not able to oxidise
þz2
FeðIVÞvO þ S ! P–FeðIVÞvO þ Sþz
P
Compound II ðLip IIÞ
þ
þz
4
(
4
-methoxybenzyl alcohol (anisyl alcohol) and 2-hydroxy-2-
4-methoxyphenyl)acetic acid (4-methoxymandelic acid,
-MMA), but these oxidations take place with good
P–FeðIVÞvO þ S þ 2H ! P–FeðIIIÞ þ S þ H O ð3Þ
2
Keywords: lignin peroxidase; mediation; redox potential; radical cation
lifetime.
1
9
efficiency in the presence of VA. An additional obser-
1
9
*
Corresponding author. Tel.: þ39-06-49913683; fax: þ39-06-490421;
vation is that other compounds (i.e. 1,4-dimethoxybenzene
2
and 2-chloro-1,4-dimethoxybenzene ) can replace VA in
8
e-mail: osvaldo.lanzalunga@uniroma1.it
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00997-3

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E. Baciocchi et al. / Tetrahedron 58 (2002) 8087–8093
Scheme 1. VA as a redox mediator in LiP-catalysed oxidation of lignin.
2. Results
2.1. Measurement of mediation efficiency
The mediation efficiency was determined as the yield of
anisaldehyde formed in the LiP-catalysed oxidation of
4
4
-MMA in the presence of the mediator (4% based on
-MMA). The enzymatic oxidation was carried out at 378C,
Scheme 2. LiP-catalysed oxidation of 4-MMA.
increasing the efficiency of LiP-promoted oxidations.
A mechanism similar to that in Scheme 2 has been proposed
in the case of the oxidation of 4-MMA.
for 10 min, under an argon atmosphere, in 50 mM sodium
tartrate buffer solution, pH 3.0, containing LiP (0.51 nmol,
0.3 U), the substrate (0.25 mmol) and the mediator
†
(0.01 mmol).
Thus, it appears that the oxidation of these recalcitrant
substrates represents a very simple system suitable to
acquire information about the relative ability of different
compounds to act as mediators in the oxidations catalysed
by LiP and about the structural factors which play a role in
this respect. Such information may also provide us further
insight about the mechanism of mediation and the enzyme–
mediator interaction.
The reaction was initiated by adding the oxidant, H
O
2 2
(0.06 mmol). The reaction mixture was subsequently
analysed by HPLC. It was first checked that in the absence
of the mediator (blank reaction) only a small amount of
anisaldehyde (2% based on the substrate) was formed and
this amount has been subtracted from the reaction yields in
the presence of the mediator.
In this paper, we report on the mediation efficiency of a
large number of substances, measured in the LiP-catalysed
oxidation of 4-MMA to 4-methoxybenzaldehyde (anisalde-
hyde) (Scheme 2).
2.2. Measurement of the redox potential
The redox potentials of the mediators have also been
determined in aqueous solution by cyclic voltammetry. The
measurements were carried out at 258C, in argon degassed
10 mM sodium acetate buffer solution, pH 4.0, with 3%
We have tested a series of substituted o-, m-, p-dimethoxy,
trimethoxy- and tetramethoxy-aromatic compounds, ring
substituted (methylthio)benzenes (thioanisoles) and trypto-
phan. Whenever possible the redox potential and the
lifetimes of the corresponding radical cations have also
been determined. These parameters should play a role in a
mechanism such as that shown in Scheme 1. Accordingly,
the mediator has to be oxidised to radical cation and only a
sufficiently long-lived radical cation (half-life of at least
CH CN as cosolvent, containing LiClO (0.12 M) as the
3 4
supporting electrolyte and the substrate (0.10 mmol). Using
a sweep scan up to 500 mV s2 only irreversible oxidation
waves were obtained.
1
Results of mediation efficiency together with the mediator’s
E values, in V vs NHE, are listed in Table 1 for di-, tri-,
p
tetramethoxyaromatics and in Table 2 for ring substituted
thioanisoles. For tryptophan, a mediation efficiency of 4.8
2
9
some milliseconds) can escape from the interaction with
the enzyme, diffuse into solution and induce the oxidation of
other substrates (diffusible redox mediator). For some
mediators, we have also measured the kinetic parameters
kcat and KM for the enzymatic oxidation to acquire
information on the enzyme–mediator interactions.
and E
¼1.09 (V vs NHE) have been determined.
p
2.3. Measurement of the kinetic constant kcat and K
M
For some of the tested mediators, the enzymatic oxidation
led to a single product (see Section 5) and it was thus
possible to measure their enzymatic kinetic constants. These
compounds are VA, its corresponding methyl ether and
1-(3,4-dimethoxyphenyl)ethanol.
†
The reaction (a) described in Scheme 2 is generally an endoergonic
process which is driven to the right by the fast fragmentation of
4
þz 19
-MMA .

E. Baciocchi et al. / Tetrahedron 58 (2002) 8087–8093
8089
Table 1. Mediation efficiency and redox potential of di-, tri- and tetramethoxy-aromatic compounds in the LiP-catalysed oxidation of 4-MMA
a
þz
b
M
a
þz
b
Mediator
Efficiency (%)
E
M
/
Mediator
Efficiency (%)
E /
M M
c
d
d
e
e
e
e
e
1
2
3
4
5
6
7
25.5
1.36
1.44
1.39
1.46
1.46
1.59
1.36
9
4.5
1.42
1.39
1.17
1.24
e
e
e
e
e
c
24.7
15.5
11.2
8.0
10
11
12
13
14
15
3.7
3.7
1.2
0
.1.60
.1.60
0.94
8.0
0
7.5
0
d
8
6.0
1.33
16
0
–
a
Yield of anisaldehyde referred to the substrate subtracted of the amount (2%) formed in the absence of mediator. Average of at least two determinations, the
error is in all cases ,^0.3.
V vs NHE, measured in aqueous solution.
Ref. 30.
Ref. 31.
b
c
d
e
p
E determined by cyclic voltammetry in aqueous solution pH 4.
Table 2. Mediation efficiency and redox potential of ring substituted
thioanisoles in the LiP-catalysed oxidation of 4-MMA
The kcat and K values were obtained following the product
M
a
þz
b
Mediator
Efficiency (%)
E /
M M
formation spectrophotometrically at the l value where the
largest difference in absorbance between the substrate and
the product is observed. The data obtained were then treated
according to the Michaelis–Menten kinetic scheme.
17
18
19
20
25.0
1.49
1.55
1.46
1.36
17.6
18.9
9.0
The kcat and KM values are reported in Table 3 where again
the mediation efficiencies are displayed.
2.4. Measurement of the half-life of the mediator radical
cations
We have measured the half-life (t1/2) of the radical cation of
the dimethoxylated aromatic compounds that displayed the
highest mediation efficiency (VA, 1,4-dimethoxybenzene,
2
1
5.4
1.30
a
Yield of anisaldehyde referred to the substrate subtracted of the amount
2%) formed in the absence of mediator. Average of at least two
determinations, the error is in all cases ,^0.3.
2-chloro-1,4-dimethoxybenzene and 3,4-dimethoxytoluene)
(
and, for comparison, the half-life of the radical cation
of 2,5-dimethoxybenzylalcohol which displayed only a
moderate mediation efficiency.
b
p
E (V vs NHE) determined by cyclic voltammetry in aqueous solution pH
4
.

8090
E. Baciocchi et al. / Tetrahedron 58 (2002) 8087–8093
M
Table 3. Mediation efficiency in the LiP-catalysed oxidation of 4-MMA and kcat and K values in the LiP-catalysed oxidation of aromatic mediators
a
b
Mediator
Efficiency (%)
k
cat
K
M
kcat/K
M
1
5
25.5
15.9
17.8
0.32
0.44
50
8.0
7.5
40
25
7
10.0
0.40
a
Yield of anisaldehyde referred to the substrate subtracted of the amount (2%) formed in the absence of mediator. Average of at least two determinations, the
error is in all cases ,^0.3.
Average of three determinations, the error is in all cases ,^2.
b
The radical cation has been generated in acidic aqueous
solution (6.5% HClO ) by oxidation with cerium(IV)
VA, is the species which displays the greatest mediation
efficiency. However, an efficiency comparable to that of VA
is also observed for some other mediators (i.e. thioanisole).
4
ammonium nitrate (CAN). Its decay has been followed in
the region around 410–470 nm (where the studied radical
cations exhibit a maximum of the absorbance) by a stopped-
flow apparatus. For all the studied compounds, the half-life
of the radical cations was determined as the time required to
halve the initial absorbance. The radical cation half-lives are
reported in Table 4.
From the data reported in Table 1, we can observe that the
þz
redox potential of the couple M /M (Table 1, M¼mediator)
is an important factor in determining the mediation
efficiency. When a series of structurally related substrates
is considered, we find that the mediation efficiency first
increases and then decreases on increasing the E
þz
p M /M
value. This trend can be easily seen in the series of 2-X-1,
-dimethoxybenzenes (Fig. 1) and of ring substituted
thioanisoles (Fig. 2).
4
3. Discussion
The results reported in Tables 1 and 2 indicate that most of
the molecules tested can act as mediators in the oxidation of
A bell shaped curve is observed in both cases with a
maximum efficiency at around 1.4–1.5 V. This result lends
further support to the hypothesis of a redox mechanism for
the mediation of LiP-induced oxidations (see above).
Accordingly, the observed trend is exactly the one expected
for such a mechanism (Scheme 1) when two contrasting
factors must be at play: the mediator must have a redox
potential sufficiently low to allow its oxidation by both LiP I
and LiP II, but at the same time it should be high enough in
order to promote the oxidative fragmentation of 4-MMA.
4
-MMA. Interestingly, it appears that the natural mediator,
Table 4. Mediation efficiency in the LiP-catalysed oxidation of 4-MMA
and lifetime of radical cations of dimethoxylated aromatic compounds
a
b
Mediator
Efficiency (%)
25.5
Half-life (s)
c
1
0.04
Even though the oxidation potential seems to play the major
role, with respect to mediation, some influence is also
exerted by other factors. Thus, the presence of an alcoholic
group has a beneficial effect on the mediation efficiency as
revealed by the fact that VA (1) is a much better mediator
than its corresponding methyl ether (7), the two compounds
2
3
24.7
15.5
8.3
2.1
exhibiting the same E þz value. Moreover, the mediation
p M /M
efficiency decreases regularly on going from primary to
secondary and tertiary alcoholic group (compare VA with
4
11.2
6.0
0.25
0.22
1-(3,4-dimethoxyphenyl)ethanol, (5) and 2-(3,4-dimethoxy-
phenyl)-2-propanol) 16; the absence of mediation with the
latter compound is probably due to the steric hindrance of
the side-chain.
8
a
Yield of anisaldehyde referred to the substrate subtracted of the amount
2%) formed in the absence of mediator. Average of at least two
determinations, the error is in all cases ,^0.3.
In the search for other properties which, in addition to the
oxidation potential, can influence the mediation efficiency,
we have investigated the role of the mediator capacity to
interact with the enzyme and therefore of the kinetic
parameters kcat and K . This investigation, however, was
M
only possible for the series of the three mediators VA, VA
(
b
c
The error is ^0.02 s for entries 1, 4, 8 and ^ 0.1 s for entries 2 and 3.
Ref. 32. The same value has been measured by Candeias and Harvey by
29
pulse radiolysis. The much shorter half-life (about 0.6 ms) measured by
is probably wrong.
33,34
Khindaria et al. by ESR,

E. Baciocchi et al. / Tetrahedron 58 (2002) 8087–8093
8091
Figure 1. Mediation efficiency vs redox potential of 2-X-1,4-dimethoxybenzenes in the LiP-catalysed oxidation of 4-MMA.
Figure 2. Mediation efficiency vs redox potential of ring substituted thioanisoles in the LiP-catalysed oxidation of 4-MMA.
methyl ether (7) and 1-(3,4-dimethoxyphenyl)ethanol (5),
whose enzymatic oxidation led to the formation of a single
product that could be monitored spectrophotometrically.
Nevertheless, from the data reported in Table 3, it would
seem that, whereas the oxidation potential remains almost
constant, the mediation efficiency increases as the kinetic
from 1.33 to 1.46 V, we have tested the role with respect to
the mediation efficiency of the half-life of the mediator
radical cation. The results reported in Table 4 show that
there is no apparent relation between the mediation
efficiency and the radical cation half-life. Indeed VA and
2-chloro-1,4-dimethoxybenzene, the best mediators, have
respectively the shortest (40 ms) and the longest (8.3 s) half-
lives, the other radical cations exhibiting intermediate
values and having lower mediation efficiencies. This finding
is of interest as it might support the suggestion that the
‡
effectiveness (kcat/K ) becomes higher. Accordingly,
M
VA, which is the best mediator among the substrates
analysed has the highest kinetic effectiveness (k /K ¼
cat
M
2
1
21
5
0 s mM ), while its methyl ether (7) (the worst
mediator) has the lowest kinetic effectiveness (k /K ¼
cat
M
2
1
21
25 s mM ). 1-(3,4-Dimethoxyphenyl)ethanol (5) shows
intermediate values of both mediation efficiency and kinetic
‡
Stopped-flow measurements have been carried out in a more acidic
medium (where the radical cations are expected to be more stable) than
the cyclic voltammetry measurements; however, we assume that the
2
1
21
effectiveness (k /K ¼40 s mM ).
cat
M
p
relative ordering of half-life and E values should be the same under these
Moreover, for a series of mediators with an E value ranging
p
conditions.

8092
E. Baciocchi et al. / Tetrahedron 58 (2002) 8087–8093
mediation is carried out by a complex between the enzyme
and the mediator radical cation, as suggested by several
accomplished on a Hewlett–Packard 1050 liquid chromato-
graph fitted with a UV–vis detector HP79853A and a
Supelcosil LC18 column (25 cm£4.6 mm). GC analyses
were carried out on a Varian 3400 GC (OV1 capillary
column, 25 m£0.2 mm). UV–vis measurement was per-
formed on a Perkin–Elmer Lambda 18 spectrophotometer.
For the cyclic voltammetry measurements, an Amel 5000
potentiostat was used, the cell was fitted with a glassy
carbon working electrode (f 3 mm) in combination with an
aqueous SCE reference electrode.
3
3–35
groups.
The half-life of such a complex might be
different (and probably longer than that of the ‘free’
mediator radical cation).
An additional observation is that the radical cations in Table
4
display very different half-lives in spite of their similar
thermodynamic stability as expressed by the Ep Mþz/M
values. This is likely due to the different reaction paths
which are available to these intermediates. Thus, the radical
cations of VA, 3,4-dimethoxytoluene (4) and 2,5-dimethoxy-
benzyl alcohol (8) are short lived as they can undergo a
relatively fast deprotonation from the benzylic position
while longer lifetimes are observed for the radical cations
of 2-chloro-1,4-dimethoxybenzene (2) and 1,4-dimethoxy-
benzene (3) which do not contain benzylic hydrogens and
probably decay by different processes like dimerization,
disproportionation or reaction with nucleophilic species.
5.2. Substrates and reagents
All the reagents and solvents were of the highest purity
available and used without further purification (unless
otherwise specified). CAN (Aldrich) was dried at 858C for
1 h. The concentration of H O (Carlo Erba Reagents) was
2
2
3
7
determined by titration with permanganate. LiP was
3
8
prepared and purified as described in the literature. The
concentration of the enzyme solution was determined
21
21 39
Finally, in view of the fact that recent work has suggested
that a tryptophan residue may be responsible for the electron
spectrophotometrically (1409 nm¼169 mM cm ).
40
3
6
exchange between the enzyme and VA, we have also
determined the mediation efficiency of tryptophan. How-
ever, this compound does not exhibit a significant mediation
efficiency almost certainly because of its low redox
potential (1.09 V vs NHE). In this respect, it would be of
interest to test tryptophan derivatives with an oxidation
potential of ca. 1.4 V. Work in this direction is under way in
our laboratory.
1-(3,4-Dimethoxy-phenyl)-ethanol (5), 2,5-dimethoxy-
4
1
42
benzenesulfonic acid (6), VA methyl ether (7), 2-(3,4-di-
4
3
methoxy-phenyl)-propan-2-ol
4
and 1,2-dimethoxy-4-
methylthiobenzene were synthesised according to the
literature.
4
5.3. Mediation efficiency
The substrate, 4-MMA, (0.25 mmol), LiP (0.51 nmol, 0.3 U)
and 4% of mediator (0.01 mmol) were magnetically stirred
in 1 mL of 50 mM sodium tartrate buffer solution, pH 3, at
4. Conclusions
378C, with 3% CH CN as cosolvent, under an Argon
3
On the basis of the results gathered in this work, several
conclusions about the efficiency of a given substrate in the
mediation of the LiP-catalysed oxidation of 4-MMA are
possible. The redox potential of the mediator is probably the
most important factor. Accordingly, in the structurally
related series examined, the highest mediation efficiency
has been reached when the Ep Mþz/M value is 1.4–1.5 V. The
atmosphere. The oxidant, H O (0.06 mmol) was added.
2 2
After 10 min, the internal standard (4-methoxyaceto-
phenone) was added and the products of the reaction were
determined by HPLC on a reversed-phase column
(Supelcosil LC18) using MeOH/H O (1/1 v/v) as the mobile
2
phase. A good recovery of material was observed (.95%)
in all the experiments.
influence of oxidation potential on the mediation efficiency
clearly supports the hypothesis of a redox mediation in LiP
induced oxidations. Another factor which appears to play
some role with respect to the mediation efficiency is the
5.4. Product analysis study
The mediator (10 mmol) and LiP (0.6 units, 1.02 nmol) were
magnetically stirred in 5 mL of 50 mM sodium tartrate
buffer solution, pH 3 containing 3% of acetonitrile at 378C
and under an Argon atmosphere. H
of buffer solution was added. After 10 min, the products of
the reaction were extracted with CH Cl , dried over Na SO
4
and characterised by GC, GC–MS, H NMR and UV–vis.
The mediators that yielded a single product were VA and
VA-methyl ether (veratraldehyde) and 1-(3,4-dimethoxy-
phenyl)ethanol (1-(3,4-dimethoxyphenyl)ethanone).
kinetic effectiveness as determined by the k /KM values,
cat
the best mediator displaying the highest kinetic effective-
ness. In contrast, the mediation efficiency is not influenced
by the lifetime of the free mediator radical cation. This
observation might support the suggestion that the actual
redox mediation is performed by a mediator–enzyme
complex with a lifetime different (and probably longer)
than that of the free mediator radical cations.
O (10 mmol) in 0.5 mL
2 2
2
2
2
1
5. Experimental
5.5. Steady-state kinetics
5
.1. Instrumentation
For VA, VA methyl ether and 1-(3,4-dimethoxyphenyl)-
ethanol H O (0.40 mmol) in 0.1 mL of buffer solution was
added in a cuvette to 2.8 mL of 50 mM sodium tartrate
buffer solution, pH 3.0, with 10% acetonitrile as cosolvent,
containing the mediator (0.04–1.20 mmol) and LiP (0.42
units, 0.71 nmol), thermostated at 258C. The formation of
2
2
1
H NMR spectra were recorded on a Bruker AC300P
spectrometer in CDCl . GC–MS analyses were performed
3
on a HP5890 GC (OV1 capillary column, 12 m£0.2 mm)
coupled with a HP5970 MSD. HPLC analyses were

E. Baciocchi et al. / Tetrahedron 58 (2002) 8087–8093
8093
the products was followed spectrophotometrically at a l
value where is the maximum difference in absorbance
between the substrate and the corresponding product
10. Renganathan, V.; Miki, K.; Gold, M. H. Arch. Biochem.
Biophys. 1986, 246, 155.
11. Harvey, P. J.; Floris, R.; Lundell, T.; Palmer, J.; Schoemaker,
H. E.; Wever, R. Biochem. Soc. Trans. 1992, 20, 345–349.
12. Schoemaker, H. E. Recl. Trav. Chim. Pay-Bas. 1990, 109, 255.
13. Haemmerli, S. D.; Leisola, M. S. A.; Sanglard, D.; Fiechter, A.
J. Biol. Chem. 1986, 261, 6900.
(
310 nm for VA and its methyl ether, 302 nm for 1-(3,4-di-
methoxyphenyl)ethanol). The kinetic parameters kcat and
KM were obtained from Lineweaver–Burke plot of the
data).
1
4. Joshi, D. K.; Gold, M. H. Biochemistry 1994, 33, 10969.
5
.6. Measurement of the mediator redox potential
15. Kersten, P. J.; Kalyanaraman, B.; Hammel, K. E.;
Reinhammer, B.; Kirk, T. K. Biochem. J. 1990, 268, 475.
16. Reddy, G. V. B.; Gelpke, M. D. S.; Gold, M. H. J. Bacteriol.
1998, 180, 5159.
The oxidation potential was determined by cyclic volt-
ammetry at 258C in argon degassed 10 mM sodium acetate
buffer solution, pH 4.0, with 3% CH CN as cosolvent,
3
containing LiClO (0.12 M) as the supporting electrolyte
4
17. Dunford, H. B. Heme Peroxidases; Wiley: New York, 1999.
18. Lundquist, K.; Kirk, T. K. Phytochemistry 1978, 17, 1676.
19. Harvey, P. J.; Schoemaker, H. E.; Palmer, J. M. FEBS Lett.
1986, 195, 242.
and the substrate (0.10 mmol). The cell was fitted with a
glassy carbon working electrode (f 3 mm) in combination
with an aqueous SCE reference electrode. The waves were
20. Goodwin, D. C.; Aust, S. D.; Grover, T. A. Biochemistry 1995,
34, 5060.
2
1
irreversible even at a 500 mV s sweep rate, so that only Ep
values are given.
21. Haemmerli, S. D.; Leisola, M. S. A.; Fiechter, A. FEMS
Microbiol. Lett. 1986, 35, 33.
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4
70 nm (where the studied radical cations exhibit a
maximum of the absorbance). The filter time constant
used varied in the range 1–100 ms. The apparatus was
thermostated at 258C.
28. Teunissen, P. J.; Sheng, M. D.; Reddy, G. V. B.; Mo e¨ nne-
Loccoz, P.; Field, J. A.; Gold, M. H. Arch. Biochem. Biophys.
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2
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According to Einstein–Smoluschowski equation of diffusion a
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Acknowledgements
30. Bietti, M.; Baciocchi, E.; Steenken, S. J. Phys. Chem. A. 1998,
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31. Candeias, L. P. Unpublished results.
1
This work was carried out into the framework of the
EU project ‘Towards Efficient Oxygen Delignification’
(Contract No. QLK5-CT-1999-01277). The authors also
thank Dr Patricia J. Harvey for providing a sample of LiP.
32. Baciocchi, E.; Bietti, M.; Gerini, M. F.; Lanzalunga, O.
Biochem. Biophys. Res. Commun. 2002, 293, 832.
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6. Doyle, W. A.; Blodig, W.; Veitch, N. C.; Piontek, K.; Smith,
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