Kinetic deuterium isotope effect in the oxidation of veratryl alcohol promoted by lignin peroxidase and chemical oxidants

Article abstract of DOI:10.1039/b104467m

The intramolecular kinetic deuterium isotope effects (k_H/k_D = 4.6-4.9) determined in the H₂O₂-induced oxidation of the racemic and enantiomeric forms of α-monodeuterated veratryl alcohol catalysed by lignin peroxidase (LiP) are very similar to those determined in the oxidation of racemic α-monodeuterated veratryl alcohol promoted either by a LiP model compound (a water soluble iron porphyrin using m-chloroperbenzoic acid as the oxidant) (k_H/k_D = 4.2) or by potassium 12-tungstocobalt(III)ate, a genuine one-electron oxidant (k_H/k_D = 4.5). These results indicate that very likely veratryl alcohol radical cation, once generated by the LiP-H₂O₂ system, is released from the enzyme and is deprotonated by the medium.

Full text of DOI:10.1039/b104467m

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Kinetic deuterium isotope effect in the oxidation of veratryl
alcohol promoted by lignin peroxidase and chemical oxidants
Enrico Baciocchi,*a Maria Francesca Gerini,b Patricia J. Harvey,c Osvaldo Lanzalungab and
Alma Prosperib
2
a Dipartimento di Chimica, Università “La Sapienza”, P.le A. Moro, 5 I-00185 Rome, Italy.
E-mail: baciocchi@uniroma1.it; Fax: ϩ39 (06) 490421
b Dipartimento di Chimica and Centro CNR di Studio sui Meccanismi di Reazione,
Università “La Sapienza”, P.le A. Moro, 5 I-00185 Rome, Italy
c School of Chemical and Life Sciences, University of Greenwich, Wellington St., Woolwich,
London, UK SE18 6PF
Received (in Cambridge, UK) 21st May 2001, Accepted 23rd July 2001
First published as an Advance Article on the web 14th August 2001
The intramolecular kinetic deuterium isotope effects (kH/kD = 4.6–4.9) determined in the H2O2-induced oxidation of
the racemic and enantiomeric forms of α-monodeuterated veratryl alcohol catalysed by lignin peroxidase (LiP) are
very similar to those determined in the oxidation of racemic α-monodeuterated veratryl alcohol promoted either by a
LiP model compound (a water soluble iron porphyrin using m-chloroperbenzoic acid as the oxidant) (kH/kD = 4.2) or
by potassium 12-tungstocobalt()ate, a genuine one-electron oxidant (kH/kD = 4.5). These results indicate that very
likely veratryl alcohol radical cation, once generated by the LiP–H2O2 system, is released from the enzyme and is
deprotonated by the medium.
of the detailed mechanism of the oxidation of VA catalysed by
Introduction
LiP is certainly essential to this discussion.
Lignin peroxidase (LiP, EC 1.11.1.7) is an extracellular heme
There is wide consensus that the oxidation of VA involves the
enzyme isolated from ligninolytic cultures of the white-rot
transfer of one electron to LiP I leading to the formation of
basidiomycetous fungus Phanerochaete chrysosporium.1,2 The
VAϩ and the reduced form of the oxo complex, compound
ؒ
importance of this enzyme is related to lignin degradation
which is a fundamental process in the pulp and paper industry.
Accordingly LiP catalyses the oxidative depolymerization of
dilute solutions of polymeric lignin in vitro.3 LiP is also able to
promote the H2O2-dependant oxidation of several classes of
compounds characterized by relatively high redox potential
(< 1.4 V).4–15 The catalytic cycle of LiP involves the initial reac-
tion of the ferric heme in the native state with H2O2 to yield
compound I, the active enzymatic species, an oxy-ferryl heme
II (eqn. 4).21,22 Subsequently VAϩ undergoes Cα-H deproton-
ؒ
(4)
porphyrin π-cation radical (Pϩ Fe()᎐O) (eqn. 1), then the
ؒ
᎐
(1)
ation,11 a typical reaction of alkylaromatic radical cations,23,24
to form a carbon centred radical which is eventually converted
to veratryl aldehyde, VAld, (eqn. 5).25
resting state of the enzyme is regenerated after two single
electron oxidations of substrates with formation of the inter-
mediate compound II, which retains the oxy-ferryl heme
(PFe()᎐O) (eqn. 2–3).
᎐
(5)
(2)
P-Fe(IV)᎐O ϩ S ϩ 2 Hϩ
P-Fe(III) ϩ Sϩ ϩ H2O (3)
ؒ
᎐
The LiP-catalysed degradation of lignin appears to require
the presence of veratryl alcohol, 3,4-dimethoxybenzyl alcohol
(VA), a secondary metabolite of the fungus Phanerochaete
chrysosporium, as a cofactor.16–18 This observation has led to a
lively discussion about the actual role played by VA in this
respect and several hypotheses (redox mediation, protection of
the enzyme from irreversible inactivation by H2O2, completion
of the catalytic cycle) have been presented.17,19,20 The knowledge
However, it has not yet been clearly established whether such
deprotonation is an enzymatic process or if it occurs in the
medium after VAϩ has been released from the enzyme. The
ؒ
latter possibility was considered the most likely being consistent
with the proposal that VA may act as a redox mediator in
LiP-catalysed oxidations.17,27,10 However, the finding that in the
LiP-catalysed oxidation of VA the formation of VAld is not
1512
J. Chem. Soc., Perkin Trans. 2, 2001, 1512–1515
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b104467m

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Table
1
Intramolecular deuterium kinetic isotope effect in the
oxidation of [α-2H1]veratryl alcohols catalysed by LiP, FeTPPSCl or
Co()Wa
b
Substrate
Oxidizing system
kH/kD
(R)-VA-d
(S)-VA-d
( )-VA-d
( )-VA-d
( )-VA-d
LiP–H2O2
LiP–H2O2
LiP–H2O2
FeTPPSCl–MCPBA
Co()W
4.6
4.9
4.7
4.2
4.5
a For the reaction conditions see the Experimental section. b Deter-
mined via GC-MS by the ratio between the corrected signal intensities
of the two molecular ions at m/z 167 and 166. Average of at least three
determinations, the error is in all cases ca. 0.2.
Fig. 1 Metalloporphyrin 5,10,15,20-tetraphenyl-21H,23H-porphine-
p,pЈ,pЉ,pٞ-tetrasulfonic acid iron() chloride.
were the only reaction products with the enzymatic25 and the
chemical oxidizing systems so indicating that deprotonation is
accompanied by that of dimeric products28 has suggested that
the only observed route for VAϩ . No products were formed
ؒ
there should be no significant diffusion of VAϩ into the reac-
ؒ
when the oxidation was carried out in the absence of the
oxidant [H2O2 or MCPBA or Co()W] or in the absence of LiP.
The intramolecular KDIE (kH/kD) values were calculated via
GC-MS analysis by the ratios of the intensities of their molecu-
lar ion peaks (166 and 167, respectively) corrected for the 13C
contribution. In order to exclude any equilibration process for
tion medium and that therefore its deprotonation probably
occurs while the radical cation is still bound to the enzyme.
Moreover, it has recently been proposed that once VAϩ is
ؒ
formed by LiP compound I, a strong VAϩ –LiP compound II
ؒ
complex is created which should be the species playing the key
role in the redox mediation process.29 Return of this complex to
VAϩ , which might affect the interpretation of our results it
ؒ
the native state may release VAϩ to the medium, however, the
ؒ
was verified that in the enzymatic oxidation of racemic VA-d
the unreacted substrate had the same deuterium content of
the starting material and that there was no enantiomeric
enrichment. The intramolecular KDIE values obtained in the
enzymatic and chemical reactions are reported in Table 1.
Inspection of the data in Table 1 immediately reveals that the
values of KDIE for the enzymatic and chemical oxidations are
very close to one another, the differences being practically
within the experimental error. This observation immediately
possibility that deprotonation takes place in the VAϩ –LiP II
ؒ
complex must be considered.29–31 On the other hand, very little
is known about the VA binding site which might be close to the
heme-edge32–34 or also, as recently proposed, on the LiP surface
in proximity of a tryptophan residue (Trp 171).35,36
In this context, we have felt that a better insight in this mech-
anistic problem might be obtained by specifically investigating
the deprotonation step, and namely one of its most impor-
tant properties, the kinetic deuterium isotope effect (KDIE).
Thus, we have determined the intramolecular KDIE of the
LiP-catalysed oxidation of both (S) and (R) forms of the
α-monodeuterated veratryl alcohol [(S)-VA-d and (R)-VA-d]
promoted by H2O2. The results of the LiP-catalysed reactions
have been compared with those obtained in the oxidation
of racemic α-monodeuterated veratryl alcohol [( )-VA-d]
promoted by potassium dodecatungstocobalt()ate, K5Co()-
W12O40, [Co()W] a genuine outer-sphere one-electron chem-
ical oxidant37,38 and by m-chloroperbenzoic acid (MCPBA) in
the presence of the water soluble metalloporphyrin 5,10,
15,20-tetraphenyl-21H,23H-porphine-p,pЈ,pЉ,pٞ-tetrasulfonic
acid iron() chloride (FeTPPSCl) (Fig. 1). The latter system is
expected to mimic the LiP reactivity,39 since the active species,
formed by reaction of FeTPPSCl with MCPBA can be
described as an iron()-oxo porphyrin radical cation and
closely resembles LiP I, the active species of LiP.
suggests that VAϩ deprotonation is very likely promoted by the
ؒ
same basic species in the oxidation catalysed by LiP and in
those promoted by FeTPPSCl and Co()W. The most reason-
able hypothesis is that this base is in all cases the reaction
medium and that therefore deprotonation of VAϩ generated by
ؒ
LiP is not an enzymatic reaction. In line with this conclusion is
also the observation that almost identical kH/kD are found in the
LiP-induced oxidations of (R)-VA-d and (S)-VA-d. Clearly, the
relative reactivity of the Cα-H and Cα-D bonds is the same
either when we compare the pro-S hydrogen with the pro-R
deuterium or vice versa, indicating that in the VAϩ deproton-
ؒ
ation process there is no differentiation between the two
enantiotopic hydrogens of the CH2OH group. This finding is
also consistent with a deprotonation reaction which does not
involve a veratryl alcohol radical cation still bound to the
enzyme. In this case, the two enantiomeric radical cations
should form two diastereomeric complexes and diastereomeric
transition states are expected for the cleavage reactions of the
prochiral hydrogens. This should lead to somewhat different
values of kH/kD for the (R) and (S) forms of VA-d, which is not
observed.
The KDIE value is expected to be significantly influenced by
the nature of the proton abstracting base. Thus, if the deproton-
ation of VAϩ in the LiP-catalysed oxidation is promoted by the
ؒ
enzyme, it is very likely that the KDIE value is different from
those determined in the chemical ([FeTPPSCl–MCPBA] or
[Co()W]) reactions. Moreover, different properties of the two
enantiomeric alcohols with respect to the KDIE value can also
Summing up, the present results allow us to conclude that a
classical enzymatic pathway for the deprotonation of veratryl
alcohol radical cation in the LiP promoted oxidation of VA is
be expected, due to the enzyme chirality.40,41 Conversely, if VAϩ
ؒ
very unlikely; even though the formation of a VAϩ –LiP II
ؒ
complex takes place, as suggested,29 such a complex should not
is deprotonated as a free radical cation in the medium no
significant differences in the KDIE values between chemical
and enzymatic reactions, as well as between the (R) and (S)
enantiomers in the enzymatic reaction, are predicted.
play any significant role in the VAϩ deprotonation process.
ؒ
This conclusion may also lend support to the suggestion that
VA binds to Trp 171 on the enzyme surface rather than to the
heme-edge.35,36 Accordingly, in the former case a fast release
from the enzyme to the medium of VAϩ , once it is formed by
ؒ
Results and discussion
a long range electron transfer, is a very reasonable possibility.
It should finally be noted that the similar kH/kD values for
the LiP and FeTPPSCl catalysed oxidation of VA confirm
the capacity of the iron porphyrin to efficiently mimic the
enzymatic reaction.
All the oxidations were carried out at pH 4 (50 mM sodium
tartrate buffer), at 25 ЊC, using equimolar amounts of the
oxidant and the substrate, under an argon atmosphere. As
expected, veratryl aldehyde and α-deuterated veratryl aldehyde
J. Chem. Soc., Perkin Trans. 2, 2001, 1512–1515
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ually added in 1 h using a syringe pump. The products of the
reaction were extracted with CH2Cl2 and dried over anhydrous
Na2SO4. The yields in VAld, referred to the starting material,
which is equimolecular with the oxidant, were as follows:
(R)-VA-d (13%), (S)-VA-d (16%), ( )-VA-d (16%).52
In the oxidation of ( )-VA-d the deuterium content of the
unreacted substrate was the same (>99%) as that of the starting
material with no enantiomeric enrichment.
Experimental
Methods
1H-NMR spectra were recorded on
a Bruker AC300P
spectrometer in CDCl3. GC-MS analyses were performed on an
HP5890 GC (OV1 capillary column, 12 m × 0.2 mm) coupled
with an HP5970 MSD. GC analyses were performed on a
Varian 3400 GC (OV1 capillary column, 25 m × 0.2 mm).
Biomimetic oxidation. The oxidant MCPBA (18 µmol) was
added to a magnetically stirred argon-degassed solution of the
substrate (18 µmol) and FeTPPSCl (0.54 µmol) in 5 mL of
50 mM sodium tartrate buffer solution, pH = 4.0, at 25 ЊC.
After 1 h, a saturated solution of NaHCO3 was added to the
reaction mixture which was extracted with CH2Cl2 and dried
over anhydrous Na2SO4. The yield in VAld, referred to the start-
ing material, which is equimolecular with the oxidant was 12%.
Substrates and reagents
All the reagents and solvents were of the highest purity
available and used without further purification (unless other-
wise specified). The concentration of H2O2 (Carlo Erba
Reagents) was determined by titration with permanganate.42
MCPBA was purified by washing it with phosphate buffer and
assayed by iodometric titration.43 5,10,15,20-Tetraphenyl-
21H,23H-porphine-p,pЈ,pЉ,pٞ-tetrasulfonic acid tetrasodium
salt dodecahydrate (Aldrich) was metallated according to the
literature procedure.44 Co()W was prepared using the liter-
ature procedure37 with some modifications.38 LiP was prepared
and purified as described in the literature.45 The concentration
of the enzyme solution was determined spectrophotometrically
(ε409nm = 169 mMϪ1 cmϪ1).46
Chemical oxidation. The alcohol (18 µmol) and the oxidant
(18 µmol) were magnetically stirred for 4 h, at 25 ЊC in 5 mL
of argon-degassed 50 mM sodium tartrate buffer solution,
pH = 4.0. The products of the reaction were extracted with
CH2Cl2 and dried over anhydrous Na2SO4. The yield in VAld,
referred to the starting material, which is equimolecular with
the oxidant was 37%.
α-Deuterated veratryl aldehyde was prepared by oxidation
of [α-2H2]veratryl alcohol (from 3,4-dimethoxybenzoic acid
and LiAlD447), with pyridinium chlorochromate (PCC) in
anhydrous CH2Cl2.48
Acknowledgements
(R)-[α-2H1]Veratryl alcohol [(R)-VA-d] and (S)-[α-2H1]-
veratryl alcohol [(S)-VA-d] were synthesized by the asym-
metric reduction of α-deuterated veratryl aldehyde with
B-(3α-pinanyl)-9-borabicyclo[3.3.1]nonanes[obtainedbyhydro-
boration of enantiomerically pure (S)-α-pinene and (R)-α-
pinene, respectively, with 9-borabicyclo[3.3.1]nonane (9-BBN)]
according to a literature procedure.49,50
E. B., M. F. G., O. L. and A. P. thank the Università degli Studi
di Roma “La Sapienza” and the Ministry for the University and
Scientific and Technological Research (MURST) for financial
support.
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J. Chem. Soc., Perkin Trans. 2, 2001, 1512–1515
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