Enzymatic oxidation of manganese ions catalysed by laccase

Article abstract of DOI:10.1016/j.bioorg.2008.09.002

The principal possibility of enzymatic oxidation of manganese ions by fungal Trametes hirsuta laccase in the presence of oxalate and tartrate ions, whereas not for plant Rhus vernicifera laccase, was demonstrated. Detailed kinetic studies of the oxidation of different enzyme substrates along with oxygen reduction by the enzymes show that in air-saturated solutions the rate of oxygen reduction by the T2/T3 cluster of laccases is fast enough not to be a readily noticeable contribution to the overall turnover rate. Indeed, the limiting step of the oxidation of high-redox potential compounds, such as chelated manganese ions, is the electron transfer from the electron donor to the T1 site of the fungal laccase.

Full text of DOI:10.1016/j.bioorg.2008.09.002

Bioorganic Chemistry 37 (2009) 1–5
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Preliminary Communication
Enzymatic oxidation of manganese ions catalysed by laccase
Marina Gorbacheva , Olga Morozova , Galina Shumakovich , Alexander Streltsov , Sergey Shleev a,b,*,
a
a
a
a
a
Alexander Yaropolov
a
Laboratory of Chemical Enzymology, A.N. Bach Institute of Biochemistry RAS, 119071 Moscow, Russia
Department of Biomedical Laboratory Science, Faculty of Health and Society, Malmö University, 20506 Malmö, Sweden
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2008
Available online 30 October 2008
The principal possibility of enzymatic oxidation of manganese ions by fungal Trametes hirsuta laccase in
the presence of oxalate and tartrate ions, whereas not for plant Rhus vernicifera laccase, was demon-
strated. Detailed kinetic studies of the oxidation of different enzyme substrates along with oxygen reduc-
tion by the enzymes show that in air-saturated solutions the rate of oxygen reduction by the T2/T3
cluster of laccases is fast enough not to be a readily noticeable contribution to the overall turnover rate.
Indeed, the limiting step of the oxidation of high-redox potential compounds, such as chelated manga-
nese ions, is the electron transfer from the electron donor to the T1 site of the fungal laccase.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords:
Laccase
Plant
Fungal
Manganese
T1 site
T2/T3 cluster
Biocatalysis
1
. Introduction
for the formation of insoluble manganese oxides by enzymatic
2
+
Mn oxidation [8]. Nevertheless, it is a well-known fact that
the substrate specificity of laccases depends on the structure
and the redox potential of the T1 site (ET1), the first electron
acceptor of the enzyme [9]. Depending on ET1 laccases can also
be classified as low- and high-redox potential enzymes [10–
12]. It is believed that only compounds with ionisation poten-
tials below ET1 can be efficiently oxidised by laccase [13]. In
Laccase (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) be-
longs to the diverse group of blue multicopper oxidases and catal-
yses one-electron oxidation of different organic substrates, as well
as some coordination compounds, coupled to the four-electron
reduction of oxygen (O
2 2
) directly to water (H O), i.e. without for-
mation of reactive oxygen species such as hydrogen peroxide
ꢀ
ꢁÅ
2+
3+
(
3
H
2
O
2
), hydroxyl radical (OH ), and superoxide radical (O ) [1–
spite of the very high-redox potential of the Mn /Mn couple
2
2
+
]. The active centre of laccase contains four copper ions classified
into three types: a mononuclear blue copper ion comprising the T1
site and three copper ions forming the trinuclear copper cluster
equal to 1510 mV [14], Mn can be quite efficiently oxidised
by both bacterial (low-redox potential enzymes [15,16]) and fun-
gal (high-redox potential enzymes [11,17]) laccases [8,18]. A
possible physiological role of laccase catalysed Mn2+ oxidation
(
T2/T3 cluster), containing one ion of type 2 (T2 site) and two cop-
per ions of type 3 (T3 site) [2,4].
2 2
is to provide H O for extracellular reactions performed by lig-
Based on the source, laccases can be classified as plant, fun-
gal, bacterial, or insect enzymes [2]. It is generally accepted that
plant laccases participate in free-radial reactions during lignin
synthesis, whereas fungal enzymes have much broader function,
e.g. they participate in lignin degradation, in maturity and mor-
phogenesis of fungi, and in pathogenesis and detoxification pro-
cesses [2,5,6]. It should be mentioned, however, that not all roles
of laccase in different living organisms are yet fully clarified
ninolytic enzymes suggesting a novel type of laccase-peroxidases
cooperation relevant to biodegradation of lignin [19]. Moreover,
2
+
enzymatic Mn oxidation might play an important role in the
function of the ligninolytic complex of wood-degrading fungi
3
+
since Mn is a strong oxidant and can oxidise some non-pheno-
lic substructures of lignin, e.g. guaiacyl (veratryl alcohol and
1-(3-methoxy-4-isopropoxyphenyl)ethanol) and syringyl (3,4,5-
trimethoxybenzyl alcohol and 1-(3,5-dimethoxy-4-isopropoxy-
phenyl)ethanol) model lignin compounds [18–20]. The
[
3,7]. For example, it has been unambiguously demonstrated
2
+
only recently that laccase-like bacterial enzymes are responsible
mechanism of the oxidation of Mn by laccase, however, is still
not understood in detail. Furthermore, a possible interaction of
2
+
plant laccases with Mn has not been studied so far. Thus, the
main objective of this work was to enhance the understanding
*
Corresponding author. Address: Department of Biomedical Laboratory Science,
Faculty of Health and Society, Malmö University, 20506 Malmö, Sweden. Fax: +46
2
+
of a possible enzymatic oxidation of Mn catalysed by plant
and fungal laccases.
4
0
0 665 8100.
E-mail address: sergey.shleev@mah.se (S. Shleev).
045-2068/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2008.09.002

2
M. Gorbacheva et al. / Bioorganic Chemistry 37 (2009) 1–5
2ꢁ
Å
2
. Experimental
2.4. Measurements of H
O
2 2
and O production
2ꢁ
Å
2.1. Materials
The formation of H
2
O
2
and O
were measured spectrophoto-
metrically using a Hitachi-557 spectrophotometer. In the case of
2 2
H O measurements a slightly modified procedure described in
0
Boric acid, sodium tartrate, 2,2 -azinobis-3-ethylbenzthiazo-
line-6-sulphonate (ABTS), nitroblue tetrazolium (NBT), KCl, and
[Mo(CN) ] were purchased from Sigma–Aldrich Chemie GmbH
Germany). Na HPO , NaOH, KOH, citric acid, 3,4-dimethoxybenzyl
alcohol (veratryl alcohol), and 3,4-dimethoxybenzaldehyde (verat-
ryl aldehyde) were from Merck (Germany). K [Fe(CN) ]—Alfa Aesar
Germany). MnSO , C , and H PO —ChemMed (Russia). All
solutions were prepared using water purified with Milli-Q system
Millipore, USA).
our previous publication was used [23]. An aliquot was taken from
the reaction mixture during enzymatic oxidation of Mn2+ in the
presence of oxalate or tartrate ions, filtered through a 12 kDa
membrane filter (Millipore) to remove laccase, and placed into a
cuvette. Then, ABTS (9 mM) and HRP (0.5 mg/ml) were added as
electron donor and catalyst, respectively, giving a final volume of
1 ml. The concentration of oxidised ABTS was measured at
K
(
4
8
2
4
4
6
(
4
2
H
O
2 4
3
4
ꢁ1
ꢁ1
(
405 nm (
centration of H
e
405 = 36,800 M cm ), which corresponds to the con-
according to the following reaction:
2 2
O
2
ꢁ
þ 2Hþ ¼ ABTSꢁÅ þ 2H
ABTS þ H
2
O
2
2
O
2.2. Enzymes
The basidiomycete Trametes hirsuta (Wulfen) Pilát (Coriolus
The formation of O2 Å was confirmed by addition of nitroblue
ꢁ
hirsutus (Wulfex Fr.) Quél.), strain T. hirsuta 56, was obtained from
the laboratory collection of the Moscow State University of Engi-
neering Ecology (Russia). The basidiomycete was grown by sub-
merged cultivation [21] and laccase was isolated from the
culture medium as described previously [17]. Partly purified Rhus
vernicifera laccase from the latex of the lacquer tree was kindly
provided by Prof. B. Reinhammar (University of Göteborg, Sweden).
The final purification for both laccases was performed by means of
HPLC on a TSK DEAE-2SW column (LKB, Sweden) using a Stayer
HPLC system (Acvilon, Russia). The enzymes were homogeneous
as judged from SDS–PAGE. Homogeneous preparations of both en-
zymes were stored in 0.1 M phosphate buffer, pH 6.5, at ꢁ20 °C.
Horseradish peroxidase (HRP Type VI, 250–330 U/mg) was pur-
chased from Sigma–Aldrich Chemie GmbH and used without fur-
ther purification.
ꢁÅ
tetrazolium, a compound sensitive to O2 , to the reaction medium
already containing T. hirsuta laccase as catalyst.
2
.5. Cyclic voltammetry measurements
Cyclic voltammograms (CVs) were recorded in a three-electrode
electrochemical cell using a three-electrode potentiostat (BAS CV-
0W) and a one-compartment electrochemical cell with a total vol-
ume of 10 ml. A glassy carbon electrode (BAS), an Ag|AgCl|3 M NaCl
electrode (BAS, 210 mV vs. NHE), and a platinum wire (1 mm diam-
eter) served as working, reference, and counter electrodes, respec-
tively. The surface of the working electrode was polished with
5
alumina FF slurry (0.1 lm, Stuers, Copenhagen, Denmark), rinsed
with double-distilled water, and allowed to dry. CVs were recorded
in 0.1 M Na-tartrate buffer, pH 5.0 in the potential range of 0–
1
200 mV vs. NHE and scan rates varied from 10 up to 100 mV/s. All
2.3. Laccase assay and kinetics studies
potentials in the present work are given vs. NHE.
Oxidation of Mn to Mn3+ by purified laccases was monitored
2+
2
2
.6. Enzymatic oxidation of veratryl alcohol
spectrophotometrically for up to 2 h by following the formation
of Mn complexes, in 0.1 M Na-oxalate or 0.1 M Na-tartrate buffer,
pH 5.0. The dependence of laccase activity on pH was performed in
3
+
.6.1. Spectrophotometric assay
The initial velocity of veratryl aldehyde formation was mea-
3+
0
.1 M Na-oxalate buffer. The concentrations of Mn -oxalate
sured by the increase in 310 nm absorbance using
9
310
e =
ꢁ
1
ꢁ1
3+
(e
270 = 55,000 mM cm
)
and
Mn -tartrate
(e
280 = 6500
ꢁ1
ꢁ1
300 M cm [24].
ꢁ1
ꢁ1
mM cm ) were determined as described in [19,22]. All spectro-
photometric measurements were carried out in air-saturated solu-
tions at 20 °C using a Hitachi-557 spectrophotometer (Japan).
The determination of the standard biocatalytic rate constant
2
.6.2. HPLC studies
Analysis of veratryl alcohol oxidation products was performed by
reverse phase hydrophobic HPLC on Luna C18 column
a
M
(kcat) and the apparent Michaelis constant (K ) of both laccases
(
250 ꢂ 4.6 mm) (Phenomenex, USA) with a linear gradient of aceto-
towards transition metal complexes and the dependence of their
activities on pH were determined in universal Britton–Robinson
buffer (40 mM boric acid–40 mM acetic acid–40 mM phosphoric
acid adjusted to the necessary pH with 0.2 M NaOH) by estima-
tion of the initial rates of O
electrode at 20 °C with constant stirring using voltammetric ana-
ꢁ1
3 4
nitrile (5–95%) in 0.086% H PO at a 1 mL min flow rate using a
Stayer-system (Acvilon, Russia). The incubation mixture contained
1
0
cases or Mn missing. The sample preparation included ultrafiltra-
tion through PM-10 membrane in an Amicon cell (USA) to remove all
proteins followed by acidification of the filtrate with 0.086% H
ꢁ7
mM veratryl alcohol, 1 mM MnSO
4
, and 10 M of the laccases in
.1 M Na-oxalate buffer, pH 5.0. The control mixture had either lac-
consumption in a Clark oxygen
2+
2
lyser CV-50W (Bioanalytical systems, USA). The concentration of
3 4
PO
O
2
in the Britton–Robinson air-saturated buffer at 20 °C was as-
sumed to be 260 M. K -values of both laccases towards O
were also determined in universal Britton–Robinson buffer con-
taining 5 mM K Fe(CN) as electron donor using the Clark O
(
v/v). The products were identified in accordance with their reten-
l
M
2
tion times. Veratryl alcohol and veratryl aldehyde were used as stan-
dards. Quantification of the products in eluate was performed
integrating the elutionpeak using theMultichromsoftware (Russia).
4
6
2
electrode thermostated at 25 °C. In these measurements the con-
centrations of R. vernicifera and T. hirsuta laccases were
3
. Results and discussion
ꢁ7
ꢁ8
0
.9 ꢂ 10 M and 3.8 ꢂ 10 M, respectively, and different con-
centrations of O inside the Clark electrode were established
2
One of the basic parameters of laccase catalysed redox reactions
using pure argon or oxygen from Venta (Russia). All kinetic
parameters were calculated using the Michaelis–Menten equa-
tion in a Microcal Origin program (version 5.0).
is the difference in the standard redox potentials of the initial elec-
tron donor (enzyme substrate, E ) and the first electron acceptor
S

M. Gorbacheva et al. / Bioorganic Chemistry 37 (2009) 1–5
3
(
the T1 site of the enzyme, ET1). The ET1-values of high-redox po-
was registered (Table 1), whereas the formation of Mn3+-com-
plexes by T. hirsuta laccase was clearly shown (e.g. oxalate in
Fig. 2). However, the rate of enzymatic oxidation of Mn2+ by T. hirs-
uta laccase is not so high. Nevertheless, the enzyme increase the
rate of manganese oxidation to approximately 10 times that of
non-biological oxidation at pH 5, in good agreement with
previously published data concerning biological rates of manga-
nese oxidation [8,30]. The sublinear dependence of the amount
tential T. hirsuta and low potential plantR. vernicifera laccases, used
in the present work as catalysts, are 780 mV (pH 6.0) [25] and
4
Mn /Mn couple (1510 mV at pH 7.0 [14]), is significantly higher
than the ET1-values of both fungal and plant blue multicopper oxi-
dases [11]. Thus, one cannot expect direct enzymatic oxidation of
Mn catalysed by laccases. The direct electrochemical method
used in this work showed that the formation of manganese com-
plexes with chelators significantly decreased the redox potential
30 mV (pH 6.8) [26], respectively. The redox potential of the
2
+
3+
2
+
3
+
of Mn -oxalate complex formed over the extended time period
is explained by the low stability of the product of enzymatic reac-
2
+
3+
3+
of the Mn /Mn couple and makes it potentially oxidiseable by
the high-redox potential enzyme. Dicarboxylic and oxycarboxylic
tion, viz. the Mn -oxalate complex (Fig. 2, inset). Kinetic studies of
2
+
enzymatic oxidation of Mn by T. hirsuta laccase showed the clas-
sical Michaelis–Menten dependence. However, the efficiency of the
enzymatic process was very low compared to the oxidation of
other substrates of the enzymes, e.g. cyanide transition metal com-
plexes (Table 1), which have much lower values of midpoint poten-
tials compared to the manganese ions (940 mV).
2+
3+
acids are good chelators of both Mn and Mn and they also pro-
vide the needed pH level for the enzymatic reaction to occur [27].
In the CV of a glassy carbon electrode submerged in 0.1 M Na-tar-
trate buffer, pH 5.0, containing 0.2 mM Mn2 , both the anodic and
cathodic peaks with potentials of 990 and 890 mV, respectively,
could be observed (Fig. 1). The peak potential difference of
+
The pH dependences of the oxidation of artificial and putative
4 8
natural substrates of T. hirsuta laccase, K [Mo(CN) ,
] and Mn2+
1
00 mV suggests a quasi-reversible redox transformation of che-
lated manganese ions. The midpoint potential of the redox process
was found to be 940 mV vs. NHE. This value corresponds to the re-
dox potential of the tartrate chelated Mn /Mn couple.
respectively, are compared in Fig. 3. Contrary to the cyanide com-
plexes, which have very acidic pH optima, the pH-optimum of the
oxidation of the chelated Mn is shifted to subacid values (pH
2+
3+
2+
Because ET1 of T. hirsuta laccase at pH 5.0 is only 130 mV lower
around 5.0), the natural pH level of the ligninolytic enzymes of
wood-degrading fungi. The rate of the oxidation of the artificial en-
zyme substrates at pH 5.0, however, is still three orders of magni-
tude higher than the rate of the oxidation of the Mn -oxalate
complex, one of the possible natural substrates of T. hirsuta laccase
(Table 1).
To test the physiological role of enzymatic oxidation of chelated
manganese ions, the oxidation of a model lignin compound, verat-
ryl alcohol, by T. hirsuta laccase in the presence of chelated Mn2+
was performed. Indeed, this possibly natural biocatalytic process
was observed spectrophotometrically at a quite significant rate,
whereas there was a complete absence of such enzymatic reaction
in the case of R. vernicifera laccase. Additional HPLC studies, how-
ever, did not confirmed without a doubt the formation of veratryl
aldehyde during alcohol oxidation. Instead, the formation of verat-
ryl acid was clearly observed. Anyhow, the rapid and deep oxida-
2+
3+
compared to the midpoint potential of the Mn /Mn tartrate cou-
ple (Table 1), thermodynamically it can be suggested that the en-
zyme would be able to slowly oxidise Mn2 in the tartrate
complex. The ET1-values of the laccases at pH 5.0 presented in Ta-
ble 1 were calculated taking into account the previously investi-
gated variation with pH (ca. 16 mV/pH) of ET1 on pH for R.
vernicifera [28] and T. hirsuta [29] laccases. Contrary to the situa-
tion with fungal laccases, a very large difference between ET1 of
+
2+
R. vernicifera Lc and the Mn2 /Mn tartrate complex (470 mV)
should result in the absence of Mn2 oxidation by the enzyme. This
simple theoretical reasoning was experimentally observed in our
studies as discussed below.
+
3+
+
As expected, no enzymatic oxidation of Mn2+-oxalate and Mn2+
-
tartrate complexes by low-redox potential R. vernicifera laccase
tion of veratryl alcohol observed in our studies might be
2
ꢁÅ
explained by the co-formation of H
2
O
2
and O2 during the enzy-
2
+
matic oxidation of Mn in the presence of oxalate or tartrate ions,
which was confirmed by the enzymatic oxidation of ABTS in the
1
0
.5
1
presence of HRP (showing H
lium (indicating the presence of O ) in the presence of T. hirsuta
2 2
O formation) or nitroblue tetrazo-
E
p.a.
ꢁÅ
2
ꢁÅ
laccase. The participation of O2 in secondary chemical reactions
results in the formation of H , a substrate of lignin peroxidases,
2 2
O
another type of enzyme forming the ligninolytic complex of white-
rot fungi.
.5
0
In order to understand the limiting step of the enzymatic oxida-
E
m
2+
tion of chelated Mn the basic kinetic parameters of T. hirsuta and
R. vernicifera laccases towards different compounds were accu-
rately measured and the results are summarised in Table 1. The
apparent Michaelis constants (K
the second natural substrate (electron acceptor) in the catalytic cy-
cle of all blue multicopper oxidases, are the lowest K -values ob-
tained in our studies. In contrast, the standard biocatalytic rate
constant (kcat) and the efficiency of O reduction by the T2/T3 clus-
ter (kcat/K ) for both laccases are the highest values (Table 1).
Approximate numerical values of the second-order rate constant
M 2
) of both enzymes towards O ,
-
-
0.5
M
E
p.c.
2
-
1
M
of O
2
reduction by the T2/T3 cluster of plant and fungal laccases
1.5
1
6
ꢁ1 ꢁ1
7
ꢁ1 ꢁ1
are 5 ꢂ 10 M
s
and 5 ꢂ 10 M
s
, respectively [31,32].
reduction by the
00
300
500
700
900
1100
1300
Thus, in air-saturated solutions the rate of O
2
Potential (mV)
T2/T3 cluster of laccases is very high and fast enough not to be a
readily noticeable contribution to the overall turnover rate (Table
Fig. 1. Cyclic voltammograms of a glassy carbon electrode recorded in 0.1 M Na-
tartrate buffer pH 5.0 in the presence (bolded line) and absence (solid line) of
1). Importantly, previously estimated second-order rate constants
0
4
.2 mM of MnSO (scan rate, 50 mV/s; starting potential, 200 mV).
for plant and fungal laccases are in good agreement with the effi-

4
M. Gorbacheva et al. / Bioorganic Chemistry 37 (2009) 1–5
Table 1
Basic kinetic parameters of redox reactions catalysed by plant and fungal laccases
Substrate
E
S
(mV)
Trametes hirsuta (ET1 ꢃ 800 mV)
cat (sꢁ1)
(mM)
Rhus vernicifera (ET1 ꢃ 460 mV)
ꢁ1
ꢁ1
ꢁ1
ꢁ1 ꢁ1
k
K
M
kcat/K
M
(M
s
)
kcat (s
)
K
M
(mM)
k
cat/K
M
(M
s )
6
1300*
0
0
0
6
O
2
940
n.d.
940
780
430
13,000*
0.04
0.08
78
ꢃ0.15
0.43
0.11
0.67
0.18
ꢃ87 ꢂ 10
93
ꢃ0.2
0
0
0
0.46
ꢃ6.5 ꢂ 10
2
+
Mn -oxalate complex
Mn -tartrate complex
0
0
0
2+
727
.
6
6
K
K
4
[Mo(CN)
[Fe(CN)
8
]
0.12 10
.
.
6
4
6
]
114
0.63 10
64
0.14 10
Notes: *, literature values [31,32,35]; n.d., not determined; redox potentials are given vs. NHE.
0
0
0
0
0
0
.11
.09
.07
.05
.03
.01
5
4
3
2
1
0
1
00
4
K
4
[Mo(CN)
8
]
2+
Mn
3
2
75
0
10
20
30
40
Time (min)
5
2
0
5
0
1
4
c
3
c
2
c
1
c
-
0.01
2.5
3
3.5
4
4.5
5
5.5
2
50
270
290
310
330
350
pH
Wavelength (nm)
Fig. 3. pH dependence of the oxidation of K
4
[Mo(CN)
8
] in 40 mM Britton–Robinson
Fig. 2. Spectral monitoring of the formation of Mn3+-oxalate complex in an
enzymatic reaction catalysed by T. hirsuta laccase (0.1 M Na-oxalate buffer, pH 5.0
containing 8 ꢂ 10 M of laccase and 1.0 mM MnSO
spectra recorded after 10, 20, 30, and 40 min of the enzymatic reaction; curves 1c,
c, 3c, and 4c—control spectra recorded after 10, 20, 30, and 40 min without
addition of T. hirsuta laccase into the reaction media. Insets: the concentration of
buffer containing 1 mM K [Mo(CN) ] and 0.074 lM laccase (solid curve; triangles)
4 8
and Mn2 in 0.1 M Na-oxalate buffer containing 1 mM MnSO
+
and 0.37 lM laccase
4
ꢁ
8
4
). Curves 1, 2, 3, and 4—UV
(bolded curve, squares) by T. hirsuta laccase.
2
3+
2+
Mn -oxalate complex vs. time during the enzymatic oxidation of Mn
.
dox potential ET1 and E
tained correlation between ET1 of fungal laccases and E
S
(Table 1), i.e. similar to the previously ob-
of
2
ciency of O reduction by the T2/T3 clusters of both enzymes cal-
culated using the K -values determined in the present study (Ta-
M
S
phenolic, energy-rich compounds, and other natural substrates of
laccases [33]. Thus, our data confirm the previously proposed
hypothesis that the electron transfer rate constant (kET) between
substrate and the T1 site of laccases is one of the major compo-
nents of kcat of blue multicopper oxidases [34]. Indeed, the limiting
step of the oxidation of very high-redox potential, energetically
ble 1).
The kcat values and the efficiency of the oxidation of both Mn2+-
2
+
oxalate and Mn -tartrate complexes by T. hirsuta laccase are the
lowest values obtained in our studies. These results are in good
agreement with previously published kinetic data about the enzy-
2
+
2
+
poor compounds, such as chelated Mn ions, is the electron trans-
fer from the electron donor to the T1 site of the fungal laccase.
In summary, both plant and fungal laccases can directly oxidise
phenolic lignin substructures, which usually have quite low-redox
potentials. However, due to the very high ET1-values, fungal lac-
cases can also indirectly oxidise non-phenolic lignin compounds,
matic oxidation of Mn -pyrophosphate complex by high-redox
potential T. versicolor laccase [18].
Based on the available spectral and kinetic investigations of lac-
cases it is widely held that neither the internal electron transfer
nor the binding of O is limiting the turnover rate of the enzyme
2
during its natural function [32]. Thus, in all likelihood, the limiting
step of the oxidation of the laccase substrate is the electron trans-
fer from the electron donor to the T1 site of the laccases. To test
3
+
e.g. veratryl alcohol, with the help of chelated Mn and reactive
oxygen species, which are formed during the enzymatic reactions
catalysed by the high-redox potential enzymes. The dramatic dif-
ference in biocatalytic behaviour between high- and low-redox po-
tential laccases is, in all likelihood, related to the different
physiological roles of these enzymes in nature, viz. polymerisation
of building blocks of lignin in the case of plant laccase and lignin
degradation by fungal laccase, one of the enzymes forming the lig-
ninolytic complex of wood-degrading fungi.
2
+
this hypothesis for enzymatic oxidation of the chelated Mn , the
effect of the redox potential on the efficiency of enzymatic catalysis
of plant and fungal laccases was investigated. For this purpose
electron donors with quite similar structure, size, and molecular
weight, but very different redox potentials, were chosen (Table
1). The kinetic studies showed that both kcat and kcat/K
M
were
highly dependent on the difference between the one-electron re-

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Acknowledgments
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579–1582.
The authors thank Dr. Curt T. Reimann (Department of Analyt-
ical Chemistry, Lund University) for critical reading and helpful
suggestions. The work has been financially supported by the Swed-
ish Research Council (Vetenskapsrådet, 621-2005-3581) and by the
Russian Foundation of Basic Research (Grant 08-04-01450a).
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