Báo cáo Y học: Production and chemiluminescent free radical reactions of glyoxal in lipid peroxidation of linoleic acid by the ligninolytic enzyme, manganese peroxidase pot

Production and chemiluminescent free radical reactions of glyoxal in
lipid peroxidation of linoleic acid by the ligninolytic enzyme,
manganese peroxidase
Takashi Watanabe
1
, Nobuaki Shirai
2
, Hitomi Okada
1
, Yoichi Honda
1
and Masaaki Kuwahara
1
1
Laboratory of Biomass Conversion, Wood Research Institute, Kyoto University, Gokasho, Uji, Japan;
2
Industrial Research Center of Shiga
Prefecture, Ritto, Kamitoyama, Japan
Glyoxal is a key compound involved in glyoxal oxidase
(GLOX)-dependent production of glyoxylate, oxalate and
H
2
O
2
by lignin-degrading basidiomycetes. In this paper, we
report that glyoxal was produced from a metabolite of
ligninolytic fungi, linoleic acid, by manganese peroxidase
(MnP)-dependent lipid peroxidation. In the absence of the
parent substrate of linoleic acid, the dialdehyde was
oxidized by MnP and Mn(III) chelate to start free radical
reactions with emission of chemiluminescence at 700–

710 nm. The spectroscopic profile of the light emission is
distinguishable from (a) singlet oxygen, (b) triplet carbonyls
from dioxetane and a-hydroxyperoxyl radicals, and (c)
biacyl triplet formed by the coupling of two acyl radicals.
The photon emission of glyoxal by MnP was activated by
co-oxidation of tartrate. The MnP-dependent oxidation of
glyoxal in tartrate buffers continued for 10 days without
addition of exogenous H
2
O
2
. The importance of these
results is discussed in relation to the free radical chemistry
of lignin biodegradation by wood rot fungi.
Keywords: Manganese peroxidase; lipid peroxidation;
Ceriporiopsis subvermispora; acyl radical.
Lignin biodegradation by white rot fungi is an extracellular
chemical event generating free radicals. Lignin-degrading
enzymes, lignin peroxidase (LiP), manganese peroxidase
(MnP) and laccase (Lac), play a key role in generating free
radicals from lignin and oxidizable fungal metabolites such
as oxalate, glyoxylate, malonate, hydroquinones and aryl
alcohols. Due to the participation of peroxidases in the
lignin breakdown, a supply of hydrogen peroxide is essential
to drive the extracellular enzymatic process. So far, several
oxidases have been proposed as the enzymes which carry
out this task. The finding that glyoxal and glyoxal oxidase
(GLOX) are secreted by white rot fungi strongly suggests
that the GLOX system plays a key role in the extracellular
H

2
O
2
production [1–6]. As GLOX is activated by
peroxidases, the peroxidase-dependent lignin-degradation
can be controlled by the combination of GLOX and its
substrate, glyoxal [2,7]. Thus, the importance of glyoxal
oxidation in wood decay has been recognized. However,
little is known about the biosynthetic route for the extra-
cellular production of glyoxal by wood rot fungi. In this
paper, we first report that a ligninolytic enzyme, MnP, is able
to catalyze formation of glyoxal from a metabolite of wood
rot fungi, linoleic acid [8], by lipid peroxidation. The
glyoxal produced by MnP can be converted to glyoxylate
and oxalate by GLOX [6] and these carboxylic acids are
further oxidized by MnP or LiP/VA to yield O
2
†
–
and
CO
2
†
–
, which in turn reduce free radicals and transition
metals like Fe(III) [9–12]. Thus, the present result
highlights the new roles of MnP-dependent lipid peroxi-
dation in free radical chemistry of wood rot fungi.
In lipid peroxidation of USFAs, it has been reported that
Mn(II) reacts with a chain-carrying radical, peroxyl radical

(LOO†), to terminate the chain reactions [13,14]. This
raises the question of how the MnP-lipid system generates
free radicals in the presence of antioxidant, Mn(II).
Recently, we reported that the chain-braking antioxidative
activity of Mn(II) is suppressed by regeneration of free
radicals by breaking down of LOOH with MnP [15]. In this
process, we found that acyl radicals were predominantly
formed. This suggests that hydrogen abstraction from
aldehydes is involved in the major chain propagation
reactions of the MnP-dependent lipid peroxidation. The
observation of acyl radicals in the MnP/lipid system
prompted us to analyze whether MnP can directly oxidize
the aldehyde intermediate in order to carry chain-reactions
Correspondence to T. Watanabe, Laboratory of Biomass Conversion,
Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto
611-0011, Japan, Fax: 181 744 38 3600,
E-mail:
Enzymes: manganese peroxidase (EC 1.11.1.13); lipoxygenase
[linoleate:oxygenoxidoreductase (EC 1.11.13)]; glyoxal oxidase
(EC 1.2.3 ).
(Received 8 May 2001, revised 24 September 2001, accepted
27 September 2001)
Abbreviations:O
2
†
–
, superoxide anion; CO
2
†
–

, formate anion
radical; MnP, manganese peroxidase; LiP, lignin peroxidase;
HRP, horseradish peroxidase; 13(S)-HPODE,
13(S)-hydroperoxy-9Z,11E-octadecadienoic acid; SFA, saturated fatty
acid; USFA, unsaturated fatty acid; 2,6-DMP, 2,6-dimethoxyphenol;
ESR, electron spin resonance; MDA, malondialdehyde; MSTFA,
N-methyl-N-trimethylsilyltrifluroacetamide; DFB, decafluorobenzene;
GLOX, glyoxal oxidase, TBARS, thiobarbituric acid reactive
substances; PFBHA, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine
hydrochloride; PFBO, pentafluorobenzyl oxime; CH
3
CN, acetonitrile;
MeOH, methyl alcohol; EtOH, ethyl alcohol; DM, n-dodecyl
b-maltoside; DHMA, dihydroxymaleic acid; EI/GC/MS, electron
ionization-gas chromatography-mass spectrometer; PAH, polycyclic
aromatic hydrocarbon.
Eur. J. Biochem. 268, 6114–6122 (2001) q FEBS 2001
without the aid of the other oxidizable substrates. We now
report the formation and chemiluminescent chain reactions
of glyoxal in MnP-dependent lipid peroxidation of linoleic
acid.
MATERIALS AND METHODS
General methods
Manganese (II) sulfate and 1,2,3-trimethoxybenzene,
decafluorobenzene, 1-dodecanal, 1-decanal, 2,4-nonadienal,
1-hexanal, 1-nonanal, 1-pentanal, 1-octanal, 1-butanal,
glyoxlic acid, glycol aldehyde, glyoxal were obtained
from Wako Pure Chemical Industries (Tokyo, Japan).
trans,trans-2,4-Decadienal, cis-4-decenal was obtained from
Aldrich Chemical Company (Milwaukee, USA). trans-2-

Hexenal, 1-undecanal, 1-heptanal, trans-2-nonenal, 1-tride-
canal, 2-butanone, 3-buten-2-one, 3-pentanone, 2-pentanone,
2-heptanone, 2-hexanone, 2-octanone was obtained from
Tokyo Kasei Kogyo (Tokyo, Japan). Linoleic acid was
purchased from Nacalai Tescque (Kyoto, Japan). The linoleic
acid was purified by passing through a Sep-Pak
TM
CN Light
cartridge (Waters, Milford, MA, USA). After dissolving in
n-hexane, the eluent from the cartridge was evaporated with a
gentle stream of N
2
gas. Milli-Q
TM
water was used
throughout the experiments. All of the chemicals used were
of analytical reagent grade. 13(S)-Hydroperoxy-9Z,11E-oc-
tadecadienoic acid [13(S)-HPODE] was prepared as
described previously [15]. Malondialdehyde (MDA) was
synthesized as described previously [16].
Enzyme preparation
Crude MnP from Ceriporiopsis subvermispora FP-90031
was collected from 7-day-old cultures grown on a wood
medium composed of beech wood (5 g), glucose (0.7 g) and
peptone (0.7 g) at 28 8C. The culture filtrate was dialyzed
against 20 m
M sodium succinate buffer (pH 4.5). The
dialyzate was concentrated by ultrafiltration, precipitated
with (NH
4

)
2
SO
4
and then purified by gel filtration on
Superdex 75 PG (1.6 Â 60 cm, Amersham Pharmacia
Biotech, Sweden) using 20 m
M sodium succinate buffer
containing 0.1
M NaCl as an eluent. Fractions showing MnP
activities were collected, and desalted with Centriprep
YM-30 (cut off, 30,000, Millipore, USA). MnP was further
purified by preparative IEF as described previously [15]
[pI 3.40, Reinheitzahl (RZ, A at l
max
/A 280) value: 3.0,
1.0 U ¼ 8.75 Â 10
211
mol]. Low molecular mass com-
pounds were removed by successive washings with Milli-
Q
TM
water with a Centricut N-10 ultrafiltration concentrator
(cut off, 10 000, Kurabo, Japan) before use. For the time
course experiments of aldehyde production, the enzyme
purified on Superdex 75 PG was desalted with distilled
water in Centricut N-10 and used without further
purification (15 U
:
mL

21
). Laccase activity in the partially
purified fraction was below 0.02 U
:
mL
21
. Glyoxal oxidase
activity was not found in all the enzyme preparations.
Enzyme assay
MnP activity was measured with 2,6-DMP. The reaction
mixture contained 0. 2 m
M 2,6-DMP, 0. 5 mM MnSO
4
,
0. 1 m
M H
2
O
2
,25mM sodium tartrate buffer (pH 3. 0) and
the enzyme solution. Reactions were started by adding H
2
O
2
and were quantified by monitoring the initial rate of
increase in absorbance at 470 nm in the presence and
absence of manganese. One unit of enzyme activity is
defined as the amount of enzyme that oxidizes 1 mmol of
2,6-DMP in 1 min. Laccase activity was measured
with 2,6-DMP under the same conditions but without

H
2
O
2
. Lipoxygenase activity was measured by O
2
uptake in
a reaction system containing 1. 5 m
M linoleic acid, 1 mM
n-dodecyl b-maltoside (DM) and 20 mM Tris/HCl buffer
(pH. 9. 0). One unit of lipoxygenase activity is defined as
the amount of enzyme that absorbs 1 mmol of O
2
in 1 min.
GLOX activity was measured by O
2
uptake in a
reaction system containing 3 m
M glyoxal in 20 mM sodium
tartrate (pH 3. 0), acetate (pH 4. 5) or phosphate (pH 6. 0)
buffers.
Electron ionization/gas chromatography/mass
spectrometetry (EI/GC/MS) analysis of oxidation
products by MnP
Linoleic acid and aldehydes were reacted with 250 mU of
the purified MnP, 0.5 m
M of Mn(II) and 50 mM of H
2
O
2

at
20 8C for 1–24 h in 10 m
M acetate, formate, lactate and
tartrate buffers (pH 4.5). After the reaction, 0.5 mL of
aqueous PFBHA (0.05
M, 200 mL) was added and reacted at
35 8C for 0.5 h [17]. To this solution, 10 mL of a 10-m
M
methanol solution of decafluorobenzene (DFB) and a
drop of 18-N-sulfuric acid were added and the mixture
was partitioned between n-hexane and H
2
O twice. The
hexane layer was dried over Na
2
SO
4
, evaporated with a
gentle stream of N
2
gas and directly injected into an EI/GC/
MS system. The EI/GC/MS analysis was done with a
Shimadzu QP-5050 A GC/MS with ionization energy of
70 eV on CP-Sil-8 (50 m  0.25 mm internal diemeter,
Chrompack, Netherlands) using helium as a carrier gas. The
column oven temperature was raised from 80 8C to 250 8C
at 5 8C
:
min
21

, and maintained at 250 8C for 20 min. The
time course of glyoxal production by MnP was analyzed as
described above after the reaction with and without linoleic
acid in formate and tartrate buffers. EI/GC/MS analyses of
authentic aldehydes and ketones were carried out using a
0.6-m
M methanol solution after derivatization with PFBHA
under the conditions described above. Tetramethylsilation
by N-methyl-N-trimethylsilyltrifluroacetamide (MSTFA)
was carried out as described previously [18].
Chemiluminescence measurements
Chemiluminescence was measured by an ultra-high sensi-
tive photon counter (ARGUS-50/VIM, Hamamatsu Photo-
nics, Hamamatsu, Japan) equipped with a charge-coupled
device (CCD) camera connected with an image intensifier
and ARGUS-50 image processor. The wavelength range of
the detector was 350–650 nm, 512 Â 483 pixels, and the
noise count was 0.15 c.p.s. The reactions were carried out in
a cuvette for a 96-well microplate reader. The conditions for
each experiment are described in the figure legends.
Inactivation of MnP was carried out by heating the MnP
in a boiling water bath for 10 min
The chemiluminescence spectra were measured by a
simultaneous multiwavelength analyzer CLA-SP2 (Tohoku
Electronic Industries Co. Ltd, Sendai, Japan) with an
q FEBS 2001 Production of glyoxal in lipid peroxidation by MnP (Eur. J. Biochem. 268) 6115
incident slit width of 1.0 mm. The wavelength range of the
spectrometer was 370–820 nm. Experimental conditions
are described in the legend of each figure.
RESULTS

Formation of glyoxal in the reaction of linoleic acid
with MnP
Lipid peroxidation by MnP is a free radical process capable
of decomposing recalcitrant PAH and nonphenolic lignin
model compounds [19 – 21]. We previously reported that the
oxidation of linoleic acid by MnP produced acyl radicals in
both tartrate and acetate buffers [15]. The formation of acyl
radicals strongly suggests that hydrogen abstraction from
aldehyde [22,23] is involved in the oxidative process. To
analyze the aldehydes formed by this reaction, linoleic acid
was reacted with MnP for 19 h at 20 8C in sodium acetate,
formate, lactate and tartrate buffers and the oxidation
products were analyzed by EI/GC/MS after derivatization
to pentafluorobenzyloxims (PFBO) with PFBHA [17]. EI/
GC/MS analysis of the reaction products and authentic 19
aldehydes and seven ketones demonstrated that glyoxal,
1-hexanal and 1-pentanal were formed from linoleic acid
by MnP in any of the buffer systems (Fig. 1). Syn and
anti-isomers of these PFBO derivatives were separated on
the GC/MS column. The mass spectrum of PFBO
derivatives of glyoxal formed from linoleic acid is shown
in Fig. 2, together with that of authentic standard. MDA, a
major peroxidation product derived from polyunsaturated
fatty acids was not detected in the reaction products of MnP
in contrast to the oxidation of linoleic acid by xantine/
xanthinoxidase/Fe(II) [24]. The mass fragments of PFBO
derivatives characteristic to saturated aldehyde (m/z 239),
2-enal (m/z 250), 2,4-dienal (m/z 276) and saturated
2-ketones (m/z 72) [25], were not observed in the spectra
of unidentified carbonyl compounds, indicating that the

MnP/Mn(II)/lipid system proceeds by complex radical
reactions involving the formation of unusual carbonyl
species. Tetramethylsilation with MSTFA did not change
the mass chromatogram at m/z 181 that originates from C–O
bond cleavage products of pentafluorobenzyl oxime [26]
(data not shown).
The reactions of MnP in four different buffers clearly
demonstrate that the formation of glyoxal was significantly
stimulated by the presence of tartrate. Therefore, the
reaction was carried out with and without linoleic acid in
sodium formate and tartrate buffers (Figs 1 and 3). GC/MS
analysis demonstrated that glyoxal was explosively
produced after 6 h in tartrate buffer containing linoleic
Fig. 1. Mass chromatograms of PFBO derivatives of products of lipid peroxidation by C. subvermipora MnP and soybean lipoxygenase at
m/z 181. (A) Products of the oxidation of linoleic acid by MnP in sodium acetate buffer for 19 h. The reaction system (500 mL) contained 3 m
M
linoleic acid, 500 mM MnSO
4
,50mM H
2
O
2
, 0.02% of Tween 20, 250 mU of purified MnP and 10 mM sodium acetate buffer (pH 4.5). (B) As (A) but
10 m
M sodium formate buffer (pH 4.5) was used instead of acetate buffer. (C), As (A) but 10 mM sodium lactate buffer (pH 4.5) was used instead of
acetate buffer. (D) As (A) but 10 m
M sodium tartrate buffer (pH 4.5) was used instead of acetate buffer. (E) As (B) but the reaction was carried out
without addition of linoleic acid. (F) As (D) but the reaction was carried out without addition of linoleic acid. (G) Products of the oxidation of linoleic
acid with soybean lipoxygenase. Linoleic acid (3 m
M) was reacted with soybean lipoxygenase (10 U) in 40 mM Tris/HCl buffer (pH 9.0) containing

0.02% of Tween 20 for 24 h at 20 8C. (H) Products of the oxidation of 13(S )HPODE by MnP in sodium lactate buffer (pH 4.5). for 3 h. The reaction
system contained 3 m
M 13(S )HPODE, 500 mM MnSO
4
,50mM H
2
O
2
, 0.02% of Tween 20, MnP (250 mU) and 10 mM sodium lactate buffer
(pH 4.5).
6116 T. Watanabe et al. (Eur. J. Biochem. 268) q FEBS 2001
acid. However, direct formation of glyoxal from tartrate was
also observed. In formate buffer, the production of glyoxal
was dependent on the presence of linoleic acid. The same
results were obtained with lactate and acetate buffers (data
not shown). In contrast to the oxidation of linoleic acid,
oxidation of 13(S )HPODE with MnP selectively produced
1-hexanal for 1–3 h (Fig. 1H). No PFBO-derivatives were
detected after the prolonged reaction of 13(S )HPODE. Thus
it was found that the formation of glyoxal was not catalyzed
by the direct oxidation of 13(S )HPODE with MnP.
Oxidation of linoleic acid with soybean lipoxyegnase
produced 1-hexanal and 1-pentanal (Fig. 1G).
Emission of chemiluminescence in lipid peroxidation
The chemiluminescence detector is a powerful tool for
analyzing the oxidation of aldehydes due to its high
sensitivity and emission spectra characteristic to chemically
excited species. Therefore, oxidation of aldehydes and
linoleic acid by MnP was analyzed by a chemiluminescence
detector, in comparison with light emission by lipoxygenase

and the Fenton reaction (Fig. 4). Lipoxygenase is an enzyme
that abstracts hydrogen from the bis-allylic position of
unsaturated fatty acids containing cis,cis-1,4-pentadienyl
moiety. In the reaction with linoleic acid, the fatty acid is
oxidized to yield a pentyl radical [27] and 12-oxododecyl-
cis-9-enoic acid [28] via b-scission of hydroperoxide
intermediates, leading to production of 1-hexanal [29] and
1-pentanal as shown in Fig. 1G. When linoleic acid was
oxidized by soybean lipoxygenase, emission of chemilumi-
nescence was close to the background level, both in the
presence and absence of Fe(II). In the Fenton system,
chemiluminescence was also below the background level,
except for a weak emission of light from linoleic acid after 2
days (Fig. 4).
In contrast to these oxidation systems, reactions of glyoxal
with MnP in tartrate buffer emitted strong chemilumines-
cence. As shown in Figs 5 and 6, intensive light emission
was observed immediately after the reaction started. The
photon emission reached a maximum (35 000 counts
:
h
21
)
within 30 min, and then decreased, but chemiluminescence
of < 9000 counts
:
h
21
was observed even after 1 h. In
lactate, formate and acetate buffers, the photon emission

was also observed within 30 min but the intensity was much
lower than that of the tartrate system. In the tartrate system,
the photon emission continued for 10 days, both in the
presence and absence of exogenous H
2
O
2
added initially
(Fig. 6). The photon emission from glyoxal was dependent
on the presence of Mn(II) and active enzyme. However, it
was found that the emission of chemiluminescence
continued for around 10 days when the reaction was started
without addition of glyoxal. This can be explained by the in
situ formation of glyoxal from tartrate with MnP (Fig. 1,3).
In the MnP-catalyzed oxidation of linoleic acid and the other
aldehydes, the light emission was not observed when Mn(II)
was omitted from the reaction system (data not shown).
When linoleic acid and seven different aldehydes were
reacted with Mn(III)–tartrate complex, strong light emission
was observed in the reaction system with glyoxal (Fig. 7).
The maximum photon emission intensity from glyoxal
reached 12 000 counts
:
h
21
. The photon emission was also
Fig. 2. Mass spectra of PFBO derivatives of glyoxal formed by the
oxidation of linoleic acid with MnP (A) and authentic standard (B).
(A) Glyoxal formed by the oxidation of linoleic acid with MnP for 19 h.
The reaction system (500 mL) contained 3 m

M linoleic acid, 500 mM
MnSO
4
,50mM H
2
O
2
, 0.02% of Tween 20, MnP (250 mU) and 10 mM
sodium acetate buffer (pH 4.5). (B) Authentic standard of glyoxal.
* 1/10 of the original signal intensity.
Fig. 3. Time course of glyoxal formation by MnP. (A) Glyoxal formed by the reaction of linoleic acid with MnP in sodium tartrate buffer. The
reaction system (500 mL) contained 3 m
M linoleic acid, 500 mM MnSO
4
,50mM H
2
O
2
, 0.02% of Tween 20, MnP (250 mU) and 10 mM sodium
tartrate buffer (pH 4.5). (B) As (A) but linoleic acid was omitted. (C) As (A) but 10 m
M sodium formate buffer (pH 4.5) was used instead of sodium
tartrate bufer. (D) As (C) but linoleic acid was omitted.
q FEBS 2001 Production of glyoxal in lipid peroxidation by MnP (Eur. J. Biochem. 268) 6117
Fig. 4. Time course of light emission during
oxidation of linoleic acid by soybean
lipoxygenase (I) and the Fenton reaction (II).
(I): (A) The reaction system (200 mL) contained
4m
M linoleic acid, 10 U of lipoxygenase, 0.05%
of Tween 20, 10 m

M Tris/HCl buffer (pH 9.0). (B)
As (A) but lipoxygenase was omitted. (C) As (A)
but 0.5 m
M FeSO
4
was added. II: (A) The reaction
system (200 mL) contained 4 m
M linoleic acid,
0.1 m
M FeSO
4
, 0.2 mM H
2
O
2
, 0.05% of Tween 20.
(B) As (A) but glyoxal was added instead of
linoleic acid. (C) As (A) but trans-2-nonenal was
added instead of linoleic acid. (D) As (A) but
1-dodecanal was added instead of linoleic acid. (E)
As (A) but 1-hexanal was added instead of linoleic
acid. (F) As (A) but 2,4-nonadienal was added
instead of linoleic acid. (G) As (A) but MDA was
added instead of linoleic acid. (H) As (A) but
linoleic acid was omitted.
Fig. 5. Chemiluminescence emitted by the
oxidation of aldehydes and linoleic acid with
MnP in sodium tartrate buffer. (A) The reaction
system (200 mL) contained 4 m
M linoleic acid,

250 mU of MnP, 500 m
M MnSO
4
, 0.2 mM H
2
O
2
,
0.05% of Tween 20 and 10 m
M sodium tartrate
buffer (pH 4.5). (B) As (A) but glyoxal was added
instead of linoleic acid. (C) As (A) but
trans-2-nonenal was added instead of linoleic acid.
(D) As (A) but 1-dodecanal was added instead of
linoleic acid. (E) As (A) but 1-hexanal was added
instead of linoleic acid. (F) As (A) but
2,4-nonadienal was added instead of linoleic acid.
(G) As (A) but MDA was added instead of linoleic
acid. (H) As (A) but linoleic acid was omitted.
Inset shows the time course of the reactions (A–H)
during 2.5 h.
Fig. 6. Chemiluminescence emitted by the
oxidation of glyoxal with MnP. (I): (A) The
reaction system (200 mL) contained 4 m
M glyoxal,
250 mU of MnP, 500 m
M MnSO
4
, 0.2 mM H
2

O
2
,
0.05% of Tween 20 and 10 m
M sodium acetate
buffer (pH 4.5); (B) As in (A) but 10 m
M sodium
formate buffer (pH 4.5) was used instead of
sodium acetate bufer. (C) As (A) but 10 m
M
sodium tartrate buffer (pH 4.5) was used instead
of sodium acetate bufer. (D) As (A) but 10 m
M
sodium lactate buffer (pH 4.5) was used instead
of sodium acetate buffer.
(II): (A) The reaction system (200 mL) contained
4m
M glyoxal, 250 mU of MnP, 500 mM MnSO
4
,
0.2 m
M H
2
O
2
, 0.05% of Tween 20 and 10 mM
sodium tartrate buffer (pH 4.5). (B) As (A) but
MnSO
4
was omitted. (C) As (A) but H

2
O
2
was
omitted. (D) As (A) but glyoxal was omitted. (E)
As (A) but inactivated MnP was used instead of
native MnP.
6118 T. Watanabe et al. (Eur. J. Biochem. 268) q FEBS 2001
observed with MDA, trans-2-nonenal and linoleic acid, but
the intenisity was less than 1/10 of that observed in the
oxidation of glyoxal. Light emissions from 1-dedecanal,
1-hexanal, 2,4-nonadienal were almost at the same level as
that observed without addition of linoleic acid/aldehyde. No
photon emission was observed in the reactions of these
oxidizable substrates with Mn(II)–tartrate complex (data
not shown). These results demonstrate that MnP catalyzes
oxidation of Mn(II) to Mn(III), which in turn reacts with
glyoxal to generate electronically excited species. Co-oxida-
tion of tartrate is essential to carry the chemiluminescent
chain reactions over several days.
Figure 8 shows emission spectra obtained by glyoxal
oxidation with (A) Mn(III)–lactate complex, and (B) MnP/
Mn(II)/H
2
O
2
in tartrate buffer. The spectra showed a broad
single peak with emission maxima at 700 and 710 nm,
respectively. Figure 2C is the chemiluminescence spectrum
of singlet oxygen formed by the reaction of HClO

–
with
H
2
O
2
. The spectrum showed two sharp emission maxima at
634 and 704 nm as reported [30]. In the spectra obtained by
the glyoxal oxidation by MnP and Mn(III)–lactate, no
shoulder peaks of the dimol emission of singlet oxygen was
observed.
DISCUSSION
Formation and oxidation of glyoxal in lipid peroxidation
of linoleic acid by MnP
It has been widely recognized that selective white rot fungi
such as Ceriporiopsis subvermispora can delignify wood
without penetration of their extracellular enzymes into wood
cell walls. Therefore, there is increasing interest in the roles
of low molecular mass compounds that generate free
radicals capable of decomposing lignin at a site far from
enzymes. Lipid peroxidation by manganese peroxidase
(MnP) is one candidate for this system because diffusible
Mn(III) chelate can react with lipid and lipid hydroperoxides
to generate free radicals [15]. When C. subvermispora was
cultivated on wood meal medium, the fungus produced
saturated fatty acids (SFAs) and unsaturated fatty acids
(USFAs) including linoleic acid at an incipient stage of
cultivation and consume them with concomitant formation
of lipid hydroperoxide and TBARS during prolonged
cultivation [8]. In the lipid peroxidation process, titres of

MnP reached a maximum around day 4 and then gradually
decreased, coincident with the peroxidation of the fatty
acids. Thus, accumulated data supports the involvement of
MnP-dependent lipid peroxidation in wood decay by white
rot fungi.
With regard to the radicals produced in the lipid
peroxidation by MnP, we reported that MnP oxidized
linoleic acid to generate acyl radicals in acetate and tartrate
buffers in the presence of Mn(II) [15]. The formation of acyl
radicals strongly suggests that hydrogen abstraction from
aldehydes is involved in the chain propagation cycle of the
MnP-dependent lipid peroxidation. Therefore, aldehydes
formed by the MnP/linoleic acid/Mn(II)/H
2
O
2
reactions
were analyzed by EI/GC/MS after derivatization to PFBO
(Figs 1–3). The GC/MS analysis demonstrated that
oxidation of linoleic acid with MnP produced glyoxal,
1-hexanal and 1-pentanal. Time course experiments of the
MnP reactions showed that glyoxal was not formed on
initiation of the lipid peroxidation but after 6 h (Fig. 3). In
contrast to the oxidation of linoleic acid, the reaction of
MnP with 13(S)-HPODE selectively produced 1-hexanal,
indicating that the glyoxal formation is not catalyzed by the
direct breakdown of lipid hydroperoxide with MnP and
Mn(III) chelates.
In wood decay, a supply of extracellular hydrogen
peroxide is essential to initiate peroxidase-dependent free

radical processes. An extracellular oxidase, GLOX, has been
identified from wood decay fungi and is considered as an
enzyme that catalyzes extracelular H
2
O
2
production.
However, little is reported about the biosynthetic pathway
of glyoxal in wood rot fungi. In 1994, Hammel et al.
Fig. 7. Chemiluminescence emitted by the
oxidation of aldehydes and linoleic acid with
Mn(III)–tartrate complex. (A) The reaction
system (200 mL) contained 4 m
M linoeic acid,
0.05% of Tween 20, and 2.5 m
M Mn(III)–tartrate.
A Mn(III) – tartrate solution (10 m
M) was prepared
by dissolving 0.1 m mol of Mn(III)–acetate in
10 mL of 0.1
M sodium tartrate buffer (pH 4.5).
The reaction was initiated by adding 50 mL of this
solution. Therefore, the final concentration of
tartrate in the reaction system was 25 m
M. (B) As
(A) but glyoxal was added instead of linoleic acid.
(C) As (A) but trans-2-nonenal was added instead
of linoleic acid. (D) As (A) but 1-dodecanal was
added instead of linoleic acid. (E) As (A) but
1-hexanal was added instead of linoleic acid. (F)

As (A) but 2,4-nonadienal was added instead of
linoleic acid. (G) As (A) but MDA was added
instead of linoleic acid. (H) As (A) but linoleic acid
was omitted.Time course of the photon emission
from glyoxal is shown separately from that of the
other oxidizable compounds due to the difference
of emission intensity.
q FEBS 2001 Production of glyoxal in lipid peroxidation by MnP (Eur. J. Biochem. 268) 6119
reported that lignin peroxidase deomposed a b-O-4 lignin
model compound with production of glycol aldehyde, a
substrate of GLOX [6]. The glycol aldehyde formed by this
process was converted to oxalate with the production of 2.8
equivalent of H
2
O
2
. Therefore, they proposed a pathway
producing oxalate from the b-O-4 lignin model compound
via glyoxal and glyoxylate. However, there has been no
direct evidence for the glyoxal formation from lignin by the
LiP/GLOX system. The finding of glyoxal formation by
MnP-dependent lipid peroxidation indicates that MnP and
Mn(III) chelate part in the formation of glyoxal, leading to
the enzymatic production of glyoxylate, oxalate and H
2
O
2
by GLOX. Mn(III) stabilized by the former two carboxylic
acids can diffuse into the wood cell wall region. At the same
time, glyoxylate and oxalate are oxidized by Mn(III) to

produce CO
2
†
–
and O
2
†
–
[11,12]. The same reaction was
also catalyzed by LiP/VA [9,10]. Due to the high reduction
potential of CO
2
†
–
, the radical catalyzes reduction of
Fe(III) [12] and reductive dehalogenation of recalcitrant
aromatic halides [31]. O
2
†
–
catalyzes oxidation of Mn(II)
and reduction of Fe(III) in addition to disproportionation
yielding H
2
O
2
. A combination of the iron reduction and
H
2
O

2
formation generates hydroxyl radicals. Thus, MnP-
dependent lipid peroxidation provides the substrate of
GLOX to produce active oxygen species in combination
with redox cycle of transition metals.
Oxidation of glyoxal by MnP
In lipid peroxidation involving aldehyde oxidation, it has
been postulated that acyl radicals are formed from aldehydes
by hydrogen abstraction with radicals (X†) [22] or transition
metals [23] according to:
X† 1 RVCHO ! XH 1 RCO† ð1Þ
M
31
ðM
21
Þ 1 RCHO ! M
21
ðM
1
Þ 1 RCO† 1 H
1
ð2Þ
With regard to the light emission from acyl radicals, four
different pathways can be discussed (Fig. 9). As shown in
Fig. 9 (pathway 2) two acyl radicals can recombine to
produce a biacyl triplet [32]. The light emission reported in
excited biacyl compounds like biacetyl (l max at 515 and
560 nm) [32] is different from the emission spectra observed
in the MnP reactions. a-Oxidation of aldehydes via a
dioxetane intermediate also produces excited triplet

carbonyls (Fig. 9, pathway 1) but the absorption maximum
of the light emission is in the range of l max 450–550 nm
[28,32]. Singlet oxygen can be formed by disproportionation
of two a-hydroxyperoxyl radicals (Fig. 9, reaction 4).
However, there are no shoulder peaks of the dimol emission
from singlet oxygen (l max 634 and 703 nm) [30] in the
spectrum of glyoxal oxidation by MnP (Fig. 8), indicating
that
1
O
2
is not the major excited species formed by the
MnP/glyoxal system. The other possible route for
Fig. 8. Chemiluminescence spectra of (A) oxidation of glyoxal by
MnP in tartrate buffer (B) oxidation of glyoxal by Mn(III)–lactate
complex and (C) singlet oxygen formed by the reaction of ClO
–
with H
2
O
2
. (A) The reaction system (500 mL) contained 3 mM glyoxal,
250 mU of MnP, 500 m
M MnSO
4
,50mM H
2
O
2
, 0.02% of Tween 20

and 10 m
M sodium tartrate buffer (pH 4.5). (B) The reaction system
(500 mL) contained 3 m
M glyoxal, 0.02% of Tween 20, and 2 mM
Mn(III)–lactate. A Mn(III)–lactate solution (10 mM) was prepared by
dissolving 0.1 m mol of Mn(III)–acetate in 10 mL of 0.1
M sodium
lactate buffer (pH 4.5). The reaction was initiated by adding 100 mLof
this solution. Therefore, the final concentration of lactate in the reaction
system was 20 m
M. (C) The reaction was started by adding 1 mL of
30% H
2
O
2
and 3 mL of 10% NaClO solution. Scanning time for (A) (B)
(C) were 20, 15, and 5 min, respectively.
6120 T. Watanabe et al. (Eur. J. Biochem. 268) q FEBS 2001
chemiluminescence emission from acyl radical is a forma-
tion of triplet carbonyls from a-hydroxyperoxyl radicals
(Fig. 9- [3]) that has been reported in the oxidation of
aetaldehyde with xantine oxidase [33]. However, the
emission maximum of the chemiluminescence by this
mechanism was lower than 500 nm [33] (Fig. 9). Thus, the
MnP-dependent light emission from glyoxal at 700 nm is a
new chemical event difficult to explain by the excited
species from acyl radicals reported before.
As shown in Fig. 8, the reaction of Mn(III)–lactate with
glyoxal emitted the chemiluminescence similar to that
observed in the reactions of MnP. Therefore, it can be

concluded that Mn(III) chelate is capable of abstracting
hydrogen from glyoxal to form the electronically excited
species, which spontaneously decay to emit the chemilumi-
nescence. As shown in Fig. 1, peroxidation of linoleic acid
with MnP produced glyoxal. However, the chemiclumines-
cence from linoleic acid was much less intensive than that of
the direct oxidation of glyoxal with MnP. This suggests that
the excited species from glyoxal react with peroxidation
intermediates from linoleic acid, leading to quenching of the
excited compound [34].
Oxidation of tartrate by MnP
In this study, we also found that tartrate is oxidized by MnP
and Mn(III) chelates in aqueous solutions to produce glyoxal
with emission of chemiluminescence. Although tartrate is
not a metabolite of ligninolytic fungi, this carboxylic acid is
widely used in studies on MnP. For tartrate, unlike oxalate
and malonate, there has been no report of MnP-catalyzed
degradation. For instance, the ratio of H
2
O
2
consumption vs.
oxidation of Mn(II) by MnP in tartrate buffer is reported to
be nonstoichiometric due to reduction of H
2
O
2
to O
2
by

Mn(III)–tartrate complex [35,36]. However, no special
attention was paid to the oxidation of tartrate itself in these
studies. This may be due to the understanding that the
reactivity of tartrate is too low to be involved in the free
radical reactions by Mn(III). For instance, Perez reported
that veratryl alcohol oxidation by LiP is not affected by the
presence of tartrate in the presence or absence of Mn(II) and
Mn(III) [37]. More recently, Collins reported that the rate
of 2,2
0
-azinobiz(3-ethylbenzo-6-thiazolinesulfonic acide)
(ABTS†
1
) reduction is enhanced by the presence of
malonate, glyoxylate and oxalate but no stimulating effects
of tartrate on the ABTS†
1
reduction was observed [38]. In
contrast, the results obtained in the present study clearly
indicate that tartrate itself was oxidized by MnP to produce
glyoxal (Figs 1–3), thereby assisting chain reactions of the
aldehyde accompanied by photon emission (Fig. 7,8). In
lipid peroxidation of linoleic acid by MnP, the consecutive
formation of aldehydes and acyl radicals was observed in
acetate and formate buffers as well as in tartrate buffer.
Therefore, we propose that the enzymatic process produces
counterpart compounds like tartrate to assist the chain
propagation reactions of acyl radicals in combination with
redox cycle of Mn(II)/Mn(III).
In conclusion, the first evidence for the production of

glyoxal from linoleic acid in MnP-dependent lipid peroxi-
dation has been presented. Glyoxal formed by this process
can be used as a substrate of GLOX and MnP to participate
in the extracellular free radical reactions of wood rot fungi.
In addition to the interest on lignin biodegradation, the
analysis of manganese-dependent glyoxal oxidation associ-
ated with tartrate oxidation will lead to the understanding of
cellular injury caused by the carcinogenic aldehyde in the
presence of catalytic amount of manganese.
ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid for Scientific Research (B)
from the Ministry of Education, Science, Sports and Culture, Japan. We
are grateful to Ms M. Nakagawa for technical assistance in the analysis
of aldehydes. We also thank Dr Rie Yamada, Tohoku Electoric Co. Ltd,
for the measurement of chemiluminescence spectra.
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