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INTRODUCTION |
Basidiomycetes that cause brown rot
of wood are the principal recyclers of lignocellulose in
coniferous forest ecosystems and also the principal cause of
biodegradation of wooden structures. These fungi degrade
polysaccharides in wood preferentially but also partially oxidize
lignin (6). Because enzymes are too large to penetrate
sound wood (1, 9, 32), most researchers believe that brown
rotters attack wood polymers by producing small, diffusible,
extracellular oxidants that operate at a distance from the hyphae. The
oxidative changes that brown rotters cause in cellulose, lignin, and
various refractory organic chemicals have led often to the proposal
that one of these oxidants is the hydroxyl radical ·OH
(15, 16, 19, 21, 22, 30, 33, 34).
In biological systems, ·OH is generally produced by the Fenton
reaction (Fig. 1, reaction 1). Therefore,
to use ·OH as an oxidant, brown rot fungi need to reduce
extracellular Fe3+ and produce extracellular
H2O2. How most of them
accomplish this is not well understood, although it has been proposed
that cellobiose dehydrogenase (14) or iron-binding
catechols (11) could initiate Fenton chemistry by reducing
Fe3+ outside the fungal mycelium.
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FIG. 1.
Reactions of the hydroquinone-driven Fenton system in
G. trabeum. Q, quinone; H2Q, hydroquinone;
HQ·, semiquinone; ·OOH, perhydroxyl radical.
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We recently identified a quinone redox cycle that the brown rotter
Gloeophyllum trabeum uses to generate extracellular
Fe2+ and
H2O2 (17).
G. trabeum produces extracellular 2,5-dimethoxyhydroquinone (2,5-DMHQ) (Fig. 2), which reduces
Fe3+ (Fig. 1, reaction 2). It is implicit that
this reaction also yields the 2,5-DMHQ semiquinone radical, which is
expected to react reversibly with O2 to yield
2,5-dimethoxy-1,4-benzoquinone (2,5-DMBQ) and the perhydroxyl radical
(·OOH) (Fig. 1, reaction 3).
H2O2 is produced when
·OOH and its conjugate base, superoxide
(O2·
), dismutate
or when either of these oxyradicals is reduced by Fe2+ (13). It is also possible, but
has not yet been established, that some of the semiquinones reduce
Fe3+ instead of O2 (Fig. 1,
reaction 4). Finally, the fungal mycelium reduces 2,5-DMBQ to 2,5-DMHQ,
thus completing the redox cycle to enable the production of additional
Fenton reagent (Fig. 1, reaction 5).
This cycle suffices to generate Fenton reagent in G. trabeum
cultures (17), but the process in vivo is probably more
complex for at least two reasons. First, G. trabeum produces
not only 2,5-DMHQ but also another hydroquinone, 4,5-dimethoxycatechol (4,5-DMC) (Fig. 2), and is able to reduce a variety of quinones (25). Therefore, it is possible that 4,5-DMC and its
oxidized form, 4,5-dimethoxy-1,2-benzoquinone (4,5-DMBQ), undergo an
additional redox cycle as described above. Second, G. trabeum produces extracellular oxalic acid (7).
Oxalate chelates Fe3+ strongly (24,
31), and Fe3+-oxalate complexes are
relatively poor oxidants whose reactivity with methoxyhydroquinones
remains to be determined. To address these questions, we have compared
the abilities of the 2,5-DMHQ-2,5-DMBQ and 4,5-DMC-4,5-DMBQ couples
to reduce Fe3+-oxalate, to reduce
O2, and to support the generation of
extracellular Fenton reagent by G. trabeum.
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MATERIALS AND METHODS |
Reagents.
4,5-DMBQ was prepared by a modified literature
method (26). Phenol (1.5 g) and dry powdered copper (100 mg, 150 mesh) were combined with 24 ml of methanol-pyridine (2:1). The
mixture was placed in a stoppered flask that was fitted with a balloon.
The flask was flushed with O2, after which it was
sealed and the balloon was inflated with about 2.5 liters of
O2. The mixture was stirred at ambient
temperature for 3 h and then removed and filtered through a
Büchner funnel. The filtrate was concentrated by rotary vacuum evaporation until it was almost dry and then redissolved in methylene chloride-methanol (985:15). It was then applied to a column (57 by 4 cm) of silica gel (SilicAR CC-4 Special; Mallinckrodt), which was
developed with the same solvent. Fractions were collected, and those
that contained 4,5-DMBQ were identified by comparing their mobility
with that of an authentic 4,5-DMBQ standard, which was kindly donated
by P. J. Kersten (USDA Forest Products Laboratory, Madison, Wis.)
(18). The crude 4,5-DMBQ thereby obtained was chromatographed again by the same procedure, and the purified compound
was recrystallized from methanol to yield 250 to 300 mg of orange
needlelike crystals that melted at 228 to 230°C (literature melting
point, 226 to 228°C [3]). The mass spectrum,
1H nuclear magnetic resonance spectrum, and
13C nuclear magnetic resonance spectrum of the
product were the same as those reported previously (26).
To prepare 4,5-DMC, 300 mg of 4,5-DMBQ was dissolved in 20 ml of
glass-distilled ether and shaken with 20 ml of cold saturated sodium
dithionite solution in an acid-washed separatory funnel. The ether
phase was kept briefly in an acid-washed beaker under a stream of
argon, while the aqueous phase was extracted again with ether. The
combined ether phases were then dried over a small column of sodium
sulfate and collected in an acid-washed beaker under a stream of argon.
The solvent was removed by rotary vacuum evaporation, yielding about
150 mg of yellowish-white crystals that melted at 114 to 115°C
(literature melting point, 114°C [3]). The product
gave a single peak by gas chromatography. Mass spectrum (m/z): 170 (M+), 155 (
CH3), 141 ( HCO), 127 ( CH3, CO), 109 ( CH3, CO, H2O). The crystalline 4,5-DMC was
stored at 20°C under argon.
Crystalline 2,5-DMBQ was purchased from TCI America. Crystalline
2,5-DMHQ, prepared by reducing 2,5-DMBQ with sodium dithionite as
described previously (17), was stored at 20°C under
argon. Polyethylene glycol (PEG) labeled with 14C
at its terminal hydroxyethyl groups (15.3 mCi
g1, 4,000 molecular weight) was obtained from
Amersham. For depolymerization experiments, this
[14C]PEG was diluted with unlabeled PEG (4,000 molecular weight) to a specific activity of 0.73 mCi
g1. All other chemicals were commercially
available, reagent-grade products.
Organism.
For most experiments, G. trabeum (ATCC
11539) was grown in stationary 125-ml Erlenmeyer flasks that contained
5 or 6 ml of Kirk's medium with the basal levels of trace elements
(20). The carbon source was glucose (55.6 mM), and the
nitrogen sources were ammonium nitrate (0.5 mM) and asparagine (0.5 mM). We refer to this medium as medium A. The medium was inoculated
with homogenized potato dextrose agar cultures of the fungus (1 g per
100 ml of medium) and incubated at 32°C under air in the dark.
The mycelial mats and extracellular medium were used for experiments 7 days after inoculation, at which time the average dry weight of one mat
was 10 mg and the average pH of the medium was 4.1. For experiments on
PEG cleavage or quinone reduction by G. trabeum mycelium,
the mats were removed from 50 cultures and shaken gently for 1 h
in 1 liter of distilled, deionized water at ambient temperature. The
mats were then transferred to fresh water, and the procedure was repeated.
In one experiment to investigate the production of extracellular
metabolites, G. trabeum was also grown under the conditions described by Paszczynski et al. (25). The cultures were
set up in stationary Roux flasks that contained 150 ml of medium A with
the following supplements: diammonium tartrate, 1.1 mM;
MnCl2, 0.1 mM; and Tween 80, 0.05%. We refer to
this medium as medium B. One mycelial mat from a 4-week Roux flask
culture was homogenized in 130 ml of sterile water, and this suspension
was used to inoculate the cultures (7 ml per 100 ml of medium). The
cultures were incubated at 24°C under air in the dark, and the
extracellular medium was analyzed 5 weeks after inoculation.
Metabolites in the culture medium.
Preliminary experiments
showed that the hydroquinones in the extracellular medium of G. trabeum cultures were oxidized rapidly if the mycelial mats were
disturbed. Therefore, a 250-µl high-performance liquid chromatography
(HPLC) syringe was used to obtain samples (200 µl) of medium from the
cultures with as little mixing as possible. A 125-µl portion of the
sample was purposely oxidized by delivering it into an Eppendorf tube
that contained 5 µl of 10 mM FeCl3. Tests with
standard solutions of 2,5-DMHQ and 4,5-DMC showed that this procedure
converted these substances to their corresponding quinones with >98% yields.
The remaining 75 µl was injected immediately, without filtration,
onto a C18 reversed-phase HPLC column (Phenomenex
Luna; 150 by 4.6 mm, 5-µm particle size). The column was eluted
isocratically with water-acetonitrile-formic acid (900:100:1) at 1.5 ml
min1 and ambient temperature. The absorbance of
the eluate was monitored at 295 nm, a wavelength at which 2,5-DMBQ and
4,5-DMBQ exhibit the same extinction coefficient. The elution times for
the metabolites were as follows: 2,5-DMHQ and 4,5-DMC, 6.5 min;
4,5-DMBQ, 7.5 min; and 2,5-DMBQ, 9.5 min.
When the HPLC analysis was completed, 75 µl of the
FeCl3-oxidized sample was chromatographed by the
same procedure. External standards of 2,5-DMBQ and 4,5-DMBQ were then
chromatographed to obtain response factors. The original concentrations
of 2,5-DMBQ and 4,5-DMBQ in the medium were calculated from their peak
areas in the chromatogram of the untreated sample. The original
concentrations of 2,5-DMHQ and 4,5-DMC were then calculated by
subtracting these peak areas from the peak areas for 2,5-DMBQ and
4,5-DMBQ in the FeCl3-oxidized sample. This
indirect method of determining the hydroquinone concentrations was
necessary because we were unable to devise an HPLC protocol that
separated 2,5-DMHQ from 4,5-DMC.
Oxalate in the extracellular medium of G. trabeum cultures
was determined enzymatically with a coupled oxalate oxidase-peroxidase assay kit according to the manufacturer's instructions (Sigma Chemical
Co., St. Louis, Mo.).
Prediction of iron species in solution.
Equilibrium
constants for the binding of oxalic acid, catechol, and
2,3-dihydroxybenzoic acid to Fe3+ were obtained
from the National Institute of Standards and Technology database
containing critically selected stability constants of metal complexes
(31). When several constants for the same equilibrium were
shown, the one for the lowest ionic strength was chosen. To model
competition between the catecholate species and oxalate for
Fe3+, 15 equations, 12 equilibria, and three mass
balance equations were solved simultaneously with the SOLVER function
of the Microsoft Excel computer program. Since the National Institute
of Standards and Technology database contains constants for the
association of only a single type of ligand with each cation, the
mixed-ligand-species constants were estimated by assuming that, for
example, Fe(oxalate)(catechol) was formed with
a constant whose logarithm is the average of the logarithm of the
constant for Fe(catechol)2 and
the logarithm of the constant for
Fe(oxalate)2. This is likely a
poor assumption, but since the mixed species represent a very small
fraction of the total chelated species, the resultant error is small.
PEG depolymerization by the mycelium.
PEG depolymerization
assays were done in 50-ml beakers that each contained 11.0 ml of sodium
oxalate buffer (1.0 mM, pH 4.1), two G. trabeum mycelial
mats, FeCl3 (50 µM),
[14C]PEG (330 mg
liter1), and various amounts of quinone.
Calculations showed that the oxalate buffer would not scavenge a
significant proportion of the ·OH produced under these
conditions (see data for oxalate and for diethylene glycol at the Notre
Dame Radiation Laboratory Radiation Chemistry Data Center
[http://allen.rad.nd.edu]). The reaction mixtures were
rotary shaken at 200 rpm and ambient temperature for 2 h in the
dark. The extracellular pH increased an average of 0.2 units during
this time. Samples (200 µl) were taken from triplicate reaction
mixtures at 1 and 2 h, and each was filtered through a
0.45-µm-pore-size membrane. More than 99% of the
14C in the sample was recovered by this
procedure. A portion (150 µl) of each filtrate was analyzed by gel
permeation chromatography (GPC) and scintillation counting of the
collected fractions as described previously (17). The
column was calibrated beforehand with PEG standards of known molecular
weight, also as previously described (17).
Calculation of the number of chain scissions in PEG.
Because
commercially available [14C]PEG is labeled only
on the ends of the polymer, its scission yields significant quantities of unlabeled polymer that are silent when analyzed by our GPC procedure. As the reaction progresses, the difference between the
molecular weight distributions of the labeled and unlabeled material
first increases and then decreases. To address this problem, we
obtained the relationship between the true molecular weight and the
molecular weight of the labeled material by computer simulation. The
program was written in Visual Basic for Microsoft Excel and is
available from the authors upon request.
From the specific activity and molecular weight of the PEG, we
determined that it had, on average, one
14C-labeled end group per polymer molecule. We
assumed that these end groups were distributed with relative
frequencies of 1:2:1 among molecules with zero, one, or two labeled end
groups, respectively. In the simulation, 105
linear molecules were defined with the same molecular weight distribution as the undegraded PEG, and then changes in the molecular weight distributions of the labeled and unlabeled materials were monitored during 6 × 105 random scission
events. From this simulation, we developed an empirical model to
determine the true molecular weight from the GPC data. Comparisons of
the simulated GPC data with the experimental data showed that they
corresponded closely, so our assumption of random chain scission in
experimental samples appears to be correct. The average number of
scissions (n) per polymer was calculated from the equation
n = (Mn0/Mnt) 1, where Mn0 is the
true number average molecular weight at time zero and Mnt is the true number
average molecular weight at time t.
Fe3+ reduction by the hydroquinones.
Acid-washed
glassware and Milli-Q water were used to prepare all solutions, and all
operations were conducted in dim light. Stock solutions of 2,5-DMHQ and
4,5-DMC (2.0 mM) were prepared in 1.0 mM sodium oxalate (final pH, 4.1)
under argon. A stock solution of FeCl3 (1.0 mM)
was prepared in 2.0 mM sodium oxalate (final pH, 4.1), also under
argon. For anoxic reactions, 65.0 ml of argon-saturated sodium oxalate
buffer (1.0 mM, pH 4.1) and 30.0 ml of argon-saturated
FeCl3 stock solution were added at 25°C to a
stirred, water-jacketed beaker, which was kept under a stream of argon.
The reaction was initiated by adding one of the anoxic hydroquinone
stock solutions (5.0 ml) to give initial concentrations of 300 µM
Fe3+ and 100 µM hydroquinone in 1.3 mM sodium
oxalate buffer. Samples (1.5 ml) were removed at various time and were
mixed rapidly with an equal volume of 4.0 mM bathophenanthroline
disulfonic acid (BPS) in 200 mM sodium oxalate (pH 4.1). The absorbance
at 535 nm was read immediately, and an extinction coefficient of 22.1 mM1 cm1 was used to
calculate the amount of Fe2+ formed
(2). Oxic reactions were performed in the same way, except
that the hydroquinone stock solutions were the only ones kept under argon.
Additional reactions were run in the presence of BPS under anoxic
conditions to determine the stoichiometry of Fe3+
reduction by the hydroquinones under irreversible conditions. The
reaction mixtures contained FeCl3 (100 µM),
2,5-DMHQ or 4,5 DMC (26 µM), and BPS (2.0 mM) in 3.0 ml of sodium
oxalate buffer (1.1 mM; final pH, 4.1) and were constantly stirred. The
hydroquinones were used to start the reactions, which were monitored at
535 nm in a Hitachi U-3010 double-beam, monochromator-equipped
UV/visible spectrum spectrophotometer. We observed that if the
reactions were done instead in a diode-array-equipped
spectrophotometer, the greater intensity of incident light resulted in
a slow but detectable photoreduction of the
Fe3+-oxalate.
Iron-dependent O2 reduction by the hydroquinones and
quinones.
O2 uptake was determined with a
Yellow Springs Instruments model 5300 oxygen electrode.
Solutions were prepared as described for the oxic
Fe3+ reduction experiments and were equilibrated
to 25°C before use. The reactions (1.8-ml reaction volumes) were
conducted in dim light at 25°C in a stirred, water-jacketed glass
chamber that had been washed with acid beforehand. Control reactions
without iron were always performed before iron-containing reactions.
For complete hydroquinone-dependent reactions, 1,170 µl of
air-saturated sodium oxalate buffer (1.0 mM, pH 4.1) and 540 µl of
air-saturated FeCl3 solution were added to the
cell. A portion of the anoxic hydroquinone stock solution was then
vortexed briefly in an acid-washed Eppendorf tube to oxygenate it, and
90 µl was injected into the cell to start the reaction. This
procedure gave initial concentrations of 300 µM
Fe3+ and 100 µM hydroquinone in 1.3 mM sodium
oxalate buffer (final pH, 4.1). Reverse reactions were conducted
similarly, with 100 µM quinone instead of 100 µM hydroquinone and
with 200 µM FeCl2 in place of 300 µM
FeCl3. Control reactions contained oxalate buffer
of the appropriate concentration in place of the hydroquinone, quinone,
or iron solution.
Reduction of 2,5-DMBQ and 4,5-DMBQ by G.
trabeum
Each assay mixture used for determination of
2,5-DMBQ or 4,5-DMBQ reduction contained three G.
trabeum mycelial mats and 50 µM quinone in 18.0 ml of sodium
oxalate buffer (1.0 mM, pH 4.1). Preliminary experiments showed that
this buffer sufficed to maintain the pH at 4.1 ± 0.1 for more
than 1 h. In some assays, 1 mM desferrioxamine was also added to
minimize iron-catalyzed cycling of the hydroquinone-quinone couples.
The reductions were done in acid-washed 50-ml beakers, which were
rotary shaken at 200 rpm and ambient temperature in the dark. Samples
(100 µl) were withdrawn with an HPLC syringe at time zero and every 7 min thereafter. They were injected immediately, without filtration,
onto the Phenomenex Luna HPLC column described above. The column was
eluted with water-acetonitrile-formic acid (850:150:1) at 1.5 ml
min1 and ambient temperature. Each hydroquinone-quinone
pair was separated with baseline resolution. Their retention times were
as follows: 2,5-DMHQ, 3.2 min; 2,5-DMBQ, 4.1 min; 4,5-DMC, 3.2 min; and
4,5-DMBQ, 3.4 min. External standards of the four metabolites were used to obtain response factors for quantitation.
Reduction of 4,5-DMBQ by 2,5-DMHQ.
Solutions used to measure
the reduction of 4,5-DMBQ by 2,5-DMHQ were prepared as described above
for the Fe3+ reduction experiments. The reaction
was monitored in a Shimadzu 1501 diode-array UV/visible
spectrophotometer. The reaction mixture contained 2,5-DMHQ (150 µM),
4,5-DMBQ (150 µM), and sodium oxalate buffer (1.0 mM, pH 4.1) in a
final volume of 3.0 ml. The reaction mixture was stirred at 25°C in
an acid-washed, 1-cm-path-length quartz cell, and spectra were recorded
for 1.5 min at 10-s intervals. The spectrum at zero time was
reconstructed from pure component spectra of 2,5-DMHQ and 4,5-DMBQ.
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RESULTS AND DISCUSSION |
Extracellular quinonoid metabolites of G.
trabeum
We confirmed the observation of Paszczynski et
al. that G. trabeum produces not only an extracellular
p-hydroquinone, 2,5-DMHQ, but also an extracellular
catechol, 4,5-DMC (25). In addition, we observed the
corresponding quinones, 2,5-DMBQ and 4,5-DMBQ (Fig.
3; Table
1). In 7-day cultures grown in medium A
under previously described conditions (17), and also in
5-week cultures grown in medium B under the conditions described by
Paszczynski et al. (25), the ratio of 2,5-DMBQ plus
2,5-DMHQ to 4,5-DMBQ plus 4,5-DMC was greater than 5:1. However, the
4,5-dimethoxy metabolite was significantly more reduced than the
2,5-dimethoxy metabolite (Table 1).
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FIG. 3.
HPLC analysis of extracellular metabolites from
G. trabeum cultures. (A) Untreated sample; (B) sample
oxidized with FeCl3. The labeled peaks consisted of
2,5-DMHQ plus 4,5-DMC (a), 4,5-DMBQ (b), and 2,5-DMBQ (c).
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Extracellular Fe3+ chelators of G.
trabeum.
The extracellular concentration of oxalate
(mean ± the standard deviation) in our 7-day cultures was
500 ± 150 µM (n = 4). In addition, the cultures
produced some unidentified, polar, UV-absorbing metabolites (Fig. 3),
some of which might be Fe3+-binding catechols of
the type reported earlier by Goodell et al. (11). With the
exception of 4,5-DMC, these catechols have not been described in
detail, but they are thought to bind Fe3+ through
two vicinal phenolate groups (as in catechol) or through vicinal
phenolate and benzoate groups (as in 2,3-dihydroxybenzoic acid).
To compare how oxalate and catechols might contribute to
Fe3+ chelation in G. trabeum cultures,
we assumed that all of the unidentified UV-absorbing material in the
chromatogram of Fig. 3A (including the void-volume peak) consisted of
compounds with properties similar to those of catechol or
2,3-dihydroxybenzoic acid. It was then possible to calculate that the
G. trabeum cultures we used contained no more than a 500 µM concentration of unidentified catecholic compounds.
We used published binding constants (31) to estimate the
concentrations of Fe3+ chelates that would be
present if 20 µM Fe3+, 500 µM oxalate, and
500 µM 2,3-dihydroxybenzoate or catechol were present simultaneously.
The results show that although both catechols compete effectively with
oxalate as Fe3+ chelators at neutral pH, neither
of them does at pH 4.1, the pH value found in G. trabeum
cultures (Table 2). Therefore, we concluded that oxalate, rather than a catechol, was the dominant chelator of Fe3+ under our culture conditions.
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TABLE 2.
Predicted chelates of Fe3+ (20 µM) in the
presence of 500 µM oxalate and a 500 µM concentration of the
indicated catechol
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Efficacy of the quinones in the extracellular Fenton system.
To compare ·OH generation by the 2,5-DMHQ-2,5-DMBQ and
4,5-DMC-4,5-DMBQ couples, we measured the rate of PEG depolymerization by G. trabeum mycelium in the presence of
Fe3+, dilute oxalate, and each of the quinones.
PEG is a useful target molecule for assays of extracellular Fenton
reagent because it does not penetrate cell membranes (23, 28,
29) and because it is cleaved by strongly oxidizing radicals
that abstract aliphatic hydrogens (5, 10, 12, 16).
The data showed that both quinones supported PEG cleavage, but the
reaction with 4,5-DMBQ was significantly slower than the reaction with
2,5-DMBQ, even when 4,5-DMBQ was supplied at concentrations higher than
those found in G. trabeum cultures (Table
3). We also examined PEG cleavage with
both quinones present simultaneously at physiological concentrations,
but we found no evidence that they acted synergistically. No PEG
cleavage occurred when the quinones were omitted. We did not perform
controls without Fe3+ because our previous data
already showed that it is essential for the reaction to proceed
(17).
When reactions 2 through 5 (Fig. 1) are considered, it is evident that
there are three possible explanations for these results. First, 4,5-DMC
might reduce Fe3+ more slowly than 2,5-DMHQ does
(reaction 2 and possibly reaction 4). Second, the 4,5-DMC semiquinone
might reduce O2, and thus produce
H2O2, more slowly than the
2,5-DMHQ semiquinone does (reaction 3). Finally, the fungal mycelium
might reduce 4,5-DMBQ more slowly than it reduces 2,5-DMBQ (reaction
5). The experiments described below were designed to assess each of
these possibilities.
Reduction of Fe3+ by 2,5-DMHQ and 4,5-DMC.
First,
we determined the stoichiometries of Fe3+
reduction by 2,5-DMHQ and 4,5-DMC in 1.1 mM oxalate buffer, with the
reactions being conducted under argon in the presence of the
colorimetric Fe2+ chelator BPS. Under these
conditions, Fe3+ reduction by the hydroquinones
is expected to be irreversible and O2 is
unavailable to react with the resulting semiquinones. Each hydroquinone
reduced 1.96 to 1.98 equivalents of Fe3+ within 1 min in this experiment (data not shown). Therefore, the potential
reductants of Fe3+ in the G. trabeum
system include not only 2,5-DMHQ and 4,5-DMC (reaction 2) but also
their corresponding semiquinones (reaction 4).
Next, we compared the rates at which 2,5-DMHQ and 4,5-DMC reduced
Fe3+ in 1.3 mM oxalate under anoxic conditions.
We omitted BPS from the reaction mixtures because colorimetric
Fe2+ chelators affect the reaction rates when
they are present during Fe3+ reductions
(2). Instead, we obtained samples at intervals during the
reaction and mixed them rapidly with concentrated oxalate buffer and
BPS, after which the absorbances were read immediately. This approach
was based on our prior observation that the rate of
Fe3+ reduction by the hydroquinones was inhibited
more than 95% by 100 mM oxalate (data not shown). Although we were
unable to take measurements rapidly enough to obtain linear initial
rates, it is evident from the results (Fig.
4) that 4,5-DMC reduces
Fe3+ in anoxic, dilute oxalate at pH 4.1 somewhat
more rapidly than 2,5-DMHQ does. Therefore, the relatively low
efficiency of the 4,5-DMC-driven Fenton system (Table 3) cannot be
attributed to a lower rate of reaction between 4,5-DMC and
Fe3+ (reactions 2 and 4).
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FIG. 4.
Fe3+ reduction by 2,5-DMHQ under argon
( ), by 2,5-DMHQ under air ( ), by 4,5-DMC under argon ( ), and
by 4,5-DMC under air ( ). In the oxic reactions, the levels of
Fe2+ declined with time (not shown). In the anoxic
reactions, the Fe2+ stabilized at a concentration of 120 µM when 2,5-DMHQ was the reductant and at 70 µM when 4,5-DMC was
the reductant. The difference between these final hydroquinone
concentrations indicates that the standard 2e reduction
potential of the 2,5-DMHQ-2,5-DMBQ couple is 32 mV more negative than
that of the 4,5-DMC-4,5-DMBQ couple, in good agreement with the
published difference of 37 mV (4, 8).
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Finally, we measured the rates at which 2,5-DMHQ and 4,5-DMC reduced
Fe3+ in the same buffer under air. Under these
conditions, Fe3+ is expected to be the only
efficient oxidant of the initial hydroquinones (reaction 2)
(27), but O2 (reaction 3) can
compete with Fe3+ (reaction 4) as an oxidant of
the semiquinones (13). As a result, the oxic reaction will
generate some ·OOH in place of Fe2+, and
some of this ·OOH will oxidize additional
Fe2+ rapidly, thereby generating
H2O2 (13).
This H2O2 will consume yet
more Fe2+ in the Fenton reaction (reaction 1),
which is rapid when oxalate is the ligand of Fe2+
(24). Therefore, if O2 oxidizes the
semiquinones efficiently, one would expect a lower rate of
Fe2+ accumulation under air than under argon. The
data (Fig. 4) showed that O2 markedly inhibited
the initial rate of Fe3+ reduction by 2,5-DMHQ
but had little effect on the initial rate of Fe3+
reduction by 4,5-DMC. This result suggested that the 2,5-DMHQ semiquinone reacts with O2 (reaction 3) more
rapidly than the 4,5-DMC semiquinone does.
Reduction of O2 by 2,5-DMHQ and 4,5-DMC.
The
results of O2 electrode experiments were
consistent with the above-stated hypothesis. In 1.3 mM oxalate buffer
at pH 4.1, the reaction of 2,5-DMHQ with Fe3+
consumed O2 more rapidly than the reaction of
4,5-DMC with Fe3+ (Fig.
5A, curves a and b). Neither hydroquinone
reacted rapidly with O2 in the absence of added
Fe3+ (curves c and d), and the uptake that did
occur was probably attributable to contamination of the reagents with
Fe3+. This result agrees with the general rule
that nonradical species react very slowly with diradical
O2 (27). The low rate of
O2 uptake that we observed in the absence of
hydroquinone (curve e) probably resulted from a photoreaction between
oxalate and Fe3+ (14).
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FIG. 5.
O2 reduction by the hydroquinone-quinone
couples in the presence of iron. (A) With hydroquinone and
Fe3+. The curves show O2 uptake with 2,5-DMHQ
plus Fe3+ (a), with 4,5-DMC plus Fe3+ (b), with
2,5-DMHQ minus Fe3+ (c), with 4,5-DMC minus
Fe3+ (d), and with Fe3+ minus hydroquinone (e).
(B) With quinone and Fe2+. The curves show O2
uptake with 2,5-DMBQ plus Fe2+ (a), with 4,5-DMBQ plus
Fe2+ (b), with Fe2+ minus quinone (c), and with
oxalate buffer alone (d).
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Further experiments showed that these reactions were reversible; i.e.,
the quinones could be used to oxidize Fe2+ with
concomitant O2 consumption.
O2 uptake was considerably faster with 2,5-DMBQ
than it was with 4,5-DMBQ (Fig. 5B, curves a and b), which again
suggests that the 2,5-DMHQ semiquinone reacts with
O2 more rapidly than the 4,5-DMC semiquinone
does. The autooxidation of Fe2+-oxalate (curve c)
and the photoreaction of oxalate with contaminating Fe3+ (curve d) made relatively small
contributions to these O2 uptake rates.
In sum, these results indicate that the extracellular hydroquinones of
G. trabeum generate Fenton reagent by the iron-dependent pathways of reactions 2 to 4 (Fig. 1). Although 4,5-DMC reduces Fe3+ more rapidly than 2,5-DMHQ does via reaction
2, it reduces O2 more slowly via reaction 3, and
consequently there is insufficient H2O2 to react with the
Fe2+ that accumulates in place of ·OOH via
reaction 4. These results explain, in part, why 2,5-DMBQ supports more
rapid PEG cleavage than 4,5-DMBQ does.
Reduction of 2,5-DMBQ and 4,5-DMBQ by G.
trabeum.
Reduction of the quinones (Fig. 1, reaction 5) is
the step that drives G. trabeum extracellular Fenton
chemistry. We had already observed that the fungal mycelium reduces
2,5-DMBQ rapidly (17), and it seemed reasonable that
4,5-DMBQ might be reduced likewise. However, we found that the rate of
4,5-DMBQ reduction by fungal mycelium (in 1.0 mM oxalate, pH 4.1) was
much lower than the rate of 2,5-DMBQ reduction (Fig.
6). This result cannot be attributed to a
rapid reoxidation of 4,5-DMC by contaminating
Fe3+, because the rate of reduction was
unaffected by desferrioxamine (1 mM), which forms with
Fe3+ a tight complex that is inert to most
reductants (13). These data provide an additional
explanation for the low rate of 4,5-DMBQ-supported PEG cleavage that we
observed in the experiment described in Table 3.
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FIG. 6.
Reduction of 2,5-DMBQ and 4,5-DMBQ by G.
trabeum. The curves show results obtained in four separate
incubations: increase in 2,5-DMHQ and decrease in 2,5-DMBQ in the
presence of desferrioxamine (), increase in 2,5-DMHQ and decrease in
2,5-DMBQ in the absence of desferrioxamine ( ), increase in 4,5-DMC
and decrease in 4,5-DMBQ in the presence of desferrioxamine (), and
increase in 4,5-DMC and decrease in 4,5-DMBQ in the absence of
desferrioxamine (). This experiment was repeated with different
mycelial mats and gave essentially the same results (data not shown).
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Reduction of 4,5-DMBQ by 2,5-DMHQ.
Even though the G. trabeum mycelium reduced 4,5-DMBQ more slowly than it reduced
2,5-DMBQ, it was evident that the fungus was able to maintain the
4,5-DMC-4,5-DMBQ couple in a more reduced state than it maintained the
2,5-DMHQ-2,5-DMBQ couple (Table 1). The only likely explanation
appeared to be that 2,5-DMHQ, rather than the mycelium, was reducing
4,5-DMBQ in fungal cultures. Spectrophotometric experiments confirmed
that 2,5-DMHQ reduced 4,5-DMBQ rapidly and quantitatively in 1.0 mM
oxalate buffer without any requirement for mycelium or exogenous
Fe3+ (Fig. 7).
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FIG. 7.
Spectrophotometric observation of 4,5-DMBQ reduction by
2,5-DMHQ. The arrows indicate the directions of change in
absorbance. The final spectrum was the same as that exhibited by 150 µM 2,5-DMBQ plus 150 µM 4,5-DMC.
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The most obvious route for this oxidoreduction is one that is catalyzed
by contaminating Fe3+ in the reagents; i.e.,
2,5-DMHQ reduces Fe3+, and the resulting
Fe2+ then reduces 4,5-DMBQ. However, we found
that the reaction between 2,5-DMHQ and 4,5-DMBQ was not stimulated by
50 µM Fe3+ and was inhibited only slightly by 1 mM desferrioxamine (data not shown). Therefore, we suspect that the
redox reaction between 2,5-DMHQ and 4,5-DMBQ is chiefly iron
independent. One possibility is that it proceeds via hydride transfer
within mixed quinhydrone complexes between 2,5-DMHQ and 4,5-DMBQ
(35).
Summary of the pathways for quinone reduction.
Even though
4,5-DMBQ is a poor substrate for the mycelial quinone-reducing system
of G. trabeum, the rapid reduction of this quinone by
2,5-DMHQ provides a route for the formation of 4,5-DMC. The fungus
evidently can reduce 4,5-DMBQ directly (Fig. 6), but we cannot rule out
the possibility that our washed mycelium contained some bound 2,5-DMBQ.
Therefore, we suspect that 4,5-DMBQ is primarily reduced outside the
mycelium through a 2,5-DMHQ-2,5-DMBQ redox shuttle. 2,5-DMBQ is
presumably reduced to 2,5-DMHQ by a mycelial quinone reductase, which
we are presently attempting to identify.
Implications for wood decay.
It remains unclear whether there
is a mechanistic reason for the production of two extracellular
hydroquinone-quinone couples by G. trabeum, but it is
possible that the fungus uses them to modulate the reactivity of its
Fenton system. When the 2,5-DMHQ-driven reaction operates alone in air,
it generates enough ·OOH and
H2O2 to oxidize all of the
Fe2+-oxalate that it produces (Fig. 4), and these
oxidations occur rapidly, with rate constants of
104 M1
s1 or higher (13, 24). Under these
conditions, the Fenton reaction probably occurs near the site where
2,5-DMHQ first reduces Fe3+-oxalate. By contrast,
excess Fe2+-oxalate is produced if the
4,5-DMC-driven reaction operates (Fig. 4). Because it autooxidizes with
a rate constant of less than 10 M1
s1 at pH 4 (24),
Fe2+-oxalate may diffuse to more remote
locations, where it could initiate Fenton chemistry by reducing
2,5-DMBQ or 4,5-DMBQ. Even if the quinones are unavailable,
Fe2+-oxalate autooxidation will lead to slow
·OH production, as proposed by Hyde and Wood (14).
Wood's group has already pointed out that secreted oxalic acid tends
to protect the hyphae of brown rot fungi from oxidative damage because
Fe2+-oxalate autooxidation is slow at low pH
(14, 24). Our results suggest that two additional
protective mechanisms probably operate in the G. trabeum
system. First, a low pH in the hyphal vicinity will inhibit the
reduction of Fe3+ by 2,5-DMHQ and 4,5-DMC because
protons are products of these reactions (Fig. 1, reactions 2 and 4).
Second, a high oxalate concentration near the hyphae will inhibit
Fe3+ reduction by the hydroquinones, because when
oxalate is present in excess at pH 4, almost all of the
Fe3+ is present as
Fe(oxalate)33, which has a
standard 1e reduction potential
(E0) of 121 mV (24).
This value is considerably more negative than those expected for
typical methoxyhydroquinones and methoxysemiquinones (13). By contrast, methoxyhydroquinones and
methoxysemiquinones are expected to be better reductants of
Fe(oxalate)2
(E0 = +181 mV) and
Fe(oxalate)+ (E0 = +430 mV), which will be more abundant in dilute oxalate at a distance
from the hyphae (24).
We are much indebted to B. Kalyanaraman for advice on quinone
free-radical chemistry. K. Hirth and D. Dietrich kindly performed spectrometric measurements on the quinone and hydroquinone standards.
This work was supported by U.S. Department of Energy grant
DE-FG02-94ER20140 to K.E.H.
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Abstract
Introduction
