Bioorganic mechanisms of the formation of free radicals catalyzed by glucose oxidase

Article abstract of DOI:10.1006/bioo.2001.1229

In this communication, we have described the activation of several xenobiotics by glucose oxidase from Aspergillus niger. The following compounds are readily reduced by D-glucose, in the presence of glucose oxidase: p-nitroso-N,N-dimethylaniline, methyl-1

Full text of DOI:10.1006/bioo.2001.1229

Bioorganic Chemistry 30, 95–106 (2002)
doi:10.1006/bioo.2001.1229, available online at http://www.idealibrary.com on
Bioorganic Mechanisms of the Formation of Free Radicals
Catalyzed by Glucose Oxidase
Svetlana Trivic´,* Vladimir Leskovac,†,1 Jasmina Zeremski,‡ Miroslav Vrvic´,§
and Gary W. Winston¶
*Faculty of Science Novi Sad, †Faculty of Technology Novi Sad, ‡Faculty of Technology
and Metallurgy Beograd, and §Faculty of Chemistry Beograd, Yugoslavia;
¶
and North Carolina State University, Raleigh, North Carolina 27695
Received June 5, 2001
In this communication, we have described the activation of several xenobiotics by glucose
oxidase from Aspergillus niger. The following compounds are readily reduced by D-glucose,
in the presence of glucose oxidase: p-nitroso-N,N-dimethylaniline, methyl-1,4-benzoquinone,
and 7,7,8,8-tetracyano-quinodimethane. In each case, the products of enzymatic reduction
undergo a dismutation reaction with the parent compound and thus afford the formation of free
radicals. In some cases, and at an appropriate pH value, the transformation of a parent compound
into free radicals is almost quantitative. Under optimal conditions, free radicals are stable for
several minutes in aqueous solutions under physiological conditions.
᭧ 2002 Elsevier Science (USA)
INTRODUCTION
Investigation of the enzyme-catalyzed formation of free radicals, in this communica-
tion, was prompted by our early observation that yeast alcohol dehydrogenase is able
to catalyze reduction of p-nitroso-N,N-dimethylaniline by NADH (Leskovac et al.,
1996); in the course of this enzymatic reaction, a considerable amount of relatively
stable quinone diimine cation radicals are formed from NDMA (Leskovac, 1999;
Leskovac et al., 2002). In this reaction, NDMA is catalytically activated by an
enzymatic reduction with NADH, followed with the formation of free radicals by the
process of dismutation.
Further, we have found that a flavoenzyme glucose oxidase from Aspergillus niger
can catalyze reduction of NDMA with glucose, followed by the same process of
radical dismutation. Glucose oxidase is able to catalyze reduction of a large number
of substrates with glucose (Wilson and Turner, 1992). In the course of this work, we
have found that, among many substrates, quinones and some quinoid compounds are
activated by glucose oxidase by the similar process of radical dismutation; considerable
amounts of stable free radicals are generated in these reactions.
Stable free radicals, formed by enzymatic activation of xenobiotics, are generally
1 To whom correspondence and reprint requests should be addressed. Fax: ϩϩ 381-21-450-413.
E-mail: leskovac@techol.ns.ac.yu.
95
0045-2068/02 $35.00
᭧ 2002 Elsevier Science (USA)
All rights reserved.

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TRIVIC ET AL.
96
toxic in living cells; the chemical and enzymatic reactions of radical dismutation are,
therefore, important in toxicology. It appeared to us, that little attention has been paid
to this type of reactions, even in the vast scientific literature dealing with the biochemi-
cal action of free radicals in living cells. For this reason, we have investigated the
activation of various quinoid compounds with glucose oxidase by the chemical process
of radical dismutation, which is described in this communication.
MATERIALS AND METHODS
Biochemicals
Kinetic measurements were performed using glucose oxidase preparations from
A. niger (EC 1.1.3.4), type VII-S, obtained from Sigma. According to Sigma, the
lyophilized powder contained approximately 80% protein; balance was phosphate
buffer and sodium chloride. The catalase impurity in Sigma preparations was sufficient
to remove rapidly the H2O2 product from solution. The specific activity of enzyme
was 100,000–200,000 units/gram solid (without added oxygen); one unit will oxidize
1.0 ␮mol of ␤-D-glucose to D-gluconic acid and H2O2 per minute, at pH 5.1 and
35ЊC. Glucose oxidase is a homodimer, composed of two identical subunits; the
molecular weight of enzyme is 150,000 Da. Each subunit carries one molecule of
tightly bound coenzyme, FAD, which is acting as a redox carrier in catalysis (Gibson
et al., 1964). The concentration of enzyme active sites was determined from its molar
extinction coefficient at 450 nm (␧ ϭ 14100 MϪ1 cmϪ1, in 0.1 M sodium acetate buffer,
pH 5.5) (Gibson et al., 1964); the concentration of enzyme in solution throughout this
work was expressed as the concentration of its active sites.
Cytochrome-c from horse heart was obtained from Sigma (C 7752). Its concentration
was determined from the molar extinction coefficient of ferricytochrome-c at 450 nm
(␧ ϭ 105000 MϪ1 cmϪ1), or from the difference between the spectrum of ferricyto-
chrome-c and ferrocytochrome-c at 550 nm (␧ ϭ 21,000 MϪ1 cmϪ1) (Dickerson and
Timkovich, 1975).
Methyl-1,4-benzoquinone was obtained from Aldrich and purified by sublimation;
since quinones are sensitive to light, the freshly prepared solutions were kept in dark.
7,7,8,8-Tetracyano-quinodimethane and p-nitroso-N,N-dimethylaniline were obtained
from Sigma, and used without further purification.
Throughout this work, 0.1 M McIlvaine citrate-phosphate buffers were used to
maintain the pH values from 2.9–6.5, except when stated otherwise.
Methods
The initial velocity studies were carried out and the absorption spectra were recorded
in a double-beam spectrophotometer, in thermostated cuvette holders at 25ЊC. The
concentrations of substrates and enzymes were estimated from their molar extinction
coefficients (Table 1).
The initial rate of cytochrome-c reduction in Figs. 5, 6, and 7, was measured at
550 nm.
RESULTS
Enzymatic Reduction of TCQD with Glucose and Glucose Oxidase
7,7,8,8-Tetracyano-quinodimethan (TCQD) is readily reduced with
D-glucose, un-
der anaerobic conditions, in the presence of glucose oxidase. In the course of this

MECHANISMS OF FREE RADICALS CATALYZED BY GOD
97
TABLE 1
Molar Extinction Coefficients in Aqueous Solutions
Compound
␭
(nm)
␧ (MϪ1 cmϪ1
)
pH
Source
max
Glucose oxidase
TCQD
TCQDH2
TCQD-radical
NDMA
DPD-Radical
MeBQ
450
395
476.2
740.7
442
550
332
253
407
14100
63600
22000
23280
35400
13450
513
18563
105000
21000
5.5
5.5
5.5
5.5
7.0
Swoboda and Massey (1966)
Acker and Herter (1962)
Svircˇevic´ (1987)
Melby et al. (1962)
Dunn and Bernhard (1971)
4.55 This work
5.5
5.5
7.0
7.4
Leskovac et al. (2002)
Leskovac et al. (2002)
Dickerson and Timkovich (1975)
Massey (1959)
Ferri-cytochrome-c
Ferro/ferri-cytochrome-c
550
enzymatic reaction, characteristic spectral changes are taking place simultaneously:
the spectrum of TCQD at 400 nm decreases gradually while the spectra of p-phenylene-
•
dimalononitrile (TCQDH2) at 476 nm and of TCQD-radical (TCQDH ) at 741 nm
are increasing, with an isosbestic point at 420 nm. Below pH 6.0, reduction of TCQD
does not take place in the absence of glucose oxidase.
Figure 1 shows the reaction progress curves at pH 6.0.
Enzymatic reduction of TCQD with glucose presumably proceeds by the follow-
ing mechanism:
E.FAD ϩ
D-Glucose → E.FADH2 ϩ ␦-Gluconolactone
(1)
FIG. 1. Reaction progress curves for the enzymatic reduction of TCQD at pH 6.0. Initial concentration
of reactants: [GOD] ϭ 16.23 nM; [TCQD] ϭ 13.11 ␮M; [Glc] ϭ 472 mM.

´
98
TRIVIC ET AL.
E.FADH2 ϩ TCQD → E.FAD ϩ TCQDH2
(2)
(3)
(4)
•
TCQDH2 ϩ TCQD s 2 TCQDH
•
Total: 2 TCQD ϩ
D
-Glucose → 2 TCQDH ϩ ␦-Gluconolactone
where E.FAD is the oxidized form of glucose oxidase, and E.FADH2 is the reduced
form of enzyme.
From Fig. 1, one can calculate that the initial rate of the disappearance of TCQD
(4.19 ␮M/min) is almost identical with the initial rate of appearance of TCQD-radicals
(4.05 ␮M/min). Thus the overall stoichiometry shown by equation (4) holds for the
initial phase of reaction, with equation (2) being the rate-limiting step of the overall
process; from the above data, one can calculate the bimolecular rate constant for this
reaction at pH 6.0, kII ϭ 330 mMϪ1 sϪ1.
Nonenzymatic Reduction of TCQD with NADH
TCQD is readily reduced with NADH, under aerobic conditions (Fig. 2).
Nonenzymatic reduction of TCQD with NADH presumably proceeds by the follow-
ing mechanism:
k
1
NADH ϩ TCQD ϩ Hϩ s NADϩ ϩ TCQDH2
(5)
(6)
k
2
k
3
•
TCQDH2 ϩ TCQD s 2 TCQDH
k
4
FIG. 2. Reaction progress curve for the nonenzymatic reduction of TCQD at pH 6.0. Initial concentra-
tion of reactants: [TCQD] ϭ 2.82 ␮M; [NADH] ϭ 5.37 ␮M.

MECHANISMS OF FREE RADICALS CATALYZED BY GOD
TABLE 2
99
Rate Constants Obtained by the Simulation of Reaction Progress Curves from Fig. 2 with a
FITSIM Program
Rate constant
mMϪ1 sϪ1
Rate constant
mMϪ1 sϪ1
k1
k2
0.825 Ϯ 0.026
1.366 Ϯ 1.833
k3
k4
63.5 Ϯ 19.3
49.0 Ϯ 15.8
Reaction progress curves in Fig. 2 were simulated with the KINSIM and FITSIM
computer programs (Barshop et al., 1983; Zimmerle and Frieden, 1989; Frieden,
1994), according to the minimal mechanism given by Eqs. (5) and (6). The simulation
procedure was performed with two files, one for the disappearance of TCQD and the
other for the appearance of the TCQD-radical, affording the following results (Table 2).
Enzymatic Reduction of NDMA with
D-Glucose and Glucose Oxidase
p-Nitroso-N,N-dimethylaniline (NDMA) is readily reduced with
D-glucose, under
anaerobic conditions, in the presence of glucose oxidase. In the course of this enzymatic
reaction, characteristic spectral changes are taking place simultaneously: the spectrum
of NDMA at 442 nm decreases gradually, while the spectrum of N,N-dimethyl-p-
phenylenediamine radical (DPD-radical) at 550 nm increases.
Figure 3 shows the reaction progress curves at pH. 4.55.
Glucose oxidase-catalyzed reduction of NDMA with D-glucose proceeds by the
FIG. 3. Reaction progress curves for the enzymatic reduction of NDMA at pH 4.55. Initial concentra-
tion of reactants: [GOD] ϭ 25.4 nM; [NDMA] ϭ 32.7 ␮M; [Glc] ϭ 99.5 mM.

´
TRIVIC ET AL.
100
following mechanism (Baetzold and Tong, 1971; Koerber et al., 1980; Leskovac and
Trivic´ 1988; Leskovac et al., 1996).
E.FAD ϩ D-Glucose → E.FADH2 ϩ ␦-Gluconolactone
NDMA ϩ E.FADH2 → QDIϩ ϩ E.FAD ϩ HOϪ
(7)
(8)
QDIϩ ϩ E.FADH2 s ADMA ϩ E.FAD ϩ Hϩ
(9)
QDIϩ ϩ ADMA ϩ Hϩ s 2 DPD-Radical
(10)
The chemical reactions involved in Eqs. (7)–(10) are shown in Scheme 1.
NDMA becomes protonated in the acid, probably on the amino nitrogen, and the
spectral peak at 442 nm is lost; pKa of this transition is 4.33. The protonated form
of NDMA is not reduced by glucose oxidase. Thus,
pK ϭ4.3
a
s
NDMA ϩ Hϩ
NDMA(Hϩ)
(11)
The rate of the formation of DPD-radicals is strongly pH-dependent (Fig. 4).
The maximal rate of production of DPD-radicals is at pH 4.55. In the acid, the
rate diminishes because NDMA becomes protonated, and in the alkaline because the
rate of radical dismutation depends on the concentration of [H+] [Eq. (10)].
SCHEME 1.

MECHANISMS OF FREE RADICALS CATALYZED BY GOD
101
FIG. 4. pH-dependence of the formation of DPD-radicals. Initial concentration of reactants:
[Glc] ϭ 195 mM; [NDMA] ϭ 13.3 ␮M; [GOD] ϭ 0.132 ␮M.
The overall stoichiometry for the enzymatic reduction of NDMA approaches the
following equations:
At neutral pH:
NDMA ϩ 2 E.FADH2 → ADMA ϩ 2 E.FAD ϩ H2O
(12)
In the acid:
2 NDMA ϩ 3 E.FADH2 ϩ 2Hϩ → 2 DPD-Radical ϩ 3 E.FAD ϩ 2H2O (13)
The molar extinction coefficient of the DPD-radical at pH 4.55 may be estimated
from the reaction progress curves, assuming that the stoichiometries [Eq. (11)] and
[Eq. (13)] are fully involved. From Fig. 3, one can see that the ratio of extinction
changes for the DPD-radical versus NDMA reaches a value of 0.38 at the beginning
of reaction; from this value, a molar extinction coefficient for the DPD-radical at pH
4.55 was calculated (␧
ϭ 13450 MϪ1 cmϪ1).
550 nm
Initial rates of NDMA-disappearance and DPD-radical-appearance were estimated
at pH 4.55, at several different concentrations of enzyme and NDMA. The bimolecular
rate constant for this reaction was found to be constant and independent of the
concentration of reactants (Table 3). Thus, it appears that the first enzymatic reaction
[Eq. (8)] is the rate-limiting step of the overall process [Eq. (13)]; the bimolecular
rate constant for the former reaction, is kII ϭ 32.6 mMϪ1 sϪ1, at pH 4.55 (Table 3).
Reduction of Methyl-1,4-benzoquinone with
D-Glucose and Glucose Oxidase
Cytochrome-c is not reduced with -glucose, in the presence of glucose oxidase,
D
at pH 3–7. Upon addition of methyl-1,4-benzoquinone (MeBQ) to this system, ferricy-
tochrome-c is readily reduced to ferrocytochrome-c.

´
102
No.
TRIVIC ET AL.
TABLE 3
The Rates of Appearance of DPD-Radicals in the Presence of Glucose (99 mM) at pH 4.55
[GOD] (nM)
[NDMA] (␮M)
v0 (␮M.sϪ1
)
kII (mMϪ1 sϪ1
)
1
2
3
4
6
5
7
8
3.37
6.675
9.915
13.10
13.35
13.40
14.00
16.85
397.4
393.6
389.7
386.9
78.7
197.7
300.0
397.4
0.040
29.87
34.525
32.30
31.33
36.54
31.36
32.65
34.35
0.0907
0.1248
0.1584
0.0384
0.08308
0.1371
0.230
Mean Ϯ SD
32.6 Ϯ 2.2
Reduction of cytochrome-c by glucose, in the presence of glucose oxidase and
methyl-1,4-benzoquinone, proceeds by the following mechanism at pH 5.5:
k
1
E.FAD ϩ
D-Glucose → E.FADH2 ϩ ␦-Gluconolactone
(14)
k
2
E.FADH2 ϩ MeBQ → E.FAD ϩ MeBQH2
(15)
(16)
(17)
MeBQH2 s MeBQHϪ ϩ Hϩ
MeBQHϪ s MeBQϭ ϩ Hϩ
FIG. 5. Dependence of the initial rate of cytochrome-c reduction on the concentration of MeBQ.
McIlvaine, pH 5.5: concentration of reactants: [Glc] ϭ 89–99 mM; [Cyt-c] ϭ 8.1 ␮M; [GOD] ϭ
0.28–10.84 ␮M. Phosphate, pH 6.5: concentration of reactants: [Glc] ϭ 93–94 mM; [Cyt-c] ϭ 8.3–8.4
␮M; [GOD] ϭ 0.33 ␮M.

MECHANISMS OF FREE RADICALS CATALYZED BY GOD
103
FIG. 6. Dependence of the initial rate of cytochrome-c reduction on the concentration of GOD.
Concentration of reactants: [Glc] ϭ 89–99 mM; [Cyt-c] ϭ 8.1 ␮M; [MeBQ] ϭ 58 Ϫ 1192 ␮M.
ϭ
Ϫ
•
MeBQ ϩ MeBQ s 2 MeBQ
(18)
(19)
MeBQ ϩ Cyt-c(Fe3ϩ) s MeBQ ϩ Cyt-c(Fe2ϩ)
Ϫ
•
Total:
D
-Glucose ϩ 2 Cyt-c(Fe3ϩ) s ␦-Lactone ϩ 2 Cyt-c(Fe2ϩ) ϩ 2 Hϩ
(20)
The initial rate of reduction of cytochrome-c increases linearly with increasing
concentrations of quinone at pH 5.5 and pH 6.5 (Fig. 5); similarly, it increases linearly
FIG. 7. Dependence of the initial rate of cytochrome-c reduction on pH. Concentration of reactants:
[Glc] ϭ 88–98 mM; [Cyt-c] ϭ 7.9–8.4 ␮M; [GOD] ϭ 0.29 ␮M; [MeBQ] ϭ 14–1192 ␮M.

´
104
TRIVIC ET AL.
TABLE 4
Bimolecular Rate Constants for Redox Reactions of Various Quinoid Compounds
Rate constant
Reaction
(mMϪ1 sϪ1
)
pH
Conditions
Figure 1
Table 1
Table 1
Table 2
E.FADH2 ϩ TCQD → E.FAD ϩ TCQDH2
330
63
49
6.0
6.0
6.0
4.55
•
TCQDH2 ϩ TCQD → 2 TCQD
•
2 TCQD → TCQDH2 ϩ TCQD
E.FADH2 ϩ NDMA ϩ H+ → E.FAD ϩ
33
QDI+ ϩ H2O
E.FADH2 ϩ MeBQ → E.FAD ϩ MeBQH2
80
260,000
80,000
1,800
5.5
Alkaline
6.5
Leskovac et al. (2002)
Ϫ
•
p-BQ= ϩ p-BQ → 2 p-BQ
Yamazaki and
Ohnishi (1966)
Yamazaki and
Ohnishi (1966)
Yamazaki and
Ohnishi (1966)
Ϫ
2 p-BQ → p-BQ ϩ p-BQ=
•
Ϫ
•
p-BQ ϩ Cyt-c(Fe3+) → p-BQ ϩ
6.5
Cyt-c(Fe2+)
with increasing concentrations of enzyme (Fig. 6). It is also apparent that the overall
reaction in the reverse direction is accelerated by protons (Fig. 7).
At pH 5.5, the bimolecular rate constants for the oxidation of glucose by enzyme
(k1 ϭ 9,6.103 MϪ1 sϪ1) and for reduction of quinone (k2 ϭ 8.104 MϪ1 sϪ1) are very
high. The Michaelis constant for methyl-1,4-benzoquinone, obtained from kinetic
measurements, is 6.0 mM at pH 5.5 (Leskovac et al., 2002).
The magnitudes of other rate constants in Eqs. (16)–(19) are not known for methyl-
1,4-benzoquinone. Therefore, it is worthwhile to discuss these reactions in terms of
analogous rate constants for p-benzoquinone, that are reported in the literature. The
bimolecular rate constant for reaction (18) with p-benzoquinone is very slow at pH 7.0
(58 MϪ1 sϪ1), while the reverse reaction is very fast (8.107 MϪ1 sϪ1); p-benzoquionone
TABLE 5
Redox Potentials in Aqueous Solutions at 25ЊC against NHE in Millivolts
Redox couple/pH
2.7
5.3
5.5
6.0
7.0
Source
GOD (FAD/FADH2)
TCQD/TCQDH2
MeBQ/MeBQH2
NAD+/NADHϩH+
Cyt-c(Fe3+)/Cyt-c-
(Fe2+)
—
125
—
Ϫ51 Ϫ59.4a
Ϫ80.5a Ϫ122.5 Hemmerich et al. (1982)
—
—
—
—
Ϫ73.0b
—
—
205
Ϫ320
Melby et al. (1962)
Wallenfels and Gerlach (1959)
Zubay (1988)
295a
—
—
Ϫ260b
270
250
Yamazaki and Ohnishi (1966)
p-BQ/p-BQH2
p-BQ/p-BQH
—
—
—
—
—
—
340
100
280
100
Yamazaki and Ohnishi (1966)
Yamazaki and Ohnishi (1966)
•
a Calculated by extrapolation.
b Calculated, assuming that E0 decreased by 60 mV for each pH unit.

MECHANISMS OF FREE RADICALS CATALYZED BY GOD
105
SCHEME 2.
dissociates with two pKa values of 9.4 and 11.2, respectively [Eqs. (16) and (17)].
The bimolecular rate constant for the oxidation of p-benzoquinone radicals with
ferricytochrome-c (reaction 19) is very high at pH 6.5 (1,8.106 MϪ1 sϪ1) (Yamazaki
and Ohnishi, 1966). The proton-dissociation constant of p-benzo-semiquionone is at
pH 4.2; thus, at pH 5.5, the semiquinone form is mostly in its unprotonated state
(Yamazaki and Piette, 1965).
Therefore, by analogy, in steady-state reactions with methyl-1,4-benzoquinone at
pH 5.5, shown in Figs. 5–7, the enzyme and quinone are present predominantly in
the reduced form, the concentration of oxidized quinone and its radical form are very
small, and the rate of the overall reaction is governed by reaction (18). An apparent
bimolecular rate constant for the overall reaction at pH 5.5 is very slow (120 MϪ1
sϪ1) (Fig. 5). Thus, it appears that the rate-limiting step of the overall reaction (20)
is the formation of radicals, because the concentration of methyl-1,4-benzoquinone
at pH 5.5 is very small.
The reaction between quinone and doubly dissociated hydroquinone is predominant
in the semiquinone formation at neutral pH (Yamazaki and Ohnishi, 1966). With
increasing pH, Eqs. (16) and (17) are shifted in favor of MeBQ=, and, therefore, the
overall reaction is accelerated in alkaline (Fig. 7).
DISCUSSION
Table 4 summarizes the bimolecular rate constants for various redox reactions with
quinoid compounds that are investigated in this work.
Normal redox potential for glucose oxidase substrates that are used in this work
are shown in Table 5. From the data in this table, one can see that the glucose oxidase
catalyzed-oxidation of glucose with TCQD at pH 6.0 and with MeBQ at pH 5.5 is
thermodynamically feasible. Normal redox potentials of NDMA and its derivatives
are not known; therefore, these reactions escape at present the thermodynamic analysis.

´
TRIVIC ET AL.
106
All redox reactions, catalyzed by glucose oxidase, which are described in this work,
are 2e-transfer reactions. Formation of free radicals always follows the initial 2e-
transfer reaction, and arises from dismutation reactions. Enzymatic reduction of
7,7,8,8-tetracyano-quinodimethane and p-nitroso-N,N-dimethylaniline afforded ini-
tially almost quantitatively stable free radicals; these radicals are stable in aqueous
solutions for 5–15 min at an appropriate pH value.
Enzymatic reduction of methyl-1,4-benzoquinone at neutral pH afforded a slow
formation of quinone-radicals by the same mechanism. A slow rate of radical formation
in neutral solutions is due to a very low steady-state concentration of doubly dissociated
quinone which dominates the semiquinone formation at neutral pH. Formation of
methyl-1,4-benzoquinone radicals is also sensitive to pH.
Scheme 2 shows the chemistry involved in radical dismutation reactions.
An ability of glucose oxidase to activate various quinoid compounds under anaerobic
conditions, and produce free radicals by redox processes, is of considerable toxicologi-
cal interest. It is especially important in view of potential application of glucose
oxidase in medicine and the recent preclinical trials with glucose oxidase in mice
(Samoszuk et al., 1993). In addition, the bioorganic reactions described in this work
illustrate the potential capability of glucose oxidase to catalyzed the formation of free
radicals by very versatile reactions of radical dismutation.
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