The Effects of Nitric Oxide on the Oxidations of
L-Dopa and Dopamine Mediated by Tyrosinase and
Peroxidase*
Anthony J.
Nappi
and
Emily
Vass
From the Department of Biology, Loyola University Chicago, Chicago,
Illinois 60626
Received for publication, October 30, 2000, and in revised form, December 18, 2000
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ABSTRACT |
The effects of nitric oxide (NO) on both
tyrosinase/O2- and horseradish
peroxidase/H2O2-mediated oxidations of dopamine
and its o-dihydric phenol precursor L-dopa were
compared with autoxidative processes and quantitatively assessed by
oxidative and reductive electrochemical detection systems. In
peroxidase/H2O2/NO-catalyzed reactions,
significantly more substrate was oxidized than in the corresponding
control incubations lacking NO. In
tyrosinase/O2/NO-promoted reactions the total amounts of
L-dopa and dopamine oxidized were significantly less than
the amounts of the substrates oxidized by enzyme alone. These data
indicate that the activity of the heme protein peroxidase was enhanced
by NO, whereas tyrosinase, a copper-containing monoxygenase, was
inhibited. The NO-mediated reduction of tyrosinase/O2
activity may be attributed to the formation of an inhibitory
copper·nitrosyl complex. An oxidized nitrodopamine derivative,
considered to be either the quinone or semiquinone of
6-nitrosodopamine, was generated in
peroxidase/H2O2/NO-mediated reactions with
dopamine along with two oxidized melanin precursors, dopamine quinone
and dopaminechrome. No corresponding nitroso compound was formed in
reactions involving L-dopa or in any of the
tyrosinase-mediated reactions. The formation of such a noncyclized nitrosodopamine represents an important alternative pathway in catecholamine metabolism, one that by-passes the formation of cytoprotective indole precursors of melanin. The results of this investigation suggest that cellular integrity and function can be
adversely affected by NO-promoted oxidations of dopamine and other
catechols, reactions that not only accelerate their conversion to
reactive quinones but also form potentially cytotoxic noncyclized nitroso derivatives. Reduced levels of dopamine in the brain through NO-enhanced oxidation of the catecholamine will almost certainly be
manifested by diminished levels of the dopamine-derived brain pigment neuromelanin.
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INTRODUCTION |
The selective degeneration of melanized nigrostriatal dopaminergic
neurons and attendant functional impairments of associated neuronal
pathways are primary neuropathological manifestations of Parkinson's
disease (PD).1 Neuronal
degeneration in PD is accompanied by the progressive depletion of the
neurotransmitter dopamine, as well as the pigment derived from its
autoxidation, neuromelanin (1-3). Several mechanisms have been
proposed to account for the selective vulnerability of the pigmented
nigrostriatal dopaminergic neurons in PD, but the pathogenesis of this
disease remains largely enigmatic. The neuropathology associated with
PD has been attributed to oxidative stress resulting from the toxic
effects of certain reactive intermediates of oxygen (ROI) (4), most
notably the hydroxyl radical (·OH), a highly reactive molecule
generated by the interaction of hydrogen peroxide
(H2O2) with superoxide anion (O
2),
transition metals (Fe2+ or Cu+), or nitric
oxide (NO) (5, 6).
The preferential targeting and destruction of nigrostriatal
dopaminergic neurons in PD implicates as cytotoxic molecules substances specifically generated as by-products of dopamine metabolism in the
brain. Dopamine autoxidation produces dopamine quinone (DAQ), an
electrophilic molecule that rapidly cyclizes to form dopaminechrome (DAC). Subsequent reactions generate indoles that polymerize to form a
brown-black pigment termed eumelanin. The autoxidation of
L-dopa, the carboxylated precursor of dopamine, is similar and results in the formation of dopaquinone (DOQ) and dopachrome (DOC)
en route to forming eumelanin (Fig. 1).
If cysteine or glutathione are present during the oxidation of the
dopamine or L-dopa, the quinones derived from these
catechols react with the thiols to form compounds that ultimately
polymerize to yield a red-yellow pigment termed pheomelanin.
Neuromelanin appears to be a heteropolymer derived in part from
dopamine and comprises pheomelanin and eumelanin (1, 7).

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Fig. 1.
Enzymatic and nonenzymatic oxidations of
L-dopa and dopamine generate the corresponding
o-quinones (DOQ, DAQ) and
semiquinones (DOSQ, DASQ) of these
o-dihydric phenols. Subsequent reactions of the
o-quinones are varied and include the formation of
noncyclizable compounds via nucleophilic additions, conjugation with
thiol compounds to form pheomelanin, and intramolecular cyclization
(via a Michael 1,4-addition) to form aminochromes
(DOC, DAC). The aminochromes evolve to eumelanin
via intermediates that include 5,6-dihydroxyindole (DHI) and
5,6-dihydroxyindole-2-carboxylic acid (DHICA). The latter
cyclized intermediates of eumelanin are viewed by some investigators as
cytoprotective molecules, and the noncyclizable intermediates are seen
as cytotoxic (58, 61).
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If the oxidation of brain dopamine represents a source of potentially
neurotoxic molecules, endogenous or exogenous factors that influence
the rate of oxidation of the brain dopamine, or that alter the
biochemistry and reactivity of its metabolites, will likely play a
critical role in neuronal homeostasis. Frequently associated with
ROI-mediated cytotoxic processes are reactive intermediates of nitrogen
(RNI) derived from nitric oxide (NO) through the action of nitric oxide
synthase. Nitric oxide is produced in virtually all cell types,
including neurons, endothelial cells, macrophages, and malignant
melanocytes. Despite a widely acknowledged beneficial role of NO in
numerous and diverse physiological processes, this diffusable free
radical has been shown to react with O2 and ROI to spawn an
array of reactive molecules that can aggressively target biomolecules.
Included in the arsenal of potentially cytotoxic RNI are nitrogen
dioxide (NO2), nitrous anhydride
(N2O3), nitrite (NO
), nitrate
(NO
), peroxynitrite anion
(ONOO
), ·OH, nitrosamines, and
S-nitrosothiols. Increased production of NO has been
implicated in several examples of neuronal injury (8). Of interest are
the recent reports that indicate that NO readily reacts with dopamine
and other catecholamines to form noncyclized 6-nitro-derivatives (9,
10) and that NO donors appear to increase tyrosinase activity and
melanin synthesis in a dose-dependent manner (11, 12).
Despite considerable interest in the involvement of NO and its
derivatives in catecholamine metabolism and melanogenesis (9-24), lacking are comparative studies of the effects of NO on the
autoxidation and enzyme-mediated oxidations of dopamine and
L-dopa. Moreover, there are conflicting reports that
document cytotoxic (8, 25) and cytoprotective (26, 27) effects of NO
and its derivatives on dopaminergic systems. These and related issues
are addressed in this investigation by employing sensitive
electrochemical detection methods to analyze the effects of NO on the
tyrosinase- and peroxidase/H2O2-mediated oxidations of dopamine and L-dopa and on the production of
reactive quinoids generated during the autoxidations of the two
catechols. Comparative and quantitative studies employing both
reductive and oxidative electrochemical methods of detection showed
that NO significantly augmented
peroxidase/H2O2-mediated oxidations but not
those promoted by tyrosinase. Dopamine nitration by
peroxidase/H2O2 and NO was manifested by the
rapid formation of a single oxidized compound considered to be a
noncyclized quinone or semiquinone of 6-nitrodopamine.
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EXPERIMENTAL PROCEDURES |
Chemicals--
The following chemicals were obtained from Sigma
Chemical Co. (St. Louis, MO): L-dopa
(L-3,4-dihydroxyphenylalanine), dopamine (3,4-dihydroxyphenylethylamine) mushroom tyrosinase (E.C. 1.14.18.1, 3870 units/mg), horseradish peroxidase (E.C. 1.11.1.7, 175 purpurogallin units/mg), and reduced glutathione (GSH).
Diethylamine·NO-generating complex (DEA) was obtained from Research
Biomedicals International (Natick, MA). Chelex 100 was purchased from
Bio-Rad Laboratories (Richmond, CA). Stock solutions of all components
were prepared daily in ultrapure reagent-grade water obtained with a
Milli-Q system (Millipore, Bedford, MA) and kept at 4 °C for a
maximum period of 3 h and then discarded.
Reaction Mixtures and Enzyme Activity--
Standard reaction
mixtures for both enzymatic and nonenzymatic oxidations of
L-dopa and dopamine were composed of 0.5 mM
substrate in a total volume of 100 µl of bicarbonate buffer (100 mM NaCl/25 mM NaHCO3; pH 7.2). For
enzyme-mediated oxidations, the reaction mixtures contained either 0.01 µg of tyrosinase (3870 units/mg) or 0.25 µg of peroxidase (175 purpurogallin units/mg) and 8 mM H2O2. Oxidations mediated by NO were initiated
by the incorporation of 2.5-10 mM DEA into the reaction
mixtures. Iron-mediated oxidations were initiated by incorporating into
the reaction mixtures 0.3 mM FeCl3·EDTA
complex. Following incubations at 22 °C for various specified
intervals, 5-µl aliquots were removed from each reaction mixture and
analyzed by high performance liquid chromatography with electrochemical
detection (HPLC-ED). Any modifications in composition or concentrations
of the standard reaction mixtures are specified where required.
Comparative and quantitative measurements of enzyme-mediated oxidations
were made using HPLC-ED to measure substrate oxidation in standard
reaction mixtures with and without NO. Specific activity was expressed
as picomoles of substrate depleted per min/µg of protein under the
standard conditions established for the study. Reduced glutathione (100 nmol) was added to reaction mixtures to note the effects of the
reductant on the production of o-quinones.
Control incubations were conducted by excluding substrate, NO, enzyme,
or iron, as was appropriate for each experiment. The possibility of
·OH production resulting from metal contaminants was excluded in experiments that compared rates of oxidation in test solutions with
those observed in identical control mixtures that were either pretreated with the metal-chelating ion exchange resin, Chelex 100, or
that contained the specific iron chelator desferrioxamine.
HPLC-ED Analyses of Catechol Oxidations--
A sensitive and
specific salicylate hydroxylation assay was used in conjunction with
HPLC-ED to monitor the enzymatic and nonenzymatic oxidations of
L-dopa and dopamine. The HPLC system consisted of a
Bioanalytical Systems (West Lafayette, IN) LC-4B amperometric detector
with a glassy carbon working electrode and an Ag/AgCl reference
electrode. The working electrode was maintained at an oxidative
potential of +650 mV to monitor the o-diphenols dopa and
dopamine, and a reductive potential of
100 mV to detect their
oxidized products, such as o-quinones, semiquinones, and eumelanochromes (e.g. dopachrome, dopaminechrome). In both
reductive and oxidative modes, instrument sensitivity was maintained at 50 nA. The solvent system was comprised of 25 mM citrate
buffer (pH 3.0) containing 2.5% acetonitrile, 0.5 mM
sodium octylsulfate, and 0.7 mM disodium EDTA. All
separations were made with an Alltech Spherisorb ODS 5-µm reverse
phase column using a flow rate of 1.0 ml/min. Known amounts of
different catecholamines occasionally were incorporated into control
reaction mixtures to serve as internal standards. The correlation
coefficient of the calibration curves established for the standards
typically was greater than 0.98.
Data Analyses--
All experiments were replicated at least
three times. Results are presented as the means ± S.D. (of four
experiments) of the determinations specified. Differences between mean
values were evaluated using the Student's t test. The
difference between two means was considered significant when
p < 0.05.
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RESULTS |
Chromatographic Analyses--
In initial experiments autoxidations
and tyrosinase/O2-mediated oxidations were analyzed by
electrochemical detection under reductive conditions (
100 mV) with
L-dopa as the substrate and incubation periods ranging from
30 s to 42 min. In subsequent experiments, assay periods ranging
from 2 to 12 min were used, under which conditions two oxidized
metabolites of L-dopa were detected in
tyrosinase/O2-mediated reactions, DOQ and DOC (Fig. 2). In reaction mixtures lacking enzyme,
the initial product formed during the autoxidation of
L-dopa was DOQ, which first appeared in 12-min incubations
(Fig. 3). The autoxidation of dopa also generated DOC, but this metabolite was detected only in reaction mixtures incubated for periods longer than 20 min (not presented). NO-mediated oxidations of dopa also generated DOQ (Fig. 3). There was
no evidence of DOC in any of the NO mixtures that were incubated for
periods up to 42 min. However, both DOQ and DOC were generated by the
iron-mediated oxidation of dopa (Fig.
4).

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Fig. 2.
Representative chromatograms showing the
time-dependent generation of two oxidized molecules,
dopachrome (DOC) and dopaquinone
(DOQ), that were derived from the tyrosinase-mediated
oxidation of dopa. Each chromatogram was obtained by injecting
5-µl samples of the reaction mixture at the specified time
post-incubation. At time zero, control samples contained 1 pmol of dopa
and 0.025 µg of enzyme. Chromatographic conditions were 100 mV, 50 nA, and a flow rate of 1 ml/min.
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Fig. 3.
Representative chromatograms comparing the
quinoids generated during the autoxidation, tyrosinase-mediated, and
NO-mediated oxidations of dopa in 12-min incubations. DOC appeared
much later (more than 20 min) in reaction mixtures undergoing
autoxidation, but was not detected in NO-mediated reactions that were
incubated for up to 42 min. Chromatographic conditions were similar to
those given in Fig. 2.
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Fig. 4.
Representative chromatograms illustrating the
production of DOC and DOQ by tyrosinase- and iron-mediated oxidations
of dopa (upper panel) and the production of DAC and
DAQ by the iron-mediated oxidation of dopamine (lower
panel). Chromatographic conditions were similar to
those given in Fig. 2.
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Two oxidized metabolites of dopamine, DAQ and DAC, were likewise
generated during autoxidation and in reactions mediated by tyrosinase/O2 (not presented) and iron (Fig. 4). The extent
of production of the two quinoids of L-dopa and
dopamine in 2-min assays was dependent on substrate concentration
(Fig. 5). The addition of GSH to the
reaction mixtures diminished the levels of the two oxidized metabolites
of dopamine, DAQ (Fig. 6) and DAC
(not shown). In tyrosinase/O2-mediated reactions the amount of DAQ was reduced by nearly 60% 6 min after the addition of 100 nmol
of GSH and by 86% after 42-min incubation (Fig. 6). GSH had a similar
reducing affect on the oxidized metabolites of dopa generated by
autoxidation and tyrosinase/O2- and iron-mediated oxidations (not presented). Interestingly, the production of the two
oxidized metabolites of dopamine were completely inhibited when GSH was
incorporated into reaction mixtures prior to the addition of enzyme.
These observations are consistent with other experimental evidence
showing that antioxidants such as GSH and ascorbate can effectively
abrogate the NO-induced oxidation of dopamine (15) and that GSH and
cysteine react readily with the o-quinones of dopa and
dopamine to form reduced glutathional and cysteinyl conjugates that can
be detected with oxidative electrochemical methods (28).

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Fig. 5.
Data showing the production of oxidized
quinoids (DAQ, DOQ) by tyrosinase and
peroxidase to be dependent on the concentration of substrate (dopa and
dopamine). Data were obtained by HPLC-ED using the chromatographic
conditions given in Fig. 2. S.D. are <4% of the means of three
identical experiments.
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Fig. 6.
Data documenting the reducibility of DAQ by
GSH. Superimposed chromatograms showing time-dependent
diminishing levels of DAQ in response to 100 nmol of GSH. DAQ is not
generated when GSH is initially incorporated into the reaction mixture
(not presented). Data, which were obtained by HPLC-ED using the
chromatographic conditions given in Fig. 2, represent the mean of four
identical experiments. S.D. are <2% of the mean value.
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Significantly (p < 0.5) more dopamine and
L-dopa were oxidized in reaction mixtures containing
peroxidase/H2O2 and NO than were oxidized by
peroxidase/H2O2 alone (Fig.
7). In 6-min incubations the percentages
of L-dopa oxidized separately by NO,
peroxidase/H2O2, and
peroxidase/H2O2/NO were 22.5, 32, and 77%,
respectively. Completely different results were obtained with
tyrosinase/O2-mediated oxidations where significantly
(p < 0.5) less substrate was consumed in reaction mixtures containing NO (Fig. 7). A series of incubations was conducted to ascertain if the NO-mediated augmentation of peroxidase activity and
the inhibition of tyrosinase activity were dependent on the concentration of NO. In 1-min incubations containing 2.5, 5, or 10 mM NO, the rates of oxidation of L-dopa by
peroxidase/H2O2 were significantly elevated
above controls (lacking NO; 0.15 nmol/min), averaging 0.19, 0.31, and
0.428 nmol/min, respectively (Fig.
8B). The augmented activity of
peroxidase/H2O2 at each concentration tested
was significantly (p < 0.05) higher than the additive
effect of NO-mediated oxidation (Fig. 8A) plus the activity
obtained by enzyme alone (Fig. 8B). With tyrosinase, the
opposite was observed. At each concentration of NO tested (2.5, 5, or
10 mM), the activity of tyrosinase/O2 was
significantly lower than control incubations lacking NO (Fig.
8B).

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Fig. 7.
Representative chromatograms depicting for
comparative purposes the diminished levels of L-dopa that
were manifested when the catechol was oxidized by tyrosinase
(TASE) or peroxidase (PASE).
Chromatographic conditions were +650 mV, 50 nA, and a flow rate of 1 ml/min.
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Fig. 8.
A, the rate of oxidation of
L-dopa in reaction mixtures containing varying
concentrations (0 = buffer, 2.5, 5, or 10 M) of NO.
B, comparative analyses of the rates of dopa oxidation by
tyrosinase/O2 (TASE) and
peroxidase/H2O2 (PASE) with and
without NO. Only in the peroxidase/H2O2 + NO
mixtures were the rates of L-dopa augmented. Data were
obtained by HPLC-ED using the chromatographic conditions given in Fig.
7 to measure substrate consumption. Each 5-µl sample of reaction
mixture analyzed by HPLC-ED initially contained 1 pmol of dopa, and
either 0.025 µg of tyrosinase or 0.5 µg of peroxidase and 0.7 nmol
of H2O2. S.D. are <1% of the mean values,
which were derived from three replicate experiments.
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The above experiments employed oxidative detection methods
(HPLC-ED, +650 mV) to monitor catechol consumption. These tests were
replicated with reductive detection methods (HPLC-ED,
100 mV) to
document the quinoid metabolites generated during the oxidations of
dopa and dopamine. Under these conditions the presence of elevated levels of quinones produced in incubations containing enzyme/NO mixtures would correlate with the amount of substrate oxidized. However, quinone production in both the tyrosinase/O2/NO-
and peroxidase/H2O2/NO-mediated reactions were
significantly lower (p < 0.05) than in reaction
control mixtures containing only enzyme (Fig.
9). Quinone levels generated in reaction
mixtures containing substrate and NO, substrate alone (autoxidation),
and substrate and H2O2 are provided for
comparison (Fig. 9).

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Fig. 9.
Data documenting lower levels of DAQ
generated in reaction mixtures composed of both enzyme and NO than
those formed in reaction mixtures containing enzyme alone. The
amount of DAQ resulting from the autoxidation of dopamine, as well as
from oxidations caused by NO and H2O2, are
provided for comparison. Data were obtained by HPLC-ED using the
chromatographic conditions given in Fig. 2 to measure the production of
oxidized metabolites of dopamine. Each 5-µl sample of reaction
mixture analyzed by HPLC-ED initially contained 1 pmol of dopamine, and
either 0.025 µg of tyrosinase, or 0.5 µg of peroxidase and 0.7 nmol
of H2O2. Reaction mixtures with NO contained 25 nmol of DEA. The amount of H2O2 in reaction
mixtures lacking enzyme was 0.7 nmol. All data points represent the
means of three replicate tests. S.D. are <4% of the mean value.
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Under the specific reductive and oxidative conditions established for
this investigation, no additional product was detected in any of the
tyrosinase/O2/NO-mediated reactions, or in the
peroxidase/H2O2/NO-mediated oxidation of
L-dopa. Of considerable interest, however, was the detection of an oxidized nitrosodopamine molecule (P#1)
generated during the
peroxidase/H2O2/NO-mediated oxidation of
dopamine (Fig. 10). The presence of
this nitro derivative indicates that horseradish peroxidase/H2O2 can promote the nitrosation of
dopamine. Under the conditions established for this investigation, the
nucleophilic addition of NO or other RNI such as
NO
to oxidized derivatives of
dopamine would most likely generate a noncyclized molecule such as
6-nitrosodopamine quinone (6NDAQ), or nitrosodopamine semiquinone
(6NDASQ) (Fig. 11). The possibility that the molecule identified only in the
peroxidase/H2O2/NO-mediated catalysis of
dopamine was derived by hydroxylation (i.e, a trihydroxyphenyl quinone,
6OHDA) instead of nitration was considered, but it was reasoned that
such a hydroxylation product also would have had to form in
peroxidase/H2O2 reactions lacking NO.
Therefore, the nitrosation of dopamine was considered to be the pathway
most likely involved in the product (P#1) seen in Fig.
10.

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Fig. 10.
Representative chromatograms depicting the
production of oxidized metabolites of dopamine in reactions promoted by
(A) tyrosinase, (B) tyrosinase/NO,
(C) peroxidase/H2O2/NO,
(D) peroxidase/H2O2, and
(E) NO. The compositions of the reaction mixtures
and the conditions under which these chromatograms were obtained are
identical to those given in Fig. 9. The assay period was 6 min. A
single, unidentified oxidized metabolite (P#1) was generated
only in peroxidase/H2O2/NO-mediated oxidations
(C). The compound is proposed to be a noncyclized
nitrosodopamine quinone (6NDAQ) or semiquinone
(6NDASQ) the structures of which are provided.
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Fig. 11.
Two competing pathways for
o-quinones involve hydroxylations via reactions with
H2O, H2O2, or hydroxyl radical
(·OH) to form potentially cytotoxic trihydroxyphenyls
(i.e. 6- hydroxydopamine, 6OHDA;
6-hydroxydopamine semiquinone, 6OHDASQ;
p-semiquinone), and nitration reactions with NO or
other RNI to form such nitrogen-containing compounds as
6- nitrosodopamine (6NDA), oxidation of which
generates the corresponding quinone
(6NDAQ) and/or semiquinone (6NDASQ).
The latter two noncyclizable compounds are potentially cytotoxic
molecules that can damage biomolecules via nucleophilic
additions.
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DISCUSSION |
This investigation documents the effects of NO on the autoxidation
and tyrosinase/O2- and
peroxidase/H2O2-mediated oxidations of dopamine
and its o-dihydric phenol precursor L-dopa. In
the absence of NO, the quinoids of dopamine (DAQ, DAC) and
L-dopa (DOQ, DOC) that formed in enzyme-mediated oxidations
were reduced in a time-dependent manner by the addition of
GSH. When GSH was incorporated into reaction mixtures prior to the
addition of enzyme, no products were observed, confirming the oxidative
nature of these molecules. In
peroxidase/H2O2/NO-catalyzed reactions, the total amounts of dopa and dopamine oxidized were significantly higher
than the additive amounts of the substrates oxidized by NO and
enzyme separately. In tyrosinase/O2/NO-catalyzed reactions, the total amount of o-diphenol substrate oxidized was less
than the amount of substrate oxidized by enzyme alone. These data
indicate that the activity of the heme protein peroxidase was enhanced by NO, whereas tyrosinase, a copper-containing monoxygenase, was inhibited.
Because NO reacts at near diffusion-controlled rates with the
iron center of heme-containing proteins (e.g. guanylyl
cyclase, oxyhemoglobin, cytochrome p450) (29), this activity likely
accounts for the enhanced peroxidase-mediated oxidation of
L-dopa and dopamine we observed in this study with
horseradish peroxidase. NO can bind reversibly with both ferric
(Fe3+) and ferrous (Fe2+) states of iron (29,
30) and has been shown to form heme·nitrosyl complexes with
horseradish peroxidase (31). However, NO has been shown to have an
inhibitory effect on such heme-containing enzymes as glutathione
peroxidase (32, 33), glutathione reductase (34), lipoxygenase (35),
lactoperoxidase (36), and thyroid peroxidase (37). Studies with
glutathione peroxidase suggest that NO and its derivatives directly
inactivate the enzyme, resulting in an increase in intracellular
peroxides that are responsible for cellular damage (33). Disparities
among reports describing the ability of NO to augment or inhibit enzyme
activity may result from differences in the concentration of NO, which
appear to influence its association/dissociation rates with the iron
redox center of some heme-containing enzymes (29, 30). Whether NO
reacts with metals at the active sites of enzymes depends on the
affinity of NO for the metal and the relative concentrations of enzyme, O2, and NO. When NO is present in much lower concentrations
than O2, the formation of nitrogen dioxide
(NO2) is initiated by the reversible reaction of NO with
O2 to form the nitrosyldioxyl radical (ONOO·)
(Reaction I). The subsequent rate-limiting step is the addition of a
second nitric oxide to ONOO· forming a compound that rapidly
decomposes by hemolytic cleavage of the O-O bond to form
NO2 (Reactions II-RIV). Hydrogen bonding with water
stabilizes ONOO· and makes it hydrophilic. The nitrosyldioxyl
radical, which possesses about the same energy as is released by the
hydrolysis of ATP under standard conditions (31), can dissociate NO
from ferrous heme proteins, thereby inactivating the enzyme.
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Thus, the reaction rate for NO binding to an enzyme must be faster
than the formation of ONOO· by the reaction of NO with
O2. When nitric oxide synthase is activated, NO will first
associate with the enzyme due to the faster reaction rate with the
metal than with O2. Because of the high O2
concentrations relative to enzyme, NO will gradually be bound as
ONOO·, and a gradual loss of enzyme activity will be manifested.
The NO-mediated inhibition of tyrosinase/O2 activity
reported in this investigation with the two phenolic precursors of
melanin as substrates may be attributed to differences in the
concentration of exogenous NO and/or to the formation of a
copper·nitrosyl complex that may diminish, rather than enhance enzyme
activity. The binding of NO to the binuclear copper center of
tyrosinase has been shown to yield a dipolar-coupled pair in which both
copper ions bind NO (38). The mechanism by which nitrosyl·copper
complexes form at the active site of tyrosinase to diminish enzyme
activity has not been established. Tyrosinase is unique in that it
catalyzes two distinct reactions, an initial hydroxylation of
monophenols to o-diphenols, and the ensuing oxidation of
o-diphenols to o-quinones. Based on chemical and
spectroscopic studies of its binuclear copper-active site, three forms
of the enzyme have been characterized; oxy-tyrosinase, deoxy-tyrosinase, and met-tyrosinase (38, 39). The oxidation of
o-diphenols to their corresponding o-quinones can
be mediated either by the oxy- or met-forms of the enzyme (40)(Fig.
12A). However, most of the
enzyme in a fresh preparation is in the met-form with the copper atoms
of the active site in the oxidized state and incapable of binding
O2. The met-form of tyrosinase can be converted to the
oxy-form by addition of H2O2 (40-43). It is
proposed that NO interferes with the normal catalytic cycle of
tyrosinase by partially transferring its unpaired electron to one of
the coppers of the met-form of the enzyme, thereby causing structural perturbations of the active site of the enzyme that result in the
formation of a transient noncatalytic, met-nitrite form (Fig. 12B). It is also possible that
NO
derived from NO can interact with
the met-form of tyrosinase to yield a noncatalytic semi-met-form of the
enzyme. In any event, such noncatalytic forms of the enzyme, even if
transient, would divert a portion of the enzyme away from its catalytic
interactions with o-diphenols and account for the inhibitory
action of NO on tyrosinase activity that we observed. Recent studies
document the existence of a half-met-form of tyrosinase that binds an
hydroxide ion in the equatorial coordination position of the
paramagnetic copper (44).

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Fig. 12.
A, tyrosinase is a dinuclear type-3
copper-containing metalloprotein that reversibly binds and activates
dioxygen. Each copper center has three histidine (His)
coordinates, and the active site is accessible to phenolic
substrates. The oxy-form of tyrosinase reacts catalytically with both
monophenols and diphenols, whereas the met-form reacts only with
diphenols. Such catalytic interactions produce transient complexes that
dissociate with the release of o-quinones. In its reduced or
deoxy-form the enzyme reversibly binds O2, generating the
oxy-form of the enzyme. The deoxy-form is reverted back to the oxy-form
by addition of O2. The met-form of tyrosinase can be
converted to the oxy-form by addition of H2O2
(40). The tyrosinase-mediated conversion of monophenols starts with
their binding to the oxy-form of the enzyme, a reaction that produces
the o-diphenol and the met-form of the enzyme. Thus, the
oxidation of o-diphenols to their corresponding
o-quinones can be mediated either by the oxy-form of
tyrosinase or the met-form. B, it is proposed that NO or its
derivative, NO , may interact with the
met-form of tyrosinase to yield transient noncatalytic forms of the
enzyme.
|
|
This investigation documents for the first time the production of an
oxidized nitrodopamine derivative generated by the
peroxidase/H2O2/NO-mediated catalysis of
dopamine. The molecule, which is considered to be either a noncyclized
quinone (6NDAQ) or semiquinone (6NDASQ) of nitrodopamine (Fig. 11), was
generated along with two oxidized melanin precursors, DAQ and DAC.
Except for the production of melanin precursors, no nitroderivative was
detected in the peroxidase/H2O2/NO-mediated catalysis of L-dopa or in any of the
tyrosinase/O2/NO-mediated reactions. These findings support
recent studies that reported a reduced 6-nitroderivative to be the
principal reaction product emanating from the activity of
catecholamines with nitrogen oxides derived from NO (9, 10, 45). The
oxidation of such a nitroderivative of dopamine by univalent or
divalent electron transfers would generate 6NDASQ and 6NDAQ,
respectively (Fig. 11), and thus account for the nitrodopamine molecule
detected in this investigation.
It is not known why substrate nitration was not evident in
peroxidase/H2O2/NO-promoted oxidations of
L-dopa, when such a reaction was manifested with dopamine,
the decarboxylated derivative of L-dopa. Because peroxidase
can use both NO and H2O2 as substrates to
catalyze the nitrosation of tyrosine residues (46), the rate of
enzyme/H2O2 oxidation of L-dopa may
exceed that of dopamine, limiting the extent of competitive involvement
of NO in nitrating L-dopa. Also, it is possible that the
electrochemical conditions established for this investigation were
inappropriate for detecting nitroderivatives of L-dopa or
that nitration of L-dopa was prevented by the side-chain
CO2H group of the catechol. The absence of substrate nitration in tyrosinase/O2/NO-mediated reactions with
either L-dopa or dopamine may document a significantly
different method of converting o-diphenols to
o-quinones than that employed by
peroxidase/H2O2. This possibility highlights
the importance of several recent studies documenting the presence of
tyrosinase in the brain (47-51) and reports that enzyme activity (11,
19, 52) and melanogenesis are enhanced by NO modulating tyrosinase gene
expression via the cGMP pathway (19, 52). In our investigations we
found that the amount of substrate oxidized in reactions containing
both tyrosinase and NO was less than additive, suggesting enzyme
inhibition. Failure to take into account the additive effects of the
separate oxidations by NO and enzyme may lead to flawed interpretations regarding enzyme activation and augmentation.
The preferential destruction of pigmented nigrostriatal dopaminergic
neurons in PD implicates as cytotoxic molecules reactive electrophilic
quinones and semiquinones derived from the metabolism of dopamine.
Exogenous and endogenous factors that either enhance the oxidation of
dopamine or chemically modify the reactivity of its oxidation products,
are likely to play critical roles in the maintenance of neuronal
homeostasis. NO has been implicated as a causative factor in
neuronal degeneration in PD and related disorders (53-55). How NO
could react to cause neuronal degeneration has not been established.
The results of this investigation suggest that the interplay between
the intracellular peroxidase and NO is critical in mediating enzyme
activity. Nitric oxide-promoted oxidations of dopamine and its
o-dihydric phenol precursor L-dopa are of
considerable interest not only because the radical can oxidize these
catechols to quinones and semiquinones but also because nitrosation
reactions generate nitroso and nitrosyl compounds, which may adversely
affect cellular integrity and function, as well as the activity of
various heme- or copper-containing enzymes. The physiological
concentration of NO in cells ranges from 10
8 to
10
7 M, but these levels may increase
significantly in response to pro-inflammatory agents. Elevated amounts
of NO may deplete GSH, thereby diminishing antioxidant protection,
deplete dopamine levels through enhanced dopamine oxidation, and form
nitration products that can damage cells. Because NO and certain of its
derivatives have the potential for engaging in redox exchange with the
metal ions at the enzyme active site, these interactions will also
modify enzyme activity. Whether the effects of NO on the
enzyme-mediated oxidations of dopamine and L-dopa are
contributory but less than additive, as we found with
tyrosinase/O2, or synergistic, as with peroxidase/H2O2, the cells in which these
reactions occur are likely to manifest significantly altered catechol
dynamics. The formation of noncyclized nitrosodopamine quinones or
semiquinones represents an important alternative pathway in the
metabolism of catecholamine, because it by-passes the formation of
melanogenic precursors, such as DHI or DHICA, which are considered to
be cytoprotective (56-58) (Fig. 1). The formation and possible role of
neuromelanin in the etiology of Parkinson's disease continue to be
issues of considerable interest (56, 59), because, as a redox-active polymer, the pigment has the capacity to engage in electron transfer processes. Depending on the prevailing intra- and extracellular environments, melanins can react with different reducing or oxidizing species and, thus, be either cytoprotective or cytotoxic (24, 60). The
capacity of neuromelanin to bind iron through
OH phenolic units (2)
reduces the involvement of the metal in .OH production by
the Fenton reaction and contributes to the antioxidant functions of the
pigment. The pro-oxidant or antioxidant functions of the pigment are
dependent on its redox equilibrium, a feature readily altered by the
presence of transition metal ions, changes in pH, and interactions with
reactive intermediates of oxygen (ROI) and nitrogen (RNI). Thus, NO
fluxes in the brain can have a significant effect on the function(s) of neuromelanin.
 |
FOOTNOTES |
*
This work was supported by Grant GM 59774 from the National
Institutes of Health and by Research Services at Loyola University Chicago.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biology,
Loyola University Chicago, Chicago, IL 60626. Tel.: 773-508-3632; Fax:
773-508-3648; E-mail: anappi@orion.it.luc.edu.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M009872200
 |
ABBREVIATIONS |
The abbreviations used are:
PD, Parkinson's
disease;
ROI, reactive intermediates of oxygen;
DAQ, dopamine quinone;
DAC, dopaminechrome;
DOC, dopachrome;
DOQ, dopaquinone;
RNI, reactive
intermediates of nitrogen;
NO, nitric oxide;
GSH, reduced glutathione;
DEA, diethylamine·NO-generating complex;
HPLC-ED, high performance
liquid chromatography with electrochemical detection;
6NDAQ, 6-nitrosodopamine quinone;
6NDASQ, nitrosodopamine semiquinone;
6OHDA, trihydroxyphenyl quinone.
 |
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