The mitochondrial amidoxime reducing component (mARC) is involved in detoxification of N-hydroxylated base analogues

Article abstract of DOI:10.1021/tx300298m

The mitochondrial Amidoxime Reducing Component (mARC) is the newly discovered fourth molybdenum enzyme in mammals. All hitherto analyzed mammals express two mARC proteins, referred to as mARC1 and mARC2. Together with their electron transport proteins cytochrome b₅ and NADH cytochrome b₅ reductase, they form a three-component enzyme system and catalyze the reduction of N-hydroxylated prodrugs. Here, we demonstrate the reductive detoxification of toxic and mutagenic N-hydroxylated nucleobases and their corresponding nucleosides by the mammalian mARC-containing enzyme system. The N-reductive activity was found in all tested tissues with the highest detectable conversion rates in liver, kidney, thyroid, and pancreas. According to the presumed localization, the N-reductive activity is most pronounced in enriched mitochondrial fractions. In vitro assays with the respective recombinant three-component enzyme system show that both mARC isoforms are able to reduce N-hydroxylated base analogues, with mARC1 representing the more efficient isoform. On the basis of the high specific activities with N-hydroxylated base analogues relative to other N-hydroxylated substrates, our data suggest that mARC proteins might be involved in protecting cellular DNA from misincorporation of toxic N-hydroxylated base analogues during replication by converting them to the correct purine or pyrimidine bases, respectively.

Full text of DOI:10.1021/tx300298m

Article
pubs.acs.org/crt
The Mitochondrial Amidoxime Reducing Component (mARC) Is
Involved in Detoxification of N‑Hydroxylated Base Analogues
Nina Krompholz,† Carmen Krischkowski,† Debora Reichmann,‡ Dieter Garbe-Schonberg,§
̈
Ralf-R. Mendel,‡ Florian Bittner,‡ Bernd Clement,*,† and Antje Havemeyer†
†Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Germany 24118
‡Department of Plant Biology, Technical University of Braunschweig, Germany 38106
§Institute of Geosciences, Christian-Albrechts-University of Kiel, Germany 24118
S
* Supporting Information
ABSTRACT: The “mitochondrial Amidoxime Reducing
Component” (mARC) is the newly discovered fourth
molybdenum enzyme in mammals. All hitherto analyzed
mammals express two mARC proteins, referred to as mARC1
and mARC2. Together with their electron transport proteins
cytochrome b5 and NADH cytochrome b5 reductase, they form
a three-component enzyme system and catalyze the reduction
of N-hydroxylated prodrugs. Here, we demonstrate the
reductive detoxification of toxic and mutagenic N-hydroxylated nucleobases and their corresponding nucleosides by the
mammalian mARC-containing enzyme system. The N-reductive activity was found in all tested tissues with the highest detectable
conversion rates in liver, kidney, thyroid, and pancreas. According to the presumed localization, the N-reductive activity is most
pronounced in enriched mitochondrial fractions. In vitro assays with the respective recombinant three-component enzyme
system show that both mARC isoforms are able to reduce N-hydroxylated base analogues, with mARC1 representing the more
efficient isoform. On the basis of the high specific activities with N-hydroxylated base analogues relative to other N-hydroxylated
substrates, our data suggest that mARC proteins might be involved in protecting cellular DNA from misincorporation of toxic N-
hydroxylated base analogues during replication by converting them to the correct purine or pyrimidine bases, respectively.
In all mammals, the respective mARC isoforms share a high
degree of sequence similarity, and after reconstitution with cyt
b5 and cyt b5R, both enzymes catalyze N-reductive reactions in
vitro with equal specificity for some substrates but different
specificities for others.9,11 The involvement of the mARC-
containing enzyme system in N-reductive drug metabolism is
well accepted,7,12 and it is assumed that the N-reductive
enzyme system plays a major role in the oxygen-independent
reduction of N-hydroxylated compounds. Therefore, we
speculate that the mARC-containing enzyme system is also
involved in the detoxification of mutagenic and toxic N-
hydroxylated aromatic amines such as N-hydroxylated
nucleobases and nucleosides.13 The latter compounds are
produced as metabolites within physiological cellular metabo-
lism by the action of cytochrome P450 enzymes.14 In addition,
chemical factors such as hydroxylamines can be involved in
these transformations. When adenine is exposed to peroxy-
radicals, the majority of the newly formed oxidation products is
N6-hydroxyadenine, a potent mutagenic base analogue.15 Such
N-hydroxylated base analogues were shown to be converted
into the deoxynucleoside triphosphates and incorporated into
replicating DNA.16 Because of their ambivalent base-pairing
INTRODUCTION
■
Until a few years ago, humans were assumed to possess only
three molybdenum-containing enzymes, that is, sulfite oxidase,
aldehyde oxidase, and xanthine oxidoreductase.1−3 Each of
these proteins is characterized by the essential need for the so-
called molybdenum cofactor (Moco), whose molybdenum
atom is coordinated to a 5,6,7,8-tetrahydropyranopterin.4
Recently, a hitherto unknown Moco-containing enzyme was
purified from the outer membrane of pig liver mitochondria.5
The new MOSC-containing (Moco sulfurase C-terminal
domain)6 protein has been found to function in concert with
the electron transport proteins NADH cytochrome b5 reductase
(cyt b5R) and cytochrome b5 (cyt b5), thereby forming a redox
chain that catalyzes the reduction of N-hydroxylated com-
pounds such as amidoximes, which are often used as
prodrugs.5,7−9 According to its subcellular localization and its
reactivity toward amidoximes the MOSC protein was renamed
into “mitochondrial Amidoxime Reducing Component”
(mARC).5 Only recently, Neve and co-workers have
demonstrated that the mARC-mediated N-reductive pathway
might play an important role in lipid synthesis in adipocyte
mitochondria.10 All fully sequenced and annotated mammalian
genomes code for two mARC proteins, which are referred to as
mARC1 and mARC2 (formerly known as MOSC1 and
MOSC2).9
Received: July 3, 2012
Published: August 27, 2012
© 2012 American Chemical Society
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(reference sequence NP_015565) were performed according to Wahl
et al.9
capacity, incorporation of these base analogues induces
mutations or DNA strand breaks.16−18
SDS-PAGE and Immunoblot Analysis. SDS-PAGE was carried
out using a separation gel containing 12.5% polyacrylamide according
to a method of Laemmli.21 The protein samples were pretreated with
β-mercaptoethanol for 5 min at 100 °C prior to loading onto the gel.
Separated proteins were blotted on a polyvinylidinefluoride transfer
membrane. For immunoblot analysis, the primary antibodies anti-
MOSC1 (Abgent, San Diego, CA), anti-MOSC2 (Santa Cruz
Biotechnology, Heidelberg, Germany, or Sigma-Aldrich), anti-
GAPDH (glycerinaldehyde-3-phosphate dehydrogenase) (Sigma-
Aldrich), anti-Calnexin (Acris antibodies, Herford, Germany), and
anti-VDAC (voltage-dependent anion channel) (Acris antibodies)
were used. Visualization was done with secondary horseradish
peroxidase-conjugated antirabbit IgG (immunoglobuline G) or
antigoat IgG antibodies (Jackson Immuno Research Laboratories,
Suffolk, United Kingdom), and detection of the chemiluminescence
was carried out by using the ECL (enhanced chemiluminescence) Plus
Western Blotting Detection System (GE Healthcare, United King-
dom) according to the manufacturer's instructions.
In the present study, the reduction of the mutagenic N-
hydroxylated nucleobases N4-hydroxycytosine and N6-hydroxy-
adenine as well as the nucleosides N4-hydroxycytidine and N6-
hydroxyadenosine by the mARC-containing enzyme system
was investigated. For precise quantification of the N-
hydroxylated base analogues and their corresponding metabo-
lites, HPLC-based analytics were developed. The reductive
detoxification of the nucleobases was characterized using
subcellular fractions from different porcine tissues and human
recombinant enzyme systems (Scheme 1). The results indeed
Scheme 1. Reduction of N-Hydroxylated Nucleobases and
Nucleosides by the mARC-Containing Three-Component
a
Enzyme System
Determination of Protein Concentration. For the determi-
nation of the protein content, a BCA (bicinchoninic acid) Protein
Assay Kit (Pierce, Rockford, United States) was used according to the
manufacturer's instructions.
Determination of Mo by Inductively Coupled Mass
Spectrometry (ICP-MS). Molybdenum contents of recombinant
human mARC1 and mARC2 proteins were determined by using ICP-
MS as described by Kotthaus et al.8 Measurements were carried out in
triplicates.
Flavin Adenine Dinucleotide (FAD) Content Determination.
To determine the FAD content of cyt b5R, samples were heated for
10 min at 100 °C. After centrifugation, the supernatant was analyzed at
450 nm.22 Measurements were carried out in duplicates.
Heme Content Determination. The heme content was estimated
by recording the spectrum of NADH-reduced protein subtracted by
the spectrum of oxidized protein.23 Measurements were carried out in
triplicates.
Determination of mARC-Catalyzed N-Reduction. Incubations
were carried out under aerobic conditions at 37 °C in a shaking water
bath. Incubation mixtures contained different substrate concentrations
as indicated, 1.0 mM NADH in a total volume of 150 μL of 20 mM 2-
morpholinoethanesulfonic acid (MES) buffer, pH 6.0 (in case of
recombinant protein), or 100 mM potassium phosphate buffer, pH 6.0
(in case of subcellular fractions). After 3 min of preincubation at
37 °C, reactions were initiated by addition of NADH and terminated
after 15 (recombinant proteins) or 20 min (subcellular fractions) by
addition of 150 μL of ice-cold methanol. Precipitated proteins were
separated by centrifugation, and the supernatant was analyzed by
HPLC. Incubations were carried out in duplicates. Incubation mixtures
with recombinant enzymes were adjusted based on their cofactors and
consisted of 76 pmol of heme (cyt b5), 7.6 pmol of FAD (cyt b5R),
and 76 pmol of Mo (mARC1 or mARC2, respectively). Incubation
mixtures with subcellular fractions contained 0.01−0.1 mg of protein.
Apparent kinetic parameters Km and Vmax were estimated using
nonlinear regression analysis (Sigma Plot 11.0; SPSS Science, Chicago,
IL). For the calculation of kinetic parameters of mARC1 and porcine
liver mitochondria with N6-hydroxyadenine, the linear plot of
Lineweaver−Burk in the range between 0.1 and 1.0 mM was used.
HPLC Analysis. The HPLC system consisted of a controller 600
from Waters (Milford, United States), equipped with an autosampler
(Waters, 717 plus) and a variable wavelength UV detector (Waters
2487 Dual λ Absorbance Detector). Peak areas were integrated using
the EZChrom Chromatography Data System (EZChrom Elite Version
2.8.3, Scientific Software Inc., San Ramon, United States). Separations
were carried out isocratically at a flow rate of 1.0 mL/min. The
injected sample volume was 10 μL. Aqueous solvents were filtered
through a Sartorius polyamide membrane filter (0.45 μm, Sartorius
AG, Goettingen, Germany), and organic solvents were of HPLC grade.
The mobile phase was degassed by sonication.
a
N4-hydroxycytosine (1a), cytosine (2a); N4-hydroxycytidine (1b),
cytidine (2b); N6-hydroxyadenine (3a), adenine (4a); and N6-
hydroxyadenosine (3b), adenosine (4b).
showed that both the native mARC-containing fractions and
the recombinant enzyme system efficiently catalyze the
detoxification of N-hydroxylated base analogues.
EXPERIMENTAL PROCEDURES
■
Chemicals. N4-hydroxycytosine was purchased from R. I.
Chemicals (Orange, United States). N4-hydroxycytidine was kindly
supplied by Dr. Schinazi (Veterans Affairs Medical Center, Decatur,
United States). N6-hydroxyadenine was purchased from MP
Biomedicals (Solon, United States). N6-hydroxyadenosine was
synthesized as described before.19 All N-hydroxylated compounds
were >97% pure as determined by HPLC analysis. Traces of the
corresponding bases and nucleosides were observed, quantified, and
taken into account for the calculation of metabolic rates (see below).
All other chemicals were purchased from Sigma-Aldrich (Munich,
Germany), Merck KGaA (Darmstadt, Germany), or Roth (Karlsruhe,
Germany) unless otherwise stated. Methanol was of HPLC grade and
purchased from J. T. Baker (Derventer, Holland).
Preparation of Subcellular Fractions. Porcine tissue was
fractionated into homogenate, mitochondria, cytosol, and microsomes.
Mitochondria were prepared by gradient centrifugation as described by
Hovius et al.20 with slight modifications. Microsomes were separated
from the cytosol by centrifugation at 140000g.
Expression and Purification of Recombinant Human
Proteins. Expression and purification of mARC1 (reference sequence
NP_073583), mARC2 (reference sequence NP_060368), mitochon-
drial cyt b5 (reference sequence NP_085056), and cyt b5R isoform 2
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Figure 1. HPLC analytic of modified bases and their reduced metabolites. Representative chromatograms of incubation mixtures with heat-
denatured protein, which were spiked with N-hydroxylated base analogue and its metabolite, are shown. (A) Separation of cytosine (2a) and N4-
hydroxycytosine (1a), (B) separation of N4-hydroxycytidine (1b) and cytidine (2b), (C) separation of N6-hydroxyadenine (3a) and adenine (4a),
and (D) separation of N6-hydroxyadenosine (3b) and adenosine (4b). The chromatographic parameters are described in detail in the Experimental
Procedures.
HPLC Method for the Separation of N4-Hydroxycytosine and
Cytosine. Separations were carried out with 95% (v/v) 20 mM
ammonium acetate buffer and 5% (v/v) methanol using a Nucleodur
100-5 C18 ec, 4.6 mm × 250 mm (Macherey-Nagel, Dueren,
Germany) with a security guard cartridge system C18, 3 mm × 4 mm
(Phenomenex, Torrance, CA) as the precolumn. The effluent was
monitored at 267 nm. The recovery rate and quantification limit of the
metabolite cytosine were determined by incubation mixtures with
defined concentrations of synthetic reference substance (from 5 to
500 μM), which were incubated and worked up under the same
conditions as the experimental samples using heat-denaturated protein.
The signals (peak areas) obtained were compared with those of the
same amount of cytosine dissolved in buffer/methanol. The standard
curves were linear in this range with regression coefficients of 0.99996
(n = 28). The recovery rate after incubation and sample workup was
102 11% after subtraction of minor contaminations of cytosine in
quantification limit of the metabolite adenine were determined by
incubation mixtures with defined concentrations of synthetic reference
substance (from 10 to 500 μM), which were incubated and worked up
under the same conditions as the experimental samples using heat-
denaturated protein. The signals (peak areas) obtained were compared
with those of the same amount of adenine dissolved in buffer/
methanol. The standard curves were linear in this range with
regression coefficients of 0.9998 (n = 36). The recovery rate after
incubation and sample workup was 106 11% after subtraction of
minor contaminations of adenine in the substrate (0.8%). The
retention times were 8.3
11.5 0.1 min (adenine).
0.1 (N6-hydroxyadenine) and
HPLC Method for the Separation of N6-Hydroxyadenosine and
Adenosine. Separations were carried out with 20 mM ammonium
acetate buffer [80% (v/v)] and methanol [20% (v/v)] using a
Nucleodur 100-5 C18 ec, 4.6 mm × 250 mm (Macherey-Nagel,
Dueren, Germany) with a security guard cartridge system C18, 3 mm
× 4 mm (Phenomenex) as the precolumn. The effluent was monitored
at 260 nm. The recovery rate and quantification limit of the metabolite
adenosine were determined by incubation mixtures with defined
concentrations of synthetic reference substance (from 1 to 500 μM),
which were incubated and worked up under the same conditions as the
experimental samples using heat-denaturated protein. The signals
(peak areas) obtained were compared with those of the same amount
of adenosine dissolved in buffer/methanol. The standard curves were
linear in this range with regression coefficients of 0.9999 (n = 36). The
recovery rate after incubation and sample workup was 114 8% after
subtraction of minor contaminations of adenosine in the substrate
(0.1%). The retention times were 4.1 0.1 (N6-hydroxyadenosine)
and 6.4 0.1 min (adenosine).
the substrate (2.2%). The retention times were 5.0
hydroxycytosine) and 3.9 0.1 min (cytosine).
0.1 (N4-
HPLC Method for the Separation of N4-Hydroxycytidine and
Cytidine. Separations were carried out with 10 mM 1-octylsulfonate
sodium salt [75% (v/v)], pH 2.0, and methanol [25% (v/v)] using a
LiChrospher 60 RP-select B column (250 mm × 4 mm, 5 μm, Merck
KGaA) with a RP select B guard precolumn (4 mm × 4 mm, Merck
KGaA). The effluent was monitored at 281 nm. The recovery rate and
quantification limit of the metabolite cytidine were determined by
incubation mixtures with defined concentrations of synthetic reference
substance (from 5 to 500 μM), which were incubated and worked up
under the same conditions as the experimental samples using heat-
denaturated protein. The signals (peak areas) obtained were compared
with those of the same amount of cytidine dissolved in buffer/
methanol. The standard curves were linear in this range with
regression coefficients of 0.9997 (n = 36). The recovery rate after
incubation and sample workup was 108 8%. The retention times
were 5.9 0.1 (N4-hydroxycytidine) and 9.5 0.1 min (cytidine).
HPLC Method for the Separation of N6-Hydroxyadenine and
Adenine. Separations were carried out with 20 mM 1-octylsulfonate
sodium salt [82% (v/v)], pH 3.0, and methanol [18% (v/v)] using a
LiChrospher 60 RP-select B column (125 mm × 4 mm, 5 μm, Merck
KGaA) with a RP select B guard precolumn (4 mm × 4 mm, Merck
KGaA). The effluent was monitored at 262 nm. The recovery rate and
RESULTS
■
HPLC Methods for Separation of Nucleobases and
Nucleosides from their Corresponding N-Hydroxylated
Analogues. In this study, HPLC methods for the separation
and quantification of N-hydroxylated nucleobases and nucleo-
sides from their reduced metabolites have been developed.
These methods are characterized by an accurate quantification
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Figure 2. In vitro reduction of N-hydroxylated nucleobases and nucleosides by porcine subcellular fractions. (A) Western blot analysis of mARC in
the subcellular hepatic fractions. Equal amounts of protein per lane were subjected to SDS-PAGE. For immunoblot analysis, anti-mARC1 antibody,
anti-mARC2 antibody, anti-GAPDH antibody, anti-VDAC antibody, or anticalnexin antibody was used. Complete blots are presented in Figure S1 in
the Supporting Information. (B) Complete incubation mixtures consisted of 0.05 mg of protein, 1 mM NADH, and 1.5−6 mM substrate in 150 μL
of 100 mM phosphate buffer, pH 6.0. Conversion rates are means SDs of four determinants. Incubation, sample preparation, and HPLC analysis
are described in detail in the Experimental Procedures (*, not detectable).
Table 1. In Vitro Reduction of N-Hydroxylated Nucleobases and Nucleosides by the Reconstituted N-Reductive Enzyme
a
System
specific activity (nmol × min−1 × mg−1 total protein)
b
mARC isoform
composition
complete
N4-hydroxycytosine
N4-hydroxycytidine N6-hydroxyadenine N6-hydroxyadenosine benzamidoxime
recombinant mARC1
118.9 26.6
ND
103.1 38.7
ND
180.8 3.6
ND
220.2 26.0
ND
159.4 36.0
ND
without NADH
without cyt b5
ND
ND
ND
ND
ND
without cyt b5R
without mARC1
limit of quantification
complete
ND
ND
ND
1.0 0.1
ND
4.4 0.4
ND
ND
ND
ND
3.5
3.5
7.0
0.7
0.7
recombinant mARC2
39.6 2.7
ND
50.2 2.8
ND
133.5 2.0
ND
105.3 8.7
ND
142.5 24.4
ND
without NADH
without cyt b5
ND
ND
ND
ND
ND
without cyt b5R
without mARC2
limit of quantification
ND
ND
ND
0.9 0.1
ND
2.4 0.2
ND
ND
ND
ND
3.7
3.7
7.3
0.7
0.7
a
A complete incubation mixture consisted of 7.6 pmol of cyt b5R, 76 pmol of cyt b5, and 76 pmol of recombinant mARC1 or mARC2, 1 mM
NADH, and 3−6 mM substrate in 150 μL of 20 mM MES buffer, pH 6.0. Conversion rates are means SDs of four determinations. Incubation,
b
sample preparation, and HPLC analysis are described in detail in the Experimental Procedures (ND, not detectable). Data previously published in
Wahl et al.9
in the range of 10−500 μM of the metabolites in the presence
of protein matrix and the cosubstrates NADH and NAD+. The
separation of all compounds was achieved within short run
times (<11.5 min) and with good resolution characteristics
(>3.2). Representative chromatograms for the separation of the
N-hydroxylated analogues and their reduced metabolites are
displayed in Figure 1, showing incubation mixtures with heat-
denaturated protein spiked with the indicated N-hydroxylated
base analogue and its respective metabolite.
Characterization of Proteins Used for in Vitro
Incubation. To assess the purity of the subcellular fractions,
Western blots using antibodies against microsomal (calnexin),24
mitochondrial (VDAC),25 and cytosolic (GAPDH)26 marker
proteins were carried out (Figure 2A). To characterize the N-
reductive activity of the protein batches used, incubations with
the well-established mARC marker substrate benzamidoxime
were performed under identical conditions as for the base
analogues (Figure 2B).
The cofactor saturation of recombinant proteins was
analyzed by different spectroscopic techniques as described in
the Experimental Procedures and showed 7% heme saturation
for cyt b5, 95% FAD saturation for cyt b5R, and 28 or 32%
molybdenum saturation for mARC1 or mARC2, respectively.
In a reconstituted enzyme system with cyt b5 and cyt b5R, both
mARC proteins were capable of reducing the model substrate
benzamidoxime to benzamidine with similar conversion rates.
The highest conversion rates were measured with the complete
enzyme system, while omitting one of the components,
including NADH, resulted in a nearly complete loss of activity
(Table 1).
N-Reductive Activity Is Enriched in Mitochondria. The
reduction of the N-hydroxylated pyrimidine and purine
nucleobases as well as their nucleosides was studied in porcine
subcellular liver fractions. Incubations with purified fractions
led to the formation of the corresponding nucleobases or
nucleosides, which were quantified by HPLC (Figure 1).
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a
Table 2. Kinetic Parameters of the Reduction of N-Hydroxylated Nucleobases and Nucleosides
substrate
N4-hydroxycytosine
mARC isoform
mARC1
Km (mM)
Vmax (nmol × min−1 × mg−1
)
catalytic efficiency (M−1 × s−1
)
0.52 0.14
1.52 0.28
0.92 0.07
0.44 0.04
5.65 0.51
1.02 0.07
0.77* 0.20
0.72 0.11
0.16* 0.02
0.98 0.11
5.28 1.26
0.63 0.15
174.1 12.6
65.3 4.4
2080
256
−
1838
117
−
3026
996
−
1774
522
−
mARC2
porcine liver mitochondria
mARC1
11.2 0.3
N4-hydroxycytidine
N6-hydroxyadenine
N6-hydroxyadenosine
130.2 3.2
111.1 5.4
8.2 0.2
mARC2
porcine liver mitochondria
mARC1
375.1* 51.5
120.2 5.7
21.9* 0.6
279.8 10.8
210.9 17.1
17.8 1.1
mARC2
porcine liver mitochondria
mARC1
mARC2
porcine liver mitochondria
a
The incubation mixture consisted of 7.6 pmol of cyt b5R, 76 pmol of cyt b5, and 76 pmol of recombinant mARC1 or mARC2 in 150 μL of 20 mM
MES buffer, pH 6.0, or 0.05 mg of porcine liver mitochondria in 100 mM phosphate buffer, pH 6.0, respectively, 1 mM NADH, and variable
concentrations of substrate. Conversion rates are means SDs of four determinants. Incubation, sample preparation, and HPLC analysis are
described in detail in the Experimental Procedures (*, calculated).
Figure 3. In vitro reduction of N-hydroxylated nucleobases and nucleosides by porcine hepatic and extrahepatic mitochondria. (A) Complete
incubation mixtures consisted of 0.01−0.1 mg of protein, 1 mM NADH, and 1.5−6 mM in 150 μL of 100 mM phosphate buffer, pH 6.0. Conversion
rates are means
SDs of four determinants. Incubation, sample preparation, and HPLC analysis are described in detail in the Experimental
Procedures (*, not detectable). (B) Western blot analysis of mARC in the porcine hepatic and extrahepatic mitochondria. Equal amounts of
mitochondrial fraction per lane were subjected to SDS-PAGE. For immunoblot analysis, anti-mARC1 or anti-mARC2 antibody was used. Complete
blots are presented in Figure S2 in the Supporting Information.
Moreover, all reactions showed strict dependency on NADH as
cosubstrate. The in vitro reduction with subcellular fractions
was most efficient in the mitochondrial fraction, whereas only
minor reduction rates were found in the microsomal and
cytosolic fractions. Because enrichment of the reductase activity
for the N-hydroxylated base analogues in the mitochondrial
fraction was consistent with the enrichment of mARC marker
activity (BAO reduction) (Figure 2B), it is indicated that all
used substrates were converted by the mARC-containing
enzyme system. This is also confirmed by the enrichment of
both mARC proteins in the mitochondrial fractions as
demonstrated by use of specific antibodies (Figure 2A).
Highest Reduction Rates in Liver, Kidney, Thyroid,
and Pancreas. As N-reductive activity was enriched in the
mitochondrial fraction, Vmax and Km were determined by
incubation of liver mitochondria at varying substrate concen-
trations of the tested base analogues. All reactions followed
Michaelis−Menten kinetics with Km values <1 mM of all tested
substrates. The highest Vmax values were observed for N6-
hydroxyadenine and its corresponding nucleoside (Table 2).
However, using N6-hydroxyadenine as the substrate, the N-
reductive activity was inhibited at concentrations above 1 mM.
Therefore, parameters of the reduction with N6-hydroxyade-
nine were calculated using the linear plot of Linewaever−Burk
in the range of 0.1−1.0 mM.
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Figure 4. Michaelis−Menten curves of N6-hydroxyadenine with mARC1 (A), mARC2 (B), and porcine liver mitochondria (C). The incubation
mixture consisted of 7.6 pmol of cyt b5R, 76 pmol of cyt b5, and 76 pmol of recombinant mARC1 or mARC2 in 150 μL of 20 mM MES buffer, pH
6.0, or 0.05 mg of porcine liver mitochondria in 100 mM phosphate buffer, pH 6.0, respectively, 1 mM NADH, and variable concentrations of
substrate. Conversion rates are means SDs of four determinants. Incubation, sample preparation, and HPLC analysis are described in detail in the
Experimental Procedures.
Furthermore, the N-reductive activities of mitochondria from
extrahepatic tissues were investigated and compared to those of
the hepatic fractions. In fact, the N-reductive activity could be
observed in all analyzed extrahepatic mitochondria. However,
depending on the substrate, mitochondria from various tissues
exhibited different conversion rates, with the highest reduction
rates in mitochondrial fractions of thyroid, kidney, pancreas,
and liver (Figure 3A). This is in agreement with the expression
pattern of both mARC isoforms (Figure 3B).
and amino acid levels.9 It has been shown earlier that both
human mARC proteins are able to reduce N-hydroxylated
substrates such as benzamidoxime. Both mARC proteins are
not active as stand-alone protein but require reconstitution with
cyt b5 and cyt b5R (Table 1). The function of cyt b5R is likely
associated with the oxidation of NADH for recruitment of
electrons, whereas cyt b5 carries the electrons from cyt b5R to
mARC and mARC represents the active site where the
respective N-hydroxylated substrate is reduced.5,8,9 mARC is
found in mitochondria,5,9 which is confirmed in the present
study by detecting and discriminating both mARC homologues
in mitochondria from various porcine tissues using isoform-
specific antibodies (Figure 3B). Thus, it is not surprising that
the N-reductive activity for the marker substrate benzamidox-
ime is enriched in mitochondrial fractions in comparison to
liver homogenates (Figure 2B). To identify a possible
contributing role of the mitochondrial three-component
enzyme system to the reduction of N-hydroxylated base
analogues and the corresponding nucleosides, coincubation
studies with porcine subcellular fractions and various N-
hydroxylated base analogues were carried out.
In fact, enrichment of N-reductive activity in the
mitochondrial fractions for N-hydroxylated base and nucleoside
analogues was comparable to that of the (mARC-specific)
marker substrate benzamidoxime, suggesting that the reduction
of benzamidoxime and N-hydroxylated base analogues is
catalyzed by identical proteins in a similar manner. In support
of this, the expression pattern of both mARC isoforms in
porcine mitochondria of distinct tissues correlates with the high
reductase activities in thyroid, pancreas, kidney, and liver
(Figure 3). We therefore conclude that the mARC-containing
enzyme system is responsible not only for reduction of the
marker substrate benzamidoxime but also for the tested N-
hydroxylated base analogues. In fact, the involvement of the
mARC-containing enzyme system in the detoxification
reactions was confirmed using the complete recombinant
human three-component system: after reconstitution with the
electron transport proteins cyt b5 and cyt b5R, both
recombinant mARC1 and mARC2 were able to reduce the
hydroxylamines, whereas incubation mixtures lacking one of
these proteins showed no or only negligible activity (Table 1).
On the basis of the arrangement of redox centers and its
similarity to eukaryotic nitrate reductases of plants, fungi, and
Detoxification of N-Hydroxylated Nucleobases and
Nucleosides Is Based on the mARC-Containing Enzyme
System. The reduction of a N-hydroxylated purine nucleobase
as well as N-hydroxylated purine and pyrimidine nucleosides
was studied using recombinantly expressed enzyme sources.
Incubations with the reconstituted enzyme system consisting of
cyt b5, cyt b5R, and either mARC1 or mARC2 led to the
formation of the corresponding nucleobases or nucleosides. As
shown for the porcine liver fractions, the reaction was strictly
dependent on NADH. Furthermore, the highest conversion
rates were only detected with the complete enzyme system,
whereas the lack of one of the components either resulted in
strongly decreased or completely abrogated activity (Table 1).
All reactions followed Michaelis−Menten kinetics, which is
shown for N6-hydroxyadenine as an example (Figure 4).
Although all tested N-hydroxylated nucleobases and nucleo-
sides were reduced by mARC1 and mARC2, differences in the
catalytic efficiency were observed. In general, Vmax values were
higher and Km values were lower when mARC1 was used,
indicating a higher affinity of mARC1 to the tested nucleobase
analogues. For both proteins, the highest Vmax values were
observed for N6-hydroxyadenine and its corresponding nucleo-
side (Table 2). As already observed with liver mitochondria
(Figure 4C), substrate concentrations above 1 mM led to a
reduction of the specific activity of mARC1 (Figure 4A) but not
of mARC2 (Figure 4B).
DISCUSSION
■
In this study, for the first time, we provide evidence for the
contribution of the mammalian molybdenum enzyme mARC to
the detoxification of toxic and mutagenic N-hydroxylated
nucleobases and nucleosides. All hitherto analyzed mammalian
genomes harbor two mARC genes, mARC1 (MOSC1) and
mARC2 (MOSC2), which are highly conserved on nucleic acid
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algae, it can be assumed that reduction of the tested N-
hydroxylated substrates by the mARC-containing enzyme
system occurs via cleavage of the N−O bond as proposed for
nitrate reductase during conversion of nitrate to nitrite.11
Kinetic studies performed with the recombinant human
enzymes revealed that all base and nucleoside analogues are
very good substrates for the mARC-containing enzyme system.
The highest conversion rates of N6-hydroxyadenine and its
nucleoside were determined with both the porcine liver
mitochondria and the reconstituted human enzyme system,
the latter presenting a specific reductase activity about 15 times
higher than that of the mitochondrial fractions. Strikingly, the
human mARC1-containing system showed higher maximum
rates (Vmax) and smaller Michaelis−Menten constants (Km)
than the enzyme system containing human mARC2 (Table 2).
In addition, Km values of mARC1 and hepatic mitochondria are
similar (Table 2), which indicates that one of the two mARC
proteins, presumably the mARC1 isoform, is of particular
relevance to the reductive detoxification pathway in vivo.
Moreover, there seems to be a slight substrate preference
comparing the reduction of nucleosides derivatives and free
bases, as reduction rates of nucleosides are faster when
reduction is catalyzed by mARC1. However, in case mARC2
is used in the incubation mixture, higher conversion rates of
free bases were determined. In this regard, another observation
is noteworthy: incubation series with concentrations above
1 mM N6-hydroxyadenine resulted in inhibition of the N-
reductive activity in both the mitochondria and the
recombinant human mARC1-containing enzyme system. In
contrast, the reconstituted system with human mARC2 was not
affected by high substrate concentrations of this purine
derivative (Figure 4A−C). We speculate that inhibition by
high substrate concentrations is a specific reaction of the
mARC1 homologue, supporting a contribution of mARC
proteins to detoxification of N-hydroxylated base analogues.
However, further experiments are needed to determine whether
both or only one mARC isoform is involved in this N-reductive
pathway of N-hydroxylated DNA bases in vivo. Using a knock-
down approach, Neve and co-workers recently postulated that
only mARC2 is involved in the N-reductive pathway in murine
cell culture.10 However, RNAi studies in HEK-293 cells
performed in our lab clearly indicate that knock-down of
human mARC1 expression significantly diminishes reductase
activity (unpublished results, manuscript in preparation).
In summary, we showed that N-hydroxylated base analogues
were converted by the mammalian enzyme system, and both
recombinant human mARC proteins are able to catalyze this
reduction in vitro. Interestingly, reduction of N6-hydroxyade-
nine has also been reported earlier for another mammalian
molybdenum enzyme, that is, xanthine oxidoreductase (in the
form of xanthine oxidase).27 However, because in the present
work the N-reductive activity was shown to be enriched in
mitochondria, the mARC-dependent pathway appears more
significant than the cytosolic pathway involving xanthine
oxidoreductase.
MOSC domain-carrying proteins were reported in other
organisms such as the green algae Chlamydomonas reinhardtii28
and E. coli.29−31 As shown in the present work, two Moco-
binding MOSC proteins have been found to participate in
detoxification of N-hydroxylated base analogues in E. coli, with
the YcbX protein together with its redox partner protein CysJ
representing the more relevant system as compared to the YiiM
protein with as yet unknown redox partner.29−31 Although
detoxification of N-hydroxylated base analogues is not
necessarily the only function of eukaryotic and bacterial
MOSC domain proteins, it is likely to represent one of the
physiologically relevant functions.
ASSOCIATED CONTENT
* Supporting Information
■
S
Western blot analysis of subcellular hepatic fractions and
Western blot analysis of mARC1 and mARC2 in the porcine
hepatic and extrahepatic mitochondria. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +49-431-8801126. Fax: +49-431-8801352. E-mail:
bclement@pharmazie.uni-kiel.de.
■
Funding
Financial support by the Deutsche Forschungsgemeinschaft
(DFG, grant number CL56/9-1, ME 1266/24-1) is greatly
appreciated.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Petra Koster, Ulrike Westernstroer, and Sven
̈
̈
Wichmann for their technical assistance.
ABBREVIATIONS
■
Moco, molybdenum cofactor; MOSC, Moco sulfurase C-
terminal domain; GAPDH, glycerinaldehyde-3-phosphate
dehydrogenase; VDAC, voltage-dependent anion channel;
IgG, immunoglobuline G; ECL, enhanced chemiluminescence;
BCA, bicinchoninic acid; MES, 2-morpholinoethanesulfonic
acid
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