NADH cytochrome b5 reductase and cytochrome b5 catalyze the microsomal reduction of xenobiotic hydroxylamines and amidoximes in humans

Article abstract of DOI:10.1124/jpet.104.072389

Hydroxylamine metabolites, implicated in dose-dependent and idiosyncratic toxicity from arylamine drugs, and amidoximes, used as pro-drugs, are metabolized by an as yet incompletely characterized NADH-dependent microsomal reductase system. We hypothesized that NADH cytochrome b₅ reductase and cytochrome b₅ were responsible for this enzymatic activity in humans. Purified human soluble NADH cytochrome b₅ reductase and cytochrome b₅ expressed in Escherichia coli, efficiently catalyzed the reduction of sulfamethoxazole hydroxylamine, dapsone hydroxylamine, and benzamidoxime, with apparent K_m values similar to those found in human liver microsomes and specific activities (V_max) 74 to 235 times higher than in microsomes. Minimal activity was seen with either protein alone, and microsomal protein did not enhance activity other than additively. All three reduction activities were significantly correlated with immunoreactivity for cytochrome b₅ in individual human liver microsomes. In addition, polyclonal antibodies to both NADH cytochrome b₅ reductase and cytochrome b₅ significantly inhibited reduction activity for sulfamethoxazole hydroxylamine. Finally, fibroblasts from a patient with type II hereditary methemoglobinemia (deficient in NADH cytochrome b₅ reductase) showed virtually no activity for hydroxylamine reduction, compared with normal fibroblasts. These results indicate a novel direct role for NADH cytochrome b₅ reductase and cytochrome b₅ in xenobiotic metabolism and suggest that pharmacogenetic variability in either of these proteins may effect drug reduction capacity.

Full text of DOI:10.1124/jpet.104.072389

0022-3565/04/3113-1171–1178$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics
JPET 311:1171–1178, 2004
Vol. 311, No. 3
72389/1179642
Printed in U.S.A.
NADH Cytochrome b5 Reductase and Cytochrome b5 Catalyze
the Microsomal Reduction of Xenobiotic Hydroxylamines and
Amidoximes in Humans
Joseph R. Kurian, Sunil U. Bajad, Jackie L. Miller, Nathaniel A. Chin, and
Lauren A. Trepanier
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin
Received June 15, 2004; accepted August 6, 2004
ABSTRACT
Hydroxylamine metabolites, implicated in dose-dependent and other than additively. All three reduction activities were signifi-
idiosyncratic toxicity from arylamine drugs, and amidoximes, cantly correlated with immunoreactivity for cytochrome b5 in
used as pro-drugs, are metabolized by an as yet incompletely individual human liver microsomes. In addition, polyclonal an-
characterized NADH-dependent microsomal reductase sys- tibodies to both NADH cytochrome b5 reductase and cyto-
tem. We hypothesized that NADH cytochrome b5 reductase chrome b5 significantly inhibited reduction activity for sulfame-
and cytochrome b5 were responsible for this enzymatic activity thoxazole hydroxylamine. Finally, fibroblasts from a patient with
in humans. Purified human soluble NADH cytochrome b5 re- type II hereditary methemoglobinemia (deficient in NADH cyto-
ductase and cytochrome b5, expressed in Escherichia coli, chrome b5 reductase) showed virtually no activity for hydrox-
efficiently catalyzed the reduction of sulfamethoxazole hydrox- ylamine reduction, compared with normal fibroblasts. These
ylamine, dapsone hydroxylamine, and benzamidoxime, with results indicate a novel direct role for NADH cytochrome b5
apparent Km values similar to those found in human liver mi- reductase and cytochrome b5 in xenobiotic metabolism and
crosomes and specific activities (Vmax) 74 to 235 times higher suggest that pharmacogenetic variability in either of these pro-
than in microsomes. Minimal activity was seen with either pro- teins may effect drug reduction capacity.
tein alone, and microsomal protein did not enhance activity
Hydroxylamine and amidoxime compounds are metabo- their parent amines by a microsomal, oxygen-insensitive,
lized in humans by an as yet incompletely characterized
NADH-dependent reductase system. Hydroxylamine me-
tabolites have been implicated in dose-dependent and id-
iosyncratic drug toxicity from sulfamethoxazole, dapsone,
procainamide, and other arylamine drugs (Uetrecht,
2002). Amidoximes and other hydroxylated amines have
been developed as prodrugs to enhance the absorption of a
wide range of antihypertensive, antiprotozoal, and anti-
thrombotic drugs (Weller et al., 1996; Hall et al., 1998;
Clement, 2002), and the reduction of these compounds is
necessary for drug bioactivation. Thus, the reduction of
hydroxylamines and amidoximes has important toxicologic
and therapeutic implications.
NADH-dependent enzyme system, variably termed NADH-
dependent N-hydroxy amine reductase, NADH-benzami-
doxime reductase, or NADH-dependent hydroxylamine re-
ductase (NDHR) (Kadlubar and Ziegler, 1974; Cribb et al.,
1995; Clement et al., 1998; Trepanier and Miller, 2000).
NDHR was originally described in the reduction of N-meth-
ylhydroxylamine, N-hydroxyamphetamine, and related com-
pounds (Kadlubar et al., 1973; Kadlubar and Ziegler, 1974;
Yamada et al., 1988). NDHR has more recently been shown
to catalyze the reduction of hydroxylamine metabolites of
sulfamethoxazole and dapsone (Cribb et al., 1995; Clement et
al., 1998), as well as the reduction of N-hydroxylated deriv-
atives of benzamidine, debrisoquine, guanabenz, metham-
phetamine, and pentamidine (Clement, 2002) (Fig. 1).
Xenobiotic hydroxylamines and amidoximes are reduced to
The complete characterization of NDHR has not been re-
ported in humans, but in the pig, a protein with sequence
homology to CYP2D6 has been proposed to be involved in the
reduction of hydroxylamines (Clement et al., 1997), along
This work was supported by National Institutes of Health Grant GM 61753.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.104.072389.
ABBREVIATIONS: NDHR, NADH-dependent hydroxylamine reductase; P450, cytochrome P450; cyt b5, cytochrome b5; b5R, NADH cytochrome
b5 reductase; PBS, phosphate-buffered saline; SMX-HA, sulfamethoxazole hydroxylamine; HPLC, high performance liquid chromatography; HSA,
human serum albumin; HLM, human liver microsome.
1171

1172
Kurian et al.
Shirabe (Oita Medical University, Japan). Initial attempts to purify
the protein using DEAE and 5Ј-AMP-Sepharose column chroma-
tography lead to poor yields, due to weak affinity of b5R for 5Ј-AMP-
Sepharose under a variety of conditions. The cDNA was inserted into
an N-terminal histidine tag expression system (pCR T7/NT-TOPO;
Invitrogen, Carlsbad, CA) to allow purification from Escherichia coli
lysate using nickel affinity chromatography. Induction with 1 mM
isopropyl ␤-D-thiogalactopyranoside was performed for 4 h at room
temperature to prevent overexpression and compartmentalization of
the protein in lipid vacuoles. Stepwise elution of lysate contaminants
followed by His-tagged b5R was accomplished with three elution
buffers: 20, 100, and 350 mM imidazole, each at pH 8.0 in binding
buffer. The eluent was collected in 1-ml fractions, which were tested
for purity by gel electrophoresis and subsequent silver staining, for
identity using immunoblotting, and for activity using potassium
ferricyanide reduction (Arinc and Cakir, 1999). The fractions con-
taining a single 36-kDa band on silver stain were pooled and dia-
lyzed against binding buffer, pH 8.0, through 10,000 mol. wt. cut-off
cassettes (Pierce Biotechnology, Rockford, IL) to remove imidazole.
The dialyzed sample was again bound to a nickel column and re-
purified and pooled as described above. The purified b5R was dia-
lyzed against PBS, pH 7.4, and concentrated to a final protein con-
centration of 2 to 3 mg/ml using 10,000 mol. wt. cut-off filtration
devices (Centricon; Millipore Corporation, Bedford, MA). Purified
protein retained maximal activity at 4°C for approximately 3 weeks.
Expression of Soluble Human cyt b5. The full-length cDNA of
human soluble cyt b5 (Lloyd et al., 1994), kindly provided by Dr.
Grant Mauk (University of British Columbia) was expressed in the
pCR T7/CT-TOPO system, using methods as described for b5R, with
some modifications. In brief, stepwise elutions using 20, 40, and 100
mM imidazole, each at pH 8.0 in binding buffer, were collected in
1-ml fractions and tested for purity by gel electrophoresis and sub-
sequent silver staining. Identity was verified with immunoblotting.
Fractions containing a single 16.5-kDa band on silver stain were
pooled, dialyzed, and re-purified as described for b5R. Purified pro-
tein retained maximal activity at 4°C for approximately 3 weeks.
Hydroxylamine Reduction by Human b5R and cyt b5. Sul-
famethoxazole-hydroxylamine (SMX-HA; Dalton Chemical Labora-
tories, Toronto, ON, Canada) in 50% dimethyl sulfoxide/3 mM ascor-
bic acid was incubated at varying concentrations with purified
soluble human recombinant b5R and cyt b5. Commercially available
cyt b5 protein (PanVera Corp., Madison WI), was used for prelimi-
nary experiments; the soluble form of cyt b5, purified to homogeneity
as described above, was used for all subsequent experiments. Reac-
tions were performed in PBS, pH 7.4, with 1 mM NADH; glutathione
and ascorbic acid (1 mM each) were included to maximally stabilize
the SMX-HA against further oxidation (Trepanier and Miller, 2000).
The final dimethyl sulfoxide concentration in all reactions was
Fig. 1. Reported substrates of oxygen-insensitive microsomal NDHR.
with NADH cytochrome b5 reductase and cytochrome b5.
NADH-dependent hydroxylamine reduction in humans, how-
ever, does not correlate with the activities of CYP2D6, nor
with the activities of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2E1,
3A4, or 3A5 (Cribb et al., 1995; Lin et al., 1996). In addition,
NDHR activity is insensitive to azide and carbon monoxide
(Kadlubar et al., 1973; Trepanier and Miller, 2000), which is
inconsistent with the involvement of electron transport
through P450 in this reaction.
NADH cytochrome b5 reductase (EC 1.6.2.2; diaphorase I;
NADH:ferricytochrome b5 oxidoreductase) and cytochrome
b5 themselves have all of the biochemical characteristics of
NDHR and, therefore, may be capable of directly mediating
NADH-dependent hydroxylamine reduction in humans.
These proteins, however, have historically been viewed as
“helper” enzymes involved in intermediate electron transfer
(Porter, 2002) and have not been shown to have a major
direct role in xenobiotic metabolism. In this report, we pro-
vide evidence that purified human recombinant NADH cyto-
chrome b5 reductase (b5R) and cytochrome b5 (cyt b5) with-
out any other microsomal components are sufficient for the
reduction of sulfamethoxazole hydroxylamine, dapsone hy-
droxylamine, and benzamidoxime to their parent amines/ 1.25%. The optimal stoichiometry of b5R/cyt b5 was determined by
measuring reduction activity using different ratios of b5R to cyt b5
while keeping the total nanomoles of protein constant (Huang, 1982).
Sulfamethoxazole hydroxylamine reduction activities were deter-
mined using isocratic HPLC with UV detection as previously de-
scribed (Trepanier and Miller, 2000). Negative controls included
human serum albumin (HSA) or protein purified from sham-trans-
formed E. coli and recombinant systems lacking NADH or substrate.
Dapsone hydroxylamine was a gift from Dr. Reginald Frye (School
of Pharmacy, University of Pittsburgh). Reduction of dapsone hy-
droxylamine was determined using HPLC as described for sulfame-
thoxazole. Retention times for dapsone and its hydroxylamine were
6.6 and 5.2 min, respectively. In addition, the reduction of benzami-
doxime [a prototypical oxime substrate also subject to NADH-depen-
dent microsomal reduction (Clement et al., 1997)] to benzamidine
was measured using a modification of a previously published HPLC
method (Clement et al., 1988). In brief, the mobile phase consisted of
water/methanol/phosphoric acid 85%/triethylamine/1-octanesulfonic
acid sodium salt (68/32/0.0036/0.0050/0.0012, v/v/v/v/M), with a flow
amidines. We show further that the apparent Km values for
reduction by the recombinant system are similar to those
seen in human liver microsomes and that addition of micro-
somal protein does not enhance activity more than addi-
tively. In addition, hydroxylamine and amidoxime reduction
activities correlate with microsomal cyt b5 content, are in-
hibited by antiserum to either b5R or cyt b5, and are absent
in fibroblasts deficient in b5R. We conclude that b5R and cyt
b5 together have an important direct role in xenobiotic re-
duction and further hypothesize that variability in the ex-
pression of either of these proteins may have important ther-
apeutic and toxicologic consequences.
Materials and Methods
Expression of Soluble Human NADH b5R. The full-length
cDNA of human soluble b5R was kindly provided by Dr. Komei

Hydroxylamine/Amidoxime Reduction
1173
rate of 1.5 ml/min. UV detection was at 229 nm, with retention times obtained with consistent purity and strong immunoreactivity
of 13.5 min for benzamidoxime and 15.1 min for benzamidine.
Estimates for apparent Km and Vmax of the expressed protein
system for the three substrates were determined by nonlinear curve
fitting using commercial software (Prism 3; GraphPad Software,
Inc., San Diego CA). For comparison of specific activity and apparent
Km values between recombinant and microsomal systems, kinetics
were also determined in pooled human liver microsomes (BD Gen-
test, Woburn, MA).
with anti-b5R antibody (Fig. 2). Preliminary studies showed
no difference in activity between tagged and untagged pro-
tein (as others have shown; Bewley et al., 2001), and since
enterokinase cleavage and processing to remove the His tag
resulted in a less pure product (data not shown), all subse-
quent studies were performed with histidine-tagged protein.
cyt b5 Protein Expression and Purification. Expres-
sion and purification of histidine-tagged human soluble cyt
b5 yielded an average of 5 mg of purified protein per liter of
Immunocorrelation Studies. For preliminary studies, rabbit
polyclonal antibody to rat b5R was kindly provided by Dr. Jose
Villalba (Universidad de Cordoba, Spain). For subsequent studies, culture. A single 16.5-kDa protein (12.5-kDa protein plus
purified human b5R and cyt b5 were used to immunize individual
rabbits using standard protocols. Immune rabbit anti-b5R and anti-
cyt b5 sera were used to probe immunoblots prepared from microso-
mal protein (40 ␮g) from individual human liver microsomes (Hu-
man Biologics International, Scottsdale AZ). Polyvinylidene
difluoride membranes (Immobilon-P; Millipore Corporation) were
blocked with 2.5% nonfat dry milk in PBS/0.1% Tween-20 and
washed with PBS/0.1% Tween-20. Primary antisera were diluted
1:10,000 for immunoblotting; horseradish peroxidase-linked second-
ary antibody (donkey anti-rabbit IgG; Amersham Biosciences, Inc.,
4-kDa tag) was obtained with consistent purity and strong
immunoreactivity with anti-cyt b5 antibody (Fig. 3).
Reduction of Hydroxylamine and Amidoxime Sub-
strates. Expressed b5R and cyt b5 catalyzed the efficient
reduction of sulfamethoxazole hydroxylamine (500 ␮M), at
rates 160 times greater than pooled human liver microsomes
(HLM; Fig. 4). No activity was seen with protein purified
with sham-transformed E. coli or in the absence of NADH,
and minimal activity was observed with either b5R or cyt b5
Piscataway, NJ) was diluted 1:10,000. An enhanced chemilumines- alone. No enhancement of activity other than an additive
cence system was used for signal detection (Amersham Biosciences,
Inc.), and was quantified using a Storm 840 imaging system with
ImageQuaNT software (Amersham Biosciences, Inc.). For each of the
individual human liver microsome samples, b5R and cyt b5 immu-
noreactivity were correlated with microsomal reduction of each sub-
strate as determined by HPLC as described above using a correlation
z test (Statview; Abacus Concepts, Berkeley, CA).
Immunoinhibition of Reduction. Antisera (150 ␮l each) to
human b5R and cyt b5 as described above were preincubated with
human liver microsomes (0.5 mg) for 30 min at room temperature,
effect was seen with the addition of 10, 100, 300, or 1000 ␮g
of pooled human liver microsomes to the recombinant system
(Fig. 5).
The optimal stoichiometry of cyt b5 to b5R was between 8:1
and 10:1 as determined by a Job plot (Huang, 1982) (data not
shown). The kinetics of sulfamethoxazole hydroxylamine re-
duction in the expressed system (Fig. 6) and in HLM were
each fit to a one-site model with similar apparent Km values
(Table 1). Dapsone hydroxylamine and benzamidoxime were
followed by quantitation of reduction of sulfamethoxazole hydroxyl- also reduced by the recombinant system with 163 and 74
amine. Preimmune rabbit serum or HSA instead of serum was used
as negative controls.
Hydroxylamine Reduction in Fibroblasts Deficient in b5R.
Fibroblasts from a patient with type II hereditary methemoglobin-
emia, a disorder in which systemic b5R activity is deficient or com-
pletely lacking, were generously provided by Drs. Wilfried Kugler
and Arnulf Pekrun (Universitats-Kinderklinik, Goettingen, Ger-
many) (Kugler et al., 2001). Normal human dermal fibroblasts were
a gift from the laboratory of Drs. Gregory MacEwen and David Vail
(University of Wisconsin-Madison). Cells were cultured in Eagle’s
minimum essential media (without phenol red, calcium, or magne-
sium; Mediatech Inc., Herndon VA), supplemented with 1 mM so-
dium pyruvate, 2 mM L-glutamine, 100 IU/ml penicillin, 100 ␮g/ml
streptomycin, and 0.25 ␮g/ml amphotericin, and incubated at 37°C in
a humidified atmosphere with 5% CO2. Cells were harvested with
trypsin containing 670 ␮M EDTA, washed with medium followed by
Hanks’ buffer, and resuspended in 15 mM HEPES, pH 7.4. Reduc-
tion activity reactions contained 1.5 ϫ 106 cells, along with 500 ␮M
of SMX-HA. Glutathione and ascorbate (1 mM each) were included in
SMX-HA reactions to prevent further oxidation of the hydroxyl-
amine. Cells were incubated at 37°C for 40 min. Reactions were
stopped on ice with half the reaction volume of methanol, then
filtered through 30-kDa filters (Millipore Corporation) prior to HPLC
analysis. Fibroblasts were also subjected to immunoblotting using
both ␣-cyt b5 and ␣-b5R.
Results
Fig. 2. Purified human soluble cytochrome b5 reductase. Panel A: lane 1,
marker; lane 2, E. coli lysate (50 ␮g) containing expressed human soluble
b5R Protein Expression and Purification. Expression
of histidine-tagged human soluble b5R in E. coli and purifi-
cation using nickel affinity chromatography yielded an aver-
age of 3 to 4 mg of purified protein per liter of culture. A
NADH cytochrome b5 reductase; lane 3, purified b5R (25 ␮g), as purified
by an N-terminal histidine tag expression system and nickel affinity
chromatography; 4 to 20% polyacrylamide gel with Coomassie stain.
Panel B: purified cytochrome b5 reductase (as shown in panel A), immu-
noblotted with rabbit anti-human b5R antisera. The 36-kDa molecular
single 36-kDa protein (32-kDa protein plus 4-kDa tag) was weight reflects the 32-kDa soluble protein with a 4-kDa histidine tag.

1174
Kurian et al.
Fig. 3. Purified human soluble cytochrome b5. Panel A: lane 1, marker;
lane 2, E. coli lysate (50 ␮g) containing expressed human soluble cyto-
chrome b5; lane 3, purified cytochrome b5 (25 ␮g), as purified by a
C-terminal histidine tag expression system and nickel affinity chroma-
tography; 4 to 20% polyacrylamide gel with Coomassie stain. Panel B:
purified cytochrome b5 (as shown in panel A), immunoblotted with rabbit
anti-human cytochrome b5 antisera. The 16.5-kDa molecular weight re-
flects the 12.5-kDa soluble protein with a 4-kDa histidine tag.
Fig. 5. Activity for reduction of SMX-HA (500 ␮M) by the recombinant
system (Recomb; b5R and cyt b5) compared with the recombinant system
plus pooled HLM (0.1 mg) is not more than additive. Note that activity is
expressed per minute and is not normalized to milligrams of protein. All
reactions are as described in Fig. 4; similar results were seen with 0.01,
0.3, and 1.0 mg of additional microsomal protein.
Fig. 6. Velocity versus substrate concentrations for reduction of SMX-HA
by purified human recombinant cytochrome b5 and NADH cytochrome b5
reductase (optimal 10:1 stoichiometry) in PBS, pH 7.4, with 1 mM NADH.
Fig. 4. Specific activity for reduction of SMX-HA is 160-fold higher by
expressed b5R with cytochrome b5 (10:1 stoichiometry, cyt b5/b5R), com-
pared with pooled HLM (0.5 mg). Negative controls include b5R alone, cyt
b5 alone, and protein purified from sham-transformed E. coli. Reactions
included SMX-HA (500 ␮M) with 1 mM NADH, glutathione, and ascor-
bate, in PBS, pH 7.4. Data are expressed as mean Ϯ S.D. for two separate
experiments each performed in duplicate.
ual human liver microsomes (Fig. 7A); however, b5R content
in these 27 commercially available samples only differed by
2.2-fold. On the other hand, microsomal cyt b5 content (as
measured by immunoreactivity to ␣-cyt b5) correlated
strongly with SMX-HA reduction activity in 19 individual
times higher specific activities, respectively, and similar ap- human liver microsomes. (r ϭ 0.75; P ϭ 0.0001; Fig. 7B) with
parent Km values for each substrate compared with HLM. 9.3-fold variability in cyt b5 immunoreactivity found in this
Immunocorrelation. Microsomal b5R content (as mea- small sample. In addition, microsomal cyt b5 content also
sured by immunoreactivity to ␣-b5R) did not correlate with correlated with benzamidoxime reduction (r ϭ 0.64; P ϭ
SMX-HA reduction activity (500 ␮M substrate) in 27 individ- 0.025; Fig. 8) and with dapsone hydroxylamine reduction (r ϭ

Hydroxylamine/Amidoxime Reduction
1175
TABLE 1
Kinetic data for the reduction of two hydroxylamines (HA) and an amidoxime by human recombinant b5R and cyt b5 compared with that seen in
pooled HLM
All reactions were performed in PBS, pH 7.4, with 1 mM NADH, under conditions approximating linear kinetics. Velocity data are expressed in nanomoles per total
milligrams of protein per minute. Data represent two to three experiments performed in duplicate for each condition, and are given as means Ϯ S.E.
Apparent Km (␮M)
Vmax (nmol/mg/min)
Substrate
HLM
b5R/cyt b5
HLM
b5R/ cyt b5
Sulfamethoxazole-HA
Dapsone-HA
Benzamidoxime
363 Ϯ 31
357 Ϯ 62
629 Ϯ 116
392 Ϯ 45
395 Ϯ 95
515 Ϯ 149
1.93 Ϯ 0.07
1.26 Ϯ 0.09
3.05 Ϯ 0.36
455 Ϯ 23
205 Ϯ 24
226 Ϯ 31
Fig. 8. Microsomal cyt b5 content correlates strongly with benzami-
doxime reduction activity (100 ␮M benzamidoxime) in 12 individual
human liver microsomes (r ϭ 0.64; P ϭ 0.025). Activity results are from
two experiments each performed in duplicate.
with 63 and 51% inhibition, respectively, compared with
preimmune serum.
Reduction Activity in b5R-Deficient Fibroblasts. Im-
munoreactive b5R was present in normal human fibroblasts
but was absent, as expected, in fibroblasts from a patient
with type II hereditary methemoglobinemia (Fig. 10). Similar
cyt b5 content was present in both cell types. Reduction of
sulfamethoxazole hydroxylamine was virtually undetectable
in the b5R-deficient fibroblasts compared with normal hu-
man dermal fibroblasts.
Discussion
Fig. 7. A, microsomal content of NADH cytochrome b5 reductase (as
measured by immunoreactivity to ␣-b5R) could not be correlated with
SMX-HA reduction activity (500 ␮M substrate) in 27 individual human
liver microsomes (r ϭ 0.05). However, there was only 2.2-fold range in
b5R content in these available microsomes. B, microsomal cytochrome b5
content (as measured by immunoreactivity to ␣-cytochrome b5) correlates
strongly with SMX-HA reduction activity (500 ␮M substrate) in 19 indi-
vidual human liver microsomes (r ϭ 0.75; P ϭ 0.001). Activity results are
from three experiments each performed in duplicate.
NADH cytochrome b5 reductase is an FAD-containing pro-
tein with soluble and membrane-bound forms, each the prod-
uct of the same gene through alternative promoters and
alternative splicing (Tomatsu et al., 1989; Leroux et al.,
2001). Both soluble (approximately 32 kDa) and membrane-
bound (approximately 35 kDa) forms of b5R have identical
catalytic domains and differ only in a hydrophobic domain at
the N terminus of the membrane-bound form. This 3-kDa
0.90; P ϭ 0.015; only 7 individual microsomes were tested N-terminal sequence is myristoylated and allows insertion of
because of limited substrate supply). the membrane-bound form into endoplasmic reticulum and
Immunoinhibition. Antisera to b5R and cyt b5 inhibited outer mitochondrial membranes of somatic cells (Ozols et al.,
SMX-HA reduction in human liver microsomes by 69 and 1984). The hemoprotein cytochrome b5 is also expressed in
58% respectively, compared with preimmune serum (Fig. 9). soluble and membrane-bound forms, although the hydropho-
This was comparable with the inhibition of SMX-HA reduc- bic anchor of membrane-bound cyt b5 is at the C terminus
tion by these antisera in the recombinant b5R/cyt b5 system, (Borgese et al., 1993). As for b5R, the microsomal and soluble

1176
Kurian et al.
al., 1971) and P450 oxidases (Hildebrandt and Estabrook,
1971) and, in erythrocytes, play a primary role in the main-
tenance of hemoglobin in its reduced state (Hultquist and
Passon, 1971). In addition, b5R mediates the reduction of the
partially oxidized form of ascorbate, ascorbyl free radical,
back to ascorbate (Ito et al., 1981; Shirabe et al., 1995).
The results of our studies indicate that b5R and cyt b5 also
have a direct role in xenobiotic metabolism. This enzyme
complex efficiently catalyzed the reduction of the xenobiotic
hydroxylamines of dapsone and sulfamethoxazole, as well as
the reduction of the prototypical amidoxime prodrug, benz-
amidoxime. The apparent Km values for reduction by the
purified system were nearly identical to those found in hu-
man liver microsomes, and microsomal activity was inhibited
by antibodies to either b5R or cyt b5 to the same degree as the
recombinant system. The microsomal content of cyt b5 cor-
related strongly with both microsomal hydroxylamine and
amidoxime reduction, and human dermal fibroblasts that
lack b5R (Kugler et al., 2001) showed virtually no activity for
the reduction of sulfamethoxazole hydroxylamine. Addition
of microsomal protein to the recombinant system did not
enhance activity more than additively, suggesting that addi-
tional microsomal proteins are not important for optimal
activity. These data together provide strong evidence that
Fig. 9. Antisera to b5R and cyt b5 (150 ␮l each) significantly inhibited the
reduction of sulfamethoxazole hydroxylamine in human liver microsomes
(by 69 and 58%, respectively) compared with preimmune serum. This was
comparable with inhibition seen with these antibodies toward the puri-
fied system containing b5R and cyt b5 (63 and 51% inhibition, respec-
tively; data not shown). Microsomes (500 ␮g) or b5R and cyt b5 (20.6 ␮g
of total protein) were preincubated with antisera for 30 min at room the enzyme complex of b5R and cyt b5 is responsible for the
temperature prior to initiation of the reaction. Data shown are the results
of two to three experiments each performed in duplicate.
reduction of hydroxylamines and amidoximes in humans.
The reduction of hydroxylamines and amidoximes repre-
sents a novel direct role for this enzyme complex in xenobiotic
metabolism. Although b5R has been shown to reduce some
chemotherapeutic agents to their cytotoxic forms (Mahmuto-
glu and Kappus, 1988; Hodnick and Sartorelli, 1993), other
reports of direct biotransformation by this enzyme complex
are lacking. Clement et al. (1997, 1998) reported that b5R
and cyt b5 were cofactors in amidoxime and hydroxylamine
reduction; however, they concluded that a third protein of
P450 origin was necessary for efficient reduction. Our data
are in disagreement with these studies, in that our purified
recombinant system containing only b5R, cyt b5, and NADH
was capable of efficient and complete reduction of one ami-
doxime and two hydroxylamine substrates. Some reasons for
the differences between our data and previous studies could
be the species studied (human for our studies compared with
pig in previous studies), the source of enzyme complex (re-
combinant expression in our studies versus biochemical pu-
rification in previous studies) or the pH of the reactions (pH
7.4 in our studies versus pH 6.3 previously). In addition, we
found an optimal stoichiometry of 8 to 10:1 for cyt b5/b5R,
whereas previous studies used b5R in excess of cyt b5 (Clem-
ent et al., 1998).
The optimal stoichiometry of 8 to 10 mol of cytochrome b5
to 1 mol of b5R for hydroxylamine reduction is interesting,
given that the reaction presumably entails two successive
single-electron reductions from b5R to cyt b5 (Iyanagi et al.,
1984). A 10:1 stoichiometry of cyt b5 to b5R has also been
found in native liver microsomes (Yang and Cederbaum,
1996) and may reflect the amount of cyt b5 required to
maintain a high turnover rate of b5R for cyt b5 reduction
(Meyer et al., 1995; Arinc and Cakir, 1999). Because our
recombinant system had an optimal stoichiometry similar to
Fig. 10. Activity for hydroxylamine reduction is virtually absent in b5R-
deficient fibroblasts. Immunoreactivity to b5R (panel A; 35-kDa protein
represents microsomal b5R that predominates in fibroblasts) and cyt b5
(panel B; 17-kDa protein represents microsomal cyt b5) antisera in nor-
mal human dermal fibroblasts (lane 1) and fibroblasts derived from a
patient with type II hereditary methemoglobinemia [lane 2 (Kugler et al.,
2001); 40 ␮g of protein loaded in each lane]. Panel C, reduction of
sulfamethoxazole hydroxylamine by normal and b5R-deficient fibro-
blasts.
isoforms of cyt b5 are the product of a single gene (Giordano that reported for native liver microsomes, the higher activity
and Steggles, 1991). Together cyt b5 and b5R mediate elec- seen with the recombinant system was most likely due to
tron transfer from NADH to fatty acid desaturases (Oshino et enrichment of these two proteins, rather than a difference in

Hydroxylamine/Amidoxime Reduction
1177
the ratio of cyt b5/b5R content between recombinant and system, variability in hydroxylamine and amidoxime metab-
native systems.
olism, and therapeutic and toxicologic outcome.
We had previously reported a model with two apparent Km
In conclusion, the results of these studies indicate that
values for the reduction of SMX hydroxylamine by HLM human recombinant NADH cytochrome b5 reductase and
(Trepanier and Miller, 2000); however, this was based upon cytochrome b5 efficiently reduce hydroxylamines and ami-
an Eadie-Hofstee transformation of reactions performed in doximes without the need for other proteins and support the
HEPES buffer. Closer examination of that data revealed an conclusion that this enzyme complex comprises the previ-
artifact (a hump in the velocity versus substrate concentra- ously reported but poorly characterized NADH-dependent
tion curve at low concentrations), which was resolved with a hydroxylamine reductase system.
switch to PBS buffer, and which then yielded a single appar-
ent Km in both HLM and the recombinant system.
Acknowledgments
Polyclonal antibodies to b5R and cyt b5 inhibited hydrox-
Special thanks to Dr. W. Wallace Cleland and Dr. George Reed
ylamine reduction activity, but not completely (Fig. 9). Al-
(Enzyme Institute, University of Wisconsin-Madison) for helpful dis-
though this could indicate the involvement of other proteins
cussions regarding enzyme kinetic mechanisms and artifacts; Dr.
in this pathway, it is more likely to reflect the imperfect
inhibitory capacity of our antisera, since our antisera inhib-
ited the purified b5R/cyt b5 system to the same incomplete
degree (63% inhibition by ␣-b5R and 51% inhibition by ␣-cyt
b5 toward the purified system). One possible mechanism for
incomplete inhibition by our sera toward even the recombi-
nant system is steric interference by antibodies directed to-
ward noninhibitory epitopes, preventing the complete bind-
ing of antibodies directed at inhibitory epitopes.
Komei Shirabe (Medical College of Oita, Japan) for soluble b5R
cDNA; Dr. Grant Mauk (University of British Columbia, Vancouver,
Canada) for soluble cyt b5 cDNA; Dr. Jose Villalba (Universidad de
Cordoba, Spain) for anti-b5R antibody used in initial experiments;
Dr. Reginald Frye (School of Pharmacy, University of Pittsburgh) for
dapsone hydroxylamine; and Drs. Wilfried Kugler and Arnulf Pek-
run (Universitats-Kinderklinik, Goettingen, Germany) for fibro-
blasts from a patient with type II hereditary methemoglobinemia.
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Address correspondence to: Dr. Lauren Trepanier, Department of Medical
Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015
Linden Drive, Madison, WI 53706. E-mail: latrepanier@svm.vetmed.wisc.edu