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Biochemical and Biophysical Research Communications 291, 220 –225 (2002)
doi:10.1006/bbrc.2002.6423, available online at http://www.idealibrary.com on
Characterization of a Cytochrome b 558 Ferric/Cupric
Reductase from Rabbit Duodenal
Brush Border Membranes
Martin Knöpfel and Marc Solioz 1
Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, 3010 Berne, Switzerland
Received January 14, 2002
Iron and probably also copper are absorbed by the
intestine in their reduced form. A b-type cytochrome,
Dcytb, has recently been cloned from mouse and has
been proposed to be the corresponding reductase.
However, the nature of the cytochrome and the reduction reaction remain unknown. Here we describe the
isolation and functional characterization of a novel
b-type cytochrome from rabbit enterocytes. The 33
kDa heme protein was solubilized from brush border
membranes with Triton X-100 and purified by successive ion exchange chromatography and hydrophobic
interaction chromatography. Spectroscopic analysis
of the heme revealed a b 558 cytochrome. The purified
hemoprotein exhibited ascorbate-stimulated reduction of iron(III) and copper(II). The rate constants, k 1,
for these reactions were 1.38 ⴞ 0.12 and 0.64 ⴞ 0.16
min ⴚ1, respectively. Cytochrome b 558 may be the rabbit
Dcytb homologue. A novel mechanism of how cytochrome b 558 could shuttle electrons from cytoplasmic
ascorbate to luminal dehydroascorbate is proposed.
© 2002 Elsevier Science (USA)
Key Words: iron; copper; cytochrome b 558; enterocytes; reductase; rabbit; membrane protein; Dcytb.
Iron and copper serve as cofactors in numerous biochemical reactions, but knowledge of their homeostasis
by eukaryotic cells is still fragmentary (see Refs. 1–9
for recent reviews). Insight into the mechanism of intestinal iron absorption has recently come from the
identification of a rat divalent cation transporter,
DCT1 (also called DMT1), by expression cloning in
Xenopus laevis oocytes (10). DCT1 is a 561-amino acid
protein with 12 putative membrane spanning helices.
It is ubiquitously expressed, but most notably in the
proximal duodenum. In enterocytes, DCT1 is expressed in brush border membranes and is upregu1
To whom correspondence and reprint requests should be addressed. Fax: ⫹41 31 632 4997. E-mail: [email protected] or
[email protected]. URL: www.ikp.unibe.ch/lab1/.
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© 2002 Elsevier Science (USA)
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lated by iron deficiency (11). This transporter thus
appears to be the key mediator of intestinal iron absorption, but has also a function in peripheral tissues.
DCT1 is a member of the “natural resistanceassociated macrophage protein” (Nramp) family (12)
and the mouse homologue was thus called Nramp2
(13). In experimental systems, the transport specificity
of DCT1 is broad, including Fe 2⫹, Zn 2⫹, Mn 2⫹, Co 2⫹,
Cd 2⫹, Cu 2⫹, Ni 2⫹ and Pb 2⫹ (10, 14 –16). It remains to be
shown whether DCT1 serves in the uptake of all these
ions or whether it has a primary role in iron absorption. In particular, DCT1 is probably not the primary
transporter for copper: Belgrade rats that harbor a
mutation in DCT1 are anemic, but exhibit normal copper status (17). Current evidence suggests that intestinal copper uptake is catalyzed by a universal copper
transporter, Ctr1, which was first identified in yeast
(18). The human homologue, hCtr1, was cloned by
complementation of a ⌬Ctr1 yeast strain with a human
cDNA library (19). The hCtr1 gene is predicted to encode a protein of 190 amino acids, containing three
transmembrane helices. Ctr1 is expressed in all cells
looked at, including enterocytes, and catalyzes the
transport of Cu(I) across the cell membrane (20).
At physiological pH and in the presence of oxygen,
iron and copper are usually present in their oxidized
iron(III) and copper(II) forms. According to the emerging concept of iron and copper transport, these ions are
however transported cross the cell membrane in their
reduced forms (16, 20, 21). Thus, corresponding reductases and oxidases working in concert with the transport proteins are required on either side of the membrane. The gene of a multicopper ferroxidase necessary
for iron egress from intestinal enterocytes into the
circulation has recently been cloned (22). The protein
encoded by this gene, hephaestin, is related to ceruloplasmin and is defective in the sex-linked anemic (sla)
mouse. Iron oxidation thus appears to be a key step in
the release of iron from enterocytes. Similarly, iron
absorption by the intestinal mucosa is expected to de-
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pend on a brush-border iron reductase. A gene that
appears to encode the corresponding reductase has
recently been cloned from mouse (23). The gene encodes a duodenal cytochrome b, Dcytb, which shares 45
to 50% sequence similarity to the cytochrome b 561 family of membrane reductases. While Dcytb induced ferric reductase activity when expressed in Xenopus oocytes and cultured cells, its spectral properties and its
in vivo function in intestinal iron absorption have not
been shown. We here report the purification and biochemical characterization of a b-type cytochrome from
rabbit brush border membranes. The protein is a b 558
cytochrome with an apparent molecular weight of 33
kDa. It stimulated ascorbate driven copper and iron
reduction in vitro and may be the rabbit Dcytb homologue.
MATERIALS AND METHODS
Materials. Triton X-100, Chaps, octylglucoside, and 4-(2aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) were
from Roche (Basel, Switzerland), trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) from Sigma (Buchs, Switzerland), and
Phenyl-, SP-, and DEAE-Sepharose fast flow from Amersham Pharmacia Biotech. Nitrilotriacetic acid (NTA) and other chemicals (all of
analytical grade) were obtained from Fluka (Buchs, Switzerland).
Protein molecular weight standards were purchased from Bio-Rad.
Preparation of brush border membrane vesicles (BBMV). Small
intestines were freshly excised prior to use or taken from frozen stock
stored at ⫺80°C. Approximately 0.5 m of proximal rabbit small
intestine were used to prepare BBMV by the method of Kessler et al.
(24), but using a MgCl 2 precipitation instead of a CaCl 2 precipitation
to avoid the activation of proteases (25). The resulting BBMV were
characterized according to established methods (25, 26).
Purification of the cytochrome. BBMV, 10 ml at ⬃20 mg
protein/ml were diluted 20-fold with 50 mM Na-Hepes, pH 7.4, 0.1 M
NaCl, and collected by centrifugation at 45,000g for 20 min at 4°C.
The pellet of washed BBMV was solubilized in 100 ml of 10 mM
Na-Hepes, pH 7.4, 0.02 M NaCl, 2% Triton X-100, 1 mM AEBSF, 10
␮M E-64. The extraction was diluted twofold and centrifuged at
130,000g for 10 min at 4°C. The supernatant containing solubilized
membrane proteins was applied to a 1.6 ⫻ 10 cm column of DEAESepharose Fast Flow, connected in series to a 0.8 ⫻ 10 cm SPSepharose column, both equilibrated with 10 mM Na-Hepes, pH 7.4,
0.02 M NaCl, 0.5% Triton X-100, 10 ␮M E-64. The columns were
washed with elution buffer containing 0.1 M NaCl to elute weekly
bound proteins, followed by elution with 200 ml of a linear NaCl
gradient from 0.1 to 0.4 M. Crude fractions from the DEAE column
were made approximately 0.6 M in NaCl and absorbed to a 1 ⫻ 5 cm
phenyl Sepharose column and eluted by stepwise lowering the salt
concentration. The cytochrome-containing fractions were collected.
All purification steps were carried out at 4°C. Patent No. 2000
0597/00, 29. 03. 2000, pending.
Measurement of reductase activities. Ferrireductase activity was
measured spectrophotometrically at 25°C in a total volume of 1 ml 50
mM Na-Hepes, pH 7.4, 0.1 M NaCl, 50 ␮M FeCl 3, 100 ␮M NTA, 100
␮M ascorbate, 50 ␮M bathophenanthroline, 0.5% Triton X-100 (⑀ for
Fe(II)-bathophenanthroline ⫽ 21 mM ⫺1cm ⫺1; Ref. 27). The reaction
was started by the addition of ascorbate. The appearance of ferrous
iron was followed by measuring the formation of the bathophenanthroline–iron(II) complex at 537 nm, using 610 nm as a reference
wavelength. Control measurements were carried out with reductase
buffer alone, and with protein in the absence of ascorbate. Cupric
FIG. 1. Absorption spectrum of three cytochrome-containing
fractions eluted from the DEAE-Sepharose column. The curves show,
from bottom to top, fractions containing protein with a heme content
of 0.2, 0.32, and 0.6 ␮M. The fractions were measured in air without
additional oxidation.
reductase was assayed similarly in 10 mM Na-Hepes, pH 7.4, 150
mM NaCl, 50 ␮M CuCl 2, 50 ␮M nitrilotriacetic acid, 100 ␮M ascorbate, 75 ␮M BCA, 0.5% Triton X-100 and following the absorption at
354 nm (⑀ for Cu(I)-BCA ⫽ 46 mM ⫺1 cm ⫺1; Ref. 28).
Miscellaneous methods. UV/VIS spectroscopy was carried out
with a single-beam Hewlett–Packard spectrophotometer. Protein
concentrations were determined by the bicinchoninic acid method of
Pierce Chemical Co. SDS–PAGE were carried out in a Mini-Protean
II dual slab cell from Bio-Rad according to the instruction manual.
RESULTS
Isolation of a cytochrome from brush border membranes (BBM). BBM contain a major b-type cytochrome. In an attempt to purify and characterize this
cytochrome, membrane extraction was evaluated with
a variety of detergents. Chaps, Triton X-100, diheptanoyl phosphatidylcholine, and n-octylglucoside were
tested for solubilization of the cytochrome. Triton
X-100 solubilized the cytochrome best. Crude membrane extracts were purified by absorption to a DEAE
Sepharose column linked in series to an SP-Sepharose
column and elution with a linear NaCl gradient. The
cytochrome component eluted between 0.25 and 0.3 M
NaCl. Yellowish to orange fractions containing the cytochrome in concentrations from 0.2 to 1.5 ␮M were
obtained (calculated on the basis of the Soret band and
assuming an absorption coefficient 58.3 mM ⫺1 䡠 cm ⫺1;
Ref. 29).
The cytochrome containing fractions from DEAE/SPSepharose columns were spectrally analyzed and contained a single, characteristic heme component (Fig. 1).
The heme containing fractions were further purified by
hydrophobic interaction chromatography on a phenyl
Sepharose column. Retention of the cytochrome on the
column was almost complete at high ionic strength and
could be eluted as a single, sharp band by lowering the
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nm. The difference spectrum, reduced-oxidized, shows
a peak at 558 nm indicative a b-type cytochrome. Ligand exchange with carbon monoxide or potassium
cyanide did not result in the band shift characteristic
for heme with a free valence on the iron center (not
shown). Taken together, these experiments suggest a
six coordinated heme iron. This cytochrome, which is
the major cytochrome component of BBM, thus appears to be a b 558 cytochrome.
FIG. 2. SDS gel electrophoresis of the BBM cytochrome component after DEAE, SP, and phenyl Sepharose chromatography. The
arrow indicates the heme containing moiety and the numbers on the
right indicate the migration of marker proteins with the corresponding molecular weights in kDa. The gel contained 10% polyacrylamide
and was stained with Coomassie blue.
NaCl concentration to below 0.2 M. This fraction contained the dominant cytochrome band with an apparent molecular weight of 33 kDa (Fig. 2). There was also
a diffuse band of co-purifying low molecular weight
material, which could be a subunit forming a complex
with the 33 kDa cytochrome, such as proteolipid component. Analysis of tryptic peptide fragments of the 33
kDa band by mass spectroscopy did not reveal significant agreement of its fragmentation pattern with any
known protein in the databases of SwissProt, PIR,
PRF, PDB, and translations from annotated coding
regions in GenBank and RefSeq (not shown). However,
these combined databases currently contain fewer
than 7 ⫻ 10 3 rabbit protein sequences. Final identification of cytochrome b 558 will have to await cloning and
sequencing of its gene.
Spectral characterization of the cytochrome. To
characterize the nature of this cytochrome from BBM,
its spectral properties were investigated. Figure 3
shows the spectra of the dithionite-reduced and the
ferricyanide-oxidized cytochrome. The reduced spectrum exhibited a Soret (␥-) band at 412 nm, and ␣- and
␤-bands at 578 and 538 nm, respectively (cf. also Fig.
1). Upon oxidation of the cytochrome, the absorption
maximum of the Soret band was decreased by 27% and
the absorption maximum shifted to the red by 11 to 423
Reductase activity measurements with cytochrome
b 558. According to the current concept of iron and copper transport across the BBM, these metal ions must,
in a first step, be reduced (8, 10, 15, 20). To test for a
possible involvement of the cytochrome b 558 in metal
reduction, we investigated the reductase activity. Measurements for iron reduction with purified cytochrome
b 558 were carried out with Fe(III)-NTA as a substrate
and bathophenanthroline as an indicator of Fe(II). No
reductase activity could be measured with NADH. If
ascorbate was used as a reductant, an ascorbateindependent reductase activity with a rate constant k 1
of 0.33 ⫾ 0.01 min ⫺1 (half time t 1/2 ⫽ 2.1 min) was
observed. This activity was stimulate 4-fold to 1.38 ⫾
0.12 min ⫺1 (t 1/2 ⫽ 0.5 min) in the presence of purified
cytochrome b 558 (Fig. 4A).
Reductase activity measurements in presence of copper gave similar results. The background reduction of
ascorbic acid in the presence of BCA as the indicator
for Cu 1⫹ exhibited a rate constant k 1 of 0.25 ⫾ 0.19
min ⫺1 (t 1/2 ⫽ 2.5 min) and was slightly lower than that
for iron. Copper reduction in the presence of cytochrome b 558 was stimulated 2.5-fold (k 1 ⫽ 0.64 ⫾ 0.16
min ⫺1; t 1/2 ⫽ 1.1 min). Thus, the cytochrome b 558 that we
have investigated here exhibits significant ascorbatestimulated iron and copper reductase activity and may
have a role in intestinal uptake of these metals.
FIG. 3. Spectral analysis of the purified cytochrome b 558. The
dithionite-reduced spectrum with a ␭ max of 423 nm and the
ferricyanide-oxidized spectrum with a ␭ max of 412 nm are shown. The
reduced-oxidized difference spectrum (bold line) exhibits the b-type
heme peak at 558 nm.
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FIG. 4. Reductase activity of cytochrome b 558. Iron reductase
activity (A) or copper reductase activity (B) was measured in either
the absence (E) or presence (䊐) of purified cytochrome b 558. Data
points were fitted and k 1 values calculated with the MacCurfit program as described previously (15). Reductase activity measurements
were carried out as described under Materials and Methods.
ascorbate could also form metal ion complexes to stabilize the reduced metals temporarily. Iron(II) can then
be taken up by DCT1 in an Fe 2⫹/H ⫹ symport mechanism, thus recycling the protons ejected in the electron
transport process by cytochrome b. Support for this
novel model is drawn from diverse observations, as
described below.
With the cloning of DCT1 it has become apparent
that this protein is the major route of iron uptake by
the small intestine (10). Functional investigations had
shown that DCT1 catalyzes cotransport of divalent
metal ions and protons, so iron is taken up as iron(II)
(16). This had suggested the participation of a reductase in the absorption process. Indeed, it had been
known for a number of years that ferric reductase
activity is associated with the intestinal brush border
membrane and probably involved in iron absorption
(30, 31). NADH- and NADPH-stimulated ferric reductase activities have been described, but we and others
failed to identify the enzyme(s) due to loss of activity
during purification (32). Conceivably, NADH-stimulated ferric reductase activity is a side-reaction of
one or several enzymes involved in other functions. A
dual-function reductase catalyzing NADH-stimulated
dihydropteridine and iron reduction has recently been
identified in pig duodenal membranes (33), but its role
in iron absorption remained unknown too. Thus, there
is no direct evidence for the involvement of an
NAD(P)H-stimulated reductase in metal ion uptake by
enterocytes.
Recently, a mouse gene encoding a putative duodenal iron reductase, Dcytb, was cloned (23). The derived
protein sequence exhibited 45 to 50% sequence similarity to b 561 cytochromes and had a calculated molecular weight of 31.5 kDa. A 30 kDa b-type cytochrome,
p-30, purified from rabbit neutrophils also shared 19 of
DISCUSSION
We here described the purification and characterization of a b 558 cytochrome which is associated with the
intestinal brush border membrane. It is the major cytochrome component of this membrane and can catalyze ascorbate-stimulated iron and copper reduction.
These and other findings suggest that this cytochrome
has a role in metal ion absorption by the duodenum.
We thus propose a speculative, but testable, model for
duodenal reduction of iron and possibly also copper
(Fig. 5). Dehydroascorbate in the lumen is reduced to
ascorbate by intracellular ascorbate via electron and
proton shuttling through cytochrome b 558. The luminal
ascorbate then reduces iron or copper ions. In addition,
FIG. 5. Model of the function of cytochrome b 558 in intestinal iron
absorption. The b-type cytochrome shuttles electrons from intracellular ascorbate (AH ⫺) across the membrane to reduce luminal dehydroascorbate (A ⫺). The ascorbate thus generated reduces luminal
iron (or copper) for transport. Proton cotransported with the electrons makes the electron transfer reaction electroneutral. The protons are recycled by metal-proton symport catalyzed by DCT1 or
similar transporters.
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the 20 known N-terminal amino acids with Dcytb (29).
Dcytb was highly expressed in brush border membranes of enterocytes and exhibited ferric reductase
activity with the artificial electron donor nitroblue tetrazolium. Dcytb had not been purified or spectrally
characterized, but it appears likely that it is the mouse
homologue of the cytochrome b 558 described here, based
on the following properties of the latter: (i) it is of
similar size, (ii) it is an integral membrane protein, (iii)
it is the major cytochrome of the brush border membrane, and (iv) it exhibits iron and copper reductase
activity. The preliminary characterization of a similar
b-type cytochrome from rabbit brush border membranes had been described by Pountney et al. (34). The
spectral characteristics of this cytochrome closely resembled those of our cytochrome b 558 and the two cytochromes are most likely identical.
Dcytb, p-30, and cytochrome b 558 thus all appear to
belong to the same family of b 561 cytochromes (23, 35).
The cytochromes of this family have putative ascorbate
and dehydroascorbate binding sites and are integral
membrane proteins. In chromaffin granules the function of cytochrome b 561 is understood in some detail.
These secretory vesicles require ascorbate to provide
reducing equivalents to peptidyl ␣-amidating enzyme,
but they are devoid of an ascorbate uptake system.
Intravesicular reducing equivalents are restored by
transmembrane electron transfer from cytoplasmic
ascorbate to dehydroascorbate on the inside of chromaffin granules by cytochrome b 561 (35). Such an electron shuttle mechanism could well operate to provide
reducing equivalents to the lumen of the duodenum. In
fact, it had been shown that Caco-2 cells can recycle
extracellular dehydroascorbate to ascorbate (36). The
demonstration by several studies that ascorbate enhances intestinal iron absorption supports a direct role
of ascorbate in iron absorption (37–39); the effect of
ascorbate on copper absorption remain to be demonstrated (40). Interestingly, ascorbate was shown to be
taken up primarily in the distal segment of the small
intestine, which would be expected if it were necessary
for iron uptake, which occurs preferentially in the
proximal segment (41).
Taken together, we here show the purification and
properties of a major 33 kDa cytochrome b 558 from
rabbit duodenal brush border membranes. It appears
to be the rabbit homologue of Dcytb and we propose a
novel function for these proteins in the shuttling of
electrons from intracellular ascorbate to luminal dehydroascorbate for metal reduction.
ACKNOWLEDGMENTS
We thank the members of the groups of Kaspar Winterhalter and
Ernesto Di Iorio for their support for characterizing the heme moiety,
Anton Lehmann for expert technical assistance, and Lorna Ebersole
for helpful discussions. This work was supported by Grant 32-
56716.99 from the Swiss National Foundation to M.S. and a grant
from the International Copper Association.
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