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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/. 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. 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- 220 Vol. 291, No. 2, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 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 221 Vol. 291, No. 2, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 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. 222 Vol. 291, No. 2, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 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. 223 Vol. 291, No. 2, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 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. REFERENCES 1. Agranoff, D. D., and Krishna, S. (1998) Metal ion homeostasis and intracellular parasitism. Mol. Microbiol. 28, 403– 412. 2. Bacon, B. R., and Schilsky, M. L. (1999) New knowledge of genetic pathogenesis of hemochromatosis and Wilson’s disease. Adv. Intern. Med. 44, 91–116, 91–116. 3. Griffiths, W., and Cox, T. (2000) Haemochromatosis: Novel gene discovery and the molecular pathophysiology of iron metabolism. Hum. Mol. Genet. 9, 2377–2382. 4. Roy, C. N., and Enns, C. A. (2000) Iron homeostasis: New tales from the crypt. Blood 96, 4020 – 4027. 5. Forbes, J. R., and Gros, P. 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