Distinct Iron Binding Property of Two Putative Iron Donors for the Iron-Sulfur Cluster Assembly
2007; Elsevier BV; Volume: 282; Issue: 11 Linguagem: Inglês
10.1074/jbc.m609665200
ISSN1083-351X
AutoresHuangen Ding, Juanjuan Yang, Liana C. Coleman, Simon S. Yeung,
Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoFrataxin, a small mitochondrial protein linked to the neurodegenerative disease Friedreich ataxia, has recently been proposed as an iron donor for the iron-sulfur cluster assembly. An analogous function has also been attributed to IscA, a key member of the iron-sulfur cluster assembly machinery found in bacteria, yeast, and humans. Here we have compared the iron binding property of IscA and the frataxin ortholog CyaY from Escherichia coli under physiological and oxidative stress conditions. In the presence of the thioredoxin reductase system, which emulates the intracellular redox potential, CyaY fails to bind any iron even at a 10-fold excess of iron in the incubation solution. Under the same physiologically relevant conditions, IscA efficiently recruits iron and transfers the iron for the iron-sulfur cluster assembly in a proposed scaffold IscU. In the presence of hydrogen peroxide, however, IscA completely loses its iron binding activity, whereas CyaY becomes a competent iron-binding protein and attenuates the iron-mediated production of hydroxyl free radicals. Hydrogen peroxide appears to oxidize the iron binding thiol groups in IscA, thus blocking the iron binding in the protein. Once the oxidized thiol groups in IscA are re-reduced with the thioredoxin reductase system, the iron binding activity of IscA is fully restored. On the other hand, hydrogen peroxide has no effect on the iron binding carboxyl groups in CyaY, allowing the protein to bind iron under oxidative stress conditions. The results suggest that IscA is capable of recruiting intracellular iron for the iron-sulfur cluster assembly under normal physiological conditions, whereas CyaY may serve as an iron chaperon to sequester redox active free iron and alleviate cellular oxidative damage under oxidative stress conditions. Frataxin, a small mitochondrial protein linked to the neurodegenerative disease Friedreich ataxia, has recently been proposed as an iron donor for the iron-sulfur cluster assembly. An analogous function has also been attributed to IscA, a key member of the iron-sulfur cluster assembly machinery found in bacteria, yeast, and humans. Here we have compared the iron binding property of IscA and the frataxin ortholog CyaY from Escherichia coli under physiological and oxidative stress conditions. In the presence of the thioredoxin reductase system, which emulates the intracellular redox potential, CyaY fails to bind any iron even at a 10-fold excess of iron in the incubation solution. Under the same physiologically relevant conditions, IscA efficiently recruits iron and transfers the iron for the iron-sulfur cluster assembly in a proposed scaffold IscU. In the presence of hydrogen peroxide, however, IscA completely loses its iron binding activity, whereas CyaY becomes a competent iron-binding protein and attenuates the iron-mediated production of hydroxyl free radicals. Hydrogen peroxide appears to oxidize the iron binding thiol groups in IscA, thus blocking the iron binding in the protein. Once the oxidized thiol groups in IscA are re-reduced with the thioredoxin reductase system, the iron binding activity of IscA is fully restored. On the other hand, hydrogen peroxide has no effect on the iron binding carboxyl groups in CyaY, allowing the protein to bind iron under oxidative stress conditions. The results suggest that IscA is capable of recruiting intracellular iron for the iron-sulfur cluster assembly under normal physiological conditions, whereas CyaY may serve as an iron chaperon to sequester redox active free iron and alleviate cellular oxidative damage under oxidative stress conditions. Frataxin is a small mitochondrial protein that has been linked to Friedreich ataxia, an autosomal recessive neurodegenerative disease (1Campuzano V. Montermini L. Molto M.D. Pianese L. Cossee M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Canizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.L. Cocozza S. Koenig M. Pandolfo M. Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2323) Google Scholar). Most Friedreich ataxia patients are homozygous for a large GAA repeat expansion in the first intron of the frataxin gene which impairs transcription and causes severe reduction in the level of frataxin in mitochondria (1Campuzano V. Montermini L. Molto M.D. Pianese L. Cossee M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Canizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. 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Chem. 2005; 280: 30432-30437Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and ease to mobilize the iron center in the protein by l-cysteine (42Ding B. Smith E.S. Ding H. Biochem. J. 2005; 389: 797-802Crossref PubMed Scopus (48) Google Scholar) led us to hypothesize that the primary function of IscA is to recruit intracellular free iron and deliver the iron for the iron-sulfur cluster assembly (44Yang J. Bitoun J.P. Ding H. J. Biol. Chem. 2006; 281: 27956-27963Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To further elucidate the role of frataxin/CyaY and IscA in biogenesis of iron-sulfur clusters, here we have compared the iron binding property of IscA and CyaY from E. coli under physiological and oxidative stress conditions. The results indicate that in the presence of the thioredoxin reductase system, which emulates the intracellular redox potential, CyaY, unlike IscA, fails to bind any iron even at a 10-fold excess of iron in the incubation solution. In the presence of hydrogen peroxide, however, CyaY becomes a competent iron-binding protein and attenuates the iron-mediated production of hydroxyl free radicals, whereas IscA completely loses its iron binding activity. The possible physiological role of IscA and CyaY in biogenesis of iron-sulfur clusters and in the intracellular iron metabolism under oxidative stresses will be discussed. Protein Preparation—The DNA fragment encoding CyaY was amplified from wild-type E. coli genomic DNA using the PCR. Two primers, CyaY-1, 5′-GATACAACCATGGACGACAGTGAA-3′, and CyaY-2, 5′-CATGCAAAGCTTGCGGAAACTGAC-3′, were used for the PCR amplification. The PCR product was digested with two restriction enzymes HindIII and NcoI and ligated into an expression vector pET28b+ as described previously (40Ding H. Clark R.J. Biochem. J. 2004; 379: 433-440Crossref PubMed Scopus (101) Google Scholar). The cloned DNA fragment was confirmed by direct sequencing using the T7 primers. Recombinant CyaY was overproduced and purified as described previously for IscA (40Ding H. Clark R.J. Biochem. J. 2004; 379: 433-440Crossref PubMed Scopus (101) Google Scholar), IscU (40Ding H. Clark R.J. Biochem. J. 2004; 379: 433-440Crossref PubMed Scopus (101) Google Scholar), and IscS (50Yang W. Rogers P.A. Ding H. J. Biol. Chem. 2002; 277: 12868-12873Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The precise molecular weight of purified CyaY was confirmed using the electrospray ionization-mass spectrometry (Chemistry Department, Louisiana State University). ApoIscA and apoCyaY (proteins devoid of any iron) were prepared by incubation with l-cysteine (2 mm) at 37 °C for 30 min followed by re-purification of the protein using a HiTrap desalting column or a Mono Q column. E. coli thioredoxin-1 and thioredoxin reductase were produced from the expression vectors pDL59 (51Veine D.M. Mulrooney S.B. Wang P.F. Williams Jr., C.H. Protein Sci. 1998; 7: 1441-1450Crossref PubMed Scopus (32) Google Scholar) and pTrR301 (52Mulrooney S.B. Protein Expression Purif. 1997; 9: 372-378Crossref PubMed Scopus (40) Google Scholar), respectively, and purified as described in Ding et al. (41Ding H. Harrison K. Lu J. J. Biol. Chem. 2005; 280: 30432-30437Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The expression vectors pDL59 (51Veine D.M. Mulrooney S.B. Wang P.F. Williams Jr., C.H. Protein Sci. 1998; 7: 1441-1450Crossref PubMed Scopus (32) Google Scholar) and pTrR301 (52Mulrooney S.B. Protein Expression Purif. 1997; 9: 372-378Crossref PubMed Scopus (40) Google Scholar) were kindly provided by Dr. Scott B. Mulrooney (University of Michigan). Both thioredoxin-1 and thioredoxin reductase were purified as the native form without any tags. The purity of purified proteins was greater than 95% as judged by the SDS-polyacrylamide gel electrophoresis analyses. The concentration of apoIscA was determined using an extinction coefficient at 260 nm of 2.4 mm–1 cm–1 (40Ding H. Clark R.J. Biochem. J. 2004; 379: 433-440Crossref PubMed Scopus (101) Google Scholar). The concentrations of apoCyaY, apoIscU, IscS, thioredoxin-1, and thioredoxin reductase were determined using extinction coefficients at 280 nm of 30.0, 11.2, 39.7, 14.2, and 17.7 mm–1 cm–1, respectively (40Ding H. Clark R.J. Biochem. J. 2004; 379: 433-440Crossref PubMed Scopus (101) Google Scholar). Iron Binding Assay—For the iron binding assay, apoIscA and apoCyaY were incubated with freshly prepared Fe(NH4)2(SO4)2 in the presence of the thioredoxin reductase system (thioredoxin-1 (5 μm), thioredoxin reductase (0.5 μm) and NADPH (500 μm)) in buffer containing NaCl (100 mm) and Tris (20 mm) (pH 8.0) in open-to-air microcentrifuge tubes at 37 °C for 30 min. CyaY and IscA were then re-purified from the incubation solutions using a Mono Q column as described in Yang et al. (44Yang J. Bitoun J.P. Ding H. J. Biol. Chem. 2006; 281: 27956-27963Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The re-purification procedure using the Mono Q column did not significantly affect the iron binding in CyaY or IscA as >90% of the iron content in the iron-bound CyaY or IscA remained after passing through the Mono Q column. The eluted proteins from the Mono Q column were analyzed using a SDS-polyacrylamide gel electrophoresis. The total iron content in the eluted fractions was determined using the inductively coupled plasma mass spectroscopy (Department of Geology and Geophysics, Louisiana State University) or an iron indicator ferrozine as described in Yang et al. (44Yang J. Bitoun J.P. Ding H. J. Biol. Chem. 2006; 281: 27956-27963Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The iron-ferrozine complex was measured at 564 nm using an extinction coefficient of 27.9 mm–1 cm–1 (53Cowart R.E. Singleton F.L. Hind J.S. Anal. Biochem. 1993; 211: 151-155Crossref PubMed Scopus (88) Google Scholar). The results from both iron analysis methods were similar to each other. Iron-Sulfur Cluster Assembly Assay—For the iron-sulfur cluster assembly assay, either IscA or CyaY was preincubated with apoIscU and IscS in the presence of the thioredoxin reductase system in buffer containing NaCl (100 mm) and Tris (20 mm) (pH 8.0) at 37 °C. The reaction solutions were purged with pure argon gas and preincubated at 37 °C for 5 min before l-cysteine was added to initiate the iron-sulfur cluster assembly reaction. The iron-sulfur cluster assembly in IscU was monitored in a Beckman DU640 UV-visible absorption spectrometer equipped with a temperature controller as described previously (41Ding H. Harrison K. Lu J. J. Biol. Chem. 2005; 280: 30432-30437Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 54Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar). Measurements of Hydroxyl Free Radicals—The iron-mediated production of hydroxyl free radicals was measured after the procedure described by Halliwell et al. (55Halliwell B. Gutteridge J.M. Aruoma O.I. Anal. Biochem. 1987; 165: 215-219Crossref PubMed Scopus (2123) Google Scholar). Briefly, hydroxyl free radicals degrade 2-deoxyribose to form a malon-dialdehyde-like compound that reacts with thiobarbituric acid to generate a chromogen. In the experiments, apoIscA or apoCyaY was preincubated with Fe(NH4)2(SO4)2 in buffer containing K2HPO4/KH2PO4 (10 mm) (pH 7.4), NaCl (60 mm), 2-deoxyribose (4 mm), and the thioredoxin reductase system at 37 °C for 10 min before hydrogen peroxide (0.5 mm) was added to initiate Fenton reaction. The reactions were continued at 37 °C for additional 25 min. A developing solution containing 1% thiobarbituric acid and 2.8% trichloroacetic acid (400 μl) was then mixed with the above incubation solutions (600 μl) and boiled for 15 min. The reaction mixtures were centrifuged at 14,000 rpm in a desktop microcentrifuge for 15 min to remove the precipitates. The relative amounts of the chromogen in the solutions were measured from the emission at a wavelength of 553 nm using an excitation wavelength of 532 nm in a PerkinElmer LS-3 fluorescence spectrophotometer (55Halliwell B. Gutteridge J.M. Aruoma O.I. Anal. Biochem. 1987; 165: 215-219Crossref PubMed Scopus (2123) Google Scholar). Measurements of the Total Free Thiol Contents—The total free thiol contents in the protein samples were analyzed using the Ellman reagent (5,5′-dithiobis(2-nitrobenzoic acid) (Sigma) (56Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar). 5,5′-Dithiobis(2-nitrobenzoic acid) (at a final concentration of 100 μm) was added to the protein samples pretreated with methanol and incubated at room temperature for 20 min followed by centrifugation to remove the precipitates. The amounts of the total free thiols in the protein samples were calculated from an absorption amplitude at 412 nm using N-acetyl-l-cysteine as a standard. EPR Measurements—The EPR spectra were recorded at X-band on a Bruker ESR-300 spectrometer using an Oxford Instruments ESR-9 flow cryostat (Chemistry Department, Louisiana State University). The routine EPR conditions were: microwave frequency, 9.45 GHz; microwave power, 20 milliwatts; modulation frequency, 100 kHz; modulation amplitude, 2.0 millitesla; sample temperature, 4.5 K; receive gain, 1.0 × 105. Iron Binding Activity of CyaY and IscA in the Presence of the Thioredoxin Reductase System—In previous studies, the frataxin ortholog CyaY and IscA from E. coli have been shown to bind iron with the iron association constants of 2.6 × 105 m–1 (13Adinolfi S. Trifuoggi M. Politou A.S. Martin S. Pastore A. Hum. Mol. Genet. 2002; 11: 1865-1877Crossref PubMed Scopus (119) Google Scholar, 15Bou-Abdallah F. Adinolfi S. Pastore A. Laue T.M. Chasteen N.D. J. Mol. Biol. 2004; 341: 605-615Crossref PubMed Scopus (111) Google Scholar) and 2.0 × 1019 m–1 (40Ding H. Clark R.J. Biochem. J. 2004; 379: 433-440Crossref PubMed Scopus (101) Google Scholar, 41Ding H. Harrison K. Lu J. J. Biol. Chem. 2005; 280: 30432-30437Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 42Ding B. Smith E.S. Ding H. Biochem. J. 2005; 389: 797-802Crossref PubMed Scopus (48) Google Scholar, 43Ding H. Clark R.J. Ding B. J. Biol. Chem. 2004; 279: 37499-37504Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 44Yang J. Bitoun J.P. Ding H. J. Biol. Chem. 2006; 281: 27956-27963Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), respectively. However, the iron binding studies for IscA and CyaY were carried out by different groups and under different experimental conditions. Because both CyaY (22Layer G. Ollagnier-de Choudens S. Sanakis Y. Fontecave M. J. Biol. Chem. 2006; 281: 16256-16263Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and IscA (40Ding H. Clark R.J. Biochem. J. 2004; 379: 433-440Crossref PubMed Scopus (101) Google Scholar, 41Ding H. Harrison K. Lu J. J. Biol. Chem. 2005; 280: 30432-30437Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 42Ding B. Smith E.S. Ding H. Biochem. J. 2005; 389: 797-802Crossref PubMed Scopus (48) Google Scholar, 43Ding H. Clark R.J. Ding B. J. Biol. Chem. 2004; 279: 37499-37504Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 44Yang J. Bitoun J.P. Ding H. J. Biol. Chem. 2006; 281: 27956-27963Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) are proposed as the potential iron donor for biogenesis of iron-sulfur clusters, it is imperative to re-evaluate the iron binding property of CyaY and IscA under the same physiologically relevant conditions. In cells the intracellular redox potential is estimated to be in the range of –260 mV to –280 mV (57Ding H. Hidalgo E. Demple B. J. Biol. Chem. 1996; 271: 33173-33175Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 58Hwang C. Sinskey A.J. Lodish H.F. Science. 1992; 257: 1496-1502Crossref PubMed Scopus (1608) Google Scholar). The relatively low intracellular redox potential is largely maintained by the redundant thiol reducing systems (59Aslund F. Beckwith J. J. Bacteriol. 1999; 181: 1375-1379Crossref PubMed Google Scholar). To emulate the intracellular redox potential we have reconstructed the thioredoxin reductase system using E. coli thioredoxin-1 (51Veine D.M. Mulrooney S.B. Wang P.F. Williams Jr., C.H. Protein Sci. 1998; 7: 1441-1450Crossref PubMed Scopus (32) Google Scholar), thioredoxin reductase (52Mulrooney S.B. Protein Expression Purif. 1997; 9: 372-378Crossref PubMed Scopus (40) Google Scholar), and NADPH as described in Ding et al. (41Ding H. Harrison K. Lu J. J. Biol. Chem. 2005; 280: 30432-30437Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In the system, NADPH provides electrons to reduce thioredoxin-1 via thioredoxin reductase (59Aslund F. Beckwith J. J. Bacteriol. 1999; 181: 1375-1379Crossref PubMed Google Scholar). To compare the iron binding activity of IscA and CyaY under the physiologically relevant conditions, we incubated apoCyaY (100 μm) and apoIscA (100 μm) with freshly prepared ferrous iron (50 μm) in the presence of the thioredoxin reductase system at 37 °C for 30 min in open-to-air micro-centrifuge tubes followed by re-purific
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