Yeast Frataxin Sequentially Chaperones and Stores Iron by Coupling Protein Assembly with Iron Oxidation
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m303158200
ISSN1083-351X
AutoresSungjo Park, Oleksandr Gakh, Heather A. O’Neill, Arianna Mangravita, Helen Nichol, Glória C. Ferreira, Grazia Isaya,
Tópico(s)Fungal and yeast genetics research
ResumoWe have investigated the mechanism of frataxin, a conserved mitochondrial protein involved in iron metabolism and neurodegenerative disease. Previous studies revealed that the yeast frataxin homologue (mYfh1p) is activated by Fe(II) in the presence of O2 and assembles stepwise into a 48-subunit multimer (α48) that sequesters >2000 atoms of iron in 2–4-nm cores structurally similar to ferritin iron cores. Here we show that mYfh1p assembly is driven by two sequential iron oxidation reactions: A ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (α → α3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding α48. Depending on the ionic environment, stepwise assembly is associated with accumulation of 50–75 Fe(II)/subunit. Initially, this Fe(II) is loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. Transfer of mYfh1p-bound Fe(II) to ferrochelatase occurs in the presence of citrate, a physiologic ferrous iron chelator, suggesting that the transfer involves an intermolecular interaction. If mYfh1p-bound Fe(II) is not transferred to a ligand, iron oxidation, and mineralization proceed to completion, Fe(III) becomes progressively less accessible, and a stable iron-protein complex is formed. Iron oxidation-driven stepwise assembly is a novel mechanism by which yeast frataxin can function as an iron chaperone or an iron store. We have investigated the mechanism of frataxin, a conserved mitochondrial protein involved in iron metabolism and neurodegenerative disease. Previous studies revealed that the yeast frataxin homologue (mYfh1p) is activated by Fe(II) in the presence of O2 and assembles stepwise into a 48-subunit multimer (α48) that sequesters >2000 atoms of iron in 2–4-nm cores structurally similar to ferritin iron cores. Here we show that mYfh1p assembly is driven by two sequential iron oxidation reactions: A ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (α → α3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding α48. Depending on the ionic environment, stepwise assembly is associated with accumulation of 50–75 Fe(II)/subunit. Initially, this Fe(II) is loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. Transfer of mYfh1p-bound Fe(II) to ferrochelatase occurs in the presence of citrate, a physiologic ferrous iron chelator, suggesting that the transfer involves an intermolecular interaction. If mYfh1p-bound Fe(II) is not transferred to a ligand, iron oxidation, and mineralization proceed to completion, Fe(III) becomes progressively less accessible, and a stable iron-protein complex is formed. Iron oxidation-driven stepwise assembly is a novel mechanism by which yeast frataxin can function as an iron chaperone or an iron store. Mitochondria require micromolar concentrations of iron to support the heme and the iron-sulfur cluster biosynthetic pathways (1Petrat F. de Groot H. Rauen U. Biochem. J. 2001; 356: 61-69Crossref PubMed Scopus (187) Google Scholar, 2Tangeras A. Biochim. Biophys. Acta. 1985; 843: 199-207Crossref PubMed Scopus (20) Google Scholar). Making this iron bioavailable while limiting its participation in free radical reactions is an essential function accomplished by mechanisms that remain largely uncharacterized (2Tangeras A. Biochim. Biophys. Acta. 1985; 843: 199-207Crossref PubMed Scopus (20) Google Scholar, 3Flatmark T. Romslo I. J. Biol. Chem. 1975; 250: 6433-6438Abstract Full Text PDF PubMed Google Scholar, 4Gattermann N. Aul C. Schneider W. Leukemia. 1993; 7: 2069-2076PubMed Google Scholar). The importance of these mechanisms is exemplified by Friedreich ataxia (FRDA), a severe neuro- and cardio-degenerative disease (5Harding A.E. Brain. 1981; 104: 589-620Crossref PubMed Scopus (794) Google Scholar) in which mitochondria lack the ability to handle iron properly (reviewed in Ref. 6Rotig A. Sidi D. Munnich A. Rustin P. Trends Mol. Med. 2002; 8: 221-224Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). FRDA is caused by defects in frataxin, a conserved nucleus-encoded mitochondrial protein of as yet unknown function (6Rotig A. Sidi D. Munnich A. Rustin P. Trends Mol. Med. 2002; 8: 221-224Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 7Campuzano V. Montermini L. Molto M.D. Pianese L. Cossee M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. et al.Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2323) Google Scholar). Studies in Saccharomyces cerevisiae have shown that the loss of frataxin results in accumulation of iron in mitochondria, widespread oxidative damage to mitochondrial and nuclear DNA via Fenton chemistry, and impaired respiration (8Babcock M. de Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (831) Google Scholar, 9Foury F. Cazzalini O. FEBS Lett. 1997; 411: 373-377Crossref PubMed Scopus (348) Google Scholar, 10Wilson R.B. Roof D.M. Nat. Genet. 1997; 16: 352-357Crossref PubMed Scopus (326) Google Scholar, 11Karthikeyan G. Lewis L.K. Resnick M.A. Hum. Mol. Genet. 2002; 11: 1351-1362Crossref PubMed Scopus (75) Google Scholar). This phenotype can be explained by new findings that yeast frataxin is required for the biosyntheses of iron-sulfur clusters (12Foury F. FEBS Lett. 1999; 456: 281-284Crossref PubMed Scopus (143) Google Scholar, 13Muhlenhoff U. Richter N. Gerber J. Lill R. J. Biol. Chem. 2002; 277: 29810-29816Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 14Muhlenhoff U. Richhardt N. Ristow M. Kispal G. Lill R. Hum. Mol. Genet. 2002; 11: 2025-2036Crossref PubMed Scopus (287) Google Scholar, 15Chen O.S. Hemenway S. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12321-12326Crossref PubMed Scopus (138) Google Scholar, 16Duby G. Foury F. Ramazzotti A. Herrmann J. Lutz T. Hum. Mol. Genet. 2002; 11: 2635-2643Crossref PubMed Scopus (107) Google Scholar) and heme (17Lesuisse E. Santos R. Matzanke B.F. Knight A.A.B. Camadro J.M. Dancis A. Hum. Mol. Genet. 2003; 12: 879-889Crossref PubMed Scopus (219) Google Scholar), two processes critical for maintenance of mitochondrial iron homeostasis (18Muhlenhoff U. Lill R. Biochim. Biophys. Acta. 2000; 1459: 370-382Crossref PubMed Scopus (181) Google Scholar, 19Ponka P. Blood. 1997; 89: 1-25Crossref PubMed Google Scholar). An open question is how frataxin influences two different iron-dependent pathways and also provides protection from iron toxicity. We have proposed that such diverse roles could be reconciled if the basic function of frataxin were to bind and store iron in a bioavailable and nontoxic form (20Patel P.I. Isaya G. Am. J. Hum. Genet. 2001; 69: 15-24Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Our studies with recombinant yeast frataxin have shown that the protein is activated by Fe(II) in the presence of O2 and forms an oligomeric species (α3) that catalyzes Fe(II) oxidation (21Park S. Gakh O. Mooney S.M. Isaya G. J. Biol. Chem. 2002; 277: 38589-38595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). When the Fe(II) concentration exceeds the iron-loading capacity of α3, stepwise assembly of α3 oligomers yields a 48-subunit multimer (α48) that sequesters ∼2,400 atoms of ferric iron. The multimer is a regular spherical particle with a hydrodynamic radius of ∼11 nm and contains small iron cores of 2–4 nm (22Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (114) Google Scholar) with Fe-O and Fe-Fe interactions similar to those found in ferritin iron cores (23Nichol H. Gakh O. O'Neill H.A. Pickering I.J. Isaya G. Graham N.G. Biochemistry. 2003; 42: 5971-5976Crossref PubMed Scopus (65) Google Scholar). Similarly, recombinant human frataxin assembles during expression in Escherichia coli yielding regular spherical particles of ∼1 MDa and ordered polymers of these particles that sequester up to 10 atoms of iron per subunit in small cores structurally identical to the yeast frataxin iron cores (23Nichol H. Gakh O. O'Neill H.A. Pickering I.J. Isaya G. Graham N.G. Biochemistry. 2003; 42: 5971-5976Crossref PubMed Scopus (65) Google Scholar). High molecular weight forms of frataxin can be detected by gel filtration and Western blotting in yeast cells or mouse cardiac tissue, and the native protein binds stoichiometric amounts of 55Fe in metabolically labeled yeast cells (24Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 25Cavadini P. O'Neill H.A. Benada O. Isaya G. Hum. Mol. Genet. 2002; 33: 217-227Crossref Google Scholar). These previous findings support the idea that frataxin, like ferritin, has an iron storage role. Here, we have tested if frataxin might also serve as a reservoir of bioavailable iron. We describe the coupled stepwise-assembly/iron-oxidation reaction of yeast frataxin and show that this mechanism is compatible with both iron chaperone and storage functions. Reagents, Solutions, and Purified Proteins—HEPES, ferrous ammonium sulfate, potassium chloride, α-α′-bipyridine (BIPY), 1The abbreviation used is: BIPY, α-α′-bipyridine. EDTA, dithionite, deuteroporphyrin IX, pyridine, sodium citrate, and bovine serum albumin were from Sigma, and bovine brain calmodulin from Calbiochem. All buffers and solutions were made with milli-Q-deionized water (18 mΩ). Stock solutions of ferrous ammonium sulfate (2–10 mm) were freshly prepared in water previously deaerated by purging with argon gas (<0.2 ppm O2). Calmodulin and albumin were desalted into the appropriate buffer using NAP-25 columns (Amersham Biosciences). The mature forms of yeast frataxin (mYfh1p and mYfh1p[C98A]) and yeast ferrochelatase were expressed in E. coli (24Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 26Gora M. Grzybowska E. Rytka J. Labbe-Bois R. J. Biol. Chem. 1996; 271: 11810-11816Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and purified as previously described (22Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (114) Google Scholar, 27Camadro J.M. Labbe P. J. Biol. Chem. 1988; 263: 11675-11682Abstract Full Text PDF PubMed Google Scholar). The construct for expression of mYfh1p[C98A] was created via PCR-mediated site-directed mutagenesis as previously described (24Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Human H- and L-chain apoferritin homopolymers (28Levi S. Santambrogio P. Cozzi A. Rovida E. Corsi B. Tamborini E. Spada S. Albertini A. Arosio P. J. Mol. Biol. 1994; 238: 649-654Crossref PubMed Scopus (163) Google Scholar) (designated H- and L-apoferritin) were a generous gift of P. Arosio (Brescia University, Brescia, Italy) and S. Levi (Ospedale San Raffaele, Milano, Italy). Protein concentration was determined from the absorbance and extinction coefficient (ϵ280 nm = 20,000/44,200/27,900/34,000 m–1 cm–1 for mYfh1p, bovine serum albumin, H- and L-chain apoferritin, respectively, and ϵ276 nm = 3,000 m–1 cm–1 for calmodulin). Iron concentration was either directly measured by inductively coupled plasma mass spectrometry (ICP-MS) at the Mayo Metals Laboratory or deduced from the concentration of Fe[BIPY]32+ (ϵ520 nm = 9,000 m–1 cm–1) (21Park S. Gakh O. Mooney S.M. Isaya G. J. Biol. Chem. 2002; 277: 38589-38595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Electrode Oximetry, Ultrafiltration, Gel Filtration, and Fluorescence Measurements—Measurements of dissolved O2 concentration were performed with a MI-730 micro-O2 electrode (Microelectrodes, Inc.) (21Park S. Gakh O. Mooney S.M. Isaya G. J. Biol. Chem. 2002; 277: 38589-38595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The drift of the electrode was ∼2 μm/60 min at 30 or 20 °C. Iron binding by mYfh1p and other proteins were measured by ultrafiltration with a molecular mass cutoff of 5 kDa (21Park S. Gakh O. Mooney S.M. Isaya G. J. Biol. Chem. 2002; 277: 38589-38595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). To analyze stepwise assembly of α48, independent samples containing identical concentrations of mYfh1p and Fe(II) were incubated at 30 °C for different periods of time. Each sample was rapidly cooled down to 4 °C to stop assembly, and analyzed by Superdex 200 or Sephacryl 300 gel filtration (22Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (114) Google Scholar). Tryptophan fluorescence intensity was measured in a Quanta Master fluorimeter (Photon Technology International, Ontario, Canada) with a monochromator bandwidth of 2–4 nm and a pathlength of 4 mm. Excitation was at 294 nm, and tryptophan emission was quantitated from the area under the emission band integrated from 300 to 400 nm. Fe[BIPY]32 + and Ferrochelatase Assays—Fe(II) was added to purified mYfh1p monomer, H- or L-apoferritin, or calmodulin in 10 mm HEPES-KOH, pH 7.3, and each sample (8 ml) was incubated at 30 °C. Two aliquots were withdrawn at the indicated time points. BIPY was added to the first aliquot (500 μl) at a final concentration of 2 mm, and after 5 min of incubation at room temperature, the concentration of Fe[BIPY]32+ was determined (21Park S. Gakh O. Mooney S.M. Isaya G. J. Biol. Chem. 2002; 277: 38589-38595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Ferrochelatase and deuteroporphyrin IX were added to the second aliquot (300 μl) at final concentrations of 2 and 118 μm, 2 and 200 μm, or 4 and 400 μm, respectively, and incubation was continued for an additional 20 min at 30 °C. The ferrochelatase reaction was stopped by adding 1 m NaOH and pyridine (176 μl each), and iron-deuteroporphyrin was measured by the pyridine hemochromogen method (29Porra R.J. Jones O.T.G. Biochem. J. 1963; 87: 181-185Crossref PubMed Scopus (188) Google Scholar) with a Δϵ(545–530) nm = 15.3 mm–1 cm–1 for the (reduced – oxidized) difference spectrum (30Porra R.J. Jones O.T.G. Biochem. J. 1963; 87: 186-192Crossref PubMed Scopus (66) Google Scholar). Competition assays were designed to test if transfer of Fe(II) from mYfh1p to ferrochelatase can occur in the presence of a Fe(II) chelator, as was done by others to study the transfer of copper from a metallochaperone to its target protein (31Huffman D.L. O'Halloran T.V. J. Biol. Chem. 2000; 275: 18611-18614Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Unlike in the transfer equilibrium between a copper chaperone and its target protein, we measured the end product of the transfer reaction, i.e. heme. Thus, upstream and downstream steps that are also susceptible to iron chelation had to be considered in choosing an appropriate chelator. We have shown that mYfh1p assembles stepwise into an 840-kDa molecule sequestering up to 50–75 Fe(II) atoms per subunit; this iron is accessible to direct chelation until it is oxidized and incorporated into a stable ferrihydrite mineral (Refs. 22Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (114) Google Scholar and 24Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar and this study). Yeast ferrochelatase is a homodimer of ∼80 kDa containing one Fe(II) substrate binding site and one protoporphyrin binding cleft per subunit; heme synthesis requires the insertion of the Fe(II) atom into the porphyrin ring (32Karlberg T. Lecerof D. Gora M. Silvegren G. Labbe-Bois R. Hansson M. Al-Karadaghi S. Biochemistry. 2002; 41: 13499-13506Crossref PubMed Scopus (48) Google Scholar). A second site in each ferrochelatase subunit is thought to be involved in the initial Fe(II) binding or enzyme regulation (32Karlberg T. Lecerof D. Gora M. Silvegren G. Labbe-Bois R. Hansson M. Al-Karadaghi S. Biochemistry. 2002; 41: 13499-13506Crossref PubMed Scopus (48) Google Scholar). Citrate is a relatively weak Fe(II) chelator (Fe(II)-binding constant = 104m–1) (33Aslamkhan A.G. Aslamkhan A. Ahearn G.A. J. Exp. Zool. 2002; 292: 507-522Crossref PubMed Scopus (38) Google Scholar) believed to represent one of the most abundant ligands to the "free" iron pool in vivo (Refs. 19Ponka P. Blood. 1997; 89: 1-25Crossref PubMed Google Scholar and 34Chen O.S. Hemenway S. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16922-16927Crossref PubMed Scopus (37) Google Scholar and Refs. therein). In ferrochelatase assays performed under strictly anaerobic conditions, Fe(II) can be provided as a ferrous citrate salt (35Ferreira G.C. Shi Z. Anal. Biochem. 2003; 318: 18-24Crossref PubMed Scopus (11) Google Scholar). Thus, citrate should not be able to remove the Fe(II) ion from ferrochelatase after the transfer or to destabilize heme, as has been shown to occur with thiol reagents (36Porra R.J. Vitols K.S. Labbe R.F. Newton N.A. Biochem. J. 1967; 104: 321-327Crossref PubMed Scopus (48) Google Scholar). Moreover, at the neutral pH and under the aerobic conditions used in our assays, citrate will promote rapid autoxidation of Fe(II) (37Minotti G. Aust S.D. Free Radic. Biol. Med. 1987; 3: 379-387Crossref PubMed Scopus (231) Google Scholar) such that any mYfh1p-bound Fe(II) mobilized by citrate will be rapidly oxidized and excluded from the reaction. Both citrate and a stronger chelator, EDTA (EDTA Fe(II)-binding constant = 1014m–1) (38Skoog D.A. West D.M. College S. Analytical Chemistry. Philadelphia, PA1980: 253Google Scholar), were used in competition assays. We used citrate/total iron ratios ranging from 0.06:1 to 166:1, and citrate/ferrochelatase ratios ranging from 1:1 to 2500:1, which encompass and exceed the ratios used in anaerobic ferrochelatase assays (citrate/Fe = 1:1; citrate/ferrochelatase = 28:1) (35Ferreira G.C. Shi Z. Anal. Biochem. 2003; 318: 18-24Crossref PubMed Scopus (11) Google Scholar). EDTA/total iron ratios ranged from 0.33:1 to 7:1 and EDTA/ferrochelatase ratios from 5:1 to 100:1. Stepwise Assembly of mYfh1p Is Coupled with Two Sequential Iron Oxidation Reactions—At Fe(II)/mYfh1p ratios ≤0.5, mYfh1p catalyzes Fe(II) oxidation with a stoichiometry of ∼2 Fe(II)/O2 and production of H2O2 (ferroxidase reaction) (21Park S. Gakh O. Mooney S.M. Isaya G. J. Biol. Chem. 2002; 277: 38589-38595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). A ∼50-kDa oligomer (α3) is responsible for this activity suggesting that three mYfh1p subunits may form one binuclear ferroxidation site (21Park S. Gakh O. Mooney S.M. Isaya G. J. Biol. Chem. 2002; 277: 38589-38595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Here, we have analyzed the iron oxidation reaction of mYfh1p at concentrations of iron (40–75 Fe(II)/mYfh1p) that encompass the iron loading capacity of mYfh1p (50–75 Fe(III)/mYfh1p depending on the ionic environment) and result in stepwise assembly of α3 to yield iron-loaded α48 (22Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (114) Google Scholar, 24Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Fig. 1A shows representative O2 consumption curves recorded when 100 μm Fe(II) was incubated in 10 mm HEPES-KOH, pH 7.3, in the absence or presence of 2 μm mYfh1p (Fe(II)/mYfh1p = 50/1). In buffer without protein there was an initial lag phase due to the time required to generate sufficient hydrolyzed Fe(III) to initiate autoxidation of Fe(II) (39Yang X. Chasteen N.D. Biochem. J. 1999; 338: 615-618Crossref PubMed Google Scholar) (Fig. 1A, black plot). The final Fe(II)/O2 stoichiometry was 3.7 ± 0.3 (n = 3) as expected for autoxidation (39Yang X. Chasteen N.D. Biochem. J. 1999; 338: 615-618Crossref PubMed Google Scholar). In the presence of mYfh1p, the initial rate of O2 consumption was faster compared with buffer, consistent with ferroxidase activity, but became slower after the first 4 min (Fig. 1A, red plot). The final Fe(II)/O2 stoichiometry was 3.6 ± 0.3 (n = 3), indicating that ferroxidation was rapidly overcome by autoxidation. We were unable to detect any H2O2 released into the solution, which should be expected given the high Fe(II)/mYfh1p ratio used in these experiments (21Park S. Gakh O. Mooney S.M. Isaya G. J. Biol. Chem. 2002; 277: 38589-38595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 40Zhao G. Bou-Abdallah F. Yang X. Arosio P. Chasteen N.D. Biochemistry. 2001; 40: 10832-10838Crossref PubMed Scopus (69) Google Scholar). In Fig. 1B, gel filtration was used to determine the speciation of mYfh1p during the iron oxidation reaction described above. Experimental conditions were similar to those employed for electrode oximetry in Fig. 1A except that both the protein and the iron concentrations were increased 2-fold to enable detection of mYfh1p by absorbance measurements. Such an increase is not expected to change the rate of α48 assembly to a significant degree (22Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (114) Google Scholar, 24Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), and therefore the gel filtration data (Fig. 1B) can be correlated with the two phases in the O2 consumption curve of mYfh1p (Fig. 1A). The initial faster phase (Fig. 1A, 0–4 min, red plot) was associated with the assembly of an oligomer of ∼50 kDa (Fig. 1B, 3 min), while the subsequent slower phase (Fig. 1A, 4–50 min, red plot) was associated with stepwise assembly of higher order oligomers (Fig. 1B, 6, 10, and 30 min), in agreement with the previously described progression, α → α3 → α6 → α12 → α24 → α48 (22Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (114) Google Scholar, 24Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The A 280 of assembled mYfh1p species increased in a time-dependent manner (Fig. 1B). At the end of the iron oxidation reaction, the A 280 of α48 was much higher than the A 280 of monomer analyzed in the absence of any added Fe(II) (Fig. 1B, peak α). These results are consistent with progressive oxidation of Fe(II) to Fe(III) and formation of a ferrihydrite-like mineral (which, unlike Fe(II), absorbs at 280 nm) within the assembled protein (23Nichol H. Gakh O. O'Neill H.A. Pickering I.J. Isaya G. Graham N.G. Biochemistry. 2003; 42: 5971-5976Crossref PubMed Scopus (65) Google Scholar). We showed previously that the mature form of Yfh1p is generated by cleavage of an N-terminal mitochondrial targeting signal between residues 51–52 (24Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 41Branda S.S. Cavadini P. Adamec J. Kalousek F. Taroni F. Isaya G. J. Biol. Chem. 1999; 274: 22763-22769Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). This cleavage eliminates one cysteine residue at position 32. The mature form of the protein (amino acids 52–174), which is the form used in our experiments, contains one cysteine residue at position 98. Thus, an alternative explanation for the results in Fig. 1B could be that chelation of iron by cysteine residues from different mYfh1p subunits leads to formation of metal-thiolate aggregates (42Reese R.N. Mehra R.K. Tarbet E.B. Winge D.R. J. Biol. Chem. 1988; 263: 4186-4192Abstract Full Text PDF PubMed Google Scholar). However, others have reported that when yeast frataxin is treated with iodoacetamide to block any exposed cysteine residues and subsequently incubated with 20 equivalents of Fe(II), iron-dependent oligomerization is not affected (43Adinolfi S. Trifuoggi M. Politou A.S. Martin S. Pastore A. Hum. Mol. Genet. 2002; 11: 1865-1877Crossref PubMed Scopus (119) Google Scholar). We obtained similar results using a mYfh1p variant in which cysteine 98 was replaced by an alanine residue (data not shown). We therefore conclude that mYfh1p assembly is driven by iron oxidation: A ferroxidase reaction catalyzed by mYfh1p is associated with the first assembly step (α → α3), followed by a slower autoxidation reaction associated with assembly of higher order oligomers to ultimately yield α48. The Ferrous Iron Sequestered by mYfh1p Is Bioavailable— The time required to complete the iron oxidation reaction of mYfh1p is in the order of hundreds of seconds (Fig. 1A), much longer than the iron oxidation reaction of ferritin, which is in the order of tens of seconds (Ref. 40Zhao G. Bou-Abdallah F. Yang X. Arosio P. Chasteen N.D. Biochemistry. 2001; 40: 10832-10838Crossref PubMed Scopus (69) Google Scholar and data not shown). At the beginning of its reaction, however, mYfh1p rapidly sequesters up to 50–75 Fe(II)/subunit, which are then progressively oxidized within the assembled protein (Ref. 21Park S. Gakh O. Mooney S.M. Isaya G. J. Biol. Chem. 2002; 277: 38589-38595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 22Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (114) Google Scholar, and 24Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar and data presented below). Given that mobilization of iron from ferritin is inefficient in the absence of reducing agents (44Crichton R. Inorganic Biochemistry of Iron Metabolism. John Wiley & Sons, LTD, New York2001: 133-165Crossref Google Scholar), we hypothesized that the coupling of a slow iron autoxidation reaction with stepwise assembly might enable mYfh1p to serve as a temporary reservoir and a chaperone for Fe(II). We therefore measured iron mobilization during mYfh1p assembly by use of α,α′-bipyridine (BIPY), a chelator that preferentially binds Fe(II) (45Richards T.D. Pitts K.R. Watt G.D. J. Inorg. Biochem. 1996; 61: 1-13Crossref PubMed Scopus (39) Google Scholar), or purified yeast ferrochelatase, a mitochondrial enzyme that catalyzes the insertion of Fe(II) into protoporphyrin IX to yield heme (reviewed in Ref. 46Ferreira G.C. Int. J. Biochem. Cell Biol. 1999; 31: 995-1000Crossref PubMed Scopus (49) Google Scholar). Iron mobilization from human H- or L-apoferritin (28Levi S. Santambrogio P. Cozzi A. Rovida E. Corsi B. Tamborini E. Spada S. Albertini A. Arosio P. J. Mol. Biol. 1994; 238: 649-654Crossref PubMed Scopus (163) Google Scholar) was analyzed in parallel. These two proteins are pre-assembled 24-subunit shells with a negatively charged inner surface that promotes iron autoxidation and mineralization; in addition, H-apoferritin has 24 dinuclear ferroxidation sites (28Levi S. Santambrogio P. Cozzi A. Rovida E. Corsi B. Tamborini E. Spada S. Albertini A. Arosio P. J. Mol. Biol. 1994; 238: 649-654Crossref PubMed Scopus (163) Google Scholar, 47Yang X. Chen-Barrett Y. Arosio P. Chasteen N.D. Biochemistry. 1998; 37: 9743-9750Crossref PubMed Scopus (130) Google Scholar). As a negative control we used calmodulin, a calcium-binding protein with a molecular mass and an isoelectric point similar to those of mYfh1p (17 versus 14 kDa, and 4.09 versus 4.34). Reactions were started by addition of a fixed concentration of Fe(II) (30 μm) to buffer in the absence or presence of protein (0.4 μm; Fe(II)/subunit = 75:1 for all proteins tested). At successive time points, an aliquot was withdrawn and divided in two parts that were immediately incubated with either BIPY (2 mm) or ferrochelatase (2 μm) and deuteroporphyrin IX (118 μm). The half-life of BIPY-accessible iron estimated from a single exponential fitting was 21.5 min in the presence of mYfh1p compared with 1.0, 4.0, 6.7, and 8.8 min in the presence of H-apoferritin, L-apoferritin, buffer only, and calmodulin, respectively (Fig. 2A). Similarly, ferrous iron was more accessible to ferrochelatase in the presence of mYfh1p relative to buffer or calmodulin (Fig. 2B). BIPY can bind Fe(III) and/or reduce Fe(III) to Fe(II) although with lower affinity compared with Fe(II) (48Schmid R. Kirchner K. Dickert F.L. Inorg. Chem. 1988; 27: 1530-1536Crossref Scopus (31) Google Scholar, 49Monsted O. Nord G. Adv. Inorg. Chem. 1991; 37: 2381-2397Google Scholar, 50Pehkonen S. Analyst. 1995; 120: 2655-2663Crossref Google Scholar), suggesting that the BIPY accessible iron mobilized from mYfh1p could represent a mixture of both ferrous and ferric iron. However, the concentrations of BIPY-accessible iron at successive time points in the presence of mYfh1p (Fig. 2A) were in the same order as the concentrations of ferrochelatase-accessible iron measured under similar conditions (Fig. 2B). We therefore conclude that the iron mobilized by direct chelation (i.e. BIPY accessible iron) during mYfh1p assembly is largely in ferrous form, becoming progressively less accessible as it is oxidized t
Referência(s)