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• W2030356875 abstract "Frataxin is required for maintenance of normal mitochondrial iron levels and respiration. The mature form of yeast frataxin (mYfh1p) assembles stepwise into a multimer of 840 kDa (α48) that accumulates iron in a water-soluble form. Here, two distinct iron oxidation reactions are shown to take place during the initial assembly step (α → α3). A ferroxidase reaction with a stoichiometry of 2 Fe(II)/O2 is detected at Fe(II)/mYfh1p ratios of ≤0.5. Ferroxidation is progressively overcome by autoxidation at Fe(II)/mYfh1p ratios of >0.5. Gel filtration analysis indicates that an oligomer of mYfh1p, α3, is responsible for both reactions. The observed 2 Fe(II)/O2 stoichiometry implies production of H2O2 during the ferroxidase reaction. However, only a fraction of the expected total H2O2 is detected in solution. Oxidative degradation of mYfh1p during the ferroxidase reaction suggests that most H2O2reacts with the protein. Accordingly, the addition of mYfh1p to a mixture of Fe(II) and H2O2 results in significant attenuation of Fenton chemistry. Multimer assembly is fully inhibited under anaerobic conditions, indicating that mYfh1p is activated by Fe(II) in the presence of O2. This combination induces oligomerization and mYfh1p-catalyzed Fe(II) oxidation, starting a process that ultimately leads to the sequestration of as many as 50 Fe(II)/subunit inside the multimer. Frataxin is required for maintenance of normal mitochondrial iron levels and respiration. The mature form of yeast frataxin (mYfh1p) assembles stepwise into a multimer of 840 kDa (α48) that accumulates iron in a water-soluble form. Here, two distinct iron oxidation reactions are shown to take place during the initial assembly step (α → α3). A ferroxidase reaction with a stoichiometry of 2 Fe(II)/O2 is detected at Fe(II)/mYfh1p ratios of ≤0.5. Ferroxidation is progressively overcome by autoxidation at Fe(II)/mYfh1p ratios of >0.5. Gel filtration analysis indicates that an oligomer of mYfh1p, α3, is responsible for both reactions. The observed 2 Fe(II)/O2 stoichiometry implies production of H2O2 during the ferroxidase reaction. However, only a fraction of the expected total H2O2 is detected in solution. Oxidative degradation of mYfh1p during the ferroxidase reaction suggests that most H2O2reacts with the protein. Accordingly, the addition of mYfh1p to a mixture of Fe(II) and H2O2 results in significant attenuation of Fenton chemistry. Multimer assembly is fully inhibited under anaerobic conditions, indicating that mYfh1p is activated by Fe(II) in the presence of O2. This combination induces oligomerization and mYfh1p-catalyzed Fe(II) oxidation, starting a process that ultimately leads to the sequestration of as many as 50 Fe(II)/subunit inside the multimer. horseradish peroxidase 2,4-dinitrophenylhydrazone mature form of yeast fretaxin The two major iron-utilizing processes in the cell, production of heme by ferrochelatase and the iron-sulfur cluster biosynthetic pathway, reside in the mitochondrial matrix. Mitochondria contain micromolar concentrations of chelatable iron (1Petrat F. de Groot H. Rauen U. Biochem. J. 2001; 356: 61-69Crossref PubMed Scopus (186) Google Scholar), i.e. iron that is not yet complexed in heme or iron-sulfur clusters and is bioavailable (2Tangeras A. Biochim. Biophys. Acta. 1985; 843: 199-207Crossref PubMed Scopus (20) Google Scholar). Keeping this iron pool in soluble and nontoxic form represents a remarkable biological challenge given the alkaline pH (3Darnell J. Lodish H. Baltimore D. Molecular Cell Biology. Scientific American Books, New York1990: 583-616Google Scholar) and the high production of O 2⨪ and H2O2(4Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, Oxford, UK1999: 1-34Google Scholar) within mitochondria. Thus, the existence of proteins capable of handling iron safely within mitochondria was first postulated several decades ago (5Flatmark T. Romslo I. J. Biol. Chem. 1975; 250: 6433-6438Abstract Full Text PDF PubMed Google Scholar). Recent studies have identified a handful of inner mitochondrial membrane proteins involved in mitochondrial iron transport (6Kispal G. Csere P. Guiard B. Lill R. FEBS Lett. 1997; 418: 346-350Crossref PubMed Scopus (245) Google Scholar, 7Lange H. Kispal G. Lill R. J. Biol. Chem. 1999; 274: 18989-18996Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 8Foury F. Roganti T. J. Biol. Chem. 2002; 277: 24475-24483Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) as well as a mitochondrial matrix ferritin involved in iron storage (9Corsi B. Cozzi A. Arosio P. Drysdale J. Santambrogio P. Campanella A. Biasiotto G. Albertini A. Levi S. J. Biol. Chem. 2002; 277: 22430-22437Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The mitochondrial matrix protein frataxin has been implicated in mitochondrial iron homeostasis (10Babcock M. de Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (817) Google Scholar, 11Foury F. Cazzalini O. FEBS Lett. 1997; 411: 373-377Crossref PubMed Scopus (342) Google Scholar), but its precise function is still not known. Frataxin was first identified as the protein deficient in Friedreich ataxia, a neuro- and cardio-degenerative disease (12Campuzano 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 (2284) Google Scholar). Studies in Saccharomyces cerevisiae have shown that defects in yeast or human frataxin are associated with the accumulation of iron in mitochondria and oxidative damage to both mitochondrial and nuclear DNA as well as to the iron-sulfur centers of mitochondrial aconitase and other respiratory enzymes (10Babcock M. de Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (817) Google Scholar, 14Cavadini P. Gellera C. Patel P.I. Isaya G. Hum. Mol. Genet. 2000; 9: 2523-2530Crossref PubMed Scopus (136) Google Scholar, 15Karthikeyan G. Lewis L.K. Resnick M.A. Hum. Mol. Genet. 2002; 11: 1351-1362Crossref PubMed Scopus (74) Google Scholar, 34Foury F. FEBS Lett. 1999; 456: 281-284Crossref PubMed Scopus (143) Google Scholar). Similarly, mouse models in which the frataxin gene is selectively inactivated in neuronal or cardiac tissue present multiple respiratory enzyme deficits and accumulate iron in mitochondria in a time-dependent manner (16Puccio H. Simon D. Cossee M. Criqui-Filipe P. Tiziano F. Melki J. Hindelang C. Matyas R. Rustin P. Koenig M. Nat. Genet. 2001; 27: 181-186Crossref PubMed Scopus (590) Google Scholar). In agreement with these observations, evidence of abnormal cellular iron homeostasis, increased oxidative damage, and respiratory enzyme deficits has been reported for the human disease (reviewed in Ref. 17Patel P.I. Isaya G. Am. J. Hum. Genet. 2001; 69: 15-24Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). It has been shown that frataxin defects result in impaired mitochondrial iron efflux (18Radisky D.C. Babcock M.C. Kaplan J. J. Biol. Chem. 1999; 274: 4497-4499Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), defective biosynthesis of iron-sulfur clusters (19Foury F. FEBS Lett. 1999; 456: 281-284Crossref PubMed Scopus (140) Google Scholar, 20Muhlenhoff U. Richter N. Gerber J. Lill R. J. Biol. Chem. 2002; 277: 29810-29816Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), loss of ATP synthesis (21Ristow M. Pfister M.F. Yee A.J. Schubert M. Michael L. Zhang C.Y. Ueki K. Michael II, M.D. Lowell B.B. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12239-12243Crossref PubMed Scopus (191) Google Scholar), and/or disabled antioxidant defenses (22Chantrel-Groussard K. Geromel V. Puccio H. Koenig M. Munnich A. Rotig A. Rustin P. Hum. Mol. Genet. 2001; 10: 2061-2067Crossref PubMed Scopus (166) Google Scholar), all conditions that could ultimately lead to mitochondrial iron accumulation and increased oxidative damage. We have proposed that the apparent involvement of frataxin in so many diverse processes could be explained if the basic function of this protein were to provide an iron storage mechanism to keep iron in a bioavailable and nontoxic form (17Patel P.I. Isaya G. Am. J. Hum. Genet. 2001; 69: 15-24Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Indeed, titration of the mature form of yeast frataxin (mYfh1p) with Fe(II) under aerobic conditions results in stepwise assembly of a 48-subunit multimer with a molecular mass of 840 kDa and a hydrodynamic radius of 11 nm that sequesters 50 atoms of iron/subunit and forms iron-rich cores with a diameter of 2–4 nm (23Adamec 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 (229) Google Scholar,24Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (113) Google Scholar). Similarly, the mature form of human frataxin assembles naturally during expression in Escherichia coli yielding regular multimers of ∼1 MDa and ordered polymers of these multimers that sequester ∼10 atoms of iron/subunit (25Cavadini P. O'Neill H.A. Benada O. Isaya G. Hum. Mol. Genet. 2002; 33: 217-227Crossref Google Scholar). Frataxin can be detected in a high molecular mass complex under native conditions in yeast or mouse heart, and both the yeast and the human protein bind stoichiometric amounts of 55Fe in metabolically labeled yeast cells (23Adamec 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 (229) Google Scholar, 25Cavadini P. O'Neill H.A. Benada O. Isaya G. Hum. Mol. Genet. 2002; 33: 217-227Crossref Google Scholar). In this study, we investigate the iron oxidation reaction of yeast frataxin. Our results support a direct role for frataxin in iron metabolism. HEPES, ferrous ammonium sulfate, 2-deoxyribose, thiobarbituric acid, and α-α′-bipyridine were from Sigma, and beef liver catalase was from Roche Molecular Biochemicals. All of the buffers and solutions were made with milli-Q deionized water. Stock solutions of ferrous ammonium sulfate (2–10 mm; pH 3.6) were freshly prepared in water previously deaerated by purging with argon gas (<0.2 ppm O2). Recombinant mYfh1p was expressed in E. coli (23Adamec 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 (229) Google Scholar) and purified as previously described (24Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (113) Google Scholar). The protein concentration was determined from the absorbance and extinction coefficient of mYfh1p monomer (ε280 = 20,000 m−1cm−1). Iron concentration measurements were carried out by inductively coupled plasma emission spectroscopy at the Metals Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN. Measurements of dissolved O2concentration were performed with a MI-730 micro-O2electrode connected to an O2-ADPT adapter (Microelectrodes, Inc., NH). A TBX-68T isothermal terminal block connected to a NI 4350 high precision voltage meter and a personal computer with the LabVIEW Base Package (National Instruments, Austin, TX) were used for data acquisition. The electrode was calibrated with HEPES-KOH buffer at each experimental pH and temperature, using buffer deaerated by extensive purging with argon gas (<0.2 ppm O2) (0% standard) and air-saturated buffer (21% standard). To determine the O2electrode response time, the electrode was equilibrated in air-saturated water and then quickly plunged into a rapidly stirred 100 mm dithionite solution or a deaerated solution, and the electrode output was measured versus time (26Yang X. Chen-Barrett Y. Arosio P. Chasteen N.D. Biochemistry. 1998; 37: 9743-9750Crossref PubMed Scopus (127) Google Scholar). The electrode response followed first order kinetics with a half-life of 6 s. The drift of the O2 electrode was not significant (∼1 μm O2/40 min). O2consumption was monitored in a 0.6-ml cell. After addition of buffer with or without protein, the cell was sealed with a rubber stopper fitted with the O2 microelectrode described above and a cone-shaped capillary (height, 4.5 cm; volume, 200 μl) with the vertex toward the inside of the cell. The capillary was partially filled with buffer and used as a port to eliminate empty air space and air bubbles. The final volume of the sample in the cell was 0.465 ml. During measurements, the sample was rapidly stirred with a microspin bar, and the temperature was maintained constant by use of a water jacket connected to a circulating water bath. The diffusion of O2 to and from the cell was determined to be insignificant over a period of 40 min. The reactions were started by adding anaerobically prepared Fe(II) stock solution (5–15 μl of total volume added) to the sample using a gas-tight syringe with a 22-gauge needle that was inserted into the cell through the buffer-filled capillary. HEPES is known to retard Fe(II) oxidation, albeit to a much lower degree compared with other Good's buffers (27Yang X. Chasteen N.D. Biochem. J. 1999; 338: 615-618Crossref PubMed Google Scholar). To minimize this effect, a relatively low concentration (10 mm) of HEPES-KOH buffer was used for O2 consumption measurements, which was sufficient to maintain the pH at 7.0–6.97 at all iron concentrations tested. An aliquot of Fe(II) stock solution was directly added to 10 mmHEPES-KOH, pH 7.0, with or without protein present. The samples (500 μl) were incubated at 30 °C for different times and immediately transferred to a Ultrafree-0.5 cell (nominal molecular weight limit = 5,000) (Millipore, Bedford, MA) and centrifuged for 5 min at 14,000 × g at 4 °C. The concentrate and the filtrate were transferred to Eppendorf tubes, a few crystals of dithionite and α-α′-bipyridine (final concentration, 2 mm) were added to each sample, and the concentration of Fe[α-α′-bipyridine]32+(ε520 = 9,000 m−1cm−1) was determined in a DU640B spectrophotometer (Beckman, Fullerton, CA) (28Richards T.D. Pitts K.R. Watt G.D. J. Inorg. Biochem. 1996; 61: 1-13Crossref PubMed Scopus (39) Google Scholar). After removal of the concentrate, insoluble iron was stripped from the Ultrafree membrane by adding 500 μl of buffer containing a few crystals of dithionite and 2 mm α-α′-bipyridine and by mixing vigorously for 1 min, followed by absorbance measurements at 520 nm as described above. For gel filtration analysis of assembly reaction products, the samples (1 ml) were centrifuged for 5 min at 20,800 × g at 4 °C and loaded onto a Superdex 200 column (23Adamec 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 (229) Google Scholar). In anaerobic experiments, the buffer and the protein stock solution were made anaerobic by purging with moisturized argon gas (<0.2 ppm O2). Protein and Fe(II) were added to 2-ml vials sealed with a rubber septum (Sherwood Medical, St. Louis, MO) (final volume, 100 μl) by a gas-tight syringe, and the assembly reaction was incubated for 1 h at 30 °C (23Adamec 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 (229) Google Scholar). After cooling of the sample at 4 °C to stop any ongoing assembly reaction (24Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (113) Google Scholar), a 50-μl aliquot was loaded onto a TSK-GEL G4000SW column (7.5 mm × 30 cm) (Tosoh Biosep, Montgomeryville, PA) using a 50-μl loop and a gas-tight syringe. The column was equilibrated with 10 mm HEPES-KOH, pH 7.3, 100 mm NaCl, previously deaerated by purging with research grade nitrogen gas (<0.5 ppm O2). The protein was eluted with 19 ml of the same buffer at a flow rate of 0.6 ml/min at 4 °C. To determine the production of H2O2 by electrode oximetry, catalase (2,600–3,900 units) was added to 0.465 ml of 96 μmmYfh1p in 10 mm HEPES-KOH, pH 7.0, either before or after the addition of Fe(II), and the O2 consumption and Fe(II)/O2 stoichiometry for the completed reaction were measured (29Zhao G. Bou-Abdallah F. Yang X. Arosio P. Chasteen N.D. Biochemistry. 2001; 40: 10832-10838Crossref PubMed Scopus (69) Google Scholar). Production of H2O2 was further measured by the Amplex Red hydrogen peroxide/peroxidase assay kit from Molecular Probes, Inc. (Eugene, OR) (29Zhao G. Bou-Abdallah F. Yang X. Arosio P. Chasteen N.D. Biochemistry. 2001; 40: 10832-10838Crossref PubMed Scopus (69) Google Scholar). All of the reactions (1 ml) were performed in vials sealed with a rubber septum according to the manufacturer's protocol except that 50 mm HEPES-KOH, pH 7.0, was used instead of 50 mm phosphate pH 7.4 buffer (29Zhao G. Bou-Abdallah F. Yang X. Arosio P. Chasteen N.D. Biochemistry. 2001; 40: 10832-10838Crossref PubMed Scopus (69) Google Scholar). The fluorescence intensity of samples in which Fe(II) was added to mYfh1p in the presence of Amplex Red and horseradish peroxidase (Amplex Red/HRP)1 was not significantly different from that of samples in which Amplex Red/HRP was added after the addition of Fe(II). In subsequent experiments, Fe(II) was therefore added to mYfh1p in the presence of Amplex Red/HRP. For standard curves, known concentrations of H2O2 were added to buffer in the presence of Amplex Red/HRP. Both H2O2 standards and unknown samples were incubated for 30 min at 30 °C and treated identically. Fluorescence emission was measured in a QuantaMaster fluorimeter (Photon Technology International, Ontario, Canada) from 570 to 610 nm with excitation at 530 nm. The concentration of H2O2 was calculated from the area under each fluorescence emission curve. Background fluorescence was determined with buffer containing Amplex Red/HRP for H2O2standards, and with buffer containing Amplex Red/HRP and the appropriate Fe(II) concentration for unknown samples. The absorbance at 530 nm was <0.02 for all samples. OxyBlot kit (Intergen, Purchase, NY) was used to detect protein oxidation. The reactions (25 μl) containing mYfh1p in the absence or presence of Fe(II) were incubated under the experimental conditions used in the Amplex Red/HRP assays described above, after which carbonyl groups were derivatized to 2,4-dinitrophenylhydrazone (DNP) by reaction with an equal volume of 2,4-dinitrophenylhydrazine for 25 min at room temperature and neutralized according to the manufacturer's protocol. The samples were analyzed by 12% SDS/PAGE and Western blotting (30Branda S.S. Yang Z.Y. Chew A. Isaya G. Hum. Mol. Genet. 1999; 8: 1099-1110Crossref PubMed Scopus (54) Google Scholar) using a polyclonal anti-DNP antiserum. Following immunodetection of carbonyls, the proteins were detected by SYPRO Ruby staining of the membrane (Molecular Probes, Inc.). In oxidative degradation assays, different combinations of 48 μm Fe(II), 24 μmH2O2, and/or 5 mm 2-deoxyribose were incubated in 10 mm HEPES-KOH, pH 7.0, in the absence or presence of 96 μm mYfh1p (final volume, 200 μl) for 30 min at 30 °C. Phosphoric acid (4%) and thiobarbituric acid (1%) were added (200 μl each), and each sample was boiled for 15 min. After cooling of the samples on ice for 3 min, 75 μl of 10% SDS was added, and a malondialdehyde-thiobarbituric acid adduct (ε532 = 1.54 × 105m−1 cm−1) was measured as described by Halliwell and Gutteridge (31Halliwell B. Gutteridge J.M. FEBS Lett. 1981; 128: 347-352Crossref PubMed Scopus (763) Google Scholar). Electrode oximetry was used to study the iron oxidation reaction of mYfh1p at low Fe(II)/mYfh1p ratios. We showed previously that these conditions result in the assembly of an oligomeric species, α3, predicted to represent the building unit of the mYfh1p multimer (23Adamec 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 (229) Google Scholar). Fig.1 A shows the O2consumption curves recorded when 24, 48, or 144 μm Fe(II) was incubated in 10 mm HEPES-KOH, pH 7.0, at 30 °C, in the absence or presence of 96 μm mYfh1p. At 24 or 48 μm Fe(II), O2 consumption was facilitated in the presence of mYfh1p (Fig. 1 A). The stoichiometric Fe(II)/O2 ratios at the end of these two reactions were 2.2 and 2.4 (Fig. 1 A). Stoichiometric Fe(II)/O2ratios of 3.5 and 3.2 were otherwise measured when 24 or 48 μm Fe(II), respectively, was incubated in buffer without mYfh1p (Fig. 1 A). At 144 μm Fe(II), similar stoichiometric Fe(II)/O2 ratios of 3.9 and 4.0 were determined in the presence or absence of mYfh1p, respectively (Fig.1 A). A Fe(II)/O2 stoichiometry of ∼2 was consistently observed in repeated measurements at 24, 36, or 48 μm Fe(II) in the presence of 96 μm mYfh1p, corresponding to a Fe(II)/mYfh1p ratio of ≤0.5 (Fig. 1 B). A Fe(II)/O2 stoichiometry of 2.3 ± 0.4 (n = 4) was similarly determined in the presence of 30 μm Fe(II) and 60 μm mYfh1p (Fe(II)/mYfh1p = 0.5) (data not shown). However, the Fe(II)/O2 stoichiometry progressively increased to a maximum value of ∼4 at a Fe(II)/mYfh1p ratio of >0.5 (Fig.1 B). In contrast, an average Fe(II)/O2stoichiometry of ∼3.6 was consistently obtained in buffer without protein at different Fe(II) concentrations (Fig. 1 B). This was also the case when 30 μm Fe(II) was incubated at 20 °C in the absence or presence of 96 μm mYfh1p (Fe(II)/mYfh1p = 0.3), yielding a Fe(II)/O2stoichiometry of 4.3 ± 0.9 (n = 3) and 2.5 ± 0.4 (n = 2), respectively (data not shown). A Fe(II)/O2 stoichiometry of 2 is typical of the ferroxidation reaction of H-ferritin (26Yang X. Chen-Barrett Y. Arosio P. Chasteen N.D. Biochemistry. 1998; 37: 9743-9750Crossref PubMed Scopus (127) Google Scholar, 32Waldo G.S. Theil E.C. Biochemistry. 1993; 32: 13262-13269Crossref PubMed Scopus (89) Google Scholar). The results reported above are therefore consistent with the presence of ferroxidase activity when ≤0.5 Fe(II) equivalents are added to mYfh1p, with autoxidation overriding ferroxidation at higher Fe(II)/mYfh1p ratios. In addition, at a Fe(II)/mYfh1p ratio ≤0.5, we have detected production of H2O2 (see below). These results together support a ferroxidation site similar to that of H-ferritin, in which H2O2 is produced from two-electron reduction of O2 at a binuclear iron center (27Yang X. Chasteen N.D. Biochem. J. 1999; 338: 615-618Crossref PubMed Google Scholar). During iron deposition in vertebrate ferritins, autoxidation overrides ferroxidation when the Fe(II) concentration exceeds 2 Fe(II)/ferroxidation site (27Yang X. Chasteen N.D. Biochem. J. 1999; 338: 615-618Crossref PubMed Google Scholar). This threshold corresponds to ≤2 Fe(II)/H-subunit, because each H-subunit contains one binuclear iron ferroxidation site (27Yang X. Chasteen N.D. Biochem. J. 1999; 338: 615-618Crossref PubMed Google Scholar). Therefore, we have assumed a binuclear iron site for mYfh1p and have estimated the number of ferroxidation sites per subunit using the following equation. Ferroxidation sites/subunit=0.5–0.6Fe(II)/subunit2Fe(II)/ferroxidation siteEquation 1 where “0.5–0.6 Fe(II)/subunit” is the smallest interval determined in Fig. 1 B within which autoxidation overcomes the ferroxidation reaction of mYfh1p, and “2 Fe(II)/ferroxidation site” is the threshold that cannot be exceeded to observe ferroxidase activity, as described for H-ferritin (27Yang X. Chasteen N.D. Biochem. J. 1999; 338: 615-618Crossref PubMed Google Scholar). The equation yields a value of 0.25–0.30, suggesting a ferroxidation site formed by four or three subunits. A three-subunit site would be consistent with the progression, α→ α3 → α6 → α12 → α24 → α48, determined previously by gel filtration analysis of the iron-dependent stepwise assembly of mYfh1p (23Adamec 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 (229) Google Scholar, 24Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (113) Google Scholar). Importantly, α3, not α, was the smallest iron-containing species detected in this progression (23Adamec 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 (229) Google Scholar). To identify the form of mYfh1p responsible for iron oxidation, monomer (96 μm) was incubated with Fe(II) at a Fe(II)/mYfh1p ratio of 0.25, 0.5, or 1.5 at 30 °C for 25 min, which is the average time required for the iron oxidation reaction to be completed under these experimental conditions (Fig. 1 A). Immediately afterward, each reaction was subjected to ultrafiltration at 14,000 × g for 5 min at 4 °C with a molecular mass cut-off of ≤5 kDa. In controls containing buffer without mYfh1p, most iron was recovered in the flow-through and/or in insoluble form (Table I). In all reactions containing mYfh1p, however, most iron was recovered in the concentrate (Table I), indicating that it was bound to the protein. The Fe(III)/mYfh1p ratios in the concentrate from the three reactions analyzed in Table I were 0.21, 0.43, and 1.2. These ratios are close to the initial Fe(II)/mYfh1p ratio of 0.25, 0.5, and 1.5, respectively, indicating that most added iron was bound to mYfh1p at the end of the oxidation reaction.Table IAnalysis of iron binding by mYfh1p oligomer (α3)Fe2+/mYfh1p ratio1-aA fixed concentration of mYfh1p (96 μm) was incubated with 24, 48, and 144 μmFe(II) for 25 min under the conditions used for electrode oximetry in Fig. 1. Iron was measured in the three indicated fractions as described under “Experimental Procedures.”0.25/1 at 24 μm Fe2+0.5/1 at 48 μm Fe2+1.5/1 at 144 μmFe2+BufferConcentrate0.6 ± 0.11-bMean ± S.D. from three independent experiments (μm iron). The total recovery at 144 μmFe(II) was only ∼83% in the presence of mYfh1p and 67% in buffer without protein due to incomplete solubilization of the precipitated ferric oxides by addition of dithionite.2.7 ± 0.71.9 ± 1.4Flow-through11.2 ± 0.516.0 ± 1.05.1 ± 0.7Insoluble iron7.5 ± 0.923.9 ± 1.090.5 ± 8.0mYfh1pConcentrate (protein-bound iron)20.5 ± 0.341.6 ± 0.1114.1 ± 2.4Flow-through (free iron)0.5 ± 0.21.1 ± 0.21.2 ± 0.1Insoluble iron1.0 ± 0.42.7 ± 0.33.9 ± 0.51-a A fixed concentration of mYfh1p (96 μm) was incubated with 24, 48, and 144 μmFe(II) for 25 min under the conditions used for electrode oximetry in Fig. 1. Iron was measured in the three indicated fractions as described under “Experimental Procedures.”1-b Mean ± S.D. from three independent experiments (μm iron). The total recovery at 144 μmFe(II) was only ∼83% in the presence of mYfh1p and 67% in buffer without protein due to incomplete solubilization of the precipitated ferric oxides by addition of dithionite. Open table in a new tab To identify the iron-binding species, duplicates of reactions Fe(II)/mYfh1p = 0.5 and Fe(II)/mYfh1p = 1.5 were incubated at 30 °C for 2 or 25 min and immediately analyzed by Superdex 200 gel filtration. In all four samples, most protein was recovered in peak α, corresponding to mYfh1p monomer (Fig.2). Inductively coupled plasma emission spectroscopy analysis demonstrated that peak α did not contain any significant levels of iron as previously reported (23Adamec 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 (229) Google Scholar, 24Gakh O. Adamec J. Gacy M.A. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (113) Google Scholar). An iron-containing peak with an apparent molecular mass of ∼50 kDa, corresponding to the α3 oligomer (23Adamec 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 (229) Google Scholar), was observed in both reactions at each time point analyzed (Fig. 2). At 25 min, the Fe(III)/mYfh1p ratio in peak α3 was 0.6 for reaction Fe(II)/mYfh1p = 0.5 and 1.7 for reaction Fe(II)/mYfh1p = 1.5. These ratios are close to those determined by ultrafiltration (see above), suggesting that α3 is the iron-binding species present at the end of the iron oxidation reaction. Consistent with this interpretation, in Fig. 2 there is a small decrease in theA 280 of peak α from 2 to 25 min, and a concomitant increase in the A 280 of peak α3 that is not completely accounted for by the decrease in peak α. Nearly identical chromatograms were obtained in two independent gel filtration analyses, one of which is shown in Fig. 2, indicating that these results are reproducible. The time-dependent increase in peak α3 intensity can therefore be explained by progressive conversion of monomer to α3 and, consistent with the time course of O2consumption in Fig. 1 A, by progressive deposition of iron oxides inside α3. These data support the conclusion that α3 forms during, and represents the main iron-binding species at the completion of, reactions Fe(II)/mYfh1p = 0.5 and Fe(II)/mYfh1p = 1.5. However, when the completed reactions are analyzed by gel filtration, peak α3 accounts for only a small fraction of the total iron and protein, whereas most protein is eluted from the Superdex 200 column as iron-free monomer (Fig. 2). This suggests that α3 disassembles during gel filtration because it is not stable. By analogy with H-ferritin (26Yan" @default.
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