The Heme Synthesis Defect of Mutants Impaired in Mitochondrial Iron-Sulfur Protein Biogenesis Is Caused by Reversible Inhibition of Ferrochelatase

Mitochondria are responsible for the synthesis of both iron-sulfur clusters and heme, but the potential connection between the two major iron-consuming pathways is unknown. Here, we have shown that mutants in the yeast mitochondrial iron-sulfur cluster (ISC) assembly machinery displayed reduced cytochrome levels and diminished activity of the heme-containing cytochrome c oxidase, in addition to iron-sulfur protein defects. In contrast, mutants in components of the mitochondrial ISC export machinery, which are specifically required for maturation of cytosolic iron-sulfur proteins, were not decreased in heme synthesis or cytochrome levels. Heme synthesis does not involve the function of mitochondrial ISC components, because immunological depletion of various ISC proteins from mitochondrial extracts did not affect the formation and amounts of heme. The heme synthesis defects of ISC mutants were found in vivo in isolated mitochondria and in mitochondrial detergent extracts and were confined to an inhibition of ferrochelatase, the enzyme catalyzing the insertion of iron into protoporphyrin IX. In support of these findings, immunopurification of ferrochelatase from ISC mutants restored its activity to wild-type levels. We conclude that the reversible inhibition of ferrochelatase is the molecular reason for the heme deficiency in ISC assembly mutants. This inhibitory mechanism may be used for regulation of iron distribution between the two iron-consuming processes. Mitochondria are responsible for the synthesis of both iron-sulfur clusters and heme, but the potential connection between the two major iron-consuming pathways is unknown. Here, we have shown that mutants in the yeast mitochondrial iron-sulfur cluster (ISC) assembly machinery displayed reduced cytochrome levels and diminished activity of the heme-containing cytochrome c oxidase, in addition to iron-sulfur protein defects. In contrast, mutants in components of the mitochondrial ISC export machinery, which are specifically required for maturation of cytosolic iron-sulfur proteins, were not decreased in heme synthesis or cytochrome levels. Heme synthesis does not involve the function of mitochondrial ISC components, because immunological depletion of various ISC proteins from mitochondrial extracts did not affect the formation and amounts of heme. The heme synthesis defects of ISC mutants were found in vivo in isolated mitochondria and in mitochondrial detergent extracts and were confined to an inhibition of ferrochelatase, the enzyme catalyzing the insertion of iron into protoporphyrin IX. In support of these findings, immunopurification of ferrochelatase from ISC mutants restored its activity to wild-type levels. We conclude that the reversible inhibition of ferrochelatase is the molecular reason for the heme deficiency in ISC assembly mutants. This inhibitory mechanism may be used for regulation of iron distribution between the two iron-consuming processes. Most of the iron acquired by eukaryotic cells is consumed by two major pathways, namely the synthesis of iron-sulfur (Fe/S) 1The abbreviations used are: Fe/S, iron-sulfur; ISC, iron-sulfur cluster; XLSA/A, X-linked sideroblastic anemia and cerebellar ataxia; yfh, yeast frataxin homologue.1The abbreviations used are: Fe/S, iron-sulfur; ISC, iron-sulfur cluster; XLSA/A, X-linked sideroblastic anemia and cerebellar ataxia; yfh, yeast frataxin homologue. clusters and of heme. Both processes are performed by mitochondria and require the energy-dependent transport of reduced iron across the mitochondrial inner membrane (1Lange H. Kispal G. Lill R. J. Biol. Chem. 1999; 274: 18989-18996Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 2Mühlenhoff U. Richhardt N. Gerber J. Lill R. J. Biol. Chem. 2002; 277: 29810-29816Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Heme synthesis is rather complex, involving several mitochondrial and cytosolic enzymes that initially convert δ-aminolevulinate to protoporphyrin IX (for review, see Ref. 3Labbe-Bois R. Labbe P. Dailey H.A. Biosynthesis of Heme and Chlorophylls. McGraw-Hill, New York1990: 235-285Google Scholar). The final step of the pathway is the incorporation of ferrous iron into the porphyrin ring and is catalyzed by ferrochelatase (Hem15p in yeast) that is located at the matrix side of the mitochondrial inner membrane (4Labbe-Bois R. Camadro J.M. Winkelmann G. Winge D.R. Metal Ions in Fungi. Marcel Dekker Inc., New York1994: 413-453Google Scholar, 5Dailey H.A. Biochem. Soc. Trans. 2002; 30: 590-595Crossref PubMed Scopus (88) Google Scholar).Iron incorporation into Fe/S clusters and their insertion into apoproteins is accomplished by the Fe/S cluster (ISC) assembly machinery located in the mitochondrial matrix (for review, see Refs. 6Lill R. Kispal G. Trends Biochem. Sci. 2000; 25: 352-356Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 7Mühlenhoff U. Lill R. Biochim. Biophys. Acta. 2000; 1459: 370-382Crossref PubMed Scopus (178) Google Scholar, 8Craig E.A. Marszalek J. Cell. Mol. Life Sci. 2002; 59: 1658-1665Crossref PubMed Scopus (85) Google Scholar, 9Frazzon J. Dean D.R. Curr. Opin. Chem. Biol. 2003; 7: 166-173Crossref PubMed Scopus (183) Google Scholar). Fe/S cluster synthesis starts with the release of sulfur from cysteine by the cysteine desulfurase Nfs1p and the assembly of a transient Fe/S cluster on the scaffold proteins, Isu1p and Isu2p (10Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (496) Google Scholar, 11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar, 12Schilke B. Voisine C. Beinert H. Craig E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10206-10211Crossref PubMed Scopus (265) Google Scholar, 13Agar J.N. Zheng L. Cash V.L. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (113) Google Scholar). A similar function may be performed by Isa1p and Isa2p as judged from biochemical investigation of their bacterial homologue, iscA (14Krebs C. Agar J.N. Smith A.D. Frazzon J. Dean D.R. Huynh B.H. Johnson M.K. 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EMBO J. 2003; 22: 4815-4825Crossref PubMed Scopus (331) Google Scholar, 23Dutkiewicz R. Schilke B. Kneiszner H. Walter W. Craig E.A. Marszalek J. J. Biol. Chem. 2003; 278: 29719-29727Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 24Rodriguez-Manzaneque M.T. Tamarit J. Belli G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (390) Google Scholar).The mitochondrial ISC assembly machinery is also involved in the maturation of Fe/S proteins outside the organelle (11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar). Mitochondria were proposed to export a still unknown component to the cytosol to support the formation of extramitochondrial Fe/S proteins (6Lill R. Kispal G. Trends Biochem. Sci. 2000; 25: 352-356Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Central components of the export pathway are the ABC transporter Atm1p (or its human orthologue ABC7) of the mitochondrial inner membrane and the sulfhydryl oxidase Erv1p/ALR of the intermembrane space (11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar, 25Bekri S. Kispal G. Lange H. Fitzsimons E. Tolmie J. Lill R. Bishop D.F. Blood. 2000; 96: 3256-3264Crossref PubMed Google Scholar, 26Lange H. Lisowsky T. Gerber J. Mühlenhoff U. Kispal G. Lill R. EMBO Rep. 2001; 2: 715-720Crossref PubMed Scopus (247) Google Scholar). In addition, the tripeptide glutathione was found to be required specifically in Fe/S protein maturation in the cytosol (27Sipos K. Lange H. Fekete Z. Ullmann P. Lill R. Kispal G. J. Biol. Chem. 2002; 277: 26944-26949Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar).Apart from the impairment of Fe/S cluster biosynthesis, several other defects have been reported to be caused by mutation or depletion of Isc proteins in yeast. For example, mitochondrial iron accumulation, reduced cytochrome levels, impaired heme synthesis, increased sensitivity to oxidative stress, and loss of mitochondrial DNA have been observed in various yeast "isc" mutants (i.e. mutants in components of the ISC assembly machinery) (11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar, 18Lange H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (241) Google Scholar, 28Wilson R.B. Roof D.M. Nat. Genet. 1997; 16: 352-357Crossref PubMed Scopus (323) Google Scholar, 29Kispal G. Csere P. Guiard B. Lill R. 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It is noteworthy that some of these effects are prominent phenotypic markers of two ISC assembly diseases in which complex symptoms are caused by frataxin depletion in Friedreich′s ataxia and ABC7 mutation in X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A) (25Bekri S. Kispal G. Lange H. Fitzsimons E. Tolmie J. Lill R. Bishop D.F. Blood. 2000; 96: 3256-3264Crossref PubMed Google Scholar, 36Rötig A. de Lonlay P. Chretien D. Foury F. Koenig M. Sidi D. Munnich A. Rustin P. Nat. Genetics. 1997; 17: 215-217Crossref PubMed Scopus (866) Google Scholar, 37Bradley J.L. Blake J.C. Chamberlain S. Thomas P.K. Cooper J.M. Schapira A.H. Hum. Mol. Genet. 2000; 9: 275-282Crossref PubMed Scopus (300) Google Scholar, 38Lill R. Kispal G. Gruson F. The NORD Guide to Rare Disorders. Lippincott, Williams & Wilkins, Philadelphia2003: 374-375Google Scholar, 39Puccio 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 (587) Google Scholar, 40Wilson R.B. J. Neurol. Sci. 2003; 207: 103-105Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Most of these effects are thought to be secondary or late consequences of the original defect in Fe/S protein maturation. In particular, this was shown for iron accumulation in a conditional frataxin gene knock-out mouse (41Cossee M. Puccio H. Gansmuller A. Koutnikova H. Dierich A. LeMeur M. Fischbeck K. Dolle P. Koenig M. Hum. Mol. Genet. 2000; 9: 1219-1226Crossref PubMed Scopus (302) Google Scholar). On the other hand, the observation of reduced cytochrome levels in Δyfh1 mutants raises the question of whether this ISC component might also play a role in heme synthesis in yeast (35Lesuisse E. Santos R. Matzanke B.F. Knight S.A. Camadro J.M. Dancis A. Hum. Mol. Genet. 2003; 12: 879-889Crossref PubMed Scopus (214) Google Scholar).We became interested in the potential connection between heme and Fe/S protein synthesis because nearly nothing is known about the mechanisms that allow a cell to direct iron into either of these two processes. A mutual cross-talk between both biochemical pathways at a posttranslational level seems reasonable because some growth conditions require a coordinated synthesis of both cofactors, whereas other situations may demand a different ratio in the synthesis of Fe/S clusters and heme. A link between heme biosynthesis and iron uptake into the cell has been reported recently (42Crisp R.J. Pollington A. Galea C. Jaron S. Yamiguchi-Iwai Y. Kaplan J. J. Biol. Chem. 2003; 278: 45499-45506Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Because mutants defective in ISC assembly greatly increase their cellular iron uptake, this may provide another argument for the proposed connection between heme and Fe/S metabolism. To test these ideas experimentally, we initiated a systematic study and investigated cytochrome levels and heme biosynthesis rates in a broad range of isc mutants that carry defects in certain steps of Fe/S protein biosynthesis. Our results indicate that cells disrupted in any step of mitochondrial Fe/S protein biosynthesis hamper iron incorporation into heme by reversible inhibition of ferrochelatase. This cross-talk between Fe/S protein and heme biosynthesis might be used for regulating the iron distribution between these two processes in mitochondria.MATERIALS AND METHODSYeast Strains and Cell Growth—The following yeast strains were used: W303a (Mata; ade2–1 ura3–1 trp1–1 his3–11,15 leu2–3,111), which served as wild-type. Published mutant strains in which the endogenous promoters of the indicated ISC genes were replaced by the galactose-inducible GAL1–10 promoter were Gal-YAH1 (18Lange H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (241) Google Scholar), Gal-NFS1 (11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar), Gal-ISU1/Δisu2 (20Gerber J. Mühlenhoff U. Lill R. EMBO Rep. 2003; 4: 906-911Crossref PubMed Scopus (309) Google Scholar), Gal-YFH1 (43Mühlenhoff U. Richhardt N. Ristow M. Kispal G. Lill R. Hum. Mol. Genet. 2002; 11: 2025-2036Crossref PubMed Scopus (285) Google Scholar), Gal-ISA1, Gal-JAC1, and Gal-SSQ1 (19Mühlenhoff U. Gerber J. Richhardt N. Lill R. EMBO J. 2003; 22: 4815-4825Crossref PubMed Scopus (331) Google Scholar). We referred to these strains as "isc" mutants. Further, we used Gal-ATM1 (11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar), Δgsh1 (27Sipos K. Lange H. Fekete Z. Ullmann P. Lill R. Kispal G. J. Biol. Chem. 2002; 277: 26944-26949Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), and erv1ts (26Lange H. Lisowsky T. Gerber J. Mühlenhoff U. Kispal G. Lill R. EMBO Rep. 2001; 2: 715-720Crossref PubMed Scopus (247) Google Scholar) cells. All cells were cultivated on galactose-containing rich medium agar plates (44Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2530) Google Scholar). To deplete cells of an Isc protein, the respective strain (precultured on glucose-containing rich medium, when necessary) was grown overnight in minimal or rich medium containing 2% glucose followed by 16 h of growth in minimal medium lacking iron chloride (for 55Fe labeling) or in lactate medium (for preparation of mitochondria). Depletion was routinely verified by immunostaining analysis. Details of the depletion procedure for each Isc protein are given in the publications describing the respective mutant cells (see above).Preparation of Intact Mitochondria and Mitochondrial Detergent Extracts—Mitochondria were prepared as described (45Diekert K. deKroon A.I.P.M. Kispal G. Lill R. Methods Cell Biol. 2001; 65: 37-51Crossref PubMed Google Scholar) from cultures grown overnight in lactate medium containing 0.1% glucose. Lysis of isolated mitochondria was performed at a protein concentration of 2 mg/ml in buffer D (1% dodecylmaltoside in 20 mm Hepes/KOH, pH 7.4, 150 mm NaCl, 10% glycerol) for 30 min at 4 °C. Aggregates were removed by centrifugation (10,000 rpm, 10 min, 4 °C).Determination of Heme Synthesis in Organello and in Vitro—50 μg of intact mitochondria, 25 μl of detergent extract corresponding to 50 μg of mitochondrial protein or immunopurified ferrochelatase derived from 50 μg of mitochondria (see below) were used per assay. All samples were analyzed in a 500-μl standard heme synthesis assay as described (1Lange H. Kispal G. Lill R. J. Biol. Chem. 1999; 274: 18989-18996Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) before using buffer A (20 mm Hepes/KOH, pH 7.4, 50 mm NaCl, 0.5 mm NADH, and 0.2 μm deuteroporphyrin). Samples containing intact organelles were supplemented with 0.6 m sorbitol. Heme synthesis reactions were started by adding 5 μl of 100 mm ascorbate and 0.1 μCi of 55FeCl3. After 5 min at 25 °C, reactions were stopped with 25 μl of 10 mm FeCl3 in 5 m HCl and extracted with 500 μl of butyl acetate. 200 μl of the organic phase were used for scintillation counting. Zinc chelatase activity was measured in mitochondrial detergent extracts by a fluorescence assay kindly provided by R. Labbe-Bois (35Lesuisse E. Santos R. Matzanke B.F. Knight S.A. Camadro J.M. Dancis A. Hum. Mol. Genet. 2003; 12: 879-889Crossref PubMed Scopus (214) Google Scholar, 46Camadro J.M. Labbe P. J. Biol. Chem. 1988; 263: 11675-11682Abstract Full Text PDF PubMed Google Scholar).Immunopurification of Ferrochelatase from Mitochondrial Extracts— Antibodies raised against Hem15p were coupled to protein A-Sepharose beads as described (47Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1998Google Scholar). 100 μl of mitochondrial extract were incubated for 30 min with 50 μl of a 50% suspension of protein A-Sepharose coupled to antisera raised against ferrochelatase (Hem15p). Immunobeads were collected by centrifugation, washed three times with buffer B (20 mm Hepes/KOH, pH 7.5, 150 mm NaCl), and eluted with sample buffer (for SDS-PAGE) or resuspended in 100 μl of buffer D for measuring ferrochelatase activity. 25 μl of this suspension corresponding to 50 μg of intact mitochondria as starting material were used to measure ferrochelatase activity. Aliquots of the supernatants and immunobeads were analyzed for depletion of ferrochelatase by SDS-PAGE and immunostaining.Biochemical depletion of Isc proteins was carried out by incubating wild-type mitochondrial extracts for 1 h at 4 °C charged with preimmune serum or with specific antibodies raised against Nfs1p, Isu1p, Yah1p, or Yfh1p. Beads were collected by centrifugation. Aliquots of the supernatants were analyzed for depletion of Isc proteins by immunostaining and for remaining ferrochelatase activity.Crude Fractionation of Mitochondrial detergent extracts prepared from wild-type or isc mutant cells were for endogenous ferrochelatase protein as described were by a membrane and by centrifugation for min to the following were used: of DNA and J. A Laboratory Manual. Cold Spring Harbor Cold Spring Harbor, Scholar), of yeast cells D. A. R.H. 20: PubMed Scopus Google Scholar), of antibodies (47Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1998Google Scholar), of cytochrome c oxidase of cytochrome B. EMBO J. 4: PubMed Scopus Google Scholar, Methods PubMed Scopus Google Scholar), iron of isolated mitochondria J. M. Knight S.A. Pain D. Dancis A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), in vivo of yeast cells with iron and of 55Fe incorporation into and into heme (1Lange H. Kispal G. Lill R. J. Biol. Chem. 1999; 274: 18989-18996Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar, U. Richhardt N. Ristow M. Kispal G. Lill R. Hum. Mol. Genet. 2002; 11: 2025-2036Crossref PubMed Scopus (285) Google in isc by Heme a connection between the two pathways, we measured the mitochondrial cytochrome and the heme synthesis rates in a of isc mutants. We used yeast ISC assembly mutant cells isc in which the endogenous promoter of the ISC genes was replaced by the GAL1–10 synthesis of the Isc proteins in the and their depletion in the of After mitochondria were isolated and the specific of the respective Isc proteins was by immunostaining isolated mitochondria were used for cytochrome A of the mitochondrial cytochrome levels was in mutants In the of Yah1p and the was In mitochondria isolated from the mutants Gal-JAC1, and Gal-ISA1, were reduced but still we measured the activity of cytochrome c oxidase, a mitochondrial enzyme that on heme as a but does not Fe/S c oxidase activity was reduced to a similar in isc mutants are with results by that a loss of in Δyfh1 cells (35Lesuisse E. Santos R. Matzanke B.F. Knight S.A. Camadro J.M. Dancis A. Hum. Mol. Genet. 2003; 12: 879-889Crossref PubMed Scopus (214) Google Scholar). this was not confined to this ISC assembly component but was observed any synthesis step of mitochondrial Fe/S In to these cytochrome levels and cytochrome c oxidase activity were of Atm1p or components of the ISC export machinery and not is the reason for the observed of and the loss of cytochrome c oxidase activity in isc To this we the de heme formation and Fe/S protein synthesis in Isc mutant cells were grown overnight in minimal medium followed by with heme was measured by organic of cell (1Lange H. Kispal G. Lill R. J. Biol. Chem. 1999; 274: 18989-18996Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). of the cell suspension was used to an with antibodies raised against the cytosolic Fe/S protein as a of cellular Fe/S protein synthesis (11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar). incorporation into heme and was by scintillation counting. of Yah1p, Nfs1p, or Isu1p displayed a to 55Fe into both heme and Fe/S a heme synthesis defect in addition to the impairment of Fe/S protein maturation In contrast, cells in components of the ISC machinery, namely and were in heme synthesis these This that Fe/S protein synthesis is the function of these ISC components and that the defect in heme synthesis is a secondary (43Mühlenhoff U. Richhardt N. Ristow M. Kispal G. Lill R. Hum. Mol. Genet. 2002; 11: 2025-2036Crossref PubMed Scopus (285) Google Scholar). It is to in this that the of the in vivo heme synthesis defect on the level of depletion of the respective ISC components and on the growth The different conditions used for 55Fe conditions and for mitochondria may the in heme levels observed in and cells in vivo and in mutants defects in de heme formation in were of the indicated Isc proteins or Atm1p and were grown overnight in minimal medium containing glucose. After with a cell was prepared and used for with antibodies against 55Fe was by scintillation and served as a direct measure for iron into this cytosolic Fe/S protein (11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar). A of the cell suspension was used for organic of heme, which was by scintillation counting. are given to for wild-type cells. the of the in or glutathione wild-type amounts of 55Fe into heme 2 and not mutants defective in cytosolic Fe/S protein but in mitochondrial synthesis of Fe/S clusters (11Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (581) Google Scholar, 26Lange H. Lisowsky T. Gerber J. Mühlenhoff U. Kispal G. Lill R. EMBO Rep. 2001; 2: 715-720Crossref PubMed Scopus (247) Google Scholar, K. Lange H. Fekete Z. Ullmann P. Lill R. Kispal G. J. Biol. Chem. 2002; 277: 26944-26949Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), were not impaired in heme synthesis, that cytochrome loss and heme synthesis deficiency are to mutants in the mitochondrial ISC assembly these that the reduced cytochrome of isc mutants at to a the of reduced heme any of Fe/S cluster synthesis in the mitochondrial matrix is followed by a defect in heme of the Heme Synthesis in isc which step of the complex heme biosynthesis pathway might be in the isc mutants. we isolated mitochondria from Isc yeast mutants and the of the intact organelles to heme from 55Fe and protoporphyrin IX Ref. H. Kispal G. Lill R. J. Biol. Chem. 1999; 274: 18989-18996Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Mitochondria isolated from isc mutants levels of as with wild-type or organelles these the amounts of ferrochelatase protein were similar in as by immunostaining that the heme defects specifically observed in the isc mutants may be caused by an impaired of porphyrin to in the ISC assembly machinery affect the step in heme Mitochondria were isolated from wild-type or the indicated isc mutant strains as described for Lysis of mitochondria was performed at a protein