Probing a Role of Subunit IV of the  -type Ubiquinol Oxidase by Deletion and Cross-linking Analyses

Saiki, Keitarou; Nakamura, H.; Mogi, Tatsushi; Anraku, Yasuhiro

doi:10.1074/jbc.271.26.15336

Cited by 75 publications

(42 citation statements)

References 54 publications

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“…Such mutants are also severely deficient in heme A but not heme O, which accumulates to different extents depending on the mutant (6). 2 The increase of heme O in cytochrome oxidase mutants was taken advantage of to determine whether the heme A content could be raised by overexpression of Cox15 and/or ferredoxin. The concentration of heme A was not significantly affected by ferredoxin but in some cases could be increased by as much as 40-fold in mutants transformed with COX15 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Heme A differs from heme B (protoheme) at the porphyrin carbons C-2, where a vinyl group is replaced by a farnesyl group, and at C-8, where there is a formyl instead of a methyl substituent (1). The first step in heme A biosynthesis is a farnesylation of the vinyl at C-2 of heme B (2). In Saccharomyces cerevisiae this reaction, resulting in heme O, is catalyzed by a mitochondrial farnesyl transferase encoded by COX10 1 (3,4).…”

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confidence: 99%

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Mitochondrial Ferredoxin Is Required for Heme A Synthesis inSaccharomyces cerevisiae

Barros¹,

Nóbrega²,

Tzagoloff³

2002

Journal of Biological Chemistry

100

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Heme A is a prosthetic group confined exclusively to mitochondrial and some bacterial cytochrome oxidases. Heme A differs from heme B (protoheme) at the porphyrin carbons C-2, where a vinyl group is replaced by a farnesyl group, and at C-8, where there is a formyl instead of a methyl substituent (1). The first step in heme A biosynthesis is a farnesylation of the vinyl at C-2 of heme B (2). In Saccharomyces cerevisiae this reaction, resulting in heme O, is catalyzed by a mitochondrial farnesyl transferase encoded by COX10 1 (3, 4). Heme O itself is a prosthetic group in some bacterial cytochrome oxidases (5). The further conversion of heme O to heme A probably involves a monooxygenase-catalyzed hydroxylation of the methyl group at C-8. The resultant alcohol would then be further oxidized to the aldehyde.In a screen of cytochrome oxidase-deficient yeast strains for lesions in heme A biosynthesis, only cox15 mutants were found to have heme O but not heme A. This observation led us to propose that Cox15p catalyzes the hydroxylation of the C-8 methyl group (6). According to this mechanism, oxidation of the methyl to the alcohol is catalyzed by a three-component monooxygenase that includes Cox15p, mitochondrial ferredoxin, and ferredoxin reductase, encoded by YAH1 and ARH1, respectively (7,8).In the present study we have further assessed the role of ferredoxin in heme A synthesis. Temperature-sensitive yah1 mutants have been used to measure heme A synthesis over a range of temperatures that elicit the increasing loss of ferredoxin activity. The ts mutants have also been used to measure the incorporation of ␦-aminolevulinic acid into heme A following the shift to the non-permissive temperature. Finally, we have examined the effect of overexpression of ferredoxin and Cox15p singly or in combination on the heme composition of various yeast mutants. The results of these studies strongly support the involvement of ferredoxin and by inference of ferredoxin reductase in heme A synthesis. MATERIALS AND METHODSYeast Strains and Media-The genotypes and sources of the strains of S. cerevisiae used in this study are listed in Table I. The following media were used to grow yeast YPD (2% glucose, 2% peptone, 1% yeast extract), YEPG (3% glycerol, 2% ethanol, 2% peptone, 1% yeast extract), and YPGal (2% galactose, 2% peptone, 1% yeast extract). Solid media contained 2% agar.Extraction and Separation of Mitochondrial Hemes-Mitochondria prepared as described previously (13) were suspended in 0.5 M sorbitol, 20 mM Tris-HCl (pH 7.5), and 0.5 mM EDTA at a protein concentration of 20 mg/ml. Total heme was extracted from 2 mg of mitochondrial protein with 0.5 ml of acetone containing 2.5% HCl. The mixture was vortexed, clarified by centrifugation, and mixed with an equal volume of 50% acetonitrile. Insoluble material was removed by a second centrifugation. The extract was adjusted to approximately pH 3.5 with 1.65 M ammonium hydroxide, clarified by centrifugation, and applied to a 3.9 ϫ 300 mm C18 Bondclone column (Phenomenex, Torrance, CA...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Mitochondrial Ferredoxin Is Required for Heme A Synthesis inSaccharomyces cerevisiae

Barros¹,

Nóbrega²,

Tzagoloff³

2002

Journal of Biological Chemistry

100

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“…The synthesis of heme A begins with farnesylation of a vinyl group at carbon C-2 of the porphyrin ring of heme B (protoheme) by the product of the COX10 gene. [1,2]. Conversion of the resultant heme O to heme A requires a further oxidation of the methyl group at C-8 to a formyl group [3].…”

Section: Introductionmentioning

confidence: 99%

Regulation of the heme A biosynthetic pathway in Saccharomyces cerevisiae

Barros

Tzagoloff

2002

FEBS Letters

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Biosynthesis of heme A, a prosthetic group of cytochrome oxidase (COX), involves an initial farnesylation of heme B. The heme O product formed in this reaction is modified by hydroxylation of the methyl group at carbon C-8 of the porphyrin ring. This reaction was proposed to be catalyzed by Cox15p, ferredoxin, and ferredoxin reductase. Oxidation of the alcohol to the corresponding aldehyde yields heme A. In the present study we have assayed heme A and heme O in yeast COX mutants

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“…COX10 is a farnesyltransferase that catalyzes the conversion of heme B (ferric protoporphyrin IX) to heme O (33,34). Heme O is subsequently converted into heme A in a biosynthetic pathway involving COX15 (35).…”

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confidence: 99%

Cytochrome c Oxidase Subassemblies in Fibroblast Cultures from Patients Carrying Mutations in COX10, SCO1, or SURF1

Williams

Valnot

Rustin

et al. 2004

Journal of Biological Chemistry

127

135

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Cytochrome c oxidase contains two redox-active copper centers (Cu A and Cu B ) and two redox-active heme A moieties. Assembly of the enzyme relies on several assembly factors in addition to the constituent subunits and prosthetic groups. We studied fibroblast cultures from patients carrying mutations in the assembly factors COX10, SCO1, or SURF1. COX10 is involved in heme A biosynthesis. SCO1 is required for formation of the Cu A center. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated severely decreased levels of holoenzyme in the patient cultures compared with controls. In addition, the blots revealed the presence of five subassemblies: three subassemblies involving the core subunit MTCO1 but apparently no other subunits; a subassembly containing subunits MTCO1, COX4, and COX5A; and a subassembly containing at least subunits MTCO1, MTCO2, MTCO3, COX4, and COX5A. As some of the subassemblies correspond to known assembly intermediates of human cytochrome c oxidase, we think that these subassemblies are probably assembly intermediates that accumulate in patient cells. The MTCO1⅐COX4⅐COX5A subassembly was not detected in COX10-deficient cells, which suggests that heme A incorporation into MTCO1 occurs prior to association of MTCO1 with COX4 and COX5A. SCO1-deficient cells contained accumulated levels of the MTCO1⅐COX4⅐COX5A subassembly, suggesting that MTCO2 associates with the MTCO1⅐COX4⅐COX5A subassembly after the Cu A center of MTCO2 is formed. Assembly in SURF1-deficient cells appears to stall at the same stage as in SCO1-deficient cells, pointing to a role for SURF1 in promoting the association of MTCO2 with the MTCO1⅐COX4⅐COX5A subassembly.

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Probing a Role of Subunit IV of the -type Ubiquinol Oxidase by Deletion and Cross-linking Analyses

Cited by 75 publications

References 54 publications

Mitochondrial Ferredoxin Is Required for Heme A Synthesis inSaccharomyces cerevisiae

Mitochondrial Ferredoxin Is Required for Heme A Synthesis inSaccharomyces cerevisiae

Regulation of the heme A biosynthetic pathway in Saccharomyces cerevisiae

Cytochrome c Oxidase Subassemblies in Fibroblast Cultures from Patients Carrying Mutations in COX10, SCO1, or SURF1

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