Electron-transfer complexes of Ascaris suum muscle mitochondria. II. Succinate-coenzyme Q reductase (complex II) associated with substrate-reducible cytochrome b-558

Takamiya, Shinzaburo; Furushima, Rieko; Okumura, Hiroshi

doi:10.1016/0005-2728(86)90165-9

Cited by 50 publications

(16 citation statements)

References 31 publications

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“…B. subtilis complex II contains two hemes b, and the heme with the higher potential (E m Ј ϭ ϩ65 mV) is reduced by succinate, while the heme with the lower potential (E m Ј ϭ Ϫ95 mV) is not (19,20). E. coli and Ascaris suum complex II have one heme b in complex II, and the heme b in E. coli complex II (E m Ј ϭ ϩ36 mV) is fully reducible by succinate (6), while the heme b in A. suum complex II (E m Ј ϭ Ϫ34 mV) is partially reduced by succinate (21,22). Bovine cytochrome b 560 (E m Ј ϭ Ϫ185 mV) is not readily reducible by succinate, and low substoichiometric amounts of heme b are found in this enzyme complex (23).…”

mentioning

confidence: 99%

Two Hydrophobic Subunits Are Essential for the Heme b Ligation and Functional Assembly of Complex II (Succinate-Ubiquinone Oxidoreductase) from Escherichia coli

Nakamura¹,

Yamaki²,

Sarada³

et al. 1996

Journal of Biological Chemistry

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Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b 556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b 556 . An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b 556 in its CO reactivity and red shift of the ␣ absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinateubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b 556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquinone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b 556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.Complex II (succinate-ubiquinone oxidoreductase) is a tricarboxylic acid cycle enzyme associated with the inner membrane of mitochondria or the cytoplasmic membrane of bacteria (1, 2). Complex II in Escherichia coli is encoded by the sdhCDAB operon (3, 4) and contains four nonidentical subunits and five prosthetic groups (5, 6). The largest hydrophilic subunit (flavoprotein; Fp), 1 encoded by sdhA, has covalently bound FAD, and the second largest subunit (iron-sulfur protein; Ip), encoded by sdhB, contains three different types of iron-sulfur clusters termed S-1[2Fe-2S], S-2[4Fe-4S], and S-3[3Fe-4S]. Two hydrophobic heme b-containing subunits are encoded by sdhC and sdhD, and at least the sdhC product is a component of cytochrome b 556 (7).Generally, the Fp and Ip subunits comprise the catalytic portion of complex II and catalyze electron transfer from suc...

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mentioning

confidence: 99%

Two Hydrophobic Subunits Are Essential for the Heme b Ligation and Functional Assembly of Complex II (Succinate-Ubiquinone Oxidoreductase) from Escherichia coli

Nakamura¹,

Yamaki²,

Sarada³

et al. 1996

Journal of Biological Chemistry

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“…Complex II was purified from Ascaris mitochondria as described by Takamiya et al [3][4][5]. Purified complex II was treated with 20% (w/v) sodium dodecyl sulfate (SDS) and then subjected to gel chromatography on a Biogel P-60 (<400) column equilibrated with 2°70 (w/v) SDS [16].…”

Section: Purification Of the Fp Subunit From Ascaris Complex Hmentioning

confidence: 99%

“…We have isolated complex II from muscle mitochondria of adult worms of Ascaris [3][4][5][6], inhabitants of the host's small intestine where the oxygen 'supply is limited. Ascaris complex II, unlike its mammalian counterparts, functions physiologically as the terminal oxidase in the NADH-fumarate reductase system, an anaerobic respiratory chain [3,5].…”

Section: Introductionmentioning

confidence: 99%

“…Ascaris complex II, unlike its mammalian counterparts, functions physiologically as the terminal oxidase in the NADH-fumarate reductase system, an anaerobic respiratory chain [3,5]. Indeed, Ascaris complex II exhibits high fumarate reductase (FRD) activity over succinate-ubiquinone oxidoreductase (SDH) activity (SDH/FRD = 0.05) [5].…”

Section: Introductionmentioning

confidence: 99%

“…Indeed, Ascaris complex II exhibits high fumarate reductase (FRD) activity over succinate-ubiquinone oxidoreductase (SDH) activity (SDH/FRD = 0.05) [5]. It is a major component of Ascaris mitochondria (8o70 of mitochondrial protein) [7] and is composed of the following four subunits: a flavoprotein (Fp) subunit containing a flavin (Mr= 68 kDa), an Ip subunit associated with iron-sulfur centers (Mr = 26 kDa) and two hydrophobic, heme bcontaining polypeptides called CybL and Cybs (Mr = 15 and 13.5 kDa, respectively) [3,5]. In the case of bacteria, the genes of complex II have been cloned and sequenced from Escherichia coil (sdh and frd) [8][9][10], Bacillus subtilis (sdh ) [11,12] and Proteus vulgaris (frd) [13].…”

Section: Introductionmentioning

confidence: 99%

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Structural studies on three flavin‐interacting regions of the flavoprotein subunit of complex II in Ascaris suum mitochondria

et al. 1990

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The flavoprotein (Fp) subunit of mitochondrial complex II contains covalently bound FAD as a prosthetic group. In this study, the primary structure of the flavin-bound tryptic peptide from the Fp subunit of Ascaris complex II was determined and found to be highly similar to those of the corresponding flavin-binding regions of bovine heart and bacterial Fp subunits. Furthermore, the Ascaris Fp subunit was shown to contain two regions exhibiting striking sequence similarity to the segments that have been predicted to interact noncovalently with the AMP moiety of FAD in bacterial Fp subunits. The conservation of these two regions also in the mitochondrial Fp subunit suggests their functional importance.

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cDNA sequence of three cysteine‐rich clusters in the iron‐sulfur subunit of complex II (succinate‐ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer

et al. 1992

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Homology probing by using mixed primers for polymerase chain reaction (PCR) and a subsequent sequence analysis by automated DNA sequencer were applied to determine a partial cDNA sequence of the iron-sulfur subunit of complex II (succinate-ubiquinone oxidoreductase). Complex II is a membrane-bound flavoenzyme, which catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle, and it is a component of the mitochondrial and bacterial respiratory chains. In this study, the partial amino acid sequence of iron-sulfur subunits in Caenorhabditis elegans mitochondria was deduced from the DNA sequence obtained from cDNA-PCR. Mixed oligonucleotide primers corresponding to two conserved regions which appear to be the binding site for the prosthetic group were used. The product of PCR was cloned into plasmid vector pUC 119 and the sequence was determined from double strand plasmid DNA by the dideoxy method using of one-dye, four-lane type the automated DNA sequencer (DSQ-1, Shimadzu). The PCR product contained 483 nucleotides and its deduced amino acid sequence was highly homologous with that in human liver (68.9%) and that of Escherichia coli sdh B product (50.3%). As expected, striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme.

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Electron-transfer complexes of Ascaris suum muscle mitochondria. II. Succinate-coenzyme Q reductase (complex II) associated with substrate-reducible cytochrome b-558

Cited by 50 publications

References 31 publications

Two Hydrophobic Subunits Are Essential for the Heme b Ligation and Functional Assembly of Complex II (Succinate-Ubiquinone Oxidoreductase) from Escherichia coli

Two Hydrophobic Subunits Are Essential for the Heme b Ligation and Functional Assembly of Complex II (Succinate-Ubiquinone Oxidoreductase) from Escherichia coli

Structural studies on three flavin‐interacting regions of the flavoprotein subunit of complex II in Ascaris suum mitochondria

cDNA sequence of three cysteine‐rich clusters in the iron‐sulfur subunit of complex II (succinate‐ubiquinone oxidoreductase) from Caenorhabditis elegans determined by automated DNA sequencer

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