Identification of the axial heme ligands of cytochrome b556 in succinate: Ubiquinone oxidoreductase from Escherichia coli

Peterson, Jim; Vibat, Cecile Rose T.; Gennis, Robert B.

doi:10.1016/0014-5793(94)01189-3

Cited by 34 publications

(19 citation statements)

References 21 publications

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“…In contrast to subunits A and B, they display generally little sequence conservation except for the pattern of hydrophobic segments (19). The so-called one-polypeptide anchors, e.g., those of B. subtilis SQR (18) and W. succinogenes QFR (32), show a common motif of five predicted transmembrane ␣-helical segments, whereas both polypeptides of two-polypeptide anchors, e.g., E. coli SdhC/D and FrdC/D, contain three predicted transmembrane segments (44). Hydrophobicity analysis of the S. acidocaldarius SdhC and SdhD subunits is depicted in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, the hydrophobic subunits of many organisms contain one (e.g., E. coli SQR [44]) or two (e.g., B. subtilis SQR [18]) b-type cytochromes, whereas other enzymes (e.g., E. coli QFR [13]) do not contain any b-type cytochromes. The function of prosthetic heme groups, particularly in internal electron transfer, has not been determined, but they are suggested to play a structural role in stabilizing the transmembrane helices and to be important in assembly of the enzyme complex (19,43).…”

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confidence: 99%

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A succinate dehydrogenase with novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization

1997

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The sdh operon of Sulfolobus acidocaldarius DSM 639 is composed of four genes coding for the 63.1-kDa flavoprotein (SdhA), the 36.5-kDa iron-sulfur protein (SdhB), and the 32.1-kDa SdhC and 14.1-kDa SdhD subunits. The four structural genes of the sdhABCD operon are transcribed into one polycistronic mRNA of 4.2 kb, and the transcription start was determined by the primer extension method to correspond with the first base of the ATG start codon of the sdhA gene. The S. acidocaldarius SdhA and SdhB subunits show characteristic sequence similarities to the succinate dehydrogenases and fumarate reductases of other organisms, while the SdhC and SdhD subunits, thought to form the membrane-anchoring domain, lack typical transmembrane ␣-helical regions present in all other succinate:quinone reductases (SQRs) and quinol:ifumarate reductases (QFRs) so far examined. Moreover, the SdhC subunit reveals remarkable 30% sequence similarity to the heterodisulfide reductase B subunit of Methanobacterium thermoautotrophicum and Methanococcus jannaschii, containing all 10 conserved cysteine residues. Electron paramagnetic resonance (EPR) spectroscopic studies of the purified enzyme as well as of membranes revealed the presence of typical S1 [2Fe2S] and S2 [4Fe4S] clusters, congruent with the deduced amino acid sequences. In contrast, EPR signals for a typical S3 [3Fe4S] cluster were not detected. However, EPR data together with sequence information implicate the existence of a second [4Fe4S] cluster in S. acidocaldarius rather than a typical [3Fe4S] cluster. These results and the fact that the S. acidocaldarius succinate dehydrogenase complex reveals only poor activity with caldariella quinone clearly suggest a unique structure for the SQR of S. acidocaldarius, possibly involving an electron transport pathway from the enzyme complex into the respiratory chain different from those for known SQRs and QFRs.Succinate:quinone reductase (SQR), a membrane-bound enzyme complex and component of the respiratory chain (complex II) of all aerobic organisms, catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and transfers the electrons directly to quinones in the membrane. The reverse reaction, the reduction of fumarate, which is the last step in fumarate respiration of anaerobic or facultative anaerobic organisms, is catalyzed by quinol:fumarate reductase (QFR). The two enzyme complexes are very similar with regard to composition of subunits, prosthetic groups, and probably structure (2, 24). SQRs and QFRs are composed of three or four subunits of which the largest protein is a flavoprotein (M r of 65,000 to 79,000) carrying covalently bound 8-␣-N(3)-histidyl-flavin adenine dinucleotide (FAD). An iron-sulfur protein (M r of 25,000 to 37,000) together with this flavoprotein subunit represents the peripheral part of the enzyme complex. The iron-sulfur protein is suggested to contain three different types of iron-sulfur clusters, each equimolar with FAD. Magnetic circular dichroism and electron paramagnetic resonance...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

A succinate dehydrogenase with novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization

1997

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“…The strategy for this study was in vivo complementation by a combination of sdh genes on different vectors, a method that was used successfully to investigate the assembly of E. coli FRD (33). Our recent analysis by EPR and MCD showed that the heme b 556 component of E. coli complex II is ligated to the protein by two histidine residues (38). Furthermore, His 84 in cybL and His 71 in cybS were determined to be possible heme axial ligands in cytochrome b 556 as shown using site-directed mutants.…”

Section: Discussionmentioning

confidence: 99%

“…We also have established a one-step protocol to purify complex II by chromatography using an E. coli strain that overproduces the enzyme by 10-fold due to the presence of a multicopy plasmid containing the cloned sdh operon (6). Using this preparation, bis-histidine ligation of the heme in cytochrome b 556 was demonstrated by EPR and MCD (38). However, it is not known whether cybL alone or cybL plus cybS provide coordination to the heme.…”

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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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“…The single heme in mammalian and E. coli SQR and the two hemes in B. subtilis SQR have bis-histidine axial ligation as demonstrated by EPR spectroscopy combined with lowtemperature near-infrared MCD spectroscopy [13][14][15]. The ligand histidine residues have been identified in the primary sequence of the B. subtilis and E. coli SQR anchor polypeptides by site directed mutagenesis analysis [12,16,17].…”

Section: The Cytoehrome B Componentmentioning

confidence: 99%

A structural moDAl for the membrane‐integral domain of succinate:quinone oxidoreductases

Hägerhäll¹,

Hederstedt²

1996

FEBS Letters

123

102

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Many succinate:quinone oxidoreductases in bacteria and mitochondria, i.e. succinate:quinone reductases and fumarate reductases, contain in the membrane anchor a cytochrome b whose structure and function is poorly understood. Based on biochemical data and polypeptide sequence information, we show that the anchors in different organisms are related despite an apparent diversity in polypeptide and heine composition. A general structural model for the membrane-integral domain of the anchors is proposed. It is an antiparallel four-helix bundle with a novel arrangement of hexa-coordinated protoheme IX. The structure can be applied to a larger group of membraneintegral cytochromes of b-type and has evolutionary and functional implications.

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Identification of the axial heme ligands of cytochrome b₅₅₆ in succinate: Ubiquinone oxidoreductase from Escherichia coli

Cited by 34 publications

References 21 publications

A succinate dehydrogenase with novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization

A succinate dehydrogenase with novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization

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

A structural moDAl for the membrane‐integral domain of succinate:quinone oxidoreductases

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