Fumarate reductase of <i>Escherichia coli</i>: an investigation of function and assembly using <i>in vivo</i> complementation

Condon, Caro; Weiner, Joël H.

doi:10.1111/j.1365-2958.1988.tb00005.x

Cited by 46 publications

(32 citation statements)

References 46 publications

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“…Moreover, this strain is unable to interact with quinone analogues, although the enzyme complex is completely functional in catalyzing fumarate reduction by reduced benzyl viologen and is localized in the membrane. In this study it was claimed that co-translational association of frdC and frdD products is required for the functional assembly of E. coli FRD (33). In contrast to FRD, we observed active complex II in membranes when both pSDHC and pSDHDAB were transformed into MK3, and the enzyme complex supported aerobic growth on succinate, indicating differences in the assembly processes of the two enzyme complexes.…”

Section: Discussioncontrasting

confidence: 41%

“…A difference in the biogenesis of the enzyme complex was also found between complex II and FRD of E. coli. The introduction of the frdABC and frdD genes on separate plasmids into E. coli MI1443, which lacks a chromosomal frd operon, fails to restore anaerobic growth on glycerol and fumarate (33). Moreover, this strain is unable to interact with quinone analogues, although the enzyme complex is completely functional in catalyzing fumarate reduction by reduced benzyl viologen and is localized in the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…The role of the two hydrophobic subunits in assembly and the amino acid residues essential for quinone binding have been analyzed (32)(33)(34). However, this enzyme complex does not contain heme b, so the function of heme b in the two-subunit cytochrome b found in most bacterial and mitochondrial complex II has not been analyzed.…”

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confidence: 99%

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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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Section: Discussioncontrasting

confidence: 41%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…The accessibility of these residues was also verified by another surface-specific iodination reagent, 1,3,4,6-tetrachloro-3ot,6a-diphenyl glycoluril (lodogen) (data not shown). We predict that DmsC anchors the DmsAB subunits to the membrane in much the same manner that FrdCD anchors FrdA and -B (9,11,25), and these radioiodination experiments suggest that at least a portion of DmsC is outside the plasma membrane domain.…”

mentioning

confidence: 99%

Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli

et al. 1990

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Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S-and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, or DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane.Dimethyl sulfoxide (DMSO) reductase of Escherichia coli (5) is a terminal enzyme expressed constitutively under anaerobic conditions and is capable of reducing DMSO and trimethylamine-N-oxide (TMAO). We have reported that the E. coli DMSO reductase is a membrane-bound iron-sulfur molybdoenzyme genetically distinct from the single or dual subunit forms of TMAO reductase (40). The structural genes encoding the enzyme have been cloned, and their DNA sequences have been determined (4, 7). The enzyme has been purified to homogeneity and shown to be composed of three nonidentical subunits: a catalytic subunit (DmsA; 82,600 daltons), an iron-sulfur subunit (DmsB; 23,600 daltons), and a membrane anchor subunit (DmsC; 22,700 daltons).The presence of one constitutive and three inducible forms of TMAO reductase have been demonstrated (32,36,42); however, the significance and precise localization of these multiple forms of TMAO reductase in the energy transduction mechanisms of the cell are not clear. The enzymes responsible for TMAO and DMSO reduction in certain marine organisms and nonphotosynthetic bacteria have been characterized in some detail (2). A single enzyme in Rhodobacter sp. has been found to be responsible for the reduction of both DMSO and TMAO (23). The reductases in these organisms were found to be localized in the periplasmic compartment.

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Fumarate reductase is essential for Helicobacter pylori colonization of the mouse stomach

Feng

Dangler

et al. 2000

Microbial Pathogenesis

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Fumarate reductase of Escherichia coli: an investigation of function and assembly using in vivo complementation

Cited by 46 publications

References 46 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

Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli

Fumarate reductase is essential for Helicobacter pylori colonization of the mouse stomach

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