Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion

Hederstedt, Lars; Magnusson, Kerstin; Rutberg, Lars

doi:10.1128/jb.152.1.157-165.1982

Cited by 27 publications

(5 citation statements)

References 25 publications

(37 reference statements)

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“…Ip subunit is found in the cytoplasm. The strain contains wild-type Cyt *555 in the membrane (Hederstedt & Rutberg, 1980;Hederstedt et al, 1982). A light absorption spectrum at 77 K of membranes from KA98011 /pBSD1300 is shown in Figure 7.…”

Section: Resultsmentioning

confidence: 99%

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II)

Hägerhäll¹,

Aasa²,

Wachenfeldt³

et al. 1992

Biochemistry

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Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide. The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.

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Section: Resultsmentioning

confidence: 99%

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II)

Hägerhäll¹,

Aasa²,

Wachenfeldt³

et al. 1992

Biochemistry

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“…The sdh operon in B. subtilis contains three genes, sdh A, sdhB, and sdhC, which "Data from this work, Hederstedt (1983), and Hederstedt et al (1985). Determination of clusters S-l, S-2, and S-3 in KA97129 by L. are transcribed in that order and code for cytochrome ¿558, Fp, and Ip, respectively, of the succinate dehydrogenase-cytochrome ¿558 complex (Hederstedt et al, 1982;Magnusson et al, 1983). Point mutations sdhBll, sdhBlOl, sdhB124, and sdhB¡29 have been located to the structural gene for the Fp subunit by classical transformation crosses, by complementation analysis and by analysis of the biochemical properties of strains containing these mutations (Hederstedt et al, 1982;Hederstedt, 1983).…”

Section: Resultsmentioning

confidence: 93%

“…Determination of clusters S-l, S-2, and S-3 in KA97129 by L. are transcribed in that order and code for cytochrome ¿558, Fp, and Ip, respectively, of the succinate dehydrogenase-cytochrome ¿558 complex (Hederstedt et al, 1982;Magnusson et al, 1983). Point mutations sdhBll, sdhBlOl, sdhB124, and sdhB¡29 have been located to the structural gene for the Fp subunit by classical transformation crosses, by complementation analysis and by analysis of the biochemical properties of strains containing these mutations (Hederstedt et al, 1982;Hederstedt, 1983). The four sdhB mutations were mapped more precisely by transforming strains containing the respective mutation to CAP resistance with plasmids pKIM57 and pKIM31.…”

Section: Resultsmentioning

confidence: 99%

“…This is supported because both mutants revert to an Sdh-positive phenotype at about the same frequency as strain KA97124. Furthermore, mutation sdhBll can be separated in reciprocal three factor transformation crosses and mapped relative to other sdhB mutations located both upstream, e.g., sdhBlOl, and downstream of sdhBll (Ohné et al, 1973;Hederstedt et al, 1982). The Fp polypeptide in both mutants lacks covalently bound flavin and is mutated at a point distant from the flavin binding histidine.…”

Section: Discussionmentioning

confidence: 99%

“…Bacillus subtilis Strains and Membrane Preparation. The B. subtilis strains 168 (prototrophic), KA98011 (trpC2 sdhBll) (Ohné et al, 1973), KA97101 (trpC2 leu-2 sdhBWl), KA97124 (trpC2 leu-2 sdhB124), and KA97129 (trpC2 leu-2 sdhB 129) (Hederstedt et al, 1982) were used. The Sdh phenotype was checked on purification agar (PA) plates (Carls & Hanson, 1971).…”

Section: Methodsmentioning

confidence: 99%

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Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme

Maguire¹,

Magnusson²,

Hederstedt³

1986

Biochemistry

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Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation.

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Ligands to the 2Fe iron‐sulfur center in succinate dehydrogenase

Aevarsson

Hederstedt

1988

FEBS Letters

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Membrane-bound succinate oxidoreductases are flavoenzymes containing one each of a 2Fe, a 3Fe and a 4Fe iron-sulfur center. Amino acid sequence homologies indicate that all three centers are located in the Ip (B) subunit. From polypeptide and gene analysis of Bacillus subtillis succinate dehydrogenase-defective mutants combined with earlier EPR spectroscopic data, we show that four conserved cysteine residues in the first half of Ip are the ligands to the [2Fe-2S] center. These four residues have previously been predicted to be the ligands. Our results also suggest that the N-terminal part of B. subtilis Ip constitutes a domain which can incorporate separately the 2Fe center and interact with Fp, the flavin-containing subunit of the dehydrogenase.

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Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion

Cited by 27 publications

References 25 publications

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II)

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II)

Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme

Ligands to the 2Fe iron‐sulfur center in succinate dehydrogenase

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