Characterization of a succinate dehydrogenase complex solubilized from the cytoplasmic membrane of Bacillus subtilis with the nonionic detergent Triton X-100

Abstract

A succinic dehydrogenase (SDH) complex has been purified from Triton X-100-solubilized membranes from Bacillus subtilis by precipitation with specific antibody. Radioactively labeled precipitated complex was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography of the gels. The complex contained equimolar amounts of three polypeptides with approximate molecular weights of 65,000, 28,000, and 19,000. Five succinic dehydrogenase-negative mutants, belonging to the citF group, contained the 65,000dalton polypeptide in a soluble form in the cytoplasm. Each 65,000-dalton polypeptide had about one molecule of flavin bound. Another citF mutant, citFIl, which lacks the 65,000-dalton polypeptide, contained a membrane-bound 28,000dalton polypeptide. The wild-type succinic dehydrogenase complex contained cytochrome, probably a cytochrome b. The 19,000-dalton polypeptide is suggested to represent the apoprotein of this cytochrome. The 65,000-dalton and the 28,000dalton polypeptides are thought to constitute succinic dehydrogenase and to correspond to the flavoprotein and the ironprotein, respectively, as described for succinic dehydrogenase isolated from beef heart mitochondria or Rhodospirillum rubrum chromatophores. The results presented suggest that in B. subtilis succinic dehydrogenase is attached to a cytochrome b in the membrane via the 28,000dalton (ironprotein) polypeptide. Succinic dehydrogenase (SDH) [EC 1.3.99.1, succinate:(acceptor) oxidoreductase] is a firmly membrane-bound enzyme in Bacillus subtilis and other aerobic bacteria. It can be solubilized with nonionic detergents in an active form (18). We have recently solubilized an SDH complex from B. subtilis membranes and characterized the solubilized proteins in crossed immunoelectrophoresis (CIE). Among a number of precipitates obtained with an antiserum prepared against whole membranes, one was identified as containing SDH. No precipitate staining for SDH was found in CIE of membranes from nine SDH mutants (18). The respective mutations of these mutants all map in a region called citF (17). An SDH-specific antiserum was obtained by immunizing rabbits with the SDH precipitate from CIE. This antiserum was used to identify a soluble SDH antigen in five of the nine citF mutants. Wild-type bacteria contain little soluble SDH antigen.In the present experiments, we have used the SDH-specific antiserum to isolate and characterize the solubilized SDH complex in B. subtilis. The experiments give information about the possible subunit structure of the enzyme and about binding of the enzyme to the cytoplasmic membrane. The experiments also indicate that SDH, in the membrane, is closely associated with cytochrome. In an accompanying paper we will show that there is a close correlation between membrane binding of SDH and synthesis of heme in B. subtilis (13).MATERIALS AND METHODS Bacteria. B. subtilis BrlO2 (hisB trpC2) was used as the wild type; it was originally obtained from J. Spizizen. The SDH mutants (citF mutants) have been...

Succinate dehydrogenase--a comparative review.

“…The cytochrome is the detergent binding, hydrophobic part of the reductase (H. Weiss, personal communication). A similar monodisperse SDH-cytochrome b complex containing SDH and a detergent binding (unpublished experiments) cytochrome bw polypeptide in an equimolar amount to SDH has also been isolated from Triton X-100-solubilized B. subtilis membranes (39,40) (Table 3). It is not known if this complex has quinone reductase activity.…”

“…These results strongly suggest the presence of a limiting number of specific SDH-binding sites in the B. subtilis cytoplasmic membrane. Evidence that cytochrome b5w is (part of) this binding site is provided by the observations that (i) cytochrome b5,m is present in stoichiometric amounts in the Triton X-100-solubilized, purified membrane SDH complex (39,40) and (ii) mutants which specifically lack cytochrome b&% contain the SDH Fp and Ip subunits in the cytoplasm (41). A model for biosynthesis and membrane binding of SDH in B. subtilis is shown in Fig.…”

Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane

“…In no case did these correspond to the major arcs containing heme. This is interesting in view of the recent reports that in Bacillus subtilis, succinate dehydrogenase, solubilized in Triton X-100, is associated with a b-type cytochrome (14). In beef heart mitochondria, the succinate-ubiquinone oxidoreductase (complex II) is also associated with a unique b cytochrome (13).…”

“…One purpose of this paper is to demonstrate the utility of both isoelectric focusing (IEF) and crossed immunoelectrophoresis (CIE) for the resolution and analysis of the E. coli cytochromes. Both techniques have been previously applied successfully to the study of membrane proteins (14,26,27,36). In our study, four heme-containing protein complexes were resolved under nondenaturing conditions in the presence of Triton X-100.…”

Succinate dehydrogenase in Rhodopseudomonas sphaeroides: subunit composition and immunocross-reactivity with other related bacteria

“…Succinate dehydrogenase [SDH; EC 1.3.99.1 succinate: (acceptor) oxidoreductase] is an enzyme which has been examined from a variety of eucaryotic and procaryotic sources (25). Most of the biochemical studies have been performed with the enzyme from beef heart mitochondria (11,16,28), but studies have also been reported on SDH from plant mitochondria (21,33), Neurospora crassa (45), Bacillus subtilis (22)(23)(24), Escherichia coli (8,34), Micrococcus luteus (9,10), Rhodospirillum rubrum (17,18), and Rhodopseudomonas sphaeroides (26). SDH from beef heart mitochondria and from Rhodospirillum rubrum can be removed from the membrane without the use of detergents by exposing the membranes either to alkaline conditions or to a chaotropic salt (25).…”