Vol. 138, No. 2JOURNAL OF BACTERIOLOGY, May 1979, p. 370-3760021-9193/79/05-0370/07$02.00/0

Characterization of a Succinate Dehydrogenase ComplexSolubilized from the Cytoplasmic Membrane of Bacillus

subtilis with the Nonionic Detergent Triton X-100LARS HEDERSTEDT, ERIK HOLMGREN, AND LARS RUTBERG*

Department ofBacteriology, Karolinska Institutet, S-104 01 Stockholn 60, Sweden

Received for publication 13 November 1978

A succinic dehydrogenase (SDH) complex has been purified from Triton X-100-solubilized membranes from Bacillus subtilis by precipitation with specificantibody. Radioactively labeled precipitated complex was analyzed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiographyof the gels. The complex contained equimolar amounts of three polypeptides withapproximate molecular weights of 65,000, 28,000, and 19,000. Five succinic dehy-drogenase-negative mutants, belonging to the citF group, contained the 65,000-dalton polypeptide in a soluble form in the cytoplasm. Each 65,000-daltonpolypeptide had about one molecule of flavin bound. Another citF mutant, citFIl,which lacks the 65,000-dalton polypeptide, contained a membrane-bound 28,000-dalton polypeptide. The wild-type succinic dehydrogenase complex containedcytochrome, probably a cytochrome b. The 19,000-dalton polypeptide is suggestedto represent the apoprotein of this cytochrome. The 65,000-dalton and the 28,000-dalton polypeptides are thought to constitute succinic dehydrogenase and tocorrespond to the flavoprotein and the ironprotein, respectively, as described forsuccinic dehydrogenase isolated from beef heart mitochondria or Rhodospirillumrubrum chromatophores. The results presented suggest that in B. subtilis succinicdehydrogenase is attached to a cytochrome b in the membrane via the 28,000-dalton (ironprotein) polypeptide.

Succinic dehydrogenase (SDH) [EC 1.3.99.1,succinate:(acceptor) oxidoreductase] is a firmlymembrane-bound enzyme in Bacillus subtilisand other aerobic bacteria. It can be solubilizedwith nonionic detergents in an active form (18).We have recently solubilized an SDH complexfrom B. subtilis membranes and characterizedthe solubilized proteins in crossed immunoelec-trophoresis (CIE). Among a number of precipi-tates obtained with an antiserum preparedagainst whole membranes, one was identified ascontaining SDH. No precipitate staining forSDH was found in CIE of membranes from nineSDH mutants (18). The respective mutations ofthese mutants all map in a region called citF(17). An SDH-specific antiserum was obtainedby immunizing rabbits with the SDH precipitatefrom CIE. This antiserum was used to identifya soluble SDH antigen in five of the nine citFmutants. Wild-type bacteria contain little solu-ble SDH antigen.

In the present experiments, we have used theSDH-specific antiserum to isolate and charac-terize the solubilized SDH complex in B. sub-tilis. The experiments give information aboutthe possible subunit structure of the enzyme and

about binding of the enzyme to the cytoplasmicmembrane. The experiments also indicate thatSDH, in the membrane, is closely associatedwith cytochrome. In an accompanying paper wewill show that there is a close correlation be-tween membrane binding of SDH and synthesisof heme in B. subtilis (13).

MATERIALS AND METHODSBacteria. B. subtilis BrlO2 (hisB trpC2) was used

as the wild type; it was originally obtained from J.Spizizen. The SDH mutants (citF mutants) have beendescribed previously (17).

General methods and media for growing the bacte-ria were described in a recent paper (18).

Preparation and solubilization of membranes.Membranes from wild-type bacteria were preparedand stored as recently described (18). To solubilize themembrane-bound SDH, membranes (10 mg of mem-brane protein per ml) were treated with 4% (vol/vol)Triton X-100 in 24 mM sodium 5,5-diethylbarbiturate(diemal) buffer (pH 8.6) for 10 min at room tempera-ture. The sample was then centrifuged at 48,200 x gfor 2 h at 4°C. The resulting supernatant was collectedand stored at -70°C until used. For radioactive label-ing, the cells were grown with 1 ,uCi of L-14C-aminoacid mixture per ml (New England Nuclear Corp.).

Preparation of soluble fraction from citF mu-

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SDH COMPLEX FROM B. SUBTILIS MEMBRANES 371

tants. The bacteria were grown in 300 ml of NSMPwith 0.33 ,uCi of L-'4C-amino acid mixture per ml ofmedium (8)' in indentated 1,600-ml Fernbach flaskswith shaking at 370C to an absorbancy at 600 nm ofabout 1. The bacteria were collected by centrifugationat 19,600 x g for 10 min at 40C. The pellet was washedonce with 50 mM Tris-hydrochloride (pH 6.6) andstored at -20°C. The next day the bacteria werethawed and suspended in 8 ml of 50 mM Tris-hydro-chloride (pH 6.6) with 10 mM Mg9l2, 300,ug of lyso-zyme per ml, and 1 ug of DNase per ml. The mixturewas incubated at 37°C with shaking for 35 min, and itwas then centrifuged at 27,000 x g for 30 min at 40C.The supernatant was collected, and the pellet waswashed once with 2 ml of 50 mM Tris-hydrochloride(pH 6.6) containing 12 mM phenylmethylsulfonyl-fluoride (Merck). The two supernatants were pooledand concentrated about eight times on a Minocon-B15 (Amicon) and then stored at -70°C until used.Preparation of antisera and immunoelectro-

phoretic techniques. The methods used for prepa-ration and purification of antisera and for immuno-electrophoresis were recently described (18).

Immunoprecipitation. Solubilized membranepreparations and soluble fractions were centrifuged at48,000 x g for 2 h at 40C before use. Appropriateamounts of antigen and antiserum were mixed andincubated for 48 h at 40C. The final concentrations ofTriton X-100 during precipitation of solubilized mem-brane SDH antigen was 1.5% (vol/vol). For precipita-tion of soluble SDH antigen from citF mutants, TritonX-100 was added to a final concentration (vol/vol) of0.6%. The precipitates were collected by centrifugationat 35,000 x g for 30 min at 40C and then washed fivetimes (when no more radioactivity could be washedaway) with 0.1 M sodium phosphate buffer (pH 8.0).During the first two washings 1% (vol/vol), Triton X-100 was included in the buffer. Samples for determi-nation of radioactivity and (in the case of wild-typebacteria) for SDH activity were taken before and afterthe first centrifugation at 35,000 x g, from the wash-ings, and from the final precipitate. The washed pre-cipitate was stored in a small volume of 0.1 M sodiumphosphate buffer (pH 8.0) at -70°C until used.SDS-PAGE. Electrophoresis in sodium dodecyl

sulfate-polyacrylamide gradient gels (SDS-PAGE)was performed as described elsewhere (18). Determi-nations of apparent molecular weights were made withthe following marker polypeptides: phosphorylase-a(rabbit muscle, Sigma), 94,000; bovine albumin(Sigma), 68,000; catalase (beef liver, Sigma), 60,000;fumarase (pig heart, Sigma), 49,000; albumin (chickenegg, Sigma), 43,000; subtilisin (Novo), 27,600; trypsin(bovine pancreas, Sigma), 23,000; myoglobin (horseheart, Sigma), 17,200.The gels were autoradiographed with Kodak X-

omat, R film XR-1.Absorption spectra. All spectra were recorded on

a Unicam SP 1700 double-beam spectrophotometer ata scan speed of 1 nm/s at room temperature. Theimmunoprecipitates were homogenized in 0.1 M so-dium phosphate buffer (pH 8.0) and then transferredto 1-cm-light-path cuvettes. Difference spectra wereobtained by placing air-oxidized samples in both cu-vettes. After recording a base line, a few grains of

Na2S204 were added to one of the cuvettes, and areduced-minus-oxidized absorption spectrum was re-corded.

Determination of non-acid-extractable flavin.Non-acid-extractable flavin was determined as de-scribed by Wilson and King (20) with an Aminco-Bowman spectrofluorometer. After precipitation andwashing with trichloroacetic acid, the immunoprecip-itates were incubated for 4 h at 370C with shakingwith subtilisin BPN type YII (Sigma) at 0.4 mg/ml,a-chymotrypsin type 1 from bovine pancreas (Sigma)at 0.075 mg/ml, and trypsin type III from bovinepancreas (Sigma) at 0.25 mg/ml. More than 90% of theradioactivity was converted to an acid-soluble formduring the incubation. Flavine adenine dinucleotide(disodium salt, grade III, Sigma) hydrolyzed overnightat 370C was used as the standard.SDH assay. SDH was assayed by a modification of

the method of Ells (7). The reaction mixture, pH 7.4,contained the following (per milliliter): 8.8 mM potas-sium phosphate buffer, 8 mM Na-succinate, 0.9 mMKCN, 17 jg of 2,6-dichlorophenolindophenol, and 0.21mg of phenazine methosulfate. Succinate-dependentreduction of 2,6-dichlorophenolindophenol was fol-lowed at 600 nm at room temperature with a UnicamSP 1700 double-beam spectrophotometer equippedwith a Unicam AR 25 linear recorder.

Protein determination. Protein was determinedby the method of Lowry et al. (14) with bovine serumalbumin (Sigma) as the standard. To avoid precipita-tion due to Triton X-100, 2% (wt/vol) SDS was in-cluded in the sodium carbonate-NaOH solution.

RESULTS

Precipitation ofthe solubilized SDH com-plex with specific antibody. We have recentlydescribed the preparation and properties of anantiserum specific for B. subtilis SDH solubi-lized with Triton X-100 (18). In CIE of solubi-lized wild-type membrane proteins, this antise-rum gave a single symmetric peak which stainedspecifically for SDH. No precipitate was foundin similar experiments with solubilized mem-brane proteins from each of nine citF mutants.However, a soluble SDH antigen was detectedin five of the citF mutants. In the followingexperiments, the SDH antiserum was used tocharacterize the SDH complex solubilized fromB. subtilis membranes with Triton X-100.

B. subtilis was grown with a mixture of 14C-labeled amino acids, and membranes were pre-pared and solubilized as described above. Thesolubilized preparations contained about 3 mg ofprotein per ml. Constant amounts of superna-tant were added to increasing amounts of SDHantiserum, the samples were incubated at 40Cfor 48 h, and the resulting precipitate was col-lected as described above. At a serum: samplevolume ratio of about 1.2, more than 90% of theenzyme activity was removed from the super-natant, and increasing amounts of activity were

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372 HEDERSTEDT, HOLMGREN, AND RUTBERG

found in the precipitate (Fig. 1). The recovery oftotal activity was less than 35%, indicating en-zyme inactivation during precipitation.

In the next experiment, about 3 mg of solubi-lized membrane protein was mixed with suffi-cient antiserum to precipitate more than 90% ofSDH enzyme activity. The precipitate, whichhad a yellow-brown color, was treated as de-scribed above. More than 90% of SDH enzymeactivity but less than 2% of the total radioactiv-ity was precipitated.The precipitate was then analyzed in SDS-

PAGE. In the same run were also included sam-ples of total membrane proteins and solubilizedmembrane proteins, with and without a smallamount of precipitated SDH added, respec-tively. The gel was stained for protein and thenautoradiographed. The results are shown in Fig.2. The precipitate contains three major radio-active polypeptides with approximate molecularweights of 65,000, 28,000, and 19,000, respec-tively (Fig. 3). The 65,000-dalton polypeptidecan easily be identified in SDS-PAGE by stain-ing total membrane proteins or solubilized mem-brane proteins with Coomassie brilliant blue,

100

C)

00

x50

U.0

1.zUC 25wiIL

0 1 2 3

RATIO ANTIBODY/SAMPLE

FIG. 1. Titration of Triton X-100-solubilized SDHfrom wild-type B. subtilis with SDH-specific antibody.Increasing amounts of antiserum were added to a

constant volume of solubilized membrane proteins.The resulting precipitate was collected as describedin the text, and the distribution of SDH enzymeactivity in the precipitate and in supernatant wasmeasured. Maximal SDH enzyme activity was 69arbitary units in the supernatant and 22 arbitaryunits in the precipitate. Symbols: 0, supernatant; 0,

precipitate.

A1 2 3

B2 3 44 5

s. ...a:....

:.:...~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..

ofltw- 65000 411

- ~ 28000

-4-.~~~ ~ ~ ~ ~ ~ ~ ~ ~ *.a:

st a....

1 9000

FIG. 2. SDS-PAGE of SDH precipitate (A) Coo-massie brilliant blue staining of the gel. (B) Autora-diography. The slots contain: 1, unlabeled total mem-brane proteins; 2, unlabeled total membrane proteinsplus radioactively labeled SDHprecipitate; 3, radio-actively labeled SDH precipitate; 4, Triton X-100-solubilized, unlabeled membraneproteinsplus radio-actively labeled SDH precipitate; 5, Triton X-100-solubilized unlabeled membrane proteins. The threebands of slot 3 were cut out, and their radioactivitywas determined as described in the text. The bandscontained 260 cpm (65,000-molecular-weight band),100 cpm (28,000-molecular-weight band), and 70 cpm(19,0()0-molecular-weight band), respectively. Thegradient was 10 to 15% (wt/vol) acrylamide-0.26% to0.40% (wt/vol) bisacrylamide.

whereas the two smaller polypeptides are moredifficult to identify. With a denser gel, however,the 28,000-dalton polypeptide can be resolved(see below). An equal area of one gel was cut outover each radioactive band from the SDH pre-cipitate, and, for background counts, over theimmunoglobulin heavy-chain band and from anarea not staining for protein. The radioactivityin each of these samples was then determined.From this experiment the molar ratio of thethree radioactive polypeptides in the SDH pre-cipitate was calculated to be 1:1:1.We conclude from the above results that the

SDH complex as solubilized from B. subtilis

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SDH COMPLEX FROM B. SUBTILIS MEMBRANES 373

membranes contains equimolar proportions ofthree polypeptides with approximate molecularweights of 65,000, 28,000, and 19,000, respec-tively.SDH precipitate contains cytochrome.

The absorption spectrum of the air-oxidizedSDH precipitate at room temperature shows anabsorption maximum at 414 nm. The differencespectrum shows an absorption maximum at 430nm and a small peak at about 560 nm (Fig. 4).These data strongly suggest that the precipitatecontains cytochrome probably of b type. Thissuggestion is strengthen by the following exper-iment. A 5-aminolevulinic acid requiring mutantof B. subtilis (13) was grown with 5-[14C]ami-

5.0

04.5

0-j

4.00

nolevulinic acid. Membranes were prepared, sol-ubilized, and run in CIE. Some 20 precipitateswere resolved in the gel by staining with Coo-massie brilliant blue. One of these precipitatesalso stained for SDH as described previously(18). On autoradiography only three peaks werefound, one of which was the SDH precipitate.As expected, the precipitate obtained in CIEwith SDH-specific antiserum was radioactive(Fig. 5).Precipitation of soluble SDH antigen

from citF mutants. The citF mutants -78, -42,-44, -12, and -83 contain a soluble antigen whichreacts with the SDH-specific antiserum (18).Each of these mutants was grown with "C-la-beled amino acids, and the SDH antigen wasprecipitated. The resulting precipitates were an-alyzed in SDS-PAGE, and the gels were stainedfor protein and then autoradiographed. The re-sults of these experiments are shown in Fig. 6.Each precipitate contains a radioactive poly-peptide of a molecular weight of about 65,000and which migrates identically with the largestpolypeptide found in the wild-type SDH com-plex. In addition the citF-12 precipitate containstwo smaller polypeptides with molecular weightsof about 51,000 and 39,000, respectively. Possiblythese represent cleavage products of the 65,000-dalton polypeptide. Absorption spectra of eachof the citF precipitates gave no evidence for thepresence of cytochrome (data not shown).Flavin content ofSDH precipitates. Flavin

0.5RELATIVE MOBILITY

FIG. 3. Relative mobilities of the three polypep-tides of the immunoprecipitated SDH complex inSDS-PAGE. The vertical bars show the range ofmolecular weights obtained in seven different gels.Apparent molecular weights given in the figure arethe mean values from these determinations.

0lC 0.005

0D io 1kA

Al B1

A

A2 B2

FIG. 5. CIE ofsolubilized membraneproteins froma 5-aminolevulinic acid-requiring mutant of B. sub-tilis (13) grown with 5-[4-14C]aminolevulinic acid(New England Nuclear Corp.). Al, Triton X-100-sol-ubilized membrane proteins run against whole mem-brane antiserum. The gel was stained with Coomassiebrilliant blue. A2, Autoradiography of the gel shownin Al. BJ, Triton X-100-solubilized membrane pro-teins run against SDH-specific antiserum. The gelwas stained with Coomassie brilliant blue. B2, Au-toradiography of the gel shown in BI.

400 SOO 600 nmWave elng t h

FIG. 4. Reduced minus oxidized absorption spec-tra of wild-type SDH immunoprecipitate.

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374 HEDERSTEDT, HOLMGREN, AND RUTBERG

was determined in wild-type and citF precipi-tates as described above. The results of theseexperiments (Table 1) show that all the precip-

Awt 42 12

ab 78 44 83

...

Bwt 42 1 2

78 44 83

65 0 00 _, __. am_

28 000

19000

FIG. 6. SDS-PAGE of antibody (ab), immunopre-cipitated wild-type (wt) (membrane-bound) SDH an-tigen, and soluble (cytoplasmic) SDH antigen fromcitF mutants. (A) Gel stained with Coomassie bril-liant blue. (B) Autoradiography of the same gel. Thegradient was 10 to 15% (wt/vol) acrylamide-0.26 to0.40% (wt/vol) bisacrylamide.

TABLE 1. Flavin content ofSDHprecipitatesNon-acid-ex-

Immunopre- tractable flavin Approx mol Flavin/cipitate bacteria po wt moleculebacterial pro-

tein)a

Wild type 10 112,000 1.1cit- 78 13 65,000 0.8cit-42 11 65,000 0.7cit-44 16 65,000 1.0cit-12 10 65,000 0.6cit-83 11 65,000 0.7

'The protein content of the acid-soluble digestsused for flavin analysis was determined from theirradioactivity. Samples were added to 5 ml of Aquasol(New England Nuclear Corp.) and counted in a Nu-clear Chicago Mark I scintillation counter. Lipids ac-count for 37% of the radioactivity in the solubilizedmembrane preparations as determined by the methodofAmes (1), whereas the cytoplasmic fraction containsless than 0.5% radioactive lipid. Protein was deter-mined on solubilized membrane preparations (14), andthe specific radioactivity of the protein was calculatedon the assumption that 63% of the radioactivity was inprotein. Triton X-100-washed precipitates were con-sidered essentially free of radioactive lipid.

itates contain flavin at a concentration of 0.6 to1.1 nmol/nmol of 65,000-dalton polypeptide.Analysis of membrane proteins in SDS-

PAGE. Since we now know the polypeptidecomposition of the wild-type SDH complex, wehave again (18) analyzed total membrane pro-teins from citF mutants in SDS-PAGE. Theseexperiments show that the 65,000-dalton poly-peptide is absent from the membrane of the citFmutants (data not shown). By using denser gelswe can identify the 28,000-dalton polypeptide insolubilized wild-type membrane proteins. A cor-responding band is found in solubilized mem-brane proteins from citF-11 but not the othercitF mutants (Fig. 7). We have not been able toresolve the 19,000-dalton polypeptide in SDS-PAGE.

DISCUSSION

The amount of detergent used to solubilizethe B. subtilis membrane is in large excess rel-ative membrane protein. It is therefore likely(19) that where several proteins are contained ina single micell this reflects some structural-func-tional relationship between these polypeptidesin the intact membrane rather than randomincorporation of polypeptides into mixed micells.This is a basic assumption for the present work.A second assumption is that the antiserum em-ployed will only react with (an) SDH antigen ina mixture of solubilized membrane proteins.This assumption seems valid in view of the factthat the phenotypically SDH-negative citF mu-tants all lack membrane antigen reacting withthe serum. Also, in the precipitation experimentswith radioactive, solubilized wild-type mem-brane proteins, more than 90% of the SDH ac-tivity is precipitated, whereas more than 98% ofthe total protein remains in solution. We con-sider the SDH precipitate to be a representationof a functional membrane-bound SDH complex.The SDH complex contains three polypep-

tides with approximate molecular weights of65,000, 28,000, and 19,000. These polypeptidesare found in equimolar amounts, which supportsthe idea of a functional-structural relation be-tween them. Hatefi and co-workers have purifiedSDH from Rhodospirillum rubrum chromato-phores (5) and from beef heart mitochondria (4).In both cases the enzyme consists oftwo unequalsubunits with molecular weights of 60,000 to70,000 and 25,000 to 27,000, with flavin cova-lently (4, 5) bound to the larger subunit. The B.subtilis SDH also contains flavin which is bolndto the 65,000-dalton polypeptide. The fact thatsome citF mutants contain this polypeptide inthe cytoplasm indicates that it plays no directrole in binding of the enzyme to the membrane.

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SDH COMPLEX FROM B. SUBTILIS MEMBRANES

42 12 8 2 74 Wt78 44 69 11 83 80 SDH~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~................-:

-65000

-28000

FIG. 7. SDS-PAGE of Triton X-100-solubilizedmembraneproteins from wild-type (wt), from mutantslacking SDH enzyme activity (citF-78 to -83), andfrom mutants with reduced SDH enzyme activity (cit-74 cit-80). Wild-type SDH immunoprecipitate is also

The 28,000-dalton polypeptide, which we sug-gest is analogous to the smaller subunit in Ha-tefi's enzymes, is located in the membrane alsowhen the bacteria do not produce the largerpolypeptide, at least in the mutant citF-11.The 19,000-dalton polypeptide in the B. sub-

tilis SDH complex has no obvious analogs in theR. rubrum or mitochondrial enzymes. This poly-peptide probably is a cytochrome apoprotein.Most likely it is a cytochrome of b type and maybe identical to the cytochrome b5we described byMiki and Okunuki (15, 16). This cytochrome isthought to be reduced by succinate via succinatedehydrogenase. An association between cyto-chrome b and succinate dehydrogenase has beensuggested for several bacterial species (9, 12). Inanalogy with complex II (succinate-ubiquinonereductase) from beef heart mitochondria (11),the B. subtilis SDH complex contains 1 mol ofcytochrome per mol of flavin. At room temper-ature the cytochrome b of complex II has an

absorption maximum in the reduced form at 560to 561 nm (6), and it has a proposed molecularweight in SDS-PAGE of 13,700 (3).

Recently, we have speculated that the B. sub-tilis SDH is composed of two subunits, one ofwhich is involved in specific binding of the en-

zyme to the membrane via a third, membrane,protein (18). The present data put this specula-tion on somewhat firmer grounds. By analogywith SDH from other organisms, it is likely thatthe two larger polypeptides of the B. subtilis

included in the gel. The gel was stained with Coo-massie brilliant blue. The gradient was 12 to 15%(wt/vol) acrylamide-0.32 to 0.40% (wt/vol) bisacryl-amide.

SDH complex constitute the enzyme proper.Several SDH mutants which do not produce the28,000-dalton polypeptide have a cytoplasmic,soluble 65,000-dalton polypeptide. One mutant,which does not produce the 65,000 polypeptide,has a membrane-bound 28,000-dalton poly-peptide. This suggests that the 28,000-daltonpolypeptide is responsible for membrane bindingof the enzyme.The 19,000-dalton polypeptide, suggested to

be cytochrome b, is proposed to represent thespecific membrane binding site for the 28,000-dalton polypeptide. Cytochrome b has beenspeculated to be involved in binding of SDH tothe mitochondrial inner membrane (2, 10). Inthe accompanying paper (13), we will show thatthere is a coupling between incorporation ofSDH in the membrane and synthesis of cyto-chromes.We do not yet have enough genetic data to

resolve the citF locus into functional parts. Thepresent results, however, support the suggestionthat the region of citF defined by mutationscitF- 74-citF-12 contains a structural gene forthe 28,000-dalton polypeptide, and the citF-69-citF-2 region includes a structural gene for the65,000-dalton polypeptide.

ACKNOWLEDGMENSThis work was supported by grants from the Swedish

Medical Research Council and from Karolinska Institutet.The expert technical assistance of Kerstin Bernholm and

Sven-Ake Franzen is gratefully acknowledged. We thank the

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Department of Biochemistry, Stockholm University, for theuse of their Aminco-Bowman spectrofluorometer.

LITERATURE CIMD

1. Ames, G. F. 1968. Lipids of Salmonella typhimurium andEscherichia coli: structure and metabolism. J. Bacte-riol. 95:833-843.

2. Bruni, A., and E. Racker. 1968. Resolution and recon-stitution of the mitochondrial electron transport sys-tem. I. Reconstitution of the succinate-ubiquinone re-ductase. J. Biol. Chem. 243:962-971.

3. Capaldi, R. A., J. Sweetland, and A. Merli. 1977.Polypeptides in the succinate-coenzyme Q reductasesegment of the respiratory chain. Biochemistry 16:5707-5710.

4. Davis, K. A., and Y. Hatefi. 1971. Succinic dehydrogen-ase. I. Purification, molecular properties, and substruc-ture. Biochemistry 10:2509-2516.

5. Davis, K. A., Y. Hatefi, I. P. Crawford, and H. Bal-tsceffsky. 1977. Purification, molecular properties, andamino acid composition of the subunits of Rhodospiril-lum rubrum succinate dehydrogenase. Arch. Biochem.Biophys. 180:459-464.

6. Davis, K. A., Y. Hatefi, K. L Poff, and W. L Butler.1973. The b-type cytochromes of bovine heart mito-chondria: absorption spectra, enzymatic properties, anddistribution in the electron transport complexes.Biochim. Biophys. Acta 325:341-356.

7. Ells, H. A. 1959. A colorimetric method for the assay ofsoluble succinic dehydrogenase and pyridinenucleotide-linked dehydrogenases. Arch. Biochem. Biophys. 85:561-562.

8. Fortnagel, P., and E. Freese. 1968. Analysis of sporu-lation mutants. II. Mutant blocked in the citric acidcycle. J. Bacteriol. 95:1431-1438.

9. Gelman, N. S., M. A. Lukoyanova, and D. N. Ostrov-skii. 1975. Bacterial membranes and the respiratorychain. Biomembranes 6:129-253.

10. Hanstein, W. G., K. A. Davis, M. A. Ghalambor, and

Y. Hatefi. 1971. Succinate dehydrogenase. II. Enzy-matic properties. Biochemistry 10:2517-2524.

11. Hatefi, Y. 1976. The enzymes and the enzyme complexesof the mitochondrial oxidative phosphorylation system,p. 3-41. In A. Martinosi (ed.) The enzymes of biologicalmembranes, vol. 4. John Wiley & Sons, New York.

12. Hendler, R. W. 1976. Respiration and energy transduc-tion in Escherichia coli, p. 75-109. In A. Martinosi(ed.), The enzymes of biological membranes. vol. 3.John Wiley & Sons, New York.

13. Holmgren, E., L Hederstedt, and L. Rutberg. 1979.Role of heme in synthesis and membrane binding ofsuccinic dehydrogenase in Bacillus subtilis. J. Bacte-riol. 138:377-382.

14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

15. Miki, K., and K. Okunuki. 1969. Cytochromes of Bacil-lus subtilis. III. Physico-chemical and enzymatic prop-erties of cytochromes C-550 and c-554. J. Biochem. 66:845-854.

16. Miki, K., I. Sekuzu, and K. Okunuki. 1967. Cyto-chromes of Bacillus subtilis. I. Cytochrome system inthe particulate fraction. Annu. Rep. Biol. Works Fac.Sci. Osaka Univ. 15:33-58.

17. Ohne, M., B. Rutberg, and J. A. Hoch. 1973. Geneticand biochemical characterization ofmutants ofBacillussubtilis defective in succinic dehydrogenase. J. Bacte-riol. 115:738-745.

18. Rutberg, B., L. Hederstedt, E. Holmgren, and L.Rutberg. 1978. Characterization of succinic dehydro-genase mutants of Bacillus subtilis with crossed im-munoelectrophoresis. J. Bacteriol. 136:304-311.

19. Tanford, C. A., and J. A. Reynolds. 1976. Characteri-zation of membrane proteins in detergent solutions.Biochim. Biophys. Acta 457:133-170.

20. Wilson, D. F., and T. E. King. 1964. The determinationof the acid-nonextractable flavin in mitochondrial prep-arations from heart muscle. J. Biol. Chem. 239:2683-2690.

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