succinate dehydrogenase-a comparative review · membrane-bound succinate dehydrogenase [sdh;...

MICROBIOLOGICAL REVIEWS, Dec. 1981, p. 542-555 Vol. 45, No. 40146-0749/81/120542-14$02.00/0

Succinate Dehydrogenase-a Comparative ReviewLARS HEDERSTEDT* AND LARS RUTBERG

Department of Bacteriology, Karolinska Institutet, S-104 01 Stockholm, Sweden

INTRODUCTION AND SCOPE ................................................ 542DETERMINATION OF SUCCINATE DEHYDROGENASE (SDH) ENZYMEACTIVITY .................................................... 542

ORIENTATION OF SDH IN MEMBRANES.................................... 543STRUCTURE .................... .. ............. .... 544GENETICS ............................ 545MEMBRANEBINDING ................................ 547RECONSTITUTION OF MEMBRANE-BOUND SDH ........................... 548BIOSYNTHESIS AND MEMBRANE BINDING OF SDH IN BACILLUSSUBTILIS.............. 549

SUMMARY AND SOME PERSPECTIVES ................. .................... 550LITERATURE CITED ...................................................... 551

INTRODUCTION AND SCOPEMembrane-bound succinate dehydrogenase

[SDH; E.C.1.3.99.1 succinate:(acceptor) oxido-reductase] is present in all aerobic cells. Eversince its discovery in 1909 (93), SDH has beenstudied intensively. The enzyme has several par-ticularly interesting properties: (i) SDH is amembrane-bound dehydrogenase linked to therespiratory chain and a member of the Krebscycle; (ii) its activity is modulated by severalactivators and inhibitors; and (iii) SDH is acomplex enzyme containing nonheme iron, acid-labile sulfur, and covalently bound flavin ade-nine dinucleotide (FAD).Most of the published work concerns mam-

malian SDH. There is considerable knowledgeabout the composition, enzymology, and mem-brane binding of the enzyme, but relatively littleis known about its genetics and biosynthesis.Mitochondrial SDH has been extensively re-viewed (3, 37, 70, 87, 88), and only the structureand some new findings on the membrane bindingof SDH will be discussed in this article. Com-pared with mitochondrial SDH, little is knownabout the corresponding procaryotic enzyme.However, we feel that sufficient knowledge onthe comparative biology, genetics, membranebinding, and biosynthesis of microbial SDH hasnow accumulated that a short review would beof value. SDH catalyzes the oxidation of succi-nate to fumarate and transfers the resultantreducing equivalents directly to the respiratorychain. The enzyme is a member of both theKrebs cycle and the respiratory chain. In bac-teria, the electron transport chains are locatedin the cytoplasmic membrane or in modificationsthereof, like the chromatophore membrane ofphotosynthetic bacteria (55).Fumarate reductase is often found in anaero-

bic or facultative organisms, where it reducesfumarate to succinate in the reverse of the SDHreaction. Fumarate reductase can be membranebound and participate in anaerobic respirationwith fumarate as the terminal electron acceptor,or it can be a soluble enzyme localized in thecytoplasm (85). SDH and fumarate reductasecatalyze the same reactions, but their equilib-riums are shifted toward succinate oxidation andfumarate reduction, respectively. In organismslike Escherichia coli containing both SDH anda membrane-bound fumarate reductase, the for-mer enzyme is repressed during anaerobicgrowth, and the latter is repressed during aerobicgrowth (45, 89). SDH is also repressed to variousextents during aerobic growth on glucose in sev-eral bacteria (68, 81).

Certain organisms contain enzymes with cat-alytic properties somewhere between SDH andfumarate reductase (85). This review will berestricted to SDH, a membrane-bound enzymewhose primary function is oxidation of succinateto fumarate. Membrane-bound fumarate reduc-tase was reviewed recently by Kroger (57).

DETERMINATION OF SUCCINATEDEHYDROGENASE (SDH) ENZYME

ACTIVITYSDH activity is conveniently assayed by the

succinate-dependent reduction of artificial elec-tron acceptors, usually dyes which change colorwhen reduced (3). In the most widely used as-say, reduction of 2,6-dichlorophenol-indophenol(DCIP), with 5-N-methyl phenazonium sulfate(PMS) as intermediate electron carrier, is meas-ured. However, SDH is inhibited at high PMSconcentrations. To estimate maximal activity itis thus important to measure activity with in-creasing PMS concentrations and extrapolate to

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SUCCINATE DEHYDROGENASE 543

infinite concentration (86). PMS and also Wiirs-ters blue (a semiquindimine radical of N,N,N',N'-tetramethyl phenylendiamine) (2, 3, 98)can accept electrons not only directly from SDH,but also from other components of the respira-tory chain.DCIP and dyes like methylene blue accept

electrons from respiratory chain componentsdownstream from SDH and not at the level ofthe enzyme. If PMS is excluded when mem-

brane-bound SDH activity is measured withDCIP, the results become quantitatively differ-ent, e.g., the velocity of transport of electrons tothe redox components of the respiratory chainthat are electron donors to DCIP and theirconcentration in the membrane will affect thevelocity by which DCIP is reduced by succinate.Ferricyanide has also been used to measure

SDH activity. The soluble purified reconstitu-tively active enzyme has two types of ferricya-nide reducing activities, a "low-Km site" and a"high-Km site" (96). Only the high-Km site isexpressed in the membrane-bound enzyme (seebelow, Reconstitution of Membrane-BoundSDH).Due to the vectorial structure of the mem-

brane, the diffusion barrier and the asymmetri-cal distribution of membrane proteins, the activ-ity measurements of membrane-bound enzymeslike SDH are more complicated than those ofsoluble enzymes. Membrane preparations whichcontain vesicles with unknown orientation ofSDH are often used. Sealed membrane vesicleshave permeability barriers to substrate and alsoelectron acceptors. Most of the activity deter-minations made on bacterial SDH are con-

founded by permeability barriers. Inefficientelectron acceptors or efficient acceptors at asingle concentration have often been used. Muchof the published results on the enzymology ofbacterial SDHs has to be interpreted with care

(34, 50, 53, 72, 77).The succinoxidase of mitochondrial (33) and

of bacterial (79) respiratory chains can be frag-mented into segments or complexes that eachshow electron transfer activity and that can of-ten be reconstructed into a functional succinox-idase. Succinate-ubiquinone (Q) reductase (of-ten called complex II) contains SDH and is themost proximal segment of the succinoxidase.Succinate-Q reductase can be measured by re-

duction of DCIP with a quinone as intermediateelectron acceptor.

ORIENTATION OF SDH IN MEMBRANESSDH is the only membrane-bound enzyme of

the Krebs cycle in both bacteria and mitochon-dria. Membranes are not freely permeable todicarboxylic acids. As the substrate, succinate,is produced and the product, fumarate, is metab-olized in the cytoplasm it is likely that the activesite ofSDH is located on the cytoplasmic side ofthe membrane. The orientation ofSDH in mem-branes from various species has been determinedby different methods. The results are compiledin Table 1.The most convincing results on the orienta-

tion of SDH are those in which sealed mem-

branes of both orientations, i.e., "right side out"and "inside out" have been used. Right-side-outbacterial membranes are easily obtained in theform of protoplasts. Chromatophore membrane

TABLE 1. Orientation ofSDH in bacterial and mitochondrial membranesOrganism or organelle Orientation Method used to determine orientation Reference

Bacillus subtilis Outside and inside Electron acceptors and trypsin treat- 56ment

Inside Antibody adsorption Hederstedt et al., un-published data

Micrococcus lysodeik- Inside Antibody adsorption 74ticus

Rhodospirillum rub- Inside Trypsin and a-chymotrypsin treat- 66rum ment, enzymatic iodination

Rhodopseudomonas Inside Electron acceptors and trypsin treat- 91sphaeroides ment

Inside Antibody adsorption 26Inside Reconstitution of succinoxidase from 47

soluble SDH and membrane vesiclesBeef heart mitochon- Matrix side DABS labeling and antibody adsorp- 63

dria tionMatrix side Reconstitution of succinoxidase from Review in 24

soluble SDH and alkali-treatedETP, electron acceptors, electronspin resonance, and availability tosuccinate

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544 HEDERSTEDT AND RUTBERG

vesicles and mitochondrial electron transportparticles (ETP) are examples of mainly inside-out membranes. When the orientation of themembrane is known, the sidedness of SDH can

be investigated by several techniques.One technique involves the use of different

electron acceptors that can accept electrons atthe level of the enzyme (and preferably at thesame site) but have different membrane perme-

abilities. Enzyme activity is then measured withthe different acceptors on a membrane prepa-

ration with a known orientation. Another ap-

proach to studying the sidedness of SDH is touse a membrane-impermeable electron acceptorsuch as ferricyanide (54). These kinds of exper-

iments thus indicate on which side of the mem-brane the electron donor site(s) is located. Pro-teolytic enzymes, impermeable to membranes,that degrade SDH have also been used to estab-lish sidedness. Fragmentation of SDH is de-tected by loss of enzymatic activity or loss ofspecific polypeptides or both. Adsorption of an-timembrane antibody by various membranepreparations and subsequent qualitative andquantitative analysis of the unadsorbed anti-bodies by crossed immunoelectrophoresis havebeen successfully used to elucidate the orienta-tion of SDH and other membrane-bound com-ponents (26, 73, 74). Immunoprecipitates con-

taining SDH can be identified by zymogramstaining. Unfortunately, the specificity of theantibodies that give rise to SDH-staining im-

munoprecipitates in crossed immunoelectropho-resis and the composition of the respective an-tigen are unknown in most cases. It is possiblethat the antibody reacts with membrane com-

ponents that are attached to SDH in the deter-gent-solubilized enzyme. It is then the orienta-tion of the attached components in the mem-brane that is determined. SDH-staining immu-noprecipitates may show heterogeneity (26, 74),

indicating inefficient solubilization or proteolyticmodification.SDH has, to our knowledge, been located ex-

clusively on the inside (cytoplasmic or matrix)of the membrane in bacteria and mitochondriain all studies except one (56) (Table 1).

In these experiments a single type of mem-brane preparation from Bacillus subtilis wasstudied. The orientation ofSDH was determinedby using two electron acceptors, PMS and 5-N-methyl phenazonium-3-sulfonate with differentmembrane permeabilities. Both were assumedto accept electrons directly from SDH. However,it has later been shown that at least PMS can

accept electrons from respiratory strain compo-nents downstream from SDH (7). The results ofantibody adsorption experiments with B. sub-tilis intact protoplasts, lysed protoplasts, andTriton X-100-solubilized membranes indicatethat SDH is located exclusively on the inside ofthe membrane (Hederstedt et al., unpublisheddata).

STRUCTUREThe simplest bacterial enzyme preparation

with SDH activity was purified from Rhodos-pirillum rubrum chromatophore membranes byHatefi and co-workers (22). SDH was releasedfrom the membranes with the chaotropic ionperchlorate in the presence of succinate anddithiotreitol as protective agents. The enzymewas fractionated and concentrated by AmSO4precipitation. The purified enzyme is water sol-uble and has a molecular weight on gel filtrationof about 100,000. It contains covalently boundFAD, nonheme iron, and acid-labile sulfur (Ta-ble 2). Its composition is very similar to theenzyme isolated from beef heart mitochondria(21). Both contain equimolar amounts of twounequal subunits noncovalently bound to eachother. The larger subunit, Mr 60K and 70K,

TABLE 2. Composition ofpurified SDHCovalently Nonheme FeSX(

Organism or organ- Compnent Mol wt bound FAD mol/mol of Sx (mol/mol Ratio Polarityelle (K) (mol/mol of protel ) of protein) (mol/mol) index (%)"

proteiproninR. rubrum SDH lOOb 1 8 8

Fp 60c 1 Present Present 1 43Ip 25c 0 Present Present 1 42

Beef heart SDH llOb 1 8 8mitochondriad Fp 70c 1 4 4 1 44

Ip 27c 0 4 4 1 48

a Calculated as the sum of the mole fractions of polar amino acids in the polypeptide as described by Capaldiand Vanderkooi (10).

b Molecular weight determined by gel filtration.c Molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.d Beef heart mitochondria data from references 16, 21, and 80.

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respectively, contains covalently bound FAD.This subunit is called Fp (flavoprotein). Thesmaller subunit, Mr 25K and 27K, respectively,is called Ip (iron protein). Upon repeated freeze-thawing of SDH in the presence of sodium tri-chloroacetate the subunits dissociate. Fp thenforms an insoluble aggregate, whereas Ip re-mains soluble. Reconstitution of active enzymefrom the dissociated subunits has not yet beenaccomplished.The amino acid composition of the SDH sub-

units of the two enzymes is very similar, al-though the mammalian enzyme is slightly larger.The bacterial Ip is less polar than the mamma-lian Ip (22). Structural and functional similari-ties between the two enzymes are also expressedin reconstitution experiments of the mammaliansuccinate-Q reductase and succinoxidase. Thebacterial SDH can substitute for the mammalianenzyme to form a hybrid reductase (35) andoxidase (34), respectively.Both Fp and Ip contain nonheme iron and

acid-labile sulfur that, together with cysteinylresidues, are the building blocks of several iron-sulfur centers. These centers render solubleSDH sensitive to inactivation by oxygen. Theenzyme should, therefore, be kept under anaer-obic or reducing conditions in the presence ofsuccinate. The exact stoichiometry, localization,and function of each iron-sulfur center in themammalian enzyme are not known (4, 6, 14, 71,84). Reports on the Rhodopseudomonas sphae-roides (47) and R. rubrum (11, 34) SDH suggestthat photosynthetic bacteria have similar sets ofiron-sulfur centers. The general view is that theFp subunit contains two Fe2SX2 (Sx indicatesacid-labile sulfur) clusters, designated S-1 andS-2. Center S-1 is reduced by succinate. CenterS-2 has a very low redox potential, and it can bereduced by dithionite. The Ip subunit probablycontains a Fe2SX4 HiPiP-type iron-sulfur center,designated S-3. Center S-3 is very susceptible todestruction by oxygen in the soluble enzyme.This center is essential for expression of the lowKm site for ferricyanide, and it is involved in theelectron transport from succinate to quinone inthe succinate-Q reductase. The substrate bind-ing site ofSDH is located in the Fp subunit (52).Reducing equivalents from the oxidation of suc-cinate are transferred via the FAD to iron-sulfurcenter S-1, S-3, and ultimately to quinone. Elec-tron transport to quinone can be inhibited by 2-thenoyltrifluoroacetone (35, 64, 87) or carbox-anilides (e.g., carboxin) (64). Both inhibitorsblock electron transfer between center S-3 andquinone, but they do not affect the reduction ofcenter S-3 by succinate. Purified SDH has no Qreductive activity, and SDH activity is not in-

hibited by 2-thenoyltrifluoroacetone or car-boxin. The binding site for these electron trans-fer inhibitors has been suggested to involve bothSDH and a membrane component (15, 35). Re-cent results by Ramsay et al. (78) indicate thatSDH does not bind carboxin. Purified beef heartsuccinate-Q reductase was photoaffinity labeledwith a carboxin analog carrying an azido group.The azidocarboxin was preferentially linked tothe hydrophobic polypeptides CI0-3 + CII-4 butnot to the SDH subunits. Isolated C0I3 and CII4were not labeled by azidocarboxin.

GENETICSWell-characterized mutants are powerful tools

in studies on the arrangement and control ofstructural genes and also in the elucidation ofenzyme structures and mechanisms of enzymeaction. Different methods have been used forthe isolation of SDH mutants in bacteria. E. coliSDH mutants can be enriched and selected forby their ability to grow on fumarate, but not onsuccinate, as the sole carbon source (45). InAgrobacterium tumefaciens, an SDH negativemutant was found among mutants able to growon hexoses but not on Krebs cycle intermediatesor pyruvate (13). A specific and elegant methodto obtain E. coli SDH mutants is to use a-ketoglutarate dehydrogenase mutants. Thesemutants cannot grow aerobically in a glucoseminimal medium without succinate or lysine andmethionine. However, double mutants that lackboth SDH and a-ketoglutarate dehydrogenaseactivity can grow on glucose alone (19). Thereason is that the low intracellular concentrationof succinate that is needed for biosynthetic pur-poses is depleted by SDH in a-ketoglutaratedehydrogenase mutants. When SDH is inacti-vated by mutation, the succinate level in the a-ketoglutarate dehydrogenase mutant will behigh enough to permit growth on glucose alone.E. coli SDH mutants show a low SDH activitydue to the membrane-bound fumarate reductaseworking in reverse (89).For isolation of B. subtilis SDH mutants acid

accumulation (12) and defective sporulation (27,83) of mutants defective in Krebs cycle enzymeshave been exploited. Sporulation defects are as-sociated with characteristic pigmentation andcolony morphology. Acid-producing bacterialcolonies can be identified on plates containingcalcium carbonate by the formation of halosaround the colony or by a change in color onplates containing a pH indicator. After primaryselection of acid-producing bacteria, SDH mu-tants are identified by the in vivo accumulationof radioactive succinate when grown in the pres-ence of radioactive glutamate (27, 83), or by in

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vitro SDH zymogram staining of lysed bacterialcolonies by a replica technique (25). The SDHnegative phenotype is finally confirmed by invitro SDH assay ofmembrane preparations. Thekinds of mutants obtained are of course influ-enced by the selection procedure and themethod used for identifying the defect. For in-stance, a mutant may have a defective SDH invivo but have a normal SDH activity with arti-ficial electron acceptors in vitro.

E. coli mutants capable ofgrowth on furmaratebut not on succinate and lacking in vitro SDHactivity were isolated by Spencer and Guest (90).Of 84 mutants, 74 had mutations in the sdh locusat 16.2 min on the E. coli chromosomal map(18). Four of the sdh mutations were nonsense,as they could be suppressed by a glutamineinserting amber suppressor gene. The polypep-tide composition of cytoplasmic membranesfrom two nonsense mutants was analyzed insodium dodecyl sulfate-polyacrylamide gel elec-trophoresis. A polypeptide, Mr 67K, was missingin the mutants, but was present in the sup-pressed mutants and the wild type. This poly-peptide is most probably the Fp subunit ofSDH.The experimental results strongly suggest thatthe sdh locus in E. coli contains the structuralgene for the Fp polypeptide.

All SDH-negative mutants isolated in B. sub-tilis carry mutations in the citF locus located at2550 on the chromosomal genetic map (44, 69,83). Two mutants with reduced in vitro SDHactivity have been described, and they havecorrespondingly reduced amounts of SDH pro-tein. The respective mutations do not map inthe citF locus (69, 82). The relative order of 11citF mutations has been established by transfor-mation crosses (69; manuscript in preparation).The presence and location of Fp and Ip havebeen determined in each mutant by using sub-unit-specific antibody (41), and the cytochromespectra of the mutant membranes have beendetermined. One mutant, citF1Ol, contains aninactive, membrane-bound SDH complex, i.e.,Fp, Ip, and cytochrome b5m (see below, Mem-brane Binding). The remaining 10 mutantseither totally lack the Fp or Ip subunits (or both)or have one or both of them located in thecytoplasm. No SDH enzyme activity was foundin the cytoplasmic fraction of any citF mutant.Based on the phenotypes of the citF mutantsand the position of the respective mutations, thecitF locus has been divided into three functionalregions (Fig. 1). Mutations in the left region(citF78 to citF12) all contain cytoplasmic Fpand Ip. The three leftmost mutants lack spec-trally detectable cytochrome bw. These mu-tants are unable to bind Fp and Ip to mem-

-~ ft A% dnaB

7842 4412l0gs698 11 2 10383i I I I I J I I I --Mutation

- TranslationCytbh55 Fp rp product

FIG. 1. Present genetic map of the citF locus in B.subtilis and the proposed citFgene products.

branes. This region of the citF locus is thoughtto contain the structural gene for apocyto-chrome b58. Mutations which lie between theleft and middle region may affect either cyto-chrome b5m or the Fp subunit. The middle regionof the citF locus (citF69 to citF2) encompassesmutants which contain cytochrome b55 butwhich lack Fp and probably Ip. This region maycode for the Fp subunit. Finally, the right region(citF103 and citF83) is characterized by piutantswhich contain cytochrome b56 and cytoplasmicFp but lack Ip. Consequently, this region issuggested to contain the structural gene for theIp subunit.

In summary, it is suggested that the citF locuscontains the structural genes for each of thethree subunits of the SDH complex, and thattheir order from left to right is cytb-Fp-Ip (Fig.1). Little is known about the genetic control ofSDH synthesis in B. subtilis or in any otherorganism. Recently, we have studied two mu-tants which lack all three subunits of the SDHcomplex. One of these mutants (isolated andkindly provided by S. A. Zahler) has phage SP-beta integrated into the citF locus. The othermutant, obtained from ethylmethanesulfonate-treated spores by the method described by Itoand Spizizen (48), most likely carries a pointmutation in the citF locus since it can revert towild type. The properties of the above mutantssupport the notion that citF contains the struc-tural genes for each of the three subunits of theSDH complex and that these genes are coordi-nately controlled.Saccharomyces cerevisiae mutants unable to

grow on nonfermentable substrates and whichhave a very low in vitro SDH activity wereisolated by De Kok et al. (23). Two mutants hadlow levels of covalently bound FAD and iron-sulfur center S-3 in the mitochondrial innermembrane. These mutants carried allelic nu-clear mutations and most probably lack mem-brane-bound Fp and Ip subunits. Another mu-tant, belonging to another complementationgroup, had a less severe reduction of covalentlybound FAD. This mutant may contain a mem-brane-bound, inactive SDH.

%a. vM so u I t r iivo,%o leuA,%o,s-,"-I

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Another approach to SDH genetics is to iso-late mutants resistant to specific enzyme or elec-tron transport inhibitors. The fungicide carboxinhas been shown to inhibit mitochondrial (64, 95)as well as bacterial (94) electron transport fromSDH to quinone. Mutants with in vitro carboxin-resistant SDH activity were isolated from theobligate aerobic ascomycete Aspergillus nidu-lans and the basidiomycete Ustilago maydis.Mutations in at least three different nucleargenes conferred resistance to carboxin in A. nid-ulans (31). It is unclear how SDH activity wasactually measured, but as carboxin inhibited theSDH activity almost completely in the wild-typemembranes, SDH was probably measured withferricyanide as the electron acceptor. A similarchromosomal mutation in U. maydis resulted ina carboxin-resistant, succinate-dependent reduc-tion of ferricyanide or DCIP (28, 29). The mu-tation has probably decreased the affinity of thebinding site for the inhibitor. However, as it isnot known whether this binding site is wholly orpartly situated on SDH or on some other mem-brane component, it cannot yet be concludedthat the above mutations are located in the SDHstructural genes.

MEMBRANE BINDINGIntegral membrane proteins are hydrophobic

or have hydrophobic domains which penetratethe lipid bilayer ofthe membrane. Such proteinscan only be extracted by procedures which de-stroy the integrity of the membrane. Detergentsare commonly used to break up membranes, andthe core lipid surrounding the hydrophobic partof a membrane protein can be substituted for bya detergent micelle. The solubilized protein thusbinds detergent, and it will aggregate on remnovalofdetergent. Hydrophilic, water-soluble proteinsgenerally do not bind detergent. Detergent bind-ing and solubility can thus be used to operation-ally classify proteins as hydrophobic or hydro-philic (92).SDHs isolated from R. rubrum chromato-

phore membranes and beef heart mitochondrialinner membranes are both soluble proteins withpolarity indexes above 40% (22). The mitochon-drial enzyme shows some dimerization at highprotein concentrations (16), but the sedimenta-tion constant of the enzyme in the ultracentri-fuge is not changed by the presence of the non-ionic detergent Triton X-100 or the more pow-

erfully disaggregating ionic detergents sodiumdodecyl sulfate and cetyldimethylethylammon-ium bromide indicating no detergent bindingand no aggregation of SDH. Based on the crite-ria of detergent binding, polarity index, and sol-

ubility, purified SDH does not have the charac-

teristics of an integral membrane protein. Re-constitution experiments (8, 32) and experimentswith cytochrome deficient mutants (41, 46), dis-cussed later in this article, strongly suggest thatSDH is bound to specific limiting sites in themembrane, rather than by hydrophobic inter-action with the lipid bilayer. The specific com-ponent(s) involved in anchoring SDH to themembrane should be hydrophobic and requiredetergent for solubilization. Proteins interactingmainly nonhydrophobically with SDH in themembrane should be solubilized by nonionicdetergent with SDH still attached to them. Ide-ally, there will be one protein per micelle whensolubilization is done in the presence of excessdetergent micelles (92).

Triton X-100 treatment ofNeurospora crassamitochondrial inner membranes at low ionicstrength results in the solubilization of a mon-odisperse succinate-Q reductase. Each reductasemolecule binds one detergent micelle and is com-posed of three different subunits (99) (Table 3).In addition to Fp and Ip subunits of SDH, thereductase contains a low-molecular-weight (Mr,14K) cytochrome b. The cytochrome is the de-tergent binding, hydrophobic part of the reduc-tase (H. Weiss, personal communication). A sim-ilar monodisperse SDH-cytochrome b complexcontaining SDH and a detergent binding (un-published experiments) cytochrome bw poly-peptide in an equimolar amount to SDH hasalso been isolated from Triton X-100-solubilizedB. subtilis membranes (39, 40) (Table 3). It isnot known if this complex has quinone reductaseactivity.Mammalian succinate-Q reductase (complex

II) extracted with bile acids from beef heartmitochondria contains four polypeptides. Twoof these are the SDH Fp and Ip subunits. Com-plex II also contains a low molecular weightcytochrome b50 which possibly is the CII-3 poly-peptide. The fourth polypeptide is called CII (9,36, 38). The four polypeptides are present inequimolar amounts. Also, the molar ratio ofcovalently bound FAD to protoheme is about 1in complex II (38). The apparent molecularweight obtained in sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of CII 3 and C114 isstrongly influenced by the buffer system used inelectrophoresis (9). These polypeptides have ahigh content of apolar amino acids, indicatingthat they are quite hydrophobic (35). Most prob-ably, the smallest polypeptides found in thedetergent-solubilized SDH complexes from N.crassa, B. subtilis, and beef heart mitochondriaare integral membrane proteins.The labeling of mitochondria and submito-

chondrial particles, which are inside-out mem-

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TABLE 3. Characterized SDH-cytochrome b complexesaMol wt (K) of: a-Absorption

Organism or organ- Detergent used to Quinone reductase maximum ofelle solubilize complex activity Fp CItochrome b cytochrome

,3p (nm at 25°C)

B. subtilisa Triton X-100 Not deternined 65 28 19 Absent 558N. crassab Triton X-100 Present 72 28 14 Absent 559Beef heart Bile salt Present 70 27 17 to 13.5 14 to 7 560

mitochondriaca B. subtilis data from references 39 and 40.b N. crassa data from reference 99.'Beef heart mitochondria data from references 9 and 38.

brane vesicles, with the water-soluble, mem-brane impermeable reagent 35S-labeled diazo-benzene sulfonate results in incorporation of la-bel into CII-3 in mitochondria (63). Photoaffinitylabeling of complex II in egg lecithin vesicleswith (arylazido) phospholipids results in cross-links between reagent and polypeptides CII-3 andCrH4 (30). Also, the Ip, but not the Fp subunit ofSDH, is linked to the hydrophobic reagent.Cross-links to these complex II polypeptideswith the (arylazido) phospholipids occur to ap-proximately the same relative extent whetherthe reactive nitrene group is located in the headgroup region or at the CH3 end of one of thefatty acid chains. Peptide CII04 can be bound toSDH in a stoichiometric amount also in theabsence of CII-3 (1). However, the CII-3 componentis necessary for expression of beef heart succi-nate-Q reductase activity. Together, these find-ings on complex II indicate that the CII-3 andCII-4 polypeptides penetrate into the membranephospholipid bilayer. The CII-3 polypeptide isexposed on the outer surface of the mitochon-drial inner membrane and spans the membraneto functionally interact with SDH (probably theIp subunit) either directly or via CII4 on theinside (30).

RECONSTITUTION OF MEMBRANE-BOUND SDH

The structural and functional combination ofpurified soluble SDH with soluble or particulatecomponents of the respiratory chain is calledreconstitution. The first successful reconstitu-tion was reported by Keilin and King (51), whoreconstituted succinoxidase activity from solubleSDH and an alkali-treated heart muscle prepa-ration. Alkali treatment (pH 9.3 at 38°C underargon) of beef heart mitochondrial ETP or com-plex II inactivates both succinate-PMS and suc-cinate-Q reductase activity. SDH is protected bythe presence of succinate in the incubationbuffer. The loss of activity after alkali treatmentis not due to release of SDH since most of the

SDH-flavin remains bound to the particles.SDH is released from ETP treated at pH 10.0,but the reconstitutive activity of the particles isdestroyed at this pH. When reconstitutively ac-tive SDH is added to alkali-treated ETP orcomplex II, the enzyme is bound to the particu-late preparations, which then regain succinoxi-dase and succinate-Q reductase activity, respec-tively. Particles reconstituted with excess SDHcontain twice as much SDH-flavin as originallypresent (32, 88). In R. sphaeroides chromato-phore membranes, SDH activity and iron-sulfurcenter are removed by a single wash at pH 9.1under anaerobic conditions and with succinatein the buffer (47). This treatment does not de-stroy the integrity of the chromatophore mem-branes, but succinoxidase activity is abolished.When the released SDH is added back to thealkali-treated membranes, succinoxidase activ-ity is reconstituted. Also, in Mycobacteriumphlei, SDH can be dissociated from ETP byalkali treatment under argon with subsequentloss of succinoxidase activity (49). The releasedSDH was fractionated by AmSO4 and chroma-tography on hydroxyapatite. Neither the puritynor the composition of the released M. phleiSDH was reported. Succinoxidase activity couldbe reconstituted by addition of the SDH prepa-ration to M. phlei ETP treated with alkali orsilicotungstate.The chaotropic ion perchlorate has been used

successfully to selectively release SDH in a re-versible manner from beef heart complex II andfrom R. rubrum chromatophore membranes, re-spectively (22, 37). A reconstitutively activeSDH is obtained when extraction is made in areducing environment and in the presence ofsuccinate. The perchlorate-extracted R. rubrumSDH can reconstitute a hybrid succinoxidasewhen mixed with alkali-treated beef heart sub-mitochondrial particles (34). Also, the mamma-lian enzyme can interact with alkali-treated R.rubrum chromatophore membranes, but in thiscase reconstitution is less efficient than with the

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homologous membrane. Attempts to extractSDH from B. subtilis (unpublished data) or

Micrococcus lysodeikticus (17) membranes withperchlorate have been unsuccessful Generally,purified SDH is found to give better reconstitu-tion when it is prepared fresh, protected againstoxygen, and kept in the presence of succinate.The reconstitutive activity of SDH does notcorrelate with the succinate-PMS activity of thesoluble enzyme. When purified SDH is exposedto oxygen the reconstitutive activity decaysfaster than does the succinate-PMS activity.SDH solubilized and purified with differentmethods often has similar activities in the PMSassay, but greatly different reconstitutive activ-ities as has been observed both with mammalianand bacterial SDH (3, 49, 88). An explanationfor this can be found in the observation that inthe mammalian enzyme, the S-3 iron-sulfur cen-

ter, the low-Km ferricyanide activity and thereconstitutive activity decay in parallel underaerobic conditions (96). This suggests that theintegrity of S-3 is necessary for reconstitution.This iron-sulfur center is very fragile in thesoluble enzyme, but it is stable in the particulateenzyme where it is protected against oxygeninactivation. The fact that SDH has a low-Kmferricyanide site exposed in the soluble enzymeand hidden in the particulate enzyme can beused to differentiate between the two states ofthe enzyme. Different preparations from beefheart mitochondria that can bind SDH and pro-tect center S-3 against oxygen inactivation andthat are active in reconstitution of 2-thenoyltri-fluoroacetone-sensitive succinate-Q reductasehave been described by several groups (1, 35, 97,101, 102). All of these preparations are particu-late and contain one or both of the low-molecu-lar-weight complex II polypeptides CII-3 andCn-4, various amounts of cytochrome b, andphospholipids. Hatefi and Galante (35) isolateda cytochrome b preparation which contains bothCII.3 and CII4 and a stoichiometric amount ofcytochrome b50. The protoheme binding com-

ponent was not identified. Reconstitution of suc-cinate-Q reductase with this preparation in-volves a structural association between cyto-chrome b and SDH. The cytochrome b50 inter-acts electronically with both SDH and quinone.These results indicate both a structural and a

functional role of cytochrome b560 in succinate-Q reductase. Ackrell et al. (1) described a prep-

aration similar to that isolated by Hatefi andGalante, but with a lower cytochrome b content.Chymotrypsin treatment selectively removesthe CII3 polypeptide. It was further shown thatCII binds SDH in the absence of CII 3, but thelatter polypeptide is essential for succinate-Q

reductase activity. A polypeptide called QPswhich shows succinate-Q reductase activitywhen mixed with soluble, reconstitutively activeSDH was described by Yu and Yu (101, 102).The most highly purified QPs gives only onemain band, Mr 15K, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a lowactivity in reconstitution. Another QPs prepa-ration with high reconstitutive activity con-tained two main polypeptides (Mr 17K and 15K,respectively) and some cytochrome b. Vinogra-dov et al. (97) have described a reconstitutivelyactive preparation in which about 80% of theprotein had a relative molecular weight less than13K.

BIOSYNTHESIS AND MEMBRANEBINDING OF SDH IN BACILLUS

SUB TISRecently, information about biosynthesis and

membrane binding in vivo of SDH has beenobtained from studies of SDH and heme mu-tants of B. subtilis (41, 46). A 5-aminolevulinicacid (5-ala) auxotroph cannot make heme and,consequently, not cytochromes when grownwithout 5-ala. The strict aerobe B. subtilis ap-parently contains an excess of cytochromes be-cause the 5-ala auxotroph grows at an undimin-ished rate for about three generations without5-ala. Membranes isolated from 5-ala-starvedauxotrophs have a strongly reduced level of cy-tochromes, especially cytochromes b and c (46).Bulk membrane protein synthesis proceeds atan undiminished rate during the first three gen-erations of growth of a 5-ala auxotroph in 5-ala-free medium (46). However, no membrane-bound SDH is made, and there is no net increasein membrane-bound SDH activity. Fp and Ipsubunits are still synthesized in the cytoplasm,but they are not associated and lack detectableenzymatic activity (41). When heme synthesis isallowed to resume, the cytoplasmic SDH sub-units bind to the membrane at an initial rateseveral times higher than the growth rate of thebacteria (46), with a concomitant rise in mem-brane-bound SDH activity. Membrane bindingof the soluble SDH subunits occurs also whenprotein synthesis is blocked by chloramphenicol.These results strongly suggest the presence of alimiting number of specific SDH-binding sites inthe B. subtilis cytoplasmic membrane. Evidencethat cytochrome b5w is (part of) this binding siteis provided by the observations that (i) cyto-chrome b5,m is present in stoichiometric amountsin the Triton X-100-solubilized, purified mem-brane SDH complex (39, 40) and (ii) mutantswhich specifically lack cytochrome b&% containthe SDH Fp and Ip subunits in the cytoplasm

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(41). A model for biosynthesis and membranebinding of SDH in B. subtilis is shown in Fig. 2.Fp and Ip subunits are synthesized as solublepolypeptides and are not associated in the cy-toplasm. The soluble subunits are precursors tothe membrane-bound enzyme (41). The solublesubunits have the same mobility in sodium do-decyl sulfate-gel electrophoresis as the mem-brane-bound SDH subunits (40, 41). It is prob-able that apocytochrome b&% is inserted into themembrane, e.g., during 5-ala starvation. In re-constitution experiments with hemeless mutantsit has been shown for Staphylococcus aureusthat the apocytochrome of b-type cytochromesis inserted into the membrane probably in con-

AAD Fp 7Ip

isdU apocytochrome b

ooooooooooooooooooooooooooooutside

FAD| lBEJLholocytochrome b

ooooooooooLiIoooooooooooooooooooooooooooooooooooo

SDH-cytoC

Coooooooooooooooooooooooo

FIG. 2. Model for the synthesis andthe B. subtilis SDH-cytochrome b5N comp

and Ip subunits are synthesized as solubFAD is covalently bound to the solubleApocytochrome b is inserted into the m,connection with its synthesis (A). As pibound to apocytochrome b a binding site(and Ip subunits is exposed (B), whichfollowed by the assembly of a functionalbound SDH-cytochrome b558 complex (C).

nection with apocytochrome synthesis (59). In-sertion ofprotoheme into membrane-bound apo-cytochrome b in B. subtilis is suggested to ex-pose a binding site for the Fp and Ip subunits ofSDH. FAD is covalently added to the Fp subunitbefore membrane binding (41). It is not knownwhen the iron-sulfur centers are incorporatedinto the subunits; consequently, their role inmembrane binding of SDH is uncertain. Theisolation of B. subtilis mutants with enzymati-cally inactive, membrane-bound SDH rules outthe possibility that enzyme activity is essentialfor membrane-binding (unpublished experi-ments). The tight association of the Fp and Ipsubunits in soluble SDH isolated from beef heartmitochondria or R. rubrum is in apparent con-trast to the free precursor subunits found in theB. subtilis cytoplasm. Also, the insolubility ofFp after chaotrop-induced dissociation of thetwo subunits of the soluble mammalian or R.rubrum SDH contrasts with the soluble Fp sub-unit found in B. subtilis. This may indicate thatthe interaction between the two subunits is dif-ferent in different species. Since the cytoplasmicsubunits are precursors to the membrane-boundenzyme, it is also possible that they lack iron-sulfur centers and that these are required for thesubunits to assume a conformation which pro-motes their tight association. In vitro reconsti-tutions of isolated Fp and Ip subunits has notbeen accomplished with SDH from any organ-ism. Interestingly, Hanstein et al. (32) have spec-ulated that the Fp subunit alone could expressfumarate reductase activity and that the Ip sub-unit functions to shift the equilibrium in a direc-tion favoring succinate oxidation.

SUMMARY AND SOME PERSPECTIVES

The structure of membrane-bound SDHseems to be quite similar in such widely differentspecies as cow, bakers' yeast (67), N. crassa, R.

)chrome b rubrum, and B. subtilis. In all of these speciesthe enzyme consists of two unequal subunits, a

complex larger flavoprotein and a smaller polypeptidewhich, at least in beef heart and R. rubrum, isan iron-sulfur protein. Most likely SDH has asimilar structure also in E. coli (90, J. L. Cowell,M. Raffeld, and I. Friedberg, Abstr. Annu. Meet.

assembly of Am. Soc. Microbiol. 1973, 157, p. 100) and M.lex. The Fp lysodeikticus (P. Owen, personal communica-5le proteins. tion). The very similar amino acid compositionFp subunit. of SDH from beef heart mitochondria and fromembrane in R. rubrum chromatophore membranes suggestsrotoheme i considerable evolutionary conservation in thes)for the Fpis rapidly enzyme. On the other hand, interaction of SDHmembrane- with a particular membrane and the nature of

the components that serve to bind the enzyme

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to that membrane seem quite different in differ-ent species.There have been numerous attempts to purify

SDH from various bacterial species (42, 43, 53,58, 60, 61, 76, 77, 79). Common to most of thiswork is the great difficulty experienced in tryingto separate SDH from cytochrome. Before thestructure of beef heart SDH was elucidated, itwas actually proposed that in Corynebacteriumdihtheriae SDH was a b cytochrome (75). Insome partially purified, cytochrome b-contain-ing, bacterial SDH preparations, the cytochromeis reduced by succinate (39, 42, 58, 61, 76),whereas in others it is not (77). Purified mem-brane-bound SDH from N. crassa is structurallyvery similar to the purified B. subtilis SDHcomplex. However, in the N. crassa complex thecytochrome b has not been shown to be reducedby succinate. In beef heart mitochondria twopolypeptides seem required to give a fully func-tional membrane-bound SDH (see above, Mem-brane Binding). The CIIn3 polypeptide, or an Mr9K fragment thereof, is necessary for the expres-sion of succinate-Q reductase activity, whereaspolypeptide CnI alone seems sufficient for mem-brane binding of SDH (1). To our knowledgethere are only two reports of binding of SDH invivo to a membrane which lacks cytochrome b.Chromatophore membranes from Chromatiumsp. strain D grown heterotrophically with suc-cinate as carbon source have a high SDH activ-ity, although the membranes do not containprotoheme, the characteristic prosthetic groupof all b type cytochromes (20). A yeast mutantthat required 5-ala to make cytochrome con-tained about 25% of the enzyme activity whenstarved for 5-ala compared with cells grown with5-ala (100).SDH is located on the cytoplasmic side of the

cytoplasmic membrane in bacteria and on thematrix side of mitochondrial inner membranes.There is an important difference, however, be-tween the biogenesis of SDH in eucaryotic andprocaryotic cells. In eucaryotic cells, or at leastin mammals (62), bakers' yeast (23), and N.crassa (25), the SDH gene(s) is nuclear. Sincethe mitochondrial protein-synthesizing machin-ery is completely separated from that of the restof the cell, SDH has to be synthesized in thecytoplasm and then transported through theouter and inner mitochondrial membrane, to beattached ultimately to the matrix side of theinner membrane. In bacteria there is no suchtransport problem, and both subunits are syn-thesized, assembled, and membrane-bound onthe same side. Soluble cytoplasmic and enzy-matically active SDH, which is suggested to bea precursor of mitochondrial SDH, has been


found in pea cotelydons (65) and in yeast (5).The function of SDH as a Krebs cycle enzymeis similar in both procaryotic and eucaryoticcells. However, the evolution of cell organellesand membrane systems in eucaryotic cells mayhave favored altemative and more complex so-lutions to the problems of membrane binding ofSDH and transfer of electrons from succinateoxidation to the respiratory chain than the seem-ingly simplest one of direct structural and func-tional coupling between SDH and cytochrome.We think that future work on SDH will focus

to a large extent on the structure, membranetopology, and biogenesis of the enzyme as wellas the succinate-Q reductase complex. Amongthe problems to be solved are the following. (i)How are the SDH structural genes organizedand controlled? (ii) How and when are the pros-thetic groups (flavin and the iron-sulfur centers)incorporated in SDH? (iii) What are the factorsinvolved in membrane binding of SDH? Someofthese and other problems can only be resolvedby using organisms in which there exist well-developed genetic systems.

ACKNOWLEDGMENTSWe are grateful for the patient secretarial help

provided by Ruth Du Rietz, Birgit Sehlberg, andYvonne Astrom.Work done in our laboratory was supported by

grants from the Swedish Medical Research Counciland from Karolinska Institutet.

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