Role of complex II in anaerobic respiration of the parasite ...

Review

Role of complex II in anaerobic respiration of the parasite mitochondriafrom Ascaris suum and Plasmodium falciparum

Kiyoshi Kita *, Hiroko Hirawake, Hiroko Miyadera, Hisako Amino, Satoru TakeoDepartment of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,

Japan

Received 26 June 2001; received in revised form 17 September 2001; accepted 12 October 2001

Abstract

Parasites have developed a variety of physiological functions necessary for existence within the specialized environment ofthe host. Regarding energy metabolism, which is an essential factor for survival, parasites adapt to low oxygen tension inhost mammals using metabolic systems that are very different from that of the host. The majority of parasites do not use theoxygen available within the host, but employ systems other than oxidative phosphorylation for ATP synthesis. In addition,all parasites have a life cycle. In many cases, the parasite employs aerobic metabolism during their free-living stage outsidethe host. In such systems, parasite mitochondria play diverse roles. In particular, marked changes in the morphology andcomponents of the mitochondria during the life cycle are very interesting elements of biological processes such asdevelopmental control and environmental adaptation. Recent research has shown that the mitochondrial complex II plays animportant role in the anaerobic energy metabolism of parasites inhabiting hosts, by acting as quinol^fumaratereductase. ß 2002 Elsevier Science B.V. All rights reserved.

Keywords: Complex II; Quinol^fumarate reductase; Anaerobic respiration; Parasite mitochondria; Plasmodium falciparum ; Ascaris suum

1. Introduction

Parasites are generally classi¢ed as either hel-minths or protozoa. Helminths are multicellular par-asites and consist of three types: nematodes (e.g.,Ascaris suum), trematodes (e.g., Schistosoma manso-ni), and cestodes (e.g., Hymenolepis diminuta). Proto-

zoa includes unicellular parasites (e.g., malaria para-site and Entamoeba histolytica), but they di¡er frombacteria in that they have a nucleus, mitochondria,vacuole and other organelles (e.g., hydrogenosomes).These parasites are able to survive and proliferate byescaping from the host defense mechanisms, and de-velop metabolic pathways for adaptation to the spe-cialized environment in the host. Studies of such par-asitic adaptation have provided extremely interestingbiological discoveries, as well as knowledge that ispotentially useful for treatment of infectious diseases.

Recent advances in biochemistry and molecularbiology have provided new insights into basic biol-ogy, and brought many revolutionary discoveriesconcerning biological evolution and diversity. Forexample, studies of variable surface glycoprotein

0005-2728 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 2 8 ( 0 1 ) 0 0 2 3 7 - 7

Abbreviations: Fp, £avoprotein; Ip, iron^sulfur protein;CybL, large subunit of cytochrome b ; CybS, small subunit ofcytochrome b ; SDH, succinate dehydrogenase; SQR, succinateûbiquinone reductase; FRD, fumarate reductase; QFR, quinol^fumarate reductase; PEPCK, phosphoenolpyruvate carboxyki-nase; OAA, oxaloacetate

* Corresponding author. Fax: +81-3-5841-3444.E-mail address: [email protected] (K. Kita).

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have led to the discoveries of RNA editing and GPIanchoring in trypanosomes and trans-splicing ofmRNA in nematodes as well as trypanosomes [1^3]. Based on these developments, a new ¢eld calledMolecular Parasitology, in which parasitism is inves-tigated at the molecular level, is being established.

The historical connection between parasites andthe research on energy metabolism described in thispaper is extremely close, and cytochrome, whichplays a major role in the aerobic respiration in mi-tochondria, was in fact discovered during research onparasites. Approximately 80 years ago, the parasitol-ogist Keilin observed horse£y larvae with a micro-spectroscope, and discovered strong absorptionbands of 604, 564, and 550 nm. This was, in e¡ect,the discovery of cytochrome a, b, and c, and settledthe dispute over cell respiration between Warburg'stheory of oxygen activation and Wieland's dehydro-genation theory, leading to the concept of oxidativephosphorylation. Keilin described the respiratory

chain in ascarids, the most widely known parasites,in his book, Òn Cytochrome, a Respiratory PigmentCommon to Animal, Yeast, and Higher Plants', pub-lished in 1925 [4], and details of the diversity of thisrespiratory chain have recently been clari¢ed at themolecular level.

In this review, we focus on recent advances in thestudy of parasite mitochondrial complex II, whichplays such an important role in the anaerobic energymetabolism of parasites.

2. Complex II of parasitic helminths

Considerable research has been conducted on theenergy metabolism of helminths, particularly in rela-tion to nematodes such as A. suum and ¢laria. In thissection, we discuss unique features of helminth com-plex II that have been extensively studied, focusingprimarily on ascarid energy metabolism.

Fig. 1. PEPCK^succinate pathway. In aerobic metabolism in mammals and A. suum larva, PEP is converted to pyruvate by pyruvatekinase (PK), and is degraded to CO2 and water via acetyl-CoA in the TCA cycle. In contrast, in adult worm, CO2 is ¢xed byPEPCK, and OAA is produced. The NADH^FRD system, which is the anaerobic electron transport system characteristic of adult A.suum mitochondria, is involved in succinate formation, which is the ¢nal step of this pathway. Complex II in adult mitochondria func-tions as QFR in this system.


K. Kita et al. / Biochimica et Biophysica Acta 1553 (2002) 123^139124

2.1. Life cycle of A. suum and changes in respiratorychain

A. suum is the most widely known parasite, andhas been studied as a representative of human andlivestock parasites [5^7]. Its large size makes it idealfor research involving biochemical analysis. Adult A.suum inhabits the small intestine of mammals, andthe female produces between 200 000 and 400 000fertilized eggs per day. Eggs are excreted with fecesand become mature eggs containing infectious 3rdstage larvae (L3) in about 2^3 weeks at normal tem-perature (the L3 stage was previously referred to asL2, until two molts in the egg were reported by Gee-nen et al. [8]). When orally ingested by a host, theeggs reach the small intestine and hatch. A hatchedlarva invades the intestinal wall, and migrates to theliver, lung, trachea, and pharyngeal region, and ¢-nally returns to the intestine via the esophagus andstomach, and becomes an adult worm. The oxygenconcentration of the small intestine is 2.5V5%, ap-proximately one-quarter of that outside the body,and thus provides an environment of low oxygentension in which the energy metabolism of the adultdi¡ers considerably from that of the larvae and thehost. The phosphoenolpyruvate carboxykinase(PEPCK)^succinate pathway, an anaerobic glycolyticpathway, operates in the adult worm (Fig. 1), pro-ducing ATP under the low oxygen conditions. Thissystem is used by many other parasites, and has alsobeen observed in the adductor muscle of oysters andother bivalves that require energy conversion underanaerobic conditions (such as in tidal areas). It istherefore considered to be a very common pathwayfor energy metabolism in adaptation to an anaerobicenvironment [9,10].

The ¢rst half of the PEPCK^succinate pathway isthe same glycolytic pathway found in mammals, inwhich phosphoenolpyruvate (PEP) is produced.Aerobic metabolism in mammals involves the con-version of PEP to pyruvate with the use of pyruvatekinase, and decomposition to CO2 and water viaacetyl CoA and the TCA cycle. In contrast, the A.suum adult ¢xes CO2 with PEPCK to produce oxa-loacetate (OAA). The OAA is converted to malateby the reverse reaction of malate dehydrogenase andtransported into the mitochondria to produce pyru-vate and fumarate. The NADH formed during pro-

duction of pyruvate from malate is used in the re-duction of fumarate to succinate. The NADH^fumarate reductase (FRD) system, which is the an-aerobic electron transport system characteristic ofadult A. suum mitochondria, is involved in succinateformation, which is the ¢nal step of this pathway.This process is described in detail below.

Cytochrome c oxidase (complex IV), a terminaloxidase, is not detected in the respiratory chain ofadult A. suum mitochondria, and the content of ubi-quinol^cytochrome c reductase complex (complexIII) is extremely low [11]. In contrast, in A. suumlarvae, which require oxygen for their development,the energy metabolism is aerobic. A biochemical in-vestigation of mitochondria isolated from aerobicallycultivated larvae showed the composition of the res-piratory system to be almost the same as that ofmammals, as shown in Fig. 2 [12].

Quinones, which are low molecular weight electroncarriers that transfer electrons between respiratorycomplexes, also markedly change during develop-ment. Larvae employ aerobic metabolism, and theubiquinone therefore functions in the same manneras in mammals. However, in the case of the adult,the major quinone is the low-potential quinone, rho-doquinone, which plays an important role as the pri-

Fig. 2. Change of the respiratory chain during the life cycle ofA. suum. Complex I (NADHûbiquinone reductase) ; complexII (SQR); complex II (QFR); complex III (ubiquinol^cyto-chrome c reductase) ; complex IV (cytochrome c oxidase) ; UQ,ubiquinone; RQ, rhodoquinone.


K. Kita et al. / Biochimica et Biophysica Acta 1553 (2002) 123^139 125

mary component of the NADH^FRD system (Fig.3) [12,13]. Thus, A. suum is able to adapt to changesin oxygen concentration in the environment duringits life cycle by dynamic change of respiratory chain.Recent research has clari¢ed the characteristics of A.suum, in particular those of the mitochondrial elec-tron transport system of the adult, at the molecularlevel.

2.2. NADH^FRD system and complex II

As described in the previous section, the ¢nal stepof the PEPCK^succinate pathway, which plays suchan important role in the anaerobic energy metabo-lism of the A. suum adult, is catalyzed by theNADH^FRD system. The reducing equivalent ofNADH is transferred to the low-potential rhodoqui-none by NADHûbiquinone reductase complex(complex I), and ¢nally succinate is produced by

FRD activity of complex II. This system can synthe-size ATP using the coupling site of complex I even inthe absence of oxygen, although its energy e¤ciencyis low.

A similar anaerobic respiration system exists in themitochondria of many other parasites, and has alsobeen found in bacteria. Studies of bacteria, includingEscherichia coli, have clari¢ed the details of this sys-tem [14,15]. In E. coli, there are two types of complexII, and quinol^fumarate reductase (QFR) encodedby the frd operon is induced under anaerobic condi-tions. The reducing equivalent of NADH and glyc-erol is transferred to menaquinone (Fig. 3), a low-potential naphthoquinone, and is ¢nally passed tofumarate by QFR. In contrast, under aerobic condi-tions, succinateûbiquinone reductase (SQR) en-coded by sdh operon that catalyzes oxidation of suc-cinate is induced [16]. SQR is a dehydrogenase in therespiratory system as well as an enzyme in the TCA

Fig. 3. (A) Structure of quinones. (B) Electron £ow through the mitochondrial respiratory chain. In the NADH^FRD of A. suumadult mitochondria, complex II catalyzes electron transfer from rhodoquinol to fumarate, while it catalyzes reverse electron £ow fromsuccinate to ubiquinone (UQ), in the aerobic respiration of larvae and their mammalian host.



cycle, and directly connects these systems in aerobicenergy metabolism.

Thus, two di¡erent enzymes (complex II) arepresent in E. coli, and the bacteria maintain homeo-stasis of the energy metabolism by controlling syn-thesis of these enzymes in response to the environ-mental oxygen supply. These ¢ndings may haveimplications for eukaryotic parasites. There is along-standing controversy concerning the enzymethat catalyzes reduction of fumarate in A. suum mi-tochondria: one is that reverse reaction of SQR oc-curs in larval mitochondria; the other is that QFR inadult mitochondria di¡ers from SQR in larval mito-chondria in a manner similar to the di¡erence be-tween E. coli QFR and SQR [17].

Complex II is an enzyme complex that catalyzesthe oxidation and reduction of succinate and fuma-rate, and that is localized in the cytoplasmic mem-brane in bacteria and in the mitochondrial innermembrane in eukaryotes [18,19]. The subunit struc-ture is highly conserved among species, and is basi-cally composed of four polypeptides (Fig. 4). Thelargest £avoprotein (Fp) subunit, with an approxi-mate molecular weight of 70 kDa, contains FADas a prosthetic group, and the relatively hydrophiliccatalytic portion is formed by this subunit, and anapproximately 30-kDa iron^sulfur protein (Ip) sub-unit containing three di¡erent types of iron^sulfurclusters.

This catalytic portion catalyzes electron transferfrom succinate to water-soluble electron acceptors

such as phenazine methosulfate in SQR (succinatedehydrogenase (SDH)). In contrast, in QFR, it cata-lyzes electron transfer from water-soluble reducedmethylviologen to fumarate (FRD). Two small hy-drophobic subunits of approximately 15 and 13 kDaare required for the localization of this catalytic por-tion in the membrane. Since many complex IIs con-tain heme b, these subunits are called cytochrome blarge and small subunits (CybL and CybS). This hy-drophobic cytochrome b is also necessary for elec-tron transfer between complex II and the hydropho-bic components of the electron transport systempresent in the membrane, such as ubiquinone andrhodoquinone. Overall, cytochrome b is localized inthe membrane as a membrane anchor, and the cata-lytic portion protrudes into the matrix side in mito-chondria and into the cytoplasm in bacteria. Thisstructure has recently been con¢rmed in bacterialQFRs by crystal structure analysis [14,15].

A. suum complex II has been puri¢ed from musclemitochondria of adult worms as SQR [20]. However,no direct evidence for the presence of two distinctenzymes, SQR and QFR, in the mitochondria ofthe same organism was obtained, although adultcomplex II showed high FRD activity [21]. To clarifythis point, complex II was isolated from larval andadult mitochondria under the same conditions, andbiochemical and antigenic properties were compared[22]. When larval and adult mitochondria were iso-lated and solubilized with sucrose monolaurate andseparated using DEAE-cellulo¢ne column chroma-

Fig. 4. Subunit structure of two complex IIs in A. suum mitochondria. Complex II of larva functions as SQR and that of adult doesas QFR. S1^S3, iron^sulfur clusters; UQ9, ubiquinone-9; RQ9, rhodoquinone-9.



tography, complex IIs were eluted at di¡erent saltconcentrations. As with the mammalian complex II,the larval complex II showed only SDH activity,whereas the adult showed both SDH activity and ahigh FRD activity (Table 1). In addition, the com-plex isolated from larvae had a higher a¤nity forsuccinate than the adult complex in SDH assay,and the complex isolated from the adult had a highera¤nity for fumarate than the larval complex in theFRD assay.

When the peptide map and antibody reactivitywere compared for each subunit, Fp and CybS incomplex II were found to di¡er between larvae andadult. Thus, A. suum mitochondria possess two typesof complex II, SQR and QFR, and A. suum deal withenvironmental changes in the life cycle by producingSQR as larvae (which use aerobic energy metabo-lism) and producing QFR as adults (which inhabitin the small intestine under low oxygen tension).

This is the ¢rst demonstration of the presence ofisoforms of complex II in mitochondria, that has notbeen found in mammals, and this ¢nding is a veryinteresting example of the diversity of complex II.

2.3. Characteristics of the A. suum adult complex II

What characteristics of the subunits and primarystructure of adult complex II are responsible for itshigh QFR activity? To answer this question, cDNAof each complex II subunit was cloned from A. suumlarvae and adults, the free-living nematode Caeno-rhabditis elegans (which uses aerobic energy metabo-lism) [7,23^25], and humans (a mammalian host)[26^28].

2.3.1. Fp subunitFp, which contains the binding site for the sub-

strates succinate and fumarate, is highly conservedfrom bacteria to humans [23]. The region aroundHis-49 (near the N-terminal), to which the prostheticgroup FAD is covalently bound, and the region thatinteracts with the AMP domain of FAD are partic-ularly well conserved (Fig. 5). Also, Arg-290 corre-sponds to Arg-301 of Wolinella succinogenes QFR[15], which has been proposed as the protondonor for fumarate reduction, and other relatedamino acid residues (His-246, His-357 and Arg-402)are all conserved. This indicates that the molecularmechanism of the succinate and fumarate conversionprocess in mitochondria is identical to that of bac-teria.

The point we wish to emphasize is that theprimary structure of adult complex II is closer tothat of SQR than to E. coli QFR, despite its highQFR activity. There were no sequences common toFp of E. coli QFR and adult A. suum Fp, and thislack of common sequences was observed not only forFp but also other subunits in adults, such as Ip[24,25].

We have recently succeeded in cloning cDNA ofthe larval Fp. Although the highest degree of homol-ogy between larvae and adults is observed in the Fpsubunit, the amino acid sequence of larval Fp ismuch closer to those of free-living C. elegans thanto adult Fp, as shown in Fig. 6. The Northern blotanalysis showed that only larval Fp mRNA (SDHA)was expressed in the larvae, indicating that stage-speci¢c expression of adult Fp gene (FRDA) is con-trolled at the transcription level (Fig. 7).

Table 1SDH and FRD activities of A. suum complex IIsa

Complex II SDHb (nmol/min/mg) Km succinate (mM) FRDc (nmol/min/mg) Km fumarate (mM)

Larvae 4.28 0.153 0.706 0.455Adult 3.53 0.608 28.9 0.143aModi¢ed from tables I and II in ref. [22].bMeasured by PMS-MTT system.cMeasured by reduced methylviologen as electron donor.

C

Fig. 5. Comparison of the amino acid sequences of Fp subunits from various species. The open arrowhead indicates FAD-binding his-tidine [14,15]. The closed arrowheads indicate histidine and arginines in the active site [15]. The references are as follows: E. coli frdA[72] ; A. suum FRDA [23] ; A. suum SDHA (our present study); Homo sapiens SDHA [27] ; E. coli sdhA [73].





2.3.2. Ip subunitThe Ip subunit contains three di¡erent iron^sulfur

clusters (2Fe^2S, 4Fe^4S, 3Fe^4S), and the regionscontributing to this binding have amino acid sequen-ces containing high numbers of cysteine (as in ferre-doxin). These cysteine residues are completely con-served in the A. suum Ip [24]. However, electronparamagnetic resonance analysis showed that the3Fe^4S cluster in the adult QFR is only partiallyreduced by succinate [29]. In comparison to the3Fe^4S cluster in mammalian SQR (+60 to +120mV), which is completely reduced by succinate, lowoxidation-reduction potential is a common feature ofbacterial QFR (320 to 370 mV). As with bacterialQFR, the low-potential 3Fe^4S cluster of A. suum Ipmay facilitate electron transfer from the low-poten-tial rhodoquinone (363 mV) to fumarate (+30 mV).

Recently, Amino et al. have found that the QFRof the parasitic adult and the SQR of free-living lar-vae share a common Ip subunit, although their com-plex IIs clearly show di¡erent enzymatic properties[24]. This is very di¡erent from the bacterial case, inwhich all subunits of SQR and QFR are enzyme-speci¢c. Two Ip genes have been reported in the

sheep nematode Haemonchus contortus [30]. Thesetwo genes are di¡erentially expressed during develop-ment, and the properties of the two enzymes theycontribute to have yet to be de¢ned.

2.3.3. Anchor subunitsThe presence of a two-subunit cytochrome b, com-

posed of one CybL (also referred to as QPs-1, com-plex II-3 or SDHC) and one CybS (also referred toas QPs-3, complex II-4 or SDHD), which acts asmembrane anchor, is a general feature of mitochon-drial complex II [19]. The cytochrome b558 of A.suum adult QFR is composed of two polypeptideswith molecular masses of 17.2 and 12.5 kDa, and isseparated from Fp and Ip by a gel permeation chro-matography in the presence of Sarkosyl [13]. In con-trast to the Fp and Ip subunits, the primary structureof which is conserved, the cytochrome b exhibits spe-cies-speci¢c characteristics. For example, polyclonalantibodies against A. suum adult CybS do not reactwith CybS of other parasitic nematodes such as ¢-larial worms (Saruta et al., unpublished observation).However, while the primary structure di¡ers consid-erably between species, the functionally importantamino acid residues and orientation within the mem-brane are conserved, as predicted from the adult

Fig. 6. Phylogenetic tree based on the deduced amino acid se-quences of various Fp subunits. The tree was constructed bymaximum-likelihood method [74]. The horizontal length indi-cates the estimated number of substitutions per site. The genesfor Fp-I and Fp-X of C. elegans are located on chromosome Iand X, respectively. Expression of both gene products was de-termined by in situ hybridization and Western blot (Amino etal., unpublished observation). The references and accessionnumbers for the each sequence are as follows: E. coli FrdA [72](AAC77114); E. coli SdhA [73] (AAC73817); A. suum SDHA(our present study); C. elegans (Fp-I) (AAB97539); C. elegans(Fp-X) [23] (BAA21637); A. suum FRDA [23] (BAA21636); B.taurus [75] (AAA30758); H. sapiens [27] (BAA06332); S. cerevi-siae [76] (S34793).

Fig. 7. Stage-speci¢c expression of genes for A. suum Fp sub-units. Northern hybridization using A. suum FRDA cDNA (A)and A. suum SDHA cDNA (B) as probes. Lane 1, adult poly-(A)+RNA (1.25 Wg); lane 2, larval poly(A)+RNA (1.28 Wg).The closed and open arrowheads indicate the FRDA andSDHA transcripts, respectively. The size marker is RNA ladder(Gibco BRL).



QFR CybL (FRDC) and CybS (FRDD) cDNA se-quences [7,25].

Both subunits of the cytochrome b anchor possessthree trans-membrane segments, and are thus highlyhydrophobic. Biochemical and molecular geneticstudies of E. coli SQR revealed that the heme bbridges the CybL and CybS subunits via two histi-dine residues [31,32]. Cytochrome b in A. suum com-plex II plays an important role in interaction withthe quinones, because it passes electrons to ubiqui-none in larvae and receives electrons from rhodoqui-none in adults. Recent analyses of larvae SQR haveshown that, as is the case with Ip, CybL of larvae isidentical to that of adults, and that CybS (SDHD) isa subunit unique to larvae (Amino and Osanai,manuscript in preparation).

While there is no direct evidence that adult QFRcytochrome b (cytochrome b558) participates in theelectron transfer from rhodoquinol to fumaratein the complex, it is rapidly reduced by succinate[20]. Using oxidation-reduction titration, it wasfound that the red-ox potential of cytochromeb558 was 334 mV, higher than the 3185 mVof bovine cytochrome b560 [33]. This property

facilitates electron transfer from the low-potentialrhodoquinol.

In addition to its role in electron transfer, the cy-tochrome b in complex II is important in the assem-bly of the subunits. A recent study of E. coli SQRusing a heme synthesis mutant has shown that hemeb is essential to the assembly of complex II [34]. Thefact that two histidine residues in A. suum QFR (His-100 in CybL and His-72 in CybS) that are thought tocoordinate heme b are conserved suggests that theassembly mechanism of A. suum QFR may be thesame as that of E. coli SQR (see Fig. 8).

2.4. Parasitic adaptation and evolution ofmitochondrial QFR

Many parasites, notably A. suum, employ theNADH^FRD system for anaerobic energy metabo-lism within the host. The mitochondrial complex IIplays an important role, acting as QFR within thissystem. As shown previously, despite their high levelof QFR activity, all four adult A. suum subunits havegreater homology with bacterial and mitochondrialSQR than they do with bacterial QFR.

Fig. 8. A possible mechanism for the assembly of A. suum QFR. The heme b is bound by axial ligands of CybL (His-100) and CybS(His-72). The heme b is essential for assembly and membrane localization of cytochrome b558 and for the attachment of the solubleFRD-dimer (Fp and Ip) to the membrane anchor subunits [13,34].



One possible interpretation of these ¢ndings is asfollows (see Fig. 9). Complex II is considered to havearisen from soluble FRD of early anaerobic bacteriathat appeared when almost no oxygen was presenton the earth [35]. Because this enzyme has low oxi-dation-reduction potential (3220 mV) for non-cova-lent FAD, succinate could not be oxidized, and onlyfumarate reduction was catalyzed. In fact, E. coliQFR is not able to oxidize succinate when the histi-dine residue that binds FAD is replaced with otheramino acids [36]. The oxidation-reduction potentialof FAD then increased (380 mV) due to the covalentbond with Fp of FRD [37], and the bacterial com-plex II (which was anchored to the membrane) thenappeared and functioned as QFR under anaerobicconditions. Ultimately, SQR activity developed asthe membrane-bound enzyme of the aerobic energymetabolism system, and the mitochondrial complexII, which links the TCA cycle directly to the respira-tory chain, was formed [19,35]. The primary struc-ture of the A. suum adult QFR indicates that it isderived from the SQR of free-living nematodes suchas C. elegans rather than the QFR of anaerobic bac-teria [7,23^25]. The fact that the primary structure ofthe aerobic A. suum larvae Fp resembles that of C.elegans rather than that of the A. suum adult stronglysupports this hypothesis (Figs. 5 and 6).

As previously described, recent research has shownthat the Ip of A. suum larvae is identical to that ofthe adult [24]. This is in contrast to the relationshipbetween bacterial SQR and bacterial QFR, in whichall the relevant subunits di¡er. Furthermore, our bio-

chemical analysis showed that at least the Fp andCybS of larval complex II di¡er from those of theadult enzyme [22]. Considering the biological signi¢-cance of the common subunit(s) in the stage-speci¢cisoforms, it may be advantageous to adapt to envi-ronmental change simply and quickly. Evolutionarychanges in the minimal subunit(s) during the transi-tion from the free-living state to parasitic life ^ e.g.,the stage-speci¢c substrate binding site of Fp (succi-nate or fumarate) and the stage-speci¢c quinonebinding sites of CybS (ubiquinone or rhodoquinol)^ may favor the establishment of parasitism in theseworms.

2.5. The role of rhodoquinone in anaerobic respiration

Rhodoquinone is a low-potential benzoquinone inwhich one methoxy group has been replaced by anamino group (Fig. 3). It plays an important role inthe NADH^FRD system in the adult mitochondria.In addition to the evolution of complex II itself, thechanges in the quinones that pass electrons directlyto the complex are of considerable interest.

When anaerobic bacterial FRD became mem-brane-bound, the reducing equivalent was transferredby menaquinone (380 mV, Fig. 3), which is a naph-thoquinone with a low oxidation-reduction potentialthat is found in the anaerobic respiratory chain ofpresent-day E. coli. As it evolved into aerobic SQR,ubiquinone (+110 mV), a high-potential benzoqui-none, was added. Then, QFR, which uses a low-potential benzoquinone called rhodoquinone (363

Fig. 9. Evolution of mitochondrial QFR. Mitochondrial QFR in adult A. suum was not directly derived from anaerobic bacterialQFR, and it may be derived from SQR of a free-living nematode such as C. elegans. Rhodoquinone may be an evolutionally new qui-none, like A. suum QFR, which has an SQR-type primary structure.



mV), found in the present-day adult A. suum, as theelectron donor, may have appeared [38]. Therefore,rhodoquinone may be an evolutionally new quinone,like A. suum QFR, which has an SQR-type primarystructure. Some bacteria are known to have a com-bination of rhodoquinone and QFR. For example,the QFR gene of Rhodoferax fermentans also encodesfor an amino acid sequence similar to the SQR ami-no acid sequence, but no regions or sequences com-mon to A. suum QFR have been found (Miyadera etal., BAA31213 in DDBJ). Based on these results, itappears that SQR-type QFR of a variety of organ-isms has evolved independently from SQR. Thestudy of these enzymes can help to elucidate the gen-eral mechanisms of enzyme evolution and molecularaspects of enzyme adaptation that generate function-al diversity.

The indispensability of low-potential rhodoqui-none in electron transfer from complex I to QFRwithin the NADH^FRD system has been clari¢edusing reconstituted systems [13]. In addition, synthe-sis of rhodoquinone derivatives with short isoprenechains has facilitated enzymatic analysis of thesecomplexes and rhodoquinone. We used decyl-rhodo-quinone when we discovered nafuredin, which com-petitively and speci¢cally inhibits the rhodoquinonebinding site of A. suum complex I [39]. Rhodoqui-none derivatives are currently being used for enzy-matic analysis of A. suum adult QFR.

While rhodoquinone plays an important physio-logical role, as described above, there is little infor-mation available about its biosynthetic pathway.Some reports indicate that it is synthesized fromthe same precursor as ubiquinone [40^42], but thestep at which the amino residue is added is unclear.Recent study has shown that the demethoxy ubiqui-none (DMQ) precursor is accumulated in place ofubiquinone in the clk-1 long-lived mutant strain ofthe free-living nematode C. elegans [43]. The amountof rhodoquinone found in this mutant strain is high-er than that in wild strain [44], indicating that thebiosynthetic pathway branches somewhere prior toDMQ.

A. suum larvae, which employ aerobic metabolism,have the same ubiquinone as mammals, but the pri-mary quinone of A. suum adults is rhodoquinone[12]. Thus, rhodoquinone holds the key to changesin energy metabolism within the life cycle, and

understanding the mechanism by which its synthesisis controlled is an important goal for future researchinto this organism's environmental adaptation.

3. Complex II of malaria parasite

Parasitic protozoa, which reproduce by division,induce acute symptoms due to their rapid rate ofreproduction, and therefore cause severe clinical fea-tures. This section focuses on the current state ofresearch on complex II of the malaria parasite, whichis associated with mortality that has been estimatedat more than two million deaths per year.

3.1. Mitochondria of parasitic protozoa

The mitochondria of parasitic protozoa that havebeen most studied are those of Trypanosoma brucei,which are the causative agents of African trypanoso-miasis in human and nagana in cattle. As is wellknown, research involving this parasite has resultedin important discoveries such as GPI anchoring, ed-iting of transcripts of mitochondrial DNA, and splic-ing of the 5P-terminal leader sequence of mRNA[1^3]. T. brucei brucei inhabits the bloodstream ofthe host mammal, ATP synthesis being handled pri-marily by the glycolytic pathway within the glyco-some [45]. The role of the electron transfer in thesingle mitochondrion is re-oxidation of NADH pro-duced in the glycosome. This is associated with op-eration of the glycerol-3-phosphate oxidase systemcomprising glycerol-3-phosphate dehydrogenase, ubi-quinone, and cyanide-insensitive terminal oxidase,and is not associated with the mammalian-type res-piratory chain [46]. This glycerol-3-phosphate oxi-dase system is essential to the survival of T. bruceibrucei, and the terminal oxidase inhibitor ascofura-none has a dramatic e¡ect on this parasite [47]. Onthe other hand, cristae develop in T. brucei bruceiwithin the Tsetse £y vector, and these contain com-ponents of the mammalian-type respiratory chain,including complex II and cytochromes, althoughmore study is required to understand the physiolog-ical function of complex II in the parasite metabo-lism [48].

Thus, parasitic protozoa living within a mamma-lian host do not generally use oxygen, and instead



synthesize ATP via the glycolytic pathway. In com-parison to research on helminths, research on mito-chondrial electron transfer of protozoa during thedevelopmental stage within the host is lacking. How-ever, as described below, mitochondria have becomea focus of chemotherapy, and research, particularlyon mitochondria of the malaria parasite, is currentlyprogressing.

3.2. Mitochondria of the malaria parasite

Malaria is one of the most serious infectious dis-eases in the developing world as a re-emerging dis-ease [49]. The presence of mitochondria in themalaria parasite was veri¢ed using the cationic£uorescent dye rhodamine 123 and microscopic ob-servation [50]. Like trypanosomes, each parasite in ared blood cell contains a single sausage-shaped mi-tochondrion [51]. The mitochondrion is always inclose proximity to the apicoplast, another organelle,and a physiological interaction may exist between thetwo. Phylogenetic analysis of the tufA gene in the 35-kb genome of Plasmodium falciparum apicoplastshows relationships with the genomes of cyanobac-teria and plastids, as evidenced by consistent cluster-ing with green algal plastids [52]. These observationsindicate that the Apicomplexa, including Plasmodi-um, acquired a plastid by secondary endosymbiosis,probably from a green alga.

Energy metabolism of malaria parasite within redblood cells was previously thought to consist primar-ily of ATP production through the glycolytic path-way, and the physiological signi¢cance of the mito-chondria was unclear. However, recent studies haveshown the presence of a functional respiratory chainin Plasmodium mitochondria, although their levels ofelectron transport activity appear to be much lowerthan those of their mammalian counterparts. Mam-malian mitochondrial DNA normally encodes 13proteins, two ribosomal RNAs, and 22 tRNAs nec-essary for oxidative phosphorylation, and has ap-proximately 16 000 base pairs in its genome. Plasmo-dium mitochondrial DNA is considerably shorter, at6000 base pairs, and encodes only cytochrome b ofcomplex III, subunits I and III of the cytochrome coxidase, and fragmented ribosomal RNA [53]. Othersubunits normally encoded in mammalian mitochon-dria are thought to be encoded in the nuclear DNA

of the malaria parasite, but this has not been con-¢rmed.

In contrast to erythrocytic stage cells, approxi-mately six mitochondria per cell have been observedin sexually di¡erentiated gametocytes, with more infemale gametocytes than in male gametocytes [54].Structure and variation of number of mitochondriaduring the life cycle, including sporozoite, is a matterof great interest in terms of physiological signi¢-cance.

3.3. The respiratory chain of Plasmodium in theerythrocytic stage

Fry et al. used a tight-¢tting Te£on pestle andglass homogenizer to break cells of Plasmodium,and obtained the mitochondrial fraction using 22%(v/v) Percoll [55]. An electron transfer from NADH,K-glycerophosphate and succinate to cytochrome cwas observed in the mitochondrial fraction. Becausethis activity was reduced by fumarate, it was hy-pothesized that FRD plays a role in this process.As with mammals, cytochrome aa3, c, b, and c+c1

were detected in the red-ox di¡erence spectrum, andthe speci¢c content of these cytochromes was high, at30^50% of the level found in bovine heart musclemitochondria. Krungkrai et al. modi¢ed Fry's meth-od to isolate the Plasmodium mitochondria, and wereable to isolate the active complex II, IV and dihy-droorotate dehydrogenase (DHOD) [56^58].

However, as pointed out by Vaidya [59], the yieldand quality of mitochondria from malaria parasitesare quite variable. For example, succinate oxidaseactivity of the mitochondria isolated from rodentmalaria by Fry et al. was 150 nmol/min/mg [55],but that of the mitochondria isolated by Krungkraiet al. was 1.45 nmol/min/mg [54]. Thus, alternativemethods for studying mitochondrial physiology areurgently needed.

Recently, using N2 cavitation, we have establisheda protocol to prepare the active mitochondria fromP. falciparum showing higher SDH activity than pre-viously reported, and a dihydroorotate-dependentrespiration [60]. DHOD is involved in the fourthstep in the synthesis of pyrimidine, in which reducingequivalent is transferred to ubiquinone. Addition offumarate partially inhibited dihydroorotate-depen-dent respiration. These results, together with pre-



vious observations, indicate that the electron transferpathway in mitochondria of malaria parasites in redblood cells is as shown in Fig. 10.

As described above, the erythrocytic stage cells ofPlasmodium lack a number of enzymes in the TCAcycle, and ATP production is dependent upon theglycolytic pathway. The question therefore arises asto the physiological function of the respiratory chainunder such conditions. One important function maybe the formation of membrane potential [61]. Themajority of mitochondrial protein is encoded in thenucleus and transported to the mitochondria follow-

ing synthesis in the cytoplasm. Details of the mech-anism of mitochondrial targeting have been exten-sively studied, and results have shown that therespiratory chain is involved in the formation ofthe membrane potential required for passage of theseproteins through the mitochondrial membrane [62].

3.4. P. falciparum complex II

P. falciparum complex II has been puri¢ed by Sar-averatum et al. [56]. Their ¢nal preparation was ob-tained without the use of detergent, and contained

Fig. 10. Possible electron transfer chain in mitochondria of erythrocytic stage P. falciparum. Reducing equivalent from dihydroorotateis transferred to oxygen via classical respiratory pathway and to fumarate via QFR activity of complex II [60].

Fig. 11. Unique primary structure of P. falciparum Fp. P. falciparum-speci¢c insertion near the second AMP-binding domain andC-terminal unicellular organism-speci¢c deletion in the amino acid sequence of Fp [63].



only Fp and Ip with molecular masses of 55 and 35kDa, respectively. This result indicates that associa-tions between the subunits in P. falciparum complexII are weak, and that the catalytic portion composedof Fp and Ip is separated from the anchor duringpuri¢cation. Catalytic e¤ciency of the PlasmodiumSDH for succinate and ubiquinone were found tobe relatively low (kcat/Km = 600 and 5000, respec-tively), and many properties were found to be di¡er-ent from those of the mammalian host's enzyme. Forexample, 2-thenoyltri£uoroacetone, a known inhibi-tor of mammalian SDH, had no inhibitory e¡ect onthe Plasmodium enzyme at a concentration of 50 WM.

To study the unique properties of PlasmodiumSDH, we have cloned and characterized the genesfor Fp (SDHA) and Ip (SDHB) [63]. This is the ¢rstreport of the primary structure of protozoan SDH.Each of the two genes contains a single open readingframe, and the genes are located on di¡erent chro-mosomes. SDHA is a sequence of 1860 nucleotideson chromosome 10, and SDHB consists of 963 nu-cleotides on chromosome 12. The expression of thesegenes in asynchronous erythrocytic-stage cells wascon¢rmed by observations of 3.3-kb and 2.4-kb tran-scripts from the SDHA and SDHB genes, respec-tively. The SDHA and SDHB genes encode proteinsof 620 (Fp) and 321 (Ip) amino acids, with molecularmasses of 69.2 and 37.8 kDa, respectively. A mito-chondrial pre-sequence essential for the import ofmitochondrial proteins encoded by nuclear DNA,along with almost all of the conserved amino acidsindispensable for substrate binding and the catalyticreaction, were found in these peptides, indicating thefunctional importance of this enzyme in the parasite.Interestingly, a P. falciparum-speci¢c insertion and aunicellular organism-speci¢c deletion were found inthe amino acid sequence of Fp, as shown in Fig. 11.Despite the fact that Fp and Ip are generally well-conserved subunits, the structure of both subunits inthe malaria parasite clearly di¡ers considerably fromthat of the host.

As described in Section 3.3, mitochondrial dihy-droorotate-dependent respiration is reduced to lessthan 70% of its normal level by the addition of fu-marate, indicating that the P. falciparum complex IIfunctions as QFR (Fig. 10). The fact that anti-senseDNA for Ip inhibits the growth of malaria parasiteclearly indicates that complex II is an enzyme essen-

tial for its survival in the erythrocyte (Wataya, per-sonal communication).

4. The parasite respiratory chain as a target forchemotherapy

The di¡erences between parasite and host mito-chondria described in this paper hold great promiseas targets for chemotherapy. For example, the anti-malarial drug Atovaquone, which recently devel-oped, acts on the mitochondrial respiratory chain[61]. Atovaquone is e¡ective against chloroquine-re-sistant strains, and is already being used for treat-ment in Africa and Thailand, where malaria is en-demic [64]. The speci¢c target is thought to becomplex III, and biochemical analysis has shownthat it acts on the ubiquinone oxidation site in thecytochrome b of complex III [65].

As shown in Fig. 10, ubiquinone plays an impor-tant role in the respiratory chain of Plasmodium mi-tochondria, and is the point of intersection betweenthe two essential metabolic pathways, pyrimidinebiosynthesis and energy metabolism. Targeting thepoint of intersection between these systems allowssimultaneous blocking of two important metabolicsystems. Many anti-malarial agents contain a qui-none ring within their structure, and the enzymegroup involved with ubiquinone in transfer of elec-trons is considered to be a promising target [66].Complex II is an enzyme essential for survival ofthe malaria parasite, and it is a promising targetfor future anti-malarial agents. In fact, a quinoneanalogue, 5-hydroxy-2-methyl-1,4-naphthoquinone(plumbagin) inhibits in vitro growth of P. falciparumas well as SDH activity of puri¢ed enzyme [56].

Such a chemotherapeutic approach is also applica-ble to the nematodes. It has been proposed that theFRD system is the target of such drugs as bithionoland thiabendazole [67,68], but there is no clear bio-chemical or pharmaceutical evidence to support thisidea. This lack of evidence is due to the fact thatdetails of this pathway have not been clari¢ed atthe protein level. However, as described in the pre-vious section, progress in the study of the NADH^FRD pathway permits screening of new anthelminticcompound. Nafuredin competitively inhibits the rho-doquinone binding sites of nematode complex I [39].



If an agent that can speci¢cally inhibit the nematodeQFR is found, it is expected to prove extremely ef-fective as an anthelmintic. Work is currently under-way on crystallization of A. suum adult QFR for thestructure-based drug design.

5. Perspective

As described above, parasites have exploited a va-riety of energy-transducing systems in their adapta-tion to the peculiar habitats in their hosts. Dynamicrearrangement of their respiratory chain during theirlife cycle is one of the key elements of this adapta-tion. However, the control mechanism responsiblefor stage-speci¢c expression of the genes of parasitesremains unclear, because research on parasites at themolecular level has only recently begun. Current re-search using the free-living nematode C. elegans hasrevealed that oxygen concentration, oxygen availabil-ity, and oxidative stress can induce a variety of in-teresting phenotypes, including di¡erent life spans,and that the respiratory chain participates in thismechanism. A single mutation in CybL of complexII at residue 71, converting glycine to glutamic acid,results in hypersensitivity to oxidative stress in the C.elegans mev-1 strain [69]. On the other hand, in thelong-lived mutant clk-1, ubiquinone biosynthesis isdramatically altered in which mitochondria do notpossess detectable levels of ubiquinone-9 and insteadcontain the ubiquinone biosynthesis intermediateDMQ [43,44]. These results indicate that the respira-tory chain plays an important role in responses tochanges in the amount of oxygen in the environment(oxygen homeostasis). In this connection, recent re-ports indicating that complex II functions as an oxy-gen sensor are of great interest [70].

Mammalian cells are able to sense decreased oxy-gen availability and activate response systems, in-cluding transcriptional activation of several genescontrolled via hypoxia-inducible factor-1 [71].Although no information on such a molecular mech-anism of gene expression has been available for para-sites, A. suum shows a very clear transition of meta-bolic systems between larvae and adults, and it isthus a very promising research model. In addition,di¡erences in energy metabolism between hosts andparasites are attractive therapeutic targets. Basic re-

search on parasitic adaptation promises to yield clin-ical applications and scienti¢c discoveries followingto Keilin's discovery of cytochrome.

Acknowledgements

This study was supported by grants-in-aid forscienti¢c research on priority areas from the Minist-ry of Education, Science, Culture and Sport,Japan (08281105, 11470065, 1147231, 12670241 and13854011) and for research on emerging and re-emerging infectious diseases from the Ministry ofHealth and Welfare.

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