Electron-transfer complexes of mitochondria in Ascaris suum

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Electron-transfer Complexes of Mitochondria in Ascaris suurn K. Kita During the past ten years, studies on the respiratory chain of mitochondria in parasites have progressed to provide new insight into the structural organization and physiological significance of the mitochondrial respiratory chain. In this review, Kiyoshi Kita focuses on studies on the respiratory chain ofAscaris mitochondria in which major advances have recently been made. These include the identification of the unique features of anaerobic respiration, the elucidation of the molecular structures of the components involved and an understanding of the evolution of the energy transducing system and of the developmental changes that occur during the life cycle of this nematode. Energy metabolism is essential for the survival, continued growth and reproduction of living organisms. Historically, the relationship between parasitology and bioenergetics has been intimate. Keilin, who discovered cytochrome in the larvae of parasitic insects of the horse, was a parasitologist, and Ascaris was one ,of the animals reported in his paper On Cytochrome, a Respiratory Pigment Common to Animals, Yeast and Higher Plants 1. Keilin's concept of the respiratory chain has subsequently been verified, and many advances have been made in elucida'dng the roles of this process in energy transduction, mainly using mammalian and yeast mitochondria, and bacteria. The major function of the aerobic respiratory chain of mammalian host mitochondria is the electrogenic translocation of protons out of the mitochondrial membrane to generate the proton motive force that drives ATP synthesis. Respiratory systems of parasites tend to show greater diversity in electron transfer pathways than those of host animals and many have exploited unique respiratory chains as adaptations to their natural habitats within their hosts. The reduction of fi~marate to succinate is important in the energy transduction system of anaerobic organisms and is a terminal step of the phosphoenolpyruvate carboxykinase-succinate (PEPCKsuccinate) pathway 2'-~. Although there is agreement that a part of the electron transport chain participates in fumarate reduction, substantial information on the enzyme catalyzing this reaction is not yet available. During the past decade, biochemical studies on this problem have progressed using Ascaris suum. Kiyoshi Kita is at the Department of Parasitology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan. (~) 1992, Elsewer Science Publishers Ltd, (UK)

NADH-fumarate reductase system Adult Ascaris resides in the mammalian small intestine, where oxygen tension is limited. Mitochondria from the body wall muscle of adult worms provide an excellent model system for the study of anaerobic energy metabolism. Spectroscopic and enzymatic studies have shown that these mitochondria contain a small amount of complex III (ubiquinol-cytochrome c oxidoreductase; b-c1 complex) and an even smaller amount of complex IV (cytochrome c oxidase). Furthermore, participation of a b-type cytochrome, designated cytochrome b558, in the fumarate reductase system has been observed at low temperature 4. The purification of complex II as succinateubiquinone oxidoreductase from Ascaris adult mitochondria initiated the biochemical and protein chemical studies of the NADH-fumarate reductase system 5. Complex II is an important enzyme complex in the tricarboxylic acid cycle and the aerobic respiratory chain of mitochondria and prokaryotic organisms. The isolated complex II of Ascaris contains succinate-reducible cytochrome b558. In the presence of Ascaris NADH-cytochrome c oxidoreductase (complex I-III), cytochrome b~ss in complex II is also reduced with N A D H and reoxidized with fumarate. The most important property of complex II of the Ascaris adult is that this enzyme shows high fumarate reductase activity (FRD), which is a reverse reaction of succinateubiquinone oxidoreductase activity (SDH) 6'7. The ratio of SDH to FRD in Ascaris adult complex II is 0.05 when fumarate reductase activity is measured using methyl viologen as the hydrogen donor. This value is comparable to that of the fumarate reductase in Escherichia coli (0.03) (Ref. 6). In contrast, complex II of aerobic mitochondria has a high ratio; the value is approximately 20 for rat liver enzyme 7. Quantitative analysis of mitochondria with high performance liquid chromatography and gel electrophoresis in the presence of sodium dodecyl sulfate shows that complex II is one of the major components of Ascaris adult mitochondria (8% of mitochondrial protein). The linear sequential order of the respiratory components in the NADH-fumarate reductase system in Ascaris adult mitochondria is shown in Fig. 1. In this anaerobic respiratory chain, reducing equivalents from N A D H are accepted by complex I and transferred to complex II via rhodoquinone, and are finally oxidized by the fumarate reductase activity of complex II. Coupling to this electron

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Complex If cybL Ip

Fp

cybS (--34 mY)

NADH1 NAD "

i Fumarate Succinate (+30 mV)

ComplexI

(-320 mY)

6 ATP Synthesis

Enoyl CoA ETF'DH

"

ETF

"* E C R

Acyl CoA (-15~-30 mV)

Fig. I. Pathway of the NADH-dependent anaerobic respiratory chain in mitochondria of adult Ascaris, with redox potentials indicated. RQ, rhodoquinone; cybL and cybS, large and small subunits of cytochrome b558;Ip, iron-sulfur subunit; Fp, flavoprotein subunit; ETF, electron-transfer flavoprotein; ETF.DH, electrontransfer flavoprotein dehydrogenase; ECR, enoyl CoA reductase.

transfer from N A D H to fumarate results in site I phosphorylation by the proton pumping in complex I. In rhodoquinone, the methyl group of ubiquinone is substituted by the amino group (2amino-3-methoxy-6-methyl-5-isoprenyl- 1,4-benzoquinone) (Fig. 2) and its redox potential, Em' (-63 mV), shows a more negative value than that of ubiquinone (+ l l0mV). That rhodoquinone is indispensable as the low-potential electron carrier in the Ascaris NADH-fumarate reductase pathway has been demonstrated by the reconstituted system made from bovine complex I, Ascaris complex II and rhodoquinone in liposomes 7. It is interesting to note that ubiquinone, which is a major quinone in aerobic mitochondria of Ascaris larvae, as described later, cannot mediate electron transfer between the two complexes. The advantage of this respiratory system is the synthesis of ATP even in the absence of oxygen. The difference in redox potential between the N A D + / N A D H couple (Em' = - 3 2 0 m V ) and the fumarate/succinate couple (Em ' = +30mV) is sufficiently high to allow for ATP formation. As described, Ascaris complex II of adult muscle mitochondria functions as the terminal oxidase and donates electrons to fumarate, which is the terminal acceptor under anaerobic conditions. What is the difference between complex II of Ascaris, which functions as fumarate reductase, and that of the mammalian host, which functions as succinateubiquinone oxidoreductase? The establishment of a protocol to purify complex II from the mitochondrial inner membrane 5'8 and the development of modern technology in biochemical and molecular biological analysis has led to the clarification of the difference between the Ascaris and mammalian enzymes.

(FAD). The second-largest, 30kDa subunit (Ip) contains three different types of iron-sulfur center. The Fp and Ip subunits are hydrophilic and form the catalytic portion of the complex that transfers the reducing equivalent from succinate to a watersoluble dye, such as dichlorophenolindophenol (DCIP), via succinate dehydrogenase, or from reduced methyl viologen to fumarate via fumarate reductase. Two small hydrophobic membraneanchoring polypeptides, cytochrome b subunits cybL and cybS (15 kDa and 13kDa, respectively) seem to be essential for the interaction between the enzyme complex and quinone species. The Fp subunit is the site of succinate-fumarate conversion and is a highly conserved subunit with regard to protein chemistry and antigenic properties. A similar amino acid sequence has been found in two segments interacting with the adenosine monophosphate (AMP) moiety of FAD in Ascaris Fp and bacterial Fp 9. One segment is at the amino terminus region of the subunit, which has been predicted to be a Rossman nucleotide-binding fold ]°. Another segment that interacts with the AMP moiety is the stretch from residues 357 to 386 in E. coli sdh A (Ref. 11) and from residues 355 to 374 in B. subtilis sdh A (Ref. 12). High sequence similarity to these bacterial segments has been found in Ascaris Fp. Conservation of the amino acid sequence around the histidyl residue through which FAD is covalently bound to the polypeptide has also been found. The amino acid sequences of these segments in Ascaris Fp are more similar to those of E. coli sdh A than to those offrd A (Ref. 13) of the same organism 9. Iron-sulfur centers are the essential prosthetic group for electron transfer in complex II, and three distinct types of iron-sulfur center are present in

O H3CO~CH3 H3CO/ Y

"F

O

H3 ~-H LCH2--CH=C--CH2Jn

H2N~CH3 o

.

co-y "r O

H3

~-H

L C H 2 _ _ C H -'- C - - CH2_.I n

C o m p l e x II acts as fumarate reductase

Complex II is generally composed of four polypeptides (Fig. 3). The largest flavoprotein subunit (Fp) of 70kDa contains covalently bound flavin

Fig. 2. Chemical structures of ubiquinone (Era' = +1 10mV) and rhodoquinone (Era' = -63 mV).

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complex II: S-1 [2Fe-2S], S-2 [4Fe-4S] and S-3 [3Fe-4S] (Ref. 14). Spectroscopic analysis with electron spin resonance has shown a relationship between high fumarate reductase activity and novel redox properties of the S-3 center in complex II of Ascaris adult is. The E m' value of the S-3 center in mammalian complex II is +65 mV, and the center has been reported to be completely reduced with succinate. In the case of Ascaris, the S-3 center is only partially reduced with succinate, indicating that the E m' value of Ascaris S-3 is lower than that of the succinate/fumarate couple (30mV). The lower E m' value of the S-3 center is a common feature of the fumarate reductase system in Wolinella succinogenes (-24mV) (Ref. 16) and E. coli (-70 mV) (Ref. 17). There is a low sensitivity to o~-thenoyl trifluoroacetone (TTFA), which is a potent inhibitor for the S-3 center of mammalian complex II, in Ascaris complex II (Ref. 7). In immunological analysis, only the Ip subunit of Caenorhabditis elegans is recognized by antibody against Ascaris Ip, and those of mammalian and bacterial complex II are not. Comparative studies on the primary structure of the Ip subunit, deduced from complementary DNAs (cDNAs), show extensive homology between mammals and nematodes despite the antigenic differences is. Striking sequence conservation is found around the three cysteine-rich clusters, which have been thought to comprise the iron-sulfur centers of the enzyme. It is interesting to note that the amino acid sequence of the Ascaris Ip subunit is more similar to those of mammalian Ip and E. coli sdh than to those of bacterial frd, even though Ascaris complex II shows a high fumarate reductase activity (H. Wang et al., unpublished). This observation, together with the conservation seen in the Fp subunit 9, suggests that Ascaris complex II evolved from the complex II of aerobic mitochondria, which has succinate dehydrogenase activity, and thus the fumarate reductase activity of Ascaris complex II must have been acquired during adaptation to parasitism. Complex II contains b-type cytochrome, the genes for which are encoded in the nuclear DNA, and the peptides are synthesized in the cytoplasm, in contrast to the b cytochrome in complex III. The participation of cytochrome b in electron transfer in complex II has not been readily accepted by workers in the field because of the observation of variable b heme content in isolated bovine heart complex II relative to its FAD content. Direct evidence for the role of cytochrome b in electron transfer in complex II has been obtained from a spectroscopic analysis of the reduction of these cytochromes in Ascaris and bacterial complex II by succinate 5'19. Cytochrome bsss of Ascaris complex II has been shown to have an Em' of-34 mV (Ref. 20). This value is much higher than that reported for mammalian cytochrome b56o of complex I! (-194mV) (Ref. 21). Reoxidation of reduced cytochrome b56o by addition of fumarate 2~ suggests that

SUCCINATE ~S

FUMARATE

f

F~DFP Sl Ip1/

Larva

M-side

Adult

C-side

Fig. 3. Molecular organization of complex II in the inner membrane of Ascarismitochondria and electron transfer in the complex. Fp, ~lavoprotein subunit; Ip, iron-sulfur subunit; SI-$3, iron-sulfur clusters I-3; Q, ubiquinone; RQ, rhodoquinone; M- and C-side, matrix and cytoplasmic side of the mitochondria, respectively.

there is a functional role for cytochrome b in mammalian complex II as well as in Ascaris. Lowpotential rhodoquinone, the low-potential S-3 center and the more positive Em' o f cytochrome b558 in complex II appear to be unique features of the anaerobic respiratory chain of Ascaris adults, indicating that electron transfer from N A D H to fumarate is favored.

Branched-chain fatty acid synthesis In addition to succinate, branched-chain fatty acids such as 2-methylbutyrate and 2-methylvalerate accumulate as predominant end products of carbohydrate catabolism in Ascaris 3,22. These compounds arise from the condensation of the acyl CoA and subsequent reduction of the product to the saturated acid by the reversal of f3-oxidation. This system is located in mitochondria and some of the enzymes have been purified and characterized, including the propionyl-CoA condensing enzyme, which catalyzes the first step and is one of the ratelimiting steps of the pathway 23. Of particular significance in this system is the participation of the electron transport chain in the reduction of the dehydroacyl CoA compound to saturated CoA esters. The NADH-dependent reduction of 2methylcrotonyl CoA or 2-methyl-2-pentenoyl CoA has been demonstrated, and this reaction is sensitive to rotenone, which is an inhibitor of complex I in the mitochondrial respiratory chain 24. The anaerobic electron transport pathway from N A D H to dehydroacyl CoA is shown in Fig. 1. The most important aspect of this pathway is that the difference in potential between the N A D + / N A D H couple (Em'=-320mV) and the enoyl CoA/acyl CoA ester couples (E,,' = - 1 5 to -30mV) is large enough to allow for ATP formation. This, indeed, may be the case in Ascaris, judging by the rotenonesensitivity of this pathway 24, although direct evidence has yet to be obtained. Two soluble flavoenzymes, electron-transfer flavoprotein (ETF)

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and enoyl CoA reductase (ECR), have been purified and well characterized24'25. ETF is composed of two polypeptides of 37kDa and 31.5kDa, and catalyzes electron transfer from the electron transport chain to ECR. ECR is a tetramer composed of identical subunits of 42.5kDa, and catalyzes the reduction of enoyl CoA to 2-methylbutyryl CoA and 2-methylvaleryl CoA. The enzymes of Ascaris differ markedly from their mammalian counterparts, not only in the reverse direction of the catalytic reaction, but also in substrate specificity and sensitivity to the inhibitors although they are similar in size and physical characteristics. In addition to these soluble enzymes, electron-transfer flavoprotein (ETF) dehydrogenase has been purified from A. suum (R. Komuiecki, pers. commun.). It is an abundant (at least 2% of the total protein) iron-sulfur flavoprotein that mediates electron transfer from rhodoquinone to ETF. This enzyme is approximately 64 kDa, and it crossreacts with antisera raised against the bovine heart enzyme. The primary role of this pathway may be the maintenance of the mitochondrial redox balance by serving as a sink for excess reducing power. It has been pointed out previously that the reversal of ~oxidation theoretically does not increase energygenerating capacity over that obtained from the formation of acetate and propionate 25.

Developmental change in the respiratorychain The life cycle of Ascaris is complex. The developmental changes in energy metabolism are interesting and important characteristics in addition to the unique features of the anaerobic metabolic pathway described above. Oxygen is required for egg development, and an active tricarboxylic acid cycle and cyanide-sensitive oxygen uptake have been observed in second-stage larvae (L2) (Ref. 26). At this stage the respiratory chain is similar to that of the mammalian host. In contrast to findings for the adult nematode, cytochrome oxidase has been detected by enzymatic and spectroscopic analysis in fertilized eggs and larvae, and its presence has been confirmed recently by studies on the nucleotide sequence of Ascaris mitochondrial DNA zT.

Table 1 summarizes the difference in respiratory chains between infective L2 and the adult. The ratio of SDH to FRD in the fertilized egg (1.05) is intermediate between those of the adult (0.05) and those of mammals (20-30) (Ref. 7). A similar result (0.87) to that of the fertilized egg has been obtained for L2 (S. Takamiya et al., unpublished). The change in the ratio of SDH to FRD during the life cycle suggests that two isoforms of complex II occur in Ascaris, as succinate-ubiquinone oxidoreductase and fumarate reductase (Fig. 3), similar to E. coli, which possesses two independent genes, sdh and frd. In contrast to adult mitochondria, which contain rhodoquinone as a major quinone, the major quinone of L2 is ubiquinone (S. Takamiya et al., unpublished and Ref. 28). Ubiquinone, with a higher potential than rhodoquinone, is a less suitable carrier of electrons to fumarate and instead donates them preferentially to the cytochrome chain in the mitochondria of the L2. The combination of SDH and ubiquinone, and FRD and low-potential quinone, such as rhodoquinone and menaquinone, are also observed in E. coli and other bacteria during adaptation to changes in oxygen supply 29'3°. Since rhodoquinone is synthesized from ubiquinone 31, induction of the enzyme catalyzing this conversion may be programmed, like the other enzymes for anaerobic respiratory systems. Synthesis of the enzymes that participate in oxidative phosphorylation in L2 may be suppressed in the adult, although some are still expressed. As shown in Table 1, all of the complexes in the mitochondrial respiratory chain except complex II, of which all the genes are encoded in the nuclei, consist of gene products from nuclear and mitochondrial DNA. In adult mitochondria, which lack cytochrome oxidase, functional complex I has been identified enzymatically and has been isolated as a super complex with complex III (Ref. 4). This finding indicates that N A D H dehydrogenase (ND) genes for complex I are expressed from mitochondrial DNA, although synthesis of cytochrome oxidase is suppressed. It is difficult to explain the selective expression of N D genes by the transfer RNA punctuation model for the maturation of

Table I. Developmental changes in the respiratory chains of Ascaris Activity Complex I

III IV

Major quinone

NADH-ubiquinone oxidoreductase Succinate-ubiquinone oxidoreductase Fumarate reductase Ubiquinol-cytochrome c oxidoreductase Cytochrome c oxidase

Presence in L2 (aerobic)

Presence in adult (anaerobic)

Number of subunits a

High

High

25 (7)

High

High

4

low High

High low

I I ( I)

High

Negligible

13 (3)

Ubiquinone (+1 l0 mV)

Rhodoquinone (-63 mY)

aNumbers of subunits coded in rnitochondrial DNA are shown in parentheses.

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messenger RNAs from the polycistronic transcription of mitochondrial D N A 32. The loss of cytochrome oxidase subunits could occur post-transcriptionally or post-translationally, or the assembly of the active complex IV could be controlled by the supply of the nuclear-encoded subunits. During the development of Ascaris, the transition from aerobic to anaerobic energy metabolism has been suggested to occur during the third moult, from L3 to L4, in the small intestine. This has been confirmed by the analysis of cultured L3 (Ref. 33). Studies on Ascaris that show dramatic changes in energy metabolism, including changes in the composition of the respiratory chain during the life cycle, will provide new light on the mechanisms regulating the aerobic-anaerobic transition and the crosstalk between nuclei and mitochondria during the biogenesis of aerobic and/or anaerobic mitochondria. Acknowledgements I would like to dedicate this short review to Professor Hiroshi Oya, who has been one of the leading inspirations in this field and who passed away on 31 July 1991. I would like to thank all those who have worked on the respiratory chain of Ascans at Juntendo University and Tokyo Unwersity. I am also grateful to S. Takamiya and R. Komuniecki for valuable recent information. Part of this work was supported by the Science Research Promotion Fund of Japan Private School Promotion Foundation and by The Mochida Memorial Foundation For Medical and Pharmaceutical Research. References

1 Keilin, D. (1925) Proc. R. Soc. London Ser. B: 98, 312-339 2 0 y a , H. and Kita, K. (1988) in Comparative Biochemistry of Parasitic Helminths (Bennet, E., Behm, C. and Bryant, C., eds), pp 35-53, Chapman & Hall

3 K6hler, P. (1991) in Physiological Strategies for Gas Exchange and Metabolism (Woakes, A.J., Grieshaber, M.K. and Bridges, C.R., eds), pp 15-34, Cambridge University Press 4 Takamiya, S., Furushima, R. and Oya, H. (1984) Mol. Biochem. Parasitol. 13, 121-134 5 Takamiya, S., Furushima, R. and Oya, H. (1986) Biochim. Biophys. Acta 848, 99-107 6 Kita, K. et al. (1988) Comp. Biochem. Physiol. B: 89, 31-34 7 Kita, K. et al. (1988) Biochim. Biophys. Acta 935, 130-140 8 Ma, Y-C. et al. (1987)Jpn. J . Parasitol. 36, 107-117 9 Furushima, R. et al. (1990) F E B S Lett. 263, 325-328 10 Wierenga, R.K., Drenth, J. and Schulz, G.E. (1983)J. Mol. Biol. 167, 725-739 11 Wood, D. et al. (1984)Biochem. J . 222, 519-534 12 Phillips, M.K. et al. (1987)J. Bacteriol. 169, 864-873 13 Cole, S.T. (1982) Eur. J . Biochem. 122,479-484 14 Ohnishi, T. (1987) Curr. Top. Bioenerg. 15, 37-65 15 Hata-Tanaka, A. et al. (1988) F E B S Lett. 242, 183-186 16 Unden, G., Albracht, S.P.J. and Kroger, A. (1984) Biochim. Biophys. Acta 767, 460-469 17 Cole, S.T. et al. (1985) Biochim. Biophys. Acta 811,381-403 18 Kita, K. et al. Electrophoresis (in press) 19 Kita, K. et al. (1989)J. Biol. Chem. 264, 2672-2677 20 Takamiya, S. et al. (1990) Biochem. Int. 21, 1073-1080 21 Yu, L. et al. (1987)J. Biol. Chem. 262, 1137-1143 22 Komuniecki, R. and Komuniecki, P.R. (1988) in Comparative Biochemistry of Parasitic Helminths (Bennet, E., Behm, C. and Bryant, C., eds), pp 1-12, Chapman & Hall 23 Suarez de Mata, Z. et al. (1991) Arch. Biochem. Biophys. 285, 158-165 24 Komuniecki, R., Fekete, S. and Thissen-Parra, J. (1985)J. Biol. Chem. 260, 4770-4777 25 Komuniecki, R. et al. (1989) Biochim. Biophys. Acta 975, 127-131 26 Oya, H., Costello, L.C. and Smith, W.M. (1963)J. Cell. Comp. Physiol. 62, 287-294 27 Wolstenholme, D.R. et al. (1987) Proc. Natl Acad. Sci. USA 84, 1324-1328 28 Kita, K. et al. (1991) Prog. Neuropathol. 7, 21-30 29 Cole, S.T. et al. (1985) Biochim. Biophys. Acta 811,381-403 30 Hiraishi, A. (1988) Arch. Microbiol. 150, 56-60 31 Powls, R. and Hemming, F.W. (1966)Phytochemistry 5, 1249-1255 32 Ojala, D., Montoya, J. and Attardi, G. (1981) Nature 290,470-474 33 Komuniecki, P.R. and Vanover, L. (1987) Mol. Biochem. Parasitol. 22, 241-248

Sexually Transmitted Diseases in Animals G. Smith and A.P. Dobson The sexual transmission of infectious agents is one of a suite of characteristics that enhance the ability of an infection to persist in low-density populations. Gary Smith and Andrew Dobson review the characteristics and control of sexually transmitted diseases in domestic animals and wildlife species. We begin our discussion of sexually transmitted diseases (STDs) in animals by considering a mysterious malady of horses that threatened to Gary Smith is at the Department of Clinical Studies (NBC), University of Pennsylvania School of Veterinary Medicine, New Bolton Center, 382 W. Street Road, Kennett Square, PA 19348, USA and Andrew Dobson is at the Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08540, USA. 1992, Elsevier Science Publishers Ltd, (UK)

disrupt commercial and military life in the nineteenth century. In 1796, a veterinarian in the district of Trakehnen, Prussia described a 'malignant disease of the generative organs' (Ref. 1) of horses. The disease attacked only stallions and mares, never foals or geldings. It spread throughout the countries of the Austro-Hungarian Empire and by 1832 it had appeared apparently for the first time in both Switzerland and France. By the latter part of the nineteenth century the disease had also been recognized in Southern Russia and North Africa, and went by various names: 'maladie du coit' in French, 'besch/ilkrankheit' in German, 'el dourine' in Arabic 2 and 'dourine' in English. The contagious nature of the dourine was recognized early on but the sporadic nature of the epidemics, which were often confined to certain