Cytochromes in the respiratory chain of helminth mitochondria

Pergamon PII:

InrernafionolJournolfor Pararirology,Vol. 27.No.6,pp. 617-630. 1997 C’ 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd Printed in Great Britain 0020-7519/97 $17.00+0.00 SOO20-7519(97)0001&7

IN VZTED RE VIEW

Cytochromes KITA,*f

KIYOSHI

in the Respiratory Mitochondria HIROKO

HIRAWAKE*

Chain of Helminth

and SHINZABURO

TAKAMIYAT

*Department of Parasitology, The Institute of Medical Science, The University of Tokyo, 4-6-l Shirokanedai, Minato-ku, Tokyo 108. Japan TDepartment of Parasitology, Juntendo University, School of Medicine, Tokyo 113, Japan (Received

12 June 1996; accepted

9 December

1996)

Abstract-Kita K., Hirawake H. & Takamiya S. 1997. Cytochromcs in the respiratory chain of hehninth mitochondria. Znternationa~ Journalfor Parasitology 27: 617-630. Parasitic hebninths exhibit greater diversity in energy metabolism than do the host animals and many have exploited unique respiratory chains as adaptations to their natural habitats. Cytochromes are Involved, not only in intracellular aerobic respiration found in free-living stages, but also in the reduction of relatively oxidized compounds such as fumarate during the adult stages of parasitic hehnintbs. In addition, most helminths retain a significant capacity to produce energy via aerobic pathways and have a mammalian type respiratory chain in their mitochondria during their development in the host. In this review, we focus on recent advances in the study of cytochromes in the respiratory chain of parasitic hehninths. These include the identification of unique features of anaerobic respiration in adult parasites, the elucidation of molecular structures of the components involved and an understanding of the developmental changes that occur during the life-cycle of these parasites. 0 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words:

ENERGY

cytochromes; helminth mitochondria; respiratory chain; energy metabolism.

METABOLISM

minths, such as the nematode Ascarissuum,the cestode Hymenolepisdiminuta,and the digenean Fasciola hepatica,which reside in areas where glucose supply is insufficient and oxygen tensions are low, exploit unique metabolic pathways as adaptations to their habitats within their host. For the establishment of this alternative pathway, designated as the phosphoenolpyruvate carboxykinase-succinate pathway (PEPCK-succinate pathway), 3 factors are critical, namely: (1) the activity ratio of pyruvate kinase/phosphoenolpyruvate carboxykinase at the branch point of the glycolytic pathway; (2) the presence of a malic enzyme and its important role in the production of NADH in parasite mitochondria; (3) the NADH-fumarate reductase system in the mitochondria at the terminal step of the pathway (Oya & Kita, 1988). The composition and the linear sequential order of the respiratory component in the NADHfumarate reductase system have been elucidated using A. suum mitochondria, as described later. These lumen-dwelling helminths exhibit a wide range of end-

OF HELMINTH

It is commonly accepted that the carbohydrate and energy metabolism of adult parasitic helminths differs from those of the vertebrate host. Furthermore, parasitic helminths exhibit a wide variation in their carbohydrate break-down pathway. The most important factors in this respect are the nutrient and oxygen supply (see reviews by Bryant & Behm, 1989; Kbhler, 1991; Tielens, 1994; Komuniecki & Harris, 1995). Blood- and tissue-dwelling helminths, such as schistosomes and filariae, convert most of their abundant supplies of environmental glucose into lactate and excrete this as an end-product of carbohydrate metabolism, despite the fact that at least one-third of energy production of adult schistosomes occurs through aerobic pathways under aerobic conditions (Van Oordt et al., 1985). In contrast, lumen-dwelling hel$To whom correspondence should be addressed. Tel: 8 lFax: 81-3-5449-5410.

3-5449-5370;

617

618

K. Kita

products of mitochondrial metabolism. The nematode, A. suum, forms a complex mixture of acetate, propionate, succinate, 2-methylbutanoate and 2methylpentanoate as end-products (Rioux & Komuniecki, 1984). The cestode, H. diminuta, forms succinate and acetate as end-products of anaerobic malate dismutation (Behm et al., 1987). In the digenean, F. hepatica, succinate is further decarboxylated to propionate (Tielens et al., 1984). The lung fluke, Paragonimus westermani, inhabits a cyst in the host lung tissue which has an oxygen tension that is much higher than that of the intestinal lumen where the adult A. suum resides. Mitochondria from adult P. westermani, unlike adult A. suum mitochondria, possess both cyanide-sensitive succinate oxidase and a NADH-fumarate reductase system, indicating that the fluke mitochondria are facultatively anaerobic (Takamiya et al., 1994). In addition to habitat, worm size plays an important role in determining the pathway used to generate energy, because size affects the availability of oxygen and glucose. A small nematode, such as Nippostrongylus braziliensis, which resides close to the gut mucosa where oxygen tensions are higher than in the lumen, has a functional aerobic respiratory chain and relies on oxygen as a terminal electron acceptor (Fry et al., 1983). The use of oxygen as a terminal electron acceptor can result in far more efficient energy metabolism than is possible by anaerobiosis. Only 2mol of ATP per glucose are generated by classical glycolysis, compared to the 38 mol of ATP generated by aerobic oxidative phosphorylation, although many facultative anaerobes are capable of employing a modified pathway that can increase significantly the yield of ATP. It might be expected that this type of parasitic helminth would show adaptation wherever possible to maximize use of any potential aerobic environment. Another unique feature of helminth energy metabolism is aerobic-anaerobic transition during their lifecycle (Tielens, 1994; Komuniecki & Harris, 1995). In general, free-living larval stages operate by aerobic energy metabolism similar to the mammalian host, with an active tricarboxylic acid (TCA) cycle and cyanide-sensitive respiration, whereas parasitic adult stages operate primarily by anaerobic metabolism. Through analysis of the end-products resulting from uptake and metabolism of radiolabelled glucose, it has been shown that free-living cercariae of S. mansoni appear to utilize largely the TCA cycle and oxidative metabolism to break down glucose aerobically (Van Oordt et al., 1989). However, after penetration of the definitive host, the schistosomula alter their carbohydrate metabolic pathway such that glucose is degraded primarily anaerobically. resulting in the production of lactate and pyruvate as end-products. In

et al. F. hepatica, the ratio of TCA cycle activity to acetate and propionate formation appears to decrease during the development of an excysted juvenile fluke into an adult (Tielens et al., 1984, 1987). A. suum also exhibits a number of alterations in carbohydrate metabolism during development from an unembryonated egg into an adult (Barrett, 1976; Komuniecki & Vanover, 1987; Takamiya et al., 1993), and is probably the best studied model system for the elucidation of energy metabolism in parasitic helminths. The present paper primarily reviews the recent progress in the study of cytochromes in the aerobic and anaerobic respiratory chains of A. suurn, as well as the unique properties of respiratory components in other helminths. Developmental changes in the respiratory chain of A. suum mitochondria Contents of cytochromes in the helminth mitochondria show a wide variation depending upon the stage of their life-cycle and habitat of each parasite (Hayashi et al., 1974; Takamiya et al., 1993). In adult parasitic helminths, a mammalian-type aerobic respiratory chain is generally absent. If present, the content of cytochromes is lower than that of its mammalian counterpart. A. suum eggs require oxygen for embryonation and develop to contain infective 2nd-stage larvae (L2) approximately 3 weeks after leaving their host. When A. suum L2 are ingested into the host, they reach the gut, moult to the 3rd~stage larvae (L3) in the liver, and then migrate to the lung. The energy metabolism of both the L2 and L3 is aerobic and sensitive to cyanide, which is an inhibitor of cytochrome c oxidase. Fourth-stage larvae (L4) become capable of synthesizing end-products typical of the adult and their motility becomes cyanide-insensitive. Thus, the transition from aerobic to anaerobic energy metabolism appears to occur during the 3rd moult, from L3 to L4, in the small intestine. This has been confirmed biochemically through analysis of cultured L3 and L4 (Komuniecki & Vanover, 1987; Takamiya et al., 1993). During the transition from L2 to adult, the components and organization of the respiratory chain change dramatically, as shown in Fig. 1. The respiratory chain of L2 mitochondria is almost identical to that of the mammalian host. Reducing equivalents from respiratory substrates such as NADH and/or succinate produced by the TCA cycle are transferred to ubiquinone via the NADH-ubiquinone dehydrogenase complexes, reductase (complex I) and succinate-ubiquinone reductase (complex II), respectively, then transferred to cytochrome c via ubiquinol-cytochrome c oxidoreductase (complex III; cytochrome bc, complex). Cytochrome c oxidase (complex IV; cytochrome aa3

Helminth cytochrome chains

(Cyt NADH +

I

Larva + Adult

*

cl

RQ++Cytc+m--,O, (Cyt b,,,.,) Fum + II * (CYt b-63) 0 (Cyt c,) (CYt b558) Fig. 1. Aerobic and anaerobic respiratory chains in larval and adult mitochondria of A. suum. I, complex I (NADHubiquinone reductase complex); II, complex II (succinateubiquinone reductase complex/ fumarate reductase); III, complex III (ubiquinone
619

of the oxidative phosphorylation system. A gene for ATPase subunit 8, common to other metazoan mtDNAs, has not been identified in nematode mtDNAs. The gene arrangements are almost identical in the C. elegans and A. suum mtDNA molecules, while the arrangement of genes in the 2 nematode mtDNAs differs extensively from those of all other metazoan mtDNAs (Fig. 2). Amino acid sequences deduced for cytochrome b (cyt b) and subunits I-III of complex IV (COI-III) from both nematodes are very similar, and most residues essential for their function and the ligation of their heme prosthetic groups are conserved. MtDNA levels are elevated in aerobic eggs but are appreciably lower in cell types with reduced respiratory activity or mitochondria populations, such as anaerobic body wall muscle (Rodrick et al., 1977). In contrast to the L2, adult worms, which reside in the host small intestine, exploit a unique anaerobic respiratory chain as an adaptation to their microaerobic habitat (Kita. 1992). The major quinone is the low potential, rhodoquinone (- 63 mV), while the major quinone in the L2 is ubiquinone (+ 110 mV) (Takamiya et al., 1993). The synthesis of enzyme complexes that participate in oxidative phosphorylation in the L2, such as cytochrome c oxidase, is suppressed in the adult stage, although some are still expressed. In the main pathway of the adult anaerobic respiratory chain, the reducing equivalents from NADH are transferred to 2 enzyme systems, the fumarate reductase of complex II and electron-transfer flavoprotein rhodoquinone oxidoreductase, via rhodoquinone (Fig. 1). Electron transfer from NADH to fumarate or enoyl CoA is coupled to ATP synthesis by a site I phosphorylation associated with the proton translocation in complex I. This NADH-fumarate reductase pathway is a final step in the PEPCK-succinate pathway as discussed previously. It should be stressed that unlike the L2 and mammalian enzymes, complex II of adult A. suum functions in the reverse direction, as a fumarate reductase rather than as a

Table I-Comparison

of cytochrome contents in mitochondria from parasitic helminths, C. elegans and bovine heart

Source

b

Bovine heart C. elegans A. .wum (L2) A. ~uurn (adult) N. brasiliensis P. westermani P. ohirai

nmol mgg’ protein

0.32 0.464 0.44

0.19 0.20 0.32 0.22

n.d., not determined.

C+C,

aa

b:

c+c,:

0.47 0.283 0.37 0.036

0.68 0.255

1 1 1 1 1 1 1

n.d. 0.42 0.21

0.15 0.00 0.08 0.20

0.11

aa3

Reference

1.5

2.1

0.61 0.83 0.19

0.34

Merle & Kadenbach (1982) Murfitt et al. (1976) Takamiya et al. (1993) Takamiya ef al. (1984) Fry et al. (1983) Takamiya et al. (1994) Fuiino et al. (1996)

n.d. 1.3 0.95

0.55 0.00 0.4 0.62 0.5

620

K. Kita et al. ND3 I

ND4L I

Human

I

I, I

I s2

Il\ ANCY ’

Q

I

ATPase

ND6

P

ND4L KL,S,

Ascaris

ND1

6

IRQF

ND2

L2

Cyt

b co111

T

CMDG

ND4

co1

H

co11

ND3

16s

ATPase

APV

ND5

WE

S2

12s

NY

AT

ND6

Fig. 2. Arrangement of genes in human (Anderson er al., 1981) and A. suum (see Okimoto et al., 1992) mitochondrial DNA. Capital letters represent 22 individual tRNA genes. COI-III, genes encoding subunits I, II and III of complex IV (cytochrome c oxidase); ND1-6, genes for subunits of complex I (NADH-ubiquinone reductase); Cyt b, gene for the apoprotein of cytochrome b in complex III (ubiquinol-cytochrome c reductase); ATPase 6 and 8, genes for subunits of ATP synthetase; 12s and 16S, genes for the small and large ribosomal RNA; AT, AT rich putative control region. succinate dehydrogenase (Kohler & Bachmann, 1980; Kita et al., 1988a). The ratios of succinate-ubiquinone reductase to fumarate reductase in the fertilized egg (1 .OS) and L2 (0.87) are intermediate between that of adult (0.05) and those of mammals (20-30) and the ratio decreases during the latter stages of development from L3 to L4 (Kita et al., 1988b; Takamiya er al., 1993). The change in the ratio suggests that 2 isoforms of complex II occur in A. suum, similar to Escherichia coli, which possesses 2 independent genes, sdh and frd. In fact, 2 different genes for the iron-sulfur cluster (Ip) subunits of Huemonchus contortus complex II have been identified. These genes are differentially expressed during the life-cycle (Roos & Tielens, 1994), although the enzymatic properties of the 2 enzymes have not yet been defined. A second flavoprotein (Fp) subunit gene has also been found in Dirojilaria immitis (see Kuramochi et al., 1995, and unpublished observations). Recently, direct evidence for the occurrence of 2 distinct stage-specific enzyme complexes in A. suum, 1 in larval mitochondria which is more similar to aerobic mammalian enzymes with low fumarate reductase activity, and the other in adult mitochondria which shows high fumarate reductase activity in addition to succinate dehydrogenase activity has been shown by the direct biochemical analysis of the isolated complexes (Saruta ef al., 1995). The presence of 2 different enzyme complexes and the combination of a succinate dehydrogenase complex with a highpotential ubiquinone, and a fumarate reductase complex with a low-potential quinone, such as rhodoquinone or menaquinone, are also observed in E. coli and other bacteria during adaptation to changes in oxygen supply (Cole et al.. 1985; Hiraishi, 1988).

Indispensability of rhodoquinone as the low-potential electron carrier in the NADH-fumarate reductase pathway has been demonstrated in a reconstituted system made from bovine complex I, complex II from adult A. suum and rhodoquinone in liposomes (Kita et af., 1988b). The high-potential ubiquinone, which is a major quinone in L2 mitochondria, cannot mediate electron transfer between the 2 complexes. Rhodoquinone is found in the mitochondria from many parasitic helminths, and also found in those from freeliving organisms that reduce fumarate under anaerobic conditions (Van Hellemond et al., 1995). Since rhodoquinone may be synthesized from ubiquinone (Parson & Rudney, 1965) expression of the enzyme catalysing this conversion during the life-cycle may be regulated, like the other enzymes for anaerobic respiratory systems. CYTOCHROME DEHYDROGENASE

b IN COMPLEX COMPLEX: REDUCTASE)

II (SUCCINATE FUMARATE

Complex II of adult A. suum purified from body muscle mitochondria exhibits high fumarate reductase activity and plays a key role as a terminal enzyme in the anaerobic electron-transport chain, the NADHfumarate reductase pathway (Takamiya et al., 1986; Kita et al., 1988a, 1988b). Complex II is generally composed of 4 polypeptides and appears to be highly conserved (Ackrell et al., 1992; Hederstedt & Ohnishi, 1992; Van Hellemond & Tielens, 1994). The largest flavoprotein (Fp) subunit has an apparent molecular mass of approximately 70 kDa and contains covalently bound flavin. The second largest subunit, an

Helminth cytochrome chains iron-sulfur protein (Ip) subunit, has an apparent molecular mass of approximately 30 kDa and contains 3 different types of iron-sulfur clusters, S-l, S-2 and S-3. The Fp and Ip subunits comprise the catalytic portion of complex II and catalyse electron transfer from succinate to artificial electron donors such as phenazine methosulfate (succinate dehydrogenase), or from reduced methylviologen to fumarate (fumarate reductase). The amino acid residues of Fp (Kuramochi et al., 1994) and Ip (Wang et al., 1992) in complex II from adult A. suum mitochondria involved in the binding of prosthetic groups and substrate recognition are highly conserved. The presence of a 2subunit cytochrome b composed of large (cybL) and small (cybS) subunits acting as a hydrophobic membrane-anchor peptide, is a general feature of complex II in mitochondria and bacteria. Exceptions are the enzymes from the Bacillus subtilis succinate dehydrogenase complex (Magnusson et al., 1986) and the Wolinella succinogenes fumarate reductase (Kiirtner et al., 1990), which contain 2 cytochrome b components bound to a single large hydrophobic subunit, and the Saccharomyces cerevisiae succinate dehydrogenase complex (Schilling et al., 1982) and the E. coli fumarate reductase (Cole et al., 1985), which do not contain heme b. The cytochrome b,,, of adult A. suum complex II can be separated from the Fp and Ip subunits, and is the typical 2-subunit cytochrome b of complex II, comprising cybL (17.2 kDa) and CybS (12.5 kDa) (Kita et al., 1988b). Cytochrome bs5* shows 2 cl-peaks at 552 and 558nm in the reduced-minus-oxidized difference spectra at 77°K and at 560nm at room temperature (Takamiya et al., 1986). A soluble cytochrome bseOand a novel b-type cytochrome, that also have split peaks at low temperatures, were purified from A. suum muscle (Cheah, 1973; Yu et al., 1996). Cytochrome bSbo is distinct from cytochrome b,,, in complex II. Their amino acid compositions are different, and the polarity of cytochrome b,,, (53%) is much higher than that ofcybL and cybS (32.5% and 28.3%, respectively) of cytochrome b,,,. The amino acid sequence of the novel b-type cytochrome deduced from cDNA contains a sequence homologous to vertebrate cytochrome b5, and is different from those of cybL and cybS. Cytochrome b,,, in complex II is a major constituent cytochrome of A. suum muscle mitochondria in the adult worm. This is consistent with the fact that complex II is one of the major components in A. suum adult mitochondria (8% of mitochondrial protein) (Kita et al., 1988a). The cytochrome b in complex II seems to be essential for interaction between the complex and quinone species. Cytochrome b,,, of adult A. suum complex II is reducible by succinate and has been shown to have an Em’

621

of -34mV (Takamiya et al., 1990), which is much higher than the Em’ of cytochrome b,,, in the bovine heart complex II (- 185 mV, reported by Yu et al., 1987). The more positive Em’ of cytochrome bSssmay facilitate electron transfer from rhodoquinone (- 63 mV) to the succinate/fumarate couple (+30mV), although direct evidence for the participation of heme b in electron transfer in the complex has not yet been demonstrated. An EPR spectrum of the air-oxidized form of cytochrome bs5* in complex II showed a ferric low-spin signal at g = 3.6, which is similar to that of cytochrome b,,, in the bovine heart complex II (Hata-Tanaka et al., 1988). Recently, the cDNA for cybL in adult A. suum complex II has been cloned and sequenced by the authors (Fig. 3). The deduced amino acid sequence shows hydrophobic characteristics as a membrane-anchor for the complex, and appears to have 3 transmembrane segments based on hydrophobicity analysis. In contrast to the highly conserved features of the Fp and Ip subunits, the amino acid sequence of A. suum cybL shows little similarity to the sequence of the other species. The similarity between A. suum cybL and the cybL of E. coli fumarate reductase is much lower (7.0% with the frd c product) than that between A. suum cybL and the cybL of E. coli succinate dehydrogenase complex (15.9% with the sdh c product), even though the adult A. suum complex II exhibits high fumarate reductase activity. It is noteworthy that the conserved His residue (His-84 in E. colisdh c product), which seems to be 1 of the axial ligands of heme b in complex II, is found in adult A. suum cybL (His-loo). Properties of A. suum cybS in adult complex II (Saruta et al., 1996) are similar to those of cybL, including lower similarity to its counterparts in other organisms, its hydrophobic nature with 3 transmembrane segments and conserved histidine residues (His-70 and His-72 in A. suum cybS). Recent analysis by EPR and near-infrared magnetic circular dichroism (MCD) suggests a bis-histidine ligation with heme b in cytochrome b,,, of the B. subtilis complex II (H&gerh&ll et al., 1995), in cytochrome b,,, of E. coli succinate dehydrogenase complex (Peterson et al., 1994), and in cytochrome bsCOof the bovine complex II (Crouse et al., 1995). These observations, in addition to the amino acid sequence data of A. suum cytochrome b,,,, indicate that heme b bridges 2 histidine residues in the cybL and cybS hetero-dimer of the 2-subunit cytochrome b in mitochondrial complex II. This idea is consistent with the fact that both cybL and cybS subunits are essential for heme b ligation to form the 2subunit cytochrome b in the E. coli succinate dehydrogenase complex (Nakamura et al., 1996). Two-subunit cytochrome b reducible by succinate is also found in purified complex II from P. westermuni (see Ma et al.. 1987).

622 Bovine C.elegans

K. Kita

et al.

Heart

A.suum

S.cerevisiae E.coli sdhC E.coli frdC BH Ce As SC

CIRN

LKNV

EcS EcF BH Ce AS SC EcS EcF

SDH3

MTT---KRKPYVM-MTSrWWKiLPFYRFYhLREGTAVPwVWFSI S---

L--0L--YLFT

----

-LFG SA-LL PGS ESHLEFVKSHCLGPALIH ;jV~I@!jF~@SWNL[CAVTA!j!j:: LIGGVGFS LPLDFTTFVEFIR#GIPWVILDTFK--ILFG SG-LLGLGLT EK SNWYHQKFSKITEWSI

-

---GILLWLLGTSLSSPEGFEQAS---AIMGSFFV --ELIFGLFALKNGPEAWAGFVD---FLQNPVIVIINL

FIM

S-

BH Ce AS SC EcS EcF Fig. 3. Comparison of the deduced amino acid sequences of cybL subunits from various species. Bovine heart (Cochran et al., 1994). C. elegans (from C. elegans database; accession L26545), A. suum (this study; DDBJ, EMBL and NCBI databases under accession Nos D78157). S. ceretlisiae (see Daignan-Fornier et al., 1994), E. coli sdh c (see Wood et al., 1984), E. coli frd c (see Grundstrdm & Jaurin. 1982). are presented. Similarity in the sequences of the various species was maximized with the computer program GENETYX. Amino acids identical to those in A. suum cybL are boxed. Conserved hi&dine residues are indicated by asterisks. The partial amino acid sequence of the A. ~uurn cybL determined from N-terminal analysis is underlined. Complex II in A. suum larval mitochondria is distinct from that in adult mitochondria (Saruta et al., 1995). Complex II purified from L2 mitochondria shows much lower fumarate reductase activity (0.706 pmol min-’ rng-‘) than that of adult complex II (28.9 pmol min-’ rng-‘), whereas succinate dehydrogenase activities of mitochondria in both stages are comparable. The complex II isolated from L2 has a higher affinity for succinate in the succinate dehydrogenase assay than the adult enzyme, while the complex isolated from the adult has a higher affinity for fumarate in the fumarate reductase assay than the L2 complex. The Fp subunit of larval complex II can be distinguished from that of adult complex II by 2dimensional gel electrophoresis and peptide mapping. The presence of 2 distinct Fps in A. mum has been confirmed by the cloning of cDNA for larval Fp (Hirawake et al., unpublished observation). Complex II of the larva also contains cytochrome b. and its Soret

band of the oxidized form (402 nm) is different from that of the adult complex II (410 nm). The Em’ value of the cytochrome b in larval complex II seems to be lower than that of adult, because cytochrome b of complex II is not detectable in larval mitochondria after succinate reduction. An antibody against cybS of the adult enzyme does not cross-react with that of the larval enzyme. These results, together with the fact that the electron acceptor from cytochrome b in the larval enzyme is the high potential quinone, ubiquinone, suggest that cytochrome b of the larval complex II is likely to be different from that of the adult enzyme, although further analysis of cybL is necessary.

COMPLEX III (UBIQUINOL-CYTOCHROME REDUCTASE) AND CYTOCHROME Complex complex,

III, also known or ubiquinol+ytochrome

as the

e c

cytochrome c reductase,

bc, cat-

Helminth cytochrome chains alyses electron transfer between 2 mobile redox carriers, ubiquinol and cytochrome c. This electron transfer couples to proton translocation, thus generating a proton-motive force which can drive ATP synthesis. The subunit composition of complex III differs slightly in various organisms. The subunits with redox centers, cytochrome b, cytochrome c, and the Reiske iron-sulfur protein, show the highest sequence similarity, including to the subunits of bacterial complexes. The presence of several additional subunits without redox groups is a characteristic feature of mitochondrial complex III. The molecular organization and function of complex III in the aerobic respiratory chain of larval and adult mitochondria from facultatively anaerobic parasites such as P. westermani appear to be the same or similar to mammalian complex III (Takamiya et al., 1994), although little is known about the helminth complex III. Complex III may also play some role in the anaerobic respiratory chain of adult mitochondria. The adult enzyme complex of A. suum muscle is the only complex III isolated from helminth mitochondria (Takamiya et al.. 1984). A. suum complex III was extracted from mitochondria using deoxycholate and KCl, and purified by ammonium acetate fractionation as a supercomplex with complex I (complex I-III). This complex contains 3 cytochromes b-559.5. b-563 and c,-550.5 and pigment-558 at concentrations of 1.28, 0.211, 1.23 and 0.321 nmol mgg ’ protein, respectively. The specific content of cytochromes b-559.5 and c,550.5 is comparable to that of the bovine heart isolated by Hatefi & Stiggall (1978), indicating the high purity of A. suum complex I-III. A. suum complex IIII exhibits NADH-cytochrome c reductase activity, and shows relatively low sensitivity to antimycin A which is a specific inhibitor of cytochrome b in complex III. At least 14 protein bands are present in SDSpolyacrylamide gel electrophoresis of isolated A. suum complex I-III, including the bands that appear to correspond to major subunits of complex III (Takamiya et al., 1984). Among the subunits of A. suum complex III, the complete amino acid sequence of only cytochrome b has been deduced because the gene for cytochrome b is encoded on mitochondrial DNA (Okimoto et al., 1992). From the extensive sequence information from different organisms. a folding model with 8 membrane-spanning domains has been predicted. Two heme b are attached to the apoprotein on opposite sides of the inner membrane of mitochondria, forming low potential cytochrome b, near the cytoplasmic side where quinol oxidation occurs (center o), and high potential cytochrome b, near the matrix side where quinone reduction occurs (center i). The arrangement of 2 cytochromes b in the membrane is essential for

623

translocation of H+ coupled to electron transfer from ubiquinol to cytochrome c in the ubiquinone-cycle (Qcycle) model, which is now widely accepted as the coupling mechanism. Oxidation of ubiquinol at center o leads to the release of 2 protons into the intermembrane space. The 2 electrons extracted from ubiquinol are divided. One passes through cytochrome c, and the FeS-protein to cytochrome c, while the other passes through cytochromes b, and b, to ubiquinone at center i. Amino acid residues involved in heme ligation and quinone binding have been revealed by studies of a site-directed mutant lacking heme b and mutants resistant to inhibitors such as antimycin A (di Rago & Colson, 1988; Daldal et al., 1989). Alignment of the A. suum 365 amino acids cytochrome b sequence with those from other organisms allowed the identification of the 4 conserved histidine residues involved in ligation of the 2 heme b, as shown in Fig. 4. Several amino acid residues that have mutations which result in resistance to inhibitors are substituted for different amino acids in A. suum cytochrome b, although most of the residues are conserved. For example, Asn-256 in yeast cytochrome b, which is critical for determining resistance to myxothiazole, is changed to methionine in A. suum. Such sequence diversity suggests that cytochrome b in complex III is a possible target for antagonists of ubiquinone in mitochondria of parasitic helminths, as well as parasitic protozoa as discussed by Vaidya et al. (1993). Cytochrome c catalyses electron transfer between complex III and complex IV (cytochrome c oxidase), and is found in the mitochondria of a variety of eukaryotic cells and bacteria. It has been sequenced from a large number of different organisms, and the 3dimensional structure of cytochrome c has been well defined from the X-ray analysis of the crystals (Dickerson et a/., 1971). Hill et al. (1971) purified and characterized cytochrome cs5,,from A. suum. The reduced form of A. suum cytochrome c550 shows c(-, p- and y-absorptions at 550, 520 and 416nm, respectively, whereas the oxidized form shows a y-absorption at 410nm. A. suum cytochrome c was also purified in order to determine the amino acid sequence for a phylogenetic study of nematodes (Vanfleteren et al., 1994). A. suum cytochrome c shows the highest homology with the free-living nematode. C. elegans (84.5%). All 4 heme-binding residues, Cys-19, Cys22, His-23 and Met-84, 2 tyrosine residues (Tyr-50 and Tyr-52), which provide a hydrogen-bond to the propionic acid side chains of the heme, and Phe-15, Phe-86 and Tyr-101, essential for the interaction between cytochrome c and complex IV. are conserved in A. suum cytochrome c. Striking sequence conservation is found in the region from Asn-74 to Met84, which is believed to form a surface domain which

624

K. Kita

70

et al. 80

A. suum

***

*

*

Human

170

A. suum

Human

+

180

FkFVLHFLVPWhLLLLVLLHLbFLHE ** ** * RFFTFHFILPFIIAALATLHLLFLHE 0

+

*

**es

190

****

11

Fig. 4. Identification of heme b ligands in cytochrome b of complex III (ubiquinolxytochrome c reductase). The amino acid sequences presented are A. ~uum (see Okimoto et al., 1992) and human (Anderson et al., 1981). bL, low potential cytochrome b at center o; b,, high potential cytochrome b at center i. Conserved histidine residues are boxed.

is also involved in the interaction with complex IV. Recently, the molecular and functional properties of cytochrome c have been studied using a highly active preparation of type-l cytochrome c, purified by a new protocol (Takamiya et al., 1996b). This preparation exhibits essentially the same spectroscopic properties as those previously reported at room temperature, and the reduced x-absorption peak splits into 2 peaks (547 and 549Snm) at low temperatures. The oxidation reduction potential of A. suum type-l cytochrome c (+248 mV) is comparable to that of cytochrome c from mammalian sources. Comparative kinetic studies have revealed species-specificity in the reaction between cytochrome c and complex IV from A. SUU~Z and bovine. The cytochrome c content in mitochondria is highest at the L2 stage, in which the respiratory chain is the most aerobic among various developmental stages of A. suum. A cDNA for the A. suum type-l cytochrome c has been cloned and sequenced (Takamiya et al., 1996a). The entire cDNA contains a reading frame of 336 nucleotides and encodes a protein consisting of 112 amino acids. Residues 2-34, deduced from the nucleotide sequence of A. suum type-l cytochrome c, are identical to the first 33 residues of the N-terminal of purified cytochrome c (Fig. 5). The first methionine is missing in the mature peptide. The amino acid sequence deduced from the cDNA differs at 8 amino acid residues from the reported sequence (Vanfleteren et al., 1994) particularly in the C-terminal region. The amino acid sequence predicted from the cDNA sequence is probably correct because the molecular mass of the apoprotein of type-l cytochrome c determined by ionspray mass spectrometry is identical to that of the mature peptide, as calculated from the deduced amino acid sequence (12 502 Da), although polymorphism of the gene cannot be ruled out.

Stage- and/or tissue-specific isoforms of mitochondrial cytochrome c have also been reported; isol- and iso-2-cytochrome c in S. cerevisiue (see Sherman et al., 1965) somatic and testis-specific cytochrome c in mouse (Henning, 1975), 2 forms of cytochrome c expressed differentially during the development of Musca domestica (see Yamanaka et al., 1980) and Drosophila melanogaster (see Limbach & Wu, 1985). The cDNA for a stage-specific isoform of cytochrome c (type-2 cytochrome c) has also been found in A. suum (Takamiya et al., 1996a and see Fig. 5). The A. suum type-2 cytochrome c shares the typical key amino acid residues for spatial organization and function that have been identified in vertebrate cytochrome c, suggesting that type-2 cytochrome c is functional. Type-2 cytochrome c is expressed only in the adult stage as a minor component, while the cytochrome c previously cloned and sequenced (type-l cytochrome c) is expressed in both adult and larval stages. The amino acid sequences of cytochrome c from other organisms show higher similarities to A. suum type- 1 cytochrome c than to type-2 cytochrome c. Sequence comparison suggests that the 2 types of A. SUUPZcytochrome c diverged approximately 650 Myr ago, thus before the divergence of the free-living C. elegans and parasitic ascarids (480-550 Myr ago). The A. suum type-2 cytochrome c may play a role in the adaptation of adult worms to low oxygen tension, although more intensive study of the physiological function of type-2 cytochrome c in the respiratory chain is required. COMPLEX

IV (CYTOCHROME OTHER TERMINAL

Most respiratory karyotic organisms

E OXIDASE) OXIDASES

AND

oxidases in eukaryotic and proare members of a superfamily of

Helminth

cytochrome

chains

625

. A. SUUIU - 2 A.s""m-1 Human

. A. sum-2 A. suumc. elegans Human

1

Fig. 5. Comparison of the amino acid sequences of cytochrome c from various species. A. suum-2, A. Suum type-2 cytochrome c; A. sum-l, A. sum type-l cytochrome c (Takamiya et al., 1996a), C. elegans (Vanfleteren et al., 1994), human (Matsubara & Smith, 1963). Identical amino acids to type-l cytochrome care boxed. v indicates axial ligands of heme iron. v indicates a nematode-specific deletion. The partial amino acid sequence of the A. suum type-l cytochrome c determined from Nterminal analysis is underlined.

enzymes that couple the redox energy available from the reduction of molecular oxygen to the pumping of protons across the membrane. Respiratory oxidases therefore play a crucial role in the physiology of virtually all aerobic organisms, including parasitic helminths that have aerobic free-living stages in their lifecycles and a functional aerobic respiratory chain even during the adult stage. Three different kinds of terminal oxidases have been reported in parasite mitochondria; complex IV (cytochrome c oxidase), cytochrome o and the alternative oxidase. Complex IV catalyses the reduction of oxygen to water using a reducing equivalent from cytochrome c and this oxidase activity is sensitive to cyanide. Depending on the source, complex IV contains 3-13 subunits, and the core structure of all complex IV is formed by 3 mitochondrially encoded subunits (I, II and III). Three of the 4 metal centers (hemes a, a3 and Cu,) reside in subunit I, whereas the fourth, Cu,, resides in subunit II. Electrons from cytochrome c are accepted by Cu, and are transferred finally to the O? binding site, which includes heme a3 and Cur,, via heme a (see review by Haltia & Wikstrdm, 1992). This enzyme is one of the most intriguing biological macromolecules in the cell. The structure of the metal sites in the bovine enzyme has recently been resolved at 2.8 A (Tsukihara et al., 1995). A comparative study of mitochondrial electron-transport in various parasitic helminths demonstrated the potential physiological importance of complex IV in the respiration of adults as well as the larval respiratory chain, which is almost same as that of the mammalian host and freeliving nematode C. elegans (Table 1). Dithionitereduced minus oxidized difference spectra of adult N. brasiliensis mitochondria showed absorption maxima at 60 1,560 and 424 nm, with shoulders at 555,550 and 440 nm (Fry et al., 1983). The absorption maximum at 601 nm and the shoulder at 440 nm are characteristic of cytochrome aal, suggesting the presence of cyto-

chrome c oxidase. The presence of complex IV in this nematode has been confirmed by the cytochemical detection of cytochrome c oxidase activity using diaminobenzidine (Fry & Beesley, 1985). It is of interest to note that there is a good correlation between the body diameter of the worm and the extent of the mammalian-type respiratory chain: the thinner the worm, the greater the extent of cyanide-sensitive respiration. Deposition of the diaminobenzidine reaction product was uniform and relatively intense in mitochondria from all tissues in N. brasiliensis. In contrast, cytochemical staining of complex IV is markedly decreased in the internal tissues of the larger helminths, H. contortus and Ascaridia galli, in the order hypodermis > muscle > gut > reproductive tissue. There are clearly morphological differences between mitochondria isolated from different tissues of A. galli and N. brasiliensis. The occurrence of different mitochondrial populations has also been shown in the Paragonimus species. The cytochemistry of cytochrome c oxidase activity and ultrastructural analysis of P. ohirai shows 2 types of parenchymal cells, PC 1 and PC 2, with morphologically and functionally different types of mitochondria that are probably involved in different modes of energy metabolism, 1 possessing an aerobic and the other an anaerobic respiratory chain (Fujino et al., 1996). The content of cytochrome aaj (see Table 1) and activity of cytochrome c oxidase of P. westermani (1.34 s-‘mgg’mll’) are significantly higher than those of P. ohirai (0.45 s-’ mgg’ mll’), suggesting that P. westermani mitochondria are more aerobic than P. ohirai mitochondria. Furthermore, expression of complex IV in adult helminths has been shown even in mitochondria from homolactic fermenters such as S. mansoni. The transcript for subunit I of complex IV from mitochondrial DNA at a relatively high level (Skelly et al., 1993) and cytochrome c oxidase activity (Bueding & Charms, 1952) are detected in adult S. mansoni,

626

K. Kita et al.

suggesting that adult schistosomes retain a significant capacity to produce energy through aerobic metabolism. The cc-absorption peak of cytochrome uaj in the mitochondria from adult A. suum, which is rather large in body size, has not been observed even with difference spectra recorded at low temperature except the report by Cheah (1975). However, the specific content (0.037 nmol mgg ’ protein) of cytochrome au, found in adult muscle mitochondria by Cheah was much lower than that of larval mitochondria. There is no direct indication of the presence of functional complex IV in adult A. suum mitochondria. Although little information on the properties of helminth complex IV is currently available because no enzyme complex has been purified from helminth mitochondria, properties of the helminth enzyme have been studied using isolated mitochondria. The apparent K,,, value for O2 in the oxidase activity of mitochondria isolated from N. brasiliensis (3SpM) is comparable to that of mammalian oxidase (2pM, reported by Chance, 1957), and helminth complex IVs show similar sensitivity to azide and cyanide that are well known inhibitors for the mammalian oxidase (Paget et al., 1987). In contrast to adult mitochondria, larval mitochondria of A. suum contain a significant amount of complex IV. The larval enzyme reacts more rapidly with A. suum cytochrome c than bovine cytochrome c when the velocity is compared at low concentrations of cytochrome c (Takamiya et al., 1996b). Amino acid sequences of core subunits, subunit I, II and III, in A. suum complex IV have been deduced from the nucleotide sequences of mitochondrial DNA, and show high similarity to those of C. eleguns complex IV (Okimoto et al., 1992). The similarity between the 2 nematodes is highest in subunit I (88.2%) which binds 3 metal prosthetic groups (2 hemes and 1 copper atom) and contains the oxygen-binding site and most of the proton-conducting channel. Recently, the fine structure of metal centers, which provide electron transfer pathways and serve as key structures for proton pumping, has been obtained from X-ray crystallography of bovine heart complex IV (Tsukihara et al., 1995). Striking conservation is found in the amino acid residues of subunits I and II in A. suum complex IV that are involved in heme and copper binding when compared to the bovine enzyme (Table 2). Subunit III, which has a physiologically important function in the correct assembly of the enzyme, also contains conserved residues including an invariant glutamate residue (Glu-86 in A. suum). This residue reacts with dicyclohexyl carbodiimide and has been a focus of interest because proton translocation in complex IV is inhibited by chemical modification of this residue. However, analysis by site-directed mutagenesis suggests that it is not crucial in proton pumping. In

Table 2-Amino acid residues involved in the binding of prosthetic groups in complex IV Prosthetic group

Subunit

Bovine”

A. swd

Heme

I

His-61 His-378 His-376 Cys-196 cys-200 His-161 His-204 Glu-198 Met-207 His-240 His-290 His-29 1

His-69 His-385 His-383 cys-199 Cys-203 His-164 His-207 Glu-201 Met-210 His-247 His-297 His-298

a

Heme a3 CL!*

1 II

%I

I

“Tsukihara et al. (1995); bOkimoto et al. (1992). general, nuclear-coded subunits of complex IV confer tissue- and/or stage-specific isoforms of the enzyme. S. cerevisiue has 2 isoforms of complex IV and cytochrome c to maintain a constant energy supply (Allen et al., 1995). Complex IV with subunit Va and iso-lcytochrome c are expressed under aerobic and hemesufficient conditions, and complex IV with subunit Vb and iso-2-cytochrome c are expressed under hypoxic and heme-deficient conditions. Kinetic analysis suggests that co-expression of the 2 complex IV isoforms with the 2 cytochrome c isoforms serves to minimize differences in cytochrome c oxidase activity, regardless of the growth conditions. Since A. suum has a stagespecific isoform of cytochrome c, type-2 cytochrome c, the nematode may possess 2 or more isoforms of complex IV and express these during its life-cycle. Cytochrome o has been defined as a b-type cytochrome that acts as a terminal oxidase in aerobic bacteria and in mitochondria from lower eukaryotes. The active cytochrome o, cytochrome bo complex, was first purified from E. coli (see Kita et al., 1984) and characterized extensively as quinol oxidase (Mogi et al., 1994). Recent studies have shown that most respiratory oxidases, including complex IV and cytochrome o, are members of a single superfamily called the hemeecopper oxidase superfamily (Calhoun et al., 1994). The hemeecopper oxidase superfamily is defined by 2 criteria: (1) a high degree of amino acid sequence similarity within the largest subunit (subunit I); (2) a unique bimetallic active site consisting of a heme and a closely associated copper atom, where molecular oxygen is reduced to water as previously described. The amino acid sequence of subunit I from E. coli cytochrome bo complex is more than 40% identical to that of bovine complex IV. It should be noted that the heme prosthetic group of cytochrome o is heme o, which differs from heme b by the replacement of a vinyl group with a hydroxyethyl farnesyl

Helminth cytochrome chains group. No information regarding the enzymatic properties and molecular structure of mitochondrial cytochrome o is currently available, although many investigators have reported CO-reactive b type cytochromes and cyanide-insensitive terminal oxidases in parasite mitochondria (Cheah, 1975; Fry et al., 1983; Mendis & Townson, 1985). The occurrence and physiological significance of helminth alternative oxidase, including cytochrome c peroxidase, are also not clear at the molecular level. In the case of protozoan parasites, a cyanide-insensitive alternative oxidase has been partially purified from bloodstream trypomastigotes of Trypanosoma brucei (Chaudhuri et al., 1995). This oxidase reduces molecular oxygen to water using ubiquinone as an electron donor, and this activity is sensitive to salicylhydroxamic acid (SHAM). The physiological function of trypanosome alternative oxidase involves the reoxidation of NADH generated during glycolysis, and the reaction is not a coupling site in oxidative phosphorylation. Characterization of N. brasiliensis electron transport has revealed at least 2 pathways: a main electron transport chain sensitive to antimycin A and cyanide, and an alternative respiratory pathway sensitive to SHAM (Fry et al., 1983). However, the molecular properties of alternative oxidase in helminth mitochondria have not yet been characterized. A full understanding of the properties and physiological role of helminth cytochrome o and alternative oxidase requires purification and a more careful characterization of the enzymes. During the past 10 years, studies on the respiratory chain in mitochondria of parasitic helminths have provided new insight into the structural organization and physiological significance of the mitochondrial respiratory chain. These include the elucidation of the molecular organization of the anaerobic NADHfumarate reductase system and an understanding of the aerobic-anaerobic transition during helminth development. However, many key areas remain unclarified. Future research on helminth mitochondria should provide more information about the detailed structure of each respiratory component and molecular mechanism involved in the control of gene expression during aerobic-anaerobic transition, thus providing a new understanding of the evolution and establishment of parasitism.

Acknowledgements-The authors are grateful to Dr Kuramochi for valuable recent information. Our research has been supported in part by a Grant-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Science and Culture of Japan (08281105), by NIG Cooperative Research Program (96-68), and by the Ohyama Health Foundation.

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