Reduction and oxidation of cytochrome C by Hymenolepis diminuta (Cestoda) mitochondria

Comp. Biochem. Physiol. Vol. 81B, No. 2, pp. 335-339, 1985 Printed in Great Britain

0305-0491/85 $3.00+0.00 © 1985PergamonPress Ltd

R E D U C T I O N A N D O X I D A T I O N OF CYTOCHROME C BY H Y M E N O L E P I S DIMINUTA (CESTODA) M I T O C H O N D R I A YOUNGHEE KIM and CARMEN F. FIORAVANTI* Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA (Tel: 419-372-0259) (Received 2 October 1984) Abstract--1. Mitochondrial membranes of adult Hymenolepis diminuta catalyzed inhibitor-sensitive

ferricytochrome c reduction. 2. Cytochrome c reductase activity was noted when NAD(P)H or succinate served as the reductant with the NADH-coupled reaction being most prominent. Both rotenone-sensitive and -insensitive reduced pyridine nucleotide-coupled activities were apparent. 3. Ferrocytochrome c oxidase activity also was catalyzed by H. diminuta mitochondrial membranes and this reaction was sensitive to azide and cyanide. 4. A cytochrome c peroxidase activity was associated primarily with the mitochondrial soluble fraction of adult H. diminuta. 5. The possibility that the activities observed may contribute to the elimination of peroxide in the helminth system is considered.

INTRODUCTION The predominantly anaerobic character of the adult intestinal cestode, Hymenolepis diminuta, is noted both in terms of in vitro cultivation and biochemical studies. H. diminuta can be cultivated in vitro, from excysted juvenile to ovigerous adult (i.e., throughout that portion of the life-cycle occurring in the mammalian host), in an atmosphere of CO2 and N 2 (Schiller, 1965; Roberts and Mong, 1969). Furthermore, the catabolism of carbohydrate by adult H. diminuta results in the accumulation of succinate as the major end-product (Fairbairn et al., 1961; Scheibel and Saz, 1966; Watts and Fairbairn, 1974). Succinate accumulation by H. diminuta is dependent upon the mitochondrial metabolism of malate which is formed in the cytosolic fraction via CO2-fixation (Scheibel and Saz, 1966; Bueding and Saz, 1968). By means of a dismutation reaction, malate serves as the anaerobic mitochondrial substrate (Saz et al., 1972). The oxidative branch of this dismutation is catalyzed by an NADP-specific "malic" enzyme (malate dehydrogenase, decarboxylating) thereby producing reducing equivalents in the form of NADPH (Prescott and Campbell, 1965; Saz et al., 1972; Li et aL, 1972; McKelvey and Fioravanti, 1984). In turn, completion of the dismutation entails the use of these reducing equivalents by the electron transport-linked fumarate reductase resulting in an anaerobic, site I-coupled generation of ATP and concomitant succinate formation (Scheibel and Saz, 1966; Scheibel et al., 1968; Saz et al., 1972). Although intramitochondrial reducing equivalents are accumulated in the form of NADPH, the H. diminuta fumarate reductase appears to be specific for NADH as the reductant (Fioravanti, 1981). Thus, the coupling of malate oxidation with electron transport necessitates a mechanism for hydride ion *To whom all correspondence should be addressed. caPla~81i2 F

transfer from NADPH to NAD. Indeed, H. diminuta mitochondria display a membrane-associated, phospholipid-dependent NADPH: NAD transhydrogenase system that links NADP-specific malate oxidation with the NADH-utilizing electron transport mechanism (Saz et al., 1972; Fioravanti and Saz, 1976; Fioravanti, 1981; Fioravanti and Kim, 1983; McKelvey and Fioravanti, 1984). In addition to the fumarate reductase, H. diminuta mitochondrial membranes exhibit a lesser, rotenonesensitive NADH oxidase activity (Fioravanti, 1981). Similar to the fumarate reductase (Saz et al., 1972; Fioravanti, 1982), this NADH-oxidizing activity is relatively insensitive to antimycin A, cyanide or azide (Fioravanti, 1982). Moreover, when oxygen serves as an acceptor, NAD(P)H as well as succinate utilization by H. diminuta mitochondrial preparations result in peroxide formation (Fioravanti, 1982; McKelvey and Fioravanti, 1984). The above fndings prompted an investigation to determine if H. diminuta mitochondrial preparations can engage in the enzymatic reduction and/or oxidation of exogenous cytochrome c. The results presented demonstrate that H. diminuta mitochondrial membranes catalyze inhibitor-sensitive, NAD(P)H- and succinate-dependent cytochrome c reductase activities. H. diminuta mitochondria also displayed a membrane-associated cytochrome c oxidase activity as well as a mitochondrial soluble cytochrome c peroxidase activity. MATERIALS AND METHODS

The maintenance of H. diminuta infections as well as the recovery and preparation of adult helminths for mitochondrial extraction were as described by Fioravanti and Saz (1976). H. diminuta mitochondria were isolated using the procedure given by Fioravanti (1981). Mitochondria were disrupted sonically by five successive 30 sec bursts (with 30 sec cooling intervals) employing a Branson Sonifier, equipped with a microtip, at a power setting of 20 W.

335

YOUNGHEEKIM and CARMENF. FIORAVANTI

336

Table 1. Mitochondrial localization of activities catalyzing the reduction or oxidation of cytochrome c by H. diminuta Total Units Reaction

Disrupted mitochondria

Membrane Soluble

Recovered

fraction

fraction

activity

(a)

(b)

% Recovered % Recovered activity

(a + b)

% Recovered

activity-

activity-

soluble

membrane-

fraction

associated

NADH:fumarate

2.02

1.38

0.13

1.51

75

9

91

NADH:cytochrome c

2.72

1.78

0.11

1.89

69

6

94

NADPH:cytochrome c

0.90

0.38

0.03

0.41

45

7

93

c

0.35

0.28

0,05

0.33

94

15

85

1.75

1.42

0,06

1,48

85

4

96

Cytochrome c peroxidase

17.15

0.66

12.29

12.95

76

95

5

Succinate:cytochrome Cytochrome c oxidase

Units express total activity in ~mol/min of disrupted mitochondria (equivalent to 40 mg protein) and fractions derived from these mitochondria. Mitochondrial membranes and mitochondrial soluble preparations were obtained by centrifugation of disrupted mitochondria at 269,0009 for 60rain. All operations were performed at 4°C. NADH-dependent fumarate reductase (NADH:fumarate) was measured spectrophotometrically by following the disappearance of NADH at 340 nm (Fioravanti, 1981). NAD(P)H- and succinate-dependent cytochrome c reductase activities (NAD(P)H:cytochrome c; succinate:cytochrome c) were assessed spectrophotometrically by measuring the accumulation of reduced cytochrome c at 550 nm essentially by the procedure of Mahler (1955). In addition to enzyme, 5.0 mg bovine serum albumin (BSA) and 0.30 mg oxidized cytochrome c, the 1.0 ml assay volume contained the following in/~mol: Tris(hydroxymethylaminomethane)HC1 (pH 7.5) 100 and NAD(P)H or succinate, 0.24. Cytochrome c oxidase activity was evaluated spectrophotometrically by following the utilization of reduced cytochrome c essentially as described by Smith (1955). In addition to enzyme, the 1.0 ml assay volume contained 5.0 mg BSA, 0.30 mg reduced cytochrome c and 100 #tool of Tris-HC1 (pH 7.5). Cytochrome c peroxidase activity was assessed by following reduced cytochrome c utilization at 550 nm employing the assay system described for cytochrome c oxidase activity with the addition of 0.012#tool hydrogen peroxide as suggested by Yonetani's work (1967). Peroxidase activity noted for disrupted organelles and membrane preparations was corrected for the utilization of reduced cytochrome c by the oxidase reaction. All spectrophotometric assays were conducted at 25°C with the aid of a Beckman Model 25 dual-beam spectrophotometer. Rotenone or antimycin A was added to the assay systems in ethanol such that ethanol was present at 0.7~. The millimolar extinction coefficients employed for the assessement of cytochrome c were 18.5 (reduced minus oxidized) or 27.7 (reduced) as given by Yonetani and Ray (1965). Protein was determined by the method of Lowry et al. (1951). Chemically reduced cytochrome c was prepared daily by using dithionite (sodium hydrosulfite) as the reductant according to Yonetani and Ray (1965). To a solution of cytochrome c (10mg/ml), prepared in 0.01 M potassium phosphate buffer (pH 7.0), sufficient dithionite was added to achieve complete reduction of the cytochrome. Excess dithionite was removed by elution of the reduced cytochrome with potassium phosphate buffer (PH 7.0) from a Sephadex G-25 column. The reduced cytochrome c so obtained was quantitated spectrophotometrically. NAD(P)H was obtained from Pharmacia P-L Biochemicals, Piscataway, N J, USA. Fumaric acid (sodium salt),

succinic acid (disodium), cytochrome c (Type VI, horseheart), rotenone, antimycin A (Type III, Streptomyces), sodium azide and Sephadex G-25 (particle size, 50-150 #m) were purchased from Sigma Chemical Company, St. Louis, MO, USA. Trishydroxymethylaminomethane (THAM), hydrogen peroxide (3~), potassium cyanide and dithionite were products of Fisher Scientific, Pittsburgh, PA, USA. Crystalline bovine serum albumin (Pentex) was obtained from Miles Laboratories, Elkart, IN, USA. RESULTS In adult H. diminuta, the mitochondrial N A D H dependent fumarate reductase is membraneassociated (Fioravanti, 1981). Therefore, this activity was employed as a marker enzyme to assess the efficacy of separation of helminth mitochondria into membrane-containing and soluble fractions. As presented in Table 1, the mitochondrial fractions were separated adequately because virtually all recoverable fumarate reductase activity was found within the membrane-containing fraction. Disrupted H, diminuta mitochondria displayed N A D ( P ) H - and succinate-dependent cytochrome c reductase activities when supplied with exogenous cytochrome c (Table I). O f the three reductants investigated, N A D H was the most effective as assessed by reduced cytochrome c accumulation (Table 1). With respect to the NAD(P)H-dependent reactions, the reduction of cytochrome c was accounted for by N A D ( P ) H utilization. The data obtained were consistent with the interpretation that the cytochrome c reductase reactions are catalyzed by membrane-associated systems (Table 1). The reason(s) for the more pronounced loss in recoverable N A D P H - d e p e n d e n t cytochrome c reductase activity was not determined. Aside from the reductase reactions, the disrupted cestode organelles catalyzed the oxidation of exogenously supplied reduced cytochrome c (Table 1). Paul and Barrett (1980) reported that H. diminuta mitochondria exhibit a cytochrome c peroxidase activity. Accordingly, the rate of cytochrome c oxidation was increased substantially by peroxide addition to the system thereby suggesting the presence of both a cytochrome c oxidase and cytochrome c peroxidase mechanism (Table 1). Based upon the

Cytochrome c reduction and oxidation

337

Table 2. Effectsof inhibitors on mitochondrialmembrane-associatedcytochrome c reductase activitiesof H. diminuta Reaction Addition

Concentration

NADH:cytochromec

NADPH:cytochromec

Succinate:cytochrome c

(M) Activity (nmol/min/mg)

None Rotenoee

I x 10-4

72.3

17.8

35.0 (52%)

13.8 (22%)

8.6 10.4

Antimycin A

3 x 10-6

43.8 (39%)

16.6

Potassium cyanide

I x 10-4

82.5

23.9

11.5

3.7 (57%)

Sodium azide

I x 10-2

95.4

20.7

16.4

Activities are expressed per mg mitochondrial membrane protein. Employed for assays was 0.11 mg protein. Numbers in parentheses indicate percent inhibition.

distribution of recovered activities, the cytochrome c oxidase reaction was associated with mitochondrial membranes while the cytochrome c peroxidase reaction was localized in the mitochondrial soluble fraction (Table 1). Subsequent washing of the mitochondrial membranes released remaining cytochrome c peroxidase activity into the soluble fraction thereby suggesting the liberation of entrapped activity. Both the reductase and cytochrome c-oxidizing activities (Table 1) were enzymatic as made evident by thermal lability and the linearity of activity with protein content. Employing H. diminuta mitochondrial membranes as the source of reductase activities, the effects of various inhibitors on these reactions were investigated. The NADH-dependent cytochrome c reductase activity exhibited a sensitivity to both rotenone and antimycin A (Table 2). The NADPHutilizing reaction was less sensitive to rotenone than was the NADPH-coupled activity and the NADPHutilizing reaction was not inhibited markedly by the inclusion of antimycin A in the assay system. Moreover, succinate-dependent reduced cytochrome c accumulation was somewhat increased by rotenone addition and inhibited significantly by the presence of antimycin A (Table 2). An increase in the rates of the

three reductase reactions was apparent in the presence of cyanide or azide which, in turn, suggested the inhibition of a reduced cytochrome c-utilizing activity (Table 2). Also assessed were the effects of inhibitors on the membrane-associated cytochrome c oxidase and the mitochondrial soluble cytochrome c peroxidase activities (Table 3). As presented in Table 3, neither rotenone nor antimycin A significantly affected the H. diminuta cytochrome e oxidase activity. However, cyanide or azide addition to the assay system markedly depressed the rate of cytochrome c utilization by mitochondrial membranes (Table 3). Similarly, the mitochondrial soluble cytochrome c peroxidase activity was essentially insensitive to either rotenone or antimycin A (Table 3). Although displaying sensitivity to cyanide, the degree of inhibition noted for the peroxidase activity was less than that observed for the corresponding oxidase activity (Table 3). In addition, the soluble peroxidase was considerably less sensitive to azide than was the cytochrome c oxidase activity (Table 3). The effects observed with cyanide or azide on the cytochrome c reductase and cytochrome c-oxidizing activities could not be accounted for by a chemical reaction between the inhibitors and cytochrome c.

Table 3. Effects of inhibitors on mitochondrial membrane-associated cytochrome c oxidase and mitochondrial soluble cytochrome c peroxidase activities of H. diminuta Reaction Addition

Concentration

Cytochrome c oxidase

Cytochromec peroxidase

(M) Activity (nmol/min/mg)

None

(~mol/min/mg)

57.2

1.0

Rotenone

i x 10-4

55.8

0.9

Antimycin A

3 x 10-6

50.7

potassium cyanide

1 x 10-4

3.6 (94%)

0.3 (70%)

Sodium azide

I x 10-2

17.2 (70%)

0.8 (22%)

1.2

Activities are expressed per mg mitochondrial membrane protein for the oxidase and per mg mitochondrial soluble protein for the peroxidase. Employed for the oxidase and peroxidase assays were 0.11 mg and 2.4 # g protein respectively.

338

YOUNGHEEKIM and CARMENF. FIORAVANTI

DISCUSSION As reviewed by Fioravanti and Saz (1980), adult H. diminuta is essentially anaerobic with respect to the physiological generation of energy. By means of an NADH-dependent, mitochondrial fumarate reductase system, this cestode engages in an electron transport-coupled, site I-linked phosphorylation with accompanying succinate accumulation (Scheibel and Saz, 1966; Saz et al., 1972; Fioravanti, 1981). Conversely, H. diminuta mitochondrial membranes also display a lesser, rotenone-sensitive N A D H oxidase activity (Fioravanti, 1981). Like the fumarate reductase (Saz et al., 1972; Fioravanti, 1982), this oxidase is not affected appreciably by antimycin A, azide or cyanide (Fioravanti, 1982). Indeed, NAD(P)H and succinate utilization by H. diminuta mitochondrial membranes result in peroxide formation (Fioravanti, 1982). Although the physiological import, if any, of these peroxide-forming reactions remains to be determined, the observation of these activities suggests the presence of a flavin-containing domain, associated with the electron transport mechanism of H. diminuta, which acts in the reduction of oxygen. The report of Kmetec and Bueding (1961) indicated that in another essentially anaerobic helminth, viz., the adult nematode Ascaris suum, the mitochondrial accumulation of peroxide involves a flavin-containing terminal oxidase mechanism. The data obtained demonstrate that despite the relative insensitivity of the oxidase reaction to antimycin A, azide or cyanide, H. diminuta mitochondrial preparations act in the enzymatic reduction of exogenously supplied cytochrome c. Of the three reductants studied, i.e., N A D H , N A D P H or succinate, the most prominent cytochrome c reductase activity was that noted with NADH. Localization experiments revealed that all three reductase activities are catalyzed by H. diminuta mitochondrial membranes. Mitochondrial cytochrome c reductase activities have been found in other adult helminths, e.g. the trematode Fasciola hepatica (Prichard and Schofield, 1971) and A. suum (Rew and Saz, 1974). Ascarid mitochondria catalyze both rotenone-sensitive and -insensitive, NADH-dependent cytochrome c reductase activities. While the former reaction is associated with the ascarid inner mitochondrial membrane, the latter activity is catalyzed by mitochondrial outer membrane preparations (Rew and Saz, 1974). Based on the inhibition by rotenone of the H. diminuta, reduced pyridine nucleotide-coupled cytochrome c reductase reactions, it appears that the cestode mitochondria catalyze both rotenone-sensitive and -insensitive activities. N A D H would be the more prominent reductant in both instances. Presumably, the antimycin A sensitivity detected, when N A D H serves as the reductant, reflects a site of inhibition associated with the rotenone-sensitive reaction. Likewise, antimycin A sensitivity of the succinatedependent cytochrome c reductase reaction would involve an inhibition at the same site. The apparent low level of rotenone-sensitive, NADPH-dependent cytochrome c reductase activity may account for the minimal inhibition observed when mitochondrial membranes were assessed for the NADPH-coupled reaction in the presence of antimycin A. Based upon oxygen consumption requiring

artificial electron donors, Read (1952) and subsequently Rahaman and Meisner (1973) reported on a cytochrome c-oxidizing mechanism in the H. diminuta system. Employing a more specific assay method, viz., the direct spectrophotometric measurement of ferrocytochrome c utilization, the data presented demonstrate that H. diminuta mitochondrial preparations can catalyze a cytochrome c-oxidizing activity. This oxidation is associated with the mitochondrial membrane fraction and is inhibited by both azide and cyanide. In accord with the report of Paul and Barrett (1980), H. diminuta mitochondria also exhibited a spectrophotometrically detectable cytochrome c peroxidase activity. However, unlike the cytochrome c oxidase, the peroxidase was localized in the mitochondrial soluble fraction of adult H. diminuta. In terms of its localization, the cestode's cytochrome c peroxidase was similar to that of mitochondria from the yeast Saccharomyces carlsbergenis (Yonetani and Ohnishi, 1966) and the hemoflagellate, Crithidiafasiculata (Kusel et al., 1973). For the H. diminuta system the localizations of the two cytochrome coxidizing activities and the differing sensitivities of these reactions to azide and cyanide suggested that these reactions are the products of two separate enzymatic mechanisms. The findings concerned with antimycin A-sensitive cytochrome c reduction by H. diminuta mitochondria do not define, nor are they intended to define, an additional site for phosphorylation. On the other hand, these findings suggested the presence of a component(s), associated with the electron transport mechanism but essentially independent of the succinate or peroxide-forming domains, which can act in the reduction of the cytochrome by H. diminuta. The cytochrome c oxidase activity, noted for H. diminuta mitochondrial membranes, may be~ aligned with the component(s) acting in the reduction of cytochrome c. It is of interest to note here that Culler and Fioravanti (1983) recently reported that cytochrome c is extractable from homogenates of adult H. diminuta. Clearly, the above considerations concerning the reduction and oxidation of cytochrome c await the intramitochondrial localizations of the activities in question and experimentation to determine if cytochrome c is contained within H. diminuta mitochondria. Orii (1982) presented evidence that under anaerobic conditions mammalian cytochrome c oxidase catalyzes a peroxidase cycle. Given an appropriate level of cytochrome c in H. diminuta mitochondria, it is possible that the cytochrome c oxidase and peroxidase activities demonstrated may act to rid these mitochondria of toxic peroxide. Acknowledgernents~ratitude is expressed to Beverly Anthony and Jonna Weaver for technical assistance and to Duane Culler and Jeffrey McKelvey for the maintenance of H. diminuta infections. Supported by grants 5-R01-AI-15597 and 5-K04-AI-00389 (C.F.) from the National Institutes of Health, United States Public Health Service. REFERENCES

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