Phoforhernisrry and Phorobiology Vol. 52, No. 5 , pp. 1011-1015, 1990 Printed in Great Britain. All rights reserved

0031 -8655/YO $03.00+0.00 Copyright 0 1990 Pergamon Press plc

INACTIVATION OF CITRIC ACID CYCLE ENZYMES AS A RESULT OF PHOTODYNAMIC SENSITIZATION BY

MITOCHONDRIAL INNER MEMBRANE JIN JUNG* and YONG-JAE KIM

Department of Agricultural Chemistry, Seoul National University, Suwon 440-744, Korea

(Received 20 February 1990; accepted 3 May 1990)

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Abstract-Exposure to blue light of mitochondria under aerobic conditions resulted in inactivation of the regulatory enzymes of the citric acid cycle (CAC) contained in the mitochondrial matrix, except citrate synthase. When “soluble mitochondrial protein” was exposed to blue light under aerobic conditions, no significant loss of activity was observed for any CAC enzymes. However, the inclusion of submitochondria particles (SMP) in the photolysis system resulted in a substantial inactivation of the CAC enzymes. Of the CAC enzymes, NAD+-specific isocitrate dehydrogenase (ICDH) appeared to be most susceptible to the membrane dependent-photoinactivation. Imidazole protected the CAC enzymes against inactivation. In contrast, superoxide dismutase failed to protect them, except Q-

ketoglutarate dehydrogenase. The photoinhibition of ICDH activity was drastically depressed in the presence of SMP whose Fe-S centers were destroyed by the mersalyl acid treatment. The results obtained in this study suggest that the photoinactivation of the CAC enzymes in situ is mediated mainly by singlet oxygen, which is photoproduced primarily by the Fe-S centers of mitochondrial membranes.

INTRODUCTION

Mitochondria, which contain a majority of the pig- ments of eucaryotic cells, except for plant leaf cells, have attracted attention from workers who were interested in natural photosensitivity in higher organisms. Epel (1973) observed that mitochondria suffer loss of respiratory activity when exposed to near-UV and visible light; this is prevented by anaerobiosis. Aggarwal et al. (1976, 1978) further confirmed this and also suggested that the photo- inhibition of mitochondrial respiration is due princi- pally to the inactivation of dehydrogenase. These observations have indicated that some visible light- absorbing chromophores of mitochondrial mem- brane-bound proteins may act as natural, internal photodynamic sensitizers.

Recently, observing that the mitochondrial inner membrane generates lo2 on exposure to visible light under aerobic conditions, we measured the action spectrum for the generation of ‘02. Based on this, the Fe-S centers of nonheme iron proteins (NHIP)?

* To whom correspondence should be addressed. t Abbreviations: A-ase, aconitase; BSA, bovine serum

albumin; CAC, citric acid cycle; CoASH, coenzyme A; CS, citrate synthase; DCIP, 2,6-dichlorophenolin- dophenol; DTNB, 5,5’-dithiobis-(2-nitrobenzoate); F- ase, fumarase; ICDH, NAD+-specific isocitrate dehydrogenase; a-KGD, a-ketoglutarate dehydrogen- ase; MA, mersalyl acid; MDH, malate dehydrogenase; NHIP, nonheme iron protein; PMS, phenazine metho- sulfate; SCS, succinyl CoA synthetase; SDH, succinate dehydrogenase; SMP, submitochondrial particles; SOD, superoxide dismutase.

appear to act as the major endogenous sensitizers in lo2 generation (Jung et al., 1990). Once ‘ 0 2 is produced in the inner membrane, it should attack primarily the substrates located in the very vicinity of its generation sites. Since lo2 penetrates quite easily through membranes and crosses the mem- brane interfaces (Valenzeno, 1987), however, it is reasonable to expect that it diffuses into the matrix and attacks functional targets there within its life- time. The possibility of lo2 diffusion into cytosol can not be excluded, but the probability of lo2

remaining in its excited state would be very small after penetrating all through the inner membrane, the intermembrane space and the outer membrane.

lo2 may find various target molecules in the matrix, each of which is of physiological importance, of courFe. In the present work, we have focused on the photodynamic inactivation of the citric acid cycle (CAC) enzymes so as to document an aspect of the detrimental effects of mitochondrial membrane- sensitization on the cellular metabolism. The results presented here clearly indicate that the photo- inactivation processes of the CAC enzymes proceed mainly via lo2 which is produced largely by the Fe- S centers of membrane-bound NHIP. Among the CAC enzymes, NAD+-specific isocitrate dehydro- genase (ICDH) appears to be the most susceptible to photodynamic inhibition.

MATERIALS AND METHODS

Soybean (Glycine rnax L.) hypocotyls were grown in the dark at about 25°C for 5-6 days. Superoxide dismutase

101 1

1012 JIN JUNG and YONG-JAE KIM

(from bovine erythrocytes) and biochemicals were pur- chased from Sigma Chemical Co. (St. Louis, MO), and all other chemicals were of reagent grade from Wako Purechemical Industries, Ltd. (Osaka, Japan). These were used without further purification.

Preparation of submitochondrial particles ( S M P ) and soluble mitochondria1 protein. The isolation of mitochon- dria from the etiolated tissue was based on the technique of Douce et al. (1972). Various aqueous media for the preparation of mitochondria and SMP are as follows: Iso- lation medium; 0.3 M mannitol, 4 mM cysteine, 1 mM EDTA, 0.2% (wtivol) defatted BSA, and 10 mM K-phos- phate (pH 7.2). Wash medium; 0.3 M mannitol, 1 mM EDTA, and 10 mM K-phosphate (pH 7.2). Suspension medium; 0.3 M mannitol, 5 mM MgCI,, 10 mM KCI, and 10 mM K-phosphate (pH 7.2). The SMP were prepared according to the procedure described in Grubmeyer et al. (1979) with a minor modification. The mitochondrial suspension was sonicated at 23 kHz for 3 X 20 s at 4°C using a Soniprep 150 ultrasonic disintegrator (MSE Scien- tific, Sussex, England), centrifuged at 13 000 g for 20 min to remove unbroken mitochondria and large mitochondrial fragments, and then centrifuged for 1 h at 110 000 g. The supernatant was used for the study of photoinactivation of the CAC enzymes and is referred to as “soluble mito- chondrial protein”. The pellet of SMP was washed and centrifuged in the suspension medium at 110 000gfor 1 h. All procedures were carried out at ca 4°C under dim room light. Protein was measured by the Lowry method (Lowry ef al., 1951) using crystalline BSA as standard.

Enzyme assay. All enzymes were assayed spectrophoto- metrically with a Cary 118-C spectrophotometer (Varian Assoc., Palo Alto, CA) at 25°C. The activity of citrate synthase (CS) was determined by measuring the appear- ance of the free SH group of the released CoASH by use of 5,5’-dithiobis-(2-nitrobenzoate) (DTNB), as described in Bogin and Wallace (1969). Aconitase (A-ase) was assayed according to Fansler and Lowenstein (1969). Fumarase (F-ase) was measured by the method of Hill and Bradshaw (1969). The activity of succinyl-CoA synthetase (SCS) was determined as described by Bridger et al. (1969). measuring the formation of thioester bonds. Succinate dehydrogenase (SDH) was assayed by the PMS-DCIP method described by Singer et al. (1973), measuring the reduction of 2,6-dichlorophenolindophenol (DCIP). The spectrophotometric measurements of the production of NADH in reaction mixtures were performed to assay NAD+-specific isocitrate dehydrogenase (ICDH) (Cox, 1969), a-ketoglutarate dehydrogenase (a-KGD) (Cho et al., 1988) and malate dehydrogenase (MDH) (Bowman et al., 1976). When ICDH and a-KGD were assayed in the presence of SMP, the experiments were carried out under anaerobic conditions attained by Nz bubbling of the reac- tion mixtures which contained 10 mM KCN in the suspen- sion medium. The anaerobiosis was required to avoid NADH oxidation by SMP. The inclusion of KCN also appeared necessary in a practical aspect in order to inhibit the NADH oxidase of SMP, as air leaking into the reaction mixtures was a source of error frequently encountered during kinetic measurements. In the case of MDH, how- ever, neither anaerobiosis nor the addition of KCN was necessary because of the extremely high activity of this enzyme compared to the NADH oxidase activity of SMP.

Phofolysis. Irradiation was performed as described previously (Jung et al., 1990) with blue light obtained from a 750 W Xe lamp (Shanghai bulb factory No.3, Shanghai, China) by use of an absorption filter with a maximum transmittance at 475 nm and an effective bandwidth of 98 nm. During irradiation, a sample contained in a long necked glass cuvette (10 mm light path) with a water (15°C) jacket was gently bubbled with either N, or air. The light fluence rates were measured with a Macam quantum/radiometer/photometer model Q. 101 (Macam Photometrics, Livingstone, Scotland).

RESULTS AND DISCUSSION

Because the mitochondrial inner membrane, on exposure to blue light, produces lo2 that can diffuse into the matrix, it is natural to expect that various ‘O,-mediated reactions take place in it. One of these may be the chemical modification of enzymes which catalyze CAC. Chemical modification can cause, in general, the inactivation of an enzyme. Thus, if an enzyme which happens t o be one of the regulatory enzymes of CAC possesses a rather high sensitivity to lo2, the photogeneration of lo2 from the mitochondrial inner membrane would result in a deleterious effect on the CAC activity. It is gener- ally known that the CAC activity is critically regu- lated at the reactions catalyzed by NAD+-specific ICDH, a-KGD, and CS. For this reason, the photo- induced inactivation of these enzymes contained in intact mitochondria was investigated first.

Mitochondria were irradiated with blue light, son- icated to release the CAC enzymes, and assayed for their enzymatic activities. Using the activities of the enzymes released from mitochondria which had been kept in the dark under a N2 atmosphere (dark/ N2) as control, we measured the relative activities of the enzymes from mitochondria bubbled with air in the dark (dark/02), those from mitochondria irradiated while being bubbled with air (Iight/O,), and those from mitochondria irradiated under a N2 atmosphere (light/N,). It turned out that blue light induced the inactivation of the regulatory enzymes in situ only when mitochondria were exposed to light under aerobic conditions; ICDH appeared to be the most susceptible to photoinactivation, whereas CS seemed very resistant to it; neither the enzymes in the dark/O*-treated mitochondria nor those in the light/N,-treated mitochondria suffered any significant loss of activity. This result, as shown in Fig. 1, indicates that photosensitization reactions occur in the matrix.

However, this does not necessarily mean that the photosensitization reactions are membrane-depen- dent; for we can assume that “soluble mitochondrial protein”, as described in Materials and Methods section, contains blue light-absorbing molecules which can act as photosensitizers. Thus, the soluble proteins were studied to ascertain whether the inac- tivation of the CAC enzymes is photoinduced by these pigments. No significant loss of activity was observed for any CAC enzymes when the soluble protein preparation was subjected to the lightlo,- treatment [Fig. 2(A)]. In contrast, the inclusion of SMP in the photolysis system resulted in a sub- stantial inactivation of the enzymes [Fig. 2(B)]. These results provide strong evidence that the photoinactivation processes of the CAC enzymes in situ proceed mainly via photodynamic sensitization by the mitochondrial inner membrane. As can be noted from Figs. 1 and 2, the CAC enzymes con- tained in intact mitochondria appear photo- dynamically less sensitive to light than the released

Photosensitization by mitochondria 1013

ICDH aKm CS Figure 1. Blue light-induced inactivation of the regulatory enzymes of CAC contained in intact mitochondria. The mitochondrial preparation was divided into four parts: two of them were irradiated with blue light (A,,,, = 425 nm, effective bandwidth = 98 nm, and intensity = 750 Wlm') for 1 h at 15°C under aerobic conditions (light/02) and under a N, atmosphere (light/N,) respectively; the other two were kept in the dark for 1 h at 15°C under aerobic conditions (dark/O,) and anaerobic conditions (darkiN,) respectively. Anoxia and aeration were achieved by gently bubbling nitrogen and air respectively into the samples. Data are presented as the percent of the activities of the

enzymes in the dark/N,-treated mitochondria.

I 1

SDH ICDH F-ase Aase M G D SCS MDH C S Figure 2. Blue light-induced inactivation of the CAC enzymes present in "soluble mitochondrial protein". After being subjected to various treatments in the absence (A) and presence (B) of SMP, the activities of all CAC enzymes were measured. The samples contained the sol- uble protein at 1.0 mg/mC and SMP at 0.33 mg protein/ mP. As to the treatment conditions and the data presen-

tation, see Fig.1.

enzymes. This might be tentatively interpreted in terms of the lo2 scavenging effect of various com- ponents at high local concentrations located in the matrix of intact mitochondria. This supposition is supported, in part at least, by a previous report that the purification of M D H from pig heart mitochon- dria results in a higher photosensitization by FMN (Codd, 1972); in agreement, Gibson et al. (1987) demonstrated that crude M D H from a tumor cell is

much less photosensitive to hematoporphyrin derivative than purified MDH.

Among the enzymes of CAC, M D H and CS showed almost no susceptibility to membrane- dependent photodynamic inactivation. It is gener- ally accepted that mitochondrial oxaloacetate must be compartmentalized by protein binding with MDH and CS (Williamson and Cooper, 1980), so it is tempting to speculate that M D H and CS are protected by the binding of oxaloacetate to their active sites. Some enzymes are protected from dye- sensitization by their substrates or products; CS purified from E . coli is partially protected by oxaloacetate from methylene blue (and Rose Benga1)-photosensitization (Danson and Weitzman, 1973); NADH-glutamate synthase from Chlamydo- monas reinhardii is protected by its substrate and product against inactivation by flavin-photo- sensitization (Gotor et al., 1987).

The superoxide radical anion (03 is less reactive than 'Oz and does not react with most proteins; however, the OH radical, as produced by the Haber-Weiss reaction, attacks proteins (Bielski et al., 1983). Several enzymes, however, are known to react with 0; leading to inactivation (Fridovich, 1986). In the previous report, the possibility of 0; generation from irradiated mitochondrial mem- branes was discussed (Jung et al., 1990). For this reason, a set of aerobic assays was carried out to ascertain whether 0; is also involved in the light- induced inactivation of the CAC enzymes. Table 1 shows that the 0; scavenger, superoxide dismutase (SOD), in contrast to the '02 quencher, imidazole, failed to protect the CAC enzymes except a-KGD. The question arises again whether the failure of SOD in the protection of the CAC enzymes against photoinactivation is because of the insufficiency of the 0; scavenging activity in the photolysis system. To answer this, the dependence of the relative activities of the CAC enzymes on the enzyme unit of SOD was determined, choosing ICDH which is most photosensitive among C A C enzymes and a- KGD which is the only CAC enzyme protected by SOD. As shown in Fig. 3 , the inclusion in the photolysis system of SOD more than 30 units did not result in further increase of the activities of both ICDH and a-KGD. Note that 30 units of SOD was contained in the system used for the experiment to investigate the effect of SOD on the photo- inactivation of the CAC enzymes (Table 1). This result implies that the processes of membrane- dependent photodynamic inactivation of the C A C enzymes occur mainly via lo2, not via 0;.

Finally, we attempted to confirm our previous observation that the Fe-S centers of NHIP bound to the mitochondrial inner membrane are most probably the sites of '02 photogeneration (Jung et a l . , 1990). If the photoinactivation of the CAC enzymes is caused by '02 generated by the Fe-S centers of NHIP, the CAC enzymes would not be inactivated on exposure to blue light in the presence

1014 JIN JUNG and YONG-JAE KIM

20 40 60 SOD (unit/ml)

Figure 3. Dependence of the photoinactivation of ICDH (0) and a-KGD (0) on the SOD concentration in the presence of SMP. The reaction mixtures were subjected to the light/02-treatment as in Fig. 1. The protein contents of the soluble protein and SMP are same as Fig. 2(B). Data are presented as the percent of the activities of the

dark/O,-treated enzymes.

Table 1. Effects of Oi scavenger and ‘02 quencher on the photoinactivation of the CAC enzymes

Enzymes Relative inactivation (YO)*

ICDH 34 18 32 SDH 32 12 30 F-ase 22 15 22 A-ase 18 8 16 a-KGD 17 10 9 scs 17 6 17

* The samples were irradiated under aerobic conditions in the presence of 8 mM imidazole (hu102/Im), in the presence of 30 units of SOD (hv1021SOD), and in the absence of the active oxygen scavengers (hv102). The samples contained “soluble mitochondrial protein” at 1.0 mg/me and SMP at 0.33 mg protein/mt, and were subjected to the light/02- treated as in Fig.1.

of SMP with the Fe-S centers destroyed. Mersalyl acid was used to destroy the mitochondrial Fe-S centers as described by Hatefi and Stempel (1969). As can be seen in Fig. 4, the photoinhibition of ICDH activity was strikingly depressed in the pres- ence of the mersalyl acid-treated SMP; the extent of the inhibition in this is about 17% of that in the presence of the “intact” SMP. This appears consistent with the relative extent of lo2 photo- generation from the Fe-S center-destroyed SMP, as presented in the previous report (Jung et al., 1990), implying that some other chromophores besides the Fe-S centers in the mitochondrial inner membrane play a minor role, acting as endogenous sensitizers, in the photodynamic inhibition of the CAC enzymes.

In conclusion, the results obtained in the present investigation suggest that the photoinactivation of the CAC enzymes is mediated primarily by ‘02 which is photoproduced largely by the Fe-S centers of membrane-bound NHIP and then diffuses into the matrix. The photosensitivities of the CAC

b I

0.1 0.3 0.5 AMOUNT OF SMP (mg Pr/ml)

Figure 4. Effect of the mersalyl acid treatment of SMP on the membrane-sensitized photoinactivation of ICDH. The dependence of the activity of the lightlo,-treated ICDH on the amount of the Fe-S center-destroyed SMP (0) was compared with that on the amount of the “intact” SMP (0). The concentration of the soluble protein was 1.0 mg/mt. The reaction mixtures were subjected to the lightlo,-treatment as in Fig. 1. Data are presented as the percent of the activity of the dark/O,-treated enzyme. For the treatment of MA, SMP suspension was diluted into 10 vol of the MA-saturated suspension medium, stored for 5 h at 4°C in the dark, and dialyzed against the suspension

medium.

enzymes contained in intact mitochondria appear to be lower than those of the enzymes released from the matrix. This is presumably due to the lo2 scav- enging effect of various molecules located in the matrix. If such is the case, the molecules which scavenge ‘02 can be chemically modified instead of the CAC enzymes, leading to damage of their own physiological functions. Furthermore, the lo2 scav- enging molecules might be important components of the metabolic pathways other than the citric acid cycle. In this respect, the possible scavenging of lo2

by some internal substrates does not necessarily imply that the metabolic activities of the matrix space are protected from the ‘02-mediated photo- dynamic reactions.

Acknowledgements-This work was supported by Korea Science and Engineering Foundation Grant No. 870509. We thank Jin-Man Kim and Yong-Uk Kim for their help in the preparation of the manuscript and technical assistance.

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