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Dihydroorotate-dependent superoxide producton in rat brain and liver
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
173, 219-224
(1976)
Dihydroorotate-Dependent Superoxide Rat Brain and Liver A Function HENRY Veterans
Administration University
of the Primary
J. FORMAN’
AND
Producton
Dehydrogenase JAMES
KENNEDY
Hospital,2 Kansas City, Missouri 64128, and the Department of Kansas Medical Center, Kansas City, Kansas 66103 Received
August
in
of Medicine,
22, 1975
Dihydroorotate dehydrogenase in rat brain mitochondria is capable of producing superoxide. The presence of a superoxide dismutase activity in brain mitochondria, similar to that found in mitochondria from chicken liver, suggests that production of superoxide may occur in uivo. Formation of superoxide is not dependent upon reduction of cytochrome b, rather, superoxide production is competitive with cytochrome b reduction. Phenazine methosulfate apparently competes with both oxygen (superoxide production) and cytochrome b as an electron carrier but does not enhance reduction of dichlorophenolindophenol or cytochrome c.
The production of superoxide radical by many flavoproteins (1) and some nonheme iron proteins (2) has been established by workers in several laboratories. Generation of superoxide within mitochondria has only recently been demonstrated, by Lo&en and coworkers (3) with succinate dehydrogenase and in our laboratory (4) with dihydroorotate dehydrogenase from rat liver. In addition, we have proposed a model for superoxide production by rat liver dihydroorotate dehydrogenase and linkage of the primary enzyme with the electron transport chain (5). It was suggested that superoxide arises at or near the primary dehydrogenase. Although autooxidation of cytochrome b,,, may produce superoxide (6), our data did not indicate this mechanism as the source of superoxide in the dihydroorotate dehydrogenase complex. Evidence is presented in the present paper which further indicates
that superoxide production is not dependent upon cytochrome b reduction in this system. Sorgato and co-workers (7) were unable to detect superoxide formation during oxidation of succinate in rat brain mitochondria and concluded that brain mitochondria do not produce superoxide. The purpose of the present paper is threefold: to show that, although the succinate dehydrogenase in brain does not produce superoxide, the dihydroorotate dehydrogenase of brain does; to indicate that a mitochondrial superoxide dismutase is present to remove the superoxide produced by the dihydroorotate dehydrogenase reaction and any other reaction that generates this free radical in brain mitochondria; and to extend our observations on the site of production of the superoxide radical by dihydroorotate dehydrogenase.
1 To whom all correspondence should dressed: Henry J. Forman, Ph.D. Veterans istration Hospital, 4801 Linwood Boulevard, City, Missouri 64128. e Project No. 488-01.
Type VI cytochrome c, antimycin A, ubiquinone50, Triton X-100, DCIP3, and PMS were obtained
MATERIALS
be adAdminKansas
AND
METHODS
a Abbreviations used: DCIP, dichlorophenolindophenol; TTFA, thenoyltrifluoroacetone; PMS, phenazine methosulfate. 219
220
FORMAN
AND
from Sigma Chemical Company. ‘ITFA was obtained from Aldrich Chemical Company. Rat brain mitochondria were prepared as described by Brody et al. (8). Preparation of rat liver mitochondria, and assays of dihydroorotate dehydrogenase and succinate dehydrogenase activities were carried out with the substitution of potassium phosphate buffer (67 mM, pH 7.8) for Tris-HCl buffer (100 mM, pH 8.5) by a modification of the procedure of Miller et al. (91 as described previously (4). Mitochondrial particles containing both dehydrogenase activities but which are deficient in cytochrome c were prepared (with the substitution of several shorter periods of sonication in an ice water bath for one long period in an ethanol bath) by Procedure II of Gregg (10). These preparations contained no endogenous superoxide dismutase as measured by the method of Misra and Fridovich (11). “Soluble” dihydroorotate dehydrogenase was prepared in the following manner. Whole mitochondria, suspended in 10 vol of 10 mM potassium phosphate buffer, pH 7.8, were frozen and thawed, and the particles collected by centrifugation at 8000g for 30 min. The particles were washed four times in buffer. The final sediment was taken up in the buffer containing 0.5% Triton X-100 (v/v) to the original 10 vol, stored overnight at 4°C and centrifuged at SOOOg for 30 min. The supernatant was then centrifuged at 144,000g for 60 min. The resulting final supernatant contained the dihydroorotate dehydrogenase activity while the precipitate contained virtually all of the cytochromes, except cytochrome c, and most of the endogenous ubiquinone. RESULTS
AND
DISCUSSION
Superoxide production in rat brain mitochondria. Loschen et al. (31, using epi-
nephrine oxidation to indicate the presence of superoxide (ll), reported the production of superoxide by the succinate dehydrogenase system in beef heart mitochondria. Subsequently, they reported that while superoxide is produced by liver succinate dehydrogenase, superoxide is not formed by the action of succinate dehydrogenase in brain. We have been able to confirm the latter finding, but have also found that rat brain dihydroorotate dehydrogenase does produce superoxide (Fig. 1). The difference in the rates of DCIP reduction in the presence and absence of added superoxide dismutase represents the rate of superoxide formation. Addition of dismutase had no effect on DCIP reduction during oxidation of succinate, but did
KENNEDY
inhibit 60% when dihydroorotate was the substrate. TTFA blocks the reduction of ubiquinone, but not the production of superoxide in rat liver mitochondria (5). As shown in Table I, superoxide dismutase is considerably more effective than TTFA as an in-
MINUTES
FIG. 1. Effect of superoxide dismutase on DCIP reduction by brain mitochondrial dehydrogenase. The reaction mixtures contained 67 mM potassium phosphate buffer, pH 7.8,4.3 x 10m2 mM DCIP, 1 mM KCN, 6.7 mM sodium succinate (lines 1 and 2) or 0.67 mM sodium dihydroorotate (lines 3 and 4) and brain mitochondrial particles (0.10 mg of protein, lines 1 and 2; 0.16 mg of protein, lines 3 and 4) in a final volume of 3 ml. The reactions were started by addition of either dihydroorotate or succinate and followed at 600 nm (4). Lines 1 and 3, superoxide dismutase absent; lines 2 and 4, superoxide dismutase present at a concentration of 2 x 10m4 mM. Ten-fold higher levels of superoxide dismutase did not increase its effect. TABLE COMPARISON
OF BRAIN
AND
I LIVER
DIHYDROOROTATE
DEHYDROGENASES~
Percentage inhibition of DCIP reduction by TTFA Superoxide TTFA plus dismutase
dismutase superoxide
Brain
Liver
37 66 100
63 39 100
D Assays as in legend of Fig. 1 for brain dihydroorotate dehydrogenase. Liver data taken from Forman and Kennedy (5). The mitochondria were freshly prepared, frozen, thawed, and washed several times with 0.01 M potassium phosphate buffer, pH 7.8, before assaying.
DIHYDROOROTATE
DEHYDROGENASE
hibitor of DCIP reduction in brain mitochondria. In liver, the converse occurs. Thus, the relative amount of superoxide production versus ubiquinone reduction is greater in brain than in liver. This might be explained by the lower endogenous level of ubiquinone in brain (37.2 wg/g wet wt or 1.92 pg/mg of N) than in liver (134.9 pglg wet wt or 4.74 pglmg of N); (12). In fact, it was observed that addition of ubiquinone to the brain assay accelerated the overall rate of DCIP reduction with a proportionate decrease in the percent of DCIP reduction dependent upon superoxide formation. Total endogenous dihydroorotate dehydrogenase activity in brain is about half that in liver on a gram wet weight basis. Superoxide dismutase in rat brain mitochondria. The production of superoxide by
brain mitochondria would imply the presence of a superoxide dismutase in this organelle to mitigate the potentially harmful effects of the superoxide radical. Evidence for the existence of such an enzyme was obtained (Fig. 2). The upper bands represent the cyanide-insensitive superoxide dismutase, which has a similar mobility to the manganese-containing superoxide dismutase from chicken liver mitochondria (13). The lower bands represent the cyanide-inhibitable copper-zinc superoxide dismutases from cytosol and/or possibly the intermembrane space of mitochondria (13, 14). We do not have direct evidence of the production of superoxide by dihydroorotate dehydrogenase in the absence of artificial electron acceptors and inhibitors of the electron transport chain. However, the presence of superoxide dismutase in the mitochondria of brain, where no other dehydrogenase has been shown as yet to produce superoxide, is consistent with the possibility that superoxide may be produced in viva by dihydroorotate dehydrogenase. Mechanism
of
superoxide
formation.
After treatment of liver mitochondrial particles with Triton, cytochrome b was separated from the dihydroorotate dehydrogenase by centrifugation (Table II). Although there was no dihydroorotate-dependent re-
AND
221
SUPEROXIDE
iii 2
3
4
FIG. 2. Superoxide dismutase in rat brain and rat liver mitochondria. Freshly prepared mitochondria from 1.5 g of tissue were suspended in 3 ml of 100 mM potassium phosphate buffer, pH 7.8, and sonicated for 5 min using an Artek Sonic 300 Dismembrator. The suspension was centrifuged at SOOOg for 20 min, and loo-p1 aliquots of supernatant fluid were used in the nitro blue tetrazolium gel staining procedure of Beauchamp and Fridovich (20). Gels 1 and 3 are unwashed and sonicated rat brain and liver mitochondria, respectively. Gels 2 and 4 are the corresponding preparations soaked in potassium cyanide before exposure of the gels to light. Cyanide inhibits the copper-zinc superoxide dismutase of cytosol, which may also be found in the inter-membrane space (13, 141. Cyanide does not inhibit the mangano enzyme of mitochondria (131.
duction of cytochrome b in the supernatant, the rate of superoxide formation remained unchanged. Virtually no cytochrome b remained in the 144,000g supernatant. Thus, it appears that superoxide production and cytochrome b reduction are not linked in this system. If cytochrome b,,, autooxidizes with concomitant production of superoxide, as is seen with certain heme proteins (15, 161, it does not account for a significant percentage of the total superoxide produced by the dihydroorotate dehydrogenase system. Moreover, the lack of production of superoxide by the brain succinate dehydrogenase system (in contrast with the production of superoxide by the liver succinate, and brain and liver dihydroorotate dehydrogenase systems) suggests that the sites of superoxide formation are in the primary dehydrogenases or closely associated proteins rather than in the electron transport chain. When reduction of DCIP is measured in the presence of cyanide, antimycin A had no effect on the rate of superoxide produc-
222
FORMAN
AND
KENNEDY
TABLE EFFECT
OF REMOVAL
II
OF CYTOCHROME
b ON SUPEROXIDE
PRODUCTION”
Cytochromes a
DCIP b
a3
c
Total supiT~dependent (nmol/min)
(PM)
Before centrifugation Reduced by dihydroorotate Total cytochromes 144,000g supernatant Reduced by dihydroorotate Total cytochromes
reduced
0 312
58 165
230 450
47 368
0.39
0.16
0 0
0 0
0 5
0 326
0.29
0.18
a Enzyme assays and determinations of spectra were carried out before and after separation of rat liver dihydroorotate dehydrogenase from cytochrome b. The latter was sedimented by centrifugation at 144,OOOg for 60 min (see Methods). Protein concentrations before centrifugation and in the 144,OOOg supernatant were 3.62 and 0.93 mg/ml, respectively. Cytochrome concentrations were determined by the method of Chance and Williams (21). Spectra were measured using 2 ml of the enzyme preparations in a total volume of 3 ml, containing 67 mM potassium phosphate buffer, pH 7.8, 50 pg of antimycin A, and in the presence and absence of 0.67 mM sodium dihydroorotate. For determinations of total cytochrome concentrations, sodium dithionite and KCN were added in excess. Dihydroorotate dehydrogenase activity was assayed using 0.1 ml of the enzyme preparations and conditions described under Fig. 1.
tion (5). However, when reduction of added cytochrome c was measured with cyanide present, antimycin A increased superoxide production, although it decreased overall cytochrome c reduction (4). One possible explanation for these phenomena is as follows: (a) If cytochrome b and oxygen compete for electrons at a flavin, antimycin A could increase superoxide-mediated cytochrome c reduction by blocking cytochrome b-mediated cytochrome c reduction; cytochrome b would be a dead end (see Fig. 3). (b) Moreover, DCIP reduction, which does not involve cytochrome b, is unaffected by the addition of antimycin A because the path of the electron flow from cytochrome b to endogenous cytochrome c is already blocked by cyanide at its terminus. In the absence of cyanide, when the pathway is not blocked, antimycin A increases DCIP reduction (4, 9). The addition of PMS, in the presence of cyanide, was found to have a negligible effect on the overall rate of DCIP reduction (Table III). The inclusion of PMS, however, eliminates production of superoxide. This is illustrated by the effects of superoxide dismutase in the presence and absence of PMS. The reduction of PMS by dihydro-
ti>o>+o,
L--------i
FIG. 3. A proposed model for the relationship of various electron carriers with dihydroorotate dehydrogenase. DHO, dihydroorotate; OA, erotic acid; DHODase, dihydroorotate dehydrogenase; SOD, superoxide dismutase; NHI, nonheme iron; UQ, ubiquinone; cyt., cytochrome; cyt. ox., cytochrome c oxidase; Anti. A, antimycin.
orotate dehydrogenase seems to occur at the same site as superoxide production. Presumably, PMS reduction is at or very close to the primary dehydrogenase. Thus, superoxide formation may also be occurring near the primary enzyme, perhaps at a flavin (with a nonheme iron being the site of ubiquinone reduction; 5). The results here are analogous with cytochrome c reduction by xanthine dehydrogenase (1’7), where addition of PMS eliminates superoxide production while replacing super-
DIHYDROOROTATE
DEHYDROGENASE
oxide as the mediator of cytochrome c reduction. Although autooxidation of reduced PMS produces superoxide, the rate of reaction with cytochrome c is apparently much higher. Consequently, in a system containing both PMS and cytochrome c, superoxide is neither produced at the primary enzyme nor as a result of the autooxidation of reduced PMS. In reduction of an electron acceptor such as nitro blue tetrazolium, which apparently reacts more slowly with reduced PMS than does oxygen or cytochrome c, the relatively faster rate of autooxidation of reduced PMS produces superoxide as an intermediate in the reaction (18). Since both PMS reduction and cytochrome b reduction are competitive with superoxide formation, PMS and cytochrome b should be competitive electron acceptors. In the presence of PMS, there seems to be no electron transfer involving cytochrome b, as evidenced by the failure of antimycin A to inhibit dihydroorotatedependent reduction of cytochrome c (Table IV). However, the possibility does exist that electrons are transferred from cytochrome b to PMS and then to cytochrome c, but it seems less likely. Transfer of electrons from cytochrome b to PMS would have to involve a cytochrome b type that is insensitive to the large change in potential induced on cytochrome b by antimycin A (19). Thus, we favor the former interpretation although either one or both are conceivable. Transfer of electrons from an antimycin-insensitive cytochrome b to PMS and competition between cytochrome b and PMS could occur simultaneously. The reduction of cytochrome c or of DCIP is partially inhibited by TTFA (5). Inhibition of TTFA is not altered by addition of PMS (Tables III and IV). The simplest explanation is that PMS and ubiquinone do not compete for electrons at the nonheme iron site. However, we have no evidence for or against the possibility that PMS acts as an intermediate carrier of electrons between ubiquinone and DCIP or added cytochrome c. As with xanthine dehydrogenase (uide supru), the involvement of a potential intermediate electron carrier
AND
223
SUPEROXIDE TABLE
III
EFFECT OF PMS ON THE REDUCTION OF DCIP LIVER DIHYDROOROTATE DEHYDROGENASE” Addition
BY
DCIP reduced (nmol/min) PMS not added
PMS added
None 0.78 0.73 0.43 TTFA 0.45 0.35 0.75 Superoxide dismutase 0 0.43 TTFA plus superoxide dismutase a Assay conditions were as indicated in Fig. 1 except that liver mitochondrial particles (0.31 mg of protein) were used. When present, the concentrations of TTFA were 0.5 mM, and of PMS, 2 x 10m2 mM, which gave the maximum effects. TABLE EFFECT
IV
OF PMS ON CYTOCHROME c REDUCTION DIHYDROOROTATE DEHYDROGENASE~ Addition
BY
Cytochrome c reduced (nmol/min)
None Antimycin A Superoxide dismutase Antimycin A + superoxide dismutase TTFA TTFA + antimycin A
PMS not added
PMS added
1.42 0.52 1.36 0.30
1.48 1.43 1.55 1.49
0.50 0.25
0.48 0.41
’ Assay conditions were as indicated for Fig. 1 with the following modifications: liver mitochondrial particles (0.31 mg of protein) were used, and 2 x lo-’ mM cytochrome c replaced DCIP. When present, the concentration of PMS was 2 x 1O-2 mM; TTFA, 0.5 mM; and antimycin A, 50 pg/incubation.
will depend on the relative rates of the reaction of the final acceptor and intermediate carrier with the electron donor. In the dihydroorotate dehydrogenase system we have not been able to compare the relative rates of reduction of DCIP, added cytochrome c, and PMS by its endogenous ubiquinone. REFERENCES 1. MASSEY, V., STRICKLAND, HOWELL, L. G., ENGEL,
S., MAYHEW, S. G., P. C., MATFHEWS, R.
224
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
FORMAN
AND
G., SCHULMAN, M., AND SULLIVAN, P. A. (1969) Biochem. Biophys. Res. Commun. 36, 891-897. MISRA, H. P., AND FRIDOVICH, I. (1971) J. Biol. Chem. 246, 6886-6890. LOSCHEN, G., AZZI, A., RICHTER, C., AND FLOHE, L. (1974) FEBS Lett. ,42, 68-72. FORMAN, H. J., AND KENNEDY, J. A. (1974) Biothem. Biophys. Res. Commun. 60,1044-1050. FORMAN, H. J., AND KENNEDY, J. (1975)J. Biol. Chem. 250,4322-4326. LOSCHEN, G. (1975) D.Sc. thesis, EberhardKarls-Universitiit zu Tubingen, Tubingen. SORGATO, M. C., SARTORELLI, L., LOSCHEN, G., AND AZZI, A. (1974) FEBS Lett. 45, 92-95. BRODY, T. M., WANG, R. I. H., AND BAIN, J. A. (1952) J. Biol. Chem. 198,821-826. MILLER, R. W., KERR, C. T., AND CURRY, J. R. (1968) Canad. J. Biochem. 46, 1099-1106. GREGG, C. T. (1967) Methods Enzymol. 10, 181185. MISRA, H. P., AND FRIDOVICH, I. (1972) J. Biol. Chem. 247,3170-3175.
KENNEDY 12. BEYER, R. E., NOBLE, W. M., AND HIRSXFELD, T. J. (1962) Biochim. Bioph.ys. Acta 57, 376379. 13. WEISIGER, R. A., AND FRIDOVICH, I. (1973) J. Biol. Chem. 248,4793-4796. 14. PANCHENKO, L. F., BRUSOV, 0. S., GERASIMOV, A. M., AND LOKTAEVA, J. D. (1975) FEBSLett. 55, 84-87. 15. MISRA, H. P., AND FRIDOVICH, I. (1972) J. Biol. Chem. 247,6960-6962. 16. CASSELL, R. H., AND FRIDOVICH, I. (1975) Biochemistry 14, 1866-1868. 17. MCCORD, J. M., AND FRIDOVICH, I. (1970) J. Biol. Chem. 245, 1374-1377. 18. NISHIKIMI, M., RAO, N. A., AND YAGI, K. (1972) Biochem. Biophys. Res. Commun. 46, 849854. 19. CHANCE, B. (1972) FEBS Lett. 23, 3-20. 20. BEAUCHAMP, C., AND FRIDOVICH, I. (1971)Anal. Biochem. 44, 276-287. 21. CHANCE, B., AND WILLIAMS, G. R. (1955) J. Biol. Chem. 217,409-427.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
173, 219-224
(1976)
Dihydroorotate-Dependent Superoxide Rat Brain and Liver A Function HENRY Veterans
Administration University
of the Primary
J. FORMAN’
AND
Producton
Dehydrogenase JAMES
KENNEDY
Hospital,2 Kansas City, Missouri 64128, and the Department of Kansas Medical Center, Kansas City, Kansas 66103 Received
August
in
of Medicine,
22, 1975
Dihydroorotate dehydrogenase in rat brain mitochondria is capable of producing superoxide. The presence of a superoxide dismutase activity in brain mitochondria, similar to that found in mitochondria from chicken liver, suggests that production of superoxide may occur in uivo. Formation of superoxide is not dependent upon reduction of cytochrome b, rather, superoxide production is competitive with cytochrome b reduction. Phenazine methosulfate apparently competes with both oxygen (superoxide production) and cytochrome b as an electron carrier but does not enhance reduction of dichlorophenolindophenol or cytochrome c.
The production of superoxide radical by many flavoproteins (1) and some nonheme iron proteins (2) has been established by workers in several laboratories. Generation of superoxide within mitochondria has only recently been demonstrated, by Lo&en and coworkers (3) with succinate dehydrogenase and in our laboratory (4) with dihydroorotate dehydrogenase from rat liver. In addition, we have proposed a model for superoxide production by rat liver dihydroorotate dehydrogenase and linkage of the primary enzyme with the electron transport chain (5). It was suggested that superoxide arises at or near the primary dehydrogenase. Although autooxidation of cytochrome b,,, may produce superoxide (6), our data did not indicate this mechanism as the source of superoxide in the dihydroorotate dehydrogenase complex. Evidence is presented in the present paper which further indicates
that superoxide production is not dependent upon cytochrome b reduction in this system. Sorgato and co-workers (7) were unable to detect superoxide formation during oxidation of succinate in rat brain mitochondria and concluded that brain mitochondria do not produce superoxide. The purpose of the present paper is threefold: to show that, although the succinate dehydrogenase in brain does not produce superoxide, the dihydroorotate dehydrogenase of brain does; to indicate that a mitochondrial superoxide dismutase is present to remove the superoxide produced by the dihydroorotate dehydrogenase reaction and any other reaction that generates this free radical in brain mitochondria; and to extend our observations on the site of production of the superoxide radical by dihydroorotate dehydrogenase.
1 To whom all correspondence should dressed: Henry J. Forman, Ph.D. Veterans istration Hospital, 4801 Linwood Boulevard, City, Missouri 64128. e Project No. 488-01.
Type VI cytochrome c, antimycin A, ubiquinone50, Triton X-100, DCIP3, and PMS were obtained
MATERIALS
be adAdminKansas
AND
METHODS
a Abbreviations used: DCIP, dichlorophenolindophenol; TTFA, thenoyltrifluoroacetone; PMS, phenazine methosulfate. 219
220
FORMAN
AND
from Sigma Chemical Company. ‘ITFA was obtained from Aldrich Chemical Company. Rat brain mitochondria were prepared as described by Brody et al. (8). Preparation of rat liver mitochondria, and assays of dihydroorotate dehydrogenase and succinate dehydrogenase activities were carried out with the substitution of potassium phosphate buffer (67 mM, pH 7.8) for Tris-HCl buffer (100 mM, pH 8.5) by a modification of the procedure of Miller et al. (91 as described previously (4). Mitochondrial particles containing both dehydrogenase activities but which are deficient in cytochrome c were prepared (with the substitution of several shorter periods of sonication in an ice water bath for one long period in an ethanol bath) by Procedure II of Gregg (10). These preparations contained no endogenous superoxide dismutase as measured by the method of Misra and Fridovich (11). “Soluble” dihydroorotate dehydrogenase was prepared in the following manner. Whole mitochondria, suspended in 10 vol of 10 mM potassium phosphate buffer, pH 7.8, were frozen and thawed, and the particles collected by centrifugation at 8000g for 30 min. The particles were washed four times in buffer. The final sediment was taken up in the buffer containing 0.5% Triton X-100 (v/v) to the original 10 vol, stored overnight at 4°C and centrifuged at SOOOg for 30 min. The supernatant was then centrifuged at 144,000g for 60 min. The resulting final supernatant contained the dihydroorotate dehydrogenase activity while the precipitate contained virtually all of the cytochromes, except cytochrome c, and most of the endogenous ubiquinone. RESULTS
AND
DISCUSSION
Superoxide production in rat brain mitochondria. Loschen et al. (31, using epi-
nephrine oxidation to indicate the presence of superoxide (ll), reported the production of superoxide by the succinate dehydrogenase system in beef heart mitochondria. Subsequently, they reported that while superoxide is produced by liver succinate dehydrogenase, superoxide is not formed by the action of succinate dehydrogenase in brain. We have been able to confirm the latter finding, but have also found that rat brain dihydroorotate dehydrogenase does produce superoxide (Fig. 1). The difference in the rates of DCIP reduction in the presence and absence of added superoxide dismutase represents the rate of superoxide formation. Addition of dismutase had no effect on DCIP reduction during oxidation of succinate, but did
KENNEDY
inhibit 60% when dihydroorotate was the substrate. TTFA blocks the reduction of ubiquinone, but not the production of superoxide in rat liver mitochondria (5). As shown in Table I, superoxide dismutase is considerably more effective than TTFA as an in-
MINUTES
FIG. 1. Effect of superoxide dismutase on DCIP reduction by brain mitochondrial dehydrogenase. The reaction mixtures contained 67 mM potassium phosphate buffer, pH 7.8,4.3 x 10m2 mM DCIP, 1 mM KCN, 6.7 mM sodium succinate (lines 1 and 2) or 0.67 mM sodium dihydroorotate (lines 3 and 4) and brain mitochondrial particles (0.10 mg of protein, lines 1 and 2; 0.16 mg of protein, lines 3 and 4) in a final volume of 3 ml. The reactions were started by addition of either dihydroorotate or succinate and followed at 600 nm (4). Lines 1 and 3, superoxide dismutase absent; lines 2 and 4, superoxide dismutase present at a concentration of 2 x 10m4 mM. Ten-fold higher levels of superoxide dismutase did not increase its effect. TABLE COMPARISON
OF BRAIN
AND
I LIVER
DIHYDROOROTATE
DEHYDROGENASES~
Percentage inhibition of DCIP reduction by TTFA Superoxide TTFA plus dismutase
dismutase superoxide
Brain
Liver
37 66 100
63 39 100
D Assays as in legend of Fig. 1 for brain dihydroorotate dehydrogenase. Liver data taken from Forman and Kennedy (5). The mitochondria were freshly prepared, frozen, thawed, and washed several times with 0.01 M potassium phosphate buffer, pH 7.8, before assaying.
DIHYDROOROTATE
DEHYDROGENASE
hibitor of DCIP reduction in brain mitochondria. In liver, the converse occurs. Thus, the relative amount of superoxide production versus ubiquinone reduction is greater in brain than in liver. This might be explained by the lower endogenous level of ubiquinone in brain (37.2 wg/g wet wt or 1.92 pg/mg of N) than in liver (134.9 pglg wet wt or 4.74 pglmg of N); (12). In fact, it was observed that addition of ubiquinone to the brain assay accelerated the overall rate of DCIP reduction with a proportionate decrease in the percent of DCIP reduction dependent upon superoxide formation. Total endogenous dihydroorotate dehydrogenase activity in brain is about half that in liver on a gram wet weight basis. Superoxide dismutase in rat brain mitochondria. The production of superoxide by
brain mitochondria would imply the presence of a superoxide dismutase in this organelle to mitigate the potentially harmful effects of the superoxide radical. Evidence for the existence of such an enzyme was obtained (Fig. 2). The upper bands represent the cyanide-insensitive superoxide dismutase, which has a similar mobility to the manganese-containing superoxide dismutase from chicken liver mitochondria (13). The lower bands represent the cyanide-inhibitable copper-zinc superoxide dismutases from cytosol and/or possibly the intermembrane space of mitochondria (13, 14). We do not have direct evidence of the production of superoxide by dihydroorotate dehydrogenase in the absence of artificial electron acceptors and inhibitors of the electron transport chain. However, the presence of superoxide dismutase in the mitochondria of brain, where no other dehydrogenase has been shown as yet to produce superoxide, is consistent with the possibility that superoxide may be produced in viva by dihydroorotate dehydrogenase. Mechanism
of
superoxide
formation.
After treatment of liver mitochondrial particles with Triton, cytochrome b was separated from the dihydroorotate dehydrogenase by centrifugation (Table II). Although there was no dihydroorotate-dependent re-
AND
221
SUPEROXIDE
iii 2
3
4
FIG. 2. Superoxide dismutase in rat brain and rat liver mitochondria. Freshly prepared mitochondria from 1.5 g of tissue were suspended in 3 ml of 100 mM potassium phosphate buffer, pH 7.8, and sonicated for 5 min using an Artek Sonic 300 Dismembrator. The suspension was centrifuged at SOOOg for 20 min, and loo-p1 aliquots of supernatant fluid were used in the nitro blue tetrazolium gel staining procedure of Beauchamp and Fridovich (20). Gels 1 and 3 are unwashed and sonicated rat brain and liver mitochondria, respectively. Gels 2 and 4 are the corresponding preparations soaked in potassium cyanide before exposure of the gels to light. Cyanide inhibits the copper-zinc superoxide dismutase of cytosol, which may also be found in the inter-membrane space (13, 141. Cyanide does not inhibit the mangano enzyme of mitochondria (131.
duction of cytochrome b in the supernatant, the rate of superoxide formation remained unchanged. Virtually no cytochrome b remained in the 144,000g supernatant. Thus, it appears that superoxide production and cytochrome b reduction are not linked in this system. If cytochrome b,,, autooxidizes with concomitant production of superoxide, as is seen with certain heme proteins (15, 161, it does not account for a significant percentage of the total superoxide produced by the dihydroorotate dehydrogenase system. Moreover, the lack of production of superoxide by the brain succinate dehydrogenase system (in contrast with the production of superoxide by the liver succinate, and brain and liver dihydroorotate dehydrogenase systems) suggests that the sites of superoxide formation are in the primary dehydrogenases or closely associated proteins rather than in the electron transport chain. When reduction of DCIP is measured in the presence of cyanide, antimycin A had no effect on the rate of superoxide produc-
222
FORMAN
AND
KENNEDY
TABLE EFFECT
OF REMOVAL
II
OF CYTOCHROME
b ON SUPEROXIDE
PRODUCTION”
Cytochromes a
DCIP b
a3
c
Total supiT~dependent (nmol/min)
(PM)
Before centrifugation Reduced by dihydroorotate Total cytochromes 144,000g supernatant Reduced by dihydroorotate Total cytochromes
reduced
0 312
58 165
230 450
47 368
0.39
0.16
0 0
0 0
0 5
0 326
0.29
0.18
a Enzyme assays and determinations of spectra were carried out before and after separation of rat liver dihydroorotate dehydrogenase from cytochrome b. The latter was sedimented by centrifugation at 144,OOOg for 60 min (see Methods). Protein concentrations before centrifugation and in the 144,OOOg supernatant were 3.62 and 0.93 mg/ml, respectively. Cytochrome concentrations were determined by the method of Chance and Williams (21). Spectra were measured using 2 ml of the enzyme preparations in a total volume of 3 ml, containing 67 mM potassium phosphate buffer, pH 7.8, 50 pg of antimycin A, and in the presence and absence of 0.67 mM sodium dihydroorotate. For determinations of total cytochrome concentrations, sodium dithionite and KCN were added in excess. Dihydroorotate dehydrogenase activity was assayed using 0.1 ml of the enzyme preparations and conditions described under Fig. 1.
tion (5). However, when reduction of added cytochrome c was measured with cyanide present, antimycin A increased superoxide production, although it decreased overall cytochrome c reduction (4). One possible explanation for these phenomena is as follows: (a) If cytochrome b and oxygen compete for electrons at a flavin, antimycin A could increase superoxide-mediated cytochrome c reduction by blocking cytochrome b-mediated cytochrome c reduction; cytochrome b would be a dead end (see Fig. 3). (b) Moreover, DCIP reduction, which does not involve cytochrome b, is unaffected by the addition of antimycin A because the path of the electron flow from cytochrome b to endogenous cytochrome c is already blocked by cyanide at its terminus. In the absence of cyanide, when the pathway is not blocked, antimycin A increases DCIP reduction (4, 9). The addition of PMS, in the presence of cyanide, was found to have a negligible effect on the overall rate of DCIP reduction (Table III). The inclusion of PMS, however, eliminates production of superoxide. This is illustrated by the effects of superoxide dismutase in the presence and absence of PMS. The reduction of PMS by dihydro-
ti>o>+o,
L--------i
FIG. 3. A proposed model for the relationship of various electron carriers with dihydroorotate dehydrogenase. DHO, dihydroorotate; OA, erotic acid; DHODase, dihydroorotate dehydrogenase; SOD, superoxide dismutase; NHI, nonheme iron; UQ, ubiquinone; cyt., cytochrome; cyt. ox., cytochrome c oxidase; Anti. A, antimycin.
orotate dehydrogenase seems to occur at the same site as superoxide production. Presumably, PMS reduction is at or very close to the primary dehydrogenase. Thus, superoxide formation may also be occurring near the primary enzyme, perhaps at a flavin (with a nonheme iron being the site of ubiquinone reduction; 5). The results here are analogous with cytochrome c reduction by xanthine dehydrogenase (1’7), where addition of PMS eliminates superoxide production while replacing super-
DIHYDROOROTATE
DEHYDROGENASE
oxide as the mediator of cytochrome c reduction. Although autooxidation of reduced PMS produces superoxide, the rate of reaction with cytochrome c is apparently much higher. Consequently, in a system containing both PMS and cytochrome c, superoxide is neither produced at the primary enzyme nor as a result of the autooxidation of reduced PMS. In reduction of an electron acceptor such as nitro blue tetrazolium, which apparently reacts more slowly with reduced PMS than does oxygen or cytochrome c, the relatively faster rate of autooxidation of reduced PMS produces superoxide as an intermediate in the reaction (18). Since both PMS reduction and cytochrome b reduction are competitive with superoxide formation, PMS and cytochrome b should be competitive electron acceptors. In the presence of PMS, there seems to be no electron transfer involving cytochrome b, as evidenced by the failure of antimycin A to inhibit dihydroorotatedependent reduction of cytochrome c (Table IV). However, the possibility does exist that electrons are transferred from cytochrome b to PMS and then to cytochrome c, but it seems less likely. Transfer of electrons from cytochrome b to PMS would have to involve a cytochrome b type that is insensitive to the large change in potential induced on cytochrome b by antimycin A (19). Thus, we favor the former interpretation although either one or both are conceivable. Transfer of electrons from an antimycin-insensitive cytochrome b to PMS and competition between cytochrome b and PMS could occur simultaneously. The reduction of cytochrome c or of DCIP is partially inhibited by TTFA (5). Inhibition of TTFA is not altered by addition of PMS (Tables III and IV). The simplest explanation is that PMS and ubiquinone do not compete for electrons at the nonheme iron site. However, we have no evidence for or against the possibility that PMS acts as an intermediate carrier of electrons between ubiquinone and DCIP or added cytochrome c. As with xanthine dehydrogenase (uide supru), the involvement of a potential intermediate electron carrier
AND
223
SUPEROXIDE TABLE
III
EFFECT OF PMS ON THE REDUCTION OF DCIP LIVER DIHYDROOROTATE DEHYDROGENASE” Addition
BY
DCIP reduced (nmol/min) PMS not added
PMS added
None 0.78 0.73 0.43 TTFA 0.45 0.35 0.75 Superoxide dismutase 0 0.43 TTFA plus superoxide dismutase a Assay conditions were as indicated in Fig. 1 except that liver mitochondrial particles (0.31 mg of protein) were used. When present, the concentrations of TTFA were 0.5 mM, and of PMS, 2 x 10m2 mM, which gave the maximum effects. TABLE EFFECT
IV
OF PMS ON CYTOCHROME c REDUCTION DIHYDROOROTATE DEHYDROGENASE~ Addition
BY
Cytochrome c reduced (nmol/min)
None Antimycin A Superoxide dismutase Antimycin A + superoxide dismutase TTFA TTFA + antimycin A
PMS not added
PMS added
1.42 0.52 1.36 0.30
1.48 1.43 1.55 1.49
0.50 0.25
0.48 0.41
’ Assay conditions were as indicated for Fig. 1 with the following modifications: liver mitochondrial particles (0.31 mg of protein) were used, and 2 x lo-’ mM cytochrome c replaced DCIP. When present, the concentration of PMS was 2 x 1O-2 mM; TTFA, 0.5 mM; and antimycin A, 50 pg/incubation.
will depend on the relative rates of the reaction of the final acceptor and intermediate carrier with the electron donor. In the dihydroorotate dehydrogenase system we have not been able to compare the relative rates of reduction of DCIP, added cytochrome c, and PMS by its endogenous ubiquinone. REFERENCES 1. MASSEY, V., STRICKLAND, HOWELL, L. G., ENGEL,
S., MAYHEW, S. G., P. C., MATFHEWS, R.
224
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
FORMAN
AND
G., SCHULMAN, M., AND SULLIVAN, P. A. (1969) Biochem. Biophys. Res. Commun. 36, 891-897. MISRA, H. P., AND FRIDOVICH, I. (1971) J. Biol. Chem. 246, 6886-6890. LOSCHEN, G., AZZI, A., RICHTER, C., AND FLOHE, L. (1974) FEBS Lett. ,42, 68-72. FORMAN, H. J., AND KENNEDY, J. A. (1974) Biothem. Biophys. Res. Commun. 60,1044-1050. FORMAN, H. J., AND KENNEDY, J. (1975)J. Biol. Chem. 250,4322-4326. LOSCHEN, G. (1975) D.Sc. thesis, EberhardKarls-Universitiit zu Tubingen, Tubingen. SORGATO, M. C., SARTORELLI, L., LOSCHEN, G., AND AZZI, A. (1974) FEBS Lett. 45, 92-95. BRODY, T. M., WANG, R. I. H., AND BAIN, J. A. (1952) J. Biol. Chem. 198,821-826. MILLER, R. W., KERR, C. T., AND CURRY, J. R. (1968) Canad. J. Biochem. 46, 1099-1106. GREGG, C. T. (1967) Methods Enzymol. 10, 181185. MISRA, H. P., AND FRIDOVICH, I. (1972) J. Biol. Chem. 247,3170-3175.
KENNEDY 12. BEYER, R. E., NOBLE, W. M., AND HIRSXFELD, T. J. (1962) Biochim. Bioph.ys. Acta 57, 376379. 13. WEISIGER, R. A., AND FRIDOVICH, I. (1973) J. Biol. Chem. 248,4793-4796. 14. PANCHENKO, L. F., BRUSOV, 0. S., GERASIMOV, A. M., AND LOKTAEVA, J. D. (1975) FEBSLett. 55, 84-87. 15. MISRA, H. P., AND FRIDOVICH, I. (1972) J. Biol. Chem. 247,6960-6962. 16. CASSELL, R. H., AND FRIDOVICH, I. (1975) Biochemistry 14, 1866-1868. 17. MCCORD, J. M., AND FRIDOVICH, I. (1970) J. Biol. Chem. 245, 1374-1377. 18. NISHIKIMI, M., RAO, N. A., AND YAGI, K. (1972) Biochem. Biophys. Res. Commun. 46, 849854. 19. CHANCE, B. (1972) FEBS Lett. 23, 3-20. 20. BEAUCHAMP, C., AND FRIDOVICH, I. (1971)Anal. Biochem. 44, 276-287. 21. CHANCE, B., AND WILLIAMS, G. R. (1955) J. Biol. Chem. 217,409-427.