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Biochimica et Biophysica Acta, 1140 (1993) 282-292 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2728/93/$06.00

BBABIO 43733

Fumarate reductase activity of bovine heart succinate-ubiquinone reductase. New assay system and overall properties of the reaction
Vera G. Grivennikova

a Eleonora V. Gavrikova a Alexander A. Timoshin b


and Andrei D. Vinogradov a

a Department of Biochemistry, School of Biology, Moscow State University, Moscow (Russia) and b Cardiology Research Center, Russian Academy of Medical Sciences, Moscow (Russia)

(Received 14 April 1992) (Revised manuscript received 20 July 1992)

Key words: Succinate-ubiquinone reductase; Ubiquinol-fumarate reductase; NAD(P)H-quinone reductase; Respiratory chain; (Bovine heart mitochondrion) A simple system for aerobic assay of the quinol-fumarate reductase reaction catalyzed by purified soluble bovine heart succinate-ubiquinone reductase in the presence of NADH, NAD(P)H-quinone reductase (DT-diaphorase) and an appropriate quinone is described. The reaction is inhibited by carboxin, suggesting that the same quinone/quinol binding site is involved in electron transfer from succinate to ubiquinone and from ubiquinol to fumarate. The kinetic properties of the reaction in both directions and comparative affinities of the substrate binding sites of the enzyme to substrates (products) and competitive inhibitors are reported. Considerable difference in affinity of the substrates binding site to oxaloacetate was demonstrated when the enzyme was assayed in the direct and reverse directions. These results were taken to indicate that the oxidized dicarboxylate-free enzyme is an intermediate during the steady-state succinate-ubiquinone reductase reaction, whereas the reduced dicarboxylate-free enzyme is an intermediate of the steady-state ubiquinol-fumarate reductase reaction. No difference in the reactivity of the substrate-protected cysteine and arginine residues was found when the pseudo-first-order rate constants for N-ethylmaleimide and phenylglyoxal inhibition were determined for oxidized and quinol-reduced enzyme. Quinol-fumarate reductase activity was reconstituted from the soluble succinate dehydrogenase and low-molecular-mass ubiquinone reactivity conferring protein(s). No reduction of cytochrome b was observed in the presence of quinol generating system, whereas S-3 low temperature EPR-detectable iron-sulfur center was completely reduced by quinol under equilibrium (without fumarate) or steady-state (in the presence of fumarate). No significant reduction of ferredoxin type iron-sulfur centers was detected during the steady-state quinol-fumarate oxidoreductase reaction. The data obtained eliminate participation of cytochrome b in the quinol-fumarate reductase reaction and show that the rate limiting step of the overall reaction lies between iron-sulfur center S-3 and lower midpoint potential redox components of the enzyme.

Introduction
S u c c i n a t e - q u i n o n e o x i d o r e d u c t a s e is t h e p o i n t w h e r e t h e di,-tricarboxylic-acid cycle is directly l i n k e d to t h e

Correspondence to: A.D. Vinogradov, Department of Biochemistry, School of Biology, Moscow State University, 119899 Moscow, Russia. Abbreviations: carboxin, 5,6-dihydro-2-methyl-l,4-oxathin-3-carboxanilide; DCIP, 2,6-dichlorophenolindophenol; DTT, dithiothreitol; NEM, N-ethylmaleimide; NQR,NAD(P)H-quinone reductase; PMS, phenazine methosulfate; OA, oxaloacetate; Q2, homolog of ubiquinone having two isoprenoid units in position 6 of the quinone ring; QPs, ubiquinone-binding protein, which confers succinateubiquinone reductase activity to the soluble succinate dehydrogenase; SDH, succinate dehydrogenase; SUR succinate-ubiquinone reductase; TTA, thenoyltrifluoroacetone; WB, (Wurster's Blue), a semiquinonediimine radical of N,N,N',N'-tetramethyl-p-phenylenediamine.

e l e c t r o n - t r a n s f e r chain in m i t o c h o n d r i a a n d n u m e r o u s p r o k a r y o t e s . This m e m b r a n e - b o u n d iron-sulfur flavop r o t e i n consists o f s u c c i n a t e d e h y d r o g e n a s e ( S D H ; E C 1.3.99.1) a n d h y d r o p h o b i c p o l y p e p t i d e s which a r e tightly b o u n d to S D H . T h e h y d r o p h o b i c p o l y p e p t i d e s serve as an a n c h o r a n d as a factor which confers t h e reactivity of t h e m e m b r a n e - b o u n d e n z y m e to the b u l k q u i n o n e p r e s e n t in the m e m b r a n e . M a m m a l i a n s u c c i n a t e - u b i q u i n o n e r e d u c t a s e (usually t e r m e d C o m p l e x II) has b e e n p u r i f i e d [ 1 - 4 ] a n d furt h e r r e s o l v e d into t w o - s u b u n i t w a t e r - s o l u b l e S D H [5] a n d c y t o c h r o m e - b - e n r i c h e d fraction (QPs) [6-9]. S D H c o n t a i n s a 70 k D a p o l y p e p t i d e carrying covalently bound 8-a-N(3)-histidyl-FAD and the substrate binding site [5] a n d a 27 k D a p o l y p e p t i d e which m o s t likely b e a r s o n e b i n u c l e a r (S-I), o n e t r i n u c l e a r (S-3) a n d one

283 tetranuclear (S-2) iron-sulfur cluster [10,11]. Both isolated subunits are catalytically inactive and no reconstitution of catalytically competent SDH from the individual subunits has been achieved so far. Properly prepared SDH (strictly anaerobic conditions, the presence of succinate) is capable of rapid oxidation of succinate by a number of artificial electron acceptors, but not ubiquinone or its homologues, and binding to the SDH-depleted membranes with complete restoration of TTA-, or carboxin-sensitive succinate-ubiquinone reductase activity [12]. The isolated SDH thus prepared is unstable in air and rapidly looses its capacity for the reconstitution of succinate-ubiquinone reductase. The reactivity of SDH with artificial electron acceptors also decreases during aerobic inactivation [13,14]. The direct correlation between inactivation of the reconstitutional capacity, loss of the S-3 EPR signal and decrease of ferricyanide reactivity has been documented [15,16], suggesting that the S-3 iron-sulfur center is responsible for the terminal one-electron transfer from the reduced SDH to ubiquinone. The membrane-bound cytochrome-b-enriched fraction (QP~), which confers succinate-ubiquinone reductase activity to the soluble SDH, has been purified [6-9]. It contains two polypeptides and cytochrome b heme. Various molecular masses of the constituent polypeptides (7-17 KDa) and various heme b content has been reported for QPs fractions obtained by several research groups [17]. QPs itself has no enzymatic activity. Addition of QP~ to soluble reconstitutively active SDH dramatically changes several properties of the latter : it becomes very stable and the TTA-, or carboxin-sensitive ubiquinone reactivity appears in parallel with disappearance of ferricyanide reductase activity [7]. The function of Complex II associated cytochrome b [18] is unknown. The stoichiometry of cytochrome b heme/covalently bound FAD in different preparations of Complex II is variable [4] and there is no simple correlation between catalytic activity of the enzyme and cytochrome b content. Cytochrome b in intact or reconstituted Complex II is not reduced by succinate, although dithionite reduced heme b can be rapidly reoxidized by fumarate [1], suggesting an electronic link between heme b and some enzyme redox component which is in equilibrium with fumarate. The enzyme seems to be very conservative in evolution and a number of succinate-quinone reductases or SDH with very similar properties have been purified and characterized, including those from Neurospora crassa [19], Escherichia coli [20], Micrococcus luteus [21], Bacillus subtilis [22], Paracoccus denitrificans [23] and Rhodospirillum rubrum [24] (see Ref. 25 for more comparative data). The major physiological function of Complex II and related enzymes in aerobic organisms is oxidation of succinate by an appropriate quinone which is further oxidized by the respiratory chain. In anaerobic or facultative organisms, similar but genetically distinct enzymes operationally called quinol-fumarate oxidoreductase or fumarate reductase (FRD; EC 1.3.99.1) exist and metabolically produced fumarate functions as the terminal electron-acceptor in anaerobic respiration. Quinol-fumarate 'anaerobic' reductases and FRD from various organisms are strikingly similar to their 'aerobic' counterparts in terms of composition and structure, but differ significantly in their kinetic properties (see Refs. 26-28 and references cited therein). Fumarate reductases rapidly reduce fumarate in the presence of low-redox-potential electron donors and slowly oxidize succinate in the presence of high-redoxpotential electron donors. Mammalian succinate-ubiquinone reductase, when operating within the inner mitochondrial membrane, catalyzes the NADH-fumarate reductase reaction which is sensitive to the specific inhibitors of Complex I and Complex II. The thermodynamic redox gap between the N A D H / N A D and the succinate/fumarate couples is large enough to provide free energy for ATP synthesis coupled with transfer of two electrons from NADH to fumarate. Indeed, the overall reaction has been demonstrated to occur in both direction (ATPproducing NADH-fumarate reductase [29,30] and succinate-supported ATP-dependent NAD reduction [31]). Mammalian SDH as well as Complex II are capable of fumarate reductase activity in the presence of strong reductants, such as FMNH 2 [32,33] and phenosafranine [34]. Some kinetic properties of the artificial donor-fumarate oxidoreductase reaction (catalytic turnover number, K m and K i for the substrate and competitive inhibitors) catalyzed by SDH have been reported [35]. However valuable the results obtained in those studies are, they seem seriously invalidated by the use of artificial electron donors and the lack of information on the reductant reactive site(s). In addition to the serious theoretical limitations in interpretation of the results obtained with artificial electron donors, the technical problems of handling anaerobic assay samples hampers routine kinetic studies of the fumarate-reductase activity. To our knowledge no TTAor carboxin-sensitive fumarate reductase reaction catalyzed by purified succinate-ubiquinone reductase has been demonstrated so far. In order to gain a better understanding of the reaction and control mechanisms involved in equilibrium between the succinate/ fumarate and the quinol/quinone redox pairs catalyzed by Complex II, we have developed a simple assay system which allows studies of the carboxin-sensitive quinol-fumarate reductase reaction. The results obtained using this system with a special emphasis on cytochrome b function and relative affinities of the enzyme substrate (products) binding sites to dicarbox-

284 ylates and quinone (quinol), as revealed in the direct and reverse reactions, are reported in this paper.
Materials and Methods

Succinate-ubiquinone reductase (SUR) was prepared [4] and assayed [4,36] using the procedures developed in this laboratory. For the stock solution, the enzyme was diluted (1 m g / ml) in 20 mM potassium phosphate (pH 7.8), 0.6% Triton X-100. The assay mixture contained 20 mM phosphate (pH 7.8), 0.2 mM EDTA, 20 mM succinate (potassium salts), 10/xM Q2 and 30 ~M WB. Two types of QPs were used, prepared as follows. All the procedures were carried out at 0-4C. Crude QPs. SUR (90 mg of protein) was diluted in 120 ml of 50 mM potassium phosphate/borate, 0.1 mM EDTA (pH 10.6) and incubated at 0C for 15 min with continuous stirring. The mixture was centrifuged (15 min at 3 0 0 0 0 g ) and the precipitated material was washed in 120 ml of the same buffer. The residues were collected, washed in 120 ml of 20 mM sodium phosphate, 0.1 mM EDTA (pH 7.4), collected by centrifugation as before and suspended in a small volume of 20 mM sodium phosphate, 0.1 mM EDTA (pH 7.4) (2 mg/ml). The preparation thus prepared showed a AA558 of 0.04 (1 mg protein/ml; dithionite reduced minus oxidized). Purified QPs. The crude QPs (approx. 45 mg) was suspended in 3.6 ml of 50 mM Tris-Cl, 5 mM DTT, 0.1 mM EDTA (pH 8.0) and 0.9 ml of 8 M NaCIO 4 was added with constant stirring. The mixture was incubated for 30 min and centrifugated (40 fnin at 200 000 g). The precipitated material was collected and the treatment with sodium perchlorate was repeated. The protein collected after centrifugation was washed with 30 ml of 50 mM Tris-C1, 5 mM DTT, 0.1 mM EDTA (pH 8.0), collected by centrifugation (30 min at 30 000 g), suspended in 3.6 ml of 0.67 M sucrose, 50 mM Tris-C1, 0.1 mM EDTA (pH 8.0) and 0.18 ml of 10% potassium deoxycholate (pH 8.0) was added. The mixture was incubated for 30 min and the loosely packed residue obtained after centrifugation (30 min at 30 000 x g) was discarded. After addition of 0.72 ml of 50% saturated ammonium acetate to the clear supernatant, the mixture was centrifuged (15 min at 30000g). The precipitated material was collected, suspended in a small volume of 20 mM sodium phosphate, 0.1 mM EDTA (pH 7.4) and stored in liquid nitrogen. The final preparation showed a AA558 of 0.1 (1 mg protein/ml; dithionite reduced minus oxidized). NAD(P)H-quinone reductase (NQR, EC 1.6.99.2) was prepared by Cibacron Blue affinity chromatography [37]. The enzyme activity was induced in rat liver by interperitoneal injection of the animals with 3-methylcholantrene for 3 days prior to killing.

EPR measurements were performed using a Varian E-109 E spectrometer with a temperature control device. Protein content was determined by the biuret method [38]. Wurster's Blue was prepared as described [39]. The special chemicals were: fumarate, oxaloacetate, phenylglyoxal, TTA from Fluka (Switzerland); EDTA, Tris, DCIP, Triton X-100, 20-methylcholanthrene from Serva (Germany); DTI', NADH from Reanal (Hungary); NEM from BDH (U.K.); Q2 PMS from Ferak (Germany). Carboxin was a kind gift from Prof. H. Lyr (Institute of Plant Protection Research, Germany). Other chemicals used were of the highest quality commercially available.
Results

One aim of the present study was to find a simple assay system which can be used for tracing the succinate-quinone (quinol-fumarate) reductase reaction in both directions under the same controlled conditions using the same substrate/product pair. An obvious difficulty in the quinol-fumarate reductase assay using ubiquinol or its water soluble homologues as the reductant is that the equilibrium between the succinate/ fumarate (E m approx. 0.0 mY) and ubiquinol/ ubiquinone (E m approx. +100 mV) redox pairs is shifted considerably to oxidation of succinate. Thus, the steady-state quinol-fumarate reductase reaction is expected to occur only under conditions where the concentration of quinone is negligible, compare to that of quinol. NAD(P)H-quinone reductase (DT-diaphorase) seemed ideally suited for the quinol regenerating system. The enzyme is simple to prepare in highly active and water soluble form [37]. It has low electronacceptor specifity and high turnover number [40]. The redox potential of the substrate (NADH/NAD +) pair is far more negative ( - 3 2 0 mV) than that of the succinate/fumarate pair (approx. 0.0 mV). The enzyme does not react with oxygen and the rate of the coupled fumarate-reductase reaction can be conventionally followed by NAD(P)H oxidation at 340 nm. Fig. 1 shows a representative example of QzH2fumarate reductase reaction catalyzed by purified SUR under aerobic conditions using NQR as the quinol regenerating system. Zero-order NADH oxidation was observed when fumarate was added to the cuvette containing NADH, Q2 and NQR. As expected, the SUR catalyzed reaction was carboxin- and malonatesensitive. Simple hyperbolic dependencies of the reaction rate on the NQR (at fixed Q2 ) and Q2 concentrations (at fixed saturating NOR) were observed, thus, allowing determination of standard kinetic parameters of Q2H2-fumarate reductase (Fig. 2). The kinetic parameters of the overall carboxin-sensitive QzH2 fumarate reductase, as compared to those of succinateQ2 reductase are summarised in Table I.

285
TABLE I

Qz

SUR Fumarate

The kinetic parameters of the ubiquinol-fumarate and succinateubiquinone reductase reactions


Assay conditions: 38C, 20 mM potassium phosphate (pH 7.8), 0.006% Triton X-100. Fumarate reduction Succinate oxidation 40 130 c (30) ~ 150 1.3 0.02 0.3 2.0 h

" A,~,oO.03

CarbnXit;

V (/~mol/min per mg) a


Kdicarboxylate b ( /k,I] m(i) .]d,..l.

1.0 30 25 e 1.0 0.2 1.5 3.0 g

-~ 30 sec ~- k_Fig. I. Q 2 H 2-fumarate reductase reaction catalyzed by coupled operation of NQR and SUR. 18 /xg NQR was placed in a 2-ml spectrophotometric cuvette containing 20 mM phosphate, 0.2 mM EDTA, 100 # M NADH (potassium salts (pH 8.0), 38C). 10 g M Qe, 20/~g SUR, 1 mM fumarate, 100 ~M carboxin and 10 mM malonate were added as indicated.

succinate fumarate malonate oxaloacetate f K 2 (IzM) Km 2H2 (/~M) K carbxin (/xM)

It has been observed that succinate dehydrogenase activity is strongly pH-dependent [41]. Several interpretations may be considered for the pH-profile of the enzyme activity, such as the effect of pH on ionization of a proton-abstracting group, on the rate of intramolecular electron transfer, on the equilibrium between active and inactive conformation of the protein. It has been reported that the fumarate reductase activity of submitochondrial particles measured with reduced phenosafranine as electron donor shows a mirror image of the pH-profile of succinate dehydrogenase activity [34]. We compared the effect of pH on the succinate-Q 2 and Q2H2-fumarate reductase activities of SUR (Fig. 3). In contrast to the mirror pH profle found for the enzyme activity revealed with different electron accepter-donor pairs, exactly the same pH-dependence was observed when the carboxin-sensitive

a Extrapolation to infinite concentration of quinone/quinol at 10 mM fumarate or succinate. b Calculated from Dixon plots for simple competitive inhibition. c Determined at 10/~M Q2. d Ks value determined by extrapolation Vma x to zero using PMS as electron accepter [47,66]. e Km for fumarate does not depend on concentration of quinol. f Determined as koff/kon ratio at 25C [47], see also Fig. 7. g Simple linear competitive with Q2H2 inhibition was found, if the carboxin-insensitive fraction of the activity was subtracted (see Fig. 4). h Mixed-type inhibition; K i value calculated from Dixon plots.

quinol-quinone reactive site was involved in the operation of the enzyme in the direct or reverse directions. The pH profile of the enzyme activity correlates with the ionization of a single group with p K a 7.0 (Fig. 3).

I00

A
,.o d
4-

6'

-~ 5 O
"4'-0

s.o

pK a = 7.0

0.5

(15 ~

2.5

f
i i i

pH

I0

.4 I102

L2

NOR bJg/ml

pM-'

Fig. 2. The dependence of the fumarate reductase activity on NQR (A) and concentration of added quinone (B). The velocities (v) were determined as described in Fig. 1 in the presence of 10/.~M Q2 (A) and 9 ~ g / m l NQR (B). The maximal rate V was 1.0/~mol/min per mg of SUR. The steady-state reduction of added Q2 (o) was determined by measuring absorption at 287 nm using a milimolar extinction coefficient of 8.4; the control experiments showed no contribution of N A D H / N A D + oxidoreduction at this wavelength.

Fig. 3. The pH-dependences of the fumarate reductase (e) and succinate-ubiquinone reductase (o) activities of SUR; the continious line is the theoretical titration curve for a fraction of deprotonated monobasic acid with PKa= 7.0. Upper curve (0) shows relative activity of NQR. The fumarate reductase activity was determined as described in Fig. 1 in the presence of 10 mM fumarate, 10 /.~M Q2 and excess of NQR. 100% activity corresponds to 0.9 and 35 p.mol/min per mg of SUR for fumarate reduction and succinate oxidation, respectively. For the assay conditions for succinate oxidation, see Materials and Methods. The NADH-Q 2 reductase activity of NOR was determined in the presence of 100/zM NADH and 10 p,M Q2 (167/.Lmol/min per mg of NQR).

286
A I00 I00

;
,,=::[

50

50

~/--o,=-lI0 Q2Hz 20 200 Carboxin pM Fumorote 30

,//'
6 I00

Succinate---- Qz

Fig. 4. Inhibition of fumarate reduction and succinate oxidation by carboxin. (A) fumarate reduction was measured in the presence of 2 (e; 1 Kn~)and 10 (o; 5 K m)/zM of Q2H2. 100% activity corresponds to 0.8 (o) and 1.33 (e) /zmol/min per mg SUR (B) succinate oxidation was measured in the presence of 0.33 (o; 1 K m) and 2 (o; 6 K m) /zM Q2.100% activity corresponds to 22 (e) and 35 () ~mol/min per mg SUR.

N o significant c h a n g e of N Q R activity was observed within the p H interval investigated. T h e m e c h a n i s m by which succinate d e h y d r o g e n a s e transfer electrons from succinate to ubiquinone is still obscure. O n e a p p r o a c h to the u n d e r s t a n d i n g of this m e c h a n i s m is the use of specific inhibitors of the reaction. T-I'A [42] and carboxin [43] are powerful inhibitors of the succinate-ubiquinone reductase reaction, whereas these c o m p o u n d s do not affect electron transfer between succinate d e h y d r o g e n a s e and artificial electron acceptors. To our knowledge, no data are available on the effect of T T A and carboxin on the f u m a r a t e - r e d u c t a s e reaction catalyzed by purified S D H or S U R , although T T A was shown to inhibit the N A D H - f u m a r a t e reductase reaction catalyzed by submitochondrial particles [29]. Thus, it seemed of interest to investigate the effect of carboxin on the quinolf u m a r a t e reductase reaction. T h e inhibition of the succinate-quinone and quinol-fumarate reductase reactions by carboxin at different concentrations of quinone (quinol) is shown in Fig. 4. T h e patterns of inhibition are quite similar for both reactions although a quantitative difference is evident. In a g r e e m e n t with the data of T r u m p o w e r and Simmons on T T A inhibition [44] and in contrast to the report of Mowery et al. [43], the inhibitory effect of carboxin on the succinateubiquinone reductase is incomplete. T h e same is true for the quinol-fumarate reductase reaction and the relative residual activities observed in the presence of saturating concentrations of the inhibitor (approx. 15%). W h e n double reciprocal plots (1/activity vs. 1 / a c c e p t o r (donor) concentrations) at different inhibitor concentrations or Dixon plots (1/activity vs. the inhibitor concentrations) were constructed, significant deviations from linearity were evident. However, the plots b e c a m e linear if the residual carboxin-insensitive

fraction was subtracted from the activities observed in the titration curves. The simple hyperbolic curves obtained after subtraction suggests the presence of a single site for carboxin inhibition or multiple sites with indistinguishable affinities. The inhibition of both reactions was competitive with quinone (quinol). A p p a r e n t affinities of carboxin to the enzyme operating in direct or reverse directions arc given in Table 1. Having a simple assay p r o c e d u r e for Q2 H 2-fumarate reductase activity, it was of interest to reconstitute the carboxin-sensitive fumarate reductase from soluble S D H and QP~ preparations with different heme b contents. Fig. 5 shows the titration curves for the reconstitution of the succinate-Q 2 reductase and Q2 H2-fumarate reductase from soluble reconstitutively active S D H and Qp~. T h e pattern of the reconstitution curves is notable in two respects. First, the elicited quinone reactivity increased linearly and in parallel for both activities up to a saturation point which is the same for quinol oxidation and reduction. This observation strongly suggests that the same c o m p o n e n t s of the reconstituted system are involved in the carboxin-sensitive succinate-Q 2 and Q 2 H 2 - f u m a r a t e reductasc reactions. Second, d e p e n d i n g on the purification of QP,, the saturation of quinone reactivity occurred at different QP~ a m o u n t s a d d e d to the reconstitution mixture. However, no quantitative correlation between relative

I00

o~ "; 50 <

0.1

0.2 QPs

0.3 rng/ml

Fig. 5. Reconstitution of ubiquinol-fumarate reductase (e) and succinate-ubiquinone reductase (o) activities from soluble SDH and QP~. SDH (0.3 mg/ml) and Qp~ (concentrations are indicated on abscissa) were added to the mixture containing 20 mM phosphate, 0.2 mM EDTA, asolectin (10 mg/ml), 2 mM succinate (potassium salts (pH 7.8)) and placed on ice. Proper amounts were withdrawn for the fumarate reduction and succinate oxidation assays. Fumarate reduction was measured as described in Fig. 1. Succinate oxidation was measured as described in Materials and Methods except for DCIP (45/zM), which was used as the terminal electron acceptor instead of WB. Curve 1 and 2, purified and crude preparations of QPs (see Materials and Methods) with specific activities of 49 and 92 #mol of succinate oxidized/min per mg of protein in the presence of SDH excess, respectively. 100% corresponds to 0,4 and 22 /~mol/min per mg of SDH for the reconstituted fumarate- and succinate-quinone reductases, respectively.

287 heme b, content in QPs preparations and the specific quinone reactivity conferring capacity was observed, as might be expected if cytochrome b is an obligatory component of electron transfer in either direction of the catalysis. Oxaloacetate (OA) has been known for many years as the most powerful inhibitor of succinate dehydrogenase [45]. It has been shown that O A also inhibits E. coli fumarate reductase [46] with parameters qualitatively similar to those for cardiac S D H [47]. An important factor which strongly influences the affinity of O A to the substrate binding site is the redox-state of the enzyme: an order of magnitude higher affinity was found for oxidized S D H and E. coil fumarate reductase than for their reduced counterparts [46,47]. It should be emphasized that the terms ' r e d u c e d ' and 'oxidized' are ambiguous for any enzyme with multiple intrinsic redox components, although the elegant experiments of Ackrell et al. have suggested that reduction of the covalently-bound F A D moiety is a prerequisite for a strong decrease of O A affinity for the substrate binding site of the enzyme [48]. In order to gain a better understanding of the molecular events involved in the exceptionally tight binding of OA, we compared the inhibitory p a r a m e t e r s of O A in Q z H 2 fumarate and succinate-Q2 reductase directions of the catalysis. Fig. 6 demonstrates the inhibition patterns of direct and reverse succinate-Q 2 reductases in the presence of O A and the corresponding substrates (succinate or fumarate) added in concentrations providing equal ratios of f r e e / s u b s t r a t e - b o u n d enzyme during steadystate zero-order catalysis (10 Km).The dramatic difference in O A sensitivity of the enzyme catalyzing the direct and reverse reactions is evident. When 0.6 p,M
,4 SUR SUR
B

>+
I

\ _=

I0

20

30

40

50 rain

Fig. 7. Kinetics of activation of oxaloacetate-inhibited SUR. SUR

(0.1 mg/ml) was preincubated at 25C with 1.8 p,M OA for 30 rain in a mixture containing 20 mM phosphate, 0.2 mM EDTA (potassium salts (pH 7.8)). At zero time, NQR (0.05 mg/ml), 10 /zM Q2, 100 p,M NADH and 5 mM malonate (line 1) or 5 mM malonate and 10 /zM Q2 (line 2) were added and the succinate-ubiquinone reductase activity at 25C (ut) was determined in the presence of 20 mM succinate, u~ is the activity of the enzyme preincubated with 20 mM succinate for 30 min at 25C. Semilogarithmic plots 1 and 2 correspond to the first-order rate constants of 0.14 rain-1 and 0.015 rain l, respectively.

,2:

*A340:0.05

Succinate ~

Q2

QzH2 ~

Fumarate

Fig. 6. Inhibition of succinate oxidation and fumarate reduction by oxaloacetate. Succinate oxidation (A) in the presence of 1.33 mM succinate (10 K m) and fumarate reduction (B) in the presence of 250 /~M fumarate (10 K m) were recorded at 25C (for other components of the reaction mixtures, see Materials and Methods and Fig. 1). Oxaloacetate (OA) was added where indicated. The reactions were started by the addition of SUR (A, 0.25/~g/ml and B, 10/xg/ml).

O A was added to enzyme catalyzing succinate-Q 2 reductase, the reaction rate was gradually inhibited down to about 20% of the control value (curve 2). The inhibition corresponds to a competitive type K~ value of 0.02 /~M, which closely fits to the dissociation constant determined previously [47]. The same concentration of O A added to enzyme catalyzing fumarate reductase in the presence of 10 K m fumarate had no detectable inhibitory effect (curve 2). A 10-times higher concentration of O A was needed to provide 80% inhibition of the fumarate reductase reaction (curve 3). The results obtained clearly demonstrate that during steady-state catalysis the substrate free enzyme is present in the reduced form when electrons are transferred from quinol to fumarate, whereas the oxidized form of free enzyme is present when the overall reaction proceeds from succinate to quinone. To assure that the reduction of S U R through the carboxin-sensitive quinol binding site provides significant decrease of O A affinity, the rate of O A dissociation from the enzyme-inhibitor complex was directly measured. Fig. 7 shows that, similar to submitochondrial particles [47] OA-inhibited S U R can be reactivated in the presence of malonate, and Q2H2 generated by N A D H and N Q R strongly accelerate the reactivation process. It has been proposed that the dicarboxylate binding site sulfhydryl-group is involved in the reaction mechanism of succinate oxidation and in stabilization of OA-binding [41,49,50]. Although direct participation of the sulfhydryl group in the reaction mechanism of succinate oxidation was seriousely questioned, if not excluded, by the results of site-specific mutation studies on E. coli fumarate reductase [51], it was of interest to measure the reactivity of the substrate-protected

288 the report of Baginsky and Hatefi [53] and in agreement with the data of Ziegler and Doeg [1], we were not able to detect any reduction of substoichiometric cytochrome b by either succinate (in the presence of Q2) or by Qa H z. The only prominent absorption change induced by succinate is a partial bleaching at 400-500 nm which most probably corresponds to the flavin and non-heme iron-sulfur components. The most striking observation is that quinol in the presence of a quinol regenerating system, i.e., under the conditions where the change in reactivity to O A was evident (Fig. 7), caused no any detectable spectral changes of the enzyme (curve 3). The optical spectra were compared with low temperature E P R data obtained after equilibration of the enzyme samples with the reducing system (quinol, N A D H , N Q R ) and those frozen during the steady-state quinol-fumarate reductase reaction (Fig. 10). SUR showed a prominent g = 2.01 signal typical for highpotential S-3 center (curve 1). The signal disappeared in the presence of Q z H 2 (curve 2) and did not reappear when fumarate was added and steady-state electron flow from Q2H2 to fumarate was initiated. Taken together, the results presented in Figs. 9 and 10 suggest that rapid equilibrium exists between reduced quinol and the S-3 center and that the rate limiting step in the overall quinol-fumarate reductase reaction is the elec-

,~ >~ 2 _.=

min

I0

Fig. 8. Inactivation of the reduced (1) and oxidized (2) SUR by NEM. (1), SUR (10 ~g/ml) was incubated at 25C in a 2 ml spectrophotometric cuvette containing 20 mM phosphate, 0.2 mM EDTA (potassium salts, (pH 7.8)), 0.006% Triton X-100, 10 /~M Q2, 100 /~M NADH, NQR (5 p.g/ml) and 100 ~M NEM. Appropriate amounts of the mixture were withdrawn for the assays of the residual succinate-ubiquinone reductase activity (o, ct). The residual fumarate reductase activity (ut) was determined after addition of 10 mM fumarate (). No substantional loss of NQR activity during incubation with NEM was detected. (2) SUR was treated as described for curve (1), except that for no NQR, NADH and Q2 were added and the residual succinate-ubiquinone reductase activities (0, vt) were measured. Co, the enzyme activities in the absence of NEM.

sulfhydryl in the 'oxidized' and ' r e d u c e d ' forms of the enzyme. The data presented in Fig. 8 show that no significant change in the sulfhydryl reactivity towards N E M was observed when alkylation was performed in the presence of Q z H 2 , i.e., under the conditions where the reduction of the enzyme strongly accelerates O A release (see Fig. 7). No change in the reactivity of the substrate protected arginine residues [52] caused by reduction was observed when oxidized and reduced enzymes were treated with phenylglyoxal under conditions similar to that described in Fig. 8 (results not shown). The results presented above show that in the presence of quinol and quinol regenerating systems, S U R has the properties of the ' r e d u c e d ' enzyme with respect to O A sensitivity. At least six distinct redox components are potentially capable of electron transfer from succinate to bulk ubiquinone (or in the opposite direction): FAD, iron-sulfur centers S-I, S-2, S-3, bound ubisemiquinone and cytochrome b. The following question arises: the reduction of what particular component is responsible for the dramatic decrease of O A affinity to the enzyme dicarboxylate binding site? Fig. 9 shows the optical difference spectra obtained when samples of S U R were treated with dithionite (curve 1, 'complete' reduction), succinate (curve 2), or Q2H2 in the presence of N Q R (curve 3) and their absorption spectra were measured vs. the enzyme treated with Q2 ('complete' oxidation). In contrast to

A A=i03

l:ll!l:!~'.,.~,".~f'~

I 2

I '~''

~r

....

. ." . . ."

~.3

I1111

I I II

a ] II

~1 I I

400

500

6 0 0 nm

Fig. 9. Differential absorption spectra of SUR. SUR (0.5 mg/ml) were added to the mixture containing 20 mM phosphate, 0.2 mM EDTA (potassium salts (pH 7.8)) and 0.3% Triton X-100. (1), few crystals of sodium dithionite; (2), 20 mM succinate, (3), NQR (0.15 mg/ml), 100 ~M NADH and 10 I~M Q2 were added to the sample cuvette.

289 limiting; (ii), there is no simple procedure for varying the substrate (ubiquinol) concentration in the membrane; (iii), very little is known about the mechanism and factors which are involved in operation of Complex I. The second system is the artificial dye-fumarate reductase reaction catalyzed by soluble SDH and membrane-bound or solubilized Complex II [32-34]. The obvious shortcomings of this assay are lack of information on the electron donor reactive site(s) (which clearly differ from the natural one) and the need for anaerobic assay conditions to prevent oxidation of the reducing dyes by oxygen. The latter seriously limits routine kinetic studies. The assay procedure described in the present paper has two advantages. First, it allows the use of water-soluble quinols as the reductant at any desired concentration: this is specifically valuable for kinetic studies. Second, as evident from carboxin sensitivity, electrons enter SUR during the steady-state fumarate reductase reaction at the same site where quinone reduction occurs during steady-state succinate-ubiquinone reductase reaction. Simple hyperbolic dependencies of the fumarate reductase (this paper) and succinate-ubiquinone reductase [36] activities on the concentration of added quinone together with quantitative agreement between the respective Km 2n2 and K 2n2 values (Table I and Ref. 36) suggest that a single ubiquinone-ubiquinol reactive site is involved in operation of the enzyme in both directions. This conclusion is in accordance with the 1:1 stoichiometry between flavin and labeled Q analogue found in binding studies with lipid-depleted Complex II [54]. It was found that oxidation/reduction of ubiquinol-ubiquinone in the mitochondrial membrane is first-order with respect to quinone [55]; this is somewhat unexpected. Due to a relatively high affinity of SUR to quinone, the reduction of quinone which is rapidly mobile and in great molar excess would be expected to exhibit transient zero-order kinetics prior to first-order kinetics. The competition between oxidized and reduced quinone with quantitatively similar affinities for the ubiquinone reactive site provides a simple kinetic control mechanism for linear dependence of the succinate oxidation rate and the thermodynamic (Q/QzH2 ratio) parameter, and explains the first-order nature of the redox reactions of ubiquinone in the respiratory chain (for further discussion see Refs. 36, 56). Exactly the same simple pH-profile was found for the reaction velocities in either direction (Fig. 3). This is somewhat unexpected, because identity of the pHprofile suggests that the same pH-dependent rate-limiting step is involved in succinate oxidation and fumarate reduction in spite of the significant difference in the enzyme turnover numbers in the two directions. A likely explanation is that a pH-dependent equilibrium

T=6K

_j
SUR+ N Q R+ NADH + Qa(2)

(2)Fumarate

g 2.01

i IOmTesla ,z

Fig. 10. Low-temperature EPR spectra of SUR. SUR (l mg/ml) in 20 mM phosphate, 0.2 mM EDTA, 0.3% Triton X-100 (potassium salts (pH 7.8)) were placed in EPR tubes at 20C and after further additions the samples were frozen and kept in liquid nitrogen. Further additions were: (1), 50 /xM PMS and 200 /zM potassium ferricyanide; (2), NQR (0.025 mg/ml), 400 /zM NADH, 50 /J.M Q2 were added and the sample was incubated for 2 rain before freezing; lowest curve, the same as (2), 20 mM fumarate was added and the sample was incubated for 30 sec before freezing. The specific fumarate reductase activity (0.05 ~.moles/min per mg of SUR) was measured under exactly the same conditions as for sample (3). It was found that the relatively low specific activity was due to the inhibitory effect of 0.3% Triton X-100 which was added to dissolve the enzyme. EPR-operating conditions : modulation frequency, 100 kHz; modulation amplitude, 6.3 gauss; microwave power, 1 roW.

tron transfer between S-3 and lower mid-point potential redox components of the enzyme.
Discussion

Two basically different assay systems have been used for fumarate reductase assay. The system which reflects the 'physiological' electron pathway is the NADH-fumarate reductase reaction catalyzed by submitochondrial particles [29,30]. Application of this system, which involves coupled operation of NADH-ubiquinone reductase (Complex I) and succinate-ubiquinone reductase (Complex II), is hampered for meaningful kinetic analysis for several reasons: (i), the enzymes involved in the overall reaction are present in the inner mitochondrial membrane at a fixed ratio and, at any given set of conditions, special precautions should be taken to assure that electron transfer from ubiquinol to fumarate is rate-

290 between active and inactive conformations of the enzyme exists independently of the occupation of the substrate binding site, and only the deprotonated form is involved in the catalytic turnover. Due to very similar pH-profiles for the enzyme activity and for inhibition by NEM [41], it seems likely that protonation-deprotonation of the sufhydryl group is involved in such an equilibrium. The kinetics and maximal extent of inhibition of succinate oxidation by carboxin and T T A were shown to be the same for a variety of enzyme preparations, thus the inhibitors are thought to act at the same site [42-44]. The results presented in this paper showed the same inhibitory patterns of succinate oxidation and fumarate reduction for carboxin: namely single site directed, incomplete and competitive with quinone inhibition. It has been proposed that T T A inhibits the enzyme-catalyzed reduction of Q H ' to Q H 2 in a sequence of electron transfers from iron-sulfur center S-3 to the exogenous quinone acceptor [44]. If this hypothesis is correct, the small portion of the carboxin-insensitive fumarate reductase is due to a slow oxidation of QH 2 to QH" at a site different from that where rapid equilibrium between bound and exogenous quinol occurs during steady-state catalysis in the absence of the inhibitor. The role of cytochrome b in electron transfer reactions catalyzed by mammalian Complex II remains puzzling. It might be proposed that cytochrome b is not an obligatory component in electron transfer from succinate to ubiquinone, but that it does participate in the ubiquinol-fumarate reductase reaction. Should this be true, a significant difference would be expected in the titration curves in the reconstitution of the two activities from soluble SDH and QP~ with different cytochrome b contents. This is not the case (Fig. 5). If cytochrome b heme is reducible by some electron-donor from the cytosolic side of the inner mitochondrial membrane, then Complex II (in addition to its succinate dehydrogenase activity) may serve as an electron shuttle for transfer of reducing equivalents from the cytosol to the mitochondrial matrix. The structural arrangement of Complex II in the mitochondrial inner membrane [57] and that of the diheme cytochrome b of similar Wolinella succinogenes fumarate reductase [58] and B. subtilis succinate-quinone reductase [59] do not contradict this proposal. The relative stabilities of OA binding to the oxidized and reduced form of cardiac SDH [47] and E. coli fumarate reductase complex [46] differ by close to an order of magnitude. The results presented in this paper show that the reactivities of the amino-acid residues which may directly or indirectly be involved in binding and subsequent stabilization of the enzyme-OA complex (arginine and cysteine [41,49,51,52]) are not changed upon reduction of the enzyme. On the other hand, OA dissociates from the complex about 10-times faster when the enzyme is reduced at the quinol binding site. Taken together these results suggest that the 'tight' enzyme-OA complexes (kof~= 0.015 min 1, /~red '~ off = 0.14 min -1, Fig. 7) are a consequence of a secondary isomerization of the primary 'rapid' enzyme-OA complexes. This mechanism agrees with the finding that succinate-ubiquinone reductase is capable of many turnovers of malate oxidation [61], providing that enoloxaloacetate (an immediate product of the reaction [61,62] is irreversibly utilized. Only iron-sulfur center S-3 was detected as the reduced component during the steady-state ubiquinolfumarate reductase reaction and under equilibrium conditions in the presence of the reduced quinol (Fig. 10). These results suggest that the rate-limiting step in the overall ubiquinol-fumarate reductase reaction is the electron transfer between center S-3 and lower midpoint potential redox components. This is consistent with E m values of +110 mV, +60 mV, approx. 0 mV, - 2 6 0 mV and - 9 0 mV for the Q / Q H z , S-3, S-l, S-2 and F A D / F A D H 2 couples, respectively [63-65]. It thus appears that the reduction of S-3 causes a decrease in OA affinity to the enzyme. Using dye-mediated redox titration of OA-deactivated soluble SDH, Ackrell et al. concluded that the reduction and protonation of the covalently bound FAD moiety and not the non-heme iron clusters is the prerequisite for a dramatic decrease of OA affinity to the substrate binding site of the enzyme [48]. A similar interpretation on the reduction of the flavin to its anionic hydroquinone form has been suggested from the redox titration of the OA-deactivated E. coli fumarate reductase complex [46]. At present, no simple explanation for such apparently contradictory results of Ackrell et al. and those reported here is evident. It may be relevant to note that an obvious difference between our experimental approach and that used by Ackrell et al. [48] is that quinol bound at the specific site was present in our system, whereas artificial redox-mediators were used in the titration experiments. The last point we would like to discuss is the strong difference in OA-sensitivity of the succinate-ubiquinone and ubiquinol-fumarate reductase reactions. During steady-state catalysis, the ubiquinol-fumarate reductase reaction responds to OA as would be expected if the dicarboxylate-free intermediate enzyme is present in a 'reduced' form. An 'oxidized' OA susceptible form appears to be an intermediate during the succinateubiquinone reductase reaction. The most likely explanation for the different intermediates is a possible difference in the kinetic mechanisms of the direct and reverse reactions. It has been shown that reduced succinate dehydrogenase is oxidized by a number of artificial electron acceptors before fumarate dissociates from the dicarboxylate binding site during steady-state

291 succinate oxidation [14,47,66]. If this is the case for operation of the enzyme in the presence of quinone as an oxidant, no free 'reduced' enzyme is expected to be an intermediate during the steady-state reaction. In the reverse reaction, a bi-,bi- ping-pong mechanism with free 'reduced' enzyme as an intermediate seems to be kinetically preferential as judged by OA-sensitivity. Should this hypothesis be true, it follows that the precise mechanisms of the direct and reverse reactions catalyzed by the mitochondrial succinate-ubiquinone reductase are not identical.
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Acknowledgements
The authors are grateful to Dr. V.D. Sled for critical comments and to Dr. R.H. Lozier for linguistic advice.

References
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