grivennikova et al fumarate reductase 93xx 120806

7/28/2019 Grivennikova Et Al Fumarate Reductase 93xx 120806

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

BBABIO 43733

F u m a r a t e r e d u c t a s e a c tiv it y o f b o v i n e h e a r t s u c c i n a t e - u b i q u i n o n er e d u c t a s e . N e w a s s a y s y s t e m a n d o v e r a l l p r o p e r t i e s

o f t h e r e a c t i o nV e r a G . G r i v e n n i ko v a a E l e o n o r a V . G a v r ik o v a a A l e x a n d e r A . T im o s h i n b

a n d A n d r e i D . V i n o g r a d o v aa Department of Biochemistry, School of Biology, Mos cow State U niversity , Mo scow (Russia) and b Cardiology Research Center,

Russian Acade my of Medical Sciences, Moscow (Russia)(Received 14 Ap ri l 1992)(Revised ma nusc ript received 20 July 1992)

Key words: Succinate-ubiquinone reductase; Ubiquinol- fum arate reductase; NAD(P)H-quinone reductase; Respi ra tory chain;(Bovine hear t mi tocho ndr ion)A s i m p l e s y s te m f o r a e r o b i c a s s a y o f t h e q u i n o l - f u m a r a t e r e d u c t a s e r e a c t i o n c a t a l y z e d b y p u r i f i e d s o l u b l e b o v in e h e a r ts u c c i n at e - u b iq u i n o n e r e d u c t a s e in t h e p r e s e n c e o f N A D H , N A D ( P ) H - q u i n o n e r e d u c t a se ( D T - d i a p h o r a s e ) a n d a n a p p r o p r i a t eq u i n o n e i s d e s c r i b e d . T h e r e a c t i o n i s i n h i b i te d b y c a r b o x i n , s u g g e s t i n g t h a t t h e s a m e q u i n o n e / q u i n o l b i n d i n g s i te i s i n v o lv e d ine l e c t r o n t r a n s f e r f r o m s u c c i n a t e t o u b i q u i n o n e a n d f r o m u b i q u i n o l t o f u m a r a t e . T h e k i n e t ic p r o p e r t i e s o f th e r e a c t i o n i n b o t hd i r e c t io n s a n d c o m p a r a t i v e a f f i n it i e s o f t h e s u b s t r a t e b i n d i n g s i t e s o f t h e e n z y m e t o s u b s t r a t e s ( p r o d u c t s ) a n d c o m p e t i t i v ei n h i b i to r s a r e r e p o r t e d . C o n s i d e r a b l e d i f f e r e n c e i n a f f in i t y o f t h e s u b s t r a t e s b i n d i n g s i te t o o x a l o a c e t a t e w a s d e m o n s t r a t e d w h e nt h e e n z y m e w a s a s s a y e d i n t h e d i r e c t a n d r e v e r s e d i r e c ti o n s . T h e s e r e s u l t s w e r e t a k e n t o i n d i c a t e th a t t h e o x i d i z e dd i c a r b o x y l a t e - f r e e e n z y m e i s a n i n t e r m e d i a t e d u r i n g t h e s t e a d y - s t a t e 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 r e a c t i o n , w h e r e a s t h er e d u c e d d i c a r b o x y l a t e - f r e e e n z y m e i s a n i n t e r m e d i a t e o f th e s t e a d y - s t a t e u b i q u i n o l - f u m a r a t e r e d u c t a s e r e a c t i o n . N o d i f f e r e n c e i nt h e r e a c t i v i ty o f t h e s u b s t r a t e - p r o t e c t e d c y s t e i n e a n d a r g i n i n e r e s i d u e s w a s f o u n d w h e n t h e p s e u d o - f i r s t - o r d e r r a t e c o n s t a n t s f o rN - e t h y l m a l e i m i d e a n d p h e n y l g l y o x a l i n h i b i ti o n w e r e d e t e r m i n e d f o r o x id i z e d a n d q u i n o l - r e d u c e d e n z y m e . Q u i n o l - f u m a r a t er e d u c t a s e a c t iv i ty w a s r e c o n s t i t u t e d f r o m t h e s o l u b l e s u c c i n a t e d e h y d r o g e n a s e a n d l o w - m o l e c u l a r - m a s s u b i q u i n o n e r e a c t i v i tyc o n f e r r i n g p r o te i n ( s ). N o r e d u c t i o n o f c y t o c h r o m e b w a s o b s e r v e d i n t h e p r e s e n c e o f q u i n o l g e n e r a t i n g s y s t e m , w h e r e a s S -3 l owt e m p e r a t u r e E P R - d e t e c t a b l e i r o n - s u l f u r c e n t e r w a s c o m p l e t e l y r e d u c e d b y q u in o l u n d e r e q u i l i b r i u m ( w i th o u t f u m a r a t e ) o rs t e a d y - s t a t e ( i n t h e p r e s e n c e o f f u m a r a t e ) . N o s i g n i fi c a n t r e d u c t i o n o f f e r r e d o x i n t y p e i r o n - s u lf u r c e n t e r s w a s d e t e c t e d d u r i n g t h es t e a d y - s t a t e q u i n o l - f u m a r a t e o x i d o r e d u c t a s e r e a c ti o n . T h e d a t a o b t a i n e d e l i m i n a t e p a r t i c i p a t i o n o f c y t o c h r o m e b i n t h eq u i n o l - f u m a r a t e r e d u c t a s e r e a c t i o n a n d s h o w t h a t t h e r a t e l i m i t in g s te p o f t h e o v e r a l l r e a c t i o n l i e s b e t w e e n i r o n - s u lf u r c e n t e r S - 3a n d l o w e r m i d p o i n t p o t e n t i a l r e d o x c o m p o n e n t s o f t h e e n z y m e .

I n t r o d u c t i o nS 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 i s t h e p o i n t w h e r et h e d i , - t r i c a r b o x y l i c - a c i d c y c l e is d i r e c t l y l in k e d t o t h e

Correspo ndence to: A .D. Vinogradov, D epar tm ent of B iochemist ry ,School of B iology, Moscow State University, 119899 Moscow, Russia.Abbreviations: carboxin, 5,6-dihydro-2-methyl-l,4-oxathin-3-carbox-anilide; DC IP, 2,6-dichlorophenolindophenol; DTT, dithioth reitol;NEM , N-ethylmaleimide; N QR,NA D(P)H-qu inone reductase; PMS,phenaz ine m e thosu l f a t e ; O A , oxa loace t a t e ; Q 2 , hom olog o fubiquinone having two isoprenoid uni ts in posi t ion 6 of the quinonering; QPs , ubiquinone-binding prote in, which confers succinate-ubiquinone redu ctase activity to the soluble succinate dehy dro-genase; SDH, succinate dehydrogenase; SUR succinate-ubiquinonereductase; TTA , thenoyl t r i f luoroacetone; WB, (W urster ' s B lue) , asemiquinonediimine r adic al of N , N , N ' , N ' - t e t r a m e t h y l - p - p h e n y l e n e -diamine.

e l e c t r o n - t r a n s f e r c h a in i n m i t o c h o n d r i a a n d n u m e r o u sp r o k a r y o t e s . T h i s m e m b r a n e - b o u n d i r o n -s u l f u r f la v o -p r o t e i n c o n s i st s o f su c c i n a t e d e h y d r o g e n a s e ( S D H ; E C1 .3 .9 9 .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 w h i c h a r et ig h t ly b o u n d t o 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 ss e r v e a s a n a n c h o r a n d a s a f a c t o r w h i c h c o n f e r s t h er e a c ti v i t y o f t h e m e m b r a n e - b o u n d e n z y m e t o t h e b u l kq u i n o n e p r e s e n t i n t h e 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 ( u s u al lyt e r m e d C o m p l e x I I ) h a s b e e n p u r i f i e d [ 1 - 4 ] a n d f u r-t h e r r e s o l v e d i n to 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 f r a c t i o n ( Q Ps ) [6 - 9 ] . S D Hc o n t a i n s a 7 0 k D a p o l y p e p t i d e c a r r y in g c o v a l e n tl yb o u n d 8 - a - N ( 3 ) - h i s t i d y l - F A D a n d t h e s u b s t r a t e b i n d i n gs i t e [ 5] a n d a 2 7 k D a p o l y p e p t i d e w h i c h m o s t l i k e lyb 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 o n e


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tetranuclear (S-2) iron-sulfur cluster [10,11]. Both iso-lated subunits a re catalytically inactive and no reconsti-tution of catalytically competent SDH from the individ-ual subunits has been achieved so far. Properly pre-pared SDH (strictly anaerobic conditions, the presenceof succinate) is capable of rapid oxidation of succinateby a number of artificial electron acceptors, but notubiquinone or its homologues, and binding to theSDH-depleted membranes with complete restorationof TTA-, or carboxin-sensitive succinate-ubiquinonereductase activity [12]. The isolated SDH thus pre-pared is unstable in air and rapidly looses its capacityfor the reconstitution of succinate-ubiquinone reduc-tase. The reactivity of SDH with artificial electronacceptors also decreases during aerobic inactivation[13,14]. The direct correlation between inactivation ofthe reconstitutional capacity, loss of the S-3 EPR signaland decrease of ferricyanide reactivity has been docu-mented [15,16], suggesting that the S-3 iron-sulfur cen-ter is responsible for the terminal one-electron transferfrom the reduced SDH to ubiquinone. Themembrane-bound cytochrome-b-enriched fraction(QP~), which confers succinate-ubiquinone reductaseactivity to the soluble SDH, has been pur ified [6-9]. Itcontains two polypeptides and cytochrome b heme.Various molecular masses of the constituent poly-peptides (7-17 KDa) and various heme b content hasbeen reported for QPs fractions obtained by severalresearch groups [17]. QPs itself has no enzymatic activ-ity. Addition of QP~ to soluble reconstitutively activeSDH dramatically changes several properties of thelatter : it becomes very stable and the TTA-, or car-boxin-sensitive ubiquinone reactivity appears in paral-lel with disappearance of ferricyanide reductase activ-ity [7].

The function of Complex II associated cytochrome b[18] is unknown. The stoichiometry of cytochrome bheme/c ovale ntly bound FAD in different preparationsof Complex II is variable [4] and there is no simplecorrelation between catalytic activity of the enzymeand cytochrome b content. Cytochrome b in intact orreconstituted Complex II is not reduced by succinate,although dithionite reduced heme b can be rapidlyreoxidized by fumarate [1], suggesting an electroniclink between heme b and some enzyme redox compo-nent which is in equilibrium with fumarate.The enzyme seems to be very conservative in evolu-tion and a number of succinate-quinone reductases orSDH with very similar properties have been purifiedand characterized, including those from N e u r o s p o r acrassa [19], E s c h e r i c h i a c o l i [20], M i c r o c o c c u s l u t e u s[21], Bac i l l us sub t i l i s [22], P a r a c o c c u s d e n i t r i f i c a n s [23]and R h o d o s p i r i l l u m r u b r u m [24] (see Ref. 25 for morecomparative data).

The major physiological function of Complex II andrelated enzymes in aerobic organisms is oxidation of

283succinate by an appropriate quinone which is furtheroxidized by the respiratory chain. In anaerobic or fac-ultative organisms, similar but genetically distinct en-zymes operationally called quinol-fumarate oxido-reductase or fumarate reductase (FRD; EC 1.3.99.1)exist and metabolically produced fumarate functions asthe terminal electron-acceptor in anaerobic respira-tion. Quinol-fumarate 'anaerobic' reductases and FRDfrom various organisms are strikingly similar to their'aerobic' counterparts in terms of composition andstructure, but differ significantly in their kinetic prop-erties (see Refs. 26-28 and references cited therein).Fumarate reductases rapidly reduce fumarate in thepresence of low-redox-potential electron donors andslowly oxidize succinate in the presence of high-redox-potential electron donors.

Mammalian succinate-ubiquinone reductase, whenoperating within the inner mitochondrial membrane,catalyzes the NADH-fumarate reductase reactionwhich is sensitive to the specific inhibitors of ComplexI and Complex II. The thermodynamic redox gap be-tween the N A D H / N A D and the succinate/fumaratecouples is large enough to provide free energy for ATPsynthesis coupled with transfer of two electrons fromNADH to fumarate. Indeed, the overall reaction hasbeen demonstrated to occur in both direction (ATP-producing NADH-fumarate reductase [29,30] and suc-cinate-supported ATP-dependent NAD reduction[31]). Mammalian SDH as well as Complex II arecapable of fumarate reductase activity in the presenceof s trong reductants, such as FMNH 2 [32,33] andphenosafranine [34]. Some kinetic properties of theartificial donor-fumarate oxidoreductase reaction (cata-lytic turnover number, K m and K i for the substrateand competitive inhibitors) catalyzed by SDH havebeen reported [35]. However valuable the results ob-tained in those studies are, they seem seriously invali-dated by the use of artificial electron donors and thelack of information on the reductant reactive site(s). Inaddition to the serious theoretical limitations in inter-pretation of the results obtained with artificial electrondonors, the technical problems of handling anaerobicassay samples hampers routine kinetic studies of thefumarate-reductase activity. To our knowledge no TTA-or carboxin-sensitive fumarate reductase reaction cat-alyzed by purified succinate-ubiquinone reductase hasbeen demonstrated so far. In order to gain a betterunderstanding of the reaction and control mechanismsinvolved in equilibrium between the succinate/fumarate and the quinol/quinone redox pairs cat-alyzed by Complex II, we have developed a simpleassay system which allows studies o f the carboxin-sensi-tive quinol-fumarate reductase reaction. The resultsobtained using this system with a special emphasis oncytochrome b function and relative affinities of theenzyme substrate (products) binding sites to dicarbox-


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284ylates and quinone (quinol), as revealed in the directand reverse reactions, are reported in this paper.M a t e r i a l s a n d M e t h o d s

Succinate-ubiquinone reductase (SUR) was pre-pared [4] and assayed [4,36] using the procedures de-veloped in this laboratory. For the stock solution, theenzyme was diluted (1 mg/ ml) in 20 mM potassiumphosphate (pH 7.8) , 0.6% Triton X-100. The assaymixture contained 20 mM phosphate (pH 7.8), 0.2 mMEDTA, 20 mM succinate (potassium salts), 10/xM Q2and 30 ~M WB. Two types of QPs were used, preparedas follows. All the procedures were carried out at0-4C.Crude QPs. SUR (90 mg of protein) was diluted in120 ml of 50 mM potassium phosphate/borate, 0.1mM EDTA (pH 10.6) and incubated at 0C for 15 minwith continuous stirring. The mixture was centrifuged(15 min at 30000g) and the precipitated materialwas washed in 120 ml of the same buffer. The residueswere collected, washed in 120 ml of 20 mM sodiumphosphate, 0.1 mM EDTA (pH 7.4), collected by cen-trifugation as before and suspended in a small volumeof 20 mM sodium phosphate, 0.1 mM EDTA (pH 7.4)(2 mg/ml). The preparation thus prepared showed aAA558 of 0.04 (1 mg protein/ml; dithionite reducedminus oxidized).Purified QPs. The crude QPs (approx. 45 mg) wassuspended in 3.6 ml of 50 mM Tris-Cl, 5 mM DTT, 0.1mM EDTA (pH 8.0) and 0.9 ml of 8 M NaCIO 4 wasadded with constant stirring. The mixture was incu-bated for 30 min and centri fugated (40 fnin at 200 000 g). The precipitated material was collected and thetreatment with sodium perchlorate was repeated. Theprotein collected after centrifugation was washed with30 ml of 50 mM Tris-C1, 5 mM DTT, 0.1 mM EDTA(pH 8.0), collected by centrifugation (30 min at 30 000g), suspended in 3.6 ml of 0.67 M sucrose, 50 mMTris-C1, 0.1 mM EDTA (pH 8.0) and 0.18 ml of 10%potassium deoxycholate (pH 8.0) was added. The mix-ture was incubated for 30 min and the loosely packedresidue obtained after centrifugation (30 min at 30 000x 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 ina small volume of 20 mM sodium phosphate, 0.1 mMEDTA (pH 7.4) and stored in liquid nitrogen. The finalpreparation 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 aff inity chromatog-raphy [37]. The enzyme activity was induced in rat liverby interperitoneal injection of the animals with 3-meth-ylcholantrene for 3 days prior to killing.

EPR measurements were performed using a VarianE-109 E spectrometer with a temperature control de-vice.Protein content was determined by the biuret

method [38]. Wurster's Blue was prepared as described[39]. The special chemicals were: fumarate, oxaloac-etate, phenylglyoxal, TTA from Fluka (Switzerland);EDTA, Tris, DCIP, Triton X-100, 20-methylcholan-threne from Serva (Germany); DTI', NADH from Re-anal (Hungary); NEM from BDH (U.K.); Q2 PMSfrom Ferak (Germany). Carboxin was a kind gift fromProf. H. Lyr (Institute of Plant Protection Research,Germany). Other chemicals used were of the highestquality commercially available.R e s u l t s

One aim of the present study was to find a simpleassay system which can be used for tracing the succi-nate-quinone (quinol-fumarate) reductase reaction inboth directions under the same controlled conditionsusing the same substrate/product pair. An obviousdifficulty in the quinol -fumarate reductase assay usingubiquinol or its water soluble homologues as the reduc-tant is that the equilibrium between the succinate/fumarate (E m approx. 0.0 mY) and ubiq uino l/ubiquinone ( E m approx. +100 mV) redox pairs isshifted considerably to oxidation of succinate. Thus,the steady-state quinol-fumarate reductase reaction isexpected to occur only under conditions where theconcentration of quinone is negligible, compare to thatof quinol. NAD(P)H-quinone reductase (DT-di-aphorase) seemed ideally suited for the quinol regener-ating system. The enzyme is simple to prepare in highlyactive and water soluble form [37]. It has low electron-acceptor specifity and high turnover number [40]. Theredox potential of the substrate (NADH/NAD +) pairis far more negative (-320 mV) than that of thesuccinate/fumarate pair (approx. 0.0 mV). The en-zyme does not react with oxygen and the rate of thecoupled fumarate-reductase reaction can be conven-tionally followed by NAD(P)H oxidation at 340 nm.Fig. 1 shows a representative example of QzH2-fumarate reductase reaction catalyzed by purified SURunder aerobic conditions using NQR as the quinolregenerating system. Zero-order NADH oxidation wasobserved when fumarate was added to the cuvettecontaining NADH, Q2 and NQR. As expected, theSUR catalyzed reaction was carboxin- and malonate-sensitive. Simple hyperbolic dependencies of the reac-tion rate on the NQR (at fixed Q 2 ) and Q 2 c o n c e n t r a -t ions (at fixed saturating NOR) were observed, thus,allowing determination of standard kinetic parametersof Q2H2-fumarate reductase (Fig. 2). The kinetic pa-rameters of the overall carboxin-sensitive QzH2 -fumarate reductase, as compared to those of succinate-Q2 reductase are summarised in Table I.


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285

Q z S U RF u m a r a t e

" A,~,oO.03 CarbnXi t ;

- ~ 30 s e c ~ - k _ -F i g . I . Q 2 H 2 - f u m a r a t e r ed u c t a s e r e ac t i o n c a t a ly z e d b y c o u p l e d o p e r -a t i o n of NQR and SUR. 18 /xg NQR was placed in a 2-ml spec-trophotometric cuvette containing 20 mM phosphate, 0.2 mM EDTA,100 #M NADH (potassium salts (pH 8.0), 38C). 10 gM Qe, 20/~gSUR, 1 mM fumarate, 100 ~M carboxin and 10 mM malonate were

added as indicated.

I t h a s b e e n o b s e r v e d t h a t s u c c i n a t e d e h y d r o g e n a s ea c t iv i t y i s s t r o n g l y p H - d e p e n d e n t [ 4 1 ]. S e v e r a l i n t e r p r e -t a t io n s m a y b e c o n s i d e r e d f o r t h e p H - p r o f i l e o f t h ee n z y m e a c t i v it y , s u c h a s t h e e f f e c t o f p H o n i o n i z a t i o no f a p r o t o n - a b s t r a c t in g g r o u p , o n t h e r a t e o f i n tr a -m o l e c u l a r e l e c t r o n t r a n s f e r , o n t h e e q u i l i b r i u m b e -t w e e n a c ti v e a n d i n a ct iv e c o n f o r m a t i o n o f t h e p r o t e i n .I t h a s b e e n r e p o r t e d t h a t t h e f u m a r a t e r e d u c t a s e a c t iv -i ty o f s u b m i t o c h o n d r i a l p a r t ic l e s m e a s u r e d w i t h r e -d u c e d p h e n o s a f r a n i n e a s e l e c t r o n d o n o r s h o w s a m i r -r o r i m a g e o f th e p H - p r o f i l e o f s u c ci n a t e d e h y d r o g e n a s ea c ti v it y [ 3 4 ]. W e c o m p a r e d t h e e f f e c t o f p H o n t h es u c c i n a t e - Q 2 a n d Q 2 H 2 - f u m a r a t e r e d u c t a s e a c ti v it ie so f S U R ( F ig . 3). I n c o n t r a s t to t h e m i r r o r p H p r o f l ef o u n d f o r t h e e n z y m e a c t iv i ty re v e a l e d w i t h d i f f e r e n te l e c t r o n a c c e p t e r - d o n o r p a ir s , e x a ct ly t h e s a m e p H - d e -p e n d e n c e w a s o b s e r v e d w h e n t h e c a r b o x i n - s e n s i t i v e

A

0.5

,.o d s.o4-

N(15 ~ 2.5

i , , , ,

5 I0N O R b Jg /m l

6'

fi i i

. 4 L 2I102 p M - '

Fig. 2. The dependence of the fumarate reductase activity on NQR(A) and concentration of added quinone (B). The velocities (v) weredetermined as described in Fig. 1 in the presence of 10/.~M Q2 (A)and 9 ~g/ml NQR (B). The maximal rate V was 1.0/~mol/min permg of SUR. The steady-state reduction of added Q2 (o) was deter-mined by measuring absorption at 287 nm using a milimolar extinc-tion coefficient of 8.4; the control experiments showed no contribu-

tion of NAD H/ NA D + oxidoreduction at this wavelength.

TABLE IThe kinetic parameters of the ubiquinol-fumarate and succinate-ubiquinone reductase reactionsAssay conditions: 38C, 20 mM potassium p hosph ate (pH 7.8), 0.006%Triton X-100.

Fumarate Succinatereduction oxidationV (/~mo l/mi n per mg) a 1.0 40K d i c a r b o x y l a t e b ( / k ,I ]m ( i ) . ] d , . . l .succinate 30 130 c (30) ~

fumarate 25 e 150malonate 1.0 1.3oxaloacetate f 0.2 0.02

K 2 (IzM) 0.3Km2H2 (/~M) 1.5K carbxin (/xM) 3.0 g 2.0 ha 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 Vmax 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.

q u i n o l - q u i n o n e r e a c t i v e s i t e w a s i n v o l v e d i n t h e o p e r a -t i o n o f th e e n z y m e i n t h e d i r e c t o r r e v e r s e d i r e c t io n s .T h e p H p r o f i l e o f t h e e n z y m e a c ti v i ty c o r r e l a t e s w i t ht h e i o n i z a t i o n o f a s i n g le g r o u p w i t h p K a 7 . 0 ( F ig . 3 ).

I 0 0

- ~ 5 O p K a = 7 . 0" 4 ' - -0

I I I I I6 7 8p HFig. 3. The pH-de pendences of the fumarate reductase (e) and

succinate-ubiquinone reductase (o) activities of SUR; the continiousline is the theoretical titration curve for a fraction of deprotonatedmonobasic acid with P Ka = 7.0. Upper curve ( 0) shows relativeactivity of NQR. The fumarate reductase activity was determined asdescribed in Fig. 1 in the p resence of 10 mM fumarate, 10 /.~M Q2and excess of NQR. 100 % activity corr esponds to 0.9 and 35p.mol/min per mg of SUR for fumarate reduction and succinateoxidation, respectively. For the assay conditions for succinate oxida-tion, see Materials and Methods. The NADH-Q 2 reductase activityof NOR was determined in the presence of 100/zM NADH and 10

p,M Q2 (167/.Lmol/min per mg of NQR).


5/11

2 8 6

I 0 0

; 5 0, , = : : [

AI0 0

~ / - - o -=-l-I 0 2 0 3 0 2 0 0

5 0

Ca r b o x in p MQ2Hz Fum orote

B

, , ,//'2 4 6 I00Su cc in a t e - - - - Qz

Fig. 4. Inhibition of fumarate reduction and succinate oxidation bycarboxin. (A) fumarate reduction was measured in the presence of 2(e; 1 Kn~) and 10 (o ; 5 K m)/zM of Q2H 2. 100% activi ty correspondsto 0 .8 (o) and 1.33 (e) / zmo l /min per m g SUR (B) succinateoxidation was measured in the presence of 0.33 (o; 1 K m) and 2 ( o;6 K m) /zM Q2.100% activi ty corresponds to 22 (e) and 35 ()~mo l / mi n pe r mg SU R .

N o s i g n i fi c a n t c h a n g e o f N Q R a c ti v it y w a s o b s e r v e dw i t h i n t h e p H i n t e r v a l i n v e s t i g a t e d .

T h e m e c h a n i s m b y w h i c h s u cc i na te d e h y d r o g e n a s et r a n s f e r e l e c t r o n s f r o m s u c c i n a t e t o u b i q u i n o n e is s ti llo b s c u r e . O n e a p p r o a c h t o t h e u n d e r s t a n d i n g o f t hi sm e c h a n i s m is t h e u s e o f s p e c if i c in h i b i t o r s o f t h er e a c t i o n . T - I ' A [ 42 ] a n d c a r b o x i n [ 43 ] a r e p o w e r f u li n h ib i to r s o f th e 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 r e a c-t io n , w h e r e a s t h e s e c o m p o u n d s d o n o t a f f ec t e le c t r o nt r a n s fe r b e t w e e n s u c c i n a te d e h y d r o g e n a s e a n d a rt if i-c ia l e l e c t r o n a c c e p t o r s . T o o u r k n o w l e d g e , n o d a t a a r ea v a i la b l e o n t h e e f f e c t o f T T A a n d c a r b o x i n o n t h ef u m a r a t e - r e d u c t a s e r e a c t io n c a t a l y z e d by p u r i f ie d S D Ho r S U R , a l t h o u g h T T A w a s s h o w n t o in h i bi t th eN A D H - f u m a r a t e r e d u c t a s e r e a c t io n c a t a ly z e d b y s u b-m i t o c h o n d r i a l p a r t i c l e s [ 29 ]. T h u s , i t s e e m e d o f in t e r e s tt o i n v e s t ig a t e t h e e f f e c t o f c a r b o x i n o n t h e q u i n o l -f u m a r a t e r e d u c t a s e r e a c t i o n . T h e i n h i b i t io n o f t h e s u c-c i n a t e - q u i n o n e a n d q u i n o l - f u m a r a t e r e d u c t a s e r e a c -t i o n s b y c a r b o x i n a t d i f f e r e n t c o n c e n t r a t i o n s o f q u i n o n e( q u i n o l ) i s s h o w n i n F ig . 4 . T h e p a t t e r n s o f i n h i b it i o na r e q u i t e s i m i la r f o r b o t h r e a c t i o n s a l t h o u g h a q u a n t i -t a ti v e d i f f e r e n c e i s e v i d e n t . I n a g r e e m e n t w i t h t h e d a t ao f T r u m p o w e r a n d S i m m o n s o n T T A i n h ib i t i o n [4 4]a n d i n c o n t r a s t t o t h e r e p o r t o f M o w e r y e t a l. [4 3 ], t h ei n h i b i t o r y e f f e c t o f c a r b o x i n o n t h e 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 is i n c o m p l e t e . T h e s a m e is t r u ef o r t h e q u i n o l - f u m a r a t e r e d u c t a s e r e a c t i o n a n d t h er e l a ti v e r e s i d u a l a c t i v it ie s o b s e r v e d i n t h e p r e s e n c e o fs a t u r a t i n g c o n c e n t r a t i o n s o f t h e i n h i b i to r ( a p p r o x .1 5 % ) . W h e n d o u b l e r e c i p r o c a l p l o t s ( 1 / a c t i v i t y v s.1 / a c c e p t o r ( d o n o r ) c o n c e n t r a t i o n s ) a t d i f f e re n t i n-h i b i t o r c o n c e n t r a t i o n s o r D i x o n p l o t s ( 1 / a c t i v i t y v s . t h ei n h i b i t o r c o n c e n t r a t i o n s ) w e r e c o n s t r u c t e d , s ig n i f ic a n td e v i a t i o n s f r o m l i n e a r it y w e r e e v i d e n t . H o w e v e r , t h ep l o t s b e c a m e l i n e a r i f t h e r e s i d u a l c a r b o x i n - i n s e n s i t iv e

f r a c t i o n w a s s u b t r a c t e d f r o m t h e a c t iv i ti e s o b s e r v e d i nt h e t i t r a t io n c u r v e s . T h e s i m p l e h y p e r b o l i c c u r v e s o b -t a i n e d a f t e r s u b t r a c t i o n s u g g e s t s t h e p r e s e n c e o f as i n g l e si t e f o r c a r b o x i n i n h i b i t i o n o r m u l t i p l e s i te s w i t hi n d i s t i n g u i s h a b l e a f f in i t ie s . T h e i n h i b i t i o n o f b o t h r e a c -t i o n s w a s c o m p e t i t i v e w i t h q u i n o n e ( q u i n o l ) . A p p a r e n ta f f in i ti e s o f c a rb o x i n t o t h e e n z y m e o p e r a t i n g i n d i r e c to r r e v e r s e d i r e c t i o n s a r c g i v e n i n T a b l e 1 .

H a v i n g a s im p l e a s s a y p r o c e d u r e f o r Q 2 H 2 -f u m a r a t er e d u c t a s e a c t iv i t y , i t w a s o f i n t e r e s t t o r e c o n s t i t u t e t h ec a r b o x i n - s e n s i ti v e f u m a r a t e r e d u c t a s e f r o m s o l u b leS D H a n d Q P ~ p r e p a r a t i o n s w i t h d i f f e r e n t h e m e bc o n t e n t s . F i g . 5 s h o w s t h e t i t r a t i o n c u r v e s f o r th er e c o n s t i t u t i o n o f t h e s u c c i n a t e - Q 2 r e d u c t a s e a n dQ 2 H 2 - f u m a r a t e r e d u c t a s e f r o m s o l u b l e r e c o n s t it u t i v e lya c t iv e S D H a n d Q p ~ . T h e p a t t e r n o f t h e r e c o n s t i t u t i o nc u r v e s is n o t a b l e i n t w o r e s p e c t s . F i r s t, t h e e l i c i te dq u i n o n e r e a c ti v i ty i n c r e a s e d l i n e ar l y a n d i n p a ra l l e l f o rb o t h a c t i v it i e s u p t o a s a t u r a t i o n p o i n t w h i c h i s t h es a m e f o r q u i n o l o x i d a t i o n a n d r e d u c t i o n . T h i s o b s e r v a -t i o n s tr o n g l y s u g g e s t s th a t t h e s a m e c o m p o n e n t s o f t h er e c o n s t i t u t e d s y s t e m a r e i n v o l v e d in th e c a r b o x i n - s e n s i -t iv e s u c c i n a t e -Q 2 a n d Q 2 H 2 - f u m a r a t e r e d u c t a s c r e a c-t io n s . S e c o n d , d e p e n d i n g o n t h e p u r i f ic a t i o n o f Q P , ,t h e s a t u r a t i o n o f q u i n o n e r e a c ti v i ty o c c u r r e d a t d if f e r-e n t Q P~ a m o u n t s a d d e d t o t h e r e c o n s t i t u t i o n m i x t u re .H o w e v e r , n o q u a n t i ta t i v e c o r r e l a t i o n b e t w e e n r e la t iv e

I 0 0

o~

" ; 5 0+ 1I

\ I_=

I 0 2 0 3 0 4 0 5 0 rainFig. 7. Kinetics of activation of oxaloacetate-inhibited SUR. SUR(0.1 m g/m l) was preincubated at 25C with 1.8 p,M OA for 30 rain ina m ixture containing 20 mM phosphate, 0.2 mM EDTA (potassiumsalts (pH 7.8)). At zero tim e, NQ R (0.05 mg /ml), 10 /zM Q2, 100p,M N AD H and 5 m M malonate (line 1) or 5 mM malonate and 10/zM Q2 (line 2) we re added and the succinate-ubiquinone reductaseactivity at 25C (ut) was determined in the presence of 20 m Msuccinate, u~ is the ac tivity of the enzym e preincub ated w ith 20 mMsuccinate for 30 m in at 25 C. Sem ilogarithm ic plots 1 and 2 corre-spond to the first-order rate constants of 0.14 rain -1 and 0.015rain l, respectiv ely.

O A w a s a d d e d t o e n z y m e c a t a l y z in g s u c c i n a t e - Q 2 re -d u c t a s e , t h e r e a c t i o n r a t e w a s g r a d u a l ly i n h i b i te d d o w nt o a b o u t 2 0 % o f t h e c o n t r o l v a l u e ( cu r v e 2 ). T h ei n h i b it i o n c o r r e s p o n d s t o a c o m p e t i t i v e t y p e K ~ v a l u eo f 0 .0 2 / ~ M , w h i c h c l o s e ly f it s t o t h e d i s s o c i a t i o n c o n -s t a n t d e t e r m i n e d p r e v i o u s l y [4 7]. T h e s a m e c o n c e n t r a -t i o n o f O A a d d e d t o e n z y m e c a ta l y z in g f u m a r a t e r e -d u c t a s e i n t h e p r e s e n c e o f 1 0 K m f u m a r a t e h a d n od e t e c t a b l e i n h i b i t o r y e f f e c t ( c u r v e 2 ). A 1 0 - t i m e s h i g h e rc o n c e n t r a t io n o f O A w a s n e e d e d t o p ro v i d e 8 0 % i nh i-b i t io n o f th e f u m a r a t e r e d u c t a s e r e a c t i o n ( c u r v e 3) .T h e r e s u lt s o b t a i n e d c l e a rl y d e m o n s t r a t e t h a t d u r i n gs t e a d y - s t a t e c a t a l y s i s t h e s u b s t r a t e f r e e e n z y m e i s p r e -s e n t in t h e r e d u c e d f o r m w h e n e l e c t r o n s a r e t r a n s -f e r r e d f r o m q u i n o l t o f u m a r a t e , w h e r e a s t h e o x i d i z e df o r m o f f r e e e n z y m e i s p r e s e n t w h e n t h e o v e r a l l re a c -t i o n p r o c e e d s f r o m s u c c i n a t e t o q u i n o n e . T o a s s u r et h a t t h e r e d u c t i o n o f S U R t h r o u g h t h e c a r b o x i n - s e n s i -t i v e q u i n o l b i n d i n g s i t e p r o v i d e s s i g n i f i c a n t d e c r e a s e o fO A a f fi n it y , t h e r a t e o f O A d i s s o c ia t i o n f r o m t h ee n z y m e - i n h i b i t o r c o m p l e x w a s d i r e c tl y m e a s u r e d . F i g . 7s h o w s t h a t , s i m i l a r t o s u b m i t o c h o n d r i a l p a r t i c l e s [4 7]O A - i n h i b it e d S U R c a n b e r ea c t i v at e d i n t h e p r e s e n c eo f m a l o n a te , a n d Q 2 H 2 g e n e r a t ed b y N A D H a n dN Q R s t r o n g l y a c c e l e r a t e t h e r e a c t i v a t i o n p r o c es s .

I t h a s b e e n p r o p o s e d t h a t t h e d i c a r b o x y l a t e b i n d i n gs i t e s u l f h y d r y l - g r o u p i s i n v o l v e d i n t h e r e a c t i o n m e c h a -n i s m o f s u c c i n a t e o x i d a t i o n a n d i n s t a b i l i z a t i o n o fO A - b i n d i n g [ 41 ,4 9 ,5 0] . A l t h o u g h d i r e c t p a r t i c i p a t io n o ft h e s u l f h y d r y l g r o u p i n t h e r e a c t i o n m e c h a n i s m o fs u c c i n a t e o x i d a t i o n w a s s e r i o u s e l y q u e s t io n e d , i f n o te x c l u d e d , b y t h e r e s u l t s o f si t e - s p e c i f i c m u t a t i o n s t u d -i e s o n E. co l i f u m a r a t e r e d u c t a s e [ 51 ], it w a s o f i n t e r e s tt o m e a s u r e t h e r e a c t iv i t y o f th e s u b s t r a t e - p r o t e c t e d


7/11

28 8

,~ 2 I >~_.=

I

I I J I J I n I I5 I 0m inFig. 8. Inactivation of the reduced (1) and ox idized (2) SUR by NEM .(1), SUR (10 ~g /m l) was incubated at 25C in a 2 m l spectrophoto-metric cuvette containing 20 mM phosphate, 0.2 mM E DTA (potas-sium sa lts, (pH 7.8)), 0.006% Triton X-10 0, 10 /~M Q 2, 100 /~MNADH, NQR (5 p.g/ml) and 100 ~M NEM. Appropriate amounts

of the mixture were withdrawn for the assays of the residual succi-nate-ubiquinone reductase activity (o, ct) . The residu al fumaratereductase activity (u ) was d etermined after ad dition of 10 mMfumarate ( ). No substantional loss of N QR activity during incuba-tion with NEM was detected. (2) SUR was treated as described forcurve (1), except that for no NQ R, NA DH and Q2 were added andthe residual succinate-ubiquinone reductase activities (0, v ) weremeasured. Co, the enzyme activities in the absence of N EM.

s u l fh y d r y l i n t h e ' o x i d i z e d ' a n d ' r e d u c e d ' f o r m s o f t h ee n z y m e . T h e d a t a p r e s e n t e d i n F i g . 8 s h o w t h a t n os i g n i f i c a n t c h a n g e i n t h e s u l f h y d r y l r e a c t i v i t y t o w a r d sN E M w a s o b s e rv e d w h e n a l ky l a ti o n w a s p e r f o r m e d i nt h e p r e s e n c e o f Q z H 2 , i . e. , u n d e r t h e c o n d i t i o n s w h e r et h e r e d u c t i o n o f t h e e n z y m e s tr o n g l y a c c e l e r a t e s O Ar e l e a s e ( s e e F i g . 7) . N o c h a n g e i n t h e r e a c t i v i t y o f t h es u b s t r a t e p r o t e c t e d a r g i n i n e r e s i d u e s [ 5 2 ] c a u s e d b yr e d u c t io n w a s o b s e r v e d w h e n o x i d iz e d a n d r e d u c e de n z y m e s w e r e t r e a t e d w i t h p h e n y l g l y o x a l u n d e r c o n d i -t i o n s s i m i l a r t o t h a t d e s c r i b e d i n F i g . 8 ( r e s u l t s n o ts h o w n ) .

T h e r e s u l t s p r e s e n t e d a b o v e s h o w t h a t i n t h e p r e s -e n c e o f q u in o l a n d q u i n o l r e g e n e r a t i n g s y s t em s , S U Rh a s t h e p r o p e r t i e s o f t h e ' r e d u c e d ' e n z y m e w i t h r e -s p e c t t o O A s e n s it i v it y . A t l e a s t s ix d i s t i n c t r e d o xc o m p o n e n t s a r e p o t e n t i al l y c a p a b l e o f e l e c tr o n t r a n s f erf r o m s u c c i n a t e t o b u l k u b i q u i n o n e ( o r in t h e o p p o s i t ed i r e c t i o n ) : F A D , i r o n - s u l f u r c e n t e r s S - I , S - 2 , S - 3 , b o u n du b i s e m i q u i n o n e a n d c y t o c h r o m e b . T h e f o ll o w i n g q u e s -t i o n a ri se s : t h e r e d u c t i o n o f w h a t p a r t i c u l a r c o m p o n e n ti s r e s p o n s i b l e f o r t h e d r a m a t i c d e c r e a s e o f O A a f f in i tyt o t h e e n z y m e d i c a r b o x y l a t e b i n d i n g s i t e ?

F i g . 9 s h o w s t h e o p t i c a l d i f f e r e n c e s p e c t r a o b t a i n e dw h e n s a m p l e s o f S U R w e r e t r e a t e d w i t h d i th i o n it e( c u r v e 1 , ' c o m p l e t e ' r e d u c t i o n ) , s u c c i n a t e ( c u r v e 2 ) , o rQ 2 H 2 i n t h e p r e s e n c e o f N Q R ( c u rv e 3 ) a n d t h e i ra b s o r p t i o n s p e c t r a w e r e m e a s u r e d v s . t h e e n z y m et r e a t e d w i t h Q 2 ( ' c o m p l e t e ' o x i d a t i o n ) . I n c o n t r a s t t o

t h e r e p o r t o f B a g i n s k y a n d H a t e f i [ 53 ] a n d i n a g r e e -m e n t w i t h t h e d a t a o f Z i e g l e r a n d D o e g [ 1 ] , w e w e r en o t a b l e t o d e t e c t a n y r e d u c t i o n o f su b s t o i c h i o m e t r i cc y t o c h r o m e b b y e i t h e r s u c c i n a t e ( in t h e p r e s e n c e o fQ 2 ) o r b y Q a H z. T h e o n l y p r o m i n e n t a b s o r p t i o n c h a n g ei n d u c e d b y s u c c i n a te i s a p a r t i al b l e a c h i n g a t 4 0 0 - 5 0 0n m w h i c h m o s t p r o b a b l y c o r r e s p o n d s t o t h e f la v i n a n dn o n - h e m e i r o n - s u lf u r c o m p o n e n t s . T h e m o s t st r ik i n go b s e r v a t i o n i s t h a t q u i n o l i n th e p r e s e n c e o f a q u in o lr e g e n e r a t i n g s y s t e m , i . e . , u n d e r t h e c o n d i t i o n s w h e r et h e c h a n g e i n r e a c t i v i t y t o O A w a s e v i d e n t ( F i g . 7 ) ,c a u s e d n o a n y d e t e c t a b l e s p e c t r a l c h a n g e s o f t h e e n -z y m e ( c u r v e 3 ) .

T h e o p t i c a l s p e c t r a w e r e c o m p a r e d w i t h l o w t e m -p e r a t u r e E P R d a t a o b t a i n e d a f t e r e q u i li b r at i on o f th ee n z y m e s a m p l e s w i th t h e r e d u c i n g s y s t e m ( q u in o l ,N A D H , N Q R ) a n d t h o s e fr o z e n d u ri n g th e s te a d y - s ta t eq u i n o l - f u m a r a t e r e d u c t a s e r e a c t i o n ( F i g . 1 0 ) . S U Rs h o w e d a p r o m i n e n t g = 2 .0 1 s i g n a l t y p i c a l f o r h i g h -p o t e n t i a l S - 3 c e n t e r ( c u r v e 1 ) . T h e s i g n a l d i s a p p e a r e di n t h e p r e s e n c e o f Q z H 2 ( c u r v e 2 ) a n d d i d n o t r e a p -p e a r w h e n f u m a r a t e w a s a d d e d a n d s t e a d y - st a te e l e c-t r o n f l ow f r o m Q 2 H 2 t o f u m a r a t e w a s i n it i at e d . T a k e nt o g e t h e r , t h e r e s u l t s p r e s e n t e d i n F i g s . 9 a n d 1 0 s u g g e s tt h a t r a p i d e q u i l i b r i u m e x i s ts b e t w e e n r e d u c e d q u i n o la n d t h e S - 3 c e n t e r a n d t h a t t h e r a t e l i m i t i n g s t e p i n t h eo v e r a l l q u i n o l - f u m a r a t e r e d u c t a s e r e a c t i o n i s t h e e l e c-

A A = i 0 3 l : l l ! l : ! ~ ' . , . ~ , " . ~ f ' ~ I 2

I ' ~ ' ' r . . . . ~ " " 3, ~ . . . . . ~ . 3

I 1 1 1 1 I I I I a ] I I I I ~ 1 I I4 0 0 5 0 0 6 0 0 n m

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


8/11

T = 6 K

_ jS U R + N Q R + N A D H + Q a ( 2 )

( 2 ) F u m a r a t e

I O m T e s l ag 2.01 i , zFig. 10. Low-temperature EPR spectra of SUR. SUR (l mg/ml) in20 mM phosphate, 0.2 mM EDTA, 0.3% Triton X-100 (potassiumsalts (pH 7.8)) were placed in EPR tubes at 20C and after furtheradditions the samples were frozen and kept in liquid nitrogen.Further additions were: (1), 50 /xM PMS and 200 /zM potassiumferricyanide; (2), NQR (0.025 mg/ml) , 400 /zM NADH, 50 /J.M Q2were added and the sample was incubated for 2 rain before freezing;lowest curve, the same as (2), 20 mM fumarate was added and thesample was incubated for 30 sec before freezing. The specific fu-marate reductase activity (0.05 ~.moles/min per mg of SUR) wasmeasur ed und er exactly the s ame conditi ons as for sample (3). It wasfound that the relatively low specific activity was due to the in-hibitory effect of 0.3% Tri ton X-100 which was added to dissolve theenzyme. EPR-operating conditions : modulation frequency, 100 kHz;

modulation ampl itude, 6.3 gauss; microwave power, 1 roW.

tron transfer between S-3 and lower mid-point poten-tial redox components of the enzyme.D i s c u s s i o n

Two basically different assay systems have beenused for fumarate reductase assay.The system which reflects the 'physiological' elec-tron pathway is the NADH-fumarate reductase reac-tion catalyzed by submitochondrial particles [29,30].Application of this system, which involves coupled op-eration of NADH-ubiquinone reductase (Complex I)and succinate-ubiquinone reductase (Complex II), ishampered for meaningful kinetic analysis for severalreasons: (i), the enzymes involved in the overall reac-tion are present in the inner mitochondrial membraneat a fixed ratio and, at any given set of conditions,special precautions should be taken to assure thatelectron transfer from ubiquinol to fumarate is rate-

289limiting; (ii), there is no simple procedure for varyingthe substrate (ubiquinol) concentration in the mem-brane; (iii), very little is known about the mechanismand factors which are involved in operation of ComplexI. The second system is the artificial dye-fumaratereductase reaction catalyzed by soluble SDH and mem-brane-bound or solubilized Complex II [32-34]. Theobvious shortcomings of this assay are lack of informa-tion on the electron donor reactive site(s) (which clearlydiffer from the natural one) and the need for anaerobicassay conditions to prevent oxidation of the reducingdyes by oxygen. The latter seriously limits routine ki-netic studies. The assay procedure described in thepresent paper has two advantages. First, it allows theuse of water-soluble quinols as the reductant at anydesired concentration: this is specifically valuable forkinetic studies. Second, as evident from carboxin sensi-tivity, electrons enter SUR during the steady-state fu-marate reductase reaction at the same site wherequinone reduction occurs during steady-state succi-nate-ubiquinone reductase reaction.

Simple hyperbolic dependencies of the fumaratereductase (this paper) and succinate-ubiquinone reduc-tase [36] activities on the concentration of addedquinone together with quantitative agreement betweenthe respective Km2n2 and K 2n2 values (Table I andRef. 36) suggest that a single ubiquinone-ubiquinolreactive site is involved in operation of the enzyme inboth directions. This conclusion is in accordance withthe 1:1 stoichiometry between flavin and labeled Qanalogue found in binding studies with lipid-depletedComplex II [54]. It was found that oxidati on/r educ tionof ubiquinol-ubiquinone in the mitochondrial mem-brane is first-order with respect to quinone [55]; this issomewhat unexpected. Due to a relatively high affinityof SUR to quinone, the reduction of quinone which israpidly mobile and in great molar excess would beexpected to exhibit transient zero-order kinetics priorto first-order kinetics. The competition between oxi-dized and reduced quinone with quantitatively similaraffinities for the ubiquinone reactive site provides asimple kinetic control mechanism for linear depen-dence of the succinate oxidation rate and the thermo-dynamic (Q /QzH 2 ratio) parameter, and explains thefirst-order nature of the redox reactions of ubiquinonein the respiratory chain (for further discussion seeRefs. 36, 56).

Exactly the same simple pH-profile was found forthe reaction velocities in either direction (Fig. 3). Thisis somewhat unexpected, because identity of the pH-profile suggests that the same pH-dependent rate-limit-ing step is involved in succinate oxidation and fumaratereduction in spite of the significant difference in theenzyme turnover numbers in the two directions. Alikely explanation is that a pH-dependent equilibrium


9/11

2 9 0b e t w e e n a c t iv e a n d i n a c t iv e c o n f o r m a t i o n s o f th e e n -z y m e e x i s t s i n d e p e n d e n t l y o f t h e o c c u p a t i o n o f t h es u b s t r a te b i n d i n g s i te , a n d o n l y t h e d e p r o t o n a t e d f o r mi s i n v o l v e d in t h e c a t a l y ti c t u r n o v e r . D u e t o v e r y s i m i la rp H - p r o f i l e s f o r t h e e n z y m e a c t i v i t y a n d f o r i n h i b i t i o nb y N E M [4 1], it s e e m s l ik e l y t h a t p r o t o n a t i o n - d e p r o t o -n a t i o n o f t h e s u f h y d r y l g r o u p i s i n v o l v e d in s u c h a ne q u i l i b r i u m .

T h e k i n e t i c s a n d m a x i m a l e x t e n t o f i n h i b i t i o n o fs u c c i n at e o x i d a ti o n b y c a r b o x in a n d T T A w e r e s h o w nt o b e t h e s a m e f o r a v a r ie t y o f e n z y m e p r e p a r a t i o n s ,t h u s t h e i n h i b i t o r s a r e t h o u g h t t o a c t a t t h e s a m e s i t e[ 4 2 -4 4 ] . T h e r e s u l t s p r e s e n t e d i n th i s p a p e r s h o w e d t h es a m e i n h i b i t o r y p a t t e r n s o f s u c c i n a t e o x i d a t i o n a n df u m a r a t e r e d u c t i o n f o r c a r b o x i n : n a m e l y s i n g l e s i t ed i r e c te d , i n c o m p l e t e a n d c o m p e t i t i v e w i t h q u i n o n e i n-h i b i t i o n . I t h a s b e e n p r o p o s e d t h a t T T A i n h i b i t s t h ee n z y m e - c a t a l y z e d r e d u c t i o n o f Q H ' t o Q H 2 in a s e -q u e n c e o f e l e c t r o n t r a n s f e rs f r o m i r o n - su l f u r c e n t e r S - 3t o t h e e x o g e n o u s q u i n o n e a c c e p t o r [4 4] . I f t h is h y p o t h -e s i s i s c o r r e c t , t h e s m a l l p o r t i o n o f t h e c a r b o x i n - i n s e n -s i t i v e f u m a r a t e r e d u c t a s e i s d u e t o a s l o w o x i d a t i o n o fQ H 2 t o Q H " a t a si t e d i f f e r e n t f r o m t h a t w h e r e r a p i de q u i li b r iu m b e t w e e n b o u n d a n d e x o g e n o u s q u in o l o c-c u r s d u r i n g s t e a d y - s t a t e c a t a ly s i s in t h e a b s e n c e o f t h ei n h i b i t o r .

T h e r o l e o f c y t o c h r o m e b i n e l e c t r o n t r a n s f e r r e a c-t io n s c a t a ly z e d b y m a m m a l i a n C o m p l e x I I r e m a i n sp u z z l i n g . I t m i g h t b e p r o p o s e d t h a t c y t o c h r o m e b i sn o t a n o b l i g a t o r y c o m p o n e n t i n e l e c t r o n t r a n s f e r f ro ms u c c i n a t e t o u b i q u i n o n e , b u t t h a t i t d o e s p a r t i c i p a t e i nt h e u b i q u i n o l - f u m a r a t e r e d u c t a s e r e a c t i o n . S h o u l d t h i sb e t r u e , a s ig n i f i ca n t d i f f e r e n c e w o u l d b e e x p e c t e d i nt h e t i t r a t i o n c u r v e s i n t h e r e c o n s t i t u t i o n o f t h e t w oa c t iv i t ie s f r o m s o l u b l e S D H a n d Q P~ w i t h d i f f e r e n tc y t o c h r o m e b c o n t e n t s . T h i s i s n o t t h e c a s e ( F i g . 5 ) . I fc y t o c h r o m e b h e m e i s r e d u c i b l e b y s o m e e l e c t r o n - d o n o rf r o m t h e c y t o s o l ic s i d e o f t h e i n n e r m i t o c h o n d r i a lm e m b r a n e , t h e n C o m p l e x I I ( i n a d d i t i o n t o i t s s u c c i -n a t e d e h y d r o g e n a s e a c ti v it y ) m a y s e r v e a s an e l e c t r o ns h u t t l e f o r t r a n s f e r o f r e d u c i n g e q u i v a l e n t s f r o m t h ec y t o s o l t o t h e m i t o c h o n d r i a l m a t r ix . T h e s t r u c t u r a la r r a n g e m e n t o f C o m p l e x I I in t h e m i t o c h o n d r i a l i n n e rm e m b r a n e [5 7] an d t h a t o f th e d i h e m e c y t o c h r o m e b o fs i m i l a r Wolinella succinogenes f u m a r a t e r e d u c t a s e [ 5 8 ]a n d B. subtilis s u c c i n a t e - q u i n o n e r e d u c t a s e [ 5 9 ] d o n o tc o n t r a d i c t t h i s p r o p o s a l .T h e r e l a t i v e s t a b i l i t i e s o f O A b i n d i n g t o t h e o x i -d i z e d a n d r e d u c e d f o r m o f c a r d i ac S D H [4 7] a n d E .coli f u m a r a t e r e d u c t a s e c o m p l e x [4 6] d i f f e r b y c l o se t oa n o r d e r o f m a g n i t u d e . T h e r e s u lt s p r e s e n t e d i n t h isp a p e r s h o w t h a t t h e r e a c t i v it i e s o f t h e a m i n o - a c i dr e s i d u e s w h i c h m a y d i r e c t l y o r i n d i r e c t l y b e i n v o l v e d inb i n d i n g a n d s u b s e q u e n t s t a b i li z a ti o n o f t h e e n z y m e - O Ac o m p l e x ( a r g i n i n e a n d c y s t e i n e [4 1 ,4 9 ,5 1 ,5 2 ] ) a r e n o tc h a n g e d u p o n r e d u c t i o n o f t h e e n z y m e . O n t h e o t h e r

h a n d , O A d i s s o c i a t e s f r o m t h e c o m p l e x a b o u t 1 0 - ti m e sf a s t e r w h e n t h e e n z y m e is r e d u c e d a t th e q u i n o l b i n d -i n g s i t e . T a k e n t o g e t h e r t h e s e r e s u l t s s u g g e s t t h a t t h e' t i g h t ' e n z y m e - O A c o m p l e x e s ( k o f ~= 0.0 15 mi n 1, /~red' ~ o f f= 0 . 1 4 m i n - 1 , F i g . 7 ) ar e a c o n s e q u e n c e o f a s e c o n d a r yi s o m e r i z at i o n o f t h e p r i m a r y ' r a p i d ' e n z y m e - O A c o m -p l e x e s. T h i s m e c h a n i s m a g r e e s w i t h th e f i n d i n g t h a ts 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 is c a p a b l e o f m a n yt u r n o v e r s o f m a l a t e o x i d a t i o n [ 6 1] , p r o v i d i n g t h a t enol-o x a l o a c e t a t e ( an i m m e d i a t e p r o d u c t o f th e r e a c t i o n[61 ,62] i s i r r eve rs ib ly u t i l i z ed .

O n l y i r o n - s u l f u r c e n t e r S - 3 w a s d e t e c t e d a s t h er e d u c e d c o m p o n e n t d u r i n g t h e s t e a d y - s t a t e u b i q u i n o l -f u m a r a t e r e d u c t a s e r e a c t i o n a n d u n d e r e q u i l i b r i u mc o n d i t i o n s i n th e p r e s e n c e o f t h e r e d u c e d q u i n o l ( F ig .1 0 ) . T h e s e r e s u l t s s u g g e s t t h a t t h e r a t e - l i m i t i n g s t e p i nt h e o v e r a l l u b i q u i n o l - f u m a r a t e r e d u c t a s e r e a c t i o n ist h e e l e c t r o n t r a n s f e r b e t w e e n c e n t e r S - 3 a n d l o w e rm i d p o i n t p o t e n t i a l r e d o x c o m p o n e n t s . T h i s is c o n s is -t e n t w i th E m v a l u e s o f + 1 1 0 m V , + 6 0 m V , a p p r o x . 0m V , - 2 6 0 m V a n d - 9 0 m V f o r t h e Q / Q H z , S -3 , S - l,S - 2 a n d F A D / F A D H 2 c o u p l es , r e s pe c t iv e l y [ 6 3 -6 5 ] . I tt h u s a p p e a r s t h a t t h e r e d u c t i o n o f S -3 c a u s e s a d e -c r e a s e i n O A a f f in i t y t o t h e e n z y m e . U s i n g d y e - m e d i a -t e d r e d o x t i t ra t i o n o f O A - d e a c t i v a t e d s o l u b le S D H ,A c k r e l l e t a l . c o n c l u d e d t h a t t h e r e d u c t i o n a n d p r o t o -n a t i o n o f t h e c o v a le n t ly b o u n d F A D m o i e t y a nd n o tt h e n o n - h e m e i r o n c l u s t e r s i s t h e p r e r e q u i s i t e f o r ad r a m a t i c d e c r e a s e o f O A a f f in i t y t o t h e s u b s t r a t e b i n d -i n g s i t e o f t h e e n z y m e [4 8 ]. A s i m i l a r i n t e r p r e t a t i o n o nt h e r e d u c t i o n o f t h e f l a v in to i ts a n i o n i c h y d r o q u i n o n ef o r m h a s b e e n s u g g e s t e d f r o m t h e r e d o x ti t ra t i o n o f t h eO A - d e a c t i v a t e d E. coli f u m a r a t e r e d u c t a s e c o m p l e x[ 4 6 ] . A t p r e s e n t , n o s i m p l e e x p l a n a t i o n f o r s u c h a p p a r -e n t l y c o n t r a d i c t o r y r e s u l t s o f A c k r e l l e t a l. a n d t h o s er e p o r t e d h e r e i s e v i d e n t . I t m a y b e r e l e v a n t t o n o t et h a t a n o b v i o u s d i f f e re n c e b e t w e e n o u r e x p e r i m e n t a la p p r o a c h a n d t h a t u s e d b y A c k r e l l e t a l . [ 4 8 ] i s t h a tq u i n o l b o u n d a t t h e s p e c i f i c s i t e w a s p r e s e n t i n o u rs y s t em , w h e r e a s a r ti f ic i al r e d o x - m e d i a t o r s w e r e u s e d i nt h e t i t r a t i o n e x p e r i m e n t s .

T h e l a s t p o i n t w e w o u l d l i k e t o d i s c u s s i s t h e s t r o n gd i f f e r e n c e i n O A - s e n s i t i v i ty o f t h e s u c c i n a t e - u b i q u i n o n ea n d u b i q u i n o l - f u m a r a t e r e d u c t a s e r e a c t i o n s . D u r i n gs t e a d y - s t a t e c a t a l y s i s , t h e u b i q u i n o l - f u m a r a t e r e d u c t a s er e a c t io n r e s p o n d s t o O A a s w o u l d b e e x p e c t e d i f t h ed i c a r b o x y l a t e - f r e e i n t e r m e d i a t e e n z y m e is p r e s e n t i n a' r e d u c e d ' f o r m . A n ' o x i d i z e d ' O A s u s c e p t i b l e f o r ma p p e a r s t o b e a n i n t e r m e d i a t e d u r i n g t h e 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 r e a c t i o n . T h e m o s t l i k el y e x p l a-n a t i o n f o r t h e d i f f e r e n t i n t e r m e d i a t e s i s a p o s s i b l ed i f f e r e n c e i n t h e k i n e t ic m e c h a n i s m s o f t h e d i r e c t a n dr e v e r s e r e a c t i o n s . I t h a s b e e n s h o w n t h a t r e d u c e ds u c c in a t e d e h y d r o g e n a s e i s o x i d iz e d b y a n u m b e r o fa r t i f i c i a l e l e c t r o n a c c e p t o r s b e f o r e f u m a r a t e d i s s o c i a t e sf r o m t h e d i c a r b o x y l a t e b i n d i n g s i t e d u r i n g s t e a d y - s t a t e


10/11

s u c c i n a t e o x i d a t i o n [ 1 4 , 4 7 ,6 6 ] . I f t h i s i s t h e c a s e f o ro p e r a t i o n o f t h e e n z y m e in t h e p r e s e n c e o f q u i n o n e a sa n o x i d a n t, n o f r e e ' r e d u c e d ' e n z y m e i s e x p e c t e d t o b ea n i n t e r m e d i a t e d u r i n g t h e s t e a d y - s t a t e r e a c ti o n . I n t h er e v e r s e r e a c t i o n , a b i - , b i - p i n g - p o n g m e c h a n i s m w i t hf r e e ' r e d u c e d ' e n z y m e a s a n i n t e r m e d i a t e s e e m s t o b ek i n e t ic a l l y p r e f e r e n t i a l a s j u d g e d b y O A - s e n s i ti v i ty .S h o u l d t h i s h y p o t h e s i s b e t r u e , i t fo l l o w s t h a t t h ep r e c i s e m e c h a n i s m s o f th e d i r e c t a n d r e v e r s e r e a c t io n sc a t a ly z e d b y t h e m i t o c h o n d r i a l s u c c i n a t e - u b i q u i n o n er e d u c t a s e a r e n o t i d e n t i c a l .Acknowledgements

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