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Resolution and reconstitution of complex II (succinate-ubiquinone reductase) by salts
ARCHIVES
OF
Resolution
BIOCHEMISTRY
and
AND
BIOPHYSICS
Reconstitution
149,
505-512 (1972)
of Complex
Reductase) K(. A. DAVIS” Department of Biochemistry,
Scripps Clinic
II (Succinate-Ubiquinone
by Salts AND
Y. HATEFI
and Research Foundation, La Jolla, California
98037
Received October l&1971; accepted January l&l972 The effects of representative chaotropic ions (NOa-, ClOa, Cl&COO-) on the resolution, and antichaotropic ions (Sod-z, HPOdm2,F-) on the reconstitution of a water-insoluble multiprotein-lipid complex (succinate-ubiquinone reductase or complex II) have been studied. Succinate dehydrogenase appears to be bound to the components of the complex mainly by hydrophobic attractions. This association is considerably stronger when the complex is suspended in D20 rather than in HsO. The resolution of complex II with respect to succinate dehydrogenase is an equilibrium process. The equilibrium can be shifted in the direction of resolution by chaotropic salts, and in the direction of reconstitution of complex II either by removal of the added chaotrope or by addition of an antichaotropic salt. The chaotrope-induced resolution of complex II has a small but significant temperature dependence. As might be expected, the equilibrium of a partially resolved complex II system can be shifted in either direction by altering the temperature of the medium. Similar to complexes I and III, the reduced form of complex II appears to be more stable to resolution than its oxidized form. Complex II is a particulat,e preparat’ion from mitochondria containing equimolar amounts of succinabe dehydrogenase and cytochrome b, 30% other proteins, and approximately 0.3 mg lipid/mg protein. The complex catalyzes electron t,ransfer from succinate to ubiquinone (Q)” or to phenazine methosulfatje at a rat’e of 40-50 pmoles succinate oxidized/min/mg protein at 38” (1, 2). The reaction to Q is inhibited by 2thenoylt rifluoroacetone (1, 3). Complex II has been resolved by the chaokopic agent h;aClOd to yield an apparently pure preparation of succinate dehydrogenase with a molecular weight of 97,000 f 5 % (4). The turnover number (10,000 moles succinat’e 1 Supported by USPHS grant AM-08126. 2 Recipient of a San Diego County Heart Association Senior Investigatorship. 3 Abbreviations: SD, succinate dehydrogenase; Q, ubiquinone; PMS, phenazine methosulfate; Cyt; cytochrome; NaTCA, sodium trichloroacetate; ETP, electron transport particles; and S, , unitary entropy.
oxidized/min/mole of enzyme at 38’) and of succinate dehydrogenase remain unchanged when the enzyme is dissociated from complex II and rendered soluble (5). The dissociated succinate dehydrogenase is essentially devoid of Q redu&ase activity. The SD-depleted complex II is particulat’c, contains approximat.ely 10 nmolcs of Cyb. b/mg protein, and is required for reconstitution of succina,be-& reductase and succinate-cytochrome c reductase (the latter in t)he presence of added complex III) activit,ies (6). The present communication describes the resolution and reconstitution of complex II according t’o Eq. (1) by clmotropic and ant,ichaotropic salts. In gcncral, these studies indicate the wide
K Z”’
chaotrope
complex
II -
SD + Cyt. b fraction
(1)
antiehaotrope
potential applicability of simple inorganic salts for the resolution and reconstitution of multiprotein and multiprotein-lipid com505
.SOG
1IAVIS
AXD
plcxes. This technique is also capable of providing kinetic and thermodynamic information regarding such rcsolut,ion-reassoci&on processes (4, 5, S, 9, 11). METHODS
ANI)
MATERIALS
Complex II and succinate dehydrogenase were prepared as before (4, 7), and stored at -70” in the presence of 20 ITIM succinate and 5 rnbr dithiothreitol. Alkali-treated complex II was prepared as described elsewhere (5). Before use in resolution-reconstitution studies, complex II was twice suspended at approximately 2.5 mg protein/ml in a solution containing 50 rnbf Tris-HCl, pH 8.0, 20 rnM succinate, 5 mM dithiothreitol and 0.2% bovine serum albumin and sedimented at 105,000 9 for 90 min. The washed pellet was suspended in 50 ml Tris-HCl, pH 8.0, and 5 mM dit,hiothreitol at a protein concentration of approximately 11 mg/ml, and stored in liquid nitrogen until used. In the experiment of Fig. 4, the buffer used was composed of 30 mM Tricine and 100 mu borate adjusted to pH 8.0 with NaOH. This buffer showed little
HATEFI temperatllre dependence in ths presence of 1 I\I ?u’aCIO1 (pH = 7.85 at, I”, = 7.91 at 29”). Unless otherwise stated, caomplex II was incubated for 2 min at; 38” in the prcscnce of 20 mtf sllccinate immediately before its use for resolution studies. This treatment resulted in 5 to 105? increase in the succinate 3 &Z and succinate -+ PMS reductase activities. These activities were assayed as before (5, 7), except. that, the concentration of suecinate in the assay mixture was 10 m&r. Ubiquinonc-10 (coenzyme Qp) was a gift of 1)r. 0. Isler, IIoffman-La I&he. Sodium trichloroacetate was prepared as described elsewhere (8). PMS was obtained from Sigma. hll other chemic:lls were reagent grade. RE,SULTS
1. IZesolutioll of Complex II chaot?“opic Ions
by
Figure 1 shows the effect’ of YaN& , NaC104 :md SaTCh on the succinate-Q reduat8aso and succinate-P&IS redu&se :w-
-60 -0
FIG.
1. Resolution of complex II with respect to succinate dehydrogenase by various chaotropes. Complex II was suspended in 50 rnM Tris-HCl, pH 8.0. After addition of 0.6 M chaotrope the concent,ration of complex II was 8 mg/ml. After addition of the salts, samples were taken at the intervals indicated and assayed for succinatc-Q and succinatc-PMS reductase activities. Solid lines, succinate-ubiquinone (Q) reductase activity; dotted line, succinate-phenazine methosulfate (PMS) reductase activity; dashed line, reconstitution of succinate-Q reductase activity upon addition of ammonium sulfate. Resolution temperature, 0”; assay temperature, 38”. The complex 11 preparations used in the experiments of this and subsequent figures had specific act.ivities between 40 and 45 pmoles Q2 reduced by succinate/min/mg protein.
SUCCISATE-UBIQUINO~E
tivities of complex II. These salts result, in t,he resolution of complex II according to Eq. (I), and consequently in a decrease of t,he succinate-Q reductase activit3y of t*he system. For comparison, the weak effect, of the perturbing agent,, urea, is also shown. It is seen that, (a) the rat.e and the extent# of resolution are bot’h functions of t,he pot’cncy of the chaot,rope used,* and (b) the cxt,ent of resolution (as shown for NaC104) increases as the chaotrope concentration is increased. That, the loss of Q reduct’ase activity is the result of the resolut’ion of complex II has been demonstrated clsewhcre by differential centrifugation and recovery of SD in the supernatant (4). In such experimcnts, there was always a correlation bctmeen the concent,ration of chaot’rope, or of complex II, added and t,he amount, of SD solubilized. Furthermore, the total Q reductase a&iv&y lost after addition of any concentration of chaotrope was equivalent t’o the total succinate dehydrogenase activity recovered after centrifugation in the solubilized SD fraction. It, might also be added that the concentration of t’he chaotropic and aut8ichaotropic salts in the assay mixture was never greater t#han 0.2 mar (see legend
to Fig.
1). At these concentrat,ions,
the added salts had no inhibitory or stimulatory effect on t,he activities of complex II or SD. In contrast
t,o its Q reduct,ase act)ivit,y,
t’hc
PMS reductase act)ivity of the system (i.e., the sum of the activities of complex IIbound and free SD) remains essentially unchanged (Fig. 1). This is to be expected because, as mentioned above, the turnover number of succinate dchydrogenase is the same in both the particle-bound and the soluble states. 2. Reconstitution of Complex II chaotropic Ions Figure
2 shows the
by Anti-
reconstitution
of
succinate-Q reductase activity upon addition of increasing amounts of ammonium
sulfate to a complex II system resolved ho the extent of 50% by the addition of 0.7 M 4 The order of potency of chaotropic ions is essentially as follows: Br&COO> Cl&COO> SCN- > ClOa- > CLHCCOO- > NOs-, Br-, F&COO- > ClH,CCOO- (8).
507
ILEl ,UCTASE
[(NH&S04]
-.
0.057M
--a---Dilution
*
40 t L’
I 0
2
4
6
8
IO
min
FIG. 2. Resolution of complex II by NaC104 , and reconstitution by increasing concentrations of ammonium sulfate as indicated. Complex II was suspended in a solution containing 50 ml1 TrisHCl, pH 8.0, 20 II~M succinate and 5 mu dithiothreitol. After the addition of NaC104 , the concentration of complex II was 8 mg/ml. Other conditions were the same as in Fig. 1.
NaC104. It is seen that the degree of rcconst’itjutjion is a function of the concent,rat#ion of added ammonium sulfate. Analysis of these data has indi&ed that the effect of ammonium sulfate concentration upon reconstitution of complex II follows a ‘Lsaturation”
curve
(50 % reconstitution
at, about.
0.14 hI ammonium sulfate). In thr experimcnt8s of Fig. 3, I’he ammonium sulfate additions were made from solutiorls of varying
concentrat,ion
so that
the
dilut,ion
of
NaC104 would be the same in all caxcs. This dilut)ion effect (from 0.7 to 0.59 M NaCl0.J is shown by the dotted line in Pig. 2, where water alone was added. The same degree of resolut)ion (40 %) was achieved when, instead of 0.7 M, 0.59 M NaC104 was added to complex II. The effect, of ammonium sulfate on t,he reconstit#ution of complex II resolved in Fig.
1. That, t,he
increase in Q reductasc activity
witJh IYaNOa is shown
is associated
lvith
rebinding
of soluble
SD t,o the Cyt.
b-
rich fraction of complex II has been ascert’ained by centrifugat)ion of the mixture, recovery of active complex II in the pellet, and t,hc loss of SD from the soluble fraction.
Figure 3A shows the effect of ammonium
508
DAVIS
AND HATEFI
sulfate and the sodium salts of phosphate (mainly HPOj- at pH 7.5), citrate, fluoride and chloride upon a 50 % resolved complex II system. In all cases the final concentration of the added salt was 0.23 M, and the dilution of NaC104 was the same as in Fig. 2. It is seen that sulfate and phosphate are equally effective, citrate and fluoride are less potent, while NaCl is without, effect. Figure 3B shows that the removal (-95%) of Clod- as its insoluble potassium salt by addition of equimolar amounts of KC1 also results in reconstitution, whereas comparable amounts of NaCl are again ineffective. As seen in Figs. 3A and 3B, the ability of various salts to reverse the resolution of complex II 0.7M NaCl04 1
is not correlated strengths.
with
their
relative
ionic
3. E$ects of Temperature and Substrate The temperature dependence of complex II resolution by NaClO* is shown in Fig. 4. It, is seen that, the effect of temperature is small (Fig. 4, left) and reversible in either direction (Fig. 4, right). This is quite unlike complex I, whose resolution by chaotropic agents is irreversible and has a very large temperature dependence (4.5-fold increase in the rate of resolution upon a temperature increase from 20 to 30” in the presence of 0.9 M NaClOJ (9). Similar to complex I (9) and complex III (lo), the reduced form of --.-.
1I& II
d w (NH&SO, Na-PO4
H&J dilution
0.23M Salt
0.59M Salt
min
FIG. 3. Resolution of complex II by NaC104 and reconstitution by (A) various antichaotropic salts, and (B) removal of C104- with K+. Conditions were the same as in Fig. 2. At the concentrations used here and in Fig. 2, the antichaotropic salts had no effect on the original activity of unresolved complex II.
SUCCIKATE-UBIQUINONE
i
REDUCTASE
NaClOa
NaCl01
FIG. 4. Effect of temperature on the resolution of complex II. Complex II was suspended in a solution containing 30 rnM Trickle, 100 mM boric acid, 20 mM succinate and 5 mM dithiothreitol. pH of t,he buffer was adjusted to 8.1 with NaOH. Complex II was resolved by addition of 1.0 M NaCIOI at the temperatures indicated. After addition of NaCIOa the concentration of complex II was 8 mg/ml. The right panel shows increased resolution and reconstitution as the temperature was changed, respectively, from 0 to 30” and from 30 to 0”. Assays were performed at 38”. TABLE
EFFECT Medium
J&O DzO Hz0 D&
N&IO*
OF D20 (Mj
I
RESOLUTION
OF COMPLEX
SUCC-+PMS activity of extract bmoles/min/~l)
0.142 0.075 0.270
0.4 0.4 0.75 0.75
LLBased on the amount extractlion and purification
ON THE
5 Hanstein, W. G., Davis, T., in preparation.
H. A., and Hatefi,
SUCC+PMS activity of SD (janloes/min/mg)
4.47 2.31 (52y0) 5.29 3.92 (627,)”
-
complex II is more stable than its oxidized form toward resolution by chaotropes. Thus, in the presence of 20 mu succinate, 0.6 u IV&104 resolves complex II to the extent, of 40 %, while in the absence of added succinate the same conditions result in 50 % resolution (see Fig. 1). This difference is also reversible inasmuch as the addition of succinate t’o the latter syst,em returns itj to 42% resolution. Although small, t,hese differences should not be considered insignificant. Other studies have shown that such stability differences are, in fact’, minimized in the presence of effective concenkations of chaotropes.”
BY NaClO~
Yield of SD (mg)
(53%)
of SD left in each complex see ref. 4.
II
II pellet
after
the first extraction.
70 65 76 76 For details
of
4. E$ect of DzO versus Hz0 Table I shows the degree of resolution of complex II and the yield of purified succinat,e dehydrogenase when in parallel experiments complex II was suspended in D20 and Hz0 and resolved by NaC104 . It is seen that the first extraction of complex II in DzO wit*h 0.4 JI NaC104 yielded only 50 % as much SD as the same experiment conducted in HzO, and that the second extraction in DzO wit’h 0.75 M NaC104 gave 62% as much SD as the parallel experiment in HtO. That. the nature of the medium had no effect upon the quality of the isolated succinate dehydrogenase is shown by the unchanged specific activity of SD in t’he parallel experi-
merits of Table I. Disc* gel elrctrophoresis also showed that the products obtained in the parallel c>xperimtlnts w(lr(A c~jually purfx.
and fluoride in reversing the chaotrop(:induced resolution of caomplex II is probabl!oxplninablo on the basis of the struct,urc,forming effect, of t,heso ions on water. Unlike DISClJ,SSION chaotropes, t)he water st’ruct,urc forming ions have very small (and ofton negative) EIP It has been shown elsewhere that, the Wpies of aqueous ions (11)) and positive effect of chaotropic ions on the destabilieation of a lipid-protein complex derived from viscosity B coefFicient,s (14, 15) ; they retard mitochondria and on increasing the wat,er wat,er self-diffusion (1 l), and have a relasolubility of nonelectrolytes can be cor- t’ively st*rong salting-out1 effcc.t, upon non(16). The cahaotropica ions arc relat,ed with t,hc abilit,y of these ions t,o dis- &%rolytes generally large monovalent ions nit’h very order water (11). In the case of haloacetates, it has been demonstrated that t,heir chao- low charge densit& whereas by caontrast t,hc tropic potency increases with their size and water structure forming ions have high charge densit#ies. polarizability and is related mainly to their It should be pointed out t#hat mechacoulombic interaction with the aqueous nist#ically, t,he type of reconstitution dophase, rather than to dispersion forces inscribed here is different from the previollB volving the haloacet’ate ions, the solvent studies involving succimtt c and the solutes (8). Thus t,he resolut8ion of reconst’itution dehydrogenase. It, was shown by lieilin and complex II by chaotropes is probably refKing (17, 18) that0 addition of a preparation erable to the disordering effect of t’hese ions of succinate dehydrogenase to alkali-inon t’he aqueous phase and the consequent particles resulted weakening of hydrophobic att’ractions be- activated Koilin-Hartrec tween the succinate dehydrogenase molecule in partial restoration of succinoxidase a(*and the remaining lipid-protein syst,em of tivity. Singer and his colleagues (19) pointed restjorat’ions should complex II. It was shown in Fig. 1 that in out that, such a&vity not be considered as true rcc:orwtit’ut,ioIl, addit,ion to the extent of resolution, t#he rate wit’h which equilibrium is at,tained (com- because alkali treatment, results in dostrucpare NaTCA with NaC104 and NaN03 in tion, but, not in removal, of the bound SD of Fig. 1) is also a fun&on of t,he potency of the particles. Consequent’ly, th(s rcconstithe chaot’rope used. Other studies (12) sug- t,ut,ed product is not identical with the ungest that, in prot,ein-protein complexes it tscated particles. Our studies showed that, might’ be the association process which is indeed, the &oichiomet,ry of this type> of is rather complicated. The more ret’arded in t’he presence of chaobropes. reconstitution reconstituted particles (made from alkaliThe results shown in Table I concerning the relat’ive effect,s of HZ0 and D,O on t*he treated ETI’ or complex II plus SD) VOW tained both actJive and inactivcx SD in chaotrope-induced resolution of complex II support t’he explanation offered above for eyuimolar amounts, and saturation of the, the mechanism of action of chaot,ropes and particles wit,h active SD required the proscnce in the mixture of 5 mole cquivalentx of t’he hydrophobic character of the SD-comadded SD (5). These complications appear plex II bond. I)20 is considered to be more t,o be associat#cd, however, wit’h the use of st’ructured than HzO. Therefore, hydrophobir associations should be st,ronger in D,O alkali-inactivat,ed particles, since in E’igs. 3 (13) and more resistant to the action of and 3 full rcconst,itution is achieved under chaot’ropes than in HzO. In general, it has conditions t,hat the stoichiomc,try of the been found in this laboratory t#hat’ in a D,O system is clearly 1: 1 with respect t’o SD and the depleted particles. As shown in Fig. :~B, medium as compared to I-It0 (a) m&iprotein-lipid complexes arc more stable, (b) removal of ClO- by I<+ results in a rapid nonelectrolytes are less soluble, and (c) reconstitut’ion of complex II. This experiment, as well as the stability of complex chaotropes are less effective in dest,abilizing t.he former and solubilizing the latter.” II it,self, indicates t’hat’ t#he equilibrium of The effect of sulfate, phosphate, citrate l
SUCCINATE;-UBI(2UINO~~:
is far in t.he dircct,ion of compkx II. The complet,eness of reconstitution in t)he experiments of Figs. 2 and 3 also suggest’s the important possibilihy that act,ivity rcsolut,ion might represent an int#ermediate stage between complex II and the physical separat,ion of SD from the particles. Thus, in the presence of chaotropes, SD would be loosely bound in complex II, thereby rcsulting in an inact’ive succinnte-Q reductase syst,cm. Bernoval of the c:haot,rope or addition of an antichaotrope would result in strongclr association of SD with the particles reconstitut,ion, whercbas %I1d in functional cont,rifugat,ion would break the inactive intBermediate and separat c soluble SD from the insoluble part,kles. In the wsc of ant’igcn-ant,ibody int,eraction, a loosely held ‘Lencountcr pair” has been proposed as an intermediate prior to complex formation (12). The possibility of a deskbilized, but not, dissociated, complex II is somewhat, unlikely however, because as ment’ioned carlicr, thn concentrat,ion of t,he added chaotropc is diminished to less than 0.2 ml1 in the assay mixt#ure. Under such condkions, :L desktbilized, but not dissociated, complex II would bc expected to recover. That such a recovery did not take place is clear from the result,s presented as well as from the fact that therfl was no act,ivat’ion of Q rcduct’ion during the 1 to 2 min time course of the individual assays. The effect, of temperature on the resolution of complex II under the conditions of Fig. 4 (data for 10, 20, 30 and 40” were used) has indicated that at lo”, AH and AS, for Eq. (I) are approximat,ely +4000 cal/mol and - 15 e.u. Because of the difficulties involved in vont,rolling the tcmperaturc during t,ransfcr of t,hc enzyme from the incubation medium to the assay mixt,ure, these vslues are very rough estimat#es. Nevertheless, they indicate the sign and t,he order of magnitude of these thermodynamic parameters. Por the reversal of Eq. (l), AX, would bc positive, and this is in agreement wit’h the compilation of Kauzmann (20) for unitary entropy changes involved in the interaction of prot,eins wit,h haptens, ant,ibodies, l&c protcin molecules, long-chain fat,ty acyl sulfak5, met,hyl orange and :Izosulfathiazolc. The
511
I:EI )CCT.N~;
magnitude of AS, is also in the range of unitary ent,rop\- changr!: in t,hc caompilation of Kauzmann. It, was shown by King (21) that, alkaline condit)ions favor the partial solubilization of succinate dehydrogenase, and this observation led to the conclusion that the linkage between SD and the respiratory chain may be due to hydrogen bonding or electrostat,ic nttrac+ions. However, our s1ml1 AH value suggest,x that the dest,abilizat,ion of SDparticle system does not involve too many ionic dissociaGons. That the attachment of SD to the respiratory chain cannot’ be mainly ionic is also support.ed by the salt, effects described above. This is because in nqucous media most’ salts tend to strengthen hydrophobic attractions, but weaken ionic bonds, and their potency is proportional t)o their charge density (20). Thus, if the SlImembrane bonds were mainly electrost,atic, ions such as sulfate, phosphate and fluoride should have dest’abilizcd these bonds. However, our data in Figs. 2 and 3 show the opposite effect,. This behavior, as mt:nt,ioncd above, is \\-hat, might b(l clxpecttad for the effect of ions \\ith high charge density on hydrophobic attractions. The authors wish to thank Dr. W. (+. Hanstein for valuable discussions, and Messrs. C. Munoz and I’. Tejada for the preparation of mitochoudria and compIex II. l~P’PEI:T’,SClM 2 1. zII‘;oLI.:R, 11. XI. (19M) Hiol. Rruct. FlLtK1. Proc. Int. Congr. Riochem. 5th 2, 253.
2.
II.Yl’EFI,
Y.
(1!%6)
COmpr.
~~Ochf??ll.
14,
1%.
3. TEETER, M. E., BIOINSTCY, M. L., AND HATEFI, Y. (1X9) Bioch.ern. Biophys. Acta 172, 331. 4. I)~vrs, K. A., .\NI) H.\I’EFI, Y. (1971) Riochemisfry 10, 2509. 5. HASSTMN, W. C+., l).\vrs, K. A., (~L~L.~J~~R, RI. A., .\ND H~TEFI, Y. (1971) Riochenktry 10, 2517. 6. I)nvrs, K. A., .\SD H.\,IxFJ, Y. (1971) Hiochem. Biophys. Res. Corrmun. 44, 1338. 7. B.~GINSKY, %\I. I,.. \NI> IFATEFI, Y. (1909) J. Biol. Chew,. 244, 5313. 8. H.\SSTE~N, W. C:., I).\vrs, Ii. A., mn H.\TI~FI, Y. (1971) Arch. Hiocheili. Bioph?/.s. 147, 534.
512
DAVIS
AS11 HATEFI
10. RIESKE, J. S., Baud, H., STONER, C. I)., AND LIPTON, S. H. (1967) J. Biol. Chem. 242, 4854. 11. HATI~FI, P., .~ND HANSTEIN, W. G. (1969) I’roc. Xal. Acad. Sci. U.S.A. 62, 1129. 12. I,SVISON, s. A., KIERSZENB.\UM, F., ASD ~~~NDL~Iu~R, w. B. (1970) BiOchemistry 9, 322. 13. KIlESHECIC, G. C., SCHNEIDER, H., AND SCHER.~G.Y, H. A. (1965) J. Phys. Chem. 69, 3132. 14. JENCICS, W. P. (1969) “Catalysis in Chemistry and Enzymology,” McGraw-Hill, New York.
15. ROBINSON, R. -$., AXD STOKES, 1~. H. (19%) “Electrolyte Solutions,” Butterworths, LondOI1.
16 17. 18. 19. 20. 21.
LOIYG, F. h., AND GDEVIT, W. F. (195%) Chem. Rev. 61, 119. KEILIS, U., .~NU KING, T. E. (19%) Nature 181, 1520. KING, T. E. (1966) Advan. Enzymol. 28,155. SINGER, T. P., (1966) Compr. Biochem. 14, 127. KXUZMANS, W. (1959) A&an. Protein Chem. 14, 1. KiNG, T. E. (1963) J. Bid. Chem. 238, 4037.
OF
Resolution
BIOCHEMISTRY
and
AND
BIOPHYSICS
Reconstitution
149,
505-512 (1972)
of Complex
Reductase) K(. A. DAVIS” Department of Biochemistry,
Scripps Clinic
II (Succinate-Ubiquinone
by Salts AND
Y. HATEFI
and Research Foundation, La Jolla, California
98037
Received October l&1971; accepted January l&l972 The effects of representative chaotropic ions (NOa-, ClOa, Cl&COO-) on the resolution, and antichaotropic ions (Sod-z, HPOdm2,F-) on the reconstitution of a water-insoluble multiprotein-lipid complex (succinate-ubiquinone reductase or complex II) have been studied. Succinate dehydrogenase appears to be bound to the components of the complex mainly by hydrophobic attractions. This association is considerably stronger when the complex is suspended in D20 rather than in HsO. The resolution of complex II with respect to succinate dehydrogenase is an equilibrium process. The equilibrium can be shifted in the direction of resolution by chaotropic salts, and in the direction of reconstitution of complex II either by removal of the added chaotrope or by addition of an antichaotropic salt. The chaotrope-induced resolution of complex II has a small but significant temperature dependence. As might be expected, the equilibrium of a partially resolved complex II system can be shifted in either direction by altering the temperature of the medium. Similar to complexes I and III, the reduced form of complex II appears to be more stable to resolution than its oxidized form. Complex II is a particulat,e preparat’ion from mitochondria containing equimolar amounts of succinabe dehydrogenase and cytochrome b, 30% other proteins, and approximately 0.3 mg lipid/mg protein. The complex catalyzes electron t,ransfer from succinate to ubiquinone (Q)” or to phenazine methosulfatje at a rat’e of 40-50 pmoles succinate oxidized/min/mg protein at 38” (1, 2). The reaction to Q is inhibited by 2thenoylt rifluoroacetone (1, 3). Complex II has been resolved by the chaokopic agent h;aClOd to yield an apparently pure preparation of succinate dehydrogenase with a molecular weight of 97,000 f 5 % (4). The turnover number (10,000 moles succinat’e 1 Supported by USPHS grant AM-08126. 2 Recipient of a San Diego County Heart Association Senior Investigatorship. 3 Abbreviations: SD, succinate dehydrogenase; Q, ubiquinone; PMS, phenazine methosulfate; Cyt; cytochrome; NaTCA, sodium trichloroacetate; ETP, electron transport particles; and S, , unitary entropy.
oxidized/min/mole of enzyme at 38’) and of succinate dehydrogenase remain unchanged when the enzyme is dissociated from complex II and rendered soluble (5). The dissociated succinate dehydrogenase is essentially devoid of Q redu&ase activity. The SD-depleted complex II is particulat’c, contains approximat.ely 10 nmolcs of Cyb. b/mg protein, and is required for reconstitution of succina,be-& reductase and succinate-cytochrome c reductase (the latter in t)he presence of added complex III) activit,ies (6). The present communication describes the resolution and reconstitution of complex II according t’o Eq. (1) by clmotropic and ant,ichaotropic salts. In gcncral, these studies indicate the wide
K Z”’
chaotrope
complex
II -
SD + Cyt. b fraction
(1)
antiehaotrope
potential applicability of simple inorganic salts for the resolution and reconstitution of multiprotein and multiprotein-lipid com505
.SOG
1IAVIS
AXD
plcxes. This technique is also capable of providing kinetic and thermodynamic information regarding such rcsolut,ion-reassoci&on processes (4, 5, S, 9, 11). METHODS
ANI)
MATERIALS
Complex II and succinate dehydrogenase were prepared as before (4, 7), and stored at -70” in the presence of 20 ITIM succinate and 5 rnbr dithiothreitol. Alkali-treated complex II was prepared as described elsewhere (5). Before use in resolution-reconstitution studies, complex II was twice suspended at approximately 2.5 mg protein/ml in a solution containing 50 rnbf Tris-HCl, pH 8.0, 20 rnM succinate, 5 mM dithiothreitol and 0.2% bovine serum albumin and sedimented at 105,000 9 for 90 min. The washed pellet was suspended in 50 ml Tris-HCl, pH 8.0, and 5 mM dit,hiothreitol at a protein concentration of approximately 11 mg/ml, and stored in liquid nitrogen until used. In the experiment of Fig. 4, the buffer used was composed of 30 mM Tricine and 100 mu borate adjusted to pH 8.0 with NaOH. This buffer showed little
HATEFI temperatllre dependence in ths presence of 1 I\I ?u’aCIO1 (pH = 7.85 at, I”, = 7.91 at 29”). Unless otherwise stated, caomplex II was incubated for 2 min at; 38” in the prcscnce of 20 mtf sllccinate immediately before its use for resolution studies. This treatment resulted in 5 to 105? increase in the succinate 3 &Z and succinate -+ PMS reductase activities. These activities were assayed as before (5, 7), except. that, the concentration of suecinate in the assay mixture was 10 m&r. Ubiquinonc-10 (coenzyme Qp) was a gift of 1)r. 0. Isler, IIoffman-La I&he. Sodium trichloroacetate was prepared as described elsewhere (8). PMS was obtained from Sigma. hll other chemic:lls were reagent grade. RE,SULTS
1. IZesolutioll of Complex II chaot?“opic Ions
by
Figure 1 shows the effect’ of YaN& , NaC104 :md SaTCh on the succinate-Q reduat8aso and succinate-P&IS redu&se :w-
-60 -0
FIG.
1. Resolution of complex II with respect to succinate dehydrogenase by various chaotropes. Complex II was suspended in 50 rnM Tris-HCl, pH 8.0. After addition of 0.6 M chaotrope the concent,ration of complex II was 8 mg/ml. After addition of the salts, samples were taken at the intervals indicated and assayed for succinatc-Q and succinatc-PMS reductase activities. Solid lines, succinate-ubiquinone (Q) reductase activity; dotted line, succinate-phenazine methosulfate (PMS) reductase activity; dashed line, reconstitution of succinate-Q reductase activity upon addition of ammonium sulfate. Resolution temperature, 0”; assay temperature, 38”. The complex 11 preparations used in the experiments of this and subsequent figures had specific act.ivities between 40 and 45 pmoles Q2 reduced by succinate/min/mg protein.
SUCCISATE-UBIQUINO~E
tivities of complex II. These salts result, in t,he resolution of complex II according to Eq. (I), and consequently in a decrease of t,he succinate-Q reductase activit3y of t*he system. For comparison, the weak effect, of the perturbing agent,, urea, is also shown. It is seen that, (a) the rat.e and the extent# of resolution are bot’h functions of t,he pot’cncy of the chaot,rope used,* and (b) the cxt,ent of resolution (as shown for NaC104) increases as the chaotrope concentration is increased. That, the loss of Q reduct’ase activity is the result of the resolut’ion of complex II has been demonstrated clsewhcre by differential centrifugation and recovery of SD in the supernatant (4). In such experimcnts, there was always a correlation bctmeen the concent,ration of chaot’rope, or of complex II, added and t,he amount, of SD solubilized. Furthermore, the total Q reductase a&iv&y lost after addition of any concentration of chaotrope was equivalent t’o the total succinate dehydrogenase activity recovered after centrifugation in the solubilized SD fraction. It, might also be added that the concentration of t’he chaotropic and aut8ichaotropic salts in the assay mixture was never greater t#han 0.2 mar (see legend
to Fig.
1). At these concentrat,ions,
the added salts had no inhibitory or stimulatory effect on t,he activities of complex II or SD. In contrast
t,o its Q reduct,ase act)ivit,y,
t’hc
PMS reductase act)ivity of the system (i.e., the sum of the activities of complex IIbound and free SD) remains essentially unchanged (Fig. 1). This is to be expected because, as mentioned above, the turnover number of succinate dchydrogenase is the same in both the particle-bound and the soluble states. 2. Reconstitution of Complex II chaotropic Ions Figure
2 shows the
by Anti-
reconstitution
of
succinate-Q reductase activity upon addition of increasing amounts of ammonium
sulfate to a complex II system resolved ho the extent of 50% by the addition of 0.7 M 4 The order of potency of chaotropic ions is essentially as follows: Br&COO> Cl&COO> SCN- > ClOa- > CLHCCOO- > NOs-, Br-, F&COO- > ClH,CCOO- (8).
507
ILEl ,UCTASE
[(NH&S04]
-.
0.057M
--a---Dilution
*
40 t L’
I 0
2
4
6
8
IO
min
FIG. 2. Resolution of complex II by NaC104 , and reconstitution by increasing concentrations of ammonium sulfate as indicated. Complex II was suspended in a solution containing 50 ml1 TrisHCl, pH 8.0, 20 II~M succinate and 5 mu dithiothreitol. After the addition of NaC104 , the concentration of complex II was 8 mg/ml. Other conditions were the same as in Fig. 1.
NaC104. It is seen that the degree of rcconst’itjutjion is a function of the concent,rat#ion of added ammonium sulfate. Analysis of these data has indi&ed that the effect of ammonium sulfate concentration upon reconstitution of complex II follows a ‘Lsaturation”
curve
(50 % reconstitution
at, about.
0.14 hI ammonium sulfate). In thr experimcnt8s of Fig. 3, I’he ammonium sulfate additions were made from solutiorls of varying
concentrat,ion
so that
the
dilut,ion
of
NaC104 would be the same in all caxcs. This dilut)ion effect (from 0.7 to 0.59 M NaCl0.J is shown by the dotted line in Pig. 2, where water alone was added. The same degree of resolut)ion (40 %) was achieved when, instead of 0.7 M, 0.59 M NaC104 was added to complex II. The effect, of ammonium sulfate on t,he reconstit#ution of complex II resolved in Fig.
1. That, t,he
increase in Q reductasc activity
witJh IYaNOa is shown
is associated
lvith
rebinding
of soluble
SD t,o the Cyt.
b-
rich fraction of complex II has been ascert’ained by centrifugat)ion of the mixture, recovery of active complex II in the pellet, and t,hc loss of SD from the soluble fraction.
Figure 3A shows the effect of ammonium
508
DAVIS
AND HATEFI
sulfate and the sodium salts of phosphate (mainly HPOj- at pH 7.5), citrate, fluoride and chloride upon a 50 % resolved complex II system. In all cases the final concentration of the added salt was 0.23 M, and the dilution of NaC104 was the same as in Fig. 2. It is seen that sulfate and phosphate are equally effective, citrate and fluoride are less potent, while NaCl is without, effect. Figure 3B shows that the removal (-95%) of Clod- as its insoluble potassium salt by addition of equimolar amounts of KC1 also results in reconstitution, whereas comparable amounts of NaCl are again ineffective. As seen in Figs. 3A and 3B, the ability of various salts to reverse the resolution of complex II 0.7M NaCl04 1
is not correlated strengths.
with
their
relative
ionic
3. E$ects of Temperature and Substrate The temperature dependence of complex II resolution by NaClO* is shown in Fig. 4. It, is seen that, the effect of temperature is small (Fig. 4, left) and reversible in either direction (Fig. 4, right). This is quite unlike complex I, whose resolution by chaotropic agents is irreversible and has a very large temperature dependence (4.5-fold increase in the rate of resolution upon a temperature increase from 20 to 30” in the presence of 0.9 M NaClOJ (9). Similar to complex I (9) and complex III (lo), the reduced form of --.-.
1I& II
d w (NH&SO, Na-PO4
H&J dilution
0.23M Salt
0.59M Salt
min
FIG. 3. Resolution of complex II by NaC104 and reconstitution by (A) various antichaotropic salts, and (B) removal of C104- with K+. Conditions were the same as in Fig. 2. At the concentrations used here and in Fig. 2, the antichaotropic salts had no effect on the original activity of unresolved complex II.
SUCCIKATE-UBIQUINONE
i
REDUCTASE
NaClOa
NaCl01
FIG. 4. Effect of temperature on the resolution of complex II. Complex II was suspended in a solution containing 30 rnM Trickle, 100 mM boric acid, 20 mM succinate and 5 mM dithiothreitol. pH of t,he buffer was adjusted to 8.1 with NaOH. Complex II was resolved by addition of 1.0 M NaCIOI at the temperatures indicated. After addition of NaCIOa the concentration of complex II was 8 mg/ml. The right panel shows increased resolution and reconstitution as the temperature was changed, respectively, from 0 to 30” and from 30 to 0”. Assays were performed at 38”. TABLE
EFFECT Medium
J&O DzO Hz0 D&
N&IO*
OF D20 (Mj
I
RESOLUTION
OF COMPLEX
SUCC-+PMS activity of extract bmoles/min/~l)
0.142 0.075 0.270
0.4 0.4 0.75 0.75
LLBased on the amount extractlion and purification
ON THE
5 Hanstein, W. G., Davis, T., in preparation.
H. A., and Hatefi,
SUCC+PMS activity of SD (janloes/min/mg)
4.47 2.31 (52y0) 5.29 3.92 (627,)”
-
complex II is more stable than its oxidized form toward resolution by chaotropes. Thus, in the presence of 20 mu succinate, 0.6 u IV&104 resolves complex II to the extent, of 40 %, while in the absence of added succinate the same conditions result in 50 % resolution (see Fig. 1). This difference is also reversible inasmuch as the addition of succinate t’o the latter syst,em returns itj to 42% resolution. Although small, t,hese differences should not be considered insignificant. Other studies have shown that such stability differences are, in fact’, minimized in the presence of effective concenkations of chaotropes.”
BY NaClO~
Yield of SD (mg)
(53%)
of SD left in each complex see ref. 4.
II
II pellet
after
the first extraction.
70 65 76 76 For details
of
4. E$ect of DzO versus Hz0 Table I shows the degree of resolution of complex II and the yield of purified succinat,e dehydrogenase when in parallel experiments complex II was suspended in D20 and Hz0 and resolved by NaC104 . It is seen that the first extraction of complex II in DzO wit*h 0.4 JI NaC104 yielded only 50 % as much SD as the same experiment conducted in HzO, and that the second extraction in DzO wit’h 0.75 M NaC104 gave 62% as much SD as the parallel experiment in HtO. That. the nature of the medium had no effect upon the quality of the isolated succinate dehydrogenase is shown by the unchanged specific activity of SD in t’he parallel experi-
merits of Table I. Disc* gel elrctrophoresis also showed that the products obtained in the parallel c>xperimtlnts w(lr(A c~jually purfx.
and fluoride in reversing the chaotrop(:induced resolution of caomplex II is probabl!oxplninablo on the basis of the struct,urc,forming effect, of t,heso ions on water. Unlike DISClJ,SSION chaotropes, t)he water st’ruct,urc forming ions have very small (and ofton negative) EIP It has been shown elsewhere that, the Wpies of aqueous ions (11)) and positive effect of chaotropic ions on the destabilieation of a lipid-protein complex derived from viscosity B coefFicient,s (14, 15) ; they retard mitochondria and on increasing the wat,er wat,er self-diffusion (1 l), and have a relasolubility of nonelectrolytes can be cor- t’ively st*rong salting-out1 effcc.t, upon non(16). The cahaotropica ions arc relat,ed with t,hc abilit,y of these ions t,o dis- &%rolytes generally large monovalent ions nit’h very order water (11). In the case of haloacetates, it has been demonstrated that t,heir chao- low charge densit& whereas by caontrast t,hc tropic potency increases with their size and water structure forming ions have high charge densit#ies. polarizability and is related mainly to their It should be pointed out t#hat mechacoulombic interaction with the aqueous nist#ically, t,he type of reconstitution dophase, rather than to dispersion forces inscribed here is different from the previollB volving the haloacet’ate ions, the solvent studies involving succimtt c and the solutes (8). Thus t,he resolut8ion of reconst’itution dehydrogenase. It, was shown by lieilin and complex II by chaotropes is probably refKing (17, 18) that0 addition of a preparation erable to the disordering effect of t’hese ions of succinate dehydrogenase to alkali-inon t’he aqueous phase and the consequent particles resulted weakening of hydrophobic att’ractions be- activated Koilin-Hartrec tween the succinate dehydrogenase molecule in partial restoration of succinoxidase a(*and the remaining lipid-protein syst,em of tivity. Singer and his colleagues (19) pointed restjorat’ions should complex II. It was shown in Fig. 1 that in out that, such a&vity not be considered as true rcc:orwtit’ut,ioIl, addit,ion to the extent of resolution, t#he rate wit’h which equilibrium is at,tained (com- because alkali treatment, results in dostrucpare NaTCA with NaC104 and NaN03 in tion, but, not in removal, of the bound SD of Fig. 1) is also a fun&on of t,he potency of the particles. Consequent’ly, th(s rcconstithe chaot’rope used. Other studies (12) sug- t,ut,ed product is not identical with the ungest that, in prot,ein-protein complexes it tscated particles. Our studies showed that, might’ be the association process which is indeed, the &oichiomet,ry of this type> of is rather complicated. The more ret’arded in t’he presence of chaobropes. reconstitution reconstituted particles (made from alkaliThe results shown in Table I concerning the relat’ive effect,s of HZ0 and D,O on t*he treated ETI’ or complex II plus SD) VOW tained both actJive and inactivcx SD in chaotrope-induced resolution of complex II support t’he explanation offered above for eyuimolar amounts, and saturation of the, the mechanism of action of chaot,ropes and particles wit,h active SD required the proscnce in the mixture of 5 mole cquivalentx of t’he hydrophobic character of the SD-comadded SD (5). These complications appear plex II bond. I)20 is considered to be more t,o be associat#cd, however, wit’h the use of st’ructured than HzO. Therefore, hydrophobir associations should be st,ronger in D,O alkali-inactivat,ed particles, since in E’igs. 3 (13) and more resistant to the action of and 3 full rcconst,itution is achieved under chaot’ropes than in HzO. In general, it has conditions t,hat the stoichiomc,try of the been found in this laboratory t#hat’ in a D,O system is clearly 1: 1 with respect t’o SD and the depleted particles. As shown in Fig. :~B, medium as compared to I-It0 (a) m&iprotein-lipid complexes arc more stable, (b) removal of ClO- by I<+ results in a rapid nonelectrolytes are less soluble, and (c) reconstitut’ion of complex II. This experiment, as well as the stability of complex chaotropes are less effective in dest,abilizing t.he former and solubilizing the latter.” II it,self, indicates t’hat’ t#he equilibrium of The effect of sulfate, phosphate, citrate l
SUCCINATE;-UBI(2UINO~~:
is far in t.he dircct,ion of compkx II. The complet,eness of reconstitution in t)he experiments of Figs. 2 and 3 also suggest’s the important possibilihy that act,ivity rcsolut,ion might represent an int#ermediate stage between complex II and the physical separat,ion of SD from the particles. Thus, in the presence of chaotropes, SD would be loosely bound in complex II, thereby rcsulting in an inact’ive succinnte-Q reductase syst,cm. Bernoval of the c:haot,rope or addition of an antichaotrope would result in strongclr association of SD with the particles reconstitut,ion, whercbas %I1d in functional cont,rifugat,ion would break the inactive intBermediate and separat c soluble SD from the insoluble part,kles. In the wsc of ant’igcn-ant,ibody int,eraction, a loosely held ‘Lencountcr pair” has been proposed as an intermediate prior to complex formation (12). The possibility of a deskbilized, but not, dissociated, complex II is somewhat, unlikely however, because as ment’ioned carlicr, thn concentrat,ion of t,he added chaotropc is diminished to less than 0.2 ml1 in the assay mixt#ure. Under such condkions, :L desktbilized, but not dissociated, complex II would bc expected to recover. That such a recovery did not take place is clear from the result,s presented as well as from the fact that therfl was no act,ivat’ion of Q rcduct’ion during the 1 to 2 min time course of the individual assays. The effect, of temperature on the resolution of complex II under the conditions of Fig. 4 (data for 10, 20, 30 and 40” were used) has indicated that at lo”, AH and AS, for Eq. (I) are approximat,ely +4000 cal/mol and - 15 e.u. Because of the difficulties involved in vont,rolling the tcmperaturc during t,ransfcr of t,hc enzyme from the incubation medium to the assay mixt,ure, these vslues are very rough estimat#es. Nevertheless, they indicate the sign and t,he order of magnitude of these thermodynamic parameters. Por the reversal of Eq. (l), AX, would bc positive, and this is in agreement wit’h the compilation of Kauzmann (20) for unitary entropy changes involved in the interaction of prot,eins wit,h haptens, ant,ibodies, l&c protcin molecules, long-chain fat,ty acyl sulfak5, met,hyl orange and :Izosulfathiazolc. The
511
I:EI )CCT.N~;
magnitude of AS, is also in the range of unitary ent,rop\- changr!: in t,hc caompilation of Kauzmann. It, was shown by King (21) that, alkaline condit)ions favor the partial solubilization of succinate dehydrogenase, and this observation led to the conclusion that the linkage between SD and the respiratory chain may be due to hydrogen bonding or electrostat,ic nttrac+ions. However, our s1ml1 AH value suggest,x that the dest,abilizat,ion of SDparticle system does not involve too many ionic dissociaGons. That the attachment of SD to the respiratory chain cannot’ be mainly ionic is also support.ed by the salt, effects described above. This is because in nqucous media most’ salts tend to strengthen hydrophobic attractions, but weaken ionic bonds, and their potency is proportional t)o their charge density (20). Thus, if the SlImembrane bonds were mainly electrost,atic, ions such as sulfate, phosphate and fluoride should have dest’abilizcd these bonds. However, our data in Figs. 2 and 3 show the opposite effect,. This behavior, as mt:nt,ioncd above, is \\-hat, might b(l clxpecttad for the effect of ions \\ith high charge density on hydrophobic attractions. The authors wish to thank Dr. W. (+. Hanstein for valuable discussions, and Messrs. C. Munoz and I’. Tejada for the preparation of mitochoudria and compIex II. l~P’PEI:T’,SClM 2 1. zII‘;oLI.:R, 11. XI. (19M) Hiol. Rruct. FlLtK1. Proc. Int. Congr. Riochem. 5th 2, 253.
2.
II.Yl’EFI,
Y.
(1!%6)
COmpr.
~~Ochf??ll.
14,
1%.
3. TEETER, M. E., BIOINSTCY, M. L., AND HATEFI, Y. (1X9) Bioch.ern. Biophys. Acta 172, 331. 4. I)~vrs, K. A., .\NI) H.\I’EFI, Y. (1971) Riochemisfry 10, 2509. 5. HASSTMN, W. C+., l).\vrs, K. A., (~L~L.~J~~R, RI. A., .\ND H~TEFI, Y. (1971) Riochenktry 10, 2517. 6. I)nvrs, K. A., .\SD H.\,IxFJ, Y. (1971) Hiochem. Biophys. Res. Corrmun. 44, 1338. 7. B.~GINSKY, %\I. I,.. \NI> IFATEFI, Y. (1909) J. Biol. Chew,. 244, 5313. 8. H.\SSTE~N, W. C:., I).\vrs, Ii. A., mn H.\TI~FI, Y. (1971) Arch. Hiocheili. Bioph?/.s. 147, 534.
512
DAVIS
AS11 HATEFI
10. RIESKE, J. S., Baud, H., STONER, C. I)., AND LIPTON, S. H. (1967) J. Biol. Chem. 242, 4854. 11. HATI~FI, P., .~ND HANSTEIN, W. G. (1969) I’roc. Xal. Acad. Sci. U.S.A. 62, 1129. 12. I,SVISON, s. A., KIERSZENB.\UM, F., ASD ~~~NDL~Iu~R, w. B. (1970) BiOchemistry 9, 322. 13. KIlESHECIC, G. C., SCHNEIDER, H., AND SCHER.~G.Y, H. A. (1965) J. Phys. Chem. 69, 3132. 14. JENCICS, W. P. (1969) “Catalysis in Chemistry and Enzymology,” McGraw-Hill, New York.
15. ROBINSON, R. -$., AXD STOKES, 1~. H. (19%) “Electrolyte Solutions,” Butterworths, LondOI1.
16 17. 18. 19. 20. 21.
LOIYG, F. h., AND GDEVIT, W. F. (195%) Chem. Rev. 61, 119. KEILIS, U., .~NU KING, T. E. (19%) Nature 181, 1520. KING, T. E. (1966) Advan. Enzymol. 28,155. SINGER, T. P., (1966) Compr. Biochem. 14, 127. KXUZMANS, W. (1959) A&an. Protein Chem. 14, 1. KiNG, T. E. (1963) J. Bid. Chem. 238, 4037.