M a m m a l i a n
By BRIAN A. C. ACKRELL, EDNA B. KEARNEV, and THOMAS P. SINGER
This article deals with four aspects of mammalian succinate dehydrogenase: (1) a critical comparison of assay methods, (2) activation-deactivation of the enzyme, (3) the active site of the enzyme, and (4) comparison of the properties of various purified preparations, including recent improvements of procedures for isolating the reconstitutively active form in high yield and with a high turnover number. Assay of Succinate Dehydrogenase
Since the catalytic turnover of succinate dehydrogenase is faster than the rate-limiting step in the respiratory chain, artificial electron acceptors are usually used for assays of the enzyme in order to ensure that full activity is being measured (a necessity in determining kinetic constants and turnover numbers, for instance). Of these, phenazine methosulfate (PMS),la with either DCIP or cytochrome c as terminal oxidant, may be used with either particulate or soluble preparations. With particle preparations such as complex II and ETP, the ubiquinone homologs Q1 and Q2, as well as the ubiquinone analog DPB, may be used in place of PMS, with the same activity, and nearly the same activity may be measured by using the free-radical form of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD', also called Wurster's blue). With soluble preparations of the enzyme, assays with PMS + DCIP, with low concentrations of ferricyanide ("low Km" ferricyanide assay) or with TMPD', all at Vmax, show the same rate of succinate oxidation. The precautions recommended for each of these procedures, their pitfalls and limitations are briefly noted at the end of each method and are more fully discussed elsewhere. 2'3 i The original studies reported here were supported by Program Project HL-16251 from the National Institutes of Health and by Grant No. PCM 76-03367 from the National Science Foundation. ia Abbreviations: DCIP, 2,6-dichlorophenolindophenol; DPB, 2,3-dimethoxy-5-methyl-6pentyl-l,4-benzoquinone; MES, 2-(N-morpholino)ethanesulfonic acid; PMS, phenazine methosulfate; TTF, thenoyltrifluoroacetone. 2 T. P. Singer, Methods Biochem. Anal. 22, 123 (1974). 3 B. A. C. Ackrell, C. J. Coles, and T. P. Singer, FEBS Lett. 75, 249 (1977).
MAMMALIAN SUCCINATE DEHYDROGENASE
Phenazine Methosulfate Assay The enzyme, reduced by its substrate, succinate, is reoxidized by PMS, which is varied in concentration to obtain V. . . . Reoxidation of the reduced PMS is accomplished by either DCIP or cytochrome c, whose reduction is monitored spectrophotometrically. The characteristics of the recording spectrophotometer required (scale expansion, recorder speed, optical density offset) have been described." Polarographic or manometric measurements of the succinate-PMS reaction are not recommended, as they are less sensitive and the rate is limited by the oxygen concentration.
Reagents 1. Potassium phosphate buffer, 200 mM, pH 7.5 (at room temperature) 2. Sodium succinate, 200 raM, adjusted to pH 7.5 3. KCN, 100 mM 4. DCIP (General Biochemicals, Inc.), 0.05% (w/v) in 100 mM potassium phosphate, pH 7.5 5. Phenazine methosulfate (Sigma), 0.33% (w/v), in glass-distilled water. Store in amber or 'qow actinic" red glassware, frozen when not in use; protect from light during use.
Procedure. If the enzyme to be assayed is not in the fully activated state, it should first be treated as described in a subsequent section. If a fully activated preparation is to be assayed, the reaction is usually started by adding the enzyme to the complete reaction mixture. Each cuvette receives 0.75 ml of phosphate buffer, 0.3 ml of succinate, water to give a final volume of 3 ml, 0.1 ml of DCIP (to give an absorbance of 1.0 to 1.25 at 600 nm in I-cm light path) and varying amounts of PMS. The latter is varied in the range of 0.3 to 0.03 ml of PMS per 3 ml of final volume. The cuvettes are brought to the temperature of assay while protected from light and placed in the spectrophotometer; 0.03 ml of KCN is added, and the enzyme immediately thereafter to start the reaction. If the reaction is started by addition of the dyes instead of the enzyme, these should be at the temperature of assay, as the volumes added are significantly large. The amount of enzyme used should cause an absorbance change corresponding to 30-50% of the chart width in 1530 sec. An absorbance range of 0-0.2 to 0-0.4 absorbance unit full scale is recommended, with a recorder chart speed of 10-12 inches/min at high PMS concentrations and 5-6 inches/min at the lower dye concentrations. The temperature of assay can be chosen for convenience but is usually 30 ° or 38 °. If it is desired to determine the extent of activation of a given
preparation, the assays should be carried out at or below 15°, since succinate does not activate the enzyme significantly during the assay in this temperature range. Activity is calculated from double reciprocal plots of absorbance change vs PMS concentration, using the millimolar extinction coefficient of 19.1 at 600 nm for DCIP.
Comments. In order to assure that no electron flux to cytochrome c and Oz via the respiratory chain occurs in the assay of membrane-bound preparations, antimycin A (1 tzg per milligram of protein) is included in the assay mixture as well as the KCN. In intact mitochondria penetration of PMS is rate-limiting. To overcome this, 1-2 /zg of crude Naja naja venom or of partially purified phospholipase A2 from this venom 4 and 750 ~ CaC12 are added to the assay mixture, and the reaction is started with dyes to allow time for phospholipase action. While the determination of activity at Vmax with respect to PMS is essential in kinetic studies, in determination of the turnover number, in studies with inhibitors, and in comparisons of soluble and particulate enzymes, because the Km for PMS is altered on extraction of the enzyme and on treatment with certain inhibitors, if only a rough estimate of the activity is desired, or if the experimental conditions do not bring about a change in Kin, the highest level of PMS recommended may be used in lieu of varying dye concentration. When membrane-bound succinate dehydrogenase is being assayed, deviations from linearity in double reciprocal plots are seen, since DCIP is reduced without the mediation of PMS. This "direct" reduction contributes significantly only at the lower concentrations of PMS. Substitution of heart muscle cytochrome c (50/xM final concentration) for the DCIP solves this problem, since cytochrome c is not reduced in the reaction without PMS, in the presence of antimycin. Although a polarographic variant of the PMS method has been used by some workers, for reasons detailed elsewhere 2 this procedure is not recommended. Ferricyanide Assay Ferricyanide has been widely used for the assay of succinate dehydrogenase. The conventional assay is spectrophotometric and uses either a fixed, high ferricyanide concentration ( - 5 mM) or a series of high ferricyanide concentrations (1.7-10 mM), with extrapolation to Vmax. Under either of these conditions the activity with ferricyanide is less 4 T. Cremona and E. B. Kearney, J. Biol. Chem. 239, 2328 (1964).
MAMMALIAN SUCCINATE DEHYDROGENASE
(-<50%) than that measured in the PMS-DCIP assay. 2'5 It has been recently reported, 5"6 however, that, when the activity is measured at a series of low ferricyanide concentrations, it very nearly equals the reactivity in the PMS-DCIP assay, at least in reconstitutively active, soluble preparations. This "low Km" reaction site for ferricyanide (Km - 200 /xM) is not evident in membrane-bound preparations, possibly because it is buried in the membrane and is thus inaccessible to ferricyanide or possibly by virtue of a configurational modification of the protein when it is bound to the membrane. The reaction site is very labile, decaying concurrently with reconstitution activity and the EPR-detectable HiPIP signal on exposure of the enzyme to air. 5,r
Reagents 1. K3Fe(CN)6, 10 mM (protect from light) 2. Tris sulfate buffer, 20 mM, pH 7.5 at the assay temperature, containing 100 ~ EDTA 3. Sodium succinate, 200 mM, pH 7.5 Procedure. Into each of a series of cuvettes are placed 2.5 ml of the buffer-EDTA solution, 0.3 ml of succinate, and H20 to give a final volume of 3.0 ml after the introduction of dye and enzyme. When temperature equilibration is complete, the cuvettes are placed in the recording spectrophotometer and ferricyanide, at the temperature of assay, is added (0.06, 0.045, 0.03, 0.02, and 0.015 ml; final concentration range 50-200 /xM). Activity is followed at 420 nm. When the blank rate has been established, enzyme is added to start the reaction. Activity is expressed a s Wmax at infinite dye concentration, using 1.0 as the millimolar extinction coefficient at 420 nm. Note that 2 mol of ferricyanide are reduced by 1 mol of succinate. Comments. It is important to adjust the amount of enzyme used and the recorder speed so that the initial reaction rate can be measured accurately. The rate of reduction of the dye decreases rapidly, in part because of the exhaustion of ferricyanide, in part because of inactivation of its reaction site (presumably the HiPIP Fe-S center) under the oxidizing conditions. Hence, scale expansion (0.1 absorbance unit full scale) and 5 A. D. Vinogradov, B. A. C. Ackrell, and T. P. Singer, Biochem. Biophys. Res. Commtln. 67, 803 (1975). 6 A. D. Vinogradov, E. V. Gavrikova, and V. G. Goloveshkina, Biochem. Biophys Res. Commun. 65, 1264 (1975). r B. A. C. Ackrell, E. B. Kearney, P. Mowery, T. P. Singer, H. Beinert, and A. D. Vinogradov, in "Iron and Copper Proteins" (K. Yasonobu, H. F. Mower, and O. Hayaishi, eds.), p. 161. Plenum, New York, 1976.
high recorder speeds (12-24 inches/min) are recommended. Activation is not required, since only preparations isolated in the presence of succinate and, hence, fully activated can be determined in this assay. TMPD" A s s a y TMPD', the free radical of N, N , N ' , N ' - t e t r a m e t h y l p-phenylenediamine, is reduced directly by succinate dehydrogenase. The reduction may be followed spectrophotometrically and provides an alternative procedure to the P M S - D C I P method for assay the activity of the enzyme. It has been reported 8 that the rate of reduction of TMPD" by succinate dehydrogenase is 2.5-fold faster than the rate measured with PMS in soluble preparations. In our hands, however, the rates measured in the P M S - D C I P and T M P D assays were the same in varying types of soluble preparations, as well as in complex II.2.9 Reagents 1. TMPD', 3.0 mM, in H20, freshly dissolved, protected from light. The radical is prepared as per Michaelis and Granick TM and is twice recrystallized from 90% ethanol. 2. Tris sulfate buffer, 20 raM, pH 7.5, containing 0.1 mM EDTA 3. Sodium succinate, 200 mM, pH 7.5 Procedure. Into each of a series of spectrophotometric cuvettes are placed 2.5 ml of Tris sulfate, 0.3 ml of succinate, and water to give 3 ml after the introduction of the e n z y m e and dye. With fully activated preparations, the reaction may be started with the enzyme; otherwise the dye (maintained at the temperature of assay) is used to initiate the reaction, following incubation of the e n z y m e with succinate, as in the P M S - D C I P assay. The dye concentration is varied between 20 and 300 tzM. In the case of membranous preparations, 1 mM K C N and 1 p~g of antimycin A per milligram of protein are included in the assay mixture. The reaction is followed at 560 nm in the range of 20-100 pA4 TMPD" and at 510 nm at 60-300 pA4 T M P D , using the molar extinction coefficients of 11,500 and 5,200 at these two wavelengths, respectively. Activity is calculated from double reciprocal plots by extrapolation to infinite dye concentration. Note that 2 tool of TMPD' are reduced per mole of succinate oxidized.
A c t i v a t i o n - D e a c t i v a t i o n of P r e p a r a t i o n s Most preparations of succinate dehydrogenase, as isolated, contain a mixture of active e n z y m e and a deactivated form of the enzyme; in some 8 A. D. Vinogradov, V. G. Goloveshkina, and E. V. Gavrikova, FEBS Lett. 73, 235 (1977). 9 B. A. C. Ackre|l, E. B. Kearney, C. J. Coles, T. P. Singer, H. Beinert, Y. Wan, and K. Folkers, Arch. Biochem. Biophys. 182, 107 (1977). 10T. P. Singer, E. B. Kearney, and W. C. Kenney, Adv. Enzymol. 37, 189 (1973).
MAMMALIAN SUCCINATE DEHYDROGENASE
cases (i.e., ETP), the enzyme may be almost entirely in the deactivated form. TM The deactivated enzyme contains a stoichiometric amount of oxaloacetate, 1'-14 bound so tightly that it is not displaced by succinate in the cold, nor removed by dialysis, gel exclusion, or ammonium sulfate precipitation. ,2 The deactivated enzyme is thus distinct from the reversibly inhibited enzyme, which arises immediately on mixing the active form of the enzyme with oxaloacetatelS.'6; it is then rapidly generated as the binding between oxaloacetate and enzyme changes to the tight, essentially irreversible, bond. This bond is thought to involve a thiohemiacetal linkage between the enol form of oxaloacetate and a sulfhydryl group at the substrate-binding site of the enzyme, '4'~7 and while oxaloacetate is so bound, the substrate cannot be bound; the enzyme is thus rendered completely inert catalytically. The extent to which this process occurs during isolation of the enzyme is dependent on the amount of oxaloacetate present in the mitochondria as a result of transaminase action or via malate dehydrogenase under state 3 conditions, or on the levels of precursors of oxaloacetate that may be converted to oxaloacetate on rupture of the mitochondria. In the presence of fumarase, invariably a contaminant in impure succinate dehydrogenase preparations, soluble or particulate, even succinate can serve as a precursor of oxaloacetate, as it is first converted to fumarate by succinate dehydrogenase, thence to malate by fumarase action, and finally to oxaloacetate. Malate has been shown to be oxidized by succinate dehydrogenase itself, TM thereby giving rise to bound oxaloacetate after one turnover of the enzyme. 12 It is extremely easy, as a consequence, to generate, even in the cold, the small amounts of oxaloacetate needed for complete deactivation from very low concentrations of succinate (10-50 /zM), as well as from malate. This is important from a practical point of view when working with succinate dehydrogenase: chance contamination
" E. B. Kearney, B. A. C. Ackrell, and M. Mayr, Biochem. Biophys. Res. Comrmm. 49, 1115 (1972). ,2 B. A. C. Ackrell, E. B. Kearney, and M. Mayr, J. Biol. Chem. 249, 2021 (1976). ':~ A. Priegnitz, O. N. Brzhevskaya, and L. Wojtczak, Biochem. Biophys. Res. Commun. 51, 1034 (1973). ,4 A. D. Vinogradov, D. B. Winter, and T. E. King, Biochem. Biophys. Res. Commun. 49, 441 (1972). 15 W. P. Zeylemaker, A. D. M. Klaasse, and E. C. Slater, Biochim. Biophys. Acta 191,229 (1969). ,6 L. Wojtczak, A. B. Wojtczak, and L. Ernster, Biochim. Biophys. Acta 191, 10 (1%9). ,7 W. C. Kenney, P. C. Mowery, R. L. Seng, and T. P. Singer, J. Biol. Chem. 251, 2369 (1976). '~ D. V. DerVartanian and C. Veeger, Biochirn. Biophys. Acta 105, 424 (1%5).
with any of these substances must be rigorously avoided, as, for example, when sampling repeatedly from a stock enzyme solution to an assay cuvette containing succinate. The addition of larger amounts of succinate (e.g., 20 mM), or of certain salts, like perchlorate, during the isolation procedure allows isolation of the enzyme in the active form, since they interfere with binding of oxaloacetate. If excess succinate must be removed from a preparation, care must be taken to avoid deactivation from traces of succinate left in the preparation, as described above. Activation of the enzyme with concurrent removal of oxaloacetate is thought to involve conformational changes in the enzyme that influence the stability of the oxaloacetate-enzyme bond. It can be effected by agents that reduce the flavin component of the enzyme,19 such as dithionite, 19"2°FMNH2, or light irradiation in the presence of EDTA, zl since the reduced enzyme binds oxaloacetate much less tightly. 19More commonly, activation is brought about by warming the enzyme in the presence of agents that aid in the displacement of oxaloacetate, 12 such as succinate, malonate, monovalent anions (e.g., Br-, CIO4-, NOa-), phosphate, and inosine di- and triphosphates.10 The inclusion of semicarbazide in activation mixtures to trap the oxaloacetate released is also useful in bringing activation to completion.lZ Succinate, although it is the natural substrate, does not act initially by reducing the enzyme, since it cannot do so until oxaloacetate is displaced, but has the advantage of preventing rebinding of oxaloacetate both competitively and by maintaining the enzyme in the reduced state. In submitochondrial particles, activation can be accomplished by incubation with NADH and cyanide or antimycin, as well as by the methods just mentioned. 22 In intact mitochondria, succinate dehydrogenase is largely activated in state 4 respiration, deactivated in state 3 and when the mitochondria are uncoupled, and fully activated when they are allowed to become anaerobic. 23 Addition of ATP can also induce activation in mitochondria, 23 but probably requires also the presence of certain endogenous substrates. Details of preferred activation techniques are given below. The converse, production of the deactivated enzyme is readily accomplished by the addition of small amounts of oxaloacetate to the enzyme (5-10 mol per mole of enzyme flavin) in the absence of competing anions, ~a B. A. C. Ackrell, E. B. K e a r n e y , and D. E. E d m o n d s o n , J. Biol. Chem. 250, 7114 (1975). 2o A. D. M. Klaasse and E. C. Slater, Z. Naturfi~rsch. B 27, 1077 (1972). 21 j. I. Salach and T. P. Singer, J. Biol. Chem. 238, 801 (1974). z2 M. G u t m a n , E. B. K e a r n e y , and T. P. Singer, Biochemistry 10, 2726 (1971). 2a M. G u t m a n , E. B. K e a r n e y , and T. P. Singer, Biochemistry 10, 4763 (1971).
M A M M A L I A N SUCC1NATE D E H Y D R O G E N A S E
at slightly alkaline pH values (pH 7.5-9.0), by adding L-(--)- or D-(+)malate and allowing the enzyme to oxidize them to oxaloacetate, or by producing it via transaminase action. The latter method is used preferentially when it is desired to label the enzyme with radioactive oxaloacetate, as this is conveniently generated from 14C-labeled aspartate. Activation by Succinate in the Assay Cuvette
The enzyme may be activated in the spectrophotometer cuvette by incubation in the complete reaction mixture, minus the dyes, at the temperature of assay. Incubation for 5-6 min at 38 °, about 10 min at 30 °, and about 20 rain at 25 ° usually suffices. KCN is then added, followed by the dyes to start the reaction. Note that excessive incubation at 38 ° can result in inactivation of purified enzyme preparations. At temperatures below 20 ° activation proceeds too slowly to be of practical use. For assays at low temperature, therefore, it is preferable to activate at higher temperature, and then bring the cuvettes to the temperature of assay. Batch Activation with Succinate or Malonate
For routine purposes, the simplest means of activating the enzyme is to incubate it with 20 mM succinate at pH 7.5, at a protein concentration of -10 mg/ml, at 25°-38 °. The time periods needed are determined by sampling the enzyme for assay at 15°, a temperature at which activation during the assay is negligible. In submitochondrial particles, where the enzyme is still linked to the respiratory chain, antimycin A (1 /zg/mg protein) or KCN (1 mM) is included to prevent substantial loss of succinate by oxidation through the chain. Stoppered containers are used to minimize loss of KCN. Alternatively, for special purposes, anaerobic conditions may be substituted to serve the same purpose, Once activated, the enzyme can be kept in the cold for several hours, provided that the level of succinate is maintained. When the presence of succinate interferes with the experimental design, malonate may replace succinate as the activating agent. It is, however, a powerful competitive inhibitor of the enzyme, and the concentration used must be chosen so as to give full activation at an acceptable rate, without causing excessive inhibition when carried over with the enzyme aliquot into the cuvette. The range is usually 1-4 mM malonate, and the pH, time, and temperature requirements are as for activation with succinate. It is not necessary, however, to add antimycin or KCN, since malonate is not oxidized. Inhibition by malonate carried over can be corrected for, if necessary, by adding the calculated amount to the assay of a sample of enzyme fully activated with succinate. Even When
the resulting concentration of malonate is very low, it is advisable to allow 30-60 sec of incubation in the cuvette in the presence of succinate to permit dissociation of the malonate-enzyme complex, prior to starting the reaction by addition of the dyes. If the malonate-activated enzyme is required for further studies, the major problem, in view of the very low dissociation constant for the malonate-enzyme complex, becomes one of removing completely the interfering malonate. It is the authors' experience that passage of a malonate-activated sample of soluble enzyme through a Sephadex G-50 column, or repeated washing of a similarly activated membrane-bound preparation, does not do this. Complete removal of the malonate is most easily accomplished if the excess malonate is first removed by ammonium sulfate precipitation of the enzyme, or centrifugation of the membranebound form of the enzyme, followed by displacement of residual malonate by successive washes with high concentrations (500 mM) of Br- or NO3(see next section). Batch Activation with Anions
The finding that succinate dehydrogenase can be activated by certain simple anions, which can then be readily removed because of their low affinity for the enzyme, has provided the best means for obtaining the fully activated, oxidized enzyme free of interfering contaminants. 12"24 Effective anions include CIO4-, NO3-, I-, Br-, CI-, formate, SO42-, and phosphate, of which Br- and NO~- are most often used, and the reaction proceeds best under mildly acid conditions. In the usual procedure the soluble enzyme is incubated at 25 °, at a protein concentration of 5-10 mg/ml, in 50 mM MES buffer, pH 6.3, with 500 mM NaBr until activation is complete, as determined by periodic assays of aliquots at 15°. This usually takes - 1 0 min. Particulate preparations, such as ETP or complex II, are activated at pH 7.0 rather than 6.3, although the reaction proceeds at somewhat lower anion concentrations at lower pH, because the particles tend to sediment at pH 6.0-6.3. For ETP the buffer used is 200 mM sucrose-50 mM HEPES, pH 7.0; for complex lI, 50 mM HEPES, pH 7.0. They are incubated in these buffers at 25 °, at protein concentrations of 10 mg/ml, with 500 mM NaBr or NaNO3 and 100 mM semicarbazide-HCl, neutralized with NaOH, until activation is complete as ascertained by measurement of activity. This takes - 3 0 min for complex II, 35-40 min for ETP.
24 E. B. Kearney, B. A. C. Ackrell, M. Mayr, and T. P. Singer, J. Biol. Chem. 249, 2016 (1974).
MAMMALIAN SUCC1NATE DEHYDROGENASE
Removal of the excess anions may be desired for certain types of work since they are themselves inhibitory at high concentration, although enough is seldom carried over into the assay mixture to cause inhibition. In order to do this without once again deactivating the enzyme by the oxaloacetate still present, the soluble enzyme is passed through two columns of Sephadex G-50, of a length at least 20 times the diameter. The first column is equilibrated with 50 mM HEPES-500 mM NaBr, pH 7.4, saturated with inert gas, such as argon or nitrogen, and serves to remove the oxaloacetate, which is prevented from rebinding to the enzyme by the Br- present. The enzyme in the excluded volume is concentrated by precipitation at 0.55 saturation of ammonium sulfate, and, after centrifugation, the precipitate is taken up in minimal volume of N2saturated 50 mM HEPES, pH 7.4, but without Br-, and passed through the second column equilibrated with the same Br -free HEPES buffer. The resulting enzyme is fully activated and free of oxaloacetate and bromide. If not used immediately, it should be once again precipitated with ammonium sulfate and stored as the precipitate in liquid N2 or under N2 at -70 °. The particulate enzymes may be freed of oxaloacetate and excess anions by centrifugal washing. The particles are washed twice in the same buffer-anion mixtures used for activation, ETP by centrifuging for 15 min and complex II for 30 min at 105,000 g, and finally in the same buffers without Br- or NO3 . The level of activation should be checked during the procedure for possible deactivation as a result of slow leakage of oxaloacetate precursors from within the vesicles. Comments
Activation of the enzyme in mitochondria occurs readily on brief incubation with succinate, or on allowing the mitochondria to become anaerobic. Nearly full activation may also be obtained under state 4 conditions. 23
Comparison of Different Preparations The differences in properties of the membrane-bound (e.g., ETP, ETPH) and soluble enzyme are numerous and distinct and are discussed in this volume. 25 Complex II, although particulate, is intermediate between inner membrane samples and the soluble enzyme in properties and complexity of composition. Four types of soluble succinate dehydrogenase preparations have been widely used: each has its advantages and disadvantages; one may
be better suited than the others for certain purposes, but none of them permits isolation of the dehydrogenase both in high purity and completely intact form. We include a brief description of a new method, which combines the advantages of two procedures and yields a nearly homogeneous preparation of the dehydrogenase, with most of the enzyme in "reconstitutively active" form. Method 1 involves extraction of dehydrogenase from acetone powders of mitochondria or of ETP by alkaline buffers, followed by fractionation. It is the earliest procedure 26"27 for obtaining the enzyme in extensively purified form. There have been several modifications, 2s the latest of which is the use of an inner membrane preparation as the starting material. 29 The advantages of the method are that it permits the isolation of relatively large quantities of the enzyme in purified form and requires no special equipment except a large blender and ultracentrifuges. The enzyme prepared by this method has the same subunit composition z9 as other purified preparations but contains only 4-6 g-atoms of Fe and labile sulfur, lacking the 4Fe-4S HiPIP center. Reconstitution activity is also lacking, although it has been reported 3° that the inclusion of succinate during extraction preserves reconstitutive capacity. It is not known whether preparations extracted from acetone powders anaerobically in the presence of succinate retain the HiPIP center as well. The enzyme isolated by method 1 is useful for sequence studies and those involving the substrate and flavin sites of the enzyme. Method 2A involves anaerobic extraction of butanol-treated KeilinHartree preparations in the presence of succinate at alkaline pH, ~' followed by a purification based on that used in method 1. In the original version of this method, 32 cyanide-treated particles were used; later cyanide was omitted, succinate was added, and anaerobic conditions were maintained during purification to aid preservation of reconstitutive activity. 3' The method yields an 8 Fe-S type enzyme, in which a large fraction of the enzyme molecules retains an EPR-detectable HiPIP center and is reconstitutively active. The enzyme prepared by this method has been useful in studies of the Fe-S composition, particularly the HiPIP center, 35 T. P. Singer and D. E. Edmondson, overview on flavoproteins, this volume . 26 T. P. Singer, E. B. Kearney, and N. Zastrow, Biochim. Biophys. Acta 17, 154 (1955). ZrT. P. Singer, E. B. Kearney, and P. Bernath, J. Biol. Chem. 223, 599 (1956). 28 p. Bernath and T. P. Singer, this series, Vol. 5, p. 597. 29 C. J. Coles, H. D. Tisdale, W. C. Kenney, and T. P. Singer, Physiol. Chem. Phys. 4, 301 (1972). 3o T. E. King, J. Biol. Chem. 238, 4037 (1963). ~' T. E. King, this series, Vol. 10, p. 322. .~2T. Y. Wang, C. L. Tsou, and Y. L. Wang, Sci. Sinica 5, 73 (1956).
MAMMALIAN SUCCINATE DEHYDROGENASE
and of the "low Km" ferricyanide activity that accompanies it, as well as requirements for reincorporating the enzyme into membranes. The disadvantages of the method are that the purity of the preparations seldom exceeds -30% (further purification results in deterioration of the reconstitution and "low Krn" ferricyanide activities); for large-scale preparations a mechanical mortar is required, which few laboratories have; and the turnover number of the enzyme in the PMS-DCIP assay is low z3 ( - 8100) as compared with other soluble preparations. 7"33This appears to reflect damage during isolation of the Keilin-Hartree particles, not during extraction and purification of the enzyme from this source, since (a) the turnover number of the enzyme in Keilin-Hartree particles is much lower 7"33 (11,000-12,500) than in other inner membrane preparations ( - 21,000) and since the same extraction procedure applied to ETP (cf. method 2B) yields succinate dehydrogenase with much higher turnover numbers 33 (12,000-14,500). The third method (2B) is a variant of 2A but uses ETP instead of a Keilin-Hartree preparation as the starting material. Its main advantage is that the resulting preparation, while comparable in purity with that obtained in method 2A, has a much higher turnover number, in accord with the fact that the starting material, unlike Keilin-Hartree preparations, appears not to contain inactivated succinate dehydrogenase?3 The procedure follows that described by King 3' with the following changes. The starting material is ETP from beef heart? 4 The vesicles are suspended in 50 mM phosphate (rather than in 50 mM borate-50 mM phosphate), pH 7.5, so as to give a final protein concentration of 10 mg/ ml after the addition of succinate. The suspension is stirred in an anaerobic vessel under a stream of water-saturated argon for -20 min at room temperature. Succinate is then added to 20-40 mM final concentration, and the suspension is stirred for 45 min longer at room temperature under argon. In the fractionation step with calcium phosphate gel the enzyme adsorbed on the gel is washed with a volume of 20 mM succinate in water equal to three-fourths of the initial aqueous layer to remove residual butanol. The enzyme is then eluted with the same volume of 100 mM potassium phosphate, pH 7.8, containing 20 mM succinate, instead of using buffer alone. The resulting enzyme shows a specific activity of 3040 ~mol of succinate per minute per milligram and a turnover number of 12,000-14,500 in the PMS-DCIP assay at 38 °. In good preparations, 8590% of the enzyme is reconstitutively active (i.e., recombines with excess alkali-treated ETP and confers succinoxidase activity). Its purity, based on histidyl flavin content, is 30-40%. 3:~ B. A. C. Ackrell, E. B. K e a r n e y , and T. P. Singer, J. Biol. Chem. 252, 1582 (1977). 34 R. L. Ringler, S. Minakami, and T. P. Singer, J. Biol. Chem. 238, 801 (1963).
Method 3 involves extraction of the enzyme from complex II with 800 mM perchlorate in the presence of succinate and dithiothreitol. 35 The method yields 75-100% homogeneous preparations of the 8 Fe-S type enzyme in a few hours' work and is thus convenient for studies where enzyme of reasonably high purity is required. There are several disadvantages to this method, however. First, complex II is laborious to make and is obtained in very low yield from mitochondria. Second, the turnover number (10,000 according to Hanstein e t a l ; z6 8300 to 11,000 in our hands 7,z~) is low, as compared with the soluble enzyme extracted with butanol from ETP (method 2B). Third, only about 15% of the enzyme population is reconstitutively active 36 and, in accord with this, the "low Km"ferricyanide activity was found to be about 20% of the PMS-DCIP activity, zr We have been able to obtain preparations where the fraction with reconstitutive capacity and "'low Kin" ferricyanide activity is as much as 50-56% of the enzyme population by maintaining rigorously anaerobic conditions during extraction and purification, a7 but the turnover number in such preparations is still low ( - 11,000). The table compares the preparations, listed with complex II included for comparison. In order to combine the advantages of the high purity afforded by material extracted from complex II (i.e., method 3) with the large fraction of reconstitutively active molecules and high turnover number obtained on butanol extraction of ETP (method 2B), we have recently combined the two procedures. 37 Complex II was extracted with butanol in the presence of succinate under argon, as in method 2B, except that 5 mM dithiothreitol was also present. The purification procedure was altered by increasing the quantity of calcium phosphate gel used from 4 to 8 mg per milliliter of extract, and precipitation with 0.3 saturated (NH4)zSO4 was omitted. Although the optimal conditions for the calcium phosphate step need to be defined in order to improve the yield from the present 30% in the overall procedure, it is clear that the procedure combines the advantages of methods 2B and 3. The purity of the preparation, based on histidyl flavin content, is comparable to that obtained in method 3, and acrylamide-SDS electrophoresis reveals <10% impurity. Reconstituted succinoxidase activity and the "low Kin" ferricyanide assay both indicate that -90% of the enzyme is reconstitutively active. The turnover number is -13,000 in the soluble form and increases to 21,900 after reincorporation into alkali-treated ETP. zr 36K. A. Davis and Y. Hatefi,Biochemistry 10, 2509 (1971). 36W. G. Hanstein, K. A. Davis, M. A. Ghalambor, and Y. Hatefi,Biochemistry 10, 2517 (1971). 37B. A. C. Ackrell, E. B. Kearney,and C. J. Coles,J. Biol. Chem. 252, 6963 (1977).
MAMMALIAN SUCCINATE DEHYDROGENASE
It seems from these results that the comparatively low reconstitution activity 3~ of the Davis-Hatefi preparation 35 (method 3) is primarily due not to preparative damage incurred in the isolation of complex II but to modification of the enzyme during extraction and purification. Further evidence for this is shown in Fig. 1, which illustrates the higher reconstitutive activity of the enzyme extracted by butanol from complex II as compared with that extracted by the perchlorate method from the same source, both under rigorously anaerobic conditions. The points marked by solid circles in curve 2 denote the fraction of the perchlorate-extracted enzyme that is active in the "low Kin" ferricyanide assay. It is seen that these points are on the same curve as the butanol-extracted samples.
Structure of the Catalytic Site
Flavin Site The flavin component of the enzyme is covalently attached to the peptide chain by way of a bond from N(3) of a histidine residue to the 8a-CH2 group of the isoalloxazine ring s y s t e m ) s The color and fluorescence of the flavin moiety permit its ready localization in the 70,000
FIG. 1. Comparison of the reconstitution activities of two types of purified succinate dehydrogenase preparations isolated from complex Ii. Curve h perchlorate-extracted preparation ( " S D B " ) modified to exclude air during extraction and purification (C)). Curve 2: butanol-extracted enzyme (~). The symbol (O) represents samples from curve 1, corrected for that fraction of the enzyme which is inactive in the "low Kin'" ferricyanide assay.
"~"W. H. Walker, T. P. Singer, S. Ghisla, P. Hemmerich, U. Hartmann, and E. Zeszotek, Eur. J. Biochern. 26, 279 (1972).
~ < Z
[.. ,-. < 0
"' :~ K,4 6
u.a. ~ . , , ~
,- ~ ' =
.o ~ u j ~
o ~ = o ~ ~
...~ . ~
>" ~ ' ~ " =
~._~ ~ = o =
~ ,'. . . : .
5= dZ -;~"== >,.=-~
= . ~ .~ ~ . ~
~ ~ ~ ~"
dalton Subunit, za'3~ as well as in flavin peptides obtained by proteolytic digestion. 39,40 A flavin pentapeptide of the structure Ser-His-Thr-Val-Ala
may be obtained by precipitating the enzyme with trichloroacetic acid, digestion with trypsin-chymotrypsin, and purification of the flavin peptide by chromatographic procedures. 4oA longer flavin peptide, containing 23 amino acids, may be obtained by tryptic digestion, followed by chromatography on Florisil, DEAE-cellulose, and phosphocellulose. 41 The amino acid sequences of both peptides have been determined. 41 The 8c~[(N)3-histidyl]-FAD moiety may be obtained by digestion of the pentapeptide with aminopeptidase? °'41 Acid digestion of the peptide results in the corresponding histidyl anhydroflavin. 4z Substrate Site
Identification of the amino acid environment at the substrate site has been difficult because no stable, covalent adducts of the enzyme with substrates or competitive inhibitors are available. Even oxaloacetate, which is extremely tightly bound to the deactivated form of the enzyme, is released on denaturation of the enzyme, 12.43,44presumably because the thiohemiacetal bond is stabilized by noncovalent interactions in the native conformation. The problem has been circumvented as follows. 44 It has been found that treatment of the enzyme with N-ethylmaleimide leads to rapid alkylation of one - - S H group on each of the 30,000- and 70,000-dalton subunits. Of these, reaction with the - - S H group on the large subunit is slower, and this is accompanied by loss of catalytic activity. Alkylation of this - - S H group and loss of activity are prevented by succinate, malonate, and oxaloacetate, whereas alkylation of the other cysteine residue is unaffected by these compounds. Thus, treatment of two succinate dehydrogenase samples with [14C]N-ethylmaleimide, one of which contains malonate as a protective agent, followed by removal of un39 E. B. Kearney, J. Biol. Chem. 235, 865 (1960). 4o j. Salach, W. H. Walker, T. P. Singer, A. Ehrenberg, P. Hemmerich, S. Ghisla, and U. Hartmann, Eur. J. Biochem. 26, 267 (1972). 41 W. C. Kenney, W. H. Walker, and T, P. Singer, J. Biol. Chem, 247, 4510 (1972). 4z D. E. Edmondson and T. P. Singer, FEBS Lett. 64, 255 (1976). 4.~D. B. Winter and T. E. King, Biochem. Biophys. Res. Commun. 56, 290 (1974). 44 W. C. Kenney and P. C. Mowery, in "Flavins and Flavoproteins" (T. P. Singer, ed.), p. 532. Elsevier, Amsterdam, 1976.
r e a c t e d i n h i b i t o r a n d p r o t e o l y t i c d i g e s t i o n , l e a d s to t h e a p p e a r a n c e o f a ~4C-labeled p e p t i d e in t h e u n p r o t e c t e d s a m p l e , w h i c h is n o t s e e n in the p r o t e c t e d o n e . I s o l a t i o n o f this p e p t i d e s h o u l d p r o v i d e t h e r e q u i s i t e m aterial f o r d e t e r m i n a t i o n o f th e a m i n o a c i d s e q u e n c e at t h e s u b s t r a t e b i n d i n g site. A s o f this w r i t i n g this s e q u e n c e has n o t b e e n a n a l y z e d b e c a u s e d i g e s t i o n w i t h v a r i o u s p r o t e o l y t i c e n z y m e s has y i e l d e d p e p t i d e s t h a t ar e f ar t o o large f o r s e q u e n c i n g by c o n v e n t i o n a l p r o c e d u r e s .
 E P R a n d Other Properties of Succinate Dehydrogenase
By TOMOKO OHNISHI and T s o o E. KING General Features of Succinate Dehydrogenase Various lipid-flee, soluble, succinate dehydrogenase preparations ( S D H ) 1 h a v e b e e n r e p o r t e d f r o m s e v e r a l l a b o r a t o r i e s ; all p r e p a r a t i o n s c a n be s u m m a r i z e d in T a b l e 12-14 w i t h c o d e s u s e d in this c h a p t e r . Abbreviations used in this article: Eh, redox potential: Era, midpoint redox potential; Fd, ferredoxin; HiPIP, high-potential iron protein; HMP, Keilin-Hartree heart muscle preparation; Q and QH2, ubiquinone and its reduced form; SDH, succinate dehydrogenase; SMP, submitochondfial particles: TTFA, trifluorotheonylacetone. 2 T. E. King, J. Biol. Chem. 238, 4037 (1963). 3 T. E. King, this series, Vol. 10, p. 322. Introduction of a water washing of the SDHabsorbed calcium phosphate gel can increase the purity of the product significantly. It is now routinely done in our laboratories. 4 T. Ohnishi, J. C. Salerno, D. B. Winter, C. A. Yu, L. Yu, and T. E. King, J. Biol. Chem. 251, 2094 (1976). '~ K. A. Davis, and Y. Hatefi, Biochemistry 10, 2509 (1971). M. L. Baginsky and Y. Hatefi, J. Biol. Chem. 244, 5313 (1969). r T. E. King, D. Winter, and W. Steel, in -Structure and Function of Oxidation-Reduction Enzymes" (A. Akeson and A. Ehrenberg. eds.), p. 519. Pergamon, Oxford, 1972. 8 D. F. Wilson and T. E. King, Biochim. Biophys. Acta 92, 173 (1964). This is a modification of the original method of Singer et al. The heart muscle preparation is used instead of mitochondria; logically HMP is a good choice as the starting material because HMP contains a powerful succinate oxidase system and, moreover, has been used successfully for other SDH preparations. Indeed, the SDH thus prepared contains flavin:iron ratio of 1:4: Singer and co-workers (see Bernath and Singer 1° and cross references cited therein) have sometimes obtained the ratio 1:2 instead of 1:4, using their starting material of acetone powder of mitochondria. H~p. Bernath and T. P. Singer, this series, Vol. 2, p. 597. H A. D. Vinogradov, E. V. Gavrikova, and V. G. Goloveshkina, Biochem. Biophys. Res. Commun. 65, 1264 (1975). v-,j. R. Kettman, Ph.D. thesis, Oregon State University, Corvallis, 1967. ~:~C. A. Yu, L. Yu, and T. E. King, Biochem. Biophys. Res. Commun. 78, 259 (1977). 14 C. A. Yu. L. Yu, and T. E. King, Biochem. Biophys. Res. Commun. 79, 939 (1977).