[48]

SUCCINATEDEHYDROGENASE

483

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 .

[48] 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).

484

[48]

FLAVOPROTE|NS TABLE I

SOLUBLE, LIPID-FREE SUCCINATE DEHYDROGENASE PREPARATIONS SDH solubilized

Code for preparation BS-SDH AS-SDH

PS-SDH B-SDH A-SDH CN-SDH AA-SDH

Solubilization methods Butanol extraction at pH 9.1 from HMP Alkali (pH 10.6) extraction from complex II Perchlorate (0.4-0.8 M) Butanol extraction at pH 9.1 from HMP Alkali (pH 8) extraction of complex II Cyanide extraction of HMP Alkali extraction from acetone powder of HMP

Prior succinate incubation

Reconstitutive activity h

Flavin : Fe : S

References

+

+

1:8:8

2, 3

+

+

1:8:8

2, 4

+" -

+ -

1: 8 : 8 1:8:8

5 2, 3

-

-

1:8:8

6

-

-

1:6:4

7, 8

-

-

1: 4 : 4

9

" Dithiothreitol is present in addition to succinate. b Among the activities toward artificial electron acceptors the succinate-KaFe(CN)6 reductase activity with low g m of ferricyanide seems to be in parallel with reconstitutive activity.11,12 H o w e v e r , this activity is lost upon the reconstitution of SDH (e.g., BS-SDH) with a Q-protein called QPs.13'~4

All preparations show electron-transfer activity from succinate to artificial redox dyes, such as phenazine methosulfate, ferricyanide, or Wurster Blue. These preparations, however, differ in their content of nonheme iron and acid-labile sulfide and also in their activity to reconstitute with the "soluble" cytochrome b¢1 complex 15 or "alkalinetreated" SMP (such as alkaline-treated HMP as originally used). The reconstituted preparations are inhibited by micromolar concentrations of TTFA and antimycin A4'1'~; the latter is in an amount equivalent to the cytochrome Cl content of the sample, as in case of succinate oxidase activity in the intact submitochondrial particles. Reconstitutive activity is the most sensitive physiological test to examine the intactness of the isolated enzyme. All enzymes extracted and purified without preincu15 C. A. Yu, L. Yu, and T. E. King, J. Biol. Chem. 249, 4905 (1974).

[48]

SUCCINATEDEHYDROGENASE

485

bation with succinate or another suitable reducing agent (cf. King 2) retain no reconstitutive activity, even though chemically determined nonheme iron and acid-labile sulfur content per flavin and specific activity of artificial dye reduction are the same as those in the active preparations. On the other hand, all enzyme preparations containing less than 8 Fe and 8 S per flavin exhibit no reconstitutive activity. Reconstitutively active SDH prepared in an essentially pure (>95% pure) form (PS-SDH) ~ from complex II '6 shows that SDH has a molecular weight (Mr) of approximately 97,000 and contains 1 flavin, 8 nonheme irons, and 8 acid-labile sulfides per molecule? It is composed of two nonidentical subunits (Fp and I~); Fp contains 1 flavin (FAD covalently bound to the peptide chain), 4 Fe, and 4 S, the other contains 4 Fe and 4 S of Mr 70,000 and 27,000, respectively. These two bands always show up on the SDS-polyacrylamide-fl-mercaptoethanol gel electrophoretic pattern in all the preparations listed in Table I including AA-SDH (D. B. Winter and T. E. King, unpublished data). Preparation of C N - S D H CN-SDH is prepared essentially as described by King e t a l . 7 taking advantage of an observation made by Wilson and King? Briefly, it may be made by the following procedure. The heart muscle preparation containing 10 mg of protein per milliliter in 50 mM sodium phosphate-borate buffer, pH 7.8, is mixed with 1 M potassium cyanide of pH 7.8 to give a final concentration of 30 mM cyanide. This mixture is stirred for 1 min, decanted, and immediately centrifuged at 140,000 g for 30 rain. The light-colored supernatant is discarded, and the pellet is suspended in 20 mM phosphate buffer, pH 7.8, up to the original volume. To it is added neutralized 1 M potassium cyanide to give a final concentration of 50 mM cyanide. This mixture is stirred under argon for 30 min at 37 °, decanted into cold centrifuge tubes, and centrifuged at 170,000 g for 30 min. The straw-colored supernatant rapidly freed from small, white, buoyant particles is carefully siphoned under a stream of argon into a flask immersed in ice. The pellet material is discarded. The supernatant is adjusted to pH 6.0 with 2 N acetic acid, and aged calcium phosphate gel is added to give a final concentration of 5 mg/ml. After gentle stirring for 15 min, the gel is collected by centrifugation at 3000 g for 10 min and the nearly colorless supernatant is discarded. The gel is washed once with water. The enzyme is then eluted from the gel by suspending the precipitate in one-third the original volume 16y. Hatefi, A. G. Haavik, and D. E. Griffiths, J. Biol. Chem. 237, 1676(1962).

486

FLAVOPROTEINS

[48]

of 80 mM phosphate buffer, pH 7.8, stirring for 15 min. The clear goldenyellow supernatant after centrifugation is slowly brought to 55% saturation with pulverized ammonium sulfate, stirred for 10 min and centrifuged for 20 min. The dark brown pellet is dissolved in 50 mM phosphate buffer of the desired pH (usually pH 7.8) and either used as such or further fractionated with a neutralized saturated ammonium sulfate to collect the precipitate between 35 and 55%. These preparations can be stored for months at liquid-nitogen temperatures without detectable loss of enzymic activity. EPR-Detectable Components in the Succinate Dehydrogenase Three distinct iron-sulfur centers (clusters) and flavin free radicals have been identified in the succinate dehydrogenase using EPR spectroscopy under various conditions in the authors' laboratories. Two ironsulfur centers show EPR signals in the reduced state, similar to plant or bacterial ferredoxins (Fd)ir; the third center is paramagnetic in the oxidized state, as in case of Chromatium high-potential iron-sulfur protein (HiPIP). 18 These Fd-type centers have been shown as two distinct centers located close to each other and designated centers S-1 TM and S-2, 2° respectively. 4 Centers S-1 and S-2 exhibit a large difference in their redox midpoint potential, as will be described later; center S-1 is reducible with succinate, whereas center S-2 can be reduced only by very low potential reductants, such as dithionite or as borohydride together with redox dyes. Thus individual spectra can be obtained utilizing the difference by redox midpoint potentials (as shown in Fig. 1). These two Fd-type centers show very similar EPR spectra of rhombic symmetry with essentially same g values (gz = 2.03, g = 1.93, gx = 1.91), but exhibit different spinrelaxation behavior. EPR signals of the HiPIP-type center (designated center S-3) is readily detectable 21 in particulate succinate-Q reductase.~6 This center shows rather symmetric signals around g = 2.01 with peak-to-peak width of ~r W. H. Orme-Johnson and R. H. Sands, in "Iron-Sulfur Proteins" (W. Lovenberg, ed.), Vol. 1, p. 195. Academic Press, New York, 1973. 18 R. G. Bartsch, in "Bacterial Photosynthesis" (H. Gest, A. San Pietro, and L. P. Vernon, eds.), p. 315. Antioch Press, Yellow Springs, Ohio, 1963. 19 H. Beinert and R. H. Sands, Biochem. Biophys. Res. Commun. 3, 41 (1960). 2o T. Ohnishi, D. B. Winter, J. Lira, and T. E. King, Biochem. Biophys. Res. Commun. 53, 231 (1973). 21 H. Beinert, B. A. C. Ackrell, E. B. Kearney, T. P. Singer, Eur. J. Biochem. 54, 185 (1975).

[48]

SUCCINATE

DEHYDROGENASE

487

B-SDH ~5, + Ddhionlte

[]3 +Suecinofe

203

t 195

t 191

C (Z~ - IB)

FIG. 1. Electron paramagnetic resonance (EPR) spectra of iron-sulfur centers S-I and S-2 of succinate dehydrogenase. Samples: Reconstitutively inactive B-SDH was used for the experiment. A slower relaxation time of center S-2 in B-SDH than in BS-SDH enables the concomitant detection of centers S-I and S-2 signals under non-power-saturated EPR conditions for both S-1 and S-2. (A) 0.35 ml of B-SDH at the protein concentration of 40 mg/ml in 0. I M phosphate buffer (pH 7.4) is reduced with excess dithionite. (B) 3.5 ~1 of 1.0 M K-succinate added to 0.35 ml of B-SDH. and incubated for 10 min at room temperature after transfer into an EPR tube. EPR operating conditions are: field modulation frequency, 100 KHz; modulation amplitude, 5 gauss; microwave frequency, 9.1 GHz; microwave power, I mW (Center S-I signal starts to saturate at 2 mW under the EPR conditions used); time constant, 0.3 sec; scanning rate, 200 gauss/min; sample temperature, 27°K. Subtraction of spectrum B from A was performed by the use of a Nicolette 1074 computer of average transients (Nicolette Instrument Corporation).

about 23 gauss as seen in Fig. 2 (spectrum shown by the solid line). Upon removal of the dehydrogenase from mitochondrial membrane, center S3 becomes extremely unstable toward oxidants such as oxygen and ferricyanide; thus a minimal concentration of K3Fe(CN)6 required to oxidize SDH, partially inactivates the enzyme and modifies EPR characteristics of this center [giving rise to overlapping signals having peaks about 20 gauss away from unmodified center S-3 signals (Fig. 2, spectrum shown by dotted line)] or partially converts it to EPR-undetectable forms.22 E P R Measurements. Since the EPR technique in general has been described in extenso elsewhere in this series, 23'24 only practical procedures relevant to the SDH experiments are described here, to assist the biochemist in avoiding some pitfalls inherent in its use. The free-radical signal of flavin has relatively long relaxation times, and signals can be 22 T. Ohnishi, J. Lim, D. B. Winter, and T. E. King, J. Biol. Chem. 251, 2105 (1976). 2.~G. Palmer, this series, Vol. 10, p. 594. 24 H. Beinert, this series, Vol. 54 [11].

488

FLAVOPROTE1NS

[48]

BS-SDH~~" FIG.. 2. Electron paramagnetic resonance (EPR) spectra of center S-3 in particulate succinate-ubiquinone reductase (complex II) and in soluble reconstitutively active BSSDH. Complex II (19 mg of protein per milliliter) and BS-SDH (18.3 mg of protein per milliliter) are oxidized with 150 ~M ferricyanide in the presence of 50 ~34 phenazine methosulfate, and with 100/xM ferricyanide and 10 b~M phenazine methosulfate, respectively. Enzymes are incubated for 1 min at room temperature. EPR operating conditions are the same as in Fig. 1, except for microwave power, 0.5 mW; sample temperature, 10.2°K [cited from T. Ohnishi, J. Lim, D. B. Winter, and T. E. King, J. Biol. Chem. 251, 2105 (1976)].

o b s e r v e d e v e n at r o o m t e m p e r a t u r e . 25 In contrast, spins in iron-sulfur centers, especially S-3 and S-2 in reconstitutively active S D H , h a v e much shorter relaxation times. E P R signals from these two centers are clearly discernible only at t e m p e r a t u r e s below 25°K with m i c r o w a v e p o w e r setting o f 1 inV. Therefore, E P R m e a s u r e m e n t s are m o s t conveniently p e r f o r m e d in the t e m p e r a t u r e range of 4.2 ° to 100°K by a controlled transfer o f liquid helium using a variable t e m p e r a t u r e cryostat s y s t e m ( E P R s p e c t r a shown here were obtained using Air Product Model L T D 3-110). Fine adjustment of the sample t e m p e r a t u r e is obtained by an O h m i c heating device, and the sample temperature is monitored with a c a r b o n resistor (for t e m p e r a t u r e s below 15°K) or a chromel vs A u - 0 . 0 7 % Fe t h e r m o c o u p l e (for higher t e m p e r a t u r e range), which are located about 1 c m b e l o w the b o t t o m of sample tube. One must be cautious about the fact that m e a s u r e d t e m p e r a t u r e is not exactly the same as the sample t e m p e r a t u r e , and the deviation depends both on the distance b e t w e e n the sample and the t e m p e r a t u r e sensor and on the helium flow rate. Thus correction can be obtained if n e c e s s a r y by parallel t e m p e r a t u r e measurem e n t using sensors inserted in an E P R sample tube. H o w e v e r , deviation 25T. E. King, R. L. Howard, and H. S. Mason, Biochem. Biophys. Res. Commun. 5, 329 (1961).

[48]

SUCC|NATE DEHYDROGENASE

489

is usually less than I°K under most conditions. In our experience, a carbon resistor can last more than 2 years in common daily usage with no special precautions taken. 2n Quartz EPR tubes used are selected for inner and outer diameters of approximately 3 mm and 4 mm, respectively. Tube size difference must be standardized (or calibrated) using EPR signals from a standard solution, such as Cu(II)-EDTA complex frozen in the individual tubes. For difference spectra or for a set of samples for one redox titration, matched EPR tubes should be used. Rapid freezing of samples in the EPR tube can be achieved by immersing samples in a freezing mixture composed of methylcyclohexane and isopentane at the volume ratio of 1:5. The freezing mixture should be chilled until slightly viscous, using a tube containing liquid nitrogen; under this condition the temperature is about 81°K. Frozen EPR samples thus prepared can be stored in liquid nitrogen until EPR experiments' are performed. EPR measures the absorption of microwave energy by unpaired electrons in an applied magnetic field. The resonance condition is described by h u = g f l H ; z6a in common practice the frequency is fixed and the field is swept. For iron-sulfur centers, a sweep range of 400 or 1000 gauss with the central field setting of around 3300 gauss is usually used. Since the position of the resonance signal depends both on u and H, it is better to specify the signal position in terms of the dimensionless constant g, which is a property of the absorbing species. The first derivative of the absorption intensity in the vertical axis is expressed in arbitrary units in contrast to spectrophotometry. This is necessary because the amount of energy absorbed by the sample depends on the quality factor (Q) of the microwave cavity, and additional factors including shape of the dewar, material and thickness of the EPR tubes, etc. The loaded Q factor can vary from instrument to instrument and even from day to day on the same machine. The optimal conditions for EPR measurement depend on both the sample and the kind of information desired. Maximization of the signal to noise ratio requires high microwave power level, since the signal is proportional to x / P below saturation. High power levels can result in saturation, causing distortion of the lineshape and loss of the signal. A large modulation amplitude is used to increase signal intensity; if the modulation level is not small compared with the linewidth, distortion of the spectra is observed. The temperature affects the spectra of iron-sulfur centers in three major ways. Slowing of relaxation processes causes the signal to sharpen to a temperature-independent linewidth, increasing the signal amplitude ~6 F. J. Kopp and T. A s h w o r t h Rev. Sci. Instrum. 43, 327 (1972). - ~ See refs. 23 and 24 for the significance of the s y m b o l s used in E P R spectrometry.

490

FLAVOPROTEINS

[48]

by decreasing the linewidth. Curie law behavior (increase in the population difference within the doublet) causes the signal to increase as a function of 1/T. At low temperatures, however, the signal may saturate at low power levels, decreasing the maximum intensity obtainable. Therefore it is necessary to examine power dependence of the signal at different temperatures. Typical examples of the EPR conditions for obtaining the individual spectra of SDH iron-sulfur centers are shown in the legends of Fig. 1 and 2. Quantitation of the number of spins in a sample is done by comparing the double integrated intensity of the sample to that of a standard, such as Cu(II) EDTA, of known concentration. Best results are obtained by using sample and standard which are in carefully matched tubes. It is preferable to use identical EPR conditions for both sample and standard: Curie law corrections demand much more accurate temperature measurements. In addition, both sample and standard signals must be nonpower saturated and a correction must be made for transition probability; 27for Cu(II) EDTA and a typical iron-sulfur center, this increases the estimated concentration by about 10%. For experiments in which only the relative concentration of a paramagnetic species is important (such as redox potentiometry) it is often advantageous to maximize the signal-to-noise ratio by using slightly saturating levels of microwave power. This practice has the additional good effect of decreasing the error caused by small changes in the temperature and power between measurements. As long as the line shape is independent of spin concentration, the relative concentration is proportional to the signal amplitude and may be estimated from the relative height or depth of any peak or trough from the base line. T h e r m o d y n a m i c Parameters of the Electron-Transfer Components in Succinate D e h y d r o g e n a s e Midpoint potentials of three iron-sulfur centers in SDH have been determined potentiometrically. All centers show titration curves for a single electron transfer (n = 1). Nernst plots of redox titration of these centers in particle-bound SDH, namely in succinate-ubiquinone reductase and succinate-cytochrome c reductase, are presented in Fig. 3. Centers S-1 and S-3 show Era7.4 value of approximately 0 and 65 mV, respectively. These values are reasonable for electron-transfer components functioning between succinate/fumarate and Q/QH2 redox couples. 27R. Aasa and T. VS.nngS.rd,J. Magn. Reson. 19, 01 (1975).

[48]

SUCCINATE DEHYDROOENASE

491

200Em't.4=65mV

~00-

!:0 mV -I00-

(Fe-S)s_j

o

-200 ( -500-

F

e

-

~

- / ~ " E~=L260 mV

-400-

-'l

6

.'1

log OX/red

FIG. 3. Nernst plots of redox titration of centers S-I, S-2, and S-3 in succinate--cytochrome e reductase (&, O, I ) and in complex II (©, []). Redox mediating dyes present are phenazine methosulfate, phenazine ethosulfate, 2-hydroxynaphthoquinone, phenosafranine, benzylviologen and methylviologen at concentrations in the range of 10 to 100 p,M. From T. Ohnishi, J. C. Salerno, D. B. Winter, C. A. Yu, L. Yu, and T. E. King, J. Biol. Chem. 251, 2094 (1976) and T. Ohnishi, J. Lim, D. B. Winter, and T. E. King, J. Biol. Chem. 251, 2105 (1976).

In contrast, center S-2 exhibits a rather low midpoint potential (approximately - 2 6 0 mV), and its physiological function is not yet established. U p o n solubilization of S D H , the Em values of centers S-1 and S-3 do not change significantly f r o m those obtained in the m e m b r a n e - b o u n d state, whereas the midpoint potential of center S-2 b e c o m e s more negative by 140 m V in all soluble d e h y d r o g e n a s e preparations, irrespective of reconstitutive activity. When B S - S D H is r e c o m b i n e d with the c y t o c h r o m e bcl c o m p l e x to reconstitute T T F A and antimycin A-sensitive s u c c i n a t e - c y t o c h r o m e c reductase, the midpoint potential of center S-2 reverts to the value obtained with the d e h y d r o g e n a s e prior to the removal f r o m the m e m b r a n e . Center S-2 of S D H , which is only a b s o r b e d to the c y t o c h r o m e bc] complex and can be readily w a s h e d off, shows an Em value o f - 4 0 0 mV. T h e s e observations indicate that during the reconstitution of s u c c i n a t e c y t o c h r o m e c reductase, S D H not only binds to the c y t o c h r o m e bcl

492

FLAVOPROTEI NS

[48]

complex, but also restores its original molecular environment around iron-sulfur center S-2. 4 Potentiometric Redox Titration. General principles of redox titration and potentiometry applied to biological systems have been amply described 28a9 and readers are referred to the chapter by Dutton 3° for the titration. In this section, description is limited to potentiometric titration combined with EPR measurements. Redox titrations are performed at room temperature in an anaerobic vessel 28"3°(as illustrated by Dutton 3°) in the presence of redox mediating dyes, which cover a wide range of redox potential (Eh) and equilibrate between respiratory chain components and electrodes. The redox potent5, BS-SOH 2 O2

x

B-SDH

2100

E] CN-SDH x2

{I]

AA-SDH

Flo. 4. Electron paramagnetic resonance (EPR) spectra of iron-sulfur center S-3 of various succinate dehydrogenases. Enzymes are oxidized with 100 ~ / K3Fe(CN)e in the presence of 43 ~ phenazine methosulfate. The final concentration of all enzyme solutions is adjusted to approximately 60 ~M. EPR operating conditions are microwave frequency, 9.14 GHz; modulation amplitude, 5 gauss; time constant, 0.3 sec; scanning rate, 200 gauss/ min; microwave power, 5 roW; sample temperature, 9.2°K. From T. Ohnishi, D. B. Winter, J. Lira, and T. E. King, Biochem. Biophys. Res. Commun. 61, 1017 (1974).

~a p. L. Dutton, Biochim. Biophys. Acta 226, 63 (1971). 29 D. F. Wilson, M. Erecihska, P. L. Dutton, and T. Tsudsuki, Biochem. Biophys. Res. Commun. 41, 1273 (1970). 30 p. L. Dutton, this series, Vol. 54 [23].

[48]

SUCCINATE DEHYDROGENASE

493

tial of the suspension is adjusted by injecting a small quantity of 0.1 M K3Fe(CN)6 or freshly prepared dilute solution of dithionite from the side arm, using syringes. EPR tubes are flushed with argon through a sampling tube connected with titration vessel. When equilibration of the system at the desired Eh is attained, an aliquot of the suspension is transferred anaerobically to the bottom of the EPR tube (by inserting the other end of the sampling tube into the suspension), which is driven by the pressure of argon gas in the vessel. Depending on the viscosity of the suspension and size of the transferring tube, pressure of the gas phase in the vessel should be properly adjusted in order to fill the sample tube in a controlled manner. EPR samples are rapidly frozen in the freezing mixture as described above, and concomitantly the Eh value of the suspension is recorded. When EPR measurements are conducted under identical conditions for a set of samples such as those of a given redox titration, the peak-topeak amplitude at g -- 1.93 or the height of the g -- 2.01 signal from the high-field base line can be used as a parameter proportional to the concentration of the iron-sulfur center in the reduced (center S-1 or S-2) or oxidized (center S-3) state, as discussed in the preceding section.

Complex IT

FIc. 5. Electron paramagnetic resonance (EPR) spectra of dithionite-reduced ferredoxintype iron-sulfur centers in reconstitutively active (BS-SDH) and inactive (B-SDH and AASDH) succinate dehydrogenases. - - . , EPR spectra shown as obtained at 5°K; . . . . , spectra obtained at 10.0°K.

494

FLAVOPROTEINS

Comparison Active

of EPR and

Characteristics

Inactive

SDH

between

[48] Reconstitutively

Preparations

As described in the preceding sections (also cf. Table I), there is a clear-cut difference between reconstitutively active and inactive SDH preparations in enzymic activity. However, in inactive preparations, such as B-SDH or A-SDH, no difference has been revealed from active preparations based on the chemical analysis of nonheme iron or acid-labile sulfide content or on spectrophotometric properties. Subtle modifications of molecular conformation around iron-sulfur centersS-2 and S-3 in the T A B L E II SUMMARY OF ELECTRON PARAMAGNETIC RESONANCE CHARACTERISTICS OF IRON-SULFUR CENTERS OF RECONSTITUTIVELY ACTIVE AND INACTIVE SUCCINATE DEHYDROGENASE

Center

Reconstitutively active dehydrogenase

Reconstitutively inactive dehydrogenase

S-I

1. Paramagnetic in reduced (succinate or dithionite) state 2. R h o m b i c s y m m e t r y , gz = 2.03, g~. = 1.93, gx = 1.91 3. Detectable at relatively high temperature, even 100°K 4. Readily saturated, especially 30°K) in reconstitutively inactive preparations, but not in active ones. (b) In all SDH preparations, centers S-1 and S-2 are closely located (< 10 ]k) and show spin-spin interactions, namely, release of power saturation of center S-I spins (longer T, component) by cross relaxation with S-2 spins (shorter T1 component). Conversely, center S2 spectra are affected by S-I spins in a low temperature range (4.2°-6°K), seen most clearly as broadening (active preparation) or splitting (inactive preparation) of the principal resonance signal, depending on the spin relaxation times of center S-24 (see Fig. 5). EPR characteristics of iron-sulfur centers in reconstitutively active and inactive succinate dehydrogenase preparations are summarized in Table II.

[49] P r e p a r a t i o n

of Monoamine Oxidase from Beef Liver Mitochondria B y JAMES I. SALACH, JR.

Two preparative procedures for monoamine oxidase have been described that yield an enzyme of high specific activity and purity. One utilizes beef kidney, 1 the other beef liver. 2 These enzyme preparations, of high specific activity, 3 are obtained in yields of 10% and 5-6%, rei H. Y. K. Chuang, D. R. Patek, and L. Hellerman, J. Biol. Chem. 249, 2381 (1974). 2 K. T. Yasunobu, I. Igaue, and B. Gomes, Adv. Pharmacol. 6, 43 (1%8); B. Gomes, I. lgaue, H. G. Kloepfer, and K. T. Yasunobu, Arch. Biochem. Biophys. 132, 16 (1%9). 3 Direct comparison of specific activities in these two preparations with the present preparation cannot be made. While the units of activity employed by these investigators may

EPR and other properties of succinate dehydrogenase.

[48] SUCCINATEDEHYDROGENASE 483 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...
622KB Sizes 0 Downloads 0 Views