Studies by electron-spin resonance of the nature of succinate dehydrogenase

Studies by electron-spin resonance of the nature of succinate dehydrogenase

660 PRELIMINARY NOTES 2440) and the American Cancer Society (P77E, The Thomas S. Mlker Memorial Grant for Cancer Research). This is publication No. ...

188KB Sizes 0 Downloads 40 Views

660

PRELIMINARY NOTES

2440) and the American Cancer Society (P77E, The Thomas S. Mlker Memorial Grant for Cancer Research). This is publication No. 212 of the Graduate Department of Biochemistry, Brandeis University, Waltham, Mass., U.S.A.

Graduate Department of Biochemistry, Brandeis University, Waltham, Mass. ( U . S . A . )

Louis A. COSTELLO NATHAN O. KAPLAN

I N. O. KAPLAN, M. M. CIOTTI, M. HAMOLSKY AND R. E. BIEBER, Science, 131 (196o) 392. R. D. CAHN, N. O. KAPLAN, L. LEVlNE AND E. ZWILLING, Science, 136 (1962) 962. 8 I. H. FINE, N. O. KAPLAN AND D. KUFTINEC, Biochemistry, 2 (1963) II6. 4 N. O. KAPLAN AND R. D. CAI-IN, Proc. Natl. Acad. Sci. U.S., 48 (1962) 2123. 6 C. L. MARKERT AND ~2~.A. URSPRUNG, Develop. Biol., 5 (1962) 363. P. J. FRITZ AND K. B. JACOBSON, Science, 14o (1963) 64. * L. A. COSTELLO AND I. H. FINE, in S. P. COLOWICE AND N. O. KAPLAN, Methods in Enzymol., Vol. O; A c a d e m i c Press, N e w York, in t h e press.

Received April I7th, 1963 Biochim. Biophys. Acga, 73 (1963) 658-66o

PN 10054

Studies by electron-spin resonance of the nature of succinate dehydrogenase I t has been reported z that two types of spectral changes were found when competitive inhibitors of obvious structural resemblance to succinate were added to the activated succinate dehydrogenase (EC 1.3.99.1) of WANG et al. ~. Malonate, fumarate, methylene succinate, maleate and acetoacetate gave an increase in absorption in the region 480-54 ° m/~ with a m a x i m u m at 51o m/z, and a decrease in the region 4o0-47 o m# with a minimum at 450 m/~. Oxaloacetate, D- and L-malate, on the other hand, gave a greater increase in absorption in the region from 500 to 75 ° m/~ with a m a x i m u m near 6o0 m/~ and a greater decrease in absorption in the region 400-480 m/~ with a minimum at 46o m/~. The increase in absorption in the 5o075o-m/~ region was thought to resemble that of a r a v i n semiquinone3, t. Electron-spin resonance (ESR) studies were undertaken to obtain additional information on the nature of the reaction of these competitive inhibitors with succinate dehydrogenase. BEINERT AND SANDS 5, using the succinate dehydrogenase preparation of SINGER et al. 6, did not detect any E S R signals in the enzyme alone at --IOO ° but found two sets of signals in the presence of succinate, a free-radical signal at g = 2.0o attributed to a r a v i n semiquinone and an asymmetric signal at g = 2.Ol and 1.94 which they postulated to represent a reduced form of iron. KING a al. ~, using the preparation of WANG et al. 2, observed the same signals even in the absence of substrate, but on the addition of succinate the signals increased ,in magnitude. They confirmed that the asymmetric signals at g ~ 2.Ol and 1.94 wore due to one species, perhaps a type of paramagnetic iron. HOLLOCHER AND COMMONER S working at 32-35 ° detected no signals in the Singer preparation in the absence of succinate, and in its presence found only a free-radical signal at g ---- 2.o0 which they attributed to a r a v i n with an unpaired electron. Biochim. Biophys. Acga, 73 (I963) 66o-662

661

PRELIMINARY NOTES g =1.94

2.00

2D1

g , 1.94

2.00

SUCCINATE

D-IMALATE 4

2.01

g=1.94

2.00

2.01

L--MALATE

'H

Fig. I. E l e c t r o n - s p i n - r e s o n a n c e s p e c t r a (first derivative) as a f u n c t i o n of m a g n e t i c field s t r e n g t h (Gauss) of a c t i v a t e d s u c c i n a t e d e h y d r o g e n a s e a t l i q u i d - n i t r o g e n t e m p e r a t u r e . T h e e n z y m e (24.8 m g / m l ) in o.i M p h o s p h a t e buffer (pH 7.6) a n d i m M E D T A w a s placed in a o.5-ml a n a e robic q u a r t z E S R s a m p l e t u b e w h i c h h a d u n d e r g o n e a t least 8 cycles of h i g h v a c u , J m followed b y purified n i t r o g e n w i t h t h e l a t t e r e n d i n g t h e cycle. T h e m e a s u r e m e n t s were p e r f o r m e d in a V a r i a n V 4 5 i o IOO k c y c l e s X - b a n d s p e c t r o m e t e r . T h e s p e c t r a were recorded w i t h t h e s a m e i n s t r u m e n t a l s e t t i n g s . T h e u p p e r records, m a d e directly after m i x i n g , give t h e s p e c t r a o b t a i n e d w i t h s u c c i n a t e (24 mM), D.-malate (I lO mM) a n d L-malate (i Io a M ) s h o w i n g t h e s i m u l t a n e o u s a p p e a r a n c e of t w o sets of signals: a t g = 2.oo a n d a t g = 2.Ol a n d 1.94. T h e lower records s h o w t h e effect of s t a n d i n g for 2 h a t r o o m t e m p e r a t u r e . I n t h e case of s u c c i n a t e a n d D-malate, t h e g = 2.oo signals h a v e decreased while t h e g = 2,Ol a n d 1.94 signals h a v e c o r r e s p o n d i n g l y increased. W i t h L-malate, t h e signals h a v e a l m o s t c o m p l e t e l y disappeared.

With the same type of activated succinate dehydrogenase preparation 2 as used b y KING et al. 7, but further purified to 75% purity (based on ferricyanide assay 2) b y two additional ammonium sulphate fractionations, and measured 5 h after the butanol-extraction step, we found no signals at --195 ° in the absence of succinate or in the presence of oxaloacetate, fumarate, or malonate. However, in the presence of succinate, D- or L-malate there appeared simultaneously two sets of signals at --195°: a free-radical signal at g = 2.00 and an asymmetric signal at g ---- 2.Ol and 1.94 (see Fig. I , upper). The signals with succinate were similar to those found b y BEINERT AND SANDSs and KING et al. ~. The distance between the points of m a x i m u m slope (/IH for m a x i m u m slope) for the g ----2.00 signal was found to be about 12 Gauss. This suggests a free radical of a conjugated aromatic system. This is in agreement, for example, with the width of the signal of an aromatic mononegative ion in a rigid glass s and is considerably less than the value obtained with succinic acid crystals irradiated with X-rays 1°. Although the same types of signals appeared with D- and L-malate and with Biochim. Biophys. Acta, 73 (1963) 66o-662

662

PRELIMINARY NOTES

succinate, there were nevertheless striking differences in their behaviour during standing in the absence of oxygen (Fig. I, lower). The signals given by L-malate almost completely disappeared within IO rain at room temperature, whereas after 2 h at room temperature the signals given by succinate or D-malate showed a slight decrease at g = 2.00 and an increase in the asymmetric g ~-- 2.Ol and 1.94 signals. In this way, the signals given by succinate, or by D-malate could be distinguished from those given by L-malate. I)L-Malate gave the same ESR signals and spectral response as the D- and Lstereoisomers. In spite of the presence of o-malate in the racemate the ESR signals declined on standing in the same way as those given by l--malate. The addition of L-malate was also found to bring about a decrease in the stability of the signal given by succinate alone. The significance of these ESR results for the spectral studies may be summarized as follows: (I) There is no unpaired electron detectable in the enzyme-inhibitor complexes with fumarate, malonate or oxaloacetate. (2) Competitive inhibitors which give a smaller decrease of absorption in the ravin region do not give an ESR signal. (3) The increase in absorption in the region 500-750 m# with oxaloacetate, Dmalate or L-malate is not due to formation of a semiquinone. Finally, since succinate dehydrogenase has been shown to be L-stereospecificn and trans 12, these studies suggest that the first hydrogen atom or hydride ion is removed non-stereospecifically and may occur at one of the two hydrogens fl to the hydroxyl group. We wish to thank Professor E. C. SLATER and Professor G. J. HOYTINK for their interest and advice. We also wish to thank Mr. R. SITTERS for assistance with the E S R measurements, and Mr. H. DE HAAN for preparation of the anaerobic quartz ESR tubes. This investigation was supported in part by Research Grant RG 6569 from the U.S. Public Health Service.

Laboratory of PhysiologicalChemistry and Laboratory of Physical Chemistry, University of Amsterdam, Amsterdam (The Netherlands)

D. V.

DERVARTANIAN

C. VEEGER

J. D. W. VAN VOORST

1 D. V. DERVARTANIAN AND C. VEEGER, Biochem. J., 84 (1962) 65P. 2 T. WANG,C. T s o u AND Y. WANG,Scientia Sinica, 5 (1956) 73. s H. BEINERT, J . Am. Chem. Sot., 78 (1956) 5323. 4 V. MASSEY,Q. H. GIBSON AND C. VEEGER, Biochem. J., 77 (196o) 341. 5 H. BEINERT AND R. H. SANDS, Biochem. Biophys. Res. Commun., 3 (196°) 41. 8 T. P. SINGER, E. B. KEARNEY AND P. BERNATI-I, J. Biol. Chem., 223 (1956) 599. T. E. KING, 1R. L. HOWARD AND H. S, MASON, Biochem. Biophys. Res. Commun., 5(1961) 329. 8 T. C. HOLLOCHER AND B. COMMONER, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1355. 9 j . D. W. VAN VOORST AND G. J. HOYTINK, to be published. xo C. HELLER AND H. M. McCoNNELL, J. Chem. Phys., 32 (196o) 1535. 11 O. GAWRON, A. J. GLAID, T. P. FONDY AND M. M. BECHTOLD, Nature, 189 (1961) lOO4. 12 T. T. TCHEN AND H. VAN MILLIGAN, J. Am. Chem. Sot., 82 (196o) 4115.

Received May I3th, 1963 Biochim. Biophys. Acta, 73 (1963) 660-662