Preliminary Note Px 61027 Reconstitution of respiratory chain enzyme systems. X V . Reconstitution of succinate oxidase using soluble succinate dehydrogenase and a "cyanide particle" Reconstitution of succinate oxidase using soluble succinate dehydrogenase (EC 126.96.36.199) and an alkali-treated heart muscle particle has been demonstratedZ, ~. In the alkali inactivation of succinate oxidase, the dehydrogenase is first dissociated from the particle and then inactivated by prolonged incubation in the presence of air 1,3. This inactivation resembles the inactivation by cyanide with respect to the decrease of particulate activities in the oxidation of succinate by molecular oxygen, cytochrome c, and methylene blue. Kinetic analysis of these two types of inactivation (alkali, refs. I, 2, 4, 5; cyanide, refs. 4, 6, 7, 8) coupled with results from inhibition studies with metal-chelating agents (cf. for example, ref. 9) suggests that the succinate-flavoprotein m a y be linked with the rest of the respiratory chain through one of the coordination bonds of non-heme iron. I f this be so, the cyanide-treated particle should be active (just like the alkali-treated preparation) in the reaction with succinate dehydrogenase for the reconstitution of succinate oxidase. The present paper reports the experimental evidence. The Keilin-Hartree preparation was prepared from beef heart 1°, and succinate dehydrogenase was prepared up to the gel eluate stage by Method I I I a (see ref. 2). The oxygen uptake was measured at room temperature, 23 °, polarographically in a G.M.E. Oxygraph. The heart muscle preparation was centrifuged for approximately 30 min at 50 ooo rev./min and the particles were suspended in 50 mM phosphate buffer (pH 7-4) to a final protein concentration of 20 mg/ml. An aliquot of the preparation was transferred to a Thunberg tube, and in the side stopper was placed an amount of lO% acetic acid just sufficient to neutralize the sodium cyanide. To the stopper was then carefully added sodium cyanide as a weighed solid to give a desired concentration. Immediately after the addition, the tube was sealed and the contents in these compartments were rapidly and thoroughly mixed. I t was found that the addition of cyanide by this scheme did not change the p H of the mixture. The mixture was then incubated for a desired length of time. At the end of the incubation, the particles were collected by centrifugation, washed with 5 ° mM phosphate buffer (pH 7-4), and then resuspended in buffer. The suspension was finally dialyzed against buffer. A control was performed in exactly the same manner with the exception that cyanide and acetic acid were omitted. The reconstitution was carried out at 2 3 ° b y mixing the particles and soluble succinate dehydrogenase in desired proportions in a medium containing (final concentrations) 50 mM phosphate buffer (pH 7.4), and approx. 20 mM succinate. The mixture was allowed to stand at room temperature for about I h, and then the activity was measured. A protocol is depicted in Fig. i. The cyanide particles could be Biochim. Biophys. Acta, 92 (1964) 173-175
PRELIMINARY NOTES I
C 0 u
Fig. I. T i t r a t i o n o f c y a n i d e particles b y soluble s u c c i n a t e d e h y d r o g e n a s e in t h e r e c o n s t i t u t i o n of s u c c i n a t e oxidase. T h e c y a n i d e particles were o b t a i n e d as detailed in S y s t e m 2, T a b l e I. T h e u n i t of abscissa is i n / ~ g of s u c c i n a t e d e h y d r o g e n a s e p r o t e i n a d d e d p e r m g of t h e c y a n i d e particle in t e r m s of protein. T h e p r o t e i n c o n t e n t s of s u c c i n a t e d e h y d r o g e n a s e a n d t h e c y a n i d e particle u s e d were o.97 a n d 17. 5 m g / m l respectively.
saturated b y soluble succinate dehydrogenase as shown; when saturation was reached, further increase of the dehydrogenase did not increase the reconstitutive activity. The oxidation of succinate b y the reconstituted particles was completely inhibited b y I mM cyanide or about 0.5 Fg antimycin A in the assay system. A number of experiments was performed by varying the cyanide concentration, the time for incubation, and the temperature. Results from some typical experiments are summarized in Table I. The reconstitutive efficiency was found to be between 65 and 85 % of the control based on the protein content of the particles, and between 50 and 80% of the control based on the cytochrome b content. The discrepancy between these two sets of values was due to the extraction b y cyanide of protein, but not cytochrome b, from the heart-muscle preparation. A part of the the cytochrome c was also apparently made soluble b y cyanide. Thus, a comparison of the reconstituted succinate oxidase with the control would be more meaningful on the basis of cytochrome b. However, the low recovery (50-80 %) must be considered provisional. By improvement of technique, higher results might be expected. Indeed, in one experiment, a lOO% reconstitution was observed. Nonetheless, an irreversible side-reaction between cyanide and the respiratory chain other than the T s o o ~ effect cannot be conclusively ruled out. That the cyanide particle possesses utilitarian applications in the study of respiratory enzymes, such as the study of inter- and intra-interactions (communications) of NADH-oxidase and succinate-oxidase chains, cannot be overemphasized. The results described do not prove the proposal that non-heme iron m a y serve as Biochim. Biophys. Acta, 92 (1964) 173-175
TABLE I O F RECONSTITUTED SUCCINATE OXIDASE A N D T H E H E A R T - M U S C L E PREPARATION W I T H RESPECT TO THEIR C Y T O C H R O M E C O N T E N T S A N D SUCCINATE OXIDASE ACTIVITY
T h e cytochrome content is expressed in m/~mole per m g of protein,, and the activity is expressed in m M of succinate oxidized per m i n at i m g of protein per ml. Cytochrome b and cytochromes c + cI were determined with dithionite-reduced samples in an A m i n c o - C h a n c e double-beam spectrophotometer at 56o-575 m/, with absorbance index of 22, and at at 553-54 o m/~ with absorbance index of 19.1, respectively. In the reconstituted system, the succinate oxidase activity was for the particles saturated b y succinate dehydrogenase, see Fig. i. Control
IO m M c y a n i d e , 14 h a t 23 ° , p a r t i c l e s w a s h e d twice, d i a l y z e d 18 h
15 m M c y a n i d e , 22 h a t 23 ° , p a r t i c l e s w a s h e d twice, d i a l y z e d 16 h
2O mM c y a n i d e , 5 h a t 29 °, p a r t i c l e s w a s h e d twice, d i a l y z e d 16 h
Cytockrome Cytochromes Succinate oxidase b c + cx activity
CytochromeCytochromes b c + cx
Succinate oxidase activity
a structural link between the succinate flavoprotein and the rest of the respiratory chain in addition to being probably an electron link or even an active redox component. Nevertheless, that cyanide acts like O H - in the disruption of the bond is in accordance with the hypothesis which m a y be useful in designing experiments for studies of the nature of this linkage. Finally it must be pointed out that we do not propose that a non-berne iron bond is the sole linkage between these two segments; other linkages probably exist. This work was supported b y grants from the National Science Foundation, the U.S. Public Health Service, the American Heart Association, the Office of Naval Research, and the Life Insurance Medical Research Fund. One of the authors (D.F.W.) was a predoctoral fellow of the U.S. Public Health Service, 1961-63.
Oregon State University, Corvallis, Oregon (U.S.A.) i D. 2 T. 8 T. 4 D. s D. s C. D. s A. 9 p. 10 T.
DAVID F. WILSON* TSOO E. KING
KEILIN AND T. E. KING, Nature, 181 (1958) 152o. E. KING, J. Biol. Chem., 238 (1963) 4037 . E. KING, Nature, 198 (1963) 366. KEILIN AND T. E. KING, Proc. Roy. So¢. London, Ser. B, 152 (196o) 163. F. WILSON, d i s s e r t a t i o n , Oregon S t a t e U n i v e r s i t y , 1964. L. T s o u , Biochem. J., 49 (1951) 512. KEILIN AND T. E. KING, Biochem. J., 69 (1958) 32 p. GIUDITTA AND T. P. SINGER, J. Biol. Chem., 234 (1959) 666. A. WHITTAKER AND E. R. REDFEARN, Biochem. J ., 88 (1963) 15 p. E. KING, J . Biol. Chem., 236 (1961) 2342.
Received August Ioth, 1964 * P r e s e n t a d d r e s s : J o h n s o n R e s e a r c h F o u n d a t i o n , U n i v e r s i t y of P e n n s y l v a n i a , P h i l a d e l phia, Pa. (U.S.A.). Biochim. Biophys. Acta, 92 (1964) 173-175