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Purification and properties of cytochromes C4 and C5 from Azotobacter vinelandii
436
PRELIMINARY NOTES
vo~. 20 (1956)
Purification and properties of cytochromes c4 and c5 f r o m Azotobacter vinelondii The p u r p o s e of this c o m m u n i c a t i o n is to describe the purification and properties of two cytoc h r o m e s from Azotobacter v i n e l a n d i i . These two c o m p o n e n t s resemble cytochrome c in several i m p o r t a n t properties and for this reason they are called here cytochromes c4 and ca. They were extracted from the cells by means of n-butanol 1. One p a r t of Azotobacter v i n e l a n d i i cells was mixed in a \Varing blender for 1-2 minutes w i t h one p a r t of n - b u t a n o l (w/v) ; the b u t a n o l was decanted off and 1.2 p a r t s of w a t e r were added to the cells, the m i x t u r e was blended again and centrifuged for 30 m i n u t e s at lO,OOO r.p.m. The clear pink layer containing the cytochromes was pipetted off and the residue was extracted once m o r e with the s a m e a m o u n t of water. The pink solution from b o t h extractions was dialysed to r e m o v e the b u t a n o l and centrifuged. The p H of the clear solution was b r o u g h t to p H 8 with o.1 N N a O H and a solution of 25 % basic lead acetate was added till no further precipitate was formed. The precipitate was centrifuged off and discarded and the excess of lead was removed b y the addition of sodium sulphate at p H 6.5, or by dialysis. Two volumes of neutralized saturated a m m o n i u m sulphate were then added at p H 7- A reddish precipitate was formed which was collected by centrifugation, dissolved in a small volume of o . o 2 M p h o s p h a t e buffer p H 7-5, dialysed first against the same buffer, then against distilled water, and freeze dried. P a p e r electrophoresis was performed on \ V h a t m a n No. 3 m m paper, with o.o5 M tris buffer p H 8 and a current of 200 volts. The pink dried material was dissolved in the m i n i m u m a m o u n t of buffer and applied as a n a r r o w b a n d across the strip of wet paper, 8 inches wide and 15 inches long. The paper, b e t w e e n the t w o large electrode vessels into which it dipped, was laid across a shallow p y r e x dish, covered w i t h a glass plate. The p a p e r was so arranged t h a t it h u n g clear of the glass plate. The p i g m e n t m o v e d t o w a r d s the positive electrode and separated into two coloured bands: the faster m o v i n g one, c y t o c h r o m e c4, comprising a b o u t 3/4 of the total pigment, had moved a b o u t io cm in 15 hours; the second one, c y t o c h r o m e cs, lagged behind by 4-5 cm. They were eluted separately. T h e c y t o c h r o m e c5 fraction often contained very small a m o u n t s of cytochrome c4 which were s e p a r a t e d by a second p a p e r electrophoresis. While the position of their joint a-absorption b a n d lies at a b o u t 552 m/* in the crude e x t r a c t or in the intact cells, t h a t of cytochrome c4 is situated a t 551 and t h a t of c y t o c h r o m e c5 at 555 mbt. The absorption s p e c t r u m of purified c y t o c h r o m e c a closely resembles t h a t of pure c y t o c h r o m e c (c/. ref.*). The a b s o r p t i o n b a n d s of the reduced c o m p o u n d have the following positions: a: 551 m/~; fl: 522 m/t; ?: 416 m/t; 3 : 3 1 5 m/~. The y - b a n d of ferricytochrome ca lies at 411 m/,. The m M extinction coefficients of the variou§ absorption bands, calculated on the basis of the iron c o n t e n t (see below) are: u: 23.8; 8: 17"6; ? (reduced): 157.2; ? (oxidized): 115.8; protein b a n d (27o m/t): 2o.5. T h u s the extinction coefficient of the u-band is lower, while t h a t of the ? - b a n d is higher t h a n the corresponding values for c y t o c h r o m e c. The extinction coefficient of the p r o t e i n b a n d is lower t h a n t h a t of c y t o c h r o m e c containing o.45 % iron (c/. ref. 2). The s a m e s a m p l e of c y t o c h r o m e c4 was dialysed for 48 hours against glass-distilled w a t e r a n d freeze dried. I r o n was estimated b y the u a'-dipyridyl m e t h o d s, and nitrogen by Kjeldahl. The iron c o n t e n t was found to be o.46 % of the dry weight or of the a m o u n t of protein. Therefore t h e molecular weight, calculated on the basis of one a t o m of iron per molecule, is a b o u t 12,ooo. These values are very similar to those obtained for c y t o c h r o m e #. T h e s p e c t r u m of c y t o c h r o m e c6 is very similar to t h a t of c 4, the whole s p e c t r u m being shifted t o w a r d s the longer wavelengths b y 4 m/~. The following properties a p p l y to b o t h c y t o c h r o m e ca and c5~ The p i g m e n t s in solution r e m a i n p a r t l y reduced after extraction from the cells. T h e y are not autoxidizable nor do t h e y c o m b i n e d with c a r b o n m o n o x i d e between p H 4.5 and 13. T h e y are not affected b y heating for xo m i n u t e s in a boiling w a t e r bath. T h e y are r e m a r k a b l y stable in alkali, no change being observed a f t e r the addition of i v o l u m e of N N a O H ; w h e n pyridine was added to this alkaline solution, the u - b a n d of either c a or ca shifted to 55 ° m/~. The p i g m e n t s are denatured when kept below p H 4.5 for several hours. Unlike c y t o c h r o m e c, the bacterial c y t o c h r o m e s do not adsorb on Amberlite IRC-5o. Between p H 8 and 5.5 the cytochromes m o v e d t o w a r d s the positive electrode on p a p e r electrophoresis, which s h o w s t h a t b o t h c a and c5 have an isoelectric point in the acid range. C y t o c h r o m e c~ lagged behind c y t o c h r o m e c a which suggests t h a t its isoelectric point is s o m e w h a t h i g h e r t h a n t h a t of ca. The oxidation-reduction potential was determined using m i x t u r e s of ferro- and ferricyanide as oxidation-reduction buffers according to DAVENPORT AND HILL5. I t was found t h a t the potential of half reduction, E0", for c y t o c h r o m e c a was + 0.30 volts between p H 5.5 and 7.3, while for c y t o c h r o m e ca it was + 0.32 volts between p H 5.8 and 7.3. The activities of c y t o c h r o m e s c a and c5 were studied in the succinic oxidase and c y t o c h r o m e oxidase s y s t e m s of Azotobacter v i n e l a n d i i preparations. The e x t r a c t was made as described previ-
VOL.
20
(1956)
PRELIMINARY NOTES
437
o u s l y s, a n d a p a r t i c u l a t e fraction, n o t s e d i m e n t e d b y 30 m i n u t e s c e n t r i f u g a t i o n at I5,ooo g b u t s e d i m e n t e d d u r i n g 60 m i n u t e s a t a b o u t x 2o,ooo g w a s obtained. It w a s w a s h e d in 0.o5 M p h o s p h a t e buffer p H 7, c e n t r i f u g e d again a n d r e s u s p e n d e d in t h e s a m e buffer. This p r e p a r a t i o n c o n t a i n s all t h e c y t o c h r o m e p i g m e n t s of t h e original cells. T h e y are all p r e s e n t in t h e oxidized f o r m a n d t h e i r a b s o r p t i o n b a n d s are t h u s n o t observable spectroscopically e x c e p t t h a t of oxidized c y t o c h r o m e a z at 645 rap. O n a d d i t i o n of a suitable r e d u c e r a s t r o n g (c, + %) b a n d a n d a w e a k b I b a n d a p p e a r at 552-552.5 m p a n d 56o m p respectively a n d t h e b a n d of c y t o c h r o m e az m o v e s to 63o rap. All t h e c o m p o n e n t s of c y t o c h r o m e c a n be r e d u c e d either b y succinate, or b y r e d u c e d d i p h o s p h o p y r i d i n e nucleotide ( D P N H ) , a n d oxidized on s h a k i n g in air. No o t h e r s u b s t r a t e w a s f o u n d to be effective. W h e n reduced purified c y t o c h r o m e (c, + on), c, or c~ are a d d e d in t h e a b s e n c e of s u b s t r a t e t h e e x o g e n o u s p i g m e n t s b e c o m e oxidized at once. If t h e n s u c c i n a t e or D P N H is a d d e d t h e e x o g e n o u s p i g m e n t s b e c o m e r e d u c e d a n d c a n u n d e r g o o x i d a t i o n on s h a k i n g in air a n d red u c t i o n on s t a n d i n g . Succinic a n d c y t o c h r o m e o x i d a s e activities were m e a s u r e d in micro differential m a n o m e t e r s a t 3 °° in t h e presence of air a n d K O H . T h e m a n o m e t r i c flask c o n t a i n e d t h e e n z y m e in o . o 5 M p h o s p h a t e buffer p H 7, a n d eithers u c c i n a t e or, in t h e case of c y t o c h r o m e oxidase, a s c o r b a t e w a s t i p p e d in from t h e side a r m to give a final c o n c e n t r a t i o n of o.o2 M. T h e t o t a l v o l u m e of t h e r e a c t i o n m i x t u r e w a s 0. 5 ml. U n d e r t h o s e conditions, t h e Q o , with s u c c i n a t e w a s a b o u t xooo. I t w a s n o t modified b y t h e addition of c y t o c h r o m e c,, cs or c. W i t h ascorbate, t h e s y s t e m did n o t t a k e u p a n y o x y g e n unless Azotobacter c y t o c h r o m e w a s added. T h e o x y g e n u p t a k e was proportional, w i t h i n c e r t a i n limits, to t h e a m o u n t of e x o g e n o u s c y t o c h r o m e c, or cs present, a n d w i t h 4" I o - S M c y t o c h r o m e c,, t h e Qo2 w a s 50o-60o. C y t o c h r o m e cs h a d a p p r o x i m a t e l y t h e s a m e effect. C y t o c h r o m e c, Rhodospirillum rubrum c y t o c h r o m e C or c y t o c h r o m e [ were ineffective. C y t o c h r o m e c4, a n d a m i x t u r e of c y t o c h r o m e c 4 a n d %, were t e s t e d m a n o m e t r i c a l l y a n d spectroscopically in t h e succinic a n d c y t o c h r o m e o x i d a s e s y s t e m s of c y t o c h r o m e c-deficient h e a r t m u s c l e p r e p a r a t i o n s 7. T h e y h a d no e ~ e c t on either of t h o s e t w o s y s t e m s , while t h e a d d i t i o n of c y t o c h r o m e c p r o d u c e d , as expected, a large o x y g e n u p t a k e . KAMEr~ AND VERNOS 8 also reported t h a t t h e c t y p e c y t o c h r o m e a n d its o x i d a s e from Azotobacter vinelandii r e a c t e d only w i t h each o t h e r a n d could n o t be linked to t h e c y t o c h r o m e c - c y t o c h r o m e o x i d a s e s y s t e m f r o m m a m m a l i a n cells. C y t o c h r o m e ca w a s also t e s t e d w i t h p a r t i c u l a t e p r e p a r a t i o n s f r o m dcetobacter peroxidans m a d e as described a b o v e for Azotobacter a n d it w a s f o u n d to h a v e no activity. I t is k n o w n t h a t a m i x t u r e of t w o c y t o c h r o m e c o m p o n e n t s , t h e a - b a n d s of w h i c h differ in position b y o n l y 4 m # , m a y s h o w a s y m m e t r i c a l a - b a n d lying at a n i n t e r m e d i a t e position* a n d it is therefore n o t s u r p r i s i n g t h a t t h e presence of t h e two c y t o c h r o m e s c a n n o t be d e t e c t e d in Azotobacter cells or in c r u d e e x t r a c t s . C y t o c h r o m e c~ differs f r o m c y t o c h r o m e ci iv t h r e e r e s p e c t s : it h a s a s o m e w h a t h i g h e r isoelectric p o i n t a n d o x i d a t i o n r e d u c t i o n potential, a n d t h e a b s o r p t i o n b a n d s lie 4 m p nearer t h e red e n d of t h e s p e c t r u m . I t is possible t h a t c5 p l a y s in Azotobacter a role similar to t h a t of c 1 in a n i m a l tissues* a n d it is conceivable t h a t t h e r e s p i r a t o r y c h a i n u s e s c5 for t h e o x i d a t i o n of s o m e specific s u b s t r a t e s . T h e s i m i l a r i t y of c y t o c h r o m e ca a n d cn w i t h c y t o c h r ~ m e c is striking. However, t h e differences in t h e isoelectric p o i n t or in t h e o x i d a t i o n - r e d u c t i o n p o t e n t i a l b e t w e e n c y t o c h r o m e c on one h a n d a n d c y t o c h r o m e c a a n d c5 o n t h e o t h e r m a y be sufficient to a c c o u n t for t h e i r specific biological properties. W e wish to t h a n k Drs. L, SMITH a n d R. HILL for s a m p l e s of Rhodospirillum rubrum c y t o c h r o m e c, a n d c y t o c h r o m e ] respectively. A. TISSfltRES Molteno Institute, University o~ Cambridge (England) R . H . BURRIS" 1 R. K. MORTOr~, Nature, x66 (I95 o) xo92. • D. KEXLIN AND E. C. SLATER, Brit. Med. Bull., 9 (I953) 89. • 1~. HILL AND D. KKILIN, Proc. Roy, Soc. (London) B., xx 4 (x933) xo44 E. MARGOLISH, Biochem. J., 56 (I954) 535s H. E. DAVENPORT ANX) R. HmL, Proc. Roy. Soc. (London) B., I39 (x952) 327 • • A. TISSI~RES, Nature, i74 (x954) x83. T C. L. T s o u , Biochem. J., 50 (x952) 493. s M. D. KAMEN AND L. P. VERNON, J. Biol. Chem., 2 I I (1954) 663. • D. KEILIN AND E. F. HARTSEE, Nature, i 7 6 (x955) 200. R e c e i v e d F e b r u a r y 24th , x956 Fellow of t h e J o h n S i m o n G u g g e n h e i m Memorial F o u n d a t i o n . P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of Wisconsin, Madison, Wis. (U.S.A.).
PRELIMINARY NOTES
vo~. 20 (1956)
Purification and properties of cytochromes c4 and c5 f r o m Azotobacter vinelondii The p u r p o s e of this c o m m u n i c a t i o n is to describe the purification and properties of two cytoc h r o m e s from Azotobacter v i n e l a n d i i . These two c o m p o n e n t s resemble cytochrome c in several i m p o r t a n t properties and for this reason they are called here cytochromes c4 and ca. They were extracted from the cells by means of n-butanol 1. One p a r t of Azotobacter v i n e l a n d i i cells was mixed in a \Varing blender for 1-2 minutes w i t h one p a r t of n - b u t a n o l (w/v) ; the b u t a n o l was decanted off and 1.2 p a r t s of w a t e r were added to the cells, the m i x t u r e was blended again and centrifuged for 30 m i n u t e s at lO,OOO r.p.m. The clear pink layer containing the cytochromes was pipetted off and the residue was extracted once m o r e with the s a m e a m o u n t of water. The pink solution from b o t h extractions was dialysed to r e m o v e the b u t a n o l and centrifuged. The p H of the clear solution was b r o u g h t to p H 8 with o.1 N N a O H and a solution of 25 % basic lead acetate was added till no further precipitate was formed. The precipitate was centrifuged off and discarded and the excess of lead was removed b y the addition of sodium sulphate at p H 6.5, or by dialysis. Two volumes of neutralized saturated a m m o n i u m sulphate were then added at p H 7- A reddish precipitate was formed which was collected by centrifugation, dissolved in a small volume of o . o 2 M p h o s p h a t e buffer p H 7-5, dialysed first against the same buffer, then against distilled water, and freeze dried. P a p e r electrophoresis was performed on \ V h a t m a n No. 3 m m paper, with o.o5 M tris buffer p H 8 and a current of 200 volts. The pink dried material was dissolved in the m i n i m u m a m o u n t of buffer and applied as a n a r r o w b a n d across the strip of wet paper, 8 inches wide and 15 inches long. The paper, b e t w e e n the t w o large electrode vessels into which it dipped, was laid across a shallow p y r e x dish, covered w i t h a glass plate. The p a p e r was so arranged t h a t it h u n g clear of the glass plate. The p i g m e n t m o v e d t o w a r d s the positive electrode and separated into two coloured bands: the faster m o v i n g one, c y t o c h r o m e c4, comprising a b o u t 3/4 of the total pigment, had moved a b o u t io cm in 15 hours; the second one, c y t o c h r o m e cs, lagged behind by 4-5 cm. They were eluted separately. T h e c y t o c h r o m e c5 fraction often contained very small a m o u n t s of cytochrome c4 which were s e p a r a t e d by a second p a p e r electrophoresis. While the position of their joint a-absorption b a n d lies at a b o u t 552 m/* in the crude e x t r a c t or in the intact cells, t h a t of cytochrome c4 is situated a t 551 and t h a t of c y t o c h r o m e c5 at 555 mbt. The absorption s p e c t r u m of purified c y t o c h r o m e c a closely resembles t h a t of pure c y t o c h r o m e c (c/. ref.*). The a b s o r p t i o n b a n d s of the reduced c o m p o u n d have the following positions: a: 551 m/~; fl: 522 m/t; ?: 416 m/t; 3 : 3 1 5 m/~. The y - b a n d of ferricytochrome ca lies at 411 m/,. The m M extinction coefficients of the variou§ absorption bands, calculated on the basis of the iron c o n t e n t (see below) are: u: 23.8; 8: 17"6; ? (reduced): 157.2; ? (oxidized): 115.8; protein b a n d (27o m/t): 2o.5. T h u s the extinction coefficient of the u-band is lower, while t h a t of the ? - b a n d is higher t h a n the corresponding values for c y t o c h r o m e c. The extinction coefficient of the p r o t e i n b a n d is lower t h a n t h a t of c y t o c h r o m e c containing o.45 % iron (c/. ref. 2). The s a m e s a m p l e of c y t o c h r o m e c4 was dialysed for 48 hours against glass-distilled w a t e r a n d freeze dried. I r o n was estimated b y the u a'-dipyridyl m e t h o d s, and nitrogen by Kjeldahl. The iron c o n t e n t was found to be o.46 % of the dry weight or of the a m o u n t of protein. Therefore t h e molecular weight, calculated on the basis of one a t o m of iron per molecule, is a b o u t 12,ooo. These values are very similar to those obtained for c y t o c h r o m e #. T h e s p e c t r u m of c y t o c h r o m e c6 is very similar to t h a t of c 4, the whole s p e c t r u m being shifted t o w a r d s the longer wavelengths b y 4 m/~. The following properties a p p l y to b o t h c y t o c h r o m e ca and c5~ The p i g m e n t s in solution r e m a i n p a r t l y reduced after extraction from the cells. T h e y are not autoxidizable nor do t h e y c o m b i n e d with c a r b o n m o n o x i d e between p H 4.5 and 13. T h e y are not affected b y heating for xo m i n u t e s in a boiling w a t e r bath. T h e y are r e m a r k a b l y stable in alkali, no change being observed a f t e r the addition of i v o l u m e of N N a O H ; w h e n pyridine was added to this alkaline solution, the u - b a n d of either c a or ca shifted to 55 ° m/~. The p i g m e n t s are denatured when kept below p H 4.5 for several hours. Unlike c y t o c h r o m e c, the bacterial c y t o c h r o m e s do not adsorb on Amberlite IRC-5o. Between p H 8 and 5.5 the cytochromes m o v e d t o w a r d s the positive electrode on p a p e r electrophoresis, which s h o w s t h a t b o t h c a and c5 have an isoelectric point in the acid range. C y t o c h r o m e c~ lagged behind c y t o c h r o m e c a which suggests t h a t its isoelectric point is s o m e w h a t h i g h e r t h a n t h a t of ca. The oxidation-reduction potential was determined using m i x t u r e s of ferro- and ferricyanide as oxidation-reduction buffers according to DAVENPORT AND HILL5. I t was found t h a t the potential of half reduction, E0", for c y t o c h r o m e c a was + 0.30 volts between p H 5.5 and 7.3, while for c y t o c h r o m e ca it was + 0.32 volts between p H 5.8 and 7.3. The activities of c y t o c h r o m e s c a and c5 were studied in the succinic oxidase and c y t o c h r o m e oxidase s y s t e m s of Azotobacter v i n e l a n d i i preparations. The e x t r a c t was made as described previ-
VOL.
20
(1956)
PRELIMINARY NOTES
437
o u s l y s, a n d a p a r t i c u l a t e fraction, n o t s e d i m e n t e d b y 30 m i n u t e s c e n t r i f u g a t i o n at I5,ooo g b u t s e d i m e n t e d d u r i n g 60 m i n u t e s a t a b o u t x 2o,ooo g w a s obtained. It w a s w a s h e d in 0.o5 M p h o s p h a t e buffer p H 7, c e n t r i f u g e d again a n d r e s u s p e n d e d in t h e s a m e buffer. This p r e p a r a t i o n c o n t a i n s all t h e c y t o c h r o m e p i g m e n t s of t h e original cells. T h e y are all p r e s e n t in t h e oxidized f o r m a n d t h e i r a b s o r p t i o n b a n d s are t h u s n o t observable spectroscopically e x c e p t t h a t of oxidized c y t o c h r o m e a z at 645 rap. O n a d d i t i o n of a suitable r e d u c e r a s t r o n g (c, + %) b a n d a n d a w e a k b I b a n d a p p e a r at 552-552.5 m p a n d 56o m p respectively a n d t h e b a n d of c y t o c h r o m e az m o v e s to 63o rap. All t h e c o m p o n e n t s of c y t o c h r o m e c a n be r e d u c e d either b y succinate, or b y r e d u c e d d i p h o s p h o p y r i d i n e nucleotide ( D P N H ) , a n d oxidized on s h a k i n g in air. No o t h e r s u b s t r a t e w a s f o u n d to be effective. W h e n reduced purified c y t o c h r o m e (c, + on), c, or c~ are a d d e d in t h e a b s e n c e of s u b s t r a t e t h e e x o g e n o u s p i g m e n t s b e c o m e oxidized at once. If t h e n s u c c i n a t e or D P N H is a d d e d t h e e x o g e n o u s p i g m e n t s b e c o m e r e d u c e d a n d c a n u n d e r g o o x i d a t i o n on s h a k i n g in air a n d red u c t i o n on s t a n d i n g . Succinic a n d c y t o c h r o m e o x i d a s e activities were m e a s u r e d in micro differential m a n o m e t e r s a t 3 °° in t h e presence of air a n d K O H . T h e m a n o m e t r i c flask c o n t a i n e d t h e e n z y m e in o . o 5 M p h o s p h a t e buffer p H 7, a n d eithers u c c i n a t e or, in t h e case of c y t o c h r o m e oxidase, a s c o r b a t e w a s t i p p e d in from t h e side a r m to give a final c o n c e n t r a t i o n of o.o2 M. T h e t o t a l v o l u m e of t h e r e a c t i o n m i x t u r e w a s 0. 5 ml. U n d e r t h o s e conditions, t h e Q o , with s u c c i n a t e w a s a b o u t xooo. I t w a s n o t modified b y t h e addition of c y t o c h r o m e c,, cs or c. W i t h ascorbate, t h e s y s t e m did n o t t a k e u p a n y o x y g e n unless Azotobacter c y t o c h r o m e w a s added. T h e o x y g e n u p t a k e was proportional, w i t h i n c e r t a i n limits, to t h e a m o u n t of e x o g e n o u s c y t o c h r o m e c, or cs present, a n d w i t h 4" I o - S M c y t o c h r o m e c,, t h e Qo2 w a s 50o-60o. C y t o c h r o m e cs h a d a p p r o x i m a t e l y t h e s a m e effect. C y t o c h r o m e c, Rhodospirillum rubrum c y t o c h r o m e C or c y t o c h r o m e [ were ineffective. C y t o c h r o m e c4, a n d a m i x t u r e of c y t o c h r o m e c 4 a n d %, were t e s t e d m a n o m e t r i c a l l y a n d spectroscopically in t h e succinic a n d c y t o c h r o m e o x i d a s e s y s t e m s of c y t o c h r o m e c-deficient h e a r t m u s c l e p r e p a r a t i o n s 7. T h e y h a d no e ~ e c t on either of t h o s e t w o s y s t e m s , while t h e a d d i t i o n of c y t o c h r o m e c p r o d u c e d , as expected, a large o x y g e n u p t a k e . KAMEr~ AND VERNOS 8 also reported t h a t t h e c t y p e c y t o c h r o m e a n d its o x i d a s e from Azotobacter vinelandii r e a c t e d only w i t h each o t h e r a n d could n o t be linked to t h e c y t o c h r o m e c - c y t o c h r o m e o x i d a s e s y s t e m f r o m m a m m a l i a n cells. C y t o c h r o m e ca w a s also t e s t e d w i t h p a r t i c u l a t e p r e p a r a t i o n s f r o m dcetobacter peroxidans m a d e as described a b o v e for Azotobacter a n d it w a s f o u n d to h a v e no activity. I t is k n o w n t h a t a m i x t u r e of t w o c y t o c h r o m e c o m p o n e n t s , t h e a - b a n d s of w h i c h differ in position b y o n l y 4 m # , m a y s h o w a s y m m e t r i c a l a - b a n d lying at a n i n t e r m e d i a t e position* a n d it is therefore n o t s u r p r i s i n g t h a t t h e presence of t h e two c y t o c h r o m e s c a n n o t be d e t e c t e d in Azotobacter cells or in c r u d e e x t r a c t s . C y t o c h r o m e c~ differs f r o m c y t o c h r o m e ci iv t h r e e r e s p e c t s : it h a s a s o m e w h a t h i g h e r isoelectric p o i n t a n d o x i d a t i o n r e d u c t i o n potential, a n d t h e a b s o r p t i o n b a n d s lie 4 m p nearer t h e red e n d of t h e s p e c t r u m . I t is possible t h a t c5 p l a y s in Azotobacter a role similar to t h a t of c 1 in a n i m a l tissues* a n d it is conceivable t h a t t h e r e s p i r a t o r y c h a i n u s e s c5 for t h e o x i d a t i o n of s o m e specific s u b s t r a t e s . T h e s i m i l a r i t y of c y t o c h r o m e ca a n d cn w i t h c y t o c h r ~ m e c is striking. However, t h e differences in t h e isoelectric p o i n t or in t h e o x i d a t i o n - r e d u c t i o n p o t e n t i a l b e t w e e n c y t o c h r o m e c on one h a n d a n d c y t o c h r o m e c a a n d c5 o n t h e o t h e r m a y be sufficient to a c c o u n t for t h e i r specific biological properties. W e wish to t h a n k Drs. L, SMITH a n d R. HILL for s a m p l e s of Rhodospirillum rubrum c y t o c h r o m e c, a n d c y t o c h r o m e ] respectively. A. TISSfltRES Molteno Institute, University o~ Cambridge (England) R . H . BURRIS" 1 R. K. MORTOr~, Nature, x66 (I95 o) xo92. • D. KEXLIN AND E. C. SLATER, Brit. Med. Bull., 9 (I953) 89. • 1~. HILL AND D. KKILIN, Proc. Roy, Soc. (London) B., xx 4 (x933) xo44 E. MARGOLISH, Biochem. J., 56 (I954) 535s H. E. DAVENPORT ANX) R. HmL, Proc. Roy. Soc. (London) B., I39 (x952) 327 • • A. TISSI~RES, Nature, i74 (x954) x83. T C. L. T s o u , Biochem. J., 50 (x952) 493. s M. D. KAMEN AND L. P. VERNON, J. Biol. Chem., 2 I I (1954) 663. • D. KEILIN AND E. F. HARTSEE, Nature, i 7 6 (x955) 200. R e c e i v e d F e b r u a r y 24th , x956 Fellow of t h e J o h n S i m o n G u g g e n h e i m Memorial F o u n d a t i o n . P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of Wisconsin, Madison, Wis. (U.S.A.).