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Cytochrome c3, a bifunctional haematohamatin
VOL. 1 8 (I955)
mOCHIMICA ET BIOPHYSICA ACTA
427
Short Communications and Preliminary Notes
Cytochrome
c 3, a b i f u n c t i o n a l
haematohaematin
The cytochrome of the anaerobic sulphate-reducing bacteria 1, 2 will be t e r m e d cytochrome c3, since its systematic name--Desulphovibrio desulphuricans c y t o c h r o m e 5 5 3 - - i s cumbersome. The protein has been extracted and purified by a procedure, to be described in detail elsewhere, involving acid-precipitation from extracts of acetone-dried cells followed by c h r o m a t o g r a p h y on cellulose and Amberlite I R C 50. Evidence for carrier activity in the reduction of sulphate and related ions has been r e p o r t e d s. The p r o d u c t was a deep red autoxidizable water-soluble powder, h o m o g e n e o u s in the ultracentrifuge and b y p a p e r electrophoresis, containing less t h a n 5 % of i m p u r i t y absorbing at 280 m/z when c h r o m a t o g r a p h e d on I R C 5 ° columns as described b y BOARDMAN AND PARTRIDGE4. The following properties h a v e been examined: (i) Iso-electric point: p a p e r electrophoresis in p h o s p h a t e buffers with pure c y t o c h r o m e c as control indicated an iso-electric point between p H lO.3 and lO.6. (ii) Redox potential: potentiometric titration with sodium dithionite in the presence of anthracene-2-sulphonic acid as a poising agent gave a value of E 0 -- - - 2 0 5 ~: 4 mV at 3 o°. (iii) Spectrum: the oxidized material had peaks at a = 535, 7 = 41° , a = 35 ° m/t, with an inflection a t ~ 5 6 8 m/t. The reduced material had peaks at a = 553.4,/3 = 525.0, 7 = 419 m/z; the f u r t h e r ultra-violet region of the reduced form was not investigated, since, owing to the low E 0 of c v only dithionite is a suitable reducing agent and this is o p a q u e b e l o w , ~ 3 8 o m/l. (iv) Specific extinction coefficient: the pure reduced material had an esp of 4.2 at its a-peak. This is r o u g h l y twice t h a t of cytochrome c, and indicates t h a t c 3 has either half the molecular weight of c, or twice as m a n y haemine groups per molecule. I n the latter case a molecular weight o f ~ I 2 , 5 o o would be expected. (v) Sedimentation coefficient: a test in the ultra-centrifuge carried out b y Dr. A. G. OGSTON at Oxford indicated a value $20,~, = 1.93. lO-13- This indicates a m i n i m u m molecular weight of lO,2OO, consistent with a double-haemin molecule similar in size to t h a t of c y t o c h r o m e c. (vi) Iron content: 8.1 mg cytochrome c 3 was wet-ashed and analysed for iron b y the op h e n a n t h r o l i n e procedure. I t contained o.92 °o Fe, roughly twice t h a t of pure cytochrome c, again consistent with a double-haemin structure. (vii) Chemical stability: cytoctlrome c3 was stable to boiling for five minutes, and no h a e m i n was released with mineral a c i d - - o r acetic-acetone. Ether-insoluble p o r p h y r i n was released from the reduced form by HC1 (3-3 N) ; its a - b a n d lay at 554 m # (c[: 553 m/z for " p o r p h y r i n c"). Hence the p i g m e n t p r o b a b l y has thio-ether links between the protein and h a e m i n similar to those of cytochrome c. (viii) Prosthetic group: t r e a t m e n t with silver sulphate and acetic acid 5 yielded a h a e m i n (Sorer peak 39o m # in KH2POa, p H 7; c/: 391 m # for a control p r e p a r a t i o n of h a e m a t o h a e m i n from c y t o c h r o m e c) which after extraction formed a pyridine h a e m o c h r o m e spectroscopically similar to pyridine h a e m a t o h a e m o c h r o m e (4o8, 517.2, 546.o mp). Reductive fission of c3 with N a / H g s yielded a p o r p h y r i n h a v i n g the s p e c t r u m of m e s o p o r p h y r i n and a chlorin, b o t h p r o d u c t s being spectroscopically like those derived from c y t o c h r o m e c. These studies lead to the conclusion t h a t cytochrome c a is a bifunctional h a e m a t o h a e m a t i n with thio-ether haemin-protein links. They will be reported in detail elsewhere. This note is published by permission of the Director, Chemical Research L a b o r a t o r y . JOHN POSTGATE
Chemical Research Laboratory, Teddington, Middlesex (England) 1 j . R. POSTGATVZ, Biochem. J., 56 (1954) xi; 58 (1954) ix. 2 M. ISHIMOTO, J. KOYAMA AND •. NAGAI, Biochem. J. (Tokyo), 41 (1955) 763 . 3 j. R. POSTGATE, Proc. 3rd. Intern. Congr. Biochem., (1955) 94. 4 N. K. BOARDMAN AND S. M. PARTRIDGE, Biochem. J., 59 (1955) 543. 5 K.-G. PAUL, .4cta Chem. Scand., 4 (195o) 239. 6 H. E. DAVENPORT, Nature, 169 (1952) 75. Received S e p t e m b e r Jst, 1955
mOCHIMICA ET BIOPHYSICA ACTA
427
Short Communications and Preliminary Notes
Cytochrome
c 3, a b i f u n c t i o n a l
haematohaematin
The cytochrome of the anaerobic sulphate-reducing bacteria 1, 2 will be t e r m e d cytochrome c3, since its systematic name--Desulphovibrio desulphuricans c y t o c h r o m e 5 5 3 - - i s cumbersome. The protein has been extracted and purified by a procedure, to be described in detail elsewhere, involving acid-precipitation from extracts of acetone-dried cells followed by c h r o m a t o g r a p h y on cellulose and Amberlite I R C 50. Evidence for carrier activity in the reduction of sulphate and related ions has been r e p o r t e d s. The p r o d u c t was a deep red autoxidizable water-soluble powder, h o m o g e n e o u s in the ultracentrifuge and b y p a p e r electrophoresis, containing less t h a n 5 % of i m p u r i t y absorbing at 280 m/z when c h r o m a t o g r a p h e d on I R C 5 ° columns as described b y BOARDMAN AND PARTRIDGE4. The following properties h a v e been examined: (i) Iso-electric point: p a p e r electrophoresis in p h o s p h a t e buffers with pure c y t o c h r o m e c as control indicated an iso-electric point between p H lO.3 and lO.6. (ii) Redox potential: potentiometric titration with sodium dithionite in the presence of anthracene-2-sulphonic acid as a poising agent gave a value of E 0 -- - - 2 0 5 ~: 4 mV at 3 o°. (iii) Spectrum: the oxidized material had peaks at a = 535, 7 = 41° , a = 35 ° m/t, with an inflection a t ~ 5 6 8 m/t. The reduced material had peaks at a = 553.4,/3 = 525.0, 7 = 419 m/z; the f u r t h e r ultra-violet region of the reduced form was not investigated, since, owing to the low E 0 of c v only dithionite is a suitable reducing agent and this is o p a q u e b e l o w , ~ 3 8 o m/l. (iv) Specific extinction coefficient: the pure reduced material had an esp of 4.2 at its a-peak. This is r o u g h l y twice t h a t of cytochrome c, and indicates t h a t c 3 has either half the molecular weight of c, or twice as m a n y haemine groups per molecule. I n the latter case a molecular weight o f ~ I 2 , 5 o o would be expected. (v) Sedimentation coefficient: a test in the ultra-centrifuge carried out b y Dr. A. G. OGSTON at Oxford indicated a value $20,~, = 1.93. lO-13- This indicates a m i n i m u m molecular weight of lO,2OO, consistent with a double-haemin molecule similar in size to t h a t of c y t o c h r o m e c. (vi) Iron content: 8.1 mg cytochrome c 3 was wet-ashed and analysed for iron b y the op h e n a n t h r o l i n e procedure. I t contained o.92 °o Fe, roughly twice t h a t of pure cytochrome c, again consistent with a double-haemin structure. (vii) Chemical stability: cytoctlrome c3 was stable to boiling for five minutes, and no h a e m i n was released with mineral a c i d - - o r acetic-acetone. Ether-insoluble p o r p h y r i n was released from the reduced form by HC1 (3-3 N) ; its a - b a n d lay at 554 m # (c[: 553 m/z for " p o r p h y r i n c"). Hence the p i g m e n t p r o b a b l y has thio-ether links between the protein and h a e m i n similar to those of cytochrome c. (viii) Prosthetic group: t r e a t m e n t with silver sulphate and acetic acid 5 yielded a h a e m i n (Sorer peak 39o m # in KH2POa, p H 7; c/: 391 m # for a control p r e p a r a t i o n of h a e m a t o h a e m i n from c y t o c h r o m e c) which after extraction formed a pyridine h a e m o c h r o m e spectroscopically similar to pyridine h a e m a t o h a e m o c h r o m e (4o8, 517.2, 546.o mp). Reductive fission of c3 with N a / H g s yielded a p o r p h y r i n h a v i n g the s p e c t r u m of m e s o p o r p h y r i n and a chlorin, b o t h p r o d u c t s being spectroscopically like those derived from c y t o c h r o m e c. These studies lead to the conclusion t h a t cytochrome c a is a bifunctional h a e m a t o h a e m a t i n with thio-ether haemin-protein links. They will be reported in detail elsewhere. This note is published by permission of the Director, Chemical Research L a b o r a t o r y . JOHN POSTGATE
Chemical Research Laboratory, Teddington, Middlesex (England) 1 j . R. POSTGATVZ, Biochem. J., 56 (1954) xi; 58 (1954) ix. 2 M. ISHIMOTO, J. KOYAMA AND •. NAGAI, Biochem. J. (Tokyo), 41 (1955) 763 . 3 j. R. POSTGATE, Proc. 3rd. Intern. Congr. Biochem., (1955) 94. 4 N. K. BOARDMAN AND S. M. PARTRIDGE, Biochem. J., 59 (1955) 543. 5 K.-G. PAUL, .4cta Chem. Scand., 4 (195o) 239. 6 H. E. DAVENPORT, Nature, 169 (1952) 75. Received S e p t e m b e r Jst, 1955