Purification and properties of cytochrome c3 of desulfovibrio vulgaris, miyazaki

214

lllOCHIMICA I4"I" BIOPHYSICA A('TA

B~,~, 35914 P U R I F I C A T I O N A N D P R O P E R T I E S OF C Y T O C H R O M E c3 01:

DESULFOVIBRIO VULGARIS, MIYAZAKI

T A T S U H I K O Y A G I AND K 1 Y O F U M I M A R U Y A M A

Department of Chemistry, Shizuoha University, Shizuoka 420 (Japan) ( Receiv ed March 22nd, 1971)

SUMMARY

Cytochrome c3 was isolated in a homogeneous state from Desulfovibrio vulgaris, Miyazaki, and its properties examined and compared with those of the cytochrome c3 from D. vulgaris, Hildenborough. The absorption spectrum of the ultraviolet region of ferrocytochrome c3 was recorded for the first time. The spectra of the cytochrome ca from the Miyazaki strain has five peaks (at 552, 523, 419, 323 and 275 nm) in the ferro-form, three peaks (at 53o with a shoulder at 56o, 41o and 35o nm) in the ferri-form, and eight isosbestic points (at 56o, 542, 532, 5o8, 432, 412, 343 and 254 nm). Upon contact with CO, the spectrum of the ferro-form changed. Its isoelectric point is near lO.6, and its redox potential, --o.29 V. The Miyazaki strain cytochrome c3 contains 4 hemes. Its N-terminus is histidine. Arginine, as well as isoleucine, is absent. These features are all different from those reported for the cytochrome c3 of D. vulgaris, Hildenborough.

INTRODUCTION

Cytochrome ca is present in sulfate-reducing bacteria (Desulfovibrio). It has been isolated from D. vulgaris, Hildenborough *,1,2 and from D. vulgaris, Miyazaki *,3-s, and is also present in other species of Desulfovibrio °-8. Its isoelectric point and molecular weight were reported to be near lO.5 and 13 ooo 2, i.e. comparable to those of mammalian and yeast cytochromes c. Its iron content was, however, reported to be o.90/o 2,5, which is twice as high as t h a t of mammalian and yeast cytochromes c. On this basis, POSTGATE2 concluded t h a t the molecule of cytochrome cz from D. vulgaris, Hildenborough, contains two hemes; HORIO AND KAMEN9 confirmed tile Abbreviation: dansyl, I-dimethylaminonaphthalene-5-sulfonyl. " D. vulgaris, H i l d e n b o r o u g h , was d e s c r i b e d as D. desulfurieans, H i l d e n b o r o u g h , in refs. i, 2, 6 a n d 9, a n d D. vulgaris, M i y a z a k i , as D. desulfuricans w i t h o u t s p e c i f y i n g a n y s t r a i n n a m e in refs.3 5 a n d I I . A c c o r d i n g to t h e r e c e n t n o m e n c l a t u r e p r o p o s e d b y POSTGATE AND C A M P B E L L TM, t h e y are to be called D. vulgaris. Since t h e l a t t e r s t r a i n w a s i s o l a t e d from a p a d d y field in M i y a z a k i , J a p a n , we propose to call t h i s o r g a n i s m , D. vulgaris, M i y a z a k i .

Biochim. Biophys. Acta, 243 (I97 I) 214 224

T. YAGI, K. MARUYAMA

215

double heine structure for their crystalline cytochrome c3 also obtained from D.

vulgaris, Hildenborough. Recently, DRUCKER et al.7,8 purified the cytochrome ca from D. vulgaris, Hildenborough, D. salexigens, and D. desulfuricans, VC, and reported that these cytochromes have three hemes. In our laboratory, cytochrome ca was highly purified from D. vulgaris, Miyazaki, and characterized as an electron carrier for hydrogenase (H2 :ferricytochrome ca oxidoreductase) n. The properties of the cytochrome have been extensively studied and the absorption spectra of the ferri- and ferro-forms of cytochrome ca in the ultraviolet as well as visible regions recorded. These properties as well as the heme content and amino acid composition, some of which are different from those reported by earlier workers, will be described in this paper. MATERIALS AND METHODS

Bacteria Desulfovibrio vulgaris, Miyazaki, was cultured on a large scale as described by YAGI et al. n. Chemicals and reagents I-Dimethylaminonaphthalene-5-sulfonyl (dansyl) chloride was purchased from Seikagaku Kogyo Co., Tokyo. Yeast cytochrome c was a generous gift from Sankyo Pharmaceutical Co., Tokyo. Highly purified hydrogenase preparations were obtained by the procedure described by YAGITM. Spotfilm for thin-layer chromatography (25o-#m-thick silicagel plate) was a product of Tokyo Kasei Kogyo Co., Tokyo.

Amino acid composition Cytochrome c3 was hydrolyzed with 6 M HC1 in evacuated glass tubes for 24 and 48 h at i i o °, and the amount of amino acids except cystine were determined with a Hitachi automatic amino acid analyzer (KLA-3B) by the procedure described by SPACKMAN et al. ~a. Corrections were made for the destruction of serine and threonine by extrapolation to zero time of hydrolysis. For assay of cysteine, heine was removed from the cytochrome and the protein moiety was treated with performic acid as described by AMBLER14. The mixture was then dried in a stream of N 2 at room temperature, and hydrolyzed with 6 M HC1 in evacuated glass tubes for 24 and 48 h at i i o °. The amount of cysteate was also determined with an amino acid analyzer and corrected for destruction by extrapolation to zero time of hydrolysis.

Identification of dansyl-amino acid This was carried out on thin-layer chromatograms on spotfilms developed in Solvent I I (toluene-chloroethano1-25% ammonia (6:1o:4, by vol.)) with synthetic dansyl-amino acids as standards, as described by GROS AND LABOUESSE15. RESULTS

Purification of cytochrome c3 Bacterial sonicate prepared from 33 g wet cells 1~ was centrifuged at 80 ooo × g for 60 rain. The supernatant fluid (240 ml) containing about 8 mg cytochrome c3 was Biochim. Biophys. Acta, 243 (i97 I) 214 224

CYTOCHROME Ca OF Desulfovibrio vulg,aris

216

15

°lo

15 20 25 30 Fraction Number (3.56 ml/tube)

Fig. I, E l u t i o n c u r v e of t h e S e p h a d e x G-5o c o l u m n c h r o m a t o g r a p h y of t h e c rude p r e p a r a t i o n of c y t o c h r o m e c 3.

passed through an Amberlite CG 50 (NH4+) column (I.3 cm × Io cm) and the adsorbed cytochromes eluted with o.I M N H 3. The eluate was concentrated to 4.8 ml by a rotary evaporator at 5o°; any precipitate formed was removed by centrifugation. The concentrated red solution whose purity index" was o.45 was applied to a column (1.8 cm × 65 cm) of Sephadex G-5o, fine. The eluting buffer was o.o5 M Tris-HCl (pH 7.3) containing o.2 M NaC1. Fig. I shows the elution pattern of the cytochrome components. As shown in this figure, 41o nm-absorbing proteins were separated into t w o " components. The elution volume of the minor component corresponded to the void volume of the column. From its behavior on a Sephadex G-2oo column, the minor component was judged to have a molecular weight of about 7 ° ooo. The elution volume of the major component which is cytochrome c3 corresponded to that of protein of molecular weight 14 ooo. This fraction, which had a purity index of 1.69, was dialyzed against o.o2 M phosphate buffer (pH 7.o) and applied to a column of Amberlite CG 5 ° (NH4+). The adsorbed cytochrome c3 was eluted with o.I M NH 3, concentrated, and rechromatographed on Sephadex G-5 o as above. The resulting cytochrome ca was dialyzed thoroughly against distilled water and concentrated. By these procedures about 3 mg of cytochrome ca was obtained whose purity index was 2.98. This material showed only one proteinaceous component upon disc electrophoresis with p H 4.o gel (ref. 17 cited in ref. 18). This highly purified preparation was used for the present experiments.

Alternate procedure for the preparation of cytoehrome ca The cytochrome could also be extracted by mere freezing and thawing of the bacterial cells (IO g wet weight) suspended in 25 ml 0.0067 M phosphate buffer (pH 7.0) (ref. 19). The purity index of the cytochrome in this extract was 0.046. The extract was passed through a column (1.8 cm × 4-5 cm) of DEAE-cellulose which had been equilibrated with 0.05 M Tris-HC1 (pH 7.3) overnight and washed with io vol. of distilled water. Cytochromes were eluted without being adsorbed on the " Purity index =

a b s o r b a n c e a t 552 n m in t h e ferro-form a b s o r b a n c e a t 28o n m in t h e ferri-form

*" Still a n o t h e r c y t o c h r o m e p r e s e n t in t h e b a c t e r i a l s o n i c a t e of D. vulgaris, M i y a z a k i , h a s been c h a r a c t e r i z e d as an e l e c t r o n a c c e p t o r for f o r m a t e d e h y d r o g e n a s e ( f o r m a t e : f e r r i c y t o c h r o m e c-553 o x i d o r e d u c t a s e ) 16. This c y t o c h r o m e was n o t a d s o r b e d on t h e i n i t i a l A m b e r l i t e c o l u m n a n d t h e r e f o r e w a s n o t p r e s e n t in t h e m a t e r i a l s u b j e c t e d t o S e p h a d e x c h r o m a t o g r a p h y .

Biochim. Biophys. Acta, 243 (1971) 214-224

CYTOCHROME C3 OF Desulfovibrio vulgaris

217

column. The p u r i t y i n d e x of the c y t o c h r o m e in the eluate was o . I I . This was lyophilized, dissolved in 3.0 ml water, a n d s u b j e c t e d to S e p h a d e x G-5o column chrom a t o g r a p h y as described in the preceding section. The elution p a t t e r n of the c y t o chromes is shown in Fig. 2. The fractions eluted in tubes between No. 21 a n d 25 were collected, a n d d i a l y z e d against 0.02 M p h o s p h a t e buffer. The p u r i t y i n d e x of c y t o c h r o m e ca in this p r e p a r a t i o n was 0.28. This p r e p a r a t i o n is b y no means a pure c y t o c h r o m e ca p r e p a r a t i o n , b u t is the p r e p a r a t i o n o b t a i n a b l e b y one of the mildest procedures available. This could be purified further b y a d s o r p t i o n on and elution from A m b e r l i t e CG 50 (NH4+) column as described in the preceding section to give a p r e p a r a t i o n with a p u r i t y index c o m p a r a b l e to t h a t of the p r e p a r a t i o n described in the preceding section. 30O c£0.5 o

200

o.4

"8 02

g

£ ioo

0,2

< 0

/.

®

° o

15 20 25 30 Froction Number (3.56 ml/tube)

i

2

3

Concentration of Cytochrome-Hem

(UM)

Fig. 2. FAution curve of the Sephadex G-5 o column c h r o m a t o g r a p h y of the c y t o c h r o m e s extracted by freezing and t h a w i n g of the bacterial cells. Fig. 3. Efficiency of c y t o c h r o m e c~ as an electron carrier for hydrogenase. The main c o m p a r t m e n t of a ~Varburg vessel contained 8.1 units purified hydrogenase and a c y t o c h r o m e c 3 p r e p a r a t i o n in 3.0 ml o.o2 M p h o s p h a t e buffer (pH 6.7). The side a r m contained 2. 4 trig Na~S204. The gas phase was N 2. The reaction was s t a r t e d b y adding Na~S204 from the side arm, and the rate of H 2 evolution was nleasured at 3 o°. (2), with the highly purified hydrogenase p r e p a r a t i o n ; O, w i t h the c y t o e h r o m e c 3 p r e p a r a t i o n obtained b y the mild procedure. The concentration of c y r o c h r o m e c3 was expressed in m o l a r i t y of heme instead of protein.

Biochemical activities of cytochrome c3 preparations A t y p i c a l biochemical a c t i v i t y of c y t o c h r o m e c3 is to act as an electron carrier for the h y d r o g e n a s e of D. vulgaris 11. The biochemical activities of the c y t o c h r o m e c3 p r e p a r a t i o n s (the h i g h l y purified p r e p a r a t i o n and the p r e p a r a t i o n o b t a i n e d b y t h e mild t r e a t m e n t ) were t h u s c o m p a r e d b y the H 2 evolution technique as described b y YAGI et al) 1 in t h e presence of excess hydrogenase. The results are i l l u s t r a t e d in Fig. 3.

Absorption spectra A b s o r p t i o n s p e c t r a of the ferri- a n d ferro-forms of the highly purified c y t o chrome c3 p r e p a r a t i o n were m e a s u r e d as follows. The side a r m of a T h u n b e r g - t y p e optical cell was charged with 34.7 ktg c y t o e h r o m e c 3 a n d o.18 unit purified h y d r o genase in 0.6 ml 0.02 M p h o s p h a t e buffer (pH 7.o), and the m a i n c o m p a r t m e n t with 2. 4 ml 0.02 M p h o s p h a t e buffer (pH 7.0). The vessel was p u r g e d with H 2 a n d filled with H 2 at 400 m m Hg, t h e n left s t a n d i n g for IO h at 4 ° to reduce the c y t o c h r o m e in the side arm. The c o n t e n t s were t h e n m i x e d a n d left s t a n d i n g for a n o t h e r few hours Biochim. Biophys. Acta, 229 (1971 ) 214 224

2I~

T.

YA(;I,

K.

MAI,~UYAM:\

to reduce the cytochrome completely; then the absorption spectrum of l\,rrocvt~chrome c3 was measured with a Hitachi i24 spectrophotometer equipped with a Hitachi Q P D 34 recorder. I~inally, the cell contents were exposed to air and the' absorption spectrum of ferricytochrome ca was recorded. The results are shown in l"ig. 4. The absorbance caused bv the added hydrogenase was about o.ooI2 at 28o nm and less in other regions of the spectra• This figure shows the first record of the absorption spectrum of ferrocytochrome c3 in the ultraviolet region, which, without purified hydrogenase, could have never been recorded because of the autoxidizability of the cytochrome and the strong absorption of Na2S204 which ordinarily is used to reduce the cvtochrome non-enzymatically. 0.6

, ....

, . , , , , ....

, , , . . , ....

, ....

, ....

0.5

0.2

*~0.i

0.

00

I , , , ,

• 250

= ,

I i , i , i J , i ,

300

350

,

~

400 450 Wavelength (nm)

500

550

600

Fig. 4. Absorption spectra of cytochrome c 3. i , f e r r i c y t o c h r o m e experimental conditions are described in the text.

c3;

2, ferrocytochrome

c.~. T h e

The absorption spectra of the ferri- and ferro-forms of the cytochrome ca obtained by the mild procedure were quite similar to those of the above mentioned spectra in visible region.

Redox potential The redox potential of cytochrome ca was calculated from measurements of the equilibrium between H 2 and cytochrome c3. When H 2 at pressure PH2 (in atm) and cytochrome c3 are in equilibrium in the presence of hydrogenase in a solution, the following relation will be attained RT E ° -cyt

E ° -H2

[H+I n iferrocytochrome]

nF

loge

PH2n/ei f e r r i c y t o c h r o m e ]

where R is the gas constant; T, the absolute temperature; F, the F a r a d a y constant; n, the n u m b e r of electrons transferred ; E°H2, the standard potential of the hydrogen electrode which is zero. From the above equation, the following equation will be derived at 3 °° . E ° = cyt

--0.060

(pH

+

1/zlogt0PH2)

+

0.060 --logto n

[ferrocytochrome] [ferricytochromel

Fig. 5 shows the plots of p H + 1/2 lOgl0 PH2 against log10 [ferrocytochrome]/

Biochim. Biophys. Acta,

243

(I971)

214-224

CYXOCHROME Ca OF Desulfovibrio vulgaris

2I 9

6.5 T Q. 6.0

+ el

5.5

o -leJ 5.C 0.5

0.0

1.0

1.5

[cyt C32+] log [cytc33÷] Fig. 5. Equilibrium between H 2 and c y t o c h r o m e c3 in the presence of hydrogenase. A T h u n b e r g type optical cell contained o.38 mg c y t o c h r o m e ca and o.2 unit purified hydrogenase in 3.o ml o.o2 M p h o s p h a t e buffer. The p H of the solution was 6.oo ( ~ ) , 6.39 (O), 7.oo (~)), or 7.1o ( 0 ) . The cell was purged with H 2 and filled w i t h H , at the pressure of o.85 atm, t h e n left s t a n d i n g for a few h o u r s to reduce the c y t o c h r o m e completely. The a-peak height of ferrocytochronle c 3 was measured spectrophotometrically. Then the H 2 pressure in the cell was reduced to the desirable pressure (PHi) and the cell was left s t a n d i n g for a n o t h e r few h o u r s for the m i x t u r e to a t t a i n equilibrium, and the a-peak height again determined. This process was repeated several times and finally the cell was filled with H~ to ascertain t h a t the initial a-peak height was recovered. The ratio of ferrocytochrome c a was calculated from the a-peak height of the spectra.

[ferricytochromel, with experimental procedures described in the legend. From the results, the redox potential of cytochrome ca was calculated to be --o.2 9 V and n to be I. Similar value for the redox potential was obtained by measuring tile redox equilibrium with redox dyes such as benzyl viologen.

Isoelectric point Cytochrome c3 and yeast cytochrome c were subjected to electrophoretic migration on cellogel at pH 9.04, IO.IO, and 11.o6. They behaved identically at these pHs. Thus, the isoelectric point of cytochrome ca is very close to that of yeast cytochrome c, i.e. lO.6 (ref. 20).

0.6

0.5

~o.4 o~

~

0.3

< 0.2

0.0

I

400

'

'

'

=

i

'

'

r

'

I-

450 500 Wavelength (nm)

i

i

i

550

Fig. 6. Absorption spectra of ferri-, ferro-, a n d CO-bound c y t o c h r o m e c 3. W h e n CO was introduced to a T h u n b e r g - t y p e optical cell containing ferrocytochrome c3 in o.o2 M p h o s p h a t e buffer (pH 7.o), the s p e c t r u m (Curve 2) changed gradually at room t e m p e r a t u r e , and in to min, the s p e c t r u m (Curve 3) was obtained. Curve I is the s p e c t r u m of ferricytochrome ca.

Biochim. Biophys. Acta, 243 (1971) 214-224

220

T. YA(;I, K. MARUYAMA

Binding of CO When CO was introduced to a Thunberg-type optical cell containing ferrocytochrome ca prepared by reducing the highly purified ferricytoehrome ca with Na2S204 at pH 7.o, the spectral change was observed (Fig. 6). The spectrum of ferrocytochrome c3 (Curve 2) was changed to Curve 3. The a, fl, and y peaks shifted toward the spectrum of the ferri-form (Curve I). However, the presumed CO-compound does not appear to be in the ferri-form, because its spectral curve does not cut the isosbestic points of the ferro- and ferricytochrome curves. The spectral change was also observed when the ferrocytochrome ca preparation obtained by the mild procedure was treated with CO. The spectrum of the CO-compound was identical with the Curve 3 in Fig. 6. POSTGATE2 previously had stated that no spectral change occurred upon treatment of cytochrome ca with CO.

Heine content (I) A cytochrome ca solution was dialyzed against distilled water (15 1, 24 h) and glass distilled water (i 1 2 times, 24 h each). The thoroughly dialyzed preparation thus obtained had an absorbance of 57-52 at 552 nm in the ferro-form. A I-ml portion of this preparation was frozen and lyophilized. Evacuation was continued for 20 h at room temperature after the cytochrome became a dry powder by visual appearance, and finally, for 3 tl at 85 °. Immediately after the vacuum was broken, the contents TABLE I THE

RESULTS

OF METAL

ANALYSES

OF CYTOCHROME

Ca

A n a l y z e d b y a t o m i c a b s o r p t i o n s p e c t r o m e t r y a t I n s t i t u t e for Solid S t a t e Physics, U n i v e r s i t y of Tokyo . Valu es are e x p r e s s e d as/*g.

Cytochrome c 3 (z.oo rag) in 0.5 ml aqueous solution Fe

I5.O

Blank (o. 5 ml diffusate of final dialysis) -4 O . O 2 5

Zn

i ,o

o.o

Cu

o.o

o.o

Mn

o.o

o.o

weighed 7.43 mg; the preparation absorbed humidity quickly and, after 2 min, it weighed 7.5o rag. The specific absorbance coefficient of the a-peak of ferrocytochrome c3 was, thus, 7.74 at pH 7.0. The compound, therefore, contains 3.9 moles heine per 14 ooo g (assumed molecular weight). (2) The results of metal analyses by atomic absorption spectrometry are summarized in Table I. These analyses indicate that cytochrome c3 contains 3.75 atoms Fe and 0.2 atoms Zn per 14 ooo g.

Amino acid composition The results of the amino acid analyses are shown in Table II.

N-terminal amino acid Dansylation of cytochrome c3 was carried out as described by GRAY22. Dansyl Biochirn. Biophys. Acta, 243 (1971) 214-224

CYTOCHROME Ca OF Desulfovibrio vulgaris

221

TABLE I I A M I N O ACID C O M P O S I T I O N OF C Y T O C H R O M E 6 3 OF

D. vulgar'is,

MIYAZAKI

(a). Moles/mole of p r o t e i n as c a l c u l a t e d from a m i n o acid a n a l y s i s . (b). Moles/mole of p r o t e i n to n e a r e s t wh ole i n t e g e r .

Residue

Moles/mole of protein

(~) Lysine Histidine Arginine Cysteate* Aspartate Threoninc Serine Glutamate Proline Glycine Alanine Half cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan T o t a l n u m b e r of resid ues Mol.wt. of p r o t e i n m o i e t y §§ Mol.wt. of 4 h e m e s Mol.wt. of c y t o c h r o m e ca

(b)

2o.9 9.5 o.o 7.7 12.5 6.5 5.o 5- i 3.9 9.7 i i, 8 6.8"* 4.5 2.8 o.o 3.o 2. 4 2. i

21

9-1o o 12-13 67 5 5 4 IO I2

5 3 o

3 2 3 2

o§ IO7--I I I II

488--I2

003

2 466 13 949-14 469

R e c o v e r e d from t h e p e r f o r m i c a c i d - t r e a t e d p r o t e i n d e r i v e d b y r e m o v a l of heroes of c y t o c h r o m e c3. ** H i g h r e c o v e r y of c y s t i n e b y mere h y d r o l y s i s of t h e n a t i v e c y t o c h r o m e c3 w a s also r e p o r t e d b y CORAL et al. 21. *** O b t a i n e d from the c y s t e a t e c o n t e n t . A s s u m e d to be zero b e c a u s e of low a b s o r b a n c e a t 28o nm. .~§ T h e p resence of i 2 - i 6 a m i d o g r o u p s is a s s u m e d from t h e a m i n o a c i d a na l ys i s .

chloride (5 mg) in 0.25 ml acetone was added to a mixture of cytochrome c3 (o.14 mg) and 240 m g urea in 0.8 ml 0.025 M NaHCOa buffer (pH 9.6), and the mixture left standing o+ernight at room temperature. The dansylated protein was precipitated by centrifugation, and washed twice with 8 ml water. The precipitate was then hydrolyzed with 0.5 ml 6 M HC1 in an evacuated glass tube for 5 h at lO5 °. The hydrolyzate was dried in a stream of N2, dissolved in water and dried. The residue was dissolved in 0.02 M acetic acid, and extracted with peroxide-free ether. The ether layer did not contain any dansyl-amino acid. The aqueous layer contained three fluorescent materials as revealed by thin-layer chromatography on spotfilm using Solvent I I of GROS AND LABOUESSE15. The material with the highest RF was dansyl-OH. That with the lowest RF had a free amino group, because, upon acetylation with acetic anhydride-pyridine (2:1, by vol.) (ref. 23), it could be extracted by ether; it was, thus, assumed to be e-dansyl-lysine. The material of intermediate RF was identified as dansyl-histidine upon cochromatography with authentic dansyl-histidine using Solvent II. Biochim. Biophys. Acta, 243 (i 97 I) :'14-224

222

"I'. Y A ( ; I , K. M A I { [ ' Y A M A

DISCUSSION

C y t o c h r o m e ca was first isolated by POSTGATE2 from D. vulgaris, Hildenborough, and b y ISHIMOTO et al. 5 from D. vulgaris, Miyazaki, in 1956- 7. Both authors reported t h a t t h e iron c o n t e n t of t h e c y t o c h r o m e was o.9°4, POSTGATE proposed a double heme s t r u c t u r e for c y t o c h r o m e ca because his molecular weight estimation was 13 ooo. Since then, s t r u c t u r a l analysis has m a i n l y been c o n d u c t e d with the c y t o c h r o m e ca of the H i t d e n b o r o u g h strain of D. vulgaris. Thus, in 1 9 6 1 , HORIO AND KAMEN 9 crystallized this c y t o c h r o m e ca and, on the basis of the results of their analysis of its heine content, s u p p o r t e d the double heme structure. In 197o, DRI:CKER ct al.7, s proposed a triple heme s t r u c t u r e for three p r e p a r a t i o n s of c y t o c h r o m e ca o b t a i n e d from D. vulgaris, H i l d e n b o r o u g h , D. salexigens, and D. desulfuricans, VC. According to the amino acid sequence r e p o r t e d b y AMBLER 2~, there are two sequences of the t y p e C y s - X Y Cys His , a n d two sequences of the t y p e Cys A B C D - C y s - H i s , a n d no SH or - S - S - linkage in the molecule for the c y t o c h r o m e ca of the H i l d e n b o r o u g h strain. W e have now o b t a i n e d c y t o c h r o m e ca of D. vulgaris, Miyazaki, in a homogeneous s t a t e a n d have e x a m i n e d its heme content, amino acid composition, and o t h e r i m p o r t a n t properties. The results are s u m m a r i z e d in Tables I I I a n d IV. I t is obvious t h a t the c y t o c h r o m e ca of D. wdgaris, Miyazaki, is different from t h a t of D. vulgaris, H i l d e n b o r o u g h , since t h e y are distinguishable in amino acid composition and o t h e r i m p o r t a n t properties. One of the most striking differences is t h a t the c y t o c h r o m c ca of D. vulgaris, Miyazaki, contains 4 hemes. This m e a n s t h a t the heine m o i e t y of this c y t o c h r o m e a m o u n t s to 17-18% of the molecule. Such h e m o p r o t e i n s have never been TABLE

111

COMPARISON

OF THE

Strain

Lysine Histidine Arginine Aspartate Threonine Serine Glutamate Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Number of residues

AMINO

ACID

COMPOSITION

OF CYTOCHROMES

Desulfovibrio vulgaris

Ca

Miyazaki

Hildenborough

D. desulfuricans, [:C

21 9-1o o 12-i 3 6-7 5 5 4 io 12 8 5 3 o 3 2 3 2 o lO7-111

20 9 1 ~2 5 6 5 4 9 io 8 8 3 o 2 3 2 o lO 7

17 9 o 7 5 (~ tI 6 8 0 8 9 i i 6 2 3 o lO8

24

7

liefs.

Biochim. Biophys. Acta,

2 4 3 ( i 9 7 I) 2 1 4 - 2 2 4

D. salexigens

22 0 o 9 6 ~2 5 5 i I 13 8 O 2 2 4 1 3 o 115 8

CYTOCHROME C3 OF

Desulfovibrio vu!garis

223

T A B L E IV C O M P A R I S O N OF T H E

PROPERTIES

OF T I l E C Y T O C H R O M E S g 3 OF

D. IJlglgar~$

D a t a c o n c e r n i n g t h e H i l d e n b o r o u g h s t r a i n are fronl the references i n d i c a t e d .

Cytochrome c3from

Refs.

M iyazaki strain

Hildenborough strain

0.52 6. 3 4 .0 1.o 5 0.77 2.98

0-54; °.51 6. 5 4.9; 4 .1

7; 9 7, 9 7; 9

0.74 2.95

9 9

Changed 4 14 ooo Histidine

Unchanged 2;3 13 5 °0 Alanine Glutamate IO-I I --0.205 V

2

Spectral data

Afl*/Aa* Ay*/Aa* AT/Aa* A6*/Aa" A6/Aa* da*/A~so U p o n c o n t a c t w i t h CO, the spectrum Heme content A p p r o x . mol. wt. N-terminus C-terminus pl Redox potential

N e a r lO.6 0.29 V

2, 9; 7 7, 24 24 24 2 2, 7

A b b r e v i a t i o n s : Aa*, Aft*, etc., a b s o r b a n c e s a t a, fl, etc., p e a k s of t h e ferro-form; AT, A6, a n d A28o, a b s o r b a n c e s a t y a n d 6 peaks, a n d a t 28o nm of t h e ferri-form; pI, isoelectric poi nt .

reported in the literature. The lower iron content a for the Miyazaki strain cytochrome ca reported earlier would be due to contamination with high molecular weight cytochrome which escaped detection until Sephadex column c h r o m a t o g r a p h y was practised (see Fig. I). The highly purified cytochrome ca combines with CO in contrast to the observations previously made b y POSTGATE2. One might suspect t h a t the cytochrome had been denatured when it was being eluted with o.I M N H a from the Amberlite column, since HORIO AND NAMER 9 reported the unstable nature of cytochrome ca against alkali. The cytochrome Ca preparation obtained b y one of the mildest procedures was also found to combine with CO. The highly purified cytochrome ca and the cytochrome Ca obtained b y the mild procedure behaved identically on Sephadex G-5o column chrom a t o g r a p h y (Figs. I and 2), had identical absorption spectra, and both of t h e m had identical biochemical activity in the hydrogenase system (Fig. 3)- It is thus evident t h a t the highly purified cytochrome ca preparation is not a denatured artifact. The N-terminus of cytochrome ca of D. vulgaris, Miyazaki, is histidine. Arginine, as well as isoleucine, is absent. These structural features are all different from the established amino acid sequence of cytochrome ca of D. vulgaris, Hildenborough 24. In spite of these differences, the amino acid compositions of both cytochromes are very similar (Table III). Both contain 8 cysteine residues, about 17 acidic amino acids (or their amides), and about 2o lysine residues. The n u m b e r of histidine, proline, methionine and aromatic amino acid units are almost the same in both cytochromes. The redox potential was estimated to be --o.2 9 V, instead of --o.2o 5 V reported for the other preparations of cytochromes ca (refs. 2, 6, 7). It is the lowest redox potential ever reported for c-cytochromes. Although the cytochrome ca contains Biochim. Biophys. Acta, 243 (I97I) 214-224

224

T. YA(;1, K. MAI{UYAMA

4 heroes, it behaves like a one-electron carrier in tile redox system as shown in Fig. 5. This means that 4 heroes nmst be surrounded by an almost identical redox environment and receive electrons independently as if they were retained in separate protein molecules. Elucidation of tile structure and of the mechanism of action of this unusual hemoprotein should help us to understand tile mechanism of electron transport on a molecular basis. ACKNOWLEDGEMENTS

We are indebted to Professor H. Inokuchi of University of Tokyo and Professor T. Ozaki of Shizuoka University who afforded us facilities used in the present experiments; to Professor E. J. Hehre of Albert Einstein College of Medicine for suggestions and advice in preparing manuscript ; to Dr. S. T a m u r a and Miss T. Miyazawa for the metal analyses; to Dr. K. Takahashi and Mr. K. Asaoka for the amino acid analyses; and to Miss A. Yagi for measuring H2-cytochrome equilibria. This work was supported in part by a grant from the Ministry of Education of Japan. REFERENCES

I J. R. POSTGATE, Biochem. J., 56 (1954) xi, 58 (1954) ix. J. R. POSTGATE, J. GeE. Microbiol., 14 (1956) 545. 3,I. ISHIMOTO, J. KOYAMA AND Y. NAGAI, Bull. Chem. Soc. Japan, 27 (1954) 565. M. ISHIMOTO, J. KOYAMA AND Y. NAGAI, J. Biochem. Tokyo, 41 (1954) 763 . M. [SHIMOTO, J. KOYAMA, T. YAGI AND M. SHIRAKI, J. Biochem. Tokyo, 44 (1957) 413. J. LE GALL, G. MAZZA AND N. DRAGONI, Bioehim. Biophys. Acta, 99 (1965) 385 • U. DRUCKER, E. B. TROUSIL, L. L. CAMPBELL, G. H. BARLOW AND E. MARGOLIASH, Biochemistry, 9 (197 °) 1515. 8 U. DRUCKER, E. B. TROUSIL AND L. L. CAMPBELL, Biochemistry, 9 (197 o) 3395. 9 T. HORIO AND M. D. KAMEN, Bioehim. Biophys. Acta, 48 (1961) 266. IO J. R. POSTGATE AND L. L. CAMPBELL, Bacteriol. Rev., 3 ° (1966) 732. I I T. YAGI, M. HONYA AND N. TAMIYA, Biochim. Biophys. Acta, 153 (1968) 699. I2 T. YAGI, J. Biochem. Tokyo, 68 (197 o) 649. I 3 D. H. SPACKMAN, V~7. H. STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. i 4 R. P. AMBLER, Biochem. J., 89 (1963) 349. 15 C. GRos AND g . LABOUESSE, Eur. J. Biochem., 7 (1969) 463 • 16 T. YAGI, J. Biochem. Tokyo, 66 (1969) 473. 17 R. A. REISFELD, [J. J. LEWIS AND D. E. WILLIAMS, Nature, 195 (1962) 281. 18 Y. NAGAI, in The Methods in Biochemical Experiments, P a r t I X , TAMPAKUSHITSU-KAKUSAN2 3 4 5 6 7

19 20 21 22 23 24

KOSO, 1967, p. 3 (in J a p a n e s e ) . M. ISHIMOTO, T. YAGI AND M. SHIRAKI, J. Biochem. Tokyo, 44 (1957) 707 . S. MINAKAMI, J. Biochem. Tokyo, 42 (1955) 749. M. L. COVAL, T. HORIO AND M. D. KAMEN, Biochim. Biophys. Aeta, 51 (1961) 246. "W. R. GRAY, Methods Enzymol., I I (1967) 139. A. A. BENSON, H. DANIEL AND J{. WISER, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1582. R. P. AMBLER, Biochem. J., lO9 (1968) 47 P.

Biochim. Biophys. Acta, 243 (1971) 214-224