Noguchi Ovexp

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ELSEVIER Biochimica et Biophysica Acta 1188 (1994) 302- 310B BB i o c h i ~ i c ~ at B iophysi ca A~ ta

Over-express ion of mem brane-bou nd cytochrom e c -551from therm ophi l ic Bac i l l u s PS3 in B ac i l l u s stearo therm oph i lu s K1041

Shunsuke Noguchi , Tamayo Yamazaki , Atsuo Yaginuma, Junshi Sakamoto, Nobuhito Sone *Department o f B iochemical Engineering and Science, Kyushu Institute of Technology, Kawazu 680-4, lizuka, Fukuoka-ken 820, Japan

Received 18 April 1994; revised 4 August 1994

A b s t r a c t

Cytochrome c -551 is a l ipopro te in o f about 10500 Da , found in the rmophi l ic Bacillus PS3 grown under a i r - l imited condi t ions .An express ion vec tor was cons truc ted from a s t ruc tura l gene of PS3 cy tochrome c -551 , sy th the t ic o l igonuc leo t ide as a p romote rfo r Bacillus stearothermophilus and a shu t t le vec tor fo r Escherichia coli a n d B. stearothermophilus. T h e t r a n s fo rme d c e ll s o f B .stearothermophilus K 1 0 41 e x p re s s e d c y to c h ro me c -55 1 a s mu c h a s 5 n mo l /m g m e mb ra n e p ro te in . T h e e f fe c ts o f o v e r -e x pre s s io non the hos t ce l ls a re ana lyzed ; a s l igh t ly s lower g rowth ra te and an inc reased syn thes is o f cy tochrome ox idase (about twofo ld)o c c u r re d . O v e r -e x p re s s e d (4 -1 0 - fo ld ) c y to c h ro me c -5 51 w e re p u r i f ie d , a n d i t s p ro p e r t i e s w e re e x a m in e d to k n o w w h e th e r th epro te in is p rocessed as in PS3 ce l ls g rown under a i r - l imited condi t ions . The molecu la r mass de te rmina t ion and t rea tment withRhizopus l ipase sugges ted tha t the same processes , c leavage of s igna l pep t idase , b lock ing of the N-te rmina l g roup and acy la t ionof g lycero l res idue b y two fa t ty acids, took p lace in the ov er-express ion sys tem. Fa t ty acy la t ion seems use fu l fo r the c y tochrom e cto be effectively oxidized.Keywords." C y to c h ro me c ; O v e r -e x p re s s io n ; cccA; Pos t- t rans la t iona l modif ica t ion ; (B. stearothermophilus)

1 . I n t r o d u c t i o n

T h e t h e r m o p h i l i c Bac i l lus P S 3 i s o l a t e d f r o mJ a p a n e s e h o t s p r i ng u s e s c y t o c h r o m e caa3- type o x i d a s ew h e n c u l t u r e d u n d e r h i g h l y a e r o b i c c o n d i t i o n s [ 1 - 3 ].T h e c e l l s a l s o s y n t h e s i z e c y t o c h r o m e b c 1 ( b 6 f ) c o m -p l e x e s [ 4 ] , a n d t h e s e t w o r e s p i r a t o r y c o m p l e x e s f o r m as u p e r - c o m p l e x w i t h o u t r e q u i r in g a n o t h e r c y t o c h r o m e ct o o x id i z e m e n a q u i n o l f o r y i e l d in g c h e m i o s m o t i c e n -e r g y f o r A T P s y n t h e s i s [ 5] . I n c o n t r a s t , P S 3 c e l l s g r o w nu n d e r a i r - l i m i t e d c o n d i t i o n s s y n t h e s i z e d h i g h e ra m o u n t s o f b -, c- a n d o - t y p e c y t o c h r o m e s , s u g g e s t i n gt h a t a d i f f e r e n t r e s p i r a t o r y c h a i n i s o p e r a t i n g [ 2,6 ]. O n eo f t h e m , s y n t h e s i z e d p r o f o u n d l y u n d e r a i r - l i m i t e d c o n -d i t i o n s , i s c y t o c h r o m e c - 5 5 1 , w h i c h i s c h o l a t e - e x t r a c t a -b l e m e m b r a n e - b o u n d s m a l l- s iz e d ( M r o f a b o u t 1 0 5 0 0)

Abbreviations: MOPS, 3-(N-morpholino)propanesulfonic acid;Tc, tetracycline; HPLC, high performance liquid chromatography;GC, gas chromatography; TMPD, N,N,N',N'-tetramethyl-p-phenyl-enediamine.* Corresponding author. Fax: +81 948 297801.0005-2728/94/$07.00 1994 Elsevier Science B.V. All rights reservedSSDI 0 0 0 5 - 2 7 2 8 ( 9 4 ) 0 0 1 3 7 - 5

c - t y p e c y t o c h r o m e [7 ]. T h e s e c h a r a c t e r i s t ic s o f r e s p i r a -t o r y c h a i n s e e m s t o b e g e n e r a l a m o n g t h e r m o p h i l i cBac i l l i , s i n c e Bac i l lus s tearo thermophi lus s h o w e d t h es i m i l a r r e s p o n s e o f r e s p i r a t o r y c h a i n v a r i a t i o n [ 8,9 ].

W e p u r i f i e d c y t o c h r o m e c - 5 5 1 f r o m P S 3 c e l l s g r o w nu n d e r a i r - l i m i t e d c o n d i t i o n s [ 7 ], a n d c l o n e d i t s s t r u c -t u r a l g e n e f r o m g e n o m i c D N A [10 ]. C o m p a r i s o n o f t h ed e d u c e d a m i n o a c i d se q u e n c e f r o m D N A w i t h c h a r a c-t e r i st i c s o f c y t o c h r o m e c - 55 1 a l l o w e d u s t o s p e c u l a t et h a t n a s c e n t c y t o c h r o m e c - 5 5 1 c o n t a i n i n g 1 1 1 a m i n oa c i d s is p r o c e s s e d t o r e l e a s e t h e s i g na l p e p t i d e o f 1 7 o r1 8 a m i n o a c i d s , a n d t h e n e w N - t e r m i n u s i s b l o c k e d .R h i z o p u s l i p a s e t r e a t m e n t a l so s u g g e s t s t h a t t h e c y s-t e i n e r e s i d u e a t t h e n e w N - t e r m i n u s w a s m o d i f i e d w i t ht w o f a t t y a c i d s p r o b a b l y v i a g l y c e r o l , a s i n t h e c a s e o fc - t y p e c y t o c h r o m e o f p h o t o s y n t h e t i c R h o d o p s e u -domonas v i r id i s r e a c t i o n c e n t e r [ 1 1 ] .

W e h a v e b e e n t r y i n g t o o v e r - e x p r e s s P S 3 c y-t o c h r o m e c - 55 1 i n o r d e r t o g e t l a r g e a m o u n t s o f th ec y t o c h r o m e f o r d e t e r m i n a t i o n o f it s s t r u c t u r e a n d f o rs e e k i n g a n i n t e r e s t i n g a p p l i c a t i o n a s a t h e r m o - s t a b l ea n d h y d r o p h o b i c s m a l l - s i z e d c y t o c h r o m e c . H e r e w er e p o r t a m e t h o d t o o v e r - e x p r e s s c y t o c h r o m e c - 5 51 i n a


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s. Noguc hi et al. / Biochirnica et Biophysica Ac ta 1188 (1994)302-310 303transformable Baci l lus s tearothermophi lus strain K1041,the effect of the expression vector on K1014 cells, andseveral properties of the expressed cytochrome, sug-gesting that the same processing observed in Baci l lusPS3 takes place in B. s tearothermophi lus K1041.

2 . M a t e r i a l s a n d m e t h o d s

2.1. Materials

the revcerse phase HPLC were applied to the capillaryby a syringe pump at a flow rate of 3 /zl/min. Thespray potential was 5 kV, and the orifice potential was100 V.SDS-polyacrylamide gel electrophoresis was carriedout according to the method of Laemmli with 15% gel[17]. Staining for hemes was carried out with o-tolui-dine [18].Other methods were the same as described previ-ously [6,11].

Baci l lus s tearothermophi lus K1041 [12], pSTE12 [13]and pJN531 [14] were kindly dona ted by Dr. Narumiand Prof. Kihara of Kansai Medical University (JichiMedical School at that time). An expression vectorpTrc99A was purchased from Pharmacia (Uppsala).The low molecular weight protein standards (bovineserum albumin, ovalbumin, carbonic anhydrase, soy-bean trypsin inhibitor and lysozyme) were purchasedfrom Bio-Rad, Richmond. Horse heart cytochrome cand MOPS were purchased from Sigma Chemicals, StLouis. Lipase from Rhi zopus de l emer , fine grade wereproducts of Seikagaku Kogyo (Tokyo). DEAE-Toyo-pearl HW-60 was a product of Tosoh (Tokyo). Otherreagents were obtained as described previously [10].2 .2 . Ana l y t i ca l metho ds

2.3 . Construct ion o f p lasm ids for over-expression of cy-t ochrome c-551The construction of the expressing plasmid,pSTEc551, is summarized Fig. 1. We used three kinds

of plasmids; pTrcc551 is a source of cytochromec-551structure gene with the initiation codon, which is de-rived from commercially-available pTrc99A, whilepJN531 is the source of promoter which has been usedto express Escherichia col i aspartate transcarbamylaseby Narumi et al. [14], and pSTE12 is a shuttle vector ofE. coli and B. s tearothermophi lus used by them [13]. Inorder to construct pTrcc551 we used 676 bp DNAcloned in pUC19 [10]. Treatment of plasmids and sev-eral methods for molecular cloning followed the meth-ods of Maniatis et al. [19].

Oxidase activity was measured polarographicallywith an oxygen electrode (YSI #4001) in a semiclosedcell, or followed with a pH meter (Beckman 4500) withascorbate (10 mM) in the medium (1.6 ml) composedof 25 mM K2504, 2.5 mM MgSO4 and 1 mM MOPS-KOH buffer (pH 6.4) at 40C as described previously[15].Absorption spectra were measured with a recordingspectrophotomete r (Beckman DU70) at room tempera-ture. The amount of cytochrome c-551 was determinedfrom the Na2S204-reduced minus oxidized differencespectrum using a millimolar extinction coefficient of20.9 at 551 [6]. The following millimolar extinctioncoefficients were used for the determination of othercytochromes; 23.2 at 604 to 630 nm for cytochrome aa 3[1], and 17.2 at 551 to 538 nm [16] for total cytochromec contents.Hydrophobicity of cytochrome c-551 was monitoredby HPLC with a C 4 column (Waters microbondasphareN10036) as described previously.Fatty acyl groups of cytochrome c-551 were meth-ylated with 5% BF3 in methanol at 70C for 30 min,and analyzed with a Shimadzu GC6BM gas chromato-graph or a GC-mass system composed of HP5QQGCand JOEL DX-303 with DA-500 Data System.Ion spray mass spectra were measured in a SciexAPI III instrument (Sciex, MDS Health Group, Thorn-hill, Canada). The samples of cytochrome c-551 from

2 .4 . Trans format ion o f B . s t earo thermoph i lu sA strain K1041 was transformed by electroporationusing A Bio-Rad Gene Pulser apparatus with a pulsecontroller by the method of Narumi et al. [12].

2.5. Culture of cellsThe small scale culture (200 ml) of B. s tearother-

moph i lu s K1041 was carried out at 55C in a 1-L flaskwith baffle by shaking vigorously (220 rpm). Themedium contained 0.8% polypeptone, 0.2% yeast ex-tract, 0.3% NaC1 and 0.05% K2HPO 4 (pH 7.2-7.8).The concentration of tetracycline, if present, was 1-2mg/1. The large scale culture (6 1) was carried out at55C in a jar fermentor (Tokyo Rika, MBF801) withvigorous aeration (1 1/min) and stirring (360 rpm). Thecells were harvested at an early stationary phase whenA650 was about 1.4. In order to follow the growth curveof cells more correctly the turbidity (light scattering at90) was monitored at 575 nm. The turbidity of 10 wasabout 1.0 A650. The membrane f ragments were pre-pared as reported previously [20].2.6. Puri f icat ion of over-expressed cytoch rom e c-551

The following procedure is simplified and modifiedfrom the previous one which was used to purified


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304 S . Noguch i e t a l . / Bio ch imica e t Biophysica Acta 1188 (1994) 3 02- 310

Pst I

~Sph I D i g e s t i o n ~Kpn I D i g e s t i o n~ B l u n t i n g ~ B l u n t i n g~ N c o I D i g e s t io n ~ N c o I D i g e s t io n~Nco IISph I f r a g m e n t ~Sty IIKpn f r a g m e n t

5 ' p G G A T C C C G G C d ~ G A C A G G T A A T G A ~ T T T C T A T A A T T T G T T A A T T A A G A G A A A ~ G A G G A C 3 '3 ' A C G T C C T A G G G C C C A A C T G T C C A T T A C T A C C T T A A A G A T A T T A A A C A A T T A A T T C T C " F F r C C C C T T C T C C T G G T A C p 5 '

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B a r n H I Eqo R I

~ E c o R I D i g e s t io n~ B l u n t i n g~ B a m H I D i g e s t i o n~Bam H I IEco R I f r a g m e n t

~ K p n I D i g e s t i o n~ B l u n t i n g~ B a m H I D i g e s t i o n~Bam H I IKpn I f r a g m e n t

E e 5 ~ / ~Fig. 1. Construction of pSTE c551 for expression in B. stearothermophilus K1041. The 676 bp PflMI-HindIII fragment of cccA gene coding forcytochrom e c-551, after b eing ligated with NcoI l inker (pGCCATGG C) was introduced into NcoI site of pTrc99A to construct pTrcc551. The666 bp NcoI-SphI (blunt) fragment of pTrcc551 was introduced into pJN531 containing the p romoter se gment to construct pJNc551, and the 745bp BamHI-EcoRI (blunt) fragment o f pJNc551 containing cccA gene after the promoter and Shine-Dalgarno sequence was introduced into themulticloning site of pSTE 12 to construct pST Ec551, which can b e am plified in E. coli and be expressed in B. stearothermophilus K1041.

c y t o c h r o m e c - 5 5 1 f r o m P S 3 c e l ls g r o w n u n d e r a i r -l i m i t e d c o n d i t i o n s [ 7 , 1 0 ] .S t e p 1 . T h e w a s h e d m e m b r a n e f r a ct io n f r o m

K 1 0 4 1 ( p S T E c 5 5 1 ) [1 0] w a s s u s p e n d e d i n a s o l u t i o nc o n t a i n i n g 2 % c h o l a t e , 0 . 5 M N a 2 S O 4 , 3 0 m M T r i s -H 2 S O 4 b u f f e r ( p H 8 .0 ) a n d 1 m M E D T A i n a fi n a lv o l u m e o f 1 0 0 m l . T h e m i x t u r e w a s s o n i c a t e d i n a n i c eb a t h f o r 1 0 m i n a t a n o u t p u t o f 5 w i t h a S o n i c a t o r( T o m y U D 2 0 1) , a n d c e n t r i f u g e d a t 1 4 0 0 0 0 g f o r 4 0m i n a f t e r 3 0 m i n s t i r r i n g .

S t e p 2 . P o l y e t h y l e n e g l y c o l # 6 0 0 0 w a s a d d e d t o t h es u p e r n a t a n t f r a c t i o n o f S t e p 1 a t a f i n a l c o n c e n t r a t i o no f 8 % . T h e s l ig h t ly t u r b i d s o l u t i o n w a s s t i r r e d f o r 3 0m i n a t r o o m t e m p e r a t u r e t h e n c e n t r i f u g e d f o r 2 0 m i na t 3 2 0 0 0 x g . T h e p i n k s u p e r n a t a n t w a s m i x e d w i t hp o l y e t h y l e n e g l y co l a n d M g S O 4 a t a f i n a l c o n c e n t r a t i o no f 3 0 % a n d 5 m M , r e s p e c t i v e l y , a n d s t i r r e d a n d c e n -t r i f u g e d a s b e f o r e a n d t h e r e d p r e c i p i t a t e w a s s o l u b i-l i z e d in 5 - 1 0 m l o f 1 0 m M T r i s - H C 1 b u f f e r ( t h e b u f f e r )c o n t a i n i n g 1 % T r i t o n X - 1 0 0 , a n d d i a l y z e d a g a i n s t 1 0m M T r i s - H C 1 b u f f e r f o r s e v e r a l h o u r s .

S t e p 3 . T h e s a m p l e f r o m S t e p 2 w a s a p p l i e d t o aD E A E - T o y o p e a r l c o l u m n ( 2 x 5 c m ) e q u i li b r a t e d w i t h

d i s t i l l e d w a t e r . T h e c o l u m n w a s t h e n w a s h e d w i t h t h eb u f f e r c o n t a in i n g 1 % T r i t o n X - 1 0 0 a n d 5 m M N a C 1( 2 0 0 m l ) . U p o n r a i s in g N a C I c o n c e n t r a t i o n t o 1 0 m Mt h e r e d b a n d m o v e d sl ow l y , a n d t h i s b a n d d u e t oc y t o c h r o m e c - 5 5 1 w a s e l u t e d b y r a i si n g t h e N a C 1 c o n -c e n t r a c t i o n t o 20 m M , w h e n t h e r e d b a n d r e a c h e d 1c m a b o v e t h e b o t t o n . T h e r e d p e a k f r a c t io n ( a b o u t 2 5m l ) w a s c o l l e c t e d .

S t e p 4 . T h i s f r a c t i o n , a f t e r d i a l y z i n g a g a i n s t 1 0 m MT r i s- H C 1 ( p H 8 . 0) , w a s a p p l i e d t o a D E A E - T o y o p e a r lc o l u m n ( 1 .5 x 6 c m ) . T h e c o l u m n w a s t r e a t e d a s i nS t e p 3 , a n d t h e r e d e l u a t e w i t h th e b u f f e r c o n t a i n i n g 2 0m M N a C 1 w a s d i a ly s e d a g a i n st w a t e r a n d a b s o r b e d o na s m a ll s iz e d D E A E T o y o p e a r l c o l u m n t o c o n c e n t r a t e .T h e c o l u m n w a s e l u t e d w i t h 1 0 m M T r i s - H C l b u f f e rc o n t a i n i n g 0 . 1 % T r i t o n X - 1 0 0 a n d 0 .1 M N a C I . T h ec y t o c h r o m e c -5 5 1 p r e p a r a t i o n ( u s ua l ly 1 - 2 m l ) w a sk e p t f r o z e n a t - 8 0 C u n ti l u s e .2 . 7 . P repara t i on o f de -acy l a t ed c y t och rom e c -551

P u r i f i e d c y t o c h r o m e c - 5 5 1 (2 0 n m o l ) w a s t r e a t e dw i t h R h i z o p u s l i p a s e ( 2 0 / z g ) i n 1 .5 m l o f 2 0 m M


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306 S . N o g u c h i e t a L / B i o c h i m i c a e t B i o p h y s ic a A c t a 1 1 8 8 ( 1 9 9 4 ) 3 0 2 - 3 1 0

LU( 2z

n-Oo 9m

TZIA=O.J.

/ i/ , i

.o

I I I J i500 550 600W A V E LE N GTH ( nm)

Fig. 3. Redox difference spectra of membrane fraction and cholate-extract. The spectrum of membrane fraction was suspended in 4%Triton X-100 containing 50 mM Tris-HCl (pH 8.0) and brieflysonicated. Reduce d (by Na2S204 addition) minus oxidized (as pre-pared) difference spectra are shown. (A) Membrane fraction fromK1041 (pSTEc551), 3.7 mg protei n/ml . (B) Membrane fracti on fromK1041, 4.2 mg prote in/ ml. (C) Cholate-extrac t of membra ne fractionfrom K1041 l(pSTEc551).

Table 3Summary of purificationStep Protein Cytochrome c Specific

(rag) (%) (nmol) cont ent(nmol/mg)

Membr ane 554 1048 (100) 1.891. Chol ate extract 140 931 (89) 6.632. Polyethylene glycol ppt. 87 578 (55) 6.73.1 st DEAE Toyopearl 40 541 (52) 13.54. 2nd DEAE Toyopearl 2.8 240 (23) 86.2

aa 3 contents. On the contrary, NADH oxidase activityof K1041(pSTEc551) was a little slower than those ofK1041 and K1041(pSTE12).3.4. Puri f icat ion of over-expressed cytoch rom e c-551

Table 3 summarizes a course of cytochrome c-551purification. The membrane fraction from the trans-formed cells with the cccA gene in the expressionplasmid (pSTEc551) were used as the starting material.The main purification step was chromatography in thepresen ce of Triton X-100. This step was re peate d againfor better purification. The final preparation was suffi-ciently concentrated (3 mg/ml) and pure. Fig. 4 showsprotein-stained SDS-PAGE patterns of membranefract ion of wild K1041 strain, that of K1041(pSTEc551),and the final c-551 preparation. We used cells har-vested from a 6-1 scale culture to prepare 500 mg

0.33 + 0.12 nm ol /m g pro tein (n = 13) and 5.3 + 1.6nmol/mg protein (n = 13) in mean + S.D., while thoseof th e wild-type were 0.18 + 0.05 (n = 8) and 0. 46 _0.17 (n = 8) nmol/mg protein. The data with TMPD-oxidase activities coincide with the data of cytochrome

Table 2A comparison of cytochrome contents and oxidation rates of mem-brane fractions from wild and transformed cellsCells Cytochrome content Oxidase activity

(nmol /mg protein) (/zg ato m/mi nper mg protein)

aa 3 total c c-551 TMPD NADHWild-type K1041 0.20 1.3 0.63 1.00 0.46K1041(pSTE) 0.17 1.2 0.62 1.10 0.44K1041(pSTEc551) 0.35 4.9 4.0 2.40 0.22The membran e fraction was prepared from vigorously aerate d cellsas described in Section 2. NADH and TMPD oxidase activities weremeasured polarographically and started by the addition 0.22 mM(final conc.) of NADH and 0,3 mM TMPD plus 10 mM ascorbate,respectively. The membranes used were 0.15-0.3 mg protein. Cy-tochrome c-551 content was the amount of cholate extractable cy-tochrome c per mg protein of the membrane fraction used for theextraction, while the total cytochrome c content was measured withTriton X-100-treated membrane. Other conditions were the same asthose shown in the legends for Fig. 3 and Table 1.

6 64 5:3 02 . 42 01 41 2 .

I Z 3 4Fig. 4. SDS-PAGE patter ns of over-expressed cytochrome c-551during its purification. The 15% gel [17] was stained with Coomassieblue R-250. Lane 1: marker proteins composed of bovine serumalbumin (66 kDa), ovatbumin (45 kDa), ca rbonic anhyd rase (30 kDa),tryps inogen (24 kDa), trypsin inhibitor (20 kDa), lysozyme (14 kDa)and horse heart cytochrome c (12.5 kDa). Lane 2: a membranefraction of wild K1041. Lane 3: a membrane fraction ofK1041(pSTEc551). Lane 4: the pur ified cyt ochrome c-551.


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S . Nog uchi e t a l. / Bioc h imica e t Biophysica Acta 1188 (1994) 3 02 -31 0 30 7protein of membrane fraction, and obtained 2.8 mg ofcytochrome c-551 by treating this membrane fraction.3.5. Structure of over-expressed cytochrome c-551

The cytochrome c-551 over-expressed in B.stearothermophilus K1041(pSTEc551) seems to be mod-ified very similarly to the naturally-occurring one whichwas prepared from PS3 cells grown under air-limitedconditions [7,10]. Over-expressed cytochrome c-551 andthat after Rhizopus lipase treatment, showed a verysimilar chromatographic pattern with a C4 column tothe PS3 cytochrome c-551 which were obta ined fromPS3 cells grown under air-limited conditions (notshown, but as shown in Fig. 6 of Ref. [10]). Theseresults thus indicate that release of two fatty acidresidues took place, as in the case of naturally occur-ring cytochrome c-551 in PS3 [9]. In order to analyzethe molecular species of fatty acids in the over-ex-pressed c-551, th e c-551 fraction of reverse phaseHPLC was extracted with CHC13/CH3OH to removeP-lipids and then converted to methyl esters with BF3.Fig. 5 shows typical gas chromatograms. The pres-ence of palmitic acid (C16:0) was prominent (2.36nmol/ cyto chrom e c-551) in cytochrome c-551 (upperpanel), while the content o f palmitic acid was low (0.34nmol/ c-551) in the deacylated cytochrome c-551 (lowerpanel). Several kinds of fatty acids, mostly branchedand saturated, were also found both in the intact c-551and t he deacylate c-551 as follows (intact c /dea cyla tedc, nmol): C15:0,iso (0.23/1.21), C16:0,iso (1.26/1.11)and C17:0,iso (0.58/1.08). These fatty acids were verysimilar to those of total P-lipids of the thermophilicBacillus PS3 [21]. It is thus likely that cytochrome c-551contains two satura ted f atty acids (mostly C16:0) in onemolecule, even though the specimen also contains small

t -

010

~- 0

C16:0

"~ C1 5:0, iso

o

0 2 4 6 8 10T I M E A F T E R I N J E C T I O N (ra in )

Fi g . 5 . G a s c h ro m a t o g ra p h i c e l u t i o n p ro f i l e o f me t h y l e s t e r s o f f a t tya c i d s e x t ra c t e d f ro m i n t a c t (u p p e r p a n e l ) a n d d e a c y l a t e d ( l o w e rpanel ) cy tochrome c-551 . The in tac t and deacyla ted species of cy-t o c h ro me c -5 5 1 w e re se p a ra t e d i n a C 4 re v e rse -p h a se c h ro ma t o g -ra p h y a s p re v i o u s l y d e sc r i b e d [1 0] . Sh i ma d z u G C 6 B M g a s c h ro ma t o -g ra p h e q u i p p e d w i t h 1 m g la s s c o l u mn p a c k e d w i t h 2 5 % e t h y l e n eglycol succinate on Shim al i te was used a t a carr ier gas f low ra te of 30m l / m i n . Th e t e m p e ra t u re w a s ra ise d f ro m 1 6 0 C a t a r a t e o f 5 C/ r a i n .

amounts of fatty acids originated from the P-lipidscontaminated.The Edman degradation of the purified over-ex-

pressed cytochrome c-551 with a gas-phase proteinsequencer did not give any peptide sequence, indicat-ing that the expressed cytochrome c in B. stearother-mophilus K1041 also had a blocked N-terminus, as hadPS3 cytochrome c-551 [10].

Fig. 6 shows the reconstructed mass spectrum of theover-expressed cytochrome c-551 and its deacylatedform. After the lipase treatment, the cytochrome

~- 100zz>

~ 50e~

0

I a I9,954B

,979

{ ~1~,014

I10,442- A

10,459

I9,800 9,900 I 0 ,000 11 ,0 0 0 10 ,40 0 I0 ,500 10,600M A S S N U M B E R M A S S N U M B E R

F ig . 6 . I on -s p ray m as s s pect ra . T he o r i g i na l m as s s pec t rum has been dec onv o lu t ed . (A ) T he i n t ac t c y t oc h rom e c -551; (B ) deac y la t ed w i t h l ipas e .


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308 S. Noguchi et aL / Biochimica et Biophysica Acta 1188 (1994) 302-31 0Table 4Effect of acylation on cytochrome c-551 as a substrate of cytochromec oxidaseSubstrate K m (~M ) Turnover

number( e - / c a a 3 )

PS3 cytochrome c-551 5.2 34.0Cytoch rome c-551 over- expressed 5.0 32.7Cytochrome c-551 deli pida ted 9.1 30.3Cytochrome c oxidase activity was follwed with a pH meter in thepresence of various concentrations of three types of cytochromesc-551. The delipidated cytochrome c-551 was prepared by treatingcytochrome c-551 with Rhizopus lipase and purified on a DEAE-Toyopearl column as in Step 4. PS3 cytochrome c-551 was preparedas described previously [10].showed a mass number of 9964, which is 442 smallerthan that before the treatment. The mass numbers ofprotein (from 19th Cys to C-terminus lllth Lys), hemec and glycerol group are 9234, 616 and 75, respectively,and t he sum minus 2 --- 9923, is a little smal ler th an thisvalue. This d ifference [41] is possibly due to a groupwhich blocks the amino group of N-terminal Cys-re-sidue. From the di fference of the mass number acetyla-tion of amino terminus is most likely, since acetylationadds 42 Da to the protein. The mass number of thenative cytochrome (10442) is attained if the two fattyacids are two molecules of C16:0.3.6. f f ec t o f a ey la t i o n o n e l ec t ro n d o n a t in g a c t i v i ty

Table 4 summarizes the effect of fatty acylation onthe cytochrome c oxidase reaction. The intact cy-tochrome c-551 showed a lower K m than that of thedeacylated cytochrome, although V~ax was the same. Itis thus likely that modification of fatty acid whichtransform the cytochrome into a lipoprotein is useful toincrease the affinity to the me mbrane-bound cy-tochrome c oxidase.3. . Hea t s t a b i l i t y o f cy to ch ro me c -5 5 1

Cytochrome c-551 was stable against heat denatura-tion. The activity of cytochrome c-551 as an oxidase

substrate was not injured up to 90C. However, thedeacylated cytochrome c-551 was less heat-tolerant;the incubation at 85C for 60 min resulted in 25%decrease, and 60% decrease aft er 90C incubation,when electron transfer-mediating activity was mea-sured at 40C with PS3 cytochrome c oxidase andascorbate (as a final electron donor). Thus the fattyacylation of the cytochrome seems to be useful forstabilization of the protein structure.

4 . Dis cus s ion

Cytochrome c-551 is an interesting cytochrome cfound in the thermophilic Ba ci l l u s PS3. This mem-brane-bound small-sized cytochrome c is synthesizedunder air-limited conditions [2,7] and thus is differentfrom the one found in subunit II of the ca a 3 - t yp eoxidase [1,22]. A similar C-type cytochrome, namedcytochrome c-550 was found in B. subt i l i s and reportedto be membrane-bound and small-sized (14 kDa)[23,24]. The deduced amino acid sequence of this cy-tochrome is clearly homologous to PS3 cytochromec-551 [10]. These Ba ci l l u s sequences are somewhatsimilar to, but dearl y different from the cytochrome c 8group of denitrifiers and the cytochrome c 6 group ofcyanobacter ia [25-27]. In spite of sequence similarity,these two Ba ci l l u s cytochromes c choose dif ferent waysto be membrane-bound; mature B. subt i l i s c-550 beingabout 14 kDa still has a signal peptide which is ahydrophobic membrane anchor [23], while PS3 c-551,being about 10 kDa, has no signal peptide moiety inthe mature form and is supposed to be modified into alipoprotein with two fatty acids, although its precisestructure has not been determined [10].We succeeded in overproducing PS3 cytochromec-551 in B. s t ea ro th ermo p h i lu s . The over-produced c-551 may be processed as in PS3 cells; the signal pep-tide part for the secretion is cleaved, and the newN-terminal amino group of the cystein residue isblocked probably with the acetyl group as well as thediacyl glycerol group being introduced to the thiol.

73

2 3 9II H20-CO-CIsH31

? H O - C O - C I s H nS

C ~ C O - N H - 1194 3 I

F e / 6 1 6 . 5

/ IC - A - S - C - H

9 2 3 9

, A _ K _ K 111 O O H

Fig. 7. A tentative struct ure of cytochrome c-551. The numbers are the molecular mass of respective portions.


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S . N o g u c h i e t a l . / B io c h imic a e t B io p h y s i ca A c ta 1 1 8 8 ( 1 99 4 ) 3 0 2 - 3 1 0 3 0 9

This derivative of the cytochrome gives a molecularmass of 10 442, and the removal of the two C16:0 fattyacids (or one C15:0,iso and one C17:0,iso) from thismodified cytochrome by lipase gives a mass of 9964.The most probable structure of cytochrome c-551 isshown in Fig. 7. The presence of diacylated N-terminalCys-residue was report ed in cytochrome-subunit havingtetra-heme of the reaction center of Rs. viridis [11] andthe E. coli lipoprotein of ou ter membrane [28], al-though these lipoproteins have a free N-terminal aminogroup.

The effects of transformation and expression on thehost cells (K1041) are summarized as follows: (1) slightretardation of growth took place in the presence ofpSTE12 as well as pSTEc551, suggesting that the retar-dation may be due to a secondary effect of presence ofthe plasmids. (2) Over-expression of cytochrome c-551was only observed in the transformant with pSTEc551and accompanied with an approx, twofold increase ofcaa3-type cytochrome oxidase. We do not know thereason why the synthesis of cytochrome oxidase in-creases upon over-expression of cytochrome c-551. Butan idea of simple induction of the catalyst upon abun-dance of its substrate seems not to be the case, sincecytochrome c-551 is not the physiological substrate forthe caa3-type oxidase. Cytochrome c-551, being ex-pressed much under air-limited conditions where thelevel of caa3-type cytochrome oxidase is reduced inwild PS3 and B. stearothermophilus cell, seems to be asubstrate of an alternative terminal oxidase [7,9]. Cy-tochromec-551 did not really accelerate the electrontransport of quinol oxidase activity due to the super-complex composed of cytochrome b c s ( b 6 f ) complexand caa3-type cytochrome oxidase [5]. It is also note-worthy that a deletion mutant of cccA gene in B.subtilis survives as before [23]. Thus this unexpect edincrease of cytochrome oxidase may indicate that bio-synthesis of the caa3-type cytochrome oxidase is ratherregulated in a sophisticated manner. For example,presence of a reduced form of cytochrome c-551 mayinduce the synthesis of caa3-type oxidase to get rid ofthe electron flow from the alternative terminal chaincomposed of cytochrome c-551 and the alternativeoxidase, which probably operates with a lower effi-ciency in forming an electrochemical proton gradientthan caa3-type oxidase does [6,29].

The lipophilic nature of cytochrome c-551 with twofatty acid groups seems important for the cytochrometo be an efficient substrate (Table 4). The cytochromeis also heat-stable. These characteristics are uniqueamong various cytochromes c which have been used asa conventional electron donor having an E m of 0.2-0.3V. In fact the reduced form of this cytochrome c-551can be used in the reduced pulse experiment [3] toshow proton pump activity of the terminal oxidase.This cytochrome is now over-produced, and will be

used in different ways. We are also studying its behav-ior in liposomes.

AcknowledgementsWe wish to thank Dr. I. Narumi and Prof. Kihara

for giving us several strains of B. stearothermophilusand thier plasmids for expression. We also wish tothank Dr. Umeda of Takara Shuzo Co. for kindlytaking the ion-spray mass spectra, and Dr. M. Nishi-hara and Prof. Koga of University of Occupational andEnvironmental Health and Dr. K. Goto of Jichi Medi-cal School for operating gas chromatography and tak-ing GC-mass spectra. This work was supported in partby Grant-in-Aids (04266217) from the ministry of Edu-cation, Science and Culture of Japan, and by a Grantfrom the Ciba-Geigy Foundation (Japan).

References[1] Sone, N. and Yanagi ta , Y. (1982) Biochim. Biophys. Acta 682,216- 226 .[2] Sone, N ., Kagawa, Y. and Or i i , Y. (1983) J. Biochem. 93,1329-13363.[3] Sone, N. and Hinkle, P .C. (1984) J. Biol . Chem. 257, 12600-12604.[4] Kutoh, E . and Sone, N. (1988) J. Biol . Chem. 263, 9020-9026.[5] Sone, N., Sekimachi , M. and Kutoh, E . (1987) J. Biol . Chem.262, 15386-15391.[6] Sone , N., Kutoh, E . a nd Sa toh, K. (1990) J. Biochem. 107,597- 602 .[7] Sone, N., Kutoh, E . and Yanagi ta , Y.(1989) Biochim. Biophys.Ac ta 977 , 329-3348 .[ 8] De Vr ij , W. , Heyn e , R . I . an d K on ings , W.N. ( 1989) E ur . J .Biochem. 178, 763-770 .[9] So ne, N. a nd F uj iwara, Y. (1991) J. Biochem. 110, 1016-1021.[ 10] Fu j iwar a , Y . , Oka , M . , Ham amoto , T . an d So ne , N . ( 1993)Biochim. Biophys. Acta 1144, 213-21 9.

[11] Weyer , K.A., Schafer , M., Lot tspeich, F . and Michel , H. (1987)Biochemist ry 26, 2909-2914.[12] Narumi, I . , Sawakami, K., Nakamoto, S . , Nakayama, N., Yanagi-sawa, T ., Takahashi , N. and Kihara, H. (1992) Biotechnol .T echn . 6 , 83 - 86 .[ 13] Nakayama, N . , Nar um i , I ., Nakamoto , S . and Kihar a , H . ( 1992)Biotechnol . Let t . 14, 649-652.[ 14] N ar umi , I . , Sawakami , K . , K imur a , T . , Nakamoto , N . , Nakayama,

N. , Yanag i sawa , T . , T akahash i , N . and Kihar a , H . ( 1992)Biotechnol . Let t . 14, 759-764.[15] Nichol ls, P . and Sone, N. (1984) Biochim. Biophys. Acta 767,240- 247 .

[16] De eb, S .S. and Hag er , L .P. (1964) J. Biol . Chem. 239, 1024-1031.[ 17] L aem ml i , U .K. ( 1970) Na tu r e 227 , 680- 685 .[ 18] Re id , G .A. an d I ng l edew, W.J . ( 1980) FE BS L e t t . 109 , 1 - 4 .[ 19] M an ia t i s , T . , F r i t sch , E .F . a nd Sam br ook , J . ( 1982) i n Molecu la rClon ing : A L abor a to r y Man ua l , Co ld Spr ing Har bor , NY.[20] Yoshida, M ., Sone, N., Hirata, H . an d Kagawa, Y. (1975) J. Biol .Chem. , 250 , 7910-7916 .[21] Sone, N., Yoshida, M., Hirata, H. and Kagawa, Y.(1975) J. Biol .Chem. 250, 7917-7923.


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31 0 S . N o g u c h i e t a l . / B io c h imic a e t B io p h y s i c a A c ta 1 1 8 8 ( 1 9 94 ) 3 0 2 - 3 1 0[22] Ishizuka, M., Machida , K., Shimada, S. , M ogi , A. , Tsuchiya , T. ,

O h mo r i ,T . , So u ma , Y . , G o n d a , M . a n d So n e , N . (1 9 9 0 ) J .B i o c h e m. 1 0 8 , 8 6 6 -8 7 3 .[23] Von Wachenfe ld t , C. and Hedersted t , L. (1990) J . Bio l . Chem.265, 13939-13948.

[24 ] V o n Wa c h e n fe l d t , C . a n d H e d e rs t e d t , L . (1 9 90 ) FEB S Le t t . 2 7 0 ,147-151.

[25] Wood, P. (1978) Eur. J . Biochem. 87 , 9 -19 .

[26] Cohn, C.L. , Hermodson , M.A. and Krogmann, D.W. (1989)Arch .Biochem. Biophys. 270 , 219-226 .

[2 7] So n e , N . a n d T o h , H . (1 99 4 ) FEM S M i c ro b io l . Le t t. 1 2 2 , 2 0 3 -210.[28] Hantke , K. and Braun , V. (1973) Eur. J . Bio ichem. 34 , 284-296 .[29] Sone, N. (1990) in The Bacteria vo l . XII (Krulwich , T.A. , ed .) ,p p . 1 -3 2 , A c a d e m i c Pre s s, N e w Y o rk .

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