The conformational changes of catalase molecule caused by ligand molecules

24

BI~)(HIMI(.K E l I~U)PHYSIt .\

.\(

i \

BBA 3532O F H E C O N F O R M A T I O N A L C H A N ( I E S O F C A T A L A S E MOI,E(iULI~ C A U S E I ) BY LIGAND MOLECULES

T A T S U Y A S A M E J I M A AND MASAI,[O I < [ T A

Ogpaft~lg~l¢ of ChemistEv, College oj Science and F.ngineering, doyama (;akuin Ut~iverst/v, Selagayo" l"okvo (Japan) ( Receiv ed A u g u s t 12th, [068)

SUMMARY

Circular dichroism a n d optical r o t a t o r y dispersion of bovine liver catalase have been m e a s u r e d over wide w a v e l e n g t h range of 2oo 65o m/~. N a t i v e catalase showed a small n e g a t i v e CD b a n d in the Soret a b s o r p t i o n region, which c o r r e s p o n d e d to a small n e g a t i v e Cotton effect. The d e n a t u r a t i o n with acid (pH 3.o), alkali (pH I2.o) a n d 8 M urea caused a l m o s t c o m p l e t e loss of this Sorer CD b a n d a n d the Cotton effect. A d d i t i o n of the ligand molecules such as KCN a n d NaN 3 to n a t i v e c a t a l a s e caused an a p p e a r a n c e of new small CD b a n d n e a r 45o m#. In the n e a r - u l t r a v i o l e t region n a t i v e molecule e x h i b i t e d a CD b a n d at 285 m#, while the d e n a t u r a t i o n s caused blue shift of the b a n d with a large e n h a n c e m e n t in i n t e n s i t y at p H I2.o as well as d i m i n u t i o n s at p H 3.o a n d b y 8 M urea. The l i g a n d e d catalase molecules showed a new b a n d n e a r 34 ° m # in a d d i t i o n to the CD b a n d at 28o 29o m#. These CD b a n d s m a y be r e l a t e d to a r o m a t i c amino acids a n d also their i n t e r a c t i o n with the heine groups in the molecules. In the u l t r a v i o l e t region all p r o t e i n s except for the d e n a t u r e d one with 8 M u r e a e x h i b i t e d two intense n e g a t i v e CD b a n d s a n d a deep t r o u g h of Cotton effect at 233 m#, which i n d i c a t e d the presence of a-helical s t r u c t u r e in their molecules. I t was concluded from these d a t a t h a t the helix c o n t e n t of the n a t i v e molecule was a b o u t ~,~ J ~ o/ / 0 , whereas the acid- a n d a l k a l i - d e n a t u r e d molecules showed the less helical c o n t e n t of 20-25°~ a n d I 5 - 2 ~ /o/ o , respectively. T r e a t m e n t with 8 M urea seemed to cause complete loss of lnelical c o n f o r m a t i o n in the molecule. However, a d d i t i o n of ligands (KCN a n d NAN3) to the heine groups of c a t a l a s e caused r e d u c t i o n of helical content in the molecule whose enzymic a c t i v i t y was c o m p l e t e l y lost. The e s t i m a t e d helical c o n t e n t was a p p r o x . 3o %.

INTRODUCTION

The c o n f o r m a t i o n of bovine liver catalase was i n v e s t i g a t e d according to measurem e n t s of o p t i c a l r o t a t o r y dispersion (ORD), which suggested t h a t n a t i v e catalase has A b b r e v i a t i o n s : ORD, o p t i c a l r o t a t o r y dispersion; CD, c i rc ul a r dichroism.

Biochim. Biophys. dcla, 275 (i909) 24 3 °

CONFORMATIONAL CHANGE OF CATALASE

25

about 5o °/o of a-helix ~. Dissociation of the molecule into two subunits by denaturation with acid at p H 3.o was accompanied by diminution of helix content to 2o-25 %o (ref. I), while alkaline treatment at pH 12.o caused a dissociation into four subunits with helical content of I~" 20 °',o (ref. 2). Thus, the use of the measurements of O R D provides us informations for conformational changes of macromolecules. Circular dichroism (CD) is another new approach to investigate the conformation of macromolecules with better resolution for optical activity in some cases. However, no study on CD of catalase has been reported as yet. Therefore, we have investigated CD of catalase and the corresponding Cotton effect for comparison in view of the conformational changes with d e n a t u r a n t s such as acid, alkali and 8 M urea. It is well known t h a t the enzymatic activity of catalase was reversibly inhibited b y addition of small ligand molecules such as cyanide and azide to iron atom in the heme groups as prosthetic group of the protein. It has been reported that addition of ligand molecules to myoglobin causes no eonformational change in the protein moiety a 5, while addition of oxygen molecule to hemoglobin causes a small reduction of a-helical content in the protein 6. However, no information has been available in respect of conformational change, if any, in the catalase molecule when it is complexed with small ligand molecules. Hence, it seemed worthwhile to investigate the conformational change of the liganded protein molecules from a viewpoint of the inhibitory mechanism for the enzyme. The present s t u d y reveals the results of CD spectra of native and denatured catalase molecules as well as the liganded molecules in comparison with the O R D curves.

MATERIALS AND METHODS

Materials The preparation of crystalline bovine liver catalase was performed according to the m e t h o d of SHIRAKAWA 7, which was briefly described in English elsewhere 8. The activity of catalase was measured b y the method of VON EULER AND JOSEPHSON 9. The 'Katalase-F~higkeit' of the purified catalase at o ° was 25 ooo-28 ooo. The concentrations of the catalase solutions were determined spectrophotometrically, with the value E ..... ~o; -- 13.5 at 4o5 m/z, which corresponds to a molar extinction coefficient of 3.24" lOS, based on a molecular weight of 24o ooo (ref. 8). The protein was dissolved in o.i M phosphate buffer at p H 7.o. Denaturation was performed by adjusting the solution at p H 3.o with o.I M KH2PO4phts dilute HC1 or at pH 12.o with o.I M Na2HPO plus NaOH, and b y adding 8 M urea to the buffered solution at neutral pH. Azide- and c y a n - c a t a l a s e complexes were prepared b y adding a large excess of the inhibitors (more than 3o times the catalase in molarity) to native catalase solutions in o.I M phosphate buffer at p H 7.o. The chemicals used were of reagent grade.

Methods The measurenlents of CD and O R D were carried out with a Jasco O R D / U V - 5 recording spectropolarimeter equipped with a CD a t t a c h m e n t at room temperature (23-27°). The instrument was calibrated with a s t a n d a r d sucrose solution and cholestane-3-one or ( + ) - I o - c a m p h o r sulfonic acid for O R D and CD, respectively 1,1°. The absorbances of the sample solutions at each wavelength were kept well below 2 to eliminate possible artifact 11. The cells were IO, ~, o.i and o.o 5 m m in length. The CD Biochim. Biophys. Acre,, 17.5 (1969) 24 3 °

"1. SAMEJIMA, M. I£.IT;\

2() and

()RD

data

were

expressed

m e a n residue r o t a t i o n , m ,

in terms

of the

mean

molar

ellipticity,

I 0 =, a n d

the

t a k i n g I29 for mean residue weight, M~ le

RESULTS

ORD and CD in the visible a~zd near-ultraviolet regio~zs Figs. I a n d 2 show the O R D curves a n d CD s p e c t r a of native, a c i d ( p H 3.0)-, a l k a l i ( p H 12.o)- a n d 8 M u r e a - d e n a t u r e d catalase as well as c y a n - a n d a z i d e - c a t a l a s e complexes. The n a t i v e catalase e x h i b i t e d a negative CD b a n d at 395 m#, which c o r r e s p o n d e d to a n e g a t i v e Soret Cotton effect having an inflection point near 395 mkt. This b a n d position is blue-shifted b y a b o u t IO m/~ from the Soret a b s o r p t i o n m a x i m u m at 4o5 m/~. Tim d e n a t u r a t i o n with acid, alkali a n d 8 M urea caused complete disapp e a r a n c e of the Soret CD b a n d a n d the Cotton effect as shown in Fig. I. On the o t h e r hand, the a d d i t i o n of the i n h i b i t o r y ligand molecules (both KCN a n d NaNa) to the n a t i v e molecule b r o u g h t a b o u t a new small n e g a t i v e b a n d n e a r 45 ° mkt besides the original n e g a t i v e b a n d at 395 m # as shown in Fig. 2. These new small b a n d s were also f a i n t l y m a n i f e s t e d in the O R D curves. I n the a b s o r p t i o n s p e c t r a the Soret a b s o r p t i o n n m x i m u m of n a t i v e catalase shifted from 405 m/~ t o w a r d 420 in/~ with a small reduction in i n t e n s i t y when it was complexed with cyanide, while the azide catalase complex 0

250

~50

350

550

~oo

., ..-' . U > -

0

ESO

~C~

t.Oo

300 i

i

/

~,

,

C09

i

i

200 j~

I00 300

/(/(

I

I

-Native ....... p~ 3

400

300

(ac,

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pH ~2

....

8 M Urea

200

/

---,KCN

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Fig. i. O R D c u r v e s a n d C1) s p e c t r a of n a t i v e , acid-, a l k a l i - a n d u r e a - d e n a t u r e d c a t a l a s e i n t h e v i s i b l e a n d n e a r - u l t r a v i o l e t r e g i o n s . T h e s o l v e n t s u s e d w e r e o.I M p h o s p h a t e b u f f e r ( p H 7.o) for n a t i v e , o. i M K H ~ P O 4 plus HC1 a t p H 3.o for a c i d - d e n a t u r e d , o. I M N % H P O 4 plus N a O H a t p H i 2 . o for a l k a l i - d e n a t u r e d , a n d o.I M p h o s p h a t e b u f f e r ( p H 7.o) plus 8 M u r e a for u r e a - d e n a tured catalase. Fig. 2. O R D c u r v e s a n d C D s p e c t r a of c y a n - a n d a z i d e c a t a l a s c c o m p l e x e s in t h e v i s i b l e a n d n e a r - u l t r a v i o l e t r e g i o n s . B o t h s o l u t i o n s c o n t a i n o. i M p h o s p h a t e b u f f e r ( p H 7.o).

Biochim. Biophvs. Acta, 175 (r969) 24 30

CONFORMATIONAL CHANGE OF CATALASE

27

displayed a maximum at 411 m# with the same intensity as that of the native protein. No absorption maximum was detected near 450 m# for the liganded molecules. However, the new CD band may indicate the presence of a weak optically active absorption in these complexes at this wavelength. In the near-ultraviolet region a positive CD band was observed at 285 m# for native molecule, which may be ascribed to amino acid groups in the protein moiety. The denaturation with acid at pH 3.0 and 8 M urea caused blue-shift of the band toward 260-27 ° m/z with decrease in intensity, whereas the denaturation with alkali at pH 12.o caused blue-shift toward 250 m/z with a great enhancement of the band intensity. This enhancement in intensity at pH 12.o may be due to ionization oftyrosyl residue in the protein moiety. The liganded eatalase molecules showed new small band near 340 m# in addition to the original band near 285 m#. It is noted that the latter band shifted to 280 m/~ in azide-catalase complexes, while cyan-catalase exhibited red-shift toward 290 m/~. In the ORD no Cotton effect was detected in this wavelength region, which indicates the better resolution of the CD than the ORD in this case. ORD and CD in the ultraviolet region In Figs. 3 and 4 the ORD curves and CD spectra of the native, denatured, and liganded eatalase are illustrated. The native catalase showed a Cotton effect with a crossover at 223 m# having a deep trough at 233 m/~ and a large peak at 198 m/~ (this peak is not shown in Fig. 3) with a magnitude of Era! approx. + 4 0 ooo, which confirmed the previous results1, ~. The corresponding CD bands were observed at 223 and 208 m/~ both in negative sign, and at 191 mF in positive one with an intensity of i01 approx. 36 ooo (this band is not shown in Fig. 3). These results indicate that native ~, 210

I

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2130

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210

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Fig. 3. T h e u l t r a v i o l e t O R D c u r v e s a n d C D s p e c t r a o f n a t i v e , acid-, a l k a l i - a n d u r e a - d e n a t u r e d c a t a l a s e . S o l v e n t s u s e d a r e t h e s a m e a s i n Fig. i. Fig. 4. T h e u l t r a v i o l e t OIRD c u r v e s a n d C D s p e c t r a o f n a t i v e c a t a l a s e , c y a n complexes.

and azide catalase

Biochim. Bioph3,s. Acta, I 7 5 (1969) 2 4 - 3 o

2~

"1. S:\MF.JIMA, 3I. t,HT.\

catalase has a significant degree of r i g h t - h a n d e d ~,-helical structure. The acid- and a l k a l i - d e n a t u r e d catalase still r e t a i n e d a large lew)rotation at the 233 m # trough and the negative CD b a n d s at 224-225 a n d 2o8--2Io m#, which indicate t h a t the d e n a t u r e d catalases still contain certain a m o u n t of helicity in spite of their gross c o n f o r m a t i o n a l changesL2. The t r e a t m e n t with 8 M urea caused a complete loss of (~-helicity judging from the profiles of the O R D a n d CD p r e s e n t e d in Fig. 3. The 233 m~ t r o u g h was v e r y shallow a n d its reduced mean residue r o t a t i o n , [m'!, corresponded to a b o u t 2ooo, while the CD s p e c t r u m no longer showed the c h a r a c t e r i s t i c two n e g a t i v e CD b a n d s for a-helix, a n d e x h i b i t e d only v e r y weak b a n d near 215 raft. The c o n f o r m a t i o n a l changes in the protein m o i e t y of c y a n a n d a z i d e - c a t a l a s e complexes are e v i d e n t from Fig. 4, which illustrates their O R D a n d CD in the u l t r a violet region. The 233 mff troughs in the Cotton effect were reduced, which correlated with the r e d u c t i o n of the negative CD b a n d s at 223 mff a n d 2o8 2 I o m#. These results indicate t h a t the c o n t e n t of~l-helix was s o m e w h a t reduced on the f o r m a t i o n of the cyana n d azide--catalase complexes. This is in close a g r e e m e n t with results of O R D analysis based on the MOFHTT YAN(; e q u a t i o n xa to be further e l a b o r a t e d on in D~SCUSSION. DISCUSSION I t has been confirmed b y the present s t u d y t h a t the a d d i t i o n of ligand molecules such as c y a n i d e a n d azide caused small c o n f o r m a t i o n a l changes in the p r o t e i n m o i e t y . The helical c o n t e n t was d i m i n i s h e d b y the complex f o r m a t i o n with ligand molecules. The absolute helical c o n t e n t according to the m a g n i t u d e of t r o u g h of the Cotton effect at 233 m/~ has been s o m e w h a t c o n t r a d i c t o r y . However, r e c e n t l y the m a g n i t u d e at 233 m/~ for perfect helical p o l y - I . - g l u t a m a t e has been a l m o s t settled to be - - I 5 ooo for the r e d u c e d m e a n residue r o t a t i o n u. Using the value of - - 2 o o o for r a n d o m coil of p o l y - L - g l u t a m a t e , t h e following forrnula can be used for t h e e s t i m a t i o n of helical content : ltelix o.,.o:=

([m']laa . 2000) X loo/I 3 ooo

The e s t i m a t e d helical contents b y this f o r m u l a are listed in T a b l e I, which are in good a g r e e m e n t with those o b t a i n e d b y the CD s p e c t r a described later. According to tile 233 m/z t r o u g h the l i g a n d e d catalase showed also less helicity of a b o u t 3 o % t h a n t h a t of n a t i v e catalase, a b o u t 50O.,o. I t was suggested t h a t the i n t e n s i t y of CD b a n d s in the u l t r a v i o l e t region is c o n f o r m a t i o n - d e p e n d e n t 1~. Therefore, we can e s t i m a t e tile c o n t e n t of a-helix b y the m e a s u r e m e n t s of the CD b a n d s near 223 or 208 m#. However, the absolute m a g n i t u d e s of these CD b a n d s have not y e t been established. F o r IOO~/o helical p o l y - L - g l u t a m a t e the value of [0], the m e a n residue ellipticity, has been r e p o r t e d as - - 3 5 ooo to approx. _ 4 0 oool.5 iv whereas the value of a r a n d o m coil is almost zero for various polyp e p t i d e s 15. However, it should be noticed t h a t proteins m a y h a v e a significant a m o u n t of b a c k g r o u n d at 222-223 m/~ p r o b a b l y due to cystine residues. There seems to be no a c c u r a t e a n d established m e t h o d for e s t i m a t i n g the helical c o n t e n t according to CD spectra. The e s t i m a t i o n was a t t e m p t e d b y assuming t h a t [0122~a n d [0j209 are - - 3 7 5 °0 a n d ---35 Ioo, respectively, for a perfect helix according to the results of HOLZWARTH AND DOTY 1'5, a n d for a r a n d o m coil of catalase is zero since the 8 M u r e a - d e n a t u r e d catalase showed v e r y small i n t e n s i t y a r o u n d this w a v e l e n g t h region. T h e e s t i m a t e d Biochim. Bioph~,s. Acta, ~75 (1969) z4 3°

C O N F O R M A T I O N A L C H A N G E OF C A T A L A S E TABLE

29

1

PARAMETERS

OF O R D

A N D H E L I C A L C O N T E N T S OF C A T A L A S E

D a t a f o r b 0 a r e b a s e d o n m e a s u r e m e n t s b e t w e e n 24o a n d 6o0 n v t for a c i d - , a l k a l i - a n d u r e a d e n a t u r e d c a t a l a s e , a n d t h e c a l c u l a t i o n s w e r e l i m i t e d t o b e t w e e n 24o a n d 34 ° m # f o r n a t i v e catalase, cyan a n d a z i d e e a t a l a s e c o m p l e x e s , a o a n d b o w e r e c a l c M a t e d f r o m t h e MOFFITTYANG e q u a t i o n 'a w i t h ).0 p r e s e t a t 212 m,u.

('alalase

Native Acid-denatured Alkali-denatured Urea denatured Cyan catalase Azide catalase

[((t)

ao

87 75 90 lie 72 85

be

19o 48o 6i o 7[0 200 34 °

32o I ~5 90 +40 200 23o

~z-helix (%) ,m']2:~ ~

CD

b~

42 i~ i2

5° IS i0

57 25 20

27 27

3o 33

47 43

helical c o n t e n t s from the values at the two wavelengths were averaged, results of which are s u m m a r i z e d in Table I t o g e t h e r with the values b y O R D analyses. The helicity from CD s p e c t r a agreed well with t h a t from the 233 m/z t r o u g h m e t h o d . The e x t e n t of the d i m i n u t i o n of a-helix in the liganded molecules was also e s t i m a t e d according to b0 value, a p a r a m e t e r of O R D a n a l y z e d b y the MOFFITT--YANG e q u a t i o n 13. The e s t i m a t i o n with MOVl~ITT-YANG e q u a t i o n is u s u a l l y p e r f o r m e d b y setting 20 - - 212 m # in the equation. F o r c y a n - a n d a z i d e - c a t a l a s e complexes as well as n a t i v e catalase, the graphic d e t e r m i n a t i o n s were l i m i t e d to the w a v e l e n g t h range of 24 ° to 34 ° m/~ because of the Cotton effects i n d u c e d b y the heine groups. URNES 3 a n d URNES AND DOTY H carefully e s t i m a t e d the r o t a t i o n due to the Soret Cotton effects a n d concluded t h a t a correction a m o u n t s to a b o u t 2 ~.~ for myoglobin. I t is not known how the Soret Cotton effects of catalase will affect the O R D in the n e a r - u l t r a violet region. However, such effect m a y be small since catalase shows much smaller r o t a t i o n a l s t r e n g t h in the Soret region t h a n t h a t of myoglobin. I t should be n o t e d t h a t the e s t i m a t e d b0 values for native, cyan a n d azide catalase were less a c c u r a t e t h a n those of the d e n a t u r e d ones because of the Soret Cotton effects. The estimnated a 0 a n d b0 values are listed in Ta])le I including those of the d e n a t u r e d catalase a n d values of [a 'n. The catalase molecule is r e g a r d e d to be c o m p l e t e l y d e n a t u r e d in 8 M urea solution, a n d non-helical b0 correction a m o u n t e d to a b o u t + 4 ° (see ] ' a b l e I) assuming a c o n s t a n t configurational r o t a t i o n due to amino acid residuesLt4. Therefore, the helical content based on b0 with non-helical b0 correction can be e s t i m a t e d b y the following fornmlal4: % t[elix

(b 0

b~;') ),." IOO/(,3o ().0

212 In[~)

where ber~' is non-helical b0 correction which, in the present case, corresponds to + 4 o. The e s t i m a t e d helicity of the l i g a n d e d catalase was found to be a b o u t 4 7 % a n d 4 3 % for c y a n - a n d a z i d e - c a t a l a s e complexes, respectively, as shown in T a b l e I t o g e t h e r w i t h those of n a t i v e a n d d e n a t u r e d catalase. The small d i s c r e p a n c y can be found b e t w e e n the values from t h e bH m e t h o d a n d the 233 m # t r o u g h m e t h o d or CD i n t e n s i t y m e t h o d . However, we can conclude from these O R D a n d CD d a t a t h a t n a t i v e c a t a l a s e Biochim. Biophys. Acla, 175 (1969) 24 3 °

3°

T. SAMEJIMA, M. KI'I'A

alters its protein confornlation by reducing helical content from 4o-5o°.,~ to 30 4 o'!, when the small ligand molecules attach to iron atoms of heine groups in the protein. The formation of the complexes causes the complete loss of enzymic activity. Removing the liganded molecules from the complexes by dialysis caused almost complete recovery of the catalatic activity, and examinations of the recovered solutions revealed that they displayed essentially identical profiles of ORD and CD to those of the native protein molecule. The problem whether this conformational change in a-helical content is an essential behavior in functioning the enzymic activity of catalase will be left to further investigations. ACKNOWLEDGEMENT

The authors would like to acknowledge the grant-in-aid for a special research project on biophysics from the Japanese Government. REFERENCES I 2 3 4 5 6 7 8 9 IO i1 12 13 14 15 16 17

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