THE ACTIVE CENTER OF BACTERIAL CATALASE INVESTIGATED BY MÖSSBAUER SPECTROSCOPY

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THE ACTIVE CENTER OF BACTERIAL CATALASE INVESTIGATED BY MÖSSBAUER SPECTROSCOPY

F. Parak, D. Bade, A. Marie

To cite this version:

F. Parak, D. Bade, A. Marie. THE ACTIVE CENTER OF BACTERIAL CATALASE INVES-

TIGATED BY MÖSSBAUER SPECTROSCOPY. Journal de Physique Colloques, 1979, 40 (C2),

pp.C2-528-C2-530. �10.1051/jphyscol:19792183�. �jpa-00218560�

JOURNAL DE PHYSIQUE

Colloque C2, supplPmen2 au no 3, Tome

40,

mars 1979, page C2-528

THE ACT1 VE CENTER OF BACTERl A L CATALASE I N V E S T 1 GATED BY MOSSBAUER SPECTROSCOPY

F. Parak, D . Bade and A.L. Yarie

Max-Planck-Institut fiir Biochemie, 8033 Martinspied, B.R.D.

and

Physik-Department

E

25 der TUM Gmching, B.R.D.

R6su1x-6.- La c a t a l a s e de Micrococcus l u t e u s e n r i c h i e au 5 7 ~ e a Q t b e t u d i b e en u t i l i s a n t l a s p e c t r o s - c o p i e Mzssbauer. A pH 6 , 3 dans un tampon a c e t a t e , l e s s p e c t r e s s o n t trGs semblables 1 ceux donnbs p a r l a metmyoglobine. Ceci suggsre que l e s niveaux e l e c t r o n i q u e s du £ e r s o n t s i m i l a i r e s dans l e s deux enzymes. L ' o p t i m i s a t i o n d'un s p e c t r e f a i t 1 4,2 K avec un champ a p p l i q u e de Hext = 0,5 kOe p a r l a mBthode des moindres c a r r b s a donne des v a l e u r s de "g" en bon agrcment avec c e l l e s obtenues l o r s de mesures EPR. Les q u a t r e sous-unitbs de l a c a t a l a s e p r b s e n t e n t un comportement i d e n t i q u e . Dans l l B c h a n t i l l o n d t u d i d en prBsence de tampon phosphate 2 pH 7 , 0 , une dewiPme espPce de Fe a Bt6 trouvbe, q u i p o u r r a i t p r o v e n i r d'une d e n a t u r a t i o n p a r t i e l l e ou d'un changement de conformation.

A b s t r a c t . - 5 7 ~ e e n r i c h e d b a c t e r i a l c a t a l a s e o b t a i n e d from Micrococcus l u t e u s was i n v e s t i g a t e d by Mzssbauer spectroscopy. At pH 6 . 3 i n an a c e t a t e b u f f e r t h e Mijssbauer s p e c t r a a r e very s i m i l a r t o

t h e s p e c t r a of metmyoglobin, i n d i c a t i n g a s i m i l a r l e v e l scheme of t h e i r o n . A l e a s t s q u a r e s f i t of a spectrum a t 4.2 K with an a p p l i e d f i e l d Hext= 0.5 kOe y i e l d e d g-values which agree w e l l w i t h EPR- measurements. The f o u r s u b u n i t s of t h e c a t a l a s e behaved i d e n t i c a l l y . I n a sample with phosphate b u f f e r a t pH 7.0 we found a second i r o n s p e c i e s , which may come from a d e n a t u r a t i o n p r o c e s s o r a conformational change.

1 . I n t r o d u c t i o n . - C a t a l a s e i s an enzyme which i s pre- s e n t i n n e a r l y a l l c e l l s c o n t a i n i n g a cytochrome sys-

tem / l / . Only s t r i c t anaerobes do n o t have t h i s enzy

me. I t c a t a l y z e s t h e decomposition of H202 i n t o 02 and H20. The molecular weight i s between 240 000 and 250 000. One molecule c o n t a i n s f o u r s u b u n i t s each of which h a s one hemegroup a t i t s a c t i v e c e n t e r . The s t r u c t u r e i s s t i l l unknown s i n c e X-ray a n a l y s i s be- comes d i f f i c u l t i f t h e molecular weight i s too l a r - ge.

I n a c t i v e c a t a l a s e t h e i r o n i s i n t h e Fe3+ s t a t e . EPR s p e c t r a from beef l i v e r c a t a l a s e 12-41, e r y t h r o - c y t e c a t a l a s e 15-71 and b a c t e r i a l c a t a l a s e / 8 / show a markedly s p l i t s i g n a l a t g = 6 and a s i g n a l a t g = 2. Although t h e s e EPR s p e c t r a a r e very s i m i l a r t o t h e s p e c t r a observed f o r methemoglobins t h e s p l i t t i n g of t h e EPR s i g n a l a t g = 6 i s g e n e r a l l y much l a r g e r f o r c a t a l a s e .

Maeda e t a1.191 have r e p o r t e d G s s b a u e r s p e c t r a from b a c t e r i a l c a t a l a s e i n f r o z e n s o l u t i o n . T h e i r r e s u l t s have been i n t e r p r e t e d a s a s u p e r p o s i t i o n of two d i f - f e r e n t s p e c t r a . The a u t h o r s suggested a p a i r w i s e d i f f e r e n t i n t e r a c t i o n of t h e i r o n atoms i n t h e f o u r a c t i v e c e n t e r s of c a t a l a s e . They concluded t h a t a t l e a s t i n two s u b u n i t s t h e hemegroups a r e l o c a t e d c l o s e t o t h e s u r f a c e of t h e p r o t e i n moiety w i t h a d i s t a n c e of 7

1

o r l e s s between each o t h e r . This model was based upon measurements on a sample a t

pH 7.0 i n 0.01 M phosphate b u f f e r . The experiments r e p o r t e d h e r e were performed on two c a t a l a s e samples one of which had t h e same pH and b u f f e r a s used i n 191. I n o r d e r t o o b t a i n some a d d i t i o n a l information t h e o t h e r sample d i f f e r e d i n b u f f e r and pH.

2. M a t e r i a l s and methods.- Micrococcus l u t e u s was grown i n a medium e n r i c h e d w i t h "Fe. The c a t a l a s e was p u r i f i e d according t o H e r b e r t and P i n s e n t 1101.

Two Mijssbauer samples were prepared. One contained 90 mg c a t a l a s e i n 0.6 m1 of 0.05 M a c e t a t e b u f f e r a t pH 6.3. The second sample was a d j u s t e d t o pH = 7.0 by 0.01 M phosphate b u f f e r .

3. R e s u l t s . - Figure 1 shows Mzssbauer s p e c t r a of t h e pH = 6.3 sample. The s p e c t r a of t h e pH P 7.0 samples a r e q u i t e s i m i l a r t o t h e s p e c t r a given i n 191. It should be noted, however, t h a t t h e two a b s o r p t i o n l i n e s o b t a i n e d a t 77 K were much b e t t e r r e s o l v e d i n our c a s e , t h a n i n f i g u r e 2 a of / g / .

The spectrum of t h e pH = 6.3 sample a t 4.2 K and an a p p l i e d magnetic f i e l d of 0.5 kOe was i n t e r p r e t e d w i t h a t h e o r y used p r e v i o u s l y f o r metmyoglobin / I l l . We assumed t h a t a l l r e l a x a t i o n processes a r e f r o z e n a t 4.2 K. The 6 ~ ,g r o u n d s t a t e has a z e r o f i e l d s p l i t t i n g s o t h a t only t h e s t a t e w i t h m = +

-

1 i s

S - 2 populated. The degeneracy of t h i s Kramers d o u b l e t i s l i f t e d by t h e e x t e r n a l l y a p p l i e d magnetic f i e l d . The e v a l u a t i o n o f t h e Mzssbauer spectrum needs an average over t h e d i f f e r e n t o r i e n t a t i o n s of t h e

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19792183

molecule i n t h e f r o z e n s o l u t i o n w i t h r e s p e c t t o t h e incoming p r a y s . S i n c e g

+

^{g /8/} t h e r e e x i s t s no

X Y

a x i a l symmetry. Thus t h e a v e r a g e was performed o v e r t h e a n g l e s 0 and 0. I n t h e n u m e r i c a l c a l c u l a t i o n s p e c t r a i n s t e p s of 10' were computed and added w i t h t h e a p p r o p r i a t e w e i g h t f a c t o r . The asymmetry p a r a m e t e r 0 was a l l o w e d t o be unequal t o z e r o .

F i g . 1 : G s s b a u e r a b s o r p t i o n s p e c t r a o f b a c t e r i a l c a t a l a s e i n 0.05 M a c e t a t e b u f f e r a t pH = 6.3.

t o p : T = 78 K Hext = 0 middle : T = 10 K Hext = 0.5 kOe bottom : T = 4 . 2 K

next

⁼ 0 . 5 kOe

S o l i d l i n e i s a f i t d e s c r i b e d i n t h e t e x t .

The l e a s t s q u a r e s f i t was performed w i t h g = 6 . 5 , gy = 5 . 5 and gZ = 2.0. Changes of 2 X of g and g

X Y

o n l y s l i g h t l y i n f l u e n c e d t h e G s s b a u e r spectrum. The l e a s t s q u a r e s f i t gave t h e p a r a m e t e r s : i s o m e r s h i f t 6 s = 0.272 - + 0.001 mm/s w i t h r e s p e c t t o a - i r o n , qua- d r u p o l e s p l i t t i n g

-

2 I e Z q q = 1.14

2

0.01 m / s ; e x p e r i - m e n t a l l i n e w i d t h

r

⁼ 0.44

+

0.04 mm/s and t h e

exp

4. D i s c u s s i o n . - The pH 6.3 sample i n a c e t a t e b u f f e r showed a q u i t e s i m i l a r b e h a v i o u r a s metmyoglobin, o n l y t h e s p l i t t i n g o f g x , g i s d i f f e r e n t . At tem-

Y

p e r a t u r e s above 4.2 K r e l a x a t i o n p r o c e s s e s t a k e p l a c e which smear o u t t h e s i x l i n e p a t t e r n . The t e r n p e r a t u r e dependence seems a g a i n t o b e s i m i l a r t o t h a t o f metmyoglobin. The r e l a x a t i o n i s f a s t e r t h a n i n myoglobin f l u o r i d e . The s i m i l a r i t i e s of t h e spec- t r a may s u g g e s t t h a t t h e l e v e l scheme o f Fe i n c a t a - l a s e and i n metmyoglobin a r e i n p r i n c i p l e t h e same.

The s p e c t r a of t h e a c e t a t e sample gave no h i n t f o r d i f f e r e n t i r o n s i t e s i n t h e c a t a l a s e ; t h e i r o n s i n t h e f o u r s u b u n i t s g i v e i d e n t i c a l s p e c t r a . T h i s a g r e e s w i t h o u r r e c e n t f i n d i n g t h a t t h e s u b u n i t s o f c a t a l a s e a r e i d e n t i c a l 112, 131. The c r y s t a l l o g r a - p h i c i n v e s t i g a t i o n s 1131 do n o t e x c l u d e a p a i r w i s e i n t e r a c t i o n of t h e s u b u n i t s b u t t h e y e x c l u d e a p a i r - w i s e d i f f e r e n t i n t e r a c t i o n a s proposed by / g / . It s h o u l d b e n o t e d , however, t h a t i n c a t a l a s e c r y s t a l s t h e s t r u c t u r e of t h e p r o t e i n is h i g h l y s t a b i l i z e d by a l a r g e s a l t c o n c e n t r a t i o n .

According t o o u r own i n v e s t i g a t i o n s and a comparison w i t h l i t e r a t u r e / 1 4 , 151 t h e c a t a l a s e h a s p r a c t i c a l -

l y i t s t o t a l a c t i v i t y a t t h e pH 6 . 3 . Only l e s s t h a n 4 % of t h e m o l e c u l e s a r e l i g a n d e d w i t h a c e t a t e . We, t h e r e f o r e , b e l i e v e t h a t c a t a l a s e i s i n t h e n a t i v e form u n d e r t h e c o n d i t i o n s r e p o r t e d above.

As a l r e a d y mentioned t h e i n v e s t i g a t i o n o f t h e pH 7.0 p h o s p h a t e b u f f e r sample y i e l d e d s i m i l a r r e s u l t s a s /9/. The s p e c t r a may b e u n d e r s t o o d a s a s u p e r p o s i - t i o n o f two d i f f e r e n t t y p e s o f h y p e r f i n e i n t e r a c - t i o n s y i e l d i n g a s i x l i n e p a t t e r n s i m i l a r t o t h a t found i n t h e pH 6.3 a c e t a t e sample and i n a d d i t i o n a q u a d r u p o l e d o u b l e t . According t o o u r e x p e r i e n c e t h e a c t i v i t y of c a t a l a s e i n phosphate b u f f e r de- c r e a s e s w i t h t i m e . T h e r e f o r e , t h e q u a d r u p o l e dou- b l e t may b e due t o a d e n a t u r a t i o n p r o c e s s . S i n c e most of t h e a c t i v i t y t e s t s on c a t a l a s e a r e performed i n p h o s p h a t e b u f f e r t h i s e x p l a n a t i o n i s n o t v e r y s a - t i s f a c t o r y .

Another e x p l a n a t i o n would b e , t h a t a t pH 7.0 i n phosphate b u f f e r a change of t h e c o n f o r m a t i o n of t h e c a t a l a s e t a k e s p l a c e , which makes two p a i r s of s u b u n i t s u n e q u i v a l e n t . For a f i n a l d e c i s i o n a d d i - t i o n a l e x p e r i m e n t s a r e needed.

T h i s work was s u p p o r t e d by t h e Deutsche Forschungs Gemeinschaft.

m a g n e t i c h y p e r f i n e c o n s t a n t A/BN = 195

t

2 kOe.

JOURNAL DE PHYSIQUE

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