MÖSSBAUER STUDY OF A NEW TYPE OF FE-S PROTEIN IN DIFFERENT OXIDATION STATES

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Submitted on 1 Jan 1980

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MÖSSBAUER STUDY OF A NEW TYPE OF FE-S PROTEIN IN DIFFERENT OXIDATION STATES

E. Bill, F. Bernhardt, V. Marathe, A. Trautwein

To cite this version:

E. Bill, F. Bernhardt, V. Marathe, A. Trautwein. MÖSSBAUER STUDY OF A NEW TYPE OF

FE-S PROTEIN IN DIFFERENT OXIDATION STATES. Journal de Physique Colloques, 1980, 41

(C1), pp.C1-485-C1-486. �10.1051/jphyscol:19801191�. �jpa-00219681�

JOURNAL DE PHYSIQUE Colloque C 1, supplt!ment au n O 1 , Tome 41, janvier 1980, page C 1-485

M~SSBAUER STUDY OF A NEW TYPE OF FE-S PROTEIN I N DIFFERENT OXIDATION STATES

E. Bill, F.H. Bernhardt, V.R. Marathe and A. Trautwein Universi ttit des SaarZandes, 66 SaarbrUcken 11, W-Germany

.

The multienzyme-system 4 - m e t h o x y b e n z o a t e - G d e m e t h y l a s e from Pseudomonas p u t i d a c a t a - l y s e s t h e o x i d a t i o n of 4-methoxybenzoate by a c t i v a t i o n of m o l e c u l a r oxygen. I t con- s i s t s o f a r e d u c t a s e and an i r o n - and a c i &

l a b i l e s u l f u r c o n t a i n i n g monooxygenase, c a l l e d Putidamonooxin 1

.

On t h i s compound we performed Mossbauer- and m o l e c u l a r - o r - b i t a l i n v e s t i g a t i o n s . I t h a s a m o l e c u l a r w e i g h t of 126 000 d a l t o n s , and i t s Fe-S- chromphores show a n e s r - s i g n a l a t g=1.90 i n t h e r e d u c e d s t a t e 1

.

I n c o n t r a s t t o o t h e r F e - S - p r o t e i n s , t h e r e i s no s i g n i f i - c a n t o p t i c a l a b s o r p t i o n peak n e a r 415 nm from t h e o x i d i z e d p r o t e i n , b u t one a t 455 nm. I n t h e p r e s e n c e o f s u b s t r a t e , con- s i d e r a b l e changes i n t h e o p t i c a l s p e c t r u m of t h e o x i d i z e d a d d u c t a p p e a r 2

.

I n t h e r e - duced s t a t e e s r - and o p t i c a l s p e c t r a do n o t show any i n f l u e n c e on s u b s t r a t e b i n - d i n g 3

.

The s p e c t r a o f t h e o x i d i z e d enzyme ( F i g . 1 ) were f i t t e d by two q u a d r u p o l e - d o u b l e t s

.

The v a l u e s

PE

and

d

i n d i c a t e two f e r r i c h i g h - s p i n Fe. The s p e c t r u m of t h e reduced

Q

enzyme c o n t a i n s an a d d i t i o n a l p a i r of li- n e s t y p i c a l f o r f e r r o u s h i g h - s p i n Fe. T h i s b e h a v i o r i s c h a r a c t e r i s t i c f o r i r o n - s u l f u r p r o t e i n s w i t h 2 ~ e - 2 ~ - c l u s t e r s ~ ' ~ . F i g . 2 shows t h e i n f l u e n c e of s u b s t r a t e b i n d i n g upon t h e s p e c t r a o b t a i n e d from t h e o x i d i - zed enzyme. F i t t i n g t h e s e s p e c t r a w i t h two q u a d r u p o l e - d o u b l e t s , we d e r i v e d i n t e n s i t y r a t i o s d i f f e r e n t from 1 : 1 , which we e x p e c t f o r an enzyme w i t h one 2Fe-2S c l u s t e r . T h e r e f o r e we c o n c l u d e t h a t Putidamonooxin c o n t a i n s more t h a n one chromophore p e r mo- l e c u l e . F i g . 3 shows c o r r e s p o n d i n g f i t s of t h e o x i d i z e d enzyme i n p r e s e n c e of 4-me- t h o x y b e z o a t e w i t h f o u r quadrupole-douh- l e t s o f e q u a l i n t e n s i t y . The spectrum of t h e r e d u c e d enzyme i n p r e s e n c e o f s u b s t r a -

t e shows no s i g n i f i c a n t d i f f e r e n c e s from t h a t o f t h e reduced p r o t e i n w i t h o u t sub- s t r a t e . For t h e i n t e r p r e t a t i o n o f cf and

A E Q

o f t h e o x i d i z e d and reduced c l u s t e r , we performed IEHTMO c a l c u l a t i o n s 6 . Because no s t r u c t u r a l d a t a a r e a v a i l a b l e f o r 2Fe-

2s

p r o t e i n s , we used t h e c e n t r o s y m m e t r i c compound

[

F ~ S ( S C H ~ ) Z ~ 6 ~ 4 J a s a model com- pound7 ( F i g . 4 ) . From t h e o r b i t a l p o p u l a t i - o n s , which a r e c o n s i s t e n t w i t h t h e diamag- n e t i s m of t h e compound we d e r i v e

A E

=

0.44 mms-' f o r b o t h i r o n s i t e s o f t h e o x i -

Q

d i z e d enzyme, which a g r e e s w e l l w i t h t h e e x p e r i m e n t a l v a l u e s of F i g . 1 . J u s t a d d i n g one e l e c t r o n i n t h e c a l c u l a t i o n d i d n o t l e a d t o d i f f e r e n t i r o n - s i t e s f o r t h e r e - duced c l u s t e r , because o f t h e f a c t t h a t t h e model compound i s c e n t r o s y m m e t r i c . S i - m u l a t i n g t h e e x p e r i m e n t a l s i t u a t i o n i n t h e reduced enzyme ( F i g . 1) r e q u i r e s t o make t h e model compound asymmetric. Correspon- d i n g changes a r e j u s t i f i e d by t h e f a c t t h a t t h e r e d u c t i o n of t h e enzyme w i t h r e - d u c t a s e works o n l y a f t e r s u b s t r a t e b i n d i n g . Geometric c h a n g e s , even by i n c r e a s i n g t h e bond d i s t a n c e s around one o f t h e two i r o n by 1 5 % d i d n o t y i e l d t h e measured d i f f e - r e n c e s of q u a d r u p o l e s p l i t t i n g s o f A E

Q -1 ( F e I I ) = 2 . 9 mms-' and

A E _Q

( F e I I I ) = 0 . 6 mms,

.

However, a d i f f e r e n c e o f 4 eV i n t h e e l e c - t r i c p o t e n t i a l o f t h e two i r o n s c o r r e s p o n - d i n g t o a change i n c h a r g e of two c y s t e i n s u l f u r s by a b o u t 0.25 e y i e l d s f a i r l y good agreement o f c a l c u l a t e d and measured

c f

and A E v a l u e s . From t h e p r e s e n t s t u d y we con- c l u d e , t h a t Putidamonooxin h a s a t l e a s t

Q

two 2Fe-2S chromophores. A f t e r b i n d i n g of s u b s t r a t e t h e enzyme undergoes a change i n c o n f o r m a t i o n , s o t h a t p a r t of t h e i r o n i s a f f e c t e d i n i t s e l e c t r i c s t r u c t u r e . I n t h e reduced s t a t e t h e n t h e chromophores a r e much l e s s s e n s i t i v e t o s u b s t r a t e b i n d i n g

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19801191

Cl-486 JOURNAL DE PHYSIQUE

t h a n i n t h e o x i d i z e d s t a t e . Most r e c e n t p r e p a r a t i o n s and measurements i n d i c a t e , t h a t under p h y s i o l o g i c a l c o n d i t i o n s an ad- d i t i o n a l i r o n i s l i n k e d t o t h e 2Fe-2s cnro- mophores and f u n c t i o n s a s t h e dioxygen b i n d i n g s i t e .

---.

1

F i g . 1 : Ox. and r e d . Putidamonooxin

C

'G 0 0.99- 2 CHROMO- .- ul

5

C 0.98-

I- 2

% 0.97-

(Z

-2

-i o

i

i

Velocity [ mm/sl

F i g . 3 : Oxidized Putidamonooxin w i t h 4-me- t h o x y b e n z o a t e ; f i t v e r s i o n w i t h f o u r qua- d r u p o l e - d o u b l e t s (two chromophores p e r mo- l e c u l e ) of e q u a l i n t e n s i t i e s

0

F i g . 4 : Model compound u s e d f o r MO-calcu- l a t i o n s

(0:

F e , O : S , @ : C , o : H)

I

Velocity [mm/sl

R e f e r e n c e s :

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2 . B e r n h a r d t , F.H., Heymann, E . , T r a y l o r , P . , E u r . J . Biochem. 92, (1978) 209 31. T w i l f l e r , H . , Gersonde, K . , B e r n h a r d t ,

F.H., H o p p e - S e y l e r ' s Z . P h y s i o l . Chem.

Bd. 360, (1979) 390

4 . Johnson, C . E . , i n T o p i c s i n Appl. Phys.

Vol. 5 e d . by U . Gonser, S p r i n g e r - V e r - l a g (1975) 139

5. Sands, R . H . , Dunham, W.R., Q u a r t . Rev.

Biophys. 7 , 4 (1975) 443

6 . G i i t l i c h , P . , Link, R . , T r a u t w e i n , A . , i n MBssbauer S p e c t r o s c o p y and T r a n s i t i - on M e t a l s C h e m i s t r y , l n o r g . ' Chemistry C o n c e p t s , Vol. 3 , Chap. VI (1978) 7. M a y e r l e , J . J . , Denmark, S.E., Depham-

p h i l i s , B . V . , I b e r s , J . A . , Holm, R . H . , JACS 97, (1975) 5

S u p p o r t e d by Deutsche Forschungsgemein- s c h a f t

.

F i g . 2 : S u b s t r a t e dependence of o x i d i z e d Putidamonooxin