THE IRON BINDING-SITES OF CHICKEN OVOTRANSFERRIN

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THE IRON BINDING-SITES OF CHICKEN OVOTRANSFERRIN

P. Lindley, R. Evans, R. Garratt, S. Hasnain

To cite this version:

P. Lindley, R. Evans, R. Garratt, S. Hasnain. THE IRON BINDING-SITES OF CHICKEN OVOTRANSFERRIN. Journal de Physique Colloques, 1986, 47 (C8), pp.C8-1189-C8-1192.

�10.1051/jphyscol:19868233�. �jpa-00226140�

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JOURNAL D E PHYSIQUE

C o l l o q u e C8, s u p p l i 5 m e n t a u n o 12, T o m e 47, d e c e m b r e 1986

THE IRON BINDING-SITES OF CHICKEN OVOTRANSFERRIN

P.F. LINDLEY', R.W. EVANS*, R.C. G A R R A T T I * and S.S. HASNAIN**

' D e p a r t m e n t of C r y s t a l l o g r a p h y , B i r k b e c k C o l l e g e , M a l e t S t r e e t , G B - L o n d o n WClE 7HX, G r e a t - B r i t a i n

* ~ i v i s i o n of B i o c h e m i s t r y , UMDS, Guy 's H o s p i t a l S t T h o m a s 's S t r e e t , G B - L o n d o n S E I 9 R T , G r e a t - B r i t a i n

**SERC D a r e s b u r y L a b o r a t o r y , G B - W a r r i n g t o n W A 4 4AD G r e a t - B r i t a i n

A b s t r a c t

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We have shown p r e v i o u s l y t h a t t h e EXAFS s p e c t r u m o f d i f e r r i c c h i c k e n o v o t r a n s f e r r i n (Fe2COT) c a n o n l y be a d e q u a t e l y s i m u l a t e d a s s u m i n g a s p l i t f i r s t s h e l l c o - o r d i n a t i o n [I]. EXAFS a n d XANES s p e c t r a o f Fe,COT m e a s u r e d i n s o l u t i o n and as a f r e e z e - d r i e d powder p r o v i d e e v i d e n c e f o r p e r t u r b a t i o n o f t h e i r o n - b i n d i n g s i t e s on f r e e z e - d r y i n g which i n v o l v e s t h e l o s s o f o n e o f t h e l o n g (-2.0483 f i r s t s h e l l l i g a n d s ( p r e s u m a b l y w a t e r ) . Measurement o f t h e XANES o f t h e C - t e r m i n a l m o n o f e r r i c COT a n d a C - t e r m i n a l domain f r a g m e n t s u g g e s t s t h a t t h e metal b i n d i n g s i t e r e m a i n s l a r g e l y u n p e r t u r b e d by t h e f r a g m e n t a t i o n p r o c e s s . The p o s s i b i l i t y o f s i t e i n t e r a c t i o n i s b r i e f l y d i s c u s s e d .

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

The r o l e o f c h i c k e n o v o t r a n s f e r r i n (COT) i n e g g w h i t e i s presumed t o be as a b a c t e r i o s t a t i c a g e n t a c t i n g v i a i t s i r o n - s e q u e s t i n g c a p a b i l i t y . E v o l u t i o n a r i l y , i t is r e l a t e d t o serum t r a n s f e r r i n , t h e p r i n c i p a l i r o n t r a n s p o r t p r o t e i n o f v e r t e b r a t e plasma.

The t r a n s f e r r i n s are monomeric g l y c o p r o t e i n s o f Mr - 8 0 , 0 0 0 c a p a b l e o f

s p e c i f i c a l l y b i n d i n g a maximum o f two f e r r i c i o n s c o n c o m i t a n t w i t h 2 ( b i l c a r b o n a t e a n i o n s a t homologous i r o n - b i n d i n g s i t e s , ( f o r r e v i e w s e e [ 2 ] ) . X-Ray d i f f r a c t i o n [ 3 ] and m o l e c u l a r f r a g m e n t a t i o n s t u d i e s have shown t h e m o l e c u l e to have a two domain s t r u c t u r e . S p e c t r o s c o p i c and c h e m i c a l m o d i f i c a t i o n e v i d e n c e s u g g e s t s t h e p r e s e n c e o f h i s t i d i n e and t y r o s i n e l i g a n d s to t h e i r o n atoms as w e l l as a

( b i ) c a r b o n a t e a n i o n a n d / o r w a t e r m o l e c u l e ( s e e [ 2 ] ) . We have shown p r e v i o u s l y u s i n g EXAFS [ I ] t h a t t h e t h e f i r s t s h e l l atoms are s p l i t i n t o two d i s t a n c e s i n v o l v i n g 2 l i g h t atoms (O/N) a t

-

¹.858. a n d a f u r t h e r f o u r l i g h t atoms a t

-

^2.04a.

On t h e b a s i s o f model compounds we a s s i g n t h e s h o r t e r d i s t a n c e t o t h e p h e n o l i c o x y g e n s o f 2 t y r o s i n e r e s i d u e s . A summary of t h e f i r s t s h e l l d i s t a n c e s f o r Fe,COT and i t s N- and C- domain i r o n - b i n d i n g f r a g m e n t s a r e g i v e n i n T a b l e 1 .

M a t e r i a l s and Methods

Chicken o v o t r a n s f e r r i n and i t s i r o n - b i n d i n g f r a g m e n t s were i s o l a t e d a s d e s c r i b e d p r e v i o u s l y [ I ] . P r o t e i n was made i r o n - s a t u r a t e d by a d d i t i o n o f e x c e s s F e ( I11 ) n i t r i l o t r i a c e t a t e and d e s a l t e d on Sephadex G25. F r e e z e - d r i e d Fe,COT was made by

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868233

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C8-1190 JOURNAL DE PHYSIQUE

s h e l l f r e e z i n g a s o l u t i o n o f i r o n - s a t u r a t e d p r o t e i n i n 50mM NH4C03 u s i n g l i q u i d n i t r o g e n and l y o p h i l i s a t i o n a t

-

5x10L2 T o r r . C-Monoferric COT was p r e p a r e d by a d d i t i o n o f 0.03 v o l of 0.6M sodium c i t r a t e , pH 5 . 0 , t o a s o l u t i o n ( 2 0 mg/ml) o f Fe,COT i n w a t e r . A f t e r t h e e x t i n c t i o n a t 470 nm had f a l l e n t o 30% o f i t s o r i g i n a l v a l u e a t h e o r e t i c a l t h r e e f o l d e x c e s s o f d e s f e r r i o x a m i n e was added and t h e

r e s u l t i n g i r o n - d e s f e r r i o x a m i n e complex removed by g e l f i l t r a t i o n on G-50. The m o n o f e r r i c p r o t e i n was f r e e z e - d r i e d p r i o r t o u s e . A l l s o l u t i o n EXAFS and XANES were r e c o r d e d i n 0.1M NaHC03

.

X-Ray a b s o r p t i o n s p e c t r a were r e c o r d e d on S t a t i o n 8 . 1 o f t h e SERC Daresbury SRS, u s i n g a double f o c u s i n g S i ( 2 2 0 ) monochromator. During d a t a c o l l e c t i o n t h e SRS beam energy was 2 GeV and m a i n t a i n e d an a v e r a g e c i r c u l a t i n g c u r r e n t o f 220 mA.

S i m u l a t i o n o f t h e EXAFS r e g i o n have i n v o l v e d an approach d e s c r i b e d e l s e w h e r e [ 4 1 .

T a b l e 1 F i r s t s h e l l EXAFS p a r a m e t e r s used i n t h e s i m u l a t i o n o f s p e c t r a from F e z COT and its N- and C- t e r m i n a l s i n g l e domain f r a g m e n t s .

N- Fragment C- Fragment Fe, COT

Atom No

~ ( 8 ) ~ ( 1 ' ) R ( X ) ,(a2) ~ ( 1 ) ~ ( 1 ' )

R e s u l t s and D i s c u s s i o n

F i g 1 shows a comparison o f t h e EXAFS o f FezCOT r e c o r d e d i n 0.1M NaHC03 b u f f e r and a s a f r e e z e - d r i e d powder. The a m p l i t u d e and f r e q u e n c y o f t h e major s h e l l c o n t r i b u t i o n is reduced i n t h e f r e e z e - d r i e d sample s u g g e s t i n g t h e l o s s o f a l i g a n d from t h e first c o - o r d i n a t i o n s p h e r e . T h i s is e v i d e n t i n t h e F o u r i e r t r a n s f o r m ( i n s e t ) where t h e f i r s t s h e l l i s s h i f t e d t o lower R and i s o f reduced i n t e n s i t y . Rearrangement o f some o f t h e o u t e r s h e l l s is a l s o a p p a r e n t from t h e 3 1 r e g i o n o f t h e F o u r i e r t r a n s f o r m .

The enhancement o f f e a t u r e s a and b ( F i g 2 ) i n t h e edge s t r u c t u r e o f t h e f r e e z e - d r i e d p r o t e i n confirm t h a t g e o m e t r i c a l changes have o c c u r r e d

a s a consequence o f f r e e z e - d r y i n g . Some e v i d e n c e [ 5 1 h a s been provided f o r a n e g a t i v e c o r r e l a t i o n between t h e i n t e n s i t y o f t h e pre-edge f e a t u r e a and t h e c o - o r d i n a t i o n number. The i n c r e a s e d i n t e n s i t y o f t h i s t r a n s i t i o n i n t h e

f r e e z e - d r i e d spectrum is t h e r e f o r e c o n s i s t e n t w i t h t h e l o s s o f a l i g a n d from t h e m e t a l b i n d i n g - s i t e s .

F i g 3 a shows t h e e x p e r i m e n t a l spectrum f o r t h e f r e e z e - d r i e d p r o t e i n superimposed upon t h e t h e o r e t i c a l s i m u l a t i o n f o r t h e s o l u t i o n sample [ I ] . Removal o f one l i g a n d from t h e 2.048 s h e l l ( F i g 3 b ) w i t h o u t r e f i n e m e n t produces an improvement i n t h e f i t i n d e x from 0 . 5 t o 0.3. Refinement o f t h i s model r e d u c e s t h e f i t i n d e x t o 0.16 ( F i g 3 c ) . We s u g g e s t t h e most p r o b a b l e e x p l a n a t i o n f o r t h e s e e f f e c t s is t h a t a co-Crdinated w a t e r molecule is l o s t on f r e e 3 r y i n g which

resets

i n mrnor l i g a n d rearrangement i n c l u d i n g t h e s h o r t e n i n g o f t h e ' l o n g 1 f i r s t s h e l l d i s t a n c e t o 2.021. E.p.r. measurements a t 77K ( n o t p r e s e n t e d h e r e ) c o n f i r m t h e observed p e r t u r b a t i o n a t t h e i r o n s i t e s a s a consequence o f f r e e z e - d r y i n g .

The n e a r edge s t r u c t u r e f o r Fe2COT, t h e N- and C- t e r m i n a l s i n g l e domain f r a g m e n t s and t h e C- m o n o f e r r i c i n t a c t molecule ( i n which t h e N- s i t e i s u n o c c u p i e d ) a r e shown i n F i g 4. The s i m i l a r i t y o f t h e C- t e r m i n a l fragment and i t s m o n o f e r r i c c o u n t e r p a r t s u g g e s t s t h a t t h e i r o n s i t e remains l a r g e l y u n p e r t u r b e d by t h e f r a g m e n t a t i o n p r o c e s s .

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F i g u r e 1 EXAFS and F o u r i e r t r a n s f o r m s F i g u r e 2 XANES s p e c t r a o f Fe2COT i n ( i n s e t ) o f Fe,COT r e c o r d e d i n G.1M NaHC03 0 . G l M NaHCQsolution ( a b o v e ) and a s a (

- --

) and a s a f r e e z e - d r i e d powder ( - - - )

.

f r e e z e - d r i e d powder ( b e l o w ) .

F i g u r e 3 S i m u l a t i o n o f t h e f r e e z e - d r i e d FsCOT EXAFS experiment ^(--), t h e o r y

( - - -

.

^{F I}i s t h e f i t i n d e x ( a ) p a r a m e t e r s a r e t h o s e f o r F q COT i n s o l u t i o n [ I ] .

( b ) assuming a 2 / 3 s p l i t f i r s t s h e l l , ( c ) a s i n ( b ) a f t e r r e f i n e m e n t .

The s i m i l a r i t y of a l l s p e c t r a shown i n F i g 4 means t h a t it i s n o t p o s s i b l e t o d e f i n i t e l y d e t e r m i n e any s t r u c t u r a l i n t e r a c t i o n between t h e s i t e s o f c h i c k e n o v o t r a n s f e r r i n , d e s p i t e t h e f a c t t h a t i n t e r d o m a i n i n t e r a c t i o n s a r e known t o o c c u r C61 and t h a t t h e p r o t e i n shows c o o p e r a t i v i t y f o r i r o n b i n d i n g [ 7 ] . However, r e c e n t l y c o l l e c t e d d a t a on human serum t r a n s f e r r i n which is r e p o r t e d e l s e w h e r e [ 8 ] h a s demonstrated t h a t f o r t h e human p r o t e i n a t l e a s t , s u c h i n t e r s i t e communication does r e s u l t i n s i g n i f i c a n t g e o m e t r i c a l changes a t t h e i r o n s i t e s which a r e

observed i n t h e edge s t r u c t u r e .

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JOURNAL DE PHYSIQUE

F i g u r e 4 XANES s p e c t r a o f Fe,COT, t h e N- and C- t e r m i n a l s i n g l e domain f r a g m e n t s and t h e C- m o n o f e r r i c i n t a c t molecule.

Acknowledgements

We g r a t e f u l l y acknowledge t h e SERC f o r f i n a n c i a l s u p p o r t and p r o v i s i o n o f f a c i l i t i e s . References

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¹⁶⁸¹

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[ 7 ] Yamamura T, I ~ e d a H , Nakazato K , Takimura M & S a t a k e K ( 1 9 8 5 ) i n ' P r o t e i n s o f i r o n s t o r a g e and t r a n s p o r t ' ( S p i k G, M o n t r e u i l J , C r i c h t o n R R & Mazurier J , e d s ) E l s e v i e r , Amsterdam, pp 53

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[ 8 ] G a r r a t t R C, Evans R W, Hasnain S S & Lindley P F ( 1 9 8 6 ) i n Proceedings of c o n f e r e n c e o n B i o p h y s i c s and Synchrotron R a d i a t i o n , F r a s c a t i .