HAL Id: jpa-00226209
https://hal.archives-ouvertes.fr/jpa-00226209
Submitted on 1 Jan 1986
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
THE FORMATION, STRUCTURE AND
DISSOLUTION OF THE FERRITIN IRON CORE STUDIED BY X-RAY ABSORPTION
SPECTROSCOPY
E. Theil, D. Sayers, C. Yang, A. Fontaine, E. Dartyge
To cite this version:
E. Theil, D. Sayers, C. Yang, A. Fontaine, E. Dartyge. THE FORMATION, STRUCTURE AND DISSOLUTION OF THE FERRITIN IRON CORE STUDIED BY X-RAY ABSORP- TION SPECTROSCOPY. Journal de Physique Colloques, 1986, 47 (C8), pp.C8-1155-C8-1157.
�10.1051/jphyscol:19868225�. �jpa-00226209�
JOURNAL DE PHYSIQUE
Colloque C8, suppl6ment au n o 12, Tome 47, d6cembre 1986
THE FORMATION, STRUCTURE AND DISSOLUTION OF THE FERRITIN IRON CORE STUDIED BY X-RAY ABSORPTION SPECTROSCOPY
E.C. THEIL, D.E. SAYERS*, C.Y. YANG*, A. FONTAINE** and E. DARTYGE* '
D e p a r t m e n t o f B i o c h e m i s t r y , N o r t h C a r o l i n a S t a t e U n i v e r s i t y , R a l e i g h , NC 2769,5, U.S.A.
" ~ e p a r t m e n t o f P h y s i c s , N o r t h C a r o l i n a S t a t e U n i v e r s i t y , R a l e i g h , NC 2 7 6 9 5 , U.S.A.
LURE, U n i v e r s i t e P a r i s - S u d , F-91405 O r s a y C e d e x , F r a n c e
ABSTRACT
F e r r i t i n i s t h e s o l u t i o n t o t h e p r o b l e m o f r u s t i n b i o l o g i c a l m a t e r i a l . Polynuclear i r o n complexes o f up t o 4500 F e ( I I 1 ) atoms r e v e r s i b l y form a hydrous f e r r i c oxide core i n s i d e a h o l l o w s p h e r i c a l p r o t e i n coat ( a p o f e r r i t i n ) t h a t p e r m i t s t h e c o n t r o l l e d release o f i r o n as needed by t h e organism. To study i n t e r m e d i a t e s i n f o r m a t i o n o f t h e i r o n c o r e (A) and f a c t o r s w h i c h may a l t e r c o r e s t r u c t u r e (B), we used EXAFS. To study t h e f a c t o r s which may i n f l u e n c e t h e k i n e t i c s o f i r o n core r e d u c t i o n and d i s s o l u t i o n (C), we used d i s p e r s i v e x - r a y a b s o r p t i o n s p e c t r o s c o p y (DXAS). The r e s u l t s show (A) t h a t t h e p r o t e i n coat appears t o c o n t r o l i n i t i a t i o n and n u c l e a t i o n o f t h e i r o n core; (B) t h a t model i r o n c o r e s t r u c t u r e s can be i n f l u e n c e d by s u l f a t e which appears t o nucleate domains o f h e m a t i t e (Fe 03) t h a t c o e x i s t i n t h e s o l u b l e complex w i t h FeOaOH; and (C) t h a t t h e r e d u c t i o n 0% core Fe i s i n f l u e n c e d by b u f f e r i o n s , s u g g e s t i n g t h a t t h e a v a i l a b i 1 i t y o f f e r r i t i n i r o n i n v i v o may be modulated by cytoplasmic changes i n small ions.
--
INTRODUCTION
I r o n i s r e q u i r e d i n p l a n t s and animals f o r a v a r i e t y of f u n c t i o n s such as e l e c t r o n t r a n s f e r , o x y g e n t r a n s p o r t a n d a c t i v a t i o n , n i t r o g e n r e d u c t i o n , a n d deoxy r i bonucl e o t i de synthesis ( f o r DNA). The i n t e r a c t i o n o f i ron and oxygen could produce r u s t i n b i o l o g i c a l systems j u s t as t h e i n t e r a c t i o n produces c o r r o s i o n o f i ron-containing m a t e r i a l s . However, i n t h e case o f l i v i n g systems, t h e o x i d a t i o n and h y d r o l y s i s o f i r o n proceeds i n s i d e a h o l l o w p r o t e i n c a l l e d a p o f e r r i t i n . F e r r i t i n , t h e complex o f h y d r o u s f e r r i c o x i d e and a p o f e r r i t i n , s t o r e s up t o 4500 F e ( I 1 I ) atoms i n a s o l u b l e , b i o a v a i l a b l e form. A l t h o u g h a g r e a t d e a l i s known a b o u t t h e s t r u c t u r e o f t h e p r o t e i n , and s o m e t h i n g i s known a b o u t t h e i r o n core, l i t t l e i s known a b o u t t h e s t e p s i n t h e f o r m a t i o n o f t h e i r o n c o r e and a b o u t t h e r e l e a s e o f i r o n f r o m t h e core. The s t e p s t h a t can b e d e f i n e d a r e i l l u s t r a t e d below:
10 Fe(I1)
ApoferritTn
_
Fe(I1)apoferritin '2,
Fe(II1)apoferritin (A)(multiple (on multiple
Fe s i t e s ) Fe(I1)
's
1 ColIgel$3t
s i t e s )/
@.Q.
(CI dc,.d a+'
F e r r i t i n (8 ) [Apoferritin
+
480 Fe(II1) as an inner core of FeO-OH]Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868225
C8-1156 JOURNAL DE PHYSIQUE
We have i n v e s t i g a t e d some o f t h e i n t e r m e d i a t e s t e p s i n t h e f o r m a t i o n and d i s s o l u t i o n o f t h e f e r r i t i n i r o n core u s i n g XAS. We selected t w o steady s t a t e s t o study by EXAFS: (A) F e ( I I I ) a p o f e r r i t i n , an e a r l y i n t e r m e d i a t e i n t h e f o r m a t i o n o f t h e core, and (B) i r o n cores formed w i t h d i f f e r e n t biopolymers ( a p o f e r r i t i n , poly- s a c c h a r i d e , and p o l y s a c c h a r i d e s u l f a t e ) . I n a d d i t i o n , we chose a k i n e t i c a l l y c h a n g i n g s t a t e t o s t u d y by d i s p e r s i v e EXAFS: (C) t h e r e d u c t i o n o f c o r e F e ( I I 1 ) t o Fe(1I) by t h i o g l y c o l i c a c i d w i t h various b u f f e r ions. The r e s u l t s p r o v i d e i n f o r m a - t i o n a b o u t (A) t h e r o l e o f t h e p r o t e i n i n c o r e f o r m a t i o n , (B) t h e e f f e c t of t h e e n v i r o n m e n t on c o r e s t r u c t u r e , and (C) t h e e f f e c t o f t h e e n v i r o n m e n t on c o r e d i s s o l u t i o n . Horse spleen f e r r i t i n was used throughout because o f i t s abundance and r e l a t i v e s i m p l i c i t y o f structure.
RESULTS AND DISCUSSION
(A) F e ( 1 I I ) A p o f e r r i t i n . a N u c l e a t i o n Complex. When F e ( I 1 ) was added t o a p o f e r r i t i n a t 10 atoms/molecule (OA/subunit) a l l o f t h e i r o n appeared t o be bound t o t h e p r o t e i n a t d i s t i n c t b i n d i n g s i t e s (1). The o x i d a t i o n o f t h e bound F e ( I 1 ) produced F e ( I I I ) , some o f which was a l s o bound by t h e p r o t e i n , and was accompanied by t h e vacancy o f some o f t h e Fe(I1) s i t e s (1). This suggested t h a t o x i d a t i o n was coupled w i t h m i g r a t i o n o f some o f t h e i r o n atoms from s o l i t a r y s i t e s on t h e p r o t e i n t o c l u s t e r e d s i t e s . To c o n f i r m t h e i d e a t h a t c l u s t e r s o f F e ( I I 1 ) formed, and t o determine whether o r n o t a1 1 F e ( I I 1 ) atoms were bound t o t h e protein, x-ray absorp- t i o n spectra were obtained and analyzed. The average Fe absorber i n t e r a c t e d w i t h t h r e e d e t e c t a b l e t y p e s o f n e i g h b r s : a l o w Z atom a t ca. 2.0W, a l o w Z atom a t ca.
2.58, and a h i g h 1 atom a t ca. 3.01. The d a t a f i t we1 1 w i t h model f o r a p r o t e i n c a r b o x y l a t e l i g a n d ( 0 a t ca. 2.08, and C a t ca. 2.51) and Fe (ca. 3.08) b r i d g e d by 0 (ca. 2.01). The i r o n s h e l l i s incomplete compared t o f u l l f e r r i t i n cores. F e ( 1 I I ) a p o f e r r i t i n t h u s appears t o be a small c l u s t e r o f Fe(I1) atoms (2-3) i n which each F e ( I I 1 ) a t o m a l s o i s l i n k e d t o t h e p r o t e i n v i a c a r b o x y l a t e - 1 i k e l i g a n d s ; t h e p o l y n u c l e a r n a t u r e o f t h e F e ( 1 I I ) i n t h e c o m p l e x was c o n f i r m e d by Mbssbauer s p e c t r o s c o p y ( w i t h B. Huynh). The f o r m a t i o n o f t h e F e ( I I 1 ) a p o f e r r i t i n complex shows t h a t t h e p r o t e i n provides t h e s i t e f o r nucleation, probably o r i e n t i n g i r o n c o r e growth toward t h e h o l l o w c e n t e r o f t h e a p o f e r r i t i n coat.
(8) I r o n Cores. I n o r d e r t o d e t e r m i n e i f a l l f e r r i t i n c o r e s w o u l d have t h e same s t r u c t u r e , we used EXAFS t o s t u d y s o l u b l e c o r e s f o r m e d u n d e r t h r e e d i f f e r e n t c o n d i t i o n s : w i t h a p o f e r r i t i n ( p r o t e i n -COOH 1 igands), w i t h dextran (polysaccharide w i t h -OH 1 ig a n d s ) , and c h o n d r o i t i n s u l f a t e ( p o l y s a c c h a r i d e w i t h -COOH, -OH, and -SO4 ligands). Only t h e i r o n cores formed i n t h e presence o f c h o n d r o i t i n s u l f a t e w e r e d i s t i n c t i v e , w i t h an i n c r e a s e d o r d e r ( l a r g e n e g a t i v e Debye Wal l e r f a c t o r compared t o f e r r i t i n ) . The r e s u l t s suggest t h a t s u l f a t e was t h e i m p o r t a n t l i g a n d f o r t h e o r d e r e d domains, s i n c e t h e o t h e r l i g a n d s were p r e s e n t i n d e x t r a n o r a p o f e r r i t i n as w e l l . Mbssbauer a n a l y s i s ( w i t h L. H. Bowen) o f t h e c h o n d r o i t i n s u l f a t e complex i n d i c a t e d t h a t t h e ordered domains were a c t u a l l y s o l u b l e h e m a t i t e (Fe 03). Thus t h e l i g a n d s present i n model experiments can determine t h e s t r u c t u r e o f %he f e r r i t i n core. D u r i n g t h e c o u r s e o f t h e s e e x p e r i m e n t s w i t h models f o r f e r r i t i n cores, e v i d e n c e f o r n a t u r a l v a r i a t i o n s i n f e r r i t i n cores, i n t h i s case associated w i t h PO4, were a l s o obtained (2).
(C) D i s p e r s i v e EXAFS A n a l y s i s o f Fe R e d u c t i o n i n F e r r i t i n . The k i n e t i c s o f r e d u c t i o n and r e l e a s e o f i r o n from t h e core o f f e r r i t i n has, i n t h e past, o n l y been s t u d i e d by i n d i r e c t a n a l y s e s , e.g. f o r m a t i o n o f t h e complex o f F e ( I 1 ) and b i p y r i d y l . It i s t h u s d i f f i c u l t t o s e p a r a t e r e d u c t i o n f r o m l i g a n d b i n d i n g . Because d i s p e r s i v e EXAFS a l l o w s spectra t o be c o l l e c t e d i n a very s h o r t t i m e (ca.
30 sec), i t i s p o s s i b l e t o f o l l o w d i r e c t l y t h e r e d u c t i o n o f i r o n i n t h e f e r r i t i n c o r e by m o n i t o r i n g t h e s h i f t i n t h e a b s o r p t i o n edge f r o m t h e p o s i t i o n f o r F e ( I I 1 ) t o t h a t of F e ( I 1 ) (Fig. 1). P r e l i m i n a r y d a t a u s i n g t h i o g l y c o l i c a c i d as t h e e l e c t r o n d o n o r s h o w e d t h a t b u f f e r i o n s [Hepes ( 4 - ( 2 - h y d r o x y e t h y 1 ) - 1 - piperazineethanesul f o n i c a c i d ) vs. T r i s ( T r i ~ ( h y d r o x y m e t h y l )ami nomethane)] a1 t e r e d t h e r a t e and t h e p a t h w a y o f r e d u c t i o n . Such d a t a s u g g e s t t h a t , i n vivo, s m a l l changes i n t h e cytoplasmic environment c o u l d modulate t h e avai l a b i 1 i t y of s t o r e d i r o n .
F i g u r e 1. D i s p e r s i v e XAS r e s u l t s f o r t h e Fe
K
edge o f t h e i r o n c o r e i n h o r s e s p l e e n f e r r i t i n d u r i n g r e d u c t i o n and d i s s o l u t i o n by t h i o g l y c o l i c a c i d (TGA) i n Hepes. Na b u f f e r a t pH 7.0. Spectra were c o l l e c t e d a t 32-sec i n t e r v a l s beginning 1 min a f t e r t h e a d d i t i o n o f t h e reductant (TGA). The spectra displayed cover a 10- min i n t e r v a l ; t h e r e a c t i o n appears complete a f t e r 1.7 min. Abscissa: PIXEL number.ACKNOWLEDGEMENT
This research has been supported, i n part, by NIH g r a n t s AM20251 and GM35675 and by DOE under c o n t r a c t DE-AS05-80ER10742. The work r e p o r t e d here was p a r t i a l l y c a r r i e d o u t a t t h e S t a n f o r d S y n c h r o t r o n R a d i a t i o n L a b o r a t o r y , w h i c h i s s u p p o r t e d b y t h e Department o f Energy and t h e O f f i c e o f Basic Energy Sciences, a t beamline X - 1 1 a t t h e National Synchrotron L i g h t Source a t Brookhaven National Laboratories, and a t LURE, Orsay, France.
REFERENCES
1. Chasteen, N. D., and Thei 1, E. C. (1982) J. B i o l . Chem. 257:7672-7677.
2. Mann, S., B a n n i s t e r , J. V., and W i l l i a m x R . . T (1986) J. Mol. B i o l . 188: 225-232.
