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SINGLE CRYSTAL STUDIES OF FERRIC MYOGLOBIN COMPOUNDS
T. Harami, Y. Maeda, Y. Morita
To cite this version:
T. Harami, Y. Maeda, Y. Morita. SINGLE CRYSTAL STUDIES OF FERRIC MYO- GLOBIN COMPOUNDS. Journal de Physique Colloques, 1979, 40 (C2), pp.C2-498-C2-499.
�10.1051/jphyscol:19792174�. �jpa-00218550�
JOURNAL DE PHYSIQUE
Colloque C2, supplhent au n
O3, Tome
40,mars 1979, page C2-498
SINGLE CRYSTAL STUDIES OF FERRIC MYOGLOBIN C3MP3UNDS
T. Harami, Y. Maeda and Y. ~ o r i t a '
Research Reactor I n s t i t u t e , Kyoto University, Osaka, Japan
' ~ e s e a r c h I n s t i t u t e for Food Science, Kyoto University, Kyoto,
JapanR6sumd.- On a 6 t u d i d p a r s p e c t r o m d t r i e Mossbauer d e s m o n o c r i s t a u x d e composds f e r r i q u e s soumis 1 u n f a i b l e champ m a g d t i q u e , a p p l i q u d p e r p e n d i c u l a i r e m e n t 1 l a d i r e c t i o n d e s r a y o n s y e t 2 l a t e m p e r a t u r e d e l ' h d l i u m l i q u i d e . P o u r d e s composds h a u t s p i n , MetMb e t YbF, d e s t e n s e u r s A i s o t r o p e s o n t 6 t d ob-
t e n u s . P o u r l e compos6 b a s s p i n Y b N , , l ' a x e d e l a p l u s g r a n d e composante du g r a d i e n t d e champ b l e c - t r i q u e e s t p r a t i q u e m e n t d a n s l e p l a n d e l'heme e t f a i t un a n g l e d e 32" a v e c une d e s q u a t r e d i r e c t i o n s Fe-N. C e c i e s t e n a c c o r d a v e c l e s r d s u l t a t s o b t e n u s p a r RPE.
A b s t r a c t . - The Mcssbauer measurements o f s i n g l e c r y s t a l s o f some f e r r i c compounds h a v e b e e n done i n t h e p r e s e n c e of a s m a l l e x t e r n a l m a g n e t i c f i e l d p e r p e n d i c u l a r t o y - r a y s a t t h e l i q u i d He t e m p e r a t u r e . F o r h i g h s p i n compounds, metMb and MbF, i s o t r o p i c A - t e n s o r s h a v e been o b t a i n e d . F o r low s p i n MbN,, t h e a x i s o f t h e l a r g e s t p r i n c i p a l component of t h e EFG l i e s n e a r l y i n t h e heme p l a n e and forms a n a n g l e of 32' w i t h one o f f o u r Fe-N d i r e c t i o n s , which c o r r e s p o n d s t o t h e r e s u l t s o f EPR e x p e r i m e n t s o f s i n g l e c r y s t a l MbN,.
I . I n t r o d u c t i o n . - D u r i n g t h e p a s t y e a r s a l a r g e num- b e r of e x p e r i m e n t a l and t h e o r e t i c a l p a p e r s have b e e n p u b l i s h e d c o n c e r n i n g t h e d e t e r m i n a t i o n o f t h e e l e c - t r o n i c s t r u c t u r e o f t h e i r o n i n v a r i o u s f e r r i c hemo- p r o t e i n s . The a n a l y s e s of e l e c t r o n p a r a m a g n e t i c re%- nance (EPR) / I - 3 / , m a g n e t i c s u s c e p t i b i l i t i e s / 4 / and p a r a m a g n e t i c a n i s o t r o p y measurements /5/ have p r o v i - ded d e t a i l e d knowledge of t h e e l e c t r o n i c s t r u c t u r e s of h i g h s p i n and low s p i n i o n s . Mossbauer s t u d i e s of h e m o p r o t e i n s h a v e b e e n done w i t h t h e i r s o l u t i o n o r p o l y c r y s t a l l i n e s a m p l e s f o r o n e d e c a d e o r s o /6-101.
We p r e s e n t M6ssbauer measurements of s i n g l e c r y s t a l s o f some f e r r i c Mb compounds i n t h e p r e s e n c e of a s m a l l e x t e r n a l m a g n e t i c f i e l d a t t h e l i q u i d He t e m p e r a t u r e , which c o n t a i n h i g h s p i n f e r r i c m e t m and MbF and low s p i n f e r r i c XbN
.
The z e r o f i e l d s p e c t r a o f f e r r i c heme compounds a r e c o m p l i c a t e d by s p i n r e l a x a t i o n and t h e l a r g e amount of s p i n t r a n s f e r t o l i g a n d s / 6 - l o / . I n a p p l i e d f i e l d s a t 4 . 2 K , how- e v e r , w e l l - r e s o l v e d p a r a m a g n e t i c h y p e r f i n e s p e c t r a were o b s e r v e d . We u s e d t h e s p i n H a m i l t o n i a n formalismt o a n a l y s e t h e I 6 s s b a u e r s p e c t r a o f t h e h i g h s p i n compounds. F o r t h e low s p i n compound, t h e M k b a u e r p a r a m e t e r s and t h e o r i e n t a t i o n s o f t h e EFG p r i n c i p a l a x e s and e f f e c t i v e m a g n e t i c f i e l d , r e l a t i v e t o t h e c r y s t a l a x e s , h a v e b e e n d e t e r m i n e d by a computer a n a l y s i s o f t h e s e s p e c t r a .
2 . E x p e r i m e n t a l . - S i n g l e c r y s t a l s o f 90 % 5 7 ~ e - e n r i c h e d metMb w e r e p r e p a r e d from sperm w h a l e s k e l e -
t a l m u s c l e and w e r e grown t o a s i z e 1 x 1 ~ 2 mm o r l a r - g e r f o r I month i n 75 % s a t u r a t e d ammonium s u l f a t e s o l u t i o n c o n t a i n i n g 0.2 M p h o s p h a t e b u f f e r , pH 6 . 8
a t 2 0 ' ~ . C r y s t a l l o g r a p h i c a n a l y s i s b e i n g p e r f o r m e d , t h e c r y s t a l s b e l o n g e d t o a m o n o c l i n i c s y s t e m , P 2 1 ,
0
w i t h l a t t i c e c o n s t a n t s , a = 64.6
i.
b = 31.1 A, c =0
34.8 A, f3 = 105.5', which w e r e i d e n t i c a l w i t h t h o s e of t y p e A c r y s t a l r e p o r t e d b y Kendrew and P a r r i s h / I ] / . S i n g l e c r y s t a l s a m p l e s of WbF and MbN3 were p r e p a r e d b y p u t t i n g c r y s t a l s of metMb i n t o 0.1 MNaF and NaN, s o l u t i o n r e s p e c t i v e l y , c o n t a i n i n g 75 % s a - t u r a t e d ammonium s u l f a t e and 0.2 M p h o s p h a t e b u f f e r pH 6 . 8 f o r a few d a y s . The c r y s t a l was v i s u a l l y a l i - gned on a l u c i t e p l a t e w i t h a s m a l l l e a d c o l l i m a t o r . The m a g n e t i c f i e l d was a p p l i e d a l o n g t h e c r y s t a l l o - g r a p h i c
a ,
b o rc
a x i s , b e c a u s e i n t h i s g e o m e t r y t h e two Fe s i t e s o f t h e m o n o c l i n i c s u b s t a n c e a r e e x p e c t e d t o show i d e n t i c a l s p e c t r a . The Mossbauer s p e c t r a were measured a t l i q u i d He t e m p e r a t u r e w i t h a s m a l l m a g n e t i c f i e l d ( 0 . 6 kG) p e r p e n d i c u l a r t o y - r a y s .3. R e s u l t s and d i s c u s s i o n s . -
3.1. liigh_-geig-c~pounds (metMb .agd_-gF_).- The ?16ss- b a u e r s p e c t r a of metMb a r e g i v e n i n t h e f i g u r e 1 ( l e f t ) . F o r h i g h s p i n compounds, we a n a l y s e d t h e e l e c t r o n i c s t r u c t u r e w i t h t h e f o l l o w i n g s p i n Hamil- t o n i a n ,
H=D(s:-s(s+I)/~) + 2BeH. S + P ~ : - I ( I + I ) / ~
We s e t up a n
xyz
c o o r d i n a t e s y s t e m f i x e d o n e a c h of t h e two heme g r o u p s w i t h d i f f e r e n t o r i e n t a t i o n s , where x andy
a x e s a r e i n t h e heme p l a n e and z a x i s i s p e r p e n d i c u l a r t o t h e heme p l a n e . We u s e d D v a l u e s from t h e r e s u l t s of m a g n e t i c s u s c e p t i b i l i t y o f t h e s eArticle published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19792174
h i g h s p i n compounds / 4 , 5 / . t h e n w i l l r e s u l t from a computer f i t t o t h e measured i n t e n s i t i e s . The EFG p r i n c i p a l a x e s i n t h e c r y s t a l
- 2 .
,-
ID! - IC I b l
6 .
a
"98. 03
F i g . 1 : Msssbauer s p e c t r a o f s i n g l e c r y s t a l metmyo- g l o b i n ( l e f t ) and myoglobin a z i d e ( r i g h t ) a t l i q u i d h e l i u m t e m p e r a t u r e . Sample o r i e n t a t i o n s a r e p a r a l l e l t o ( a ) H
N a ,
y1 1 ~ ' ;
( b ) HN
b , yI/
c ' ;( c ) Ha
Cf/
c, y / / a 1 . The s o f i d l i n e s a r e s p e c t r a c a l c u l a t e d a s d e s c r i b e d i n t h e t e x t .Then t h e p a r a m e t e r s w e r e o b t a i n e d a s
metMb : D = 10.0 cm-', P = 0.55 mm/s, A. = 1.31 m/s, MbF : D = 7 . 0 cm-', P = 0 . 3 5 m / s , A = 1.39 m / s . The v a l u e s o f i s o m e r s h i f t a r e 0 . 4 3 2 0.05 m/s f o r metMb and 0 . 4 6 f 0 . 0 5 mm/s f o r MbF. I n t h e f i g u r e
( l e f t ) , t h e c a l c u l a t e d s p e c t r a of met% a r e shown w i t h s o l i d l i n e s . The e l e c t r o n i c c o n f i g u r a t i o n o f h i g h s p i n i o n s i s 65. However, Lang e t a l . / 6 , 7 / s u g g e s t e d t h a t t h e i n t e r a c t i o n s which p r o d u c e non- z e r o D m i g h t a l s o r e d u c e A-tensor t o a x i a l symmetry and t h a t A, i s a b o u t 13 % less t h a n A Z , which i s e s t i m a t e d by t h e v a l u e o f t h e q u a d r u p o l e s p l i t t i n g o f metMb. Our s i n g l e c r y s t a l measurements w i t h t h e m a g n e t i c f i e l d a p p l i e d a l o n g t h e a a x i s d i d n o t show
t h e a n i s o t r o p y o f A - t e n s o r .
3 . 2 . & g ~ - g p i n compound (MbN2)
. -
F o r t h i s compound, t h e MGssbauer e x p e r i m e n t a l s p e c t r a a r e shown i n t h e f i g u r e ( r i g h t ) . The Mijssbauer e f f e c t p a r a m e t e r s h a v e b e e n d e t e r m i n e d by t h e p o s i t i o n s and t h e l i n e i n t e n - s i t i e s o f t h e s p e c t r a 112,131. The H a m i l t o n i a n o f t h e h y p e r f i n e i n t e r a c t i o n w r i t t e n i n t h e p r i n c i p a l a x e s s y s t e m o f t h e EFG i s :where
I
VzzI 2 I
VyyI
)I
VnI
and 05
Q2
1 . O and41
a r e p o l a r a n g l e s o f t h e d i r e c t i o n o f t h e e f f e c t i v e m a g n e t i c f i e l d H e f f r e s p e c t i v e t o t h e p r i n - c i p a l a x e s s y s t e m o f t h e EFG. The v a l u e s s i n 2 8 , c o s 2 @ and eQVzz c a n b e c a l c u l a t e d u n i q u e l y a s f u n c - t i o n s o f I) and t h e s i g n o f eQVZZ. These p a r a m e t e r ss y s t e m c a n b e l o c a t e d from t h r e e measurements w i t h d i f f e r e n t o r i e n t a t i o n s o f t h e c r y s t a l . The r e s u l t s a r e i n d e t a i l : asymmetry p a r a m e t e r ~l = 0 . 4 f 0 . 1 , q u a d r u p o l e s p l i t t i n g AE = 2.19 f 0 . 0 5 m / s , e Q V Z z < O ,
Q
i s o m e r s h i f t = 0 . 3 0 f 0 . 0 5 mm/s. The e f f e c t i v e f i e l d Heff i s 184.0 2 5 . 0 kG (Ha / / a ) , 133.0 2 5 . 0 kG
(Ha / / b ) , 9 9 . 0 2 5 . 0 kG (Ha / / e l . The z a x i s of t h e EFG p r i n c i p a l s y s t e m i s r a t h e r c l o s e t o t h e heme p l a n e and d e v i a t e s by 32' f r o m o n e o f f o u r Fe-N d i r e c t i o n s . I n 1971, H o r i / 3 / measured t h e d i r e c t i o n s o f t h e p r i n c i p a l a x e s o f t h e g - t e n s o r s t o t h e heme p l a n e . Then t h e d i r e c t i o n of t h e z a x i s ( p r i n c i p a l a x i s o f t h e g - t e n s o r c o r r e s p o n d i n g t o t h e minimal g - v a l u e ) i s n o t p a r a l l e l t o t h e p l a n e o f t h e l i n k e d h i s t i d i n e r i n g n o r t o one o f Fe-N d i r e c t i o n s , b u t i s r o t a t e d a b o u t t h e heme normal by a n a n g l e 31". From o u r s i n g l e c r y s t a l e x p e r i m e n t s o f low s p i n MbN3, we found t a h t t h e d i r e c t i o n s o f t h e x a x i s f o r b o t h g- t e n s o r and EFG t e n s o r w e l l c o i n c i d e d w i t h e a c h o t h e r . We c a l c u l a t e d t h e Mgssbauer s p e c t r a of t h i s low s p i n compounds w i t h t h e wave f u n c t i o n s and t h e e n e r g y l e v e l s w h i c h were o b t a i n e d from t h e m a g n e t i c d a t a and t o o k t h e same method t h a t O o s t e r h u i s and Lang made u s e o f t o p r e d i c t ~ G s s b a u e r s p e c t r a o f K,Fe(CN), 1141. I n t h e f i g u r e ( r i g h t ) , t h e s o l i d l i n e s show t h e c a l c u l a t e d X s s s b a u e r s p e c t r a . I t was found t h a t t h e wave f u n c t i o n s and t h e e n e r g y l e v e l s o b t a i n e d f r o m EPR were w e l l f i t t e d t o t h e Msssbauer s p e c t r a a p a r t from t h e d e t a i l s .
R e f e r e n c e s
/ I / K o t a n i , M. and Yorimoto, H.,"Magnetic Resonance i n B i o l o g i c a l s y s t e m s w (Pergamon P r e s s New York)
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