EXAFS AND XANES OF SUPEROXIDE DISMUTASE : A COPPER PROTEIN

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EXAFS AND XANES OF SUPEROXIDE DISMUTASE : A COPPER PROTEIN

S. Hasnain, L. Alagna, N. Blackburn, R. Strange

To cite this version:

S. Hasnain, L. Alagna, N. Blackburn, R. Strange. EXAFS AND XANES OF SUPEROXIDE DISMU- TASE : A COPPER PROTEIN. Journal de Physique Colloques, 1986, 47 (C8), pp.C8-1129-C8-1136.

�10.1051/jphyscol:19868220�. �jpa-00226133�

JOURNAL DE PHYSIQUE

C o l l o q u e C8, s u p p l 6 m e n t a u n o 1 2 , Tome 47, d 6 c e m b r e 1986

EXAFS AND XANES OF SUPEROXIDE DISMUTASE : A COPPER PROTEIN

S.S. HAsNAIN*, L . ALAGNA**, N . J . BLACKBURN*** a n d

R.W. STRANGE*,"'

"synchrotron Radiation Source, SERC Daresbury Laboratory, GB-Warrington W A 4 4AD, Great-Britain

Consiglio Nazionale delle Ricerche, Via Salaria, K M 29.500, Casella Postale 10, 1-00016 Monterotondo Stazione, Roma, Italy

" " ~ e p a r t m e n t of Chemistry, UMIST, P.O. Box 88, GB-Manchester

M60 l Q V , Great-Britain

Abstract

EXAFS and XANES have provided uriique and s p e c i f i c s t r u c t u r a l information f o r a number of copper p r o t e i n s . Some of t h i s work has r e c e n t l y been reviewed ill. We r e p o r t here t h e EXAFS and XANES work on superoxide dismutase i n some d e t a i l to high- l i g h t some of t h e s t r u c t u r a l information which EXAFS and XANES have provided, and a l s o to r e p o r t on some of t h e r e c e n t developments f o r c a l c u l a t i n g multiple s c a t t e r - i n g e f f e c t s i n the EXAFS region. An attempt has been made to understand the high r e s o l u t i o n XANES region of superoxide dismutase, which shows t h e presence of i n t e r - ligand s c a t t e r i n g i n a d d i t i o n to the strong m u l t i p l e s c a t t e r i n g from the atoms of an imidazole r i n g [21. This o f f e r s an e x c i t i n g p o s s i b i l i t y of i n v e s t i g a t i n g t h e stereochemical changes which accompany ligand binding o r reduction of t h e protein.

1. EXAFS and XANES

The x-ray absorption spectrum has been t r a d i t i o n a l l y divided i n t o an EXAFS and a XANES region; s t r u c t u r e observed a t some 50-60 eV above the absorption threshold has been r e f e r r e d t o a s t h e EXAFS region while s t r u c t u r e below it a s t h e XANES. A s we s h a l l see i n the following s e c t i o n , t h e EXAFS region can be extended to lower values of photoelectron energies i f use i s made of t h e s p h e r i c a l wave method and t h e m u l t i p l e s c a t t e r i n g e f f e c t s a r e considered from atoms of a r i n g such a s an

imidazole. A t high photoelectron energies, the s i m p l i f i e d plane wave approximation f o r EXAFS i s v a l i d [3 I .

A t lower e l e c t r o n energies, t h e curvature of t h e e l e c t r o n wave cannot be neglected and thus t h e plane wave approximation breaks down and leads t o i n c o r r e c t determina- t i o n of interatomic distances. Furthermore, it i s t h i s low energy p a r t which con- t a i n s most of t h e information, p a r t i c u l a r l y from the more d i s t a n t s h e l l . Also, f o r metalloproteins where t h e s c a t t e r e r s a r e generally weak (0, N e t c ) , t h e data i s most

r e l i a b l e a t lower energies. The low energy p a r t of t h e EXAFS spectrum can be used i f use of t h e exact theory given by Lee and Pendry [41 i s made, which takes account of t h e curvature of e l e c t r o n wave and thus has been namcd t h e 'curved wave method'.

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868220

JOURNAL DE PHYSIQUE

I n g e n e r a l t h i s s i n g l e s c a t t e r i n g t h e o r y i s s u f f i c i e n t f o r i n t e r p r e t a t i o n of EXAFS d a t a . However, s t r o n g m u l t i p l e s c a t t e r i n g e f f e c t s can r e s u l t from g e o m e t r i c a l arrangement of l i g a n d s e.g. i n an imidazole l i g a n d s t r o n g m u l t i p l e s c a t t e r i n g e x i s t s due to an almost c o l l i n e a r geometry of Cu-Na-C

.

[ 5 ] . T h i s s h o r t e x i n 9 of t h e bond r e s u l t s from a phase m o d i f i c a t i o n due to t h e i n t e r v e n i n g atom. Recently t h e s i n g l e s c a t t e r i n g curved-wave EXAFS f u n c t i o n h a s been modified 161 t o i n c l u d e t h e double- and t r i p l e - s c a t t e r i n g c o n t r i b u t i o n s , which a r e expected t o be t h e o n l y o t h e r c o n t r i b u t i o n s of s i g n i f i c a n c e in t h e EXAFS r e g i o n .

These m u l t i p l e s c a t t e r i n g c o n t r i b u t i o n s and h i g h e r o r d e r s c a t t e r i n g become even more important in t h e x-ray a b s o r p t i o n near-edge r e g i o n (XANES) [71. I n t h e XANES, m u l t i p l e s c a t t e r i n g c o n t r i b u t i o n s a r e s t r o n g even i n t h e open s t r u c t u r e and t h u s o f f e r a p o s s i b i l i t y of o b t a i n i n g a f u l l e r s t e r e o c h e m i c a l p i c t u r e of t h e metal s i t e in p r o t e i n s . Recent t h e o r e t i c a l developments have made t h i s r e g i o n of t h e spectrum t r a c t a b l e t o t h e o r e t i c a l i n t e r p r e t a t i o n and o f f e r s a unique method of o b t a i n i n g h i g h e r c o r r e l a t i o n f u n c t i o n s in aqueous p r o t e i n samples.

2. Superoxide dismutase ( Cu-Zn)

A g r e a t d e a l of s t r u c t u r a l information i s a v a i l a b l e f o r t h e bovine Cu-Zn SOD;

i n c l u d i n g i t s amino a c i d sequence, and t h e c r y s t a l s t r u c t u r e d e t e r m i n a t i o n a t high r e s o l u t i o n [ a ] . It i s a dimeric p r o t e i n , of molecular weight

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2.1 P r o t e i n s o l u t i o n and f r e e z e - d r i e d powder

Blackburn e t al have used t h e EXAFS and XANES technique f o r s t u d y i n g t h e Cu and Zn s i t e s of t h e f r e e z e - d r i e d powder 151 and s o l u t i o n [ I 03 Cu-Zn bovine superoxide dismutase. They were a l s o a b l e t o show t h a t t h e 4 Cu-N d i s t a n c e f o r t h e p r o t e i n was 2.00 2 0.02 A, which i s t y p i c a l f o r [ C ~ ( i m i d ) ~ ] * + complexes. A t t h e time t h e 2 8

c r y s t a l s t r u c t u r e d a t a w i t h r e s i d u a l f a c t o r R

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[131. Thus, a comparative s t u d y o f s o l u t i o n and f r e e z e - d r i e d p r o t e i n may prove h e l p f u l i n o t h e r c a s e s t o o f o r d e f i n i n g t h e p r e s e n c e of t h e c o o r d i n a t e d water. The o b s e r v a t i o n of Cu-0 a t 2.24 f 0.03 8 provided c o n f i r m a t i o n to t h e c o n s i d e r a b l e s p e c t r o s c o p i c evidence t h a t copper i n s u p e r o x i d e dismutase i s f i v e c o o r d i n a t e , t h e f i f t h c o o r d i n a t i o n p o s i t i o n being taken up by a water molecule. For t h e Zn s i t e , 3 Zn-N were e s t a b l i s h e d a t 2.01 2 0 . 0 2 8 and Zn-O a t 2.04 2 0.03 8. We note t h a t , given t h e d a t a range, t h e d i s t i n c t i o n between Zn-N and Zn-0 c o n t r i b u t i o n s i s n o t p o s s i b l e .

2 . 2 Oxidised v s reduced p r o t e i n

Blumberg e t a 1 [I41 used t h e Zn edge XANES of t h e n a t i v e and t h e reduced p r o t e i n t o show t h a t t h e n e a r edge spectrum of t h e two were v e r y s i m i l a r , t h u s s u g g e s t i n g t h a t t h e b r i d g i n g imidazole from His-61 i s c o o r d i n a t e d to Zn i n b o t h c a s e s .

Blackburn e t a 1 [I 0 I extended t h i s study t o show t h a t w h i l e t h e Zn-edge spectrum d i d not change, t h e Cu-edge XANES and EXAFS changed.

A comparison of t h e Cu K-edge EXAFS f o r t h e two forms showed t h e o v e r a l l ampli- t u d e decreased by

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c o o r d i n a t i o n . I n a d d i t i o n t o a d e c r e a s e i n c o o r d i n a t i o n number, t h e y f i n d t h a t t h e Cu-N bond l e n g t h i s decreased t o 1.94 f 0.02 A, which i s again i n d i c a t i v e of a c o o r d i n a t i o n number lower t h a n i n t h e n a t i v e p r o t e i n . These r e s u l t s have provided t h e f i r s t d i r e c t s t r u c t u r a l i n f o r m a t i o n on t h e copper s i t e of t h e reduced p r o t e i n . 2.3 M u l t i p l e s c a t t e r i n g e f f e c t s

It has been r e a l i s e d f o r some time t h a t t h e o u t e r atomic s h e l l s of an imidazole r i n g cause s t r o n g m u l t i p l e s c a t t e r i n g over an extended k-range i n t h e EXAFS, and t h a t t h e EXAFS c a n be s i m u l a t e d o n l y by allowing t h e s e atcans t o t a k e a s u b s t a n t i a l l y s h o r t e r p o s i t i o n Ca. 0.2-0.3 A under t h e s i n g l e s c a t t e r i n g scheme. We p r o v i d e (elsewhere in t h e proceedings) t h e f i r s t q u a n t i t a t i v e examination o f m u l t i p l e s c a t t e r i n g c o n t r i b u t i o n from t h e atoms of an imidazole r i n g . Here, f o r completeness, some of t h e r e l e v a n t r e s u l t s a r e d e s c r i b e d .

I ^-

1

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I

Figure 1. Cu K-edge EXAFS of f r e e z e d r i e d superoxide dismutase and a t h e o r e t i c a l s i m u l a t i o n based on parameters given i n t a b l e 1. The m u l t i p l e s c a t t e r i n g e f f e c t s from o u t e r s h e l l atoms of t h e imidazole r i n g s have been included. The experimental spectrum has been s h i f t e d by 18 eV t o match t h e t h e o r e t i c a l c a l c u l a t i o n .

C8-1132 JOURNAL DE PHYSIQUE

An a n a l y s i s of EXAFS d a t a from s e v e r a l c r y s t a l l o g r a p h i c a l l y w e l l c h a r a c t e r i s e d compounds has been c a r r i e d o u t t o d e f i n e c l e a r l y t h e m u l t i p l e s c a t t e r i n g e f f e c t s o f an imidazole r i n g . We f i n d t h a t i f t h e m u l t i p l e s c a t t e r i n g e f f e c t s a r e included, t h e t h o e r e t i c a l c a l c u l a t i o n s c a n b e extended t o t h e experimental k = 0 8-I to f i t t h e o v e r a l l shape of t h e XANES spectrum. F i g u r e 1 i s t h e b e s t s i m u l a t i o n f o r t h e Cu K-edge EXAFS of t h e f r e e z e - d r i e d form of superoxide dismutase. T h i s i s o b t a i n e d w i t h t h e parameters given in t a b l e 1, which a r e compared with t h e parameters f o r

s i n g l e s c a t t e r i n g s i m u l a t i o n s . I t i s c l e a r t h a t a r i g o r o u s a n a l y s i s of t h e EXAFS spectrum, when m u l t i p l e s c a t t e r i n g e f f e c t s a r e taken i n t o account, y i e l d s c o r r e c t d i s t a n c e s of a l l t h e atoms of an imidazole r i n g . I n a d d i t i o n t h e s t r o n g a n g u l a r dependence of t h e m u l t i p l e s c a t t e r i n g along with t h e s p l i t t i n g of C2/C5 atoms should a l l o w a d e t e r m i n a t i o n of t h e o r i e n t a t i o n of an imidazole r i n g around Cu-N.

T a b l e 1. P a r a m e t e r s f o r b e s t s i m u l a t i o n s of t h e Cu K-edge EXAFS spectrum of t h e f r e e z e - d r i e d powder of Cu-Zn s u p e r o x i d e dismutase

Atoms S i n g l e s c a t t e r i n g M u l t i p l e s c a t t e r i n g c o r r e c t e d

R ( a ) a2 ( a 2 ) R ( a ) 02

2.4 S i g n i f i c a n c e of s t r u c t u r e in XANES

We [21 have r e c e n t l y c a r r i e d o u t d e t a i l e d m u l t i p l e s c a t t e r i n g c a l c u l a t i o n s t o understand t h e XANES o f a number of copper imidazole compounds which show EXAFS and XANES s p e c t r a a k i n to t h e copper s i t e o f t h e f r e e z e - d r i e d and aqueous superoxide dismutase. F i g u r e 2 shows t h e experimental XANES f o r t h r e e imidazole complexes each with two a x i a l l i g a n d s to t h e CuN4 e q u a t o r i a l plane. Also shown a r e t h e s p e c t r a f o r t h e Cu s i t e of superoxide dismutase. Each of t h e s p e c t r a show a number of s p e c t r a l f e a t u r e s , with v a r y i n g degree of r e s o l u t i o n , s u g g e s t i n g s i g n i f i c a n t s t e r e o c h e m i c a l d i f f e r e n c e s between them. We have shown t h a t each o f t h e s e f e a t u r e s , t h e i r r e l a t i v e i n t e n s i t y and r e s o l u t i o n a r e a s i g n a t u r e of a p a r t i c u l a r a s p e c t of t h e s t r u c t u r e . For example, t h e peak marked 'b' a r i s e s due to t h e a x i a l l i g a n d s ; i t s i n t e n s i t y and p o s i t i o n i s r e l a t e d to t h e number and p o s i t i o n o f t h e s e l i g a n d s . S i m i l a r l y , f e a t u r e s marked 'd' and ' e ' (and a s s o c i a t e d f e a t u r e s ' f ' and ' g'

,

2.5 Axial l i g a n d s

The f i r s t of t h e s e can be simply demonstrated by a s e t of s i n g l e - s h e l l c a l c u l a - t i o n s , f i g . 3. I n f i g . 3a, s c a t t e r i n g c o n t r i b u t i o n s w i t h t h e four n i t r o g e n s a t 2.01

a

Energy (Hartrees)

F i g u r e 2. Experimental XANES of t h r e e copper i m i d a i o l e complexes and f o r t h e Cu s i t e o f Cu-Zn superoxide dismutase in t h e f r e e z e - d r i e d and aqueous s t a t e . 1 H a r t r e e = 27.2 eV.

shows up most in t h e n-component. The p o l a r i s a t i o n - a v e r a g e d s p e c t r m i s shown in t h e right-hand panel. F i g u r e 3b shows t h e s e c o n t r i b u t i o n s w i t h two oxygen l i g a n d s included a t 2.56

a.

,+

3 2

due to a r e d u c t i o n i n t h e number of a x i a l lxgands and i t s p o s i t i o n ( f i g s . 3b and 3 d ) . This i s c o n s i s t e n t with t h e EXAFS r e s u l t of superoxide dismutase [I41 which shows t h a t t h e oxygen atom i s a t 2.24

a

C ~ ( I m i d ) ~ (No ) a t an average d i s t a n c e of 2.56 8. We n o t e t h a t t h e p s i t i o n of t h i s f e a t u r e 'b' w l l l 3.2 n o t be a f f e c t e d s i g n i f i c a n t l y when t h e a x i a l l i g a n d ' s d i s t a n c e i s

(28-1134 JOURNAL DE PHYSIQUE

i n c r e a s e d beyond

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Energy (Hartrees)

Figure 3. P a n e l s on t h e l e f t hand s i d e a r e t h e two p o l a r i s a t i o n components, p ( s o l i d curve) and n (dashed c u r v e ) . The r i g h t hand panel g i v e s t h e averaged spectrum. ( a ) One-shell c a l c u l a t i o n with Cu-N where Cu-N i s a t 2.01 ,fi; ( b ) w i t h CuN402, where oxygen atoms a r e p l a c e d a t 2.56 %'normal to t h e CuN4 p l a n e . m e arrowed f e a t u r e corresponds t o t h e experimental f e a t u r e marked 'b', in f i g . 2; ( c ) w i t h CuN40, where Cu-0 i s a t 2.56 ,fi and ( d l where Cu-0 i s a t 2.24 A.

2.6 ~ e l a t i v e o r i e n t a t i o n of d i f f e r e n t groups

F i g u r e 4 shows two c a l c u l a t i o n s with a c l u s t e r of 22 atoms o r i g i n a t i n g from t h e f o u r imidazole groups and two a x i a l oxygen atoms a t 2.56 A. I n ( a ) , t h e two imidazole r i n g s a r e p e r p e n d i c u l a r and two n e a r l y f l a t w i t h r e s p e c t to t h e CuN4 plane. For (b), a l l t h e four imidazole r i n g s a r e n e a r l y p e r p e n d i c u l a r +a t h e CuN4 plane. The two c a l c u l a t e d s p e c t r a a r e almost i d e n t i c a l over t h e whole XANES e x c e p t i n t h e r e g i o n around experimental f e a t u r e s ' f ' and 'g'. I n t h e c a s e of ( a ) both fep.tures are reproduced, whereas t h e y merge when a l l t h e r i n g s a r e p e r p e n d i c u l a r t o t h e p l a n e . Thus, t h e f a c t t h a t t h e XANES s p e c t r a a r e s e n s i t i v e to t h e r e l a t i v e o r i e n t a t i o n of t h e imidazole r i n g s o f f e r s t h e e x c i t i n g p o s s i b i l i t y t h a t r e l a t i v e o r i e n t a t i o n of d i f f e r e n t groups around a metal s i t e can b e determined u s i n g high r e s o l u t i o n XANES s p e c t r a .

Energy (Hartrees)

F i g u r e 4. Three s h e l l c a l c u l a t i o n s f o r CuN(Imid)402. ( a ) 2 imidazole r i n g s Lr and two II t o t h e CuN4 p l a n e ; ( b ) a l l 4 imidazole r i n g s n e a r l y l r . D i f f e r e n c e s i n t h e two t h e o r e t i c a l s p e c t r a between 1.2-2.00 H a r t r e e s a r e evidence f o r t h e i n t e r - l i g a n d s c a t t e r i n g .

2.7 XANES s p e c t r a of t h e superoxide dismutase

F i g u r e 5a shows t h e e x p e r i m e n t a l XANES spectrum of superoxide dismutase and f i g . 5b-d a r e t h e v a r i o u s s i m u l a t i o n s u s i n g d i f f e r e n t geometrical arrangements around t h e Cu atom. F i g u r e 5b i s based on t h e c r y s t a l s t r u c t u r e c o o r d i n a t e s a v a i l a b l e 20 months ago i n t h e Brookhaven P r o t e i n Databank. F i g u r e 5d was o b t a i n e d from t h e c o o r d i n a t e s o b t a i n e d u s i n g t h e EXAFS d a t a of Cu and Zn s i t e s i n c o n j u n c t i o n w i t h t h e c r y s t a l s t r u c t u r e d a t a of t h e p r o t e i n w i t h t h e molecular g r a p h i c s f a c i l i t y a t Birkbeck College. D e t a i l s of t h i s work have been p u b l i s h e d elsewhere [151.

F i g u r e 5c was o b t a i n e d with r e v i s e d c r y s t a l s t r u c t u r e c o o r d i n a t e s , which show an obvious improvement. These c o o r d i n a t e s were o b t a i n e d i n December 1985 from a f u r t h e r c r y s t a l l o g r a p h i c refinement, R = 19% a t 2

a

comparison of f i g s . 5b and 5d with t h e experimental spectrum c l e a r l y shows t h a t t h e EXAFS-generated Cu s i t e r e s u l t s i n a c l o s e s i m u l a t i o n of t h e XANES spectrum. T h i s opens up an e x c i t i n g p o s s i b i l i t y where use i s made of EXAFS and XANES f o r o b t a i n i n g a more r e f i n e d s t r u c t u r e of a metal s i t e i n a p r o t e i n , but more i m p o r t a n t l y i n o b t a i n i n g t h i s i n f o r m a t i o n f o r p r o t e i n i n s o l u t i o n and a l s o when t h e p r o t e i n i s modified e i t h e r by l i g a n d binding, change of pH o r o x i d a t i o n s t a t e . F i g u r e 6 shows t h e s e n s i t i v i t y of t h i s r e g i o n where t h e s p e c t r a f o r t h e Cu-site of SOD i n i t s d i f f e r e n t forms a r e shown. Thus, t h e importance of understanding t h e XANES r e g i o n cannot be over emphasised.

Acknowledgement

We would l i k e t o thank t h e Science and Engineering Research Council f o r t h e i r s u p p o r t of t h i s work. We a r e p l e a s e d t o acknowledge D r . Paul Durham f o r s e v e r a l d i s c u s s i o n s on t h e use of XANES. D r . N. B i n s t e d ' s c o n t r i b u t i o n t o t h i s work i s i n v a l u a b l e , a s without h i s programme package MCURVE most of t h e work p r e s e n t e d h e r e would not have been p o s s i b l e .

CS-1136 JOURNAL DE PHYSIQUE

I I

0.2 0.6 1.0 1 4 1.8 2.2

Energy (Hartrees)

F i g u r e 5. Experimental XANES of an o x i d i s e d s o l u t i o n of superoxide d i s - mutase w i t h a number of 3 - s h e l l calcu- l a t i o n s ( s e e t e x t f o r d e t a i l s ) .

8960 8980 9000 9020 9040 Photon energy (eV)

F i g u r e 6 . Experimental XANES of super- oxide dismutase s o l u t i o n p r o t e i n i n i t s n a t i v e , reduced and cyano-bound form.

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