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CHANGES IN THE METAL COORDINATION SPHERE OF AZURIN AND STELLACYANIN
UNDER INFLUENCE OF pH,
OXIDATION/REDUCTION AND METAL SUBSTITUTION
M. Feiters, S. Hasnain, C. Groeneveld, G. Canters, J. van Rijn, J. Reedijk, S.
Dahlin, B. Reinhammar
To cite this version:
M. Feiters, S. Hasnain, C. Groeneveld, G. Canters, J. van Rijn, et al.. CHANGES IN THE METAL COORDINATION SPHERE OF AZURIN AND STELLACYANIN UNDER INFLUENCE OF pH, OXIDATION/REDUCTION AND METAL SUBSTITUTION. Journal de Physique Colloques, 1986, 47 (C8), pp.C8-1173-C8-1176. �10.1051/jphyscol:19868229�. �jpa-00226213�
Colloque C8, supplBment au no 12, Tome 47, dkcembre 1986
CHANGES I N THE METAL COORDINATION SPHERE OF AZURIN AND STELLACYANIN UNDER INFLUENCE OF pH, OXIDATION/REDUCTION AND METAL SUBSTITUTION
M.C. FEITERS',"', S.S. H A S N A I N * * , C.M. GROENEVELDI*',
G.W. C A N T E R S * * * , J. VAN RIJN*"', J. REEDIJK**', S. DAHLIN' and B. REINHAMMAR'
'~epartment of Cllemistry, University of Manchester, GB-Manchester M13 9PL, Great-Britain
Synchrotron Radiation Source, SERC Daresbury Laboratory, GB-Warrington WA4 4 A D , Great-Britain
*.* Gorlaeus Laboratories, State University, P.O. Box 9502, NL-2300 RA Leiden, The Netherlands
'Department of Biochemistry and Biophysics, Chalmers University of Technology/University of Gothenburg, S-41296 Gothenburg, Sweden
Rksun6
La c o o r d i n a t i o n de mktal dans des p r o t g i n e s 2 c u i v r e bleu, a z u r i n e e t
s t e l l a c y a n i n e , a 6 t 6 k t u d i 6 par EXAFS. Le pH n ' a pas beaucoup d ' i n f l u e n c e sur l a c o o r d i n a t i o n d e c u i v r e dans a z u r i n e . D a n s a z u r i n e comme dans s t e l l a c y a n i n e , l16chelonnement d e s d i s t a n c e s Cu-N (His) g r a n d i t avec r a u c t i o n , e t un l i g a n t s o u f r e o u t r e l e l i g a n t S(Cys) e s t observe.
A b s t r a c t
The m e t a l c o o r d i n a t i o n sphere in t h e b l u e copper p r o t e i n s , a z u r i n and
s t e l l a c y a n i n , h a s been s t u d i e d by EXAFS. The pH h a s l i t t l e i n f l u e n c e on t h e copper c o o r d i n a t i o n i n a z u r i n . I n both a z u r i n and s t e l l a c y a n i n , t h e s p r e a d i n t h e
Cu-N(His) bonds i n c r e a s e s upon r e d u c t i o n and a sulphur l i g a n d i n a d d i t i o n t o S(Cys) i s observed.
The e l e c t r o n t r a n s f e r p r o t e i n s , t h e b l u e copper p r o t e i n s , have l i g a n d s and l i g a n d geometries p o i s e d between t h o s e p r e f e r r e d by Cu(1) and C u ( I I ) , t h u s f a c i l i t a t i n g e l e c t r o n uptake and r e l e a s e . C r y s t a l l o g r a p h i c s t u d i e s p o i n t t o c o o r d i n a t i o n by two N(His), one S(Cys) and one l o n g - d i s t a n t S(Met) i n what i s b e s t d e s c r i b e d i s a d i s t o r t e d t e t r a h e d r a l l i g a n d geometry in Populus n i g r a p l a s t o c y a n i n [11 and Pseudomonas a e r u g i n o s a a z u r i n [21. I n Rhus v e r n i c i f e r a s t e l l a c y a n i n , t h e r e i s no methionine r e s i d u e 131 and p o s s i b l e l i g a n d s in a d d i t i o n t o t h e two N(His) and one
S(Cys) have n o t y e t been p o s i t i v e l y i d e n t i f i e d .
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868229
C8-1174 JOURNAL DE PHYSIQUE
For p l a s t o c y a n i n , t h e dependence of t h e copper c o o r d i n a t i o n s p h e r e on pH and o x i d a t i o n s t a t e has been s t u d i e d c r y s t a l l o g r a p h i c a l l y [41. Such s t u d i e s a r e n o t f e a s i b l e f o e Pseudomonas aeruginosa a z u r i n , which has l e s s r o b u s t c r y s t a l s , nor f o r
Fig. 1 EXAFS (upper p a n e l ) and phase-corrected modulus of t h e F o u r i e r t r a n s f o r m ( lower p a n e l ) of Cu(1 )bbdhp. - experimental, --- s i m u l a t i o n 1 and 2 i n Pigs. l a and I b r e s p e c t i v e l y . - - - - and - - - in F i g . l b r e p r e s e n t t h e i n t e r f e r i n g sulphur and carbon c o n t r i b u t i o n s , r e s p e c t i v e l y , a t 3 8.
Table 1. R a d i a l d i s t r i b u t i o n around Cu in Cu(I)bbdhp, r a d i i in 8
C r y s t a l EXAFS EXAFS
S t r u c t u r e 171 S i m u l a t i o n 1 (Fig. l a ) S i m u l a t i o n 2 (Fig. l b )
s t e l l a c y a n i n a t n e u t r a l pH.
Table 2 Simulations f o r a z u r i n and s t e l l a c y a n i n Cu-EXAFS, main s h e l l s . D i s t a n c e s i n 8 , Debye-Waller f a c t o r s , i n p a r e n t h e s e s , a s a = 2 cr2, i n a2.
Azur i n S t e l l a c y a n i n
CU(I1) CU(I1) CU(1) CU(1) CU(I1) C U ( I )
pH 9.1 pH 4.1 pH 9.2 pH 5.5
C8-1176 JOURNAL DE PHYSIQUE
Rhus v e r n i c i f e r a s t e l l a c y a n i n , which does n o t c r y s t a l l i z e a t a l l , due to i t s h i g h c a r b o h y d r a t e c o n t e n t . EXAFS i s t h e r e f o r e t h e method of c h o i c e t o probe t h e copper environments in t h e s e p r o t e i n s . I t i s a l s o a p p l i c a b l e t o t h e Co(I1) and N I ( I 1 ) s t e l l a c y a n i n d e r i v a t i v e s , which have been s t u d i e d by lH-NMR 151.
I n our EXAFS s t u d i e s , we have i n c l u d e d model compounds with metal-nitrogen and metal-sulphur bonds o f v a r y i n g bond l e n g t h s [6,71, t o t e s t our a n a l y s i s procedures and t h e f e a s i b i l i t y of observing c e r t a i n metal-ligand i n t e r a c t i o n s in EXAFS, p a r t i c u l a r l y l o n g metal-sulphur bonds. I n t h i s l a s t r e s p e c t , t h e a n a l y s i s of Cu(1)bbdhp 171 i s i n t e r e s t i n g , a s i t s EXAFS c a n be well accounted f o r without s u l p h u r s , and t h e i r i n c l u s i o n l e a d s t o p a r t i c u l a r e f f e c t s i n t h e refinement, a s t h e s u l p h u r and carbon b a c k s c a t t e r i n g amplitudes a r e approximately o u t of phase when p l a c e d a t t h e same d i s t a n c e ( F i g . 1, Table 1 ) .
We f i n d small changes i n d i s t a n c e s and l a r g e changes in Debye-Waller f a c t o r s f o r t h e copper l i g a n d s i n a z u r i n when t h e pH and o x i d a t i o n s t a t e a r e v a r i e d ( T a b l e 2 ) . I n p a r t i c u l a r t h e i n c r e a s e in Cu-N(His) Debye-Waller f a c t o r upon r e d u c t i o n i s s t r o n g and s u g g e s t s t h a t o n e n i t r o g e n l i g a n d has come c l o s e r w h i l e t h e o t h e r h a s moved out.
I n reduced a z u r i n , we f i n d evidence f o r s u l p h u r l i g a t i o n a t 2.7 8 , with s t r o n g l y i n c r e a s i n g Debye-Waller f a c t o r a t higher pH. T h i s l o n g - d i s t a n t sulphur i s
i l l - d e f i n e d in t h e o x i d i z e d p r o t e i n , where it probably moves o u t t o i n t e r f e r e with t h e i n n e r imidazole carbon s h e l l .
The s t e l l a c y a n i n copper s i t e a t n e u t r a l pH i s i d e n t i c a l to t h a t in a z u r i n a t low pH ( F i g . 2, Table 2 ) . I n t e r e s t i n g l y , i n t h e reduced p r o t e i n EXAFS, a s i n t h e a z u r i n d a t a , a s u l p h u r atom shows up a t 2.7 8 , g i v i n g evidence f o r a d d i t i o n a l c y s t e i n e o r d i s u l p h i d e b r i d g e c o o r d i n a t i o n i n s t e l l a c y a n i n . The a n a l y s i s o f N i ( I 1 ) and C o ( I 1 ) s t e l l a c y a n i n EXAFS i s in progress. P r e l i m i n a r y r e s u l t s s u g g e s t t h a t b o t h a r e s i m i l a r t o C u ( I 1 ) s t e l l a c y a n i n b u t t h a t N i ( I 1 ) has perhaps an a d d i t i o n a l n i t r o g e n o r oxygen l i g a n d and t h a t t h e a d d i t i o n a l s u l p h u r l i g a n d h a s come c l o s e r to Co(I1).
Acknowledgement
The a u t h o r s thank t h e D i r e c t o r of t h e SERC Daresbury Laboratory f o r p r o v i d i n g experimental f a c i l i t i e s under t h e agreements with Manchester U n i v e r s i t y (EXAFS s e r v i c e ) , t h e Dutch Organization f o r t h e Advancement of Pure Research (ZWO) and t h e Swedish N a t u r a l Science Research Council.
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