Spectroscopic Studies of Active Sites - ACS Symposium Series (ACS

16 Spectroscopic Studies of Active Sites Blue Copper and Electronic Structural Analogs

Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: April 30, 1986 | doi: 10.1021/bk-1986-0307.ch016

Edward I. Solomon, Andrew A. Gewirth, and Susan L. Cohen Department of Chemistry, Stanford University, Stanford, CA 94305

An understanding of the electronic structure of metal ion active sites is essential in understanding their high reactivity. An important example of the contributions of spectroscopic, crystallographic and theoretical studies in elucidating electronic structure is in investigations of the blue copper active site in plastocyanin. These studies underscore the large role of covalent delocalization in determining the electronic and spectro scopic properties of the site. Further confirmation of the role of covalent delocalization comes from photoemission studies of small molecule spectral analogs. I n o r g a n i c s p e c t r o s c o p y has e v o l v e d t o t h e p o i n t where a g r e a t d e a l o f i n s i g h t i n t o e l e c t r o n i c s t r u c t u r e and i t s c o n t r i b u t i o n t o r e a c t i v i t y can be o b t a i n e d from d e t a i l e d s t u d i e s on h i g h symmetry t r a n s i t i o n m e t a l complexes. A t t e n t i o n c a n now be d i r e c t e d toward some r a t h e r u n u s u a l i n o r g a n i c complexes which a r e a c t i v e s i t e s i n v o l v e d i n c a t a lysis. These i n c l u d e m e t a l l o p r o t e i n s i n v o l v e d i n enzymatic c a t a l y s i s and m e t a l i o n s on s u r f a c e s i n v o l v e d i n heterogenous c a t a l y s i s . These a c t i v e s i t e s o f t e n e x h i b i t unique s p e c t r a l f e a t u r e s compared t o h i g h symmetry i n o r g a n i c complexes. These unique f e a t u r e s g e n e r a l l y d e r i v e from u n u s u a l g e o m e t r i c and e l e c t r o n i c s t r u c t u r e s imposed on the m e t a l by t h e b i o p o l y m e r o r the s u r f a c e . An u n d e r s t a n d i n g o f t h e s e geomet r i c and e l e c t r o n i c s t r u c t u r e s s h o u l d p r o v i d e s i g n i f i c a n t i n s i g h t i n t o the h i g h l y s p e c i f i c r e a c t i v i t y o f t h e s e a c t i v e s i t e s . A c l e a r example o f t h e c o n t r i b u t i o n o f i n o r g a n i c s p e c t r o s c o p y i n u n d e r s t a n d i n g the unique p r o p e r t i e s a s s o c i a t e d w i t h an a c t i v e s i t e i s the b l u e copper c e n t e r (1-3) i n p l a s t o c y a n i n . The b l u e copper s i t e e x h i b i t s unique s p e c t r a l p r o p e r t i e s when compared w i t h those o f normal copper complexes. These s p e c t r a l f e a t u r e s i n c l u d e an u n u s u a l l y s m a l l copper h y p e r f i n e s p l i t t i n g o f t h e EPR s i g n a l in^thej|„ r e g i o n (A„ < 70x10 cm as compared t o A_L = 150x10 cm f o r normal t e t r a g o n a l copper) [ F i g u r e 1] and an e x t r e m e l y i n t e n s e low e n e r g y ^ a b s o r p t i o n band ( V = 600 nm, ε * 4000 M cm compared t o ε - 100 M cm f o r normal copper complexes). T h e o r i g i n a l g o a l o f s p e c t r o s c o p y on the b l u e copper s i t e was t o under0097-6156/ 86/ 0307-0236508.75/ 0 © 1986 A m e r i c a n C h e m i c a l Society

In Excited States and Reactive Intermediates; Lever, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


16.

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Spectroscopic Studies of Active Sites

237

stand these f e a t u r e s and use them to g e n e r a t e a " s p e c t r o s c o p i c a l l y e f f e c t i v e " working model o f the a c t i v e s i t e . In p a r t i c u l a r , i n f r a r e d c i r c u l a r d i c h r o i s m (IRCD) s t u d i e s (4-5) [ F i g u r e 2] demonstrated t h a t a t l e a s t t h r e e d-d t r a n s i t i o n s e x i s t e d i n b l u e copper p r o t e i n s to e n e r g i e s below 5000 cm . A l i g a n d f i e l d a n a l y s i s (4-5) o f t h e s e t r a n s i t i o n s then i n d i c a t e d t h a t the s i t e s h o u l d have a geometry c l o s e to t e t r a h e d r a l and t h a t a l l d-d t r a n s i t i o n s o c c u r a t e n e r g i e s below 800 nm. T h e r e f o r e , the i n t e n s e 600 nm a b s o r p t i o n band must i n v o l v e a charge t r a n s f e r (CT) t r a n s i t i o n which, based on o t h e r c h e m i c a l and s p e c t r o s c o p i c s t u d i e s ( 6 - 8 ) , p r o b a b l y d e r i v e d from c y s t e i n e l i g a t i o n a t the s i t e . In 1978, h i g h r e s o l u t i o n s t r u c t u r e s (9-10) appeared which c o n f i r m e d the g e n e r a l t e t r a h e d r a l geometry and c y s t e i n e l i g a t i o n and demonstrated t h a t the r e m a i n i n g l i g a n d s a r e two imidazoles o f h i s t i d i n e and a t h i o e t h e r from m e t h i o n i n e . While the length of the i m i d a z o l e to copper bond i s f a i r l y normal when compared to model complexes, the Cu-S ( t h i o l a t e ) bond i s found to be q u i t e s h o r t (2.1 Â ) and the Cu-S ( t h i o e t h e r ) bond i s q u i t e l o n g (2.9 À ) [ F i g u r e 3 ] . With establishment o f the c r y s t a l s t r u c t u r e , t h r e e major features concerning the e l e c t r o n i c s t r u c t u r e o f the b l u e copper s i t e can be a d d r e s s e d . These f e a t u r e s are 1) the n a t u r e o f the t h i o l a t e and t h i o e t h e r bonds, 2) the n a t u r e o f the ground s t a t e w a v e f u n c t i o n and 3) t h e e x t e n t o f c o v a l e n c y . We h a v e a l s o become s t r o n g l y i n v o l v e d i n u s i n g p h o t o e l e c t r o n s p e c t r o s c o p y as a p o w e r f u l approach toward d e t e r m i n i n g c o v a l e n c y i n t r a n s i t i o n m e t a l complexes. These w i l l be d i s c u s s e d i n t u r n . T h i o l a t e and

Thioether

Bonds

The f i r s t e x p e r i m e n t s to be d i s c u s s e d i n v o l v e d p o l a r i z e d s i n g l e c r y s t a l o p t i c a l ( 11) s t u d i e s on the charge t r a n s f e r r e g i o n o f p l a s t o c y anin. The p l a s t o c y a n i n c r y s t a l has f o u r symmetry r e l a t e d m o l e c u l e s i n the Ρ 2.2^2. ( o r t h o r h o m b i c ) u n i t c e l l . The c r y s t a l morphology combined w i t h the o p t i c a l p r o p e r t i e s o f c r y s t a l s a l l o w e d p o l a r i z e d s p e c t r a t o be o b t a i n e d p a r a l l e l and p e r p e n d i c u l a r to the a, a x i s o f the (011) f a c e . The s p e c t r a i n F i g u r e 4A a r e o b s e r v e d to be s t r o n g e s t i n the p a r a l l e l ( t o "a") polarization. As the Cu-S thioether bond i s o r i e n t e d approximately along the ς, a x i s , CT t r a n s i t i o n s a s s o c i a t e d w i t h t h i s l i g a n d s h o u l d appear d o m i n a t e l y i n the perpen d i c u l a r ( t o "a") p o l a r i z a t i o n . Thus, Cu-S t h i o e t h e r CT t r a n s i t i o n s c o n t r i b u t e a t most weakly to the a b s o r p t i o n s p e c t r a , a f e a t u r e which r a i s e d s i g n i f i c a n t c o n c e r n w i t h r e s p e c t to the n a t u r e o f a copper t h i o e t h e r bond. The absence o f a l o n g bond between the copper and the t h i o e t h e r a t the b l u e s i t e has, i n f a c t , been c o n s i d e r e d as a p o s s i b l i t y based on resonance Raman (12) and EXAFS (13) s t u d i e s . A c o m b i n a t i o n o f v a r i a b l e temperature a b s o r p t i o n , CD and MCD spectroscopies (4-5) i n d i c a t e d t h a t a t l e a s t f i v e t r a n s i t i o n s are present i n the CT r e g i o n o f p l a s t o c y a n i n . A c o r r e l a t i o n o f these w i t h the p o l a r i z e d s i n g l e c r y s t a l a b s o r p t i o n s p e c t r a gave the pos s i b l e band assignments shown i n F i g u r e 4B. C l e a r l y , the imidazole contributes in this region. In a d d i t i o n , t h e s e r e s u l t s i n d i c a t e t h a t t h r e e t h i o l a t e to Cu CT t r a n s i t i o n s may be p r e s e n t . O r i g i n a l l y , two were c o n s i d e r e d : a low energy d o u b l y d e g e n e r a t e p i s e t and a h i g h e r energy, more i n t e n s e sigma t r a n s i t i o n (bands 5 and 4, r e s p e c t i v e l y ) . T h i s p a t t e r n however c o n s i d e r s o n l y the bonding o f a s u l f u r ^ to a copper. I f the C-S-Cu a n g l e i s s i g n i f i c a n t l y l e s s than 180 , the


238

EXCITED STATES AND REACTIVE INTERMEDIATES

"Blue" Copper ( "Normal" Copper


3000

1000

2A000

16000 Energy (cm ) -1

8000

2500

3300

2900 Field (gauss)

F i g u r e 1. O p t i c a l ( l e f t ) and EPR ( r i g h t ) s p e c t r a o f a b l u e copper p r o t e i n ( s o l i d l i n e ) and a t e t r a g o n a l copper s i t e (dashed l i n e ) . Reproduced from R e f . 1. C o p y r i g h t W i l e y .

5.0

0.50

0.25

w

0.0

-2.5

-0.50

14000

4000

F i g u r e 2. N e a r - i n f r a r e d c i r c u l a r d i c r h o i s m spectrum o f p l a s t o c y a n i n i n D 2 0 a t 290 K. Spectrum A c o r r e s p o n d s t o s c a l e on left. Spectrum Β c o r r e s p o n d s t o s c a l e on r i g h t . Reproduced from Ref. 5. C o p y r i g h t 1980, American C h e m i c a l S o c i e t y .



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239

F i g u r e 3· The b l u e copper s i t e i n p l a s t o c y a n i n as determined by X-ray c r y s t a l l o g r a p h y . L i g a n d s (and c o p p e r - l i g a n d bond l e n g t h s ) are h i s t i d i n e 37 (2.04 A ) , c y s t e i n e 84 (2.13 A ) , h i s t i d i n e 87 (2.10 A) and m e t h i o n i n e 92 ( 2.90 A ) . Reproduced w i t h p e r m i s s i o n from Ref. 9. C o p y r i g h t 1983, J o u r n a l o f M o l e c u l a r B i o l o g y .



240

EXCITED STATES AND

REACTIVE INTERMEDIATES

F i g u r e 4. A) Room-temperature o p t i c a l spectrum o f a s i n g l e c r y s t a l o f p l a s t o c y a n i n o b t a i n e d w i t h l i g h t i n c i d e n t on the ( 0 , 1 , 1 ) f a c e and p o l a r i z e d p a r a l l e l ( s o l i d l i n e ) and perpendi c u l a r (dashed l i n e ) to a. ( f r o m Ref, 1 1 ) . B) G a u s s i a n r e s o l u t i o n of the 35 Κ v i s i b l e a b s o r p t i o n spectrum of a p l a s t o c y a n i n f i l m w i t h suggested assignments; the symbols ( · ) r e p r e s e n t the e x p e r i m e n t a l a b s o r p t i o n spectrum. Right: plastocyanin unit c e l l p r o j e c t e d on the (0,1,1) p l a n e , showing the p o s i t i o n s o f the f o u r s y m m e t r y - r e l a t e d Cu atoms a t t h e i r f i r s t c o o r d i n a t i o n shells.



16.

SOLOMON ET AL.


241

strong i n t e r a c t i o n o f the s u l f u r with the carbon o f the c y s t e i n e r e s i d u e would r e s u l t i n t h r e e t r a n s i t i o n s . The R-S-Cu a n g l e i n t h e p l a s t o c y a n i n s t r u c t u r e i s i n f a c t 107 . These r e s u l t s have l e d us t o a more q u a n t i t a t i v e e v a l u t a t i o n o f the bonding i n the b l u e copper s i t e through a many e l e c t r o n SCF-Xa-SW c a l c u l a t i o n (14). The s t r u c t u r e s c a l c u l a t e d i n c l u d e the f r e e l i g a n d s and a p p r o x i m a t i o n s t o the s i t e shown i n F i g u r e 5 w i t h t h e Xa-SW parameters as i n d i c a t e d . F i r s t c o n s i d e r i n g the t h i o l a t e bond, t h e v a l e n c e o r b i t a l e o f t h e f r e e t h i o l a t e l i g a n d i n c l u d e the h i g h e s t energy o c c u p i e d d o u b l y de g e n e r a t e 2e l e v e l c o n s i s t i n g o f s u l f u r ρ o r b i t a l e o r i e n t e d perp e n d i c u l a r t o t h e S-C bond, and t o a p p r o x i m a t e l y 2 eV d e e p e r b i n d i n g energy, t h e 3a^ l e v e l which i s t h e s u l f u r ρ o r b i t a l involved i n sigma bonding w i t h t h e c a r b o n . C o o r d i n a t i o n o f the t h i o l a t e l i g a n d t o the copper s p l i t s t h e s e v a l e n c e o r b i t a l s i n t o t h r e e r o u g h l y e q u a l l y spaced l e v e l s , [ F i g u r e 6] each w i t h s i g n i f i c a n t bonding interac t i o n s w i t h t h e o r b i t a l s on the copper (7a"-35%, 9a'-43%, 7a'-20%). T h i s s p l i t t i n g r e s u l t s from one o f t h e s u l f u r ρ o r b i t a l s o f the f r e e t h i o l a t e b e i n g s i g n i f i c a n t l y s t a b i l i z e d due éof^onding and mixed w i t h t h e C-Sp o r b i t a l , which has the same symmetry. The c o n t o u r diagrams a s s o c i a t e d w i t h these t h r e e bonding levels are given i n F i g u r e 7. The h i g h e s t energy o c c u p i e d o r b i t a l (7a") i s the s u l f u r ρ which i s p e r p e n d i c u l a r t o the C-S-Cu p l a n e and i n v o l v e d i n a s t r o n g p i bond w i t h t h e d 2_ 2 o r b i t a l on t h e copper. The m i d d l e level i n v o l v e s t h e s u l f u r p J which i s i n p l a n e and mixes w i t h t h e s u l f u r ρ , forming a pseudo-Jigma bond w i t h t h e copper. Here, t h e e l e c t r o n d e n s i t y i s no l o n g e r maximized a l o n g t h e S-Cu bond. The l e v e l to d e e p e s t b i n d i n g energy i n v o l v e s a m o l e c u l a r o r b i t a l which i s sigma bonding w i t h copper b u t a l s o s i g n i f i c a n t l y d e l o c a l i z e d i n t o t h e S-C bond. χ

X

The l o n g (2.9 A) C u - S ( t h i o e t h e r ) bond i s next c o n s i d e r e d . The v a l e n c e o r b i t a l s o f t h e f r e e l i g a n d a r e 2b^ which i s a ρ o r b i t a l o f the s u l f u r , p e r p e n d i c u l a r t o the C-S-C p l a n e , and, t o 2.^1 eV deeper b i n d i n g energy, t h e 4a^ l e v e l which i s t h e p o r b i t a l o f t h e s u l f u r i n v o l v e d i n a sigma bonding i n t e r a c t i o n w i t h t h e symmetric c o m b i n a t i o n o f methyl valence o r b i t a l s . C o o r d i n a t i o n o f the t h i o e t h e r to the copper a t the same d i s t a n c e and a n g l e as i n t h e p r o t e i n l e a d s t o a s t a b i l i z a t i o n o f t h e Ua^ o r b i t a l by 0.4 eV r e l a t i v e t o o t h e r v a l e n c e o r b i t a l s due t o bonding w i t h about 36% d e r e a l i z a t i o n o f t h e wavef u n c t i o n onto t h e copper [ F i g u r e 8 ] , The n a t u r e o f t h e bonding i n t e r a c t i o n i s shown i n F i g u r e 9. Here the s u l f u r ρ o r b i t a l i s i n v o l v e d i n a pseudo sigma type bond i n t o t h e d 2 o r b i t a l o f t h e cop per. As mentioned p r e v i o u s l y , a number o f p h y s i c a l methods have r a i s e d the q u e s t i o n as t o whether t h e r e i s a Cu-S ( m e t h i o n i n e ) bond. The l a c k o f CT i n t e n s i t y i s now seen t o be a consequence o f t h e o r i e n t a t i o n o f t h e S ( t h i o e t h e r ) ρ donor o r b i t a l which i s o r t h o g o n a l t o the h a l f - o c c u p i e d d 2_ 2 a c c e p t o r ( v i d a i n f r a ) . The c a l c u l a t i o n does, however, indicate s p e c t r a l f e a t u r e s which a r e s e n s i t i v e to t h i o e t h e r i n t e r a c t i o n with the copper. The e f f e c t o f a x i a l t h i o e t h e r c o o r d i n a t i o n i n the Χ α c a l c u l a t i o n s i s p r e s e n t e d i n F i g u r e 10. Here, the r e l a t i v e c a l c u l a t e d energy o f t h e copper d o r b i t a l s a r e g i v e n f o r the copper s i t e i n C symmetry w i t h and w i t h o u t t h e t h i o e t h e r . Upon a d d i t i o n o f t h e a x i a l l i g a n d , t h r e e o f t h e f o u r l e v e l s go down s l i g h t l y i n energy r e l a t i v e t o t h e h a l f - o c c u p i e d l e v e l . The d^2 l e v e l , however, goes up i n energy due t o a n t i b o n d i n g i n t e r a c t i o n s z




242

Χα PARAMETERS FOR Cu(S(CH ) )(SCH3)(NH ) 3

L

2

3

2

OVERLAP

MAX

OUTER SPHERE

3

Cu

-

S(THIOLATE)

2%

Cu

2

Cu

-

S(THIOETHER)

0%

S

1

Cu - N

Ν

1

C

1

H

0

0%

CS SYMMETRY CONVERGED AFTER 29

ITERATIONS

(EPS=0.009; -2T/V=0.9998) Figure tion.

5. Top: A p p r o x i m a t i o n s c o n s i d e r e d by S C F - Χ α -SW c a l c u l a Bottom: Χ α parameters f o r Cu(S(CH ) )(SCH ) ( N H ) . q


SOLOMON ET AL.


Cu(SCH )(NH )


3

2 e

3

2

l

(PxV

2.163 eV

i 7a'

ChL 2e

9

1

Λ Λ

(Cu-S-C)

CH

Cu 7a"

35°ki, 7°ip

9a

43°ki, 2°ip

1

7a'

\3%ά,

9°is

3%

32%P , V

l o ° i p , , 21f p , , 3%s z

23°ip

3

0

1 l°ip . w

10% 31%

F i g u r e 6. R e p r e s e n t a t i o n o f t h e i n t e r a c t i o n o f t h r e e h i g h e s t energy o r b i t a l s o f m e t h y l t h i o l a t e w i t h a c o p p e r ( I I ) i o n . Top: s h i f t s i n e n e r g i e s r e l a t i v e t o t h e s u l f u r 2s o r b i t a l . Bottom: c h a r a c t e r o f t h e o r b i t a l s i n terms o f a t o m i c o r b i t a l s . Primed c o o r d i n a t e s o f t h e copper-bound s u l f u r ρ o r b i t a l s i n d i c a t e a l i g a n d - b a s e d c o o r d i n a t e system. Reproduced from Ref. 14. C o p y r i g h t 1985, American C h e m i c a l S o c i e t y .




244

F i g u r e 7. C o n t o u r s o f t h e t h r e e bonding o r b i t a l s w i t h s u b s t a n t i a l t h i o l a t e s u l f u r 3p c h a r a c t e r and t h e geometry o f Cu(SCH^)(NH >2 with copper and t h i o l a t e based c o o r d i n a t e systems indicated. A l lnuclei indicated are i n the planes of the f i g u r e s e x c e p t f o r t h e c o n t o u r o f t h e 7a" o r b i t a l i n t h e xy plane. O n l y t h e copper n u c l e u s i s i n t h e p l a n e o f t h i s f i g u r e . V a l u e s o f t h e c o n t o u r s a r e +0.003, +0.009, +0.027 and +0.081 Reproduced from Ref. 14. C o p y r i g h t 1985, American Chemical Society.


SOLOMON ET AL.

S(CH )


3

2b

2


245

Cu(S(CH ) )(SCH )(NH ) 3

2

2

3

3

2

V 18a'

(p ) yl

2.125 eV

2.506 eV 4a (S-C) 1

Î 1.140 eV

14a' (Cu-S-C)

t

i

0.673 eV 9a" (S-C)

i

CH

0

92.ρ

Q\ y

64 p , 3 s z

3b,

47"'ρ

31.. 48

18a' 14a' 9a"

6 d 2, 2 d 28'd 2, 5'',d z

lod

, 2%s

80%p , y

6%

39%p ,, l%s

18%

55Zp ,

44%

z

v

F i g u r e 8. I n t e r a c t i o n o f the three h i g h e s t energy occupied orbitals of dimethylsulfide with copper. Top: s h i f t s i n e n e r g i e s r e l a t i v e t o t h e s u l f u r 2s o r b i t a l . Bottom: c h a r a c t e r of t h e o r b i t a l s i n terms o f atomic o r b i t a l s . Primed c o o r d i n a t e s o f t h e copper-bound s u l f u r ρ o r b i t a l s i n d i c a t e a l i g a n d - b a s e d c o o r d i n a t e system. Reproduced from Ref. 14. C o p y r i g h t 1985, American Chemical S o c i e t y .


246



CU(S(CH3)2)(SCH3)(NH3)2

1-

14A'

f

F i g u r e 9. Contour o j the 14a l e v e l and t h e g e o m e t r y o f Cu(S(CH ) )(SCH )(NH ) w i t h copper and t h i o e t h e r based c o o r d i n a t e systems i n d i c a t e d . I n the c o n t o u r , a l l n u c l e i i n d i c a t e d a r e i n t h e p l a n e o f the diagram e x c e p t f o r t h o s e o f the amine n i t r o g e n s and t h i o e t h e r c a r b o n s . V a l u e s of the c o n t o u r s a r e the same as i n F i g u r e 7. Reproduced from R e f . 14. C o p y r i g h t 1985, American C h e m i c a l S o c i e t y . 3

2

3


16.

SOLOMON ET AL.


WITH THIOETHER

WITHOUT THIOETHER


247

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Y

hUG.V

50°

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-OK:

d

ο

χ -y2

xy

10000

20000

J

F i g u r e 10. C a l c u l a t e d e f f e c t s o f a x i a l t h i o e t h e r l i g a t i o n upon copper d o r b i t a l s . The r e l a t i v e e n e r g i e s o f t h e a n t i b o n d i n g o r b i t a l s and t h e i r predominant copper d c h a r a c t e r a r e i n d i c a t e d f o r t h e two s i t e s shown. The e n e r g i e s o f t h e h a l f - o c c u p i e d l e v e l have been s e t t o z e r o . Reproduced from R e f . 14. C o p y r i g h t 1985, American C h e m i c a l S o c i e t y .

American Chemical Society Library 1155 16th St., N.W. In Excited States and Reactive Intermediates; Washington, DC. 20036Lever, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

EXCITED STATES AND

248

REACTIVE INTERMEDIATES

w i t h the s u l f u r ρ o r b i t a l o f the t h i o e t h e r . Thus, the d-d t r a n s i t i o n a s s o c i a t e d w f t h the d 2 o r b i t a l s h o u l d go down i n energy. W h i l e removing the t h i o e t h e r from the b l u e copper s i t e v i a s i t e d i r e c t e d m u t a g e n e s i s and a t the same time r e t a i n i n g the r e m a i n i n g geometric f e a t u r e s i s u n r e a l i s t i c , copper c h l o r i d e s p e c t r a l analog s t u d i e s do c l e a r l y demonstrate d e s t a b i l i z a t i o n o f the d 2 o r b i t a l due to a n t i bonding i n t e r a c t i o n s w i t h c h l o r i d e l i g a n d s a t a p p r o x i m a t e l y 3 % d i s tance as shown i n F i g u r e 11. A t the top o f the f i g u r e i s the l i g a n d f i e l d spectrum o f bis(N-methylphenethylammonium) C u C l ^ (15) , a square p l a n a r complex which c o n t a i n s no a x i a l l i g a n d . At the bottom i s the spectrum o f b i s ( e t h y l ammonium) C u C l ^ (16) which does c o n t a i n a x i a l chlorides. The a s s i g n e d t r a n s i t i o n s , based on p o l a r i z e d s p e c t r a f o r each complex are i n d i c a t e d a t the top o f the a b s o r p t i o n bands i n F i g u r e 12. Upon a d d i t i o n o f a p i c a l c h l o r i d e s , ( F i g u r e 11 top to b o t tom), t h e ^ t r a n s i t i o n frqm the d 2 o r b i t a l d e c r e a s e s i n energy from 16000 cm t o 11000 cm , w h i l e the o t h e r t r a n s i t i o n s o c c u r a t ap p r o x i m a t e l y the same e n e r g i e s i n b o t h complexes. Thus, a d i s t a n t a p i c a l l i g a n d has s i g n i f i c a n t e f f e c t upon the energy o f the d 2 orb i t a l o f the complex.


Z

2

Ground S t a t e W a v e f u n c t i o n and

Covalency

S i n g l e c r y s t a l EPR s t u d i e s (_11) o f p l a s t o c y a n i n i n c o n j u n c t i o n w i t h a l i g a n d ^ . e l d c a l c u l a t i o n enabled a d e t e r m i n a t i o n o f the o r i e n t a t i o n o f the g t e n s o r r e l a t i v e to the copper s i t e . F i g u r e 12 p r e s e n t s the EPR s p e c t r a o f a s i n g l e c r y s t a l o f p l a s t o c y a n i n , o r i e n t e d w i t h the magnetic f i e l d (H) p e r p e n d i c u l a r to a, and r o t a t e d about the a. a x i s . An a p p r o x i m a t e l y g„ spectrum i s observed when H i s a l o n g c. i n d i c a t i n g t h a t g„ i s o r i e n t a t e d i n the g e n e r a l d i r e c t i o n o f the t h i o e t h e r - C u bond. S i m u l a t i o n o f f o u r d i f f e r e n t r o t a t i o n s f o r the f o u r m o l e c u l e s i n the u n i t c e l l demonstrate t h a t g and A a r e c o l i n e a r and t h a t g ο ο ζ ζ ζ is 8 o f f £, and 5 out o f t h i s p l a n e . T h u s , the d 2_ 2 o r b i t a l , which i s p e r p e n d i c u l a r to g and c o n t a i n s the unpaired* eïectron, i s l e s s than 15 d e g r e e s above tfie plane formed by the S ( c y s ) and the two Ν ( h i s ) l i g a n d s . T h i s o r i e n t a t i o n o f the d 2_ 2 o r b i t a l i s reproduced i n F i g u r e 13, a l o n g w i t h the energy l e v e l 5ia§ram a s s o c i a t e d w i t h the ligand f i e l d of p l a s t o c y a n i n . W h i l e t h i s energy l e v e l diagram r e f l e c t s a low symmetry, r h o m b i c a l l y d i s t o r t e d s i t e , i t i s o f import ance to c o n s i d e r the a x i a l l i m i t s so as t o e v a l u a t e the c l o s e to a x i a l n a t u r e o f the e x p e r i m e n t a l X-band EPR spectrum, shown i n F i g u r e 1. Two a x i a l subgroups o f a t e t r a h e d r o n are p o s s i b l e , D^j and C^ « I f the rhombic s p l i t t i n g o f the d o r b i t a l s i s removed from t h a t g i v e n i n F i g u r e 13 o n l y the energy l e v e l o r d e r i n g i s r e a s o n a b l e . This energy o r d e r i n g i s c o n s i s t e n t w i t h an e l o n g a t e d C ^ s t r u c t u r e , the l o n g a x i s b e i n g a l o n g the Cu t h i o e t h e r bond, as shown i n F i g u r e 13. This elongated C^ e f f e c t i v e symmetry o f the b l u e copper s i t e r a i s e s a s i g n i f i c a n t problem w i t h r e s p e c t to the p r e s e n t i n t e r p r e t a t i o n s (17-18) o f the s m a l l copper h y p e r f i n e s p l i t t i n g observed i n the EPR spectrum shown i n F i g u r e 1. The s m a l l s p l i t t i n g had been a t t r i b u t e d to a D m i x i n g o f Cu 4 p i n t o the

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