Electronic Structures of Active Sites in Copper Proteins and Their

5 Electronic Structures of Active Sites in Copper Proteins and Their Contributions to Reactivity Edward I. Solomon, Michael D . Lowery, David E . Root, and Brooke L . Hemming

Downloaded by PURDUE UNIV on April 9, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/ba-1995-0246.ch005

Department of Chemistry, Stanford University, Stanford, CA 94305

Many active sites in copper proteins exhibit unique spectral features in comparison to small inorganic complexes. These spectral features are becoming well-understood and reflect unusual electronic struc tures that make key contributions to the reactivity of these sites in biology. In blue copper proteins, the unique spectral features reflect a ground-state, redox-active wave function that has high anisotropic covalency involving a cysteine ligand that activates this residue for rapid, directional, long-range electron transfer. For hemocyanin and tyrosinase, the characteristic spectral features reflect a per oxide-binuclear cupric bond that has very strong σ-donor and πacceptor interactions that stabilize the oxy site with respect to loss of peroxide. In tyrosinase, this bonding mode activates peroxide for hydroxylation of phenolic substrates by making the peroxide less negative but with an unusually weak O-O bond. Finally, for the multicopper oxidases, magnetic circular dichroism and X-ray absorption studiesfirstshowed the presence of a trinuclear copper cluster site, which is the minimal structural unit required for the multielectron reduction of dioxygen to water. A peroxide level in termediate in this reduction has been obtained and is found to have strikingly different spectral features than those associated with bound peroxide in oxyhemocyanin and oxytyrosinase. This dem onstrates a fundamentally different electronic and geometric struc ture for peroxide binding in the multicopper oxidases that promotes the further reduction of peroxide to water at the trinuclear copper cluster site.

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A N Y CLASSES O F ACTIVE SITES i n c o p p e r p r o t e i n s e x h i b i t u n i q u e

spectral features w h e n c o m p a r e d to s i m p l e , h i g h - s y m m e t r y t r a n s i t i o n 0065-2393/95/0246-0121/$12.32/0 © 1995 American Chemical Society

Thorp and Pecoraro; Mechanistic Bioinorganic Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1996.

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m e t a l c o m p l e x e s . T h e s e a c t i v e sites d e r i v e f r o m t h e u n u s u a l g e o m e t r i c and electronic structures that can be i m p o s e d o n the metal i o n i n a p r o t e i n e n v i r o n m e n t . It has b e e n a g e n e r a l g o a l o f o u r r e s e a r c h t o u n d e r s t a n d these e l e c t r o n i c structures a n d to evaluate t h e i r c o n t r i b u t i o n s t o t h e r e a c t i v i t i e s o f t h e s e a c t i v e sites i n c a t a l y s i s ( 1 - 3 ) . O u r p r o g r e s s i n f o u r areas w i l l b e s u m m a r i z e d (4, 5). F i r s t , i f o n e is to u n d e r s t a n d the o r i g i n o f these u n i q u e spectral features, one must understand the electronic structure of " n o r m a l " high-symmetry, trans i t i o n - m e t a l c o m p l e x e s . S q u a r e - p l a n a r c u p r i c c h l o r i d e has s e r v e d as a n e l e c t r o n i c s t r u c t u r a l m o d e l c o m p l e x a n d is n o w o n e o f t h e b e s t u n d e r s t o o d m o l e c u l e s i n i n o r g a n i c c h e m i s t r y (6). Its s p e c t r a l f e a t u r e s a n d t h e e l e c t r o n i c structure these reflect w i l l be b r i e f l y d e s c r i b e d i n the f o l l o w i n g s e c t i o n (4). H a v i n g p r o v i d e d t h i s d e s c r i p t i o n o f a " n o r m a l " c o p p e r s i t e , t h e u n i q u e s p e c t r a l f e a t u r e s o f t h e b l u e c o p p e r a c t i v e site w i l l t h e n be addressed. A n u n d e r s t a n d i n g of these features provides insight into g r o u n d a n d e x c i t e d state c o n t r i b u t i o n s t o t h e r a p i d r a t e o f l o n g - r a n g e electron transfer observed i n this family of proteins. N e x t w e focus o n the c o u p l e d binuclear c o p p e r proteins, hemocyanin, and tyrosinase. T h e s e p r o t e i n s h a v e s i m i l a r a c t i v e sites t h a t g e n e r a t e t h e same o x y i n t e r m e d i a t e i n v o l v i n g p e r o x i d e b o u n d to t w o copper(II) ions. T h e h e m o c y a n i n s r e v e r s i b l y b i n d d i o x y g e n a n d f u n c t i o n as o x y g e n c a r r i e r s i n arthropods a n d molluscs, whereas the tyrosinases have h i g h l y accessible a c t i v e sites t h a t b i n d p h e n o l i c s u b s t r a t e s a n d o x y g e n a t e t h e m t o orthod i p h e n o l s . T h e i r o x y sites e x h i b i t u n i q u e e x c i t e d state s p e c t r a l f e a t u r e s that reflect n o v e l p e r o x i d e - c o p p e r b o n d i n g interactions that m a k e a s i g n i f i c a n t e l e c t r o n i c c o n t r i b u t i o n to t h e b i n d i n g a n d a c t i v a t i o n o f d i o x y g e n b y t h e s e sites. I n t h e final s e c t i o n , s p e c t r o s c o p i c s t u d i e s o f t h e m u l t i c o p p e r oxidases, w h i c h i n c l u d e laccase, ascorbate oxidase, a n d cer u l o p l a s m i n , are s u m m a r i z e d . T h e s e e n z y m e s catalyze the four-electron r e d u c t i o n o f d i o x y g e n to w a t e r . S p e c t r a l studies have d e m o n s t r a t e d that the m u l t i c o p p e r oxidases c o n t a i n a f u n d a m e n t a l l y different c o u p l e d b i n u c l e a r c o p p e r site ( c a l l e d t y p e 3) w h e n c o m p a r e d w i t h h e m o c y a n i n a n d t y r o s i n a s e . T h e t y p e 3 site is p a r t o f a t r i n u c l e a r c o p p e r c l u s t e r t h a t plays the key role i n the m u l t i e l e c t r o n reduction of dioxygen b y this i m p o r t a n t class o f e n z y m e s .

Normal Copper Complexes P l a c i n g a c u p r i c i o n w i t h its n i n e d e l e c t r o n s i n a n o c t a h e d r a l l i g a n d field p r o d u c e s a E g r o u n d state ( F i g u r e 1). T h i s g r o u n d state is u n s t a b l e to a J a h n - T e l l e r d i s t o r t i o n that l o w e r s the s y m m e t r y a n d e n e r g y o f the c o m p l e x . T h e J a h n - T e l l e r d i s t o r t i o n n o r m a l l y o b s e r v e d is a t e t r a g o n a l e l o n g a t i o n a l o n g t h e z-axis a n d c o n t r a c t i o n i n t h e e q u a t o r i a l x,y p l a n e t h a t u l t i m a t e l y r e s u l t s i n a s q u a r e - p l a n a r l i g a n d e n v i r o n m e n t as i n D h2

g

4



h

Χ

4h

°

2

4

e

6

g 10Dq

h

O

\

2

Mr

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W

/xy

2

4h

D

/x -y

r f

Levels

Energy

a

b

1g

ig

C 4

-CuCI 2-

HOMO

4 / J

39%Clp 0

x

y

Ψ =61 % C u d 2 _ 2

Adjusted S C F - X a - S W

D

+

j

D

E P R

4h

Η

2

A^^xlO^cnr

1

x

\ V

g = 2.00

g = 2.040

C u : 1 = 3/2/. 21+1=4

g ^ g ^ 2.221

Spectrum

4

Figure 1. The ground-state spectral features of normal copper complexes. A: Jahn-Teller tetragonal elongation of an octahedral CuL complex to the square planar limit. B: Energy level correlation diagram for the JahnTeller distortion depicted in A. C: SCF-Xa-SW wave function contour and charge decomposition for the HOMO ofO -CuCl ~ (18, 19). D: X-hand EPR spectrum for tetragonal Cu(II) with O -CuCl ~ parameters.

4,

ι d>

A

?

Y

JahnTeller

Ψ

0

ο

Cupric Geometric Structure

o

A


124

M E C H A N I S T I C BIOINORGANIC

CHEMISTRY

C U C 1 " ( F i g u r e 1 A ) . A k e y f e a t u r e o f l i g a n d field t h e o r y ( 7 - 1 1 ) is t h a t t h e d o r b i t a l s p l i t t i n g is v e r y s e n s i t i v e t o t h e e n v i r o n m e n t o f t h e l i g a n d s around the metal center. T h e d orbital splitting experimentally deter m i n e d u s i n g o p t i c a l s p e c t r o s c o p y (12) f o r D -CuC\ ~ is g i v e n i n F i g u r e I B . T h e h a l f - o c c u p i e d d 2. 2 o r b i t a l is at h i g h e s t e n e r g y as i t h a s t h e largest repulsive interaction w i t h t h e ligands i n t h e equatorial plane. A m o r e c o m p l e t e d e s c r i p t i o n o f this h a l f - o c c u p i e d g r o u n d state is p r o v i d e d b y molecular orbital ( M O ) theory. I n particular, self-consistent fieldΧ α - s c a t t e r e d w a v e ( S C F - X a - S W ) c a l c u l a t i o n s (13-17), a d j u s t e d t o g r o u n d - s t a t e p a r a m e t e r s (18-21) as w i l l b e d e s c r i b e d , a r e i n g o o d a g r e e ment w i t h spectral data over many orders o f magnitude i n energy. These c a l c u l a t i o n s (18, 19) g e n e r a t e a d e s c r i p t i o n o f t h e g r o u n d state o f D CuCl t h a t has 6 1 % C u d 2. 2 c h a r a c t e r w i t h t h e r e m a i n i n g p a r t o f t h e w a v e f u n c t i o n b e i n g d e l o c a l i z e d e q u i v a l e n t l y i n t o t h e f o u r ρσ o r b i t a l s of the c h l o r i d e ligands that are i n v o l v e d i n a n t i b o n d i n g interactions w i t h the metal i o n (Figure 1 C ) . T h e u n p a i r e d electron i n this w a v e function produces the electron paramagnetic resonance (EPR) spectrum shown i n F i g u r e I D , i n w h i c h g\\ ( c o r r e s p o n d i n g t o t h e m a g n e t i c field o r i e n t e d a l o n g t h e z-axis o f t h e c o m p l e x ) > g > 2 . 0 0 , t h a t is c h a r a c t e r i s t i c o f t h i s d 2. 2 g r o u n d state. A d d i t i o n a l l y , c o p p e r h a s a n u c l e a r s p i n ( i = %) t h a t c o u p l e s t o t h e e l e c t r o n s p i n t o p r o d u c e a f o u r - l i n e h y p e r f i n e s p l i t t i n g (A) o f t h e E P R s p e c t r u m . T e t r a g o n a l c u p r i c c o m p l e x e s g e n e r a l l y h a v e a l a r g e h y p e r f i n e s p l i t t i n g i n t h e r e g i o n (A > 1 3 0 Χ 1 0 " c m " ) ; t h a t o f D -CuC\ is 1 6 4 Χ 1 0 " c m " (22). 4

2

2

4h

x

4

y


4h

4

2 -

x

y

±

x

y

N

4

l{

2

4h

4

4

1

1

W i t h r e s p e c t t o e x c i t e d states, o p t i c a l e x c i t a t i o n o f e l e c t r o n s f r o m t h e filled C u d o r b i t a l s t o t h e h a l f - f i l l e d d 2. 2 o r b i t a l ( F i g u r e 2 A ) p r o d u c e s L a p o r t é - f o r b i d d e n l i g a n d - f i e l d t r a n s i t i o n s (23). T h e s e t r a n s i t i o n s a r e w e a k i n t h e a b s o r p t i o n s p e c t r u m w i t h m o l a r e x t i n c t i o n c o e f f i c i e n t s (e) o f 3 0 - 5 0 M c m " i n t h e 1 2 , 0 0 0 - 1 6 , 0 0 0 c m " r e g i o n (12) ( F i g u r e 2 B , r i g h t ) . O b s e r v e d at h i g h e r e n e r g y i n t h e a b s o r p t i o n s p e c t r u m a r e t h e L a p o r t é - a l l o w e d l i g a n d - t o - m e t a l c h a r g e - t r a n s f e r t r a n s i t i o n s t h a t a r e at least t w o o r d e r s o f m a g n i t u d e m o r e i n t e n s e t h a n t h e l i g a n d - f i e l d t r a n sitions (24) ( F i g u r e 2 B , left). T h e e n e r g i e s a n d i n t e n s i t i e s o f t h e s e c h a r g e transfer (CT) transitions allow one to p r o b e t h e specific b o n d i n g i n t e r a c t i o n s o f t h e l i g a n d w i t h t h e m e t a l c e n t e r (24). C h l o r i d e h a s t h r e e v a l e n c e 3p o r b i t a l s t h a t s p l i t i n t o t w o sets o n b i n d i n g t o c o p p e r ( F i g u r e 2 C ) . T h e ρσ o r b i t a l is o r i e n t e d a l o n g t h e C l - C u b o n d a n d is s t a b i l i z e d to h i g h e r b i n d i n g e n e r g y d u e t o s t r o n g o v e r l a p w i t h t h e C u ion. T h e t w o c h l o r i d e ρπ o r b i t a l s a r e p e r p e n d i c u l a r t o t h e C l - C u b o n d a n d h e n c e a r e m o r e w e a k l y i n t e r a c t i n g w i t h t h e m e t a l a n d at l o w e r b i n d i n g e n e r g y . T h e intensity associated w i t h charge-transfer excitation o f an elec t r o n f r o m t h e s e filled l i g a n d o r b i t a l s i n t o t h e h a l f - o c c u p i e d C u d 2. 2 o r b i t a l also r e f l e c t s m e t a l - l i g a n d b o n d i n g . C h a r g e - t r a n s f e r i n t e n s i t y is p r o p o r t i o n a l t o ( R S ) , w h e r e S is t h e o v e r l a p o f t h e d o n o r a n d a c c e p t o r x

1

1

y

1

2 +

x

2


y


C

Clo

cin

2

2

_

U

PJJ

ll

S

C

2

4

d x

u

2_

I « (SR)

= Wcu

4h

>

C

y 2

allowed

L

>

T

d->d forbidden

4h

Β

2

4

45000

6000

38000

24000

Energy (cm )

31000

Charge Transfer

17000

10000

Ligand Field

Electronic Absorption Spectrum

y

Figure 2. Excited-state spectral features ofO -CuCl ~. A: Energy level diagram showing the ligand-field (a a) and charge-transfer (CT) optical transitions. The intensity of the transitions is approximated by the thickness of the arrow with the very weak ligand-field transitions represented as a dotted arrow. B: Electronic absorption spectrum for O -CuCl ~ (12). C: Schematic of the σ and π bonding modes between the Cu 3d 2. 2 and CI 3p orbitals.

x

x y

x z > y z

dz

d -JL. d

2

dx -y

Metal-Ligand Bonding

-f-

Lb-

Α Α Α Α"

Energy Level Diagram


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CHEMISTRY

o r b i t a l s i n v o l v e d i n t h e c h a r g e - t r a n s f e r t r a n s i t i o n a n d R is t h e m e t a l l i g a n d b o n d l e n g t h (25). T h u s , t h e C l ρσ C u d 2. 2 c h a r g e - t r a n s f e r t r a n s i t i o n is at h i g h e n e r g y a n d i n t e n s e f r o m l a r g e o v e r l a p , w h i l e t h e C l ρπ -> d 2_ 2 c h a r g e t r a n s f e r t r a n s i t i o n is at l o w e r e n e r g y a n d w e a k e r ( F i g u r e 2 C ) . ( N o t e that the l i g a n d orbitals are actually l i n e a r c o m b i nations of the orbitals f r o m the four c h l o r i d e ligands. O n e c o m b i n a t i o n o f t h e C l ρσ o r b i t a l s has e s y m m e t r y . T h e C T t r a n s i t i o n f r o m t h i s o r b i t a l is e l e c t r i c d i p o l e a l l o w e d a n d r e s p o n s i b l e f o r t h e i n t e n s e b a n d at 3 6 , 0 0 0 c m . T h e C l ρπ set also c o n t a i n s a l i n e a r c o m b i n a t i o n h a v i n g e s y m m e t r y . C o n f i g u r a t i o n a l i n t e r a c t i o n w i t h e (pa) c o n t r i b u t e s to t h e i n t e n s i t y o f t h e ρπ d 2_ 2 b a n d at 2 6 , 5 0 0 c m ) . T h e k e y p o i n t s to b e e m p h a s i z e d h e r e are that t h e c h a r g e - t r a n s f e r t r a n s i t i o n s s e n s i t i v e l y p r o b e t h e l i g a n d m e t a l b o n d a n d that for " n o r m a l " c o m p l e x e s o n e s h o u l d o b s e r v e a l o w e r energy weak π and higher-energy intense σ charge-transfer transition as is o b s e r v e d e x p e r i m e n t a l l y f o r C u C l " i n F i g u r e 2 B (left). x

x

y

y

u

- 1

u

u


x

- 1

y

4

2

Blue Copper Proteins A s p r e d i c t e d b y s p e c t r o s c o p y (26), t h e b l u e c o p p e r site has a s t r u c t u r e very different from the n o r m a l tetragonal g e o m e t r y of c u p r i c complexes. T h e c o p p e r site i n p l a s t o c y a n i n (27) has a d i s t o r t e d t e t r a h e d r a l s t r u c t u r e w i t h a thiolate sulfur of cysteine (Cys) 84 b o u n d w i t h a short C u - S b o n d l e n g t h of 2.13 À, a t h i o e t h e r sulfur of m e t h i o n i n e (Met) 9 2 b o u n d w i t h a l o n g C u - S b o n d l e n g t h of 2.90 À, a n d t w o fairly n o r m a l histidine (His) N - C u l i g a n d s ( F i g u r e 3 A ) . T h i s site has c h a r a c t e r i s t i c s p e c t r a l f e a t u r e s (1-3) t h a t i n c l u d e a n i n t e n s e a b s o r p t i o n b a n d (e ~ 3 , 0 0 0 - 5 , 0 0 0 M " cm" ) i n the 6 0 0 n m ligand-field region (Figure 3B) and a small parallel h y p e r f i n e s p l i t t i n g (A < 7 0 X 1 0 ~ c m " ) ( F i g u r e 3 C ) . T h e s e u n u s u a l s p e c t r a l features are n o w w e l l - u n d e r s t o o d a n d h e l p to d e f i n e t h e g r o u n d state w a v e f u n c t i o n o f t h e b l u e c o p p e r s i t e . T h i s is e x t r e m e l y i m p o r t a n t i n t h a t t h i s is t h e h a l f - o c c u p i e d o r b i t a l t h a t takes u p a n d t r a n s f e r s t h e electron i n the redox f u n c t i o n i n g of this center. A d e t a i l e d e x p e r i m e n t a l a n d t h e o r e t i c a l d e s c r i p t i o n o f t h e g r o u n d state p r o v i d e s f u n d a m e n t a l i n s i g h t i n t o t h e a c t i v e site c o n t r i b u t i o n to t h e l o n g - r a n g e e l e c t r o n - t r a n s f e r r e a c t i v i t y e x h i b i t e d b y t h e b l u e c o p p e r p r o t e i n s (2, 3, 28). 1

1

N

4

1

T h e E P R spectrum of the blue c o p p e r protein plastocyanin (Figure 3 C ) has g|| > g± > 2 . 0 0 , a n d t h u s t h e c o p p e r site m u s t h a v e a d 2_ 2 g r o u n d state. F i r s t , w e are i n t e r e s t e d i n d e t e r m i n i n g t h e o r i e n t a t i o n o f t h e d 2. 2 o r b i t a l r e l a t i v e to t h e d i s t o r t e d t e t r a h e d r a l g e o m e t r y o b s e r v e d i n the protein crystal structure. Single crystal E P R spectroscopy allowed us to o b t a i n t h i s o r i e n t a t i o n a n d l o c a t e d t h e u n i q u e (i.e., z) d i r e c t i o n i n this d i s t o r t e d site (29). P l a s t o c y a n i n c r y s t a l l i z e s i n a n o r t h o r h o m b i c s p a c e group w i t h four symmetry related molecules i n the unit cell. T h e o r i e n t a t i o n o f t h e p l a s t o c y a n i n c o p p e r sites i n t h e u n i t c e l l a r e s h o w n i n x

x

y

y



A

Cys 84

4h

Met 92

2

4

Β

Normal Copper

100

200

1

Energy (cm )

25000200001500010000

1000h

Λ

300 -

H400

4000h

600 H500

1 Blue Copper

π

5000h

6ΟΟΟ1

Absorption

2950

Γ

x

2.00->d l 2 y

3175 3400

Γ"

Field (gauss)

2725 g>g>

2500

c CD

Blue Copper

1

E P R

4h

4

2

Figure 3. Blue copper proteins. A: X-ray structure of poplar plastocyanin (27). B: Absorption spectrum of plastocyanin and "normal" O -CuCl ~ (e scale expanded by 10). C: X-band EPR spectrum of plastocyanin and O -CuCl ~.

His 87

Poplar Plastocyanin X-ray Structure


128


F i g u r e 4 A (27). F i g u r e 4 B p r e s e n t s t h e E P R s p e c t r a o b t a i n e d i f o n e r o t a t e s t h e c r y s t a l a r o u n d t h e a axis w i t h t h e m a g n e t i c field i n t h e b/c p l a n e . T h e k e y f e a t u r e to n o t e i n t h e figure is t h a t o n e o b s e r v e s a n ~ g u E P R s i g n a l ( w i t h f o u r p a r a l l e l h y p e r f i n e c o m p o n e n t s ) w i t h t h e field a l o n g t h e c axis a n d a n ~g s p e c t r u m as t h e field is r o t a t e d p e r p e n d i c u l a r to t h i s d i r e c t i o n . T h u s , g| is n e a r l y c o l i n e a r w i t h t h e c axis. R e f e r e n c i n g to t h e f o u r b l u e c o p p e r sites i n t h e u n i t c e l l , e a c h has its M e t S - C u b o n d a p p r o x i m a t e l y a l o n g t h e c axis. T h e r e f o r e , g is a p p r o x i m a t e l y a l o n g t h e l o n g M e t S - C u b o n d . A m o r e q u a n t i t a t i v e fit o f t h e E P R d a t a s h o w s t h a t gli, w h i c h d e f i n e s t h e z-axis o f t h e s i t e , is ~ 5 ° off t h e M e t S - C u b o n d a n d p l a c e s t h e d 2. 2 o r b i t a l p e r p e n d i c u l a r to t h i s d i r e c t i o n a n d w i t h i n 15° of the plane defined b y the thiolate S and two imidazole Ν ligands. ±

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x

y

T h e n e x t f e a t u r e o f t h e g r o u n d - s t a t e w a v e f u n c t i o n to b e d i s c u s s e d is t h e o r i g i n o f t h e s m a l l p a r a l l e l h y p e r f i n e s p l i t t i n g (A\\ < 7 0 X 1 0 ~ c m ) . D i s t o r t e d t e t r a h e d r a l c u p r i c sites, for e x a m p l e D r C u C l ~ , o f t e n e x h i b i t s m a l l A v a l u e s s i m i l a r to t h e b l u e c o p p e r p r o t e i n s a n d t h e m e c h a n i s m f o r r e d u c i n g t h e p a r a l l e l h y p e r f i n e v a l u e has b e e n t h o u g h t to have a c o m m o n origin. In D r C u C l ~ , the small v a l u e has b e e n a t t r i b u t e d to t h e effect o f C u 4 p m i x i n g i n t o t h e d 2. 2 o r b i t a l , w h i c h is a l l o w e d i n l o w e r - s y m m e t r y m e t a l sites (30). I n D d s y m m e t r y , t h e 4p o r b i t a l is a l l o w e d b y g r o u p t h e o r y to m i x i n t o t h e d 2. 2 o r b i t a l . T h e s p i n dipolar interaction of the 4 p orbital w i t h the c o p p e r nuclear spin op p o s e s t h a t o f t h e e l e c t r o n s p i n i n t h e d 2. 2 o r b i t a l a n d r e d u c e s t h e An v a l u e (4p m i x i n g is f o r b i d d e n i n D ^ - s y m m e t r y ) . T w e l v e p e r c e n t 4p m i x i n g is r e q u i r e d to l o w e r t h e A | v a l u e to t h a t v a l u e o b s e r v e d f o r D dCuCl a n d p l a s t o c y a n i n (31, 3 2 ) . - 1

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z

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ι

z =C u - S . . Met ; E||z j (1S->4p ) ;

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Figure 6. X-ray absorption spectroscopy. A: Energy level diagram depicting a Cu Is -*> HOMO transition at ~ g±> 2 . 0 0 , w h i c h is c o n s i s t e n t w i t h a s p i n - o r b i t m i x e d d 2. 2 g r o u n d state. H o w e v e r , t h e c a l c u l a t e d v a l u e s a r e l a r g e r t h a n t h e e x p e r i m e n t a l v a l u e s . T h i s is d u e to t h e fact that t h e l i g a n d field c a l c u l a t i o n s use p u r e d o r b i t a l s that h a v e t o o m u c h o r b i t a l a n g u l a r m o m e n t u m . C o valent d e r e a l i z a t i o n of the u n p a i r e d electron onto the ligands reduces t h e o r b i t a l c o n t r i b u t i o n to t h e g v a l u e s .


x

y

S C F - X a - S W c a l c u l a t i o n s (33, 36) w e r e p u r s u e d t o d e s c r i b e t h e b o n d i n g i n t h e b l u e c o p p e r site. T h e w a v e f u n c t i o n s o b t a i n e d f r o m t h e s e c a l c u l a t i o n s w e r e u s e d to c a l c u l a t e t h e g r o u n d state g v a l u e s a n d e v a l u a t e the covalent d e s c r i p t i o n g e n e r a t e d b y these calculations relative to ex p e r i m e n t . T h e g v a l u e c a l c u l a t i o n i n c l u d e d a l l t h e a n t i b o n d i n g (d) a n d b o n d i n g ( c h a r g e transfer) l e v e l s a n d i n c l u d e d s p i n - o r b i t m i x i n g f r o m b o t h t h e m e t a l a n d t h e l i g a n d s . A Z e e m a n o p e r a t o r w a s a p p l i e d to t h e s p i n - o r b i t c o r r e c t e d g r o u n d state m a k i n g n o a s s u m p t i o n c o n c e r n i n g t h e o r i e n t a t i o n o f t h e p r i n c i p a l axes. A g t e n s o r w a s g e n e r a t e d a n d d i a g o n a l i z e d to o b t a i n t h e p r i n c i p a l c o m p o n e n t g v a l u e s that c a n b e c o m p a r e d to e x p e r i m e n t ( f o u r t h c o l u m n ) . It is o b s e r v e d t h a t a l t h o u g h t h e g v a l u e s are r e d u c e d f r o m those o b t a i n e d f r o m the l i g a n d - f i e l d c a l c u l a t i o n due to t h e i n c l u s i o n o f c o v a l e n c y , t h e y a r e c l o s e r to 2 . 0 0 t h a n is o b t a i n e d e x p e r i m e n t a l l y . T h u s , the S C F - X a - S W calculations are p r o d u c i n g too c o v a l e n t a d e s c r i p t i o n o f t h e a c t i v e s i t e . T h e r e is o n e set o f a d j u s t a b l e p a r a m e t e r s i n t h i s c a l c u l a t i o n , w h i c h is t h e s p h e r e sizes u s e d i n t h e scattered w a v e solutions. T h o s e e m p l o y e d i n this i n i t i a l c a l c u l a t i o n are the standard spheres n o r m a l l y used that are d e f i n e d b y the N o r m a n c r i t e r i a (37). W e s y s t e m a t i c a l l y v a r i e d t h e s e s p h e r e s ( i n c r e a s i n g t h e m e t a l s p h e r e i n c r e a s e s its e l e c t r o n d e n s i t y , l o w e r s its e f f e c t i v e n u c l e a r c h a r g e , a n d r e d u c e s its i n t e r a c t i o n w i t h t h e l i g a n d s ) , a n d i t e r a t i v e l y repeated this g value p r o t o c o l u n t i l the calculated values w e r e i n g o o d a g r e e m e n t w i t h e x p e r i m e n t (33, 36). T h i s a p p r o a c h p r o v i d e d t h e e x p e r i m e n t a l l y a d j u s t e d d e s c r i p t i o n o f t h e g r o u n d state w a v e f u n c t i o n o f t h e b l u e c o p p e r site t h a t is g i v e n i n F i g u r e 7 A . 2

T h e s e Χ α c a l c u l a t i o n s p r o v i d e a d e s c r i p t i o n o f t h e g r o u n d state o f t h e b l u e c o p p e r site t h a t is h i g h l y c o v a l e n t . T h e c o v a l e n c y is s t r o n g l y anisotropic w i t h d e r e a l i z a t i o n predominantly into the Spx orbital of t h e t h i o l a t e ( F i g u r e 7 A ) . W e h a v e b e e n a b l e to e x p e r i m e n t a l l y test t h e k e y features o f this g r o u n d state u s i n g a v a r i e t y o f s p e c t r o s c o p i c m e t h o d s . First, the high covalency can be p r o b e d b y c o p p e r L - e d g e spectroscopy (38). T h e e l e c t r i c d i p o l e i n t e n s i t y o f t h e C u 2p Ψ Ο Μ Ο ( H O M O is t h e h i g h e s t o c c u p i e d m o l e c u l a r o r b i t a l ) t r a n s i t i o n at 9 3 0 e V r e f l e c t s t h e C u 2p C u 3d t r a n s i t i o n p r o b a b i l i t y a n d p r o b e s t h e a m o u n t o f C u d 2. 2 c h a r a c t e r i n t h e g r o u n d - s t a t e w a v e f u n c t i o n . F r o m F i g u r e 7 C , i t is o b Η

x


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Cu

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1.2%N s"

Cu

15% Spa

tetb

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925 |Q

4h

1 4

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/ tet b \ / ' \ / a -15% y

2

\

/ \ plastocyanin / \ a -38%

S K-edge

930 935 940 2468 2469 2470 2471 Energy (eV) Energy (eV)

«

M: ιM ·

II : plastocyanin

;;D CuCI 61%

ι

Cu L-edge

•Sulfur 1s

- ^ - C o p p e r 2p

- α S(Cys) 3p

^

25000

5000 h

15000 10000 1

Energy (cm*)

20000

5000

Figure 7. Ground-state wave function of plastocyanin. A: HOMO wave function contour for plastocyanin (28). B: HOMO wave function contour for the thiolate copper complex tetb (34). C: Copper L-edge (38) and sulfur K-edge (34) spectra as probes of metal-ligand covalency. D: Absorption, single-crystal polarized absorption, and low-temperature MCD spectra of plastocyanin. The absorption spectrum has been Gaussian resolved into its component bands as in reference 33.

6% Ν

66%

6% Ν

σ-antibonding HOMO, Cu-S(thiolate) = 2.36 A

Β

42%

Q

Plastocyanin Experimental Probes of Covalency

π-antibonding HOMO Cu-S(Cys) = 2.13A

A


g

w

ο S? g S ο ο > ζ Ω ο χ

1

2 η >

5.

Active Sites in Copper Proteins

S O L O M O N E T AL.

s e r v e d t h a t t h e 9 3 0 - e V p e a k i n p l a s t o c y a n i n has 6 7 % o f t h e i n t e n s i t y o f D -CuC\ ~, w h i c h is k n o w n to h a v e 6 1 % C u d 2_ 2 c h a r a c t e r ( F i g u r e 1 C ) . T h u s , t h e g r o u n d state o f t h e b l u e c o p p e r site is e s t i m a t e d f r o m e x p e r i m e n t to h a v e 4 1 % C u d 2. 2 c h a r a c t e r . T h i s is i n g o o d a g r e e m e n t w i t h the adjusted S C F - X a - S W calculations (42%). Second, the sulfur c o n t r i b u t i o n to t h e H O M O c a n b e s t u d i e d u s i n g s u l f u r K - e d g e s p e c t r o s c o p y (34) i n w h i c h t h e e l e c t r i c d i p o l e i n t e n s i t y n o w r e f l e c t s t h e Sis -*> S3p c h a r a c t e r i n t h e H O M O . F r o m F i g u r e 7 C , p l a s t o c y a n i n e x h i b i t s a n i n t e n s e s u l f u r p r e - e d g e f e a t u r e at 2 4 6 9 e V . It has 2 . 6 t i m e s t h e i n t e n s i t y o f t h e tet b m o d e l c o m p l e x o f S c h u g a r (39), w h i c h c o n t a i n s a n o r m a l 2 . 3 6 À c o p p e r - t h i o l a t e s u l f u r b o n d a n d has 1 5 % s u l f u r ρ c h a r a c t e r i n t h e g r o u n d state ( F i g u r e 7 B ) . T h u s , t h e b l u e c o p p e r site is also e x p e r i m e n t a l l y e s t i m a t e d to h a v e 3 8 % s u l f u r ρ c h a r a c t e r f r o m t h e c y s t e i n e l i g a n d , a g a i n i n g o o d a g r e e m e n t w i t h t h e Xa c a l c u l a t i o n s ( 3 6 % ) . 4h

2

4

x

x


135

y

y

T h e final f e a t u r e o f t h e g r o u n d - s t a t e w a v e f u n c t i o n is e l u c i d a t e d through the assignment of the characteristic excited-state absorption s p e c t r a l f e a t u r e s o f p l a s t o c y a n i n ( F i g u r e 7 D ) . A l t h o u g h t h e r e a r e i n fact e i g h t b a n d s r e q u i r e d to fit a c o m b i n a t i o n o f a b s o r p t i o n ( A b s ) , c i r c u l a r dichroism ( C D ) , and magnetic circular dichroism ( M C D ) spectra of the b l u e c o p p e r site (33), at l o w r e s o l u t i o n t h e a b s o r p t i o n s p e c t r u m w a s r e g a r d e d o r i g i n a l l y as h a v i n g a l o w - e n e r g y w e a k a n d h i g h e r - e n e r g y i n t e n s e (i.e., t h e 6 0 0 n m , 1 6 , 0 0 0 c m ) b a n d p a t t e r n (1, 26). P o l a r i z e d s i n g l e c r y s t a l s p e c t r a l s t u d i e s o v e r t h i s r e g i o n (29) s h o w e d t h e s a m e p o l a r i z a t i o n ratio for b o t h bands, w h i c h r e q u i r e d that b o t h bands b e a s s o c i a t e d w i t h t h e C y s S - C u ( I I ) b o n d . T h u s , i n p a r a l l e l to t h e C l ~ Cu(II) charge-transfer assignment presented earlier, these w e r e assigned as l o w - e n e r g y w e a k π a n d h i g h e r - e n e r g y i n t e n s e σ c h a r g e t r a n s f e r t r a n sitions i n v o l v i n g the thiolate sulfur. H o w e v e r , M C D spectroscopy s h o w e d that this assignment was not correct. A l l four o f the l o w - e n e r g y bands ( 5 - 8 i n F i g u r e 7 D ) that c o m p r i s e this r e g i o n are w e a k i n the absorption spectrum but quite intense i n the low-temperature M C D s p e c t r u m (33). B e c a u s e M C D C - t e r m i n t e n s i t y f o r C u ( I I ) r e q u i r e s s p i n o r b i t c o u p l i n g , a n d h e n c e d o r b i t a l c h a r a c t e r , this leads to t h e a s s i g n m e n t o f b a n d s 5 - 8 as d -> d t r a n s i t i o n s . T h u s , t h e 6 0 0 - n m b a n d (4), t h a t is intense i n the absorption spectrum and weak i n the low-temperature M C D s p e c t r u m , is t h e l o w e s t - e n e r g y c h a r g e - t r a n s f e r t r a n s i t i o n f r o m t h e t h i o l a t e a n d m u s t b e t h e C y s ρπ -> C u d 2. 2 c h a r g e - t r a n s f e r t r a n s i t i o n . T h e C y s ρσ C u d 2_ 2 is a w e a k b a n d at h i g h e r e n e r g y . T h e k e y p o i n t is t h a t f o r t h e b l u e c o p p e r site o n e has a l o w - e n e r g y i n t e n s e π a n d h i g h e r e n e r g y w e a k σ c h a r g e - t r a n s f e r t r a n s i t i o n t o t h e C u d 2. 2 o r b i t a l . I n a s m u c h as c h a r g e - t r a n s f e r i n t e n s i t y r e f l e c t s o r b i t a l o v e r l a p , t h i s o v e r l a p r e q u i r e s that t h e d 2_ 2 o r b i t a l h a v e its l o b e s b i s e c t e d b y t h e C y s S - C u b o n d (Figure 7A) and thus be i n v o l v e d i n a strong π a n t i b o n d i n g inter- 1

x

x

y

y

x

x

y

y


136


a c t i o n w i t h t h e t h i o l a t e as also o b t a i n e d f r o m t h e Χ α c a l c u l a t i o n s . T h e s t r o n g π i n t e r a c t i o n r o t a t e s t h e d 2. 2 o r b i t a l b y 4 5 ° r e l a t i v e to its u s u a l o r i e n t a t i o n a l o n g t h e l i g a n d - c o p p e r b o n d as, f o r e x a m p l e , i n t h e tet b m o d e l c o m p l e x ( F i g u r e 7 B ) . T h i s r o t a t i o n o f t h e d 2. 2 o r b i t a l d e r i v e s f r o m the q u i t e short b l u e c o p p e r C y s S - C u b o n d l e n g t h o f 2.13 À. T h u s the S C F - X a - S W calculations are p r o d u c i n g an accurate d e s c r i p t i o n o f t h e g r o u n d state o f t h e b l u e c o p p e r site a n d o n e c a n n o w c o r r e l a t e t h i s w i t h c r y s t a l s t r u c t u r e i n f o r m a t i o n to o b t a i n s i g n i f i c a n t insight into function. I n p a r t i c u l a r , the X - r a y structure of ascorbate oxi d a s e (40) s h o w s t h a t t h e c y s t e i n e l i g a n d o f a b l u e c o p p e r site i n t h i s m u l t i c o p p e r o x i d a s e is flanked o n e i t h e r s i d e i n t h e s e q u e n c e b y h i s t i d i n e s t h a t a r e l i g a n d s to t w o o f t h e c o p p e r s i n a t r i n u c l e a r c o p p e r c l u s t e r site ( d i s c u s s e d i n t h e l a t e r s e c t i o n , Multicopper Oxidases) (41). T h i s b l u e c o p p e r site t r a n s f e r s a n e l e c t r o n r a p i d l y i n t h e r e d u c t i o n o f 0 at t h e trinuclear c o p p e r center. A s can be seen from the Χ α calculated wave function contour that w e have s u p e r i m p o s e d on the crystal structure of t h e b l u e c e n t e r i n a s c o r b a t e o x i d a s e ( F i g u r e 8), t h e g r o u n d - s t a t e w a v e function provides a highly anisotropic covalent pathway involving the cysteine sulfur. T h e covalency activates this residue for d i r e c t i o n a l elec tron transfer. I n addition, the low-energy, intense C y s π C u d 2. 2 charge-transfer transition i n the blue copper absorption spectrum p r o v i d e s a n efficient h o l e s u p e r e x c h a n g e p a t h w a y for r a p i d e l e c t r o n t r a n s f e r b e t w e e n t h e b l u e a n d t r i n u c l e a r c o p p e r c l u s t e r sites (2). C l e a r l y , as s h o w n i n F i g u r e 8, t h e u n i q u 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 b l u e c o p p e r center reflects a ground-state w a v e f u n c t i o n that plays a c r i t i c a l r o l e i n its f u n c t i o n i n g o f r a p i d l o n g - r a n g e e l e c t r o n t r a n s f e r to a s p e c i f i c l o c a t i o n in or on the protein. x

y


x

y

2

x

Coupled Binuclear

y

Copper Proteins

T h e binuclear c o p p e r proteins h e m o c y a n i n and tyrosinase reversibly b i n d d i o x y g e n a n d i n t h e case o f t y r o s i n a s e a c t i v a t e i t f o r h y d r o x y l a t i o n o f p h e n o l to o r f / i o - d i p h e n o l a n d f u r t h e r o x i d a t i o n to o r f / i o - q u i n o n e ( F i g u r e 9) (42). B o t h p r o t e i n s h a v e e s s e n t i a l l y t h e s a m e o x y a c t i v e sites (42, 43) t h a t i n v o l v e t w o C u ( I I ) i o n s ( s h o w n b y X - r a y a b s o r p t i o n e d g e d a t a (44, 45)) a n d b o u n d p e r o x i d e ( s h o w n b y t h e u n u s u a l l y l o w O - O s t r e t c h ing frequency of 750 c m observed i n the resonance R a m a n spectrum (46-48)). A s w i l l b e s u m m a r i z e d i n t h i s s e c t i o n , t h e u n i q u e v i b r a t i o n a l a n d g r o u n d a n d e x c i t e d state e l e c t r o n i c s p e c t r a l f e a t u r e s o f t h i s o x y site are n o w u n d e r s t o o d . T h e s e g e n e r a t e a d e t a i l e d d e s c r i p t i o n o f t h e p e r o x i d e - c o p p e r b o n d that p r o v i d e s f u n d a m e n t a l i n s i g h t i n t o t h e r e v e r s i b l e b i n d i n g a n d activation of d i o x y g e n b y this site. T h e g r o u n d state o f o x y h e m o c y a n i n e x h i b i t s n o E P R s i g n a l . T h i s results from a strong antiferromagnetic c o u p l i n g of the t w o Cu(II) ions - 1



Figure 8. Proposed electron transfer pathway in blue copper proteins. The plastocyanin wave function contours have been superimposed on the blue copper (type 1) site in ascorbate oxidase (40). The contour shows the substantial electron delocalization onto the cysteine Sp-κ orbital that activates electron transfer to the trinuclear copper cluster at 12.5 Â from the blue copper site. This low-energy, intense Cys Sp Cu charge-transfer transition provides an effective hole superexchange mechanism for rapid long-range electron transfer between these sites (2, 3, 28).


138


Reactivity Hemocyanin: [Cu(l)Cu(l)] + 0 deoxy Tyrosinase:

2

[Cu(l)Cu(l)] + 0 deoxy

2

^ [Cu(ll)Cu(ll)]0 oxy 2 2

^ [Cu(ll)Cu(ll)]0 oxy v = 750 cm 2 2

-1

0 0

[Cu(ll)Cu(ll)]0 -+ phenol +2H+ ^ oxytyrosinase Downloaded by PURDUE UNIV on April 9, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/ba-1995-0246.ch005

2 2

[Cu(ll)Cu(ll)]+o-diphenol ^

[Cu(ll)Cu(l!)]+o-diphenol+H 0 2

[Cu(l)Cu(l)] + o-quinone + 2H+ deoxytyrosinase

Ground State No EPR ->

Antiferromagnetic coupling (#= -2J SyS2) -Op " 2

[Cu(ii)Cu(ii)]o 2

2

— ? — >

Cu(ll)

Cu(ll) 500 cm" Excited States

Methemocyanin -2J > 500 cm"

1

1

0 " -> Cu(ll) CT Transitions 2

2

Wavelength (nm) Figure 9. Coupled binuclear copper proteins; ground- and excited-state spectral features.


5.


139


(—2/ > 5 0 0 c m " ) , (49, 50) h e n c e its c l a s s i f i c a t i o n as a coupled b i n u c l e a r c o p p e r site ( 5 i ) . D i s p l a c e m e n t o f t h e p e r o x i d e p r o d u c e s a m e t d e r i v a t i v e t h a t also has t w o C u ( I I ) i o n s t h a t a r e s t r o n g l y a n t i f e r r o m a g n e t i c a l l y c o u p l e d (-2J > 5 0 0 c m ) ( W i l c o x , D . E . ; W e s t m o r e l a n d , T . D . ; S a n d u s k y , P . O . ; S o l o m o n , Ε. I . , u n p u b l i s h e d r e s u l t s ) . T h u s , t h e r e m u s t b e a n e n dogenous bridge present i n the met derivative. T h e crystal structure of d e o x y h e m o c y a n i n (52) s h o w s n o p r o t e i n r e s i d u e s c a p a b l e o f b r i d g i n g t h e c o p p e r ions i n t h e v i c i n i t y o f t h e b i n u c l e a r c o p p e r site so this b r i d g i n g l i g a n d is l i k e l y to b e h y d r o x i d e . W i t h r e s p e c t to t h e e x c i t e d state s p e c troscopy, oxyhemocyanin exhibits a moderately intense b a n d i n the ab s o r p t i o n s p e c t r u m at ~ 6 0 0 n m (e ~ 1 0 0 0 M " c m " ) a n d a n e x t r e m e l y i n t e n s e b a n d at ~ 3 5 0 n m (e ~ 2 0 , 0 0 0 M " c m " ) . D i s p l a c e m e n t o f p e r o x i d e o n g o i n g to t h e m e t d e r i v a t i v e ( s o l i d to d a s h e d s p e c t r u m i n F i g u r e 9, b o t t o m ) e l i m i n a t e s t h e s e f e a t u r e s as w e l l as a b a n d at 4 8 0 n m t h a t is p r e s e n t i n t h e C D b u t n o t t h e a b s o r p t i o n s p e c t r u m (53). T h e s e t h r e e b a n d s c a n b e a s s i g n e d as p e r o x i d e - t o - c o p p e r c h a r g e t r a n s f e r t r a n s i t i o n s a n d w i l l b e s e e n to p r o v i d e a d e t a i l e d p r o b e o f t h e p e r o x i d e c o p p e r b o n d . W e a r e p a r t i c u l a r l y i n t e r e s t e d i n (1) t h e fact t h a t t h e r e a r e t h r e e c h a r g e t r a n s f e r b a n d s , (2) t h e s e l e c t i o n r u l e s a s s o c i a t e d w i t h t h e p r e s e n c e o f a b a n d i n t h e C D b u t n o t a b s o r p t i o n s p e c t r u m , a n d (3) the h i g h energy and intensity of the 3 5 0 - n m band. 1

- 1

1

1


1

1

W e first c o n s i d e r p e r o x i d e b o u n d e n d - o n t o a s i n g l e C u (II) i o n ( F i g u r e 1 0 A ) . T h e valence orbitals of p e r o x i d e i n v o l v e d i n b o n d i n g are t h e π* set. T h e s e o r b i t a l s s p l i t i n t o t w o n o n d e g e n e r a t e l e v e l s ( l a b e l e d 7τ * a n d 7τ *) o n b o n d i n g to t h e m e t a l i o n . T h e π * o r b i t a l is o r i e n t e d a l o n g t h e C u - O b o n d a n d has s t r o n g o v e r l a p w i t h t h e d 2_ 2 o r b i t a l p r o d u c i n g a higher-energy, intense charge-transfer transition. T h e peroxide 7Γ * o r b i t a l is v e r t i c a l to t h e C u - O b o n d a n d w e a k l y π i n t e r a c t i n g w i t h the copper, p r o d u c i n g a lower-energy, relatively weak transition. T h u s , e n d - o n p e r o x i d e b o n d i n g is d o m i n a t e d b y t h e σ d o n o r i n t e r a c t i o n o f t h e 0 ~ 7τ * o r b i t a l w i t h t h e d 2. 2 o r b i t a l . T h i s p r e d i c t e d l o w - e n e r g y w e a k / h i g h - e n e r g y i n t e n s e c h a r g e - t r a n s f e r s p e c t r u m is o b s e r v e d e x p e r i m e n t a l l y f o r t h e [ C u ( X Y L - 0 - ) ( 0 ) ] c o m p l e x p r e p a r e d b y K a r l i n (54) t h a t has 0 ~ e n d - o n b o u n d to a s i n g l e C u ( I I ) i o n ( b a s e d o n m i x e d i s o t o p e effects o n its r e s o n a n c e R a m a n s p e c t r u m ) ( F i g u r e 1 0 B ) (55). N o t e , h o w ever, that there are only t w o bands i n the c h a r g e - t r a n s f e r s p e c t r u m of this m o n o m e r i c c o m p l e x a n d that t h e π * t r a n s i t i o n is c o n s i d e r a b l y l o w e r i n e n e r g y ( 5 0 0 n m ) a n d w e a k e r i n i n t e n s i t y (e ~ 5 0 0 0 M " c m " ) t h a n the 350 n m 0 " charge-transfer b a n d i n oxyhemocyanin. σ

σ

ν

x

y

ν

2

2

x

σ

2

2

y

2

+

2

σ

1

2

1

2

T h e fact t h a t t h r e e p e r o x i d e - t o - c o p p e r c h a r g e - t r a n s f e r t r a n s i t i o n s a r e o b s e r v e d i n o x y h e m o c y a n i n a n d o x y t y r o s i n a s e l e d us to c o n s i d e r t h e s p e c t r a l effects o f b r i d g i n g p e r o x i d e b e t w e e n t w o C u ( I I ) i o n s . A transition-dipole v e c t o r - c o u p l i n g ( T D V C ) m o d e l was d e v e l o p e d that p r e d i c t s t h a t e a c h c h a r g e - t r a n s f e r state i n a C u - p e r o x i d e m o n o m e r


140


|End-on Peroxide-Cu(ll) Bonding]0 ' -> Cu(ll) CT Spectrum 2

2

8000

L

,o-o

d2.y8 X

„ D

d2-y2 X


Cud

400

500 600 700 Wavelength (nm)

800

Ν ~-»Cu(ll)CT Spectra

i t

Transition Dipole Vector Coupling Model Monomer

Dimer

Figure 10. Peroxide charge-transfer transitions in copper monomers and dimers. A: Orbital interactions involved in end-on peroxide-copper bonding and predicted charge-transfer transitions (thickness of arrow indicates rel ative intensity). B: Charge-transfer absorption spectrum of peroxide bound to a single Cu(ll) ion (Adapted from ref. 55). C: Ground-state and chargetransfer excited state splittings due to dimer interactions in a peroxide bridged copper dimer. Κ is the coulomb dimer interaction, ] is the excitedstate magnetic exchange, and I and L are the coulomb and exchange con tributions to the excitation transfer between halves of the dimer, respectively. D: Azide-to-copper charge-transfer spectra of model complexes and met azide hemocyanin (Adapted from ref 56). ex


5.


141


c o m p l e x w i l l s p l i t i n t o f o u r states i n a d i m e r ( F i g u r e I O C ) ( 5 3 , 56-58). W e h a v e f u r t h e r d e v e l o p e d a g e n e r a l m o d e l o f e x c i t e d state d i m e r i n teractions, the valence bond-configurational interaction ( V B C I ) m o d e l . This m o d e l reduces to t h e T D V C m o d e l i n the in-state limit b u t t h e V B C I treatment gives a quantitative d e s c r i p t i o n o f the d i m e r splittings i n terms o f parameters that c a n b e e v a l u a t e d u s i n g S C F - Χ α m o l e c u l a r o r b i t a l c a l c u l a t i o n s (58-60). F i r s t , t h e r e is a s i n g l e t - t r i p l e t a n t i f e r r o m a g n e t i c s p l i t t i n g i n t h e e x c i t e d state j u s t as t h e r e is i n t h e g r o u n d state b u t c o n s i d e r a b l y l a r g e r i n m a g n i t u d e (58). I n a d d i t i o n , b o t h t h e s i n g l e t a n d t r i p l e t states a r e s p l i t f u r t h e r i n t o t w o states t h a t c o r r e s p o n d t o symmetric and antisymmetric combinations o f the 0 ~ Cu(II) chargetransfer transition to each c o p p e r i n t h e b r i d g e d d i m e r . A s t h e antiferr o m a g n e t i c a l l y c o u p l e d g r o u n d state is a s i n g l e t , o n l y t h e t w o t r a n s i t i o n s t o t h e s i n g l e t e x c i t e d states s h o u l d h a v e a b s o r p t i o n i n t e n s i t y . T h i s p r e d i c t e d s p l i t t i n g i n t o t w o b a n d s is o b s e r v e d (56) i n a s e r i e s o f a z i d e m o d e l c o m p l e x e s p r e p a r e d b y S o r r e l l (61), R e e d (62, 63), a n d K a r l i n (64) ( F i g u r e 1 0 D ) . A z i d e b o u n d t o a s i n g l e C u ( I I ) i o n e x h i b i t s a 7r Cu(II) c h a r g e - t r a n s f e r t r a n s i t i o n t h a t is a n a l o g o u s t o t h e p e r o x i d e π * Cu(II) c h a r g e - t r a n s f e r t r a n s i t i o n . A s p r e d i c t e d , b r i d g i n g t h e a z i d e i n a cis μ1,3 g e o m e t r y b e t w e e n t w o C u ( I I ) i o n s r e s u l t s i n a s p l i t t i n g o f t h e m o n o mer charge-transfer transition into t w o bands, the symmetric ( A l i n the C dimer symmetry) and antisymmetric ( B l ) components of the π * charge-transfer transition. N o t e i n F i g u r e 1 0 D (bottom) that b i n d i n g N ~ to t h e met h e m o c y a n i n de r i va t i ve p r o d u c e s the same A l / B l charget r a n s f e r i n t e n s i t y p a t t e r n i n d i c a t i n g t h a t a z i d e also b r i d g e s i n a cis μ1,3 g e o m e t r y i n m e t h e m o c y a n i n (56).


2

2

ff

nb

σ

2v

σ

3

T h e p r e c e d i n g discussion shows that the presence o f m o r e t h a n t w o p e r o x i d e - t o - c o p p e r C T transitions i n o x y H c requires that this l i g a n d b r i d g e t h e c o p p e r c e n t e r s . I n 1 9 8 9 , K i t a j i m a o b t a i n e d t h e first c r y s t a l structure of a side-on bridging peroxide i n transition-metal chemistry (65, 66) a n d , i n 1 9 9 2 , M a g n u s d e t e r m i n e d t h a t L i m u l u s p o l y p h e m u s o x y H c has t h e s i d e - o n b r i d g i n g s t r u c t u r e s h o w n i n F i g u r e 1 1 (top) (67). T h e V B C I m o d e l can then b e used to predict the energy splittings a n d symmetries a n d hence the selection rules for the peroxide-to-copper C T transitions i n t h e effective D h s y m m e t r y o f the s i d e - o n b r i d g e d site (the trans a x i a l H i s l i g a n d s r e d u c e t h e site s y m m e t r y t o C h b u t h a v e a s m a l l effect o n t h e s p e c t r u m ) ( 5 9 , 60). B r i d g i n g t h e p e r o x i d e i n t h e s i d e 2

2

on structure results i n a splitting o f the π * into t w o components. T h e l o w - e n e r g y c o m p o n e n t is e l e c t r i c d i p o l e a l l o w e d (z) a n d s h o u l d a p p e a r i n t h e a b s o r p t i o n s p e c t r u m , b u t w i t h l i m i t e d i n t e n s i t y i n a s m u c h as i t is a 7T * c h a r g e - t r a n s f e r t r a n s i t i o n . T h i s c a n b e a s s o c i a t e d w i t h t h e 6 0 0 n m a b s o r p t i o n b a n d . T h e s e c o n d c o m p o n e n t o f t h e p e r o x i d e 7r * c h a r g e t r a n s f e r t r a n s i t i o n is p r e d i c t e d t o b e o n l y m a g n e t i c d i p o l e a l l o w e d ( R ) and thus it should contribute to the C D b u t not absorption spectrum. ν

V

v

y


142


Oxyhemocyanin and Oxytyrosinase Limulus Oxyhemocyanin X-ray Crystal Structure

: c u r ~ / ° ^ c u : —

2

K. Magnus, H. Ton-That J. Inorg. Biochem. 47, 20(1992)

ι

2


Side-on μ-η :η Bridge Excited

State Spectral Assignments

Monomer

D h 2

Dimer ζ

7C

C

Β2g

V

C Ο

Β3u

c

J/\^

Monomer

B^iRy) B (z) ^ _ o 1u

2 h

Dimer

20,000h oxy (Abs.)

E υ 2 ω 350

400

400 500 V Wavelength (nm)

700

800

Figure 11. Excited-state spectral assignments for the D h μ-η :*! peroxocopper unit in oxyhemocyanin and oxytyrosinase. 2

2

2


5.


143


T h e 4 8 0 - n m C D f e a t u r e c a n b e a s s i g n e d to t h i s t r a n s i t i o n . T h e 7Γ * also splits into t w o bands w i t h the l o w e r - e n e r g y c o m p o n e n t b e i n g e l e c t r i c dipole allowed and having the dominant absorption intensity. This can b e associated w i t h t h e 3 5 0 - n m a b s o r p t i o n b a n d i n o x y h e m o c y a n i n . T h u s , the side-on b r i d g i n g peroxide produces the three observed chargetransfer transitions w i t h one b e i n g present i n the C D b u t not the ab s o r p t i o n s p e c t r u m . H o w e v e r , o n e m u s t s t i l l a c c o u n t for t h e h i g h i n t e n s i t y a n d e n e r g y o f t h e 3 5 0 - n m 0 ~ ττ * c h a r g e - t r a n s f e r t r a n s i t i o n a n d t h e l o w v i b r a t i o n a l f r e q u e n c y o f t h e O - O s t r e t c h . T h u s w e p r o c e e d to evaluate quantitatively the electronic structure associated w i t h the side-on b r i d g i n g p e r o x i d e a n d c o m p a r e this to the m o r e c o m m o n l y ob served end-on bridging peroxide structure both theoretically and experimentally. σ


2

2

σ

Broken-symmetry, spin-unrestricted S C F - X a - S W M O calculations w e r e p e r f o r m e d to d e s c r i b e t h e e l e c t r o n i c s t r u c t u r e s a s s o c i a t e d w i t h both the end-on and side-on b r i d g i n g peroxide geometric structures (68, 69). T h e s e c a l c u l a t i o n s a r e a p p r o p r i a t e f o r a n t i f e r r o m a g n e t i c a l l y c o u p l e d d i m e r s (70, 71). I n F i g u r e 1 2 , w e f o c u s o n t h e i n t e r a c t i o n o f the highest o c c u p i e d molecular orbital ( H O M O ) and the lowest unoc c u p i e d m o l e c u l a r o r b i t a l ( L U M O ) , that are the s y m m e t r i c a n d a n t i s y m m e t r i c c o m b i n a t i o n s o f d 2_ 2 o r b i t a l s o n e a c h c o p p e r , w i t h t h e v a l e n c e orbitals of the peroxide. F o r the end-on b r i d g e d structure (Figure 12, left), t h e b o n d i n g is c o n s i s t e n t w i t h t h e q u a l i t a t i v e d e s c r i p t i o n p r e s e n t e d e a r l i e r . T h e p e r o x i d e π * o r b i t a l is s t a b i l i z e d t h r o u g h a b o n d i n g i n t e r action w i t h the L U M O on b o t h coppers. T h u s , i n the end-on b r i d g e d g e o m e t r y p e r o x i d e acts as a σ d o n o r l i g a n d w i t h o n e b o n d i n g i n t e r a c t i o n w i t h e a c h o f t h e t w o c o p p e r s . A v e r y d i f f e r e n t b o n d i n g d e s c r i p t i o n is o b t a i n e d for t h e s i d e - o n b r i d g e d p e r o x i d e . I n t h i s s t r u c t u r e , t h e π * o r b i t a l is a g a i n s t a b i l i z e d b y a σ d o n o r i n t e r a c t i o n w i t h t h e L U M O o n b o t h coppers. In the side-on structure the bonding/antibonding inter a c t i o n o f t h e 7τ * o r b i t a l is l a r g e r t h a n i n t h e e n d - o n s t r u c t u r e b e c a u s e the peroxide n o w occupies two coordination positions on each of the t w o c o p p e r s . T h u s , p e r o x i d e b e h a v e s as a n e x t r e m e l y s t r o n g σ d o n o r i n t h e s i d e - o n s t r u c t u r e . F u r t h e r m o r e , t h e s i d e - o n p e r o x i d e is p r e d i c t e d to h a v e a n a d d i t i o n a l b o n d i n g i n t e r a c t i o n w i t h t h e d 2_ 2 o r b i t a l s o n t h e c o p p e r s . T h i s i n v o l v e s s t a b i l i z a t i o n o f t h e H O M O t h r o u g h its i n t e r a c t i o n w i t h t h e h i g h - e n e r g y u n o c c u p i e d σ* o r b i t a l o n t h e p e r o x i d e . T h i s a d d i t i o n a l b o n d i n g i n t e r a c t i o n shifts e l e c t r o n d e n s i t y f r o m t h e c o p p e r i o n s o n t o t h e p e r o x i d e . T h u s , p e r o x i d e also acts as a π a c c e p t o r l i g a n d u s i n g t h i s h i g h l y a n t i b o n d i n g σ* o r b i t a l . x

y

σ

σ

σ

x

y

It w a s o f c r i t i c a l i m p o r t a n c e to e v a l u a t e e x p e r i m e n t a l l y t h i s u n u s u a l e l e c t r o n i c structure d e s c r i p t i o n for the side-on b r i d g e d p e r o x i d e a n d its r e l a t i o n t o t h e s p e c t r a l f e a t u r e s o f t h e μ-η :η model complex and o x y h e m o c y a n i n . T h i s e v a l u a t i o n was a c c o m p l i s h e d t h r o u g h a series o f 2

2


144



CHEMISTRY

-θ!σ* LUMP .LUMO

Y

1 ' iCu^y CUfld^y

/

u HOMO \

fr** ** 6

-4C"a*V

- 4 — \ ^ Λ ν ^ '\

Figure 12. Electronic structures of the end-on cis-μ-1,2 (0 ) and side-on μ-η :η (Ό 0 models of the oxyhemocyanin active site. Wave function con tours of the HOMO and LUMO and energy level diagrams showing dominant orbital contributions. 2υ

2

2

2


5.



145

studies of the charge-transfer a n d v i b r a t i o n a l spectral features of e n d o n (72) a n d s i d e - o n (73) b o u n d p e r o x i d e - c o p p e r m o d e l c o m p l e x e s p r e p a r e d b y K a r l i n (74) a n d K i t a j i m a (65, 66). T h e σ - d o n o r a b i l i t y o f p e r o x i d e c a n b e r e l a t e d to t h e i n t e n s i t y ( a n d e n e r g y (58)) o f t h e 7Γ * -> C u ( I I ) c h a r g e - t r a n s f e r t r a n s i t i o n ( F i g u r e 1 3 ) . T h e i d e a is t h a t as t h e w a v e f u n c t i o n o f t h e o c c u p i e d 0 ~ 7τ * o r b i t a l g a i n s c o p p e r c h a r a c t e r , α (i.e., t h e c o e f f i c i e n t f o r t h e a m o u n t o f c o p p e r c h a r a c t e r i n t h e w a v e f u n c t i o n is a ) , its σ - d o n o r i n t e r a c t i o n w i t h t h e c o p p e r i n c r e a s e s . T h i s wave function gives an a p p r o x i m a t i o n for the ligand-to-metal charget r a n s f e r i n t e n s i t y t h a t ( a l o n g w i t h g e o m e t r i c factors) is p r o p o r t i o n a l t o a . T h u s , the peroxide π * Cu(II) charge-transfer intensity increases as its σ - d o n o r i n t e r a c t i o n w i t h t h e c o p p e r i n c r e a s e s . I f w e n o r m a l i z e t o t h e 7τσ* c h a r g e - t r a n s f e r i n t e n s i t y o f t h e e n d - o n p e r o x i d e m o n o m e r c o m p l e x s h o w n i n F i g u r e 1 0 B , t h e 0 ~ c h a r g e - t r a n s f e r i n t e n s i t y o f t h e trans μ-1,2 e n d - o n b r i d g e d c o m p l e x increases b y a factor o f t w o , consistent w i t h p e r o x i d e b i n d i n g to e a c h o f t w o C u ( I I ) i o n s . A l t h o u g h n o cis m o d e l c o m p l e x exists, o u r Χ α calculations i n d i c a t e that p e r o x i d e b i n d i n g i n this g e o m e t r y s h o u l d h a v e a s i m i l a r σ - d o n o r i n t e r a c t i o n w i t h t h e c o p p e r s as p e r o x i d e b r i d g e d i n t h e trans c o m p l e x . H o w e v e r , t h e s i d e - o n b r i d g e d μ-η :η c o m p l e x a n d o x y h e m o c y a n i n exhibit an e x t r e m e l y intense π * charge-transfer transition. T h e i n t e n s i t y o f this t r a n s i t i o n quantitates to ~ 4 times the σ-donor i n t e r a c t i o n of p e r o x i d e b o u n d to a single Cu(II) i o n . T h i s is c o n s i s t e n t w i t h t h e Χ α c a l c u l a t i o n s a n d t h e fact t h a t i n t h i s g e o m e t r y p e r o x i d e has f o u r b o n d i n g i n t e r a c t i o n s w i t h t h e t w o C u ( I I ) ions. T h e e x t r e m e l y h i g h intensity of the 3 5 0 - n m b a n d i n o x y h e m o c y a n i n also q u a n t i t a t e s t o h a v i n g ~ 4 σ - d o n o r i n t e r a c t i o n s w i t h t h e b i n u c l e a r c o p p e r site c o n s i s t e n t w i t h t h e s i d e - o n p e r o x i d e b r i d g e d s t r u c t u r e o f oxyhemocyanin. σ

2


2

2

σ

σ

2

2

2

2

σ

O n e c a n p r o b e t h e 7r-acceptor a b i l i t y o f t h e p e r o x i d e t h r o u g h a s t u d y o f its i n t r a l i g a n d s t r e t c h i n g f o r c e c o n s t a n t ( a n d h e n c e O - O b o n d s t r e n g t h ) , w h i c h is o b t a i n e d f r o m a n o r m a l c o o r d i n a t e a n a l y s i s ( N C A ) o f v i b r a t i o n a l spectra ( F i g u r e 13). O n e w o u l d expect this force constant t o i n c r e a s e as t h e σ - d o n o r i n t e r a c t i o n o f t h e p e r o x i d e w i t h t h e c o p p e r increases, because this increased interaction removes the e l e c t r o n d e n s i t y f r o m a π - a n t i b o n d i n g o r b i t a l o n t h e p e r o x i d e t h a t i n c r e a s e s its i n t r a l i g a n d b o n d s t r e n g t h . T h i s i n c r e a s e i n b o n d s t r e n g t h is o b s e r v e d e x p e r i m e n t a l l y b y c o m p a r i n g t h e e n d - o n m o n o m e r t o t h e trans e n d - o n d i m e r , i n w h i c h the O - O v i b r a t i o n a l f r e q u e n c y increases f r o m 8 0 3 to 8 3 2 c m . T h i s i n c r e a s e i n v i b r a t i o n a l f r e q u e n c y is c o n s i s t e n t w i t h t h e trans e n d - o n d i m e r h a v i n g σ - d o n o r i n t e r a c t i o n s w i t h t w o c o p p e r s (72). H o w e v e r , o n g o i n g to the s i d e - o n p e r o x o b r i d g i n g g e o m e t r y , the O - O s t r e t c h i n g f r e q u e n c y d r a m a t i c a l l y d e c r e a s e s i n t h e μ-η :η m o d e l c o m p l e x a n d o x y h e m o c y a n i n , y e t i n t h i s g e o m e t r y t h e p e r o x i d e is t h e s t r o n g e s t σ donor, based on the h i g h charge-transfer intensity associated w i t h four - 1

2

2



2

1 / 2 x

2 2

2.9 (803)

1 (0.105)

K. Karlin

Cu

/ Ο — 0

2

y

a

3.1 ( 8 3 2 )

1.9 ( 0 . 2 5 2 )

K. Karlin

C U ^ N Q ^ C U

/ / ° - ° \ \ \Cu X Cu J

4A + 2H 0

2

2

Number of Centers


Multicopper Oxidases:

Laccase Ascorbate Oxidase Ceruloplasmin

Type 1 (Blue)

Type 2 (Normal)

1 2 2

1 2 1

Total Cu Type 3 (Coupled Binuclear) 4 1 2 8 5-7 1 or 2

Laccase Derivatives: Type 2 Depleted (T2D) 1 Type 1 Hg Sub. (T1 Hg) Hg2+ Figure 15.

1

1 1

3 3

Multicopper oxidases: reactivity and stoichiometry.

this e n z y m e is a c o m p l e x p r o b l e m , a n d t w o d e r i v a t i v e s have s e r v e d to simplify this system. I n t y p e 2 d e p l e t e d ( T 2 D ) laccase, the t y p e 2 c o p p e r is r e v e r s i b l y r e m o v e d l e a v i n g t h e t y p e 1 a n d t y p e 3 c e n t e r s (81). T h e t y p e 1 m e r c u r y s u b s t i t u t e d d e r i v a t i v e ( T l H g ) is f o r m e d b y r e p l a c i n g the type 1 copper w i t h the spectroscopic and redox innocent mercuric i o n (82). T h e goal o f o u r r e s e a r c h o n t h e m u l t i c o p p e r oxidases has b e e n to d e t e r m i n e t h e s p e c t r a l f e a t u r e s o f t h e t y p e 3 ( a n d t y p e 2) c e n t e r s , t o use these spectral features t o define g e o m e t r i c a n d e l e c t r o n i c s t r u c t u r a l differences relative to h e m o c y a n i n a n d tyrosinase, a n d to u n d e r s t a n d h o w these structural differences c o n t r i b u t e to t h e i r v a r i a t i o n i n b i o l o g i c a l function. T h e hemocyanins a n d tyrosinases reversibly b i n d a n d activate d i o x y g e n whereas t h e m u l t i c o p p e r oxidases catalyze its f o u r - e l e c t r o n reduction to water. W e start b y d e f i n i n g t h e s p e c t r a l f e a t u r e s a s s o c i a t e d w i t h e a c h t y p e of c o p p e r i n native a n d T 2 D laccase ( F i g u r e 16). T h e E P R s p e c t r u m o f t h e n a t i v e e n z y m e c o n t a i n s c o n t r i b u t i o n s f r o m t w o d i s t i n c t c u p r i c sites (Figure 16A); one w i t h a large a n d a second w i t h a small parallel h y perfine splitting. These have been assigned to the type 2 a n d type 1 c o p p e r sites, r e s p e c t i v e l y . T h e E P R s p e c t r u m o f t h e T 2 D d e r i v a t i v e c o n t a i n s a s i n g l e c o m p o n e n t , w i t h a s m a l l h y p e r f i n e c o u p l i n g , t h a t is a s s i g n e d t o t h e t y p e 1 c o p p e r c e n t e r (83). T h e t y p e 3 c o p p e r a t o m s a r e E P R nondetectable and, b y analogy to h e m o c y a n i n and tyrosinase, c a n



Figure 16. Spectral features of native and T2D laccase. A: EPR. B: Visible absorption. G X-ray absorption spectra of native, T2D, and T2D laccase following reaction with hydrogen peroxide.


5.



151

b e c o n s i d e r e d to b e a c o u p l e d b i n u c l e a r c o p p e r site. T h e a b s o r p t i o n s p e c t r u m of native a n d T 2 D laccase ( F i g u r e 16B) exhibits an intense thiolate S C u ( I I ) c h a r g e - t r a n s f e r t r a n s i t i o n at 6 0 0 n m , w h i c h is ass i g n e d t o t h e t y p e 1 c o p p e r c e n t e r (4). T h e o n l y s p e c t r a l f e a t u r e t h a t has b e e n a s s o c i a t e d w i t h t h e t y p e 3 c e n t e r is t h e a b s o r p t i o n b a n d c e n t e r e d at 3 3 0 n m (e ~ 3 0 0 0 M " c m " ) . T h i s b a n d d e c r e a s e s i n i n t e n s i t y w i t h r e d u c t i o n o f t h e p r o t e i n b y t w o e l e c t r o n s (at t h e s a m e p o t e n t i a l ) . T h e 3 3 0 - n m s p e c t r a l r e g i o n is e x p e c t e d t o c o n t a i n h i s t i d i n e a n d h y d r o x i d e - t o - t y p e 3 C u ( I I ) c h a r g e - t r a n s f e r t r a n s i t i o n s (84).


1

1

T h e assignment of the 3 3 0 - n m absorption b a n d i n native laccase to the type 3 center, h o w e v e r , was c o m p l i c a t e d b y the absence of a 3 3 0 n m b a n d i n the absorption s p e c t r u m of T 2 D laccase ( w h i c h still contains a t y p e 3 site) ( F i g u r e 1 6 B ) . W e d i s c o v e r e d a k e y r e a c t i o n t h a t c l a r i f i e d the assignment of the 3 3 0 - n m absorption b a n d . A d d i t i o n o f p e r o x i d e to T 2 D l a c c a s e l e a d s t o t h e r e a p p e a r a n c e o f t h e 3 3 0 - n m b a n d (85). T h i s i n d i c a t e s t h a t t h e t y p e 3 site i n T 2 D l a c c a s e w a s r e d u c e d ( e v e n w h e n e x p o s e d to d i o x y g e n ) b u t t h a t t h e s t r o n g e r o x i d a n t , p e r o x i d e , w a s c a p a b l e o f o x i d i z i n g t h e t y p e 3 c e n t e r . T h i s finding w a s c o n f i r m e d u s i n g X - r a y a b s o r p t i o n s t u d i e s at t h e C u K - e d g e ( 9 0 0 0 e V ) (86). T h e T 2 D d e r i v a t i v e e x h i b i t s a p e a k at 8 9 8 4 e V ( F i g u r e 1 6 C ) t h a t is c h a r a c t e r i s t i c o f C u ( I ) i n a t h r e e - c o o r d i n a t e site. T h e m a g n i t u d e o f the 8 9 8 4 - e V b a n d c o u l d b e q u a n t i t a t e d u s i n g a n o r m a l i z e d e d g e m e t h o d that w e h a v e d e v e l o p e d . W e d e t e r m i n e d that the T 2 D derivative c o n t a i n e d a fully r e d u c e d t y p e 3 site. A d d i t i o n of p e r o x i d e e l i m i n a t e d the 8 9 8 4 - e V peak, w h i c h i n d i cates t h a t p e r o x i d e f u l l y o x i d i z e s t h e t y p e 3 c e n t e r t o f o r m a m e t T 3 site. H a v i n g d e f i n e d t h e T 2 D d e r i v a t i v e , w e c o u l d s t u d y t h e t y p e 3 site i n t h e a b s e n c e o f t h e t y p e 2 c o p p e r a n d c o m p a r e it t o t h e c o u p l e d b i n u c l e a r site i n h e m o c y a n i n a n d t y r o s i n a s e ( F i g u r e s 1 7 a n d 1 8 ) . F i r s t , as d e m o n s t r a t e d f r o m t h e X - r a y e d g e s i n F i g u r e 1 6 C , t h e f u l l y r e d u c e d t y p e 3 site is s t r i k i n g l y d i f f e r e n t f r o m t h a t o f h e m o c y a n i n a n d t y r o s i n a s e i n a s m u c h as i t d o e s n o t r e a c t w i t h d i o x y g e n (85, 86). P e r o x i d e d o e s o x i d i z e the site, a n d w e can further c o m p a r e this met t y p e 3 center i n l a c c a s e t o m e t h e m o c y a n i n . A s w i t h h e m o c y a n i n , t h e m e t t y p e 3 site i n t h e m u l t i c o p p e r o x i d a s e s is s t r o n g l y a n t i f e r r o m a g n e t i c a l l y c o u p l e d (49, 50, 75). T h i s i n d i c a t e s t h e p r e s e n c e o f a n e n d o g e n o u s h y d r o x i d e b r i d g e , w h i c h has b e e n c o n f i r m e d b y X - r a y c r y s t a l l o g r a p h y (87). One-electron reduction of met derivatives of hemocyanin and T 2 D l a c c a s e p r o d u c e s t h e m i x e d - v a l e n t h a l f - m e t sites t h a t e x h i b i t d r a m a t i c d i f f e r e n c e s ( F i g u r e 18). I n p a r t i c u l a r , h a l f - m e t h e m o c y a n i n has v e r y u n u s u a l c o o r d i n a t i o n c h e m i s t r y w i t h respect to exogenous l i g a n d b i n d i n g . F o r e x a m p l e , a z i d e b i n d s to t h i s h a l f - m e t a c t i v e site w i t h a n e q u i l i b r i u m b i n d i n g c o n s t a n t t h a t is m o r e t h a n t w o o r d e r s o f m a g n i t u d e greater t h a n that o f a z i d e b i n d i n g to aqueous C u ( I I ) , a n d this b i n d i n g



2

1

[Cu(ll)Cu(ll)]0|-

T2D:[Cu(l)Cu(l)] + 0 2

H

o

Cu(ll)

Cu(ll) o

H

Cu(ll)

1

> No Reaction

Figure 17. Comparison of the reactivity and magnetism of deoxy and met hemocyanin and the laccase type 3 copper site in the T2D derivative.

Cu(ll)

2

2

> [Cu(ll)Cu(ll)] T2D:[Cu(l)Cu(l)] + H 0 > [Cu(ll)Cu(ll)] met met Met-[Cu(ll) Cu(ll)] -2J > 500 cm" -2J > 500 cm"

2

[Cu(l)Cu(l)] + H 0

2

Type 3 Copper

Coupled Binuclear

[Cu(l)Cu(l)] + 0

Laccase

Hemocyanin

Deoxy-[Cu(l) Cu(l)]



Ο Η

3X0 .

3000

1«

\1 w

/

-> OxyHc

Cu(l)

3250

\

.(X4)

m

. ι %

t

y ~

Field (gauss)

Deoxy He + O2

Cu(ll)

2750

1

I

*/

*

0x

r

L

Ο Η

exo

3000

Cu(l)

3250

3500

> No Reaction

Field (gauss)

Cu(ll)

2750

Laccase - Type 3

T2D:[T1 T3 ed] + O2

3500 2500

Β

Figure 18. Comparison of half-met hemocyanin with the half-met type 3 (in T2D) laccase copper sites. A: EPR spectra and binding constants of exogenous azide binding. B: Spectroscopically effective structural models for exogenous ligand binding to the half-met derivatives and their relation to differences in dioxygen reactivity.

2500

4

K>10 M~V

aquo ^ /

1

Hemocyanin - Coupled Binuclear

Half-Met [Cu(ll) Cu(l)]


154


r e s u l t s i n q u i t e u n u s u a l m i x e d - v a l e n t s p e c t r a l f e a t u r e s (88). W e h a v e studied this u n u s u a l half-met h e m o c y a n i n c h e m i s t r y a n d spectroscopy i n s o m e d e t a i l (88) a n d d e t e r m i n e d t h a t t h e s e d e r i v e f r o m t h e fact t h a t exogenous ligands b r i d g e b e t w e e n the Cu(II) and Cu(I) of this m i x e d v a l e n t s i t e ( F i g u r e 1 8 A , b o t t o m ) . A l t e r n a t i v e l y , t h e h a l f - m e t t y p e 3 site i n T 2 D laccase exhibits n o r m a l Cu(II) E P R spectra for a l l exogenous l i g a n d - b o u n d f o r m s a n d has a n e q u i l i b r i u m b i n d i n g c o n s t a n t c o n s i s t e n t w i t h aqueous Cu(II) c h e m i s t r y , i n d i c a t i n g that the exogenous ligands b i n d t e r m i n a l l y to t h e C u ( I I ) o f t h e h a l f - m e t t y p e 3 site (84). T h i s d i f f e r e n c e i n exogenous l i g a n d b i n d i n g m o d e s ( b r i d g i n g vs. t e r m i n a l ) d i rectly correlates w i t h differences i n 0 reactivity of these b i n u c l e a r c o p p e r sites as d e s c r i b e d a b o v e i n t h a t o n l y t h e d e o x y h e m o c y a n i n s i t e reversibly binds dioxygen (Figure 18A).


2

T h e c o m b i n a t i o n of the type 3 w i t h the type 2 center does, of course, r e a c t w i t h d i o x y g e n i n t h e n a t i v e e n z y m e . T h i s r e a c t i o n l e d us to c o n s i d e r exogenous ligand interactions w i t h b o t h the type 3 and type 2 coppers i n n a t i v e laccase. A n a p p r o p r i a t e s p e c t r a l m e t h o d to s t u d y t h e i n t e r a c t i o n o f e x o g e n o u s l i g a n d s w i t h e a c h c e n t e r is l o w - t e m p e r a t u r e M C D s p e c troscopy, w h i c h allows c o r r e l a t i o n of excited-state features w i t h g r o u n d state p r o p e r t i e s (89-91). I n p a r t i c u l a r , t h e p a r a m a g n e t i c t y p e 2 c o p p e r exhibits v e r y different l o w - t e m p e r a t u r e M C D features relative to the antiferromagnetically c o u p l e d t y p e 3 center ( F i g u r e 19). F o r the t y p e 2 c e n t e r , b o t h t h e g r o u n d a n d e x c i t e d states h a v e S = fa a n d s p l i t i n a m a g n e t i c field. T h e s e l e c t i o n r u l e s f o r M C D s p e c t r o s c o p y p r e d i c t t h a t t h e r e s h o u l d b e t w o t r a n s i t i o n s t o a g i v e n e x c i t e d state t h a t a r e o f e q u a l magnitude but of opposite sign. A s the Z e e m a n splitting w i l l be on the order of 10 c m " a n d absorption bands are o n the o r d e r o f a few t h o u s a n d l

1

c m " broad, the positive and negative bands w i l l mostly cancel and p r o d u c e a b r o a d , w e a k , d e r i v a t i v e - s h a p e d M C D s i g n a l k n o w n as a n A - t e r m . T h i s is o b s e r v e d i f b o t h c o m p o n e n t s o f t h e g r o u n d state a r e e q u a l l y p o p u l a t e d . H o w e v e r , as o n e l o w e r s t h e t e m p e r a t u r e t h e B o l t z m a n n p o p u l a t i o n o f t h e h i g h e r - e n e r g y c o m p o n e n t is r e d u c e d , c a n c e l l a t i o n n o l o n g e r occurs, a n d one observes intense, l o w - t e m p e r a t u r e M C D signals k n o w n as C - t e r m s . T h e s e c a n b e t w o t o t h r e e o r d e r s o f m a g n i t u d e m o r e intense t h a n the h i g h - t e m p e r a t u r e M C D signals. 1

F o r the t y p e 3 c e n t e r , the a n t i f e r r o m a g n e t i c c o u p l i n g leads to an S = 0 g r o u n d state t h a t c a n n o t s p l i t i n a m a g n e t i c field. T h u s , t h i s s i t e does not exhibit C - t e r m intensity a n d the l o w - t e m p e r a t u r e M C D spect r u m o f n a t i v e l a c c a s e w i l l b e d o m i n a t e d b y t h e i n t e n s e C - t e r m s assoc i a t e d w i t h p a r a m a g n e t i c c o p p e r c e n t e r s (89, 90). L o w - t e m p e r a t u r e M C D s p e c t r o s c o p y w a s u s e d t o p r o b e t h e effects o f b i n d i n g t h e e x o g e n o u s l i g a n d a z i d e to n a t i v e laccase (89, 90). T i t r a t i o n of the native enzyme w i t h azide produces two N ~ Cu(II) charget r a n s f e r t r a n s i t i o n s : o n e at 5 0 0 n m a n d a s e c o n d m o r e i n t e n s e b a n d at 3


5.


S O L O M O N ET AL.

B

T 2 ( S = 1/2)

155

T 3 ( S = 0 , 1)

+ 1/2 Ψ ( δ = 1/2)

*e(S«i)

θ

Φ


=

RCP

RCP φ

Am = - 1

=

Δτη«-1

(S = 1)

V S = 1/2)

Vg(S

= 0) -2J > 500 cm"

H

1

H Low-Temperature C-Term

sî CL

I High-Temperature ATerm

•hv

û- I ° 1

hv

Figure 19. Model for the temperature dependence of the MCD bands of native laccase. A: Transitions and band profiles associated with type 2 copper. B: Transitions and band profiles associated with type 3 copper. Note the difference in temperature dependence of the MCD signal as described in the text. 4 0 0 n m ( F i g u r e 2 0 A ) . T h e i n t e n s i t y o f t h e 4 0 0 - n m b a n d as a f u n c t i o n o f a z i d e c o n c e n t r a t i o n is p l o t t e d as a d a s h e d l i n e i n F i g u r e 2 0 C . O n e can use l o w - t e m p e r a t u r e M C D to correlate these excited-state features t o s p e c i f i c c o p p e r c e n t e r s . T h e 5 0 0 - n m a b s o r p t i o n b a n d has a n e g a t i v e l o w - t e m p e r a t u r e M C D s i g n a l a s s o c i a t e d w i t h i t at 4 8 5 n m t h a t i n c r e a s e s in magnitude with increasing azide concentration (Figure 20B). T h e



3

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_

_

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\\.«

—

nm 385

/ /

\

/ ι / / \/

' .

I

I

2

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1

1

u 1t / -

485 nm

L

[azide]/[protein] ω Ν Τδ Ε λ_ ο

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3

Abs.

- Titration

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Τ"

Τ2

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LTMCD (385 nm) Uncoupled-|J Τ3 (

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2 M ο χ

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157

i n t e n s i t y o f t h i s M C D f e a t u r e is p l o t t e d as a s o l i d l i n e i n F i g u r e 2 0 C . T h e 5 0 0 - n m a b s o r p t i o n b a n d has a c o r r e s p o n d i n g l o w - t e m p e r a t u r e M C D s i g n a l ; t h u s i t m u s t b e a s s o c i a t e d w i t h a z i d e b i n d i n g to t h e p a r a m a g n e t i c t y p e 2 c e n t e r . T h e r e is also a n M C D s i g n a l i n t h e r e g i o n o f t h e 4 0 0 - n m a b s o r p t i o n b a n d ; h o w e v e r , i t d o e s n o t e x h i b i t t h e s a m e b e h a v i o r as t h e a b s o r p t i o n i n t e n s i t y ( F i g u r e 2 0 B ) . T h e 3 8 5 - n m M C D s i g n a l first i n creases a n d t h e n d e c r e a s e s i n i n t e n s i t y w i t h i n c r e a s i n g a z i d e c o n c e n t r a t i o n . Its m a g n i t u d e is p l o t t e d as t h e d o t - d a s h l i n e i n F i g u r e 2 0 C . A l t h o u g h the l o w - t e m p e r a t u r e M C D signal does not correlate w i t h the 4 0 0 - n m a b s o r p t i o n b a n d , i t d o e s c o r r e l a t e w i t h a n u n u s u a l g = 1.86 signal i n the E P R s p e c t r u m ( F i g u r e 2 0 D ) , w h i c h w e h a v e s h o w n to b e a s s o c i a t e d w i t h < 1 0 % o f t h e t y p e 3 sites t h a t b e c o m e p r o t o n a t i v e l y u n c o u p l e d (and h e n c e paramagnetic) u p o n b i n d i n g azide. T h u s , the i n t e n s e 4 0 0 - n m a b s o r p t i o n b a n d has n o l o w - t e m p e r a t u r e M C D s i g n a l ass o c i a t e d w i t h i t , a n d it m u s t c o r r e s p o n d t o a z i d e b o u n d t o t h e M C D silent c o u p l e d t y p e 3 center. T h e l o w - t e m p e r a t u r e M C D a n d absorption titration studies ( F i g u r e 10) h a v e d e t e r m i n e d t h a t a z i d e b i n d s t o b o t h t h e t y p e 2 a n d t y p e 3 c e n t e r s w i t h s i m i l a r b i n d i n g c o n s t a n t s . A series o f c h e m i c a l p e r t u r b a t i o n s a n d s t o i c h i o m e t r y s t u d i e s h a v e s h o w n t h a t t h e s e effects a r e a s s o c i a t e d w i t h the same azide. T h i s demonstrates that one N ~ b r i d g e s b e t w e e n the t y p e 2 a n d type 3 centers i n laccase. T h e s e a n d other results f r o m M C D s p e c t r o s c o p y first d e f i n e d t h e p r e s e n c e o f a t r i n u c l e a r c o p p e r c l u s t e r a c t i v e site i n b i o l o g y (89). A t h i g h e r a z i d e c o n c e n t r a t i o n , a s e c o n d a z i d e b i n d s to t h e t r i n u c l e a r site i n l a c c a s e . M e s s e r s c h m i d t et a l . h a v e d e t e r m i n e d f r o m X - r a y crystallography that a t r i n u c l e a r c o p p e r cluster site is also p r e s e n t i n a s c o r b a t e o x i d a s e (87, 92) a n d h a v e o b t a i n e d a 3

c r y s t a l s t r u c t u r e f o r a t w o - a z i d e - b o u n d d e r i v a t i v e (87). It a p p e a r s t h a t s o m e d i f f e r e n c e s exist b e t w e e n t h e t w o - a z i d e - b o u n d l a c c a s e a n d asc o r b a t e o x i d a s e d e r i v a t i v e s , a n d it w i l l b e i m p o r t a n t to s p e c t r o s c o p i c a l l y c o r r e l a t e b e t w e e n t h e s e sites. H a v i n g d e m o n s t r a t e d t h a t t h e t y p e 3 c e n t e r m u s t b e v i e w e d as p a r t of a t r i n u c l e a r c o p p e r cluster, i n c l u d i n g the t y p e 2 center, it was i m portant to d e t e r m i n e w h i c h c o p p e r s are r e q u i r e d for the r e a c t i v i t y o f the m u l t i c o p p e r oxidases w i t h d i o x y g e n . W e h a d already d e m o n s t r a t e d using X - r a y absorption edges (Figure 1 6 C ) that a r e d u c e d type 3 center i n the presence of an o x i d i z e d type 1 center does not react w i t h 0 (85, 2

86). W e n e x t l o o k e d at t h e r e a c t i v i t y o f t h e f u l l y r e d u c e d T 2 D [ T l i T 3 d ] d e r i v a t i v e w i t h 0 . T h i s h a d b e e n g e n e r a l l y v i e w e d as t h e c o m b i n a t i o n of c o p p e r centers i n laccase r e q u i r e d for d i o x y g e n r e a c t i v i t y i n t h e m e c h a n i s t i c p r o p o s a l s i n t h e l i t e r a t u r e (93, 94). F r o m F i g u r e 2 1 A it is c l e a r t h a t t h e 8 9 8 4 - e V - r e d u c e d C u K - e d g e p e a k d o e s n o t c h a n g e o n exposure o f fully r e d u c e d T 2 D laccase to 0 . T h i s indicates that the t y p e 2 c e n t e r is r e q u i r e d f o r d i o x y g e n r e a c t i v i t y . T h u s , t h e C u K - e d g e r e c

re

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Figure 21. Laccase copper centers required for dioxygen reactivity. A: XAS of fully reduced T2D laccase and fully reduced T2D laccase following exposure to dioxygen. B: XAS of reduced TlHg laccase and reduced TlHg laccase following exposure to dioxygen. C: Summary of the reactivity of deoxy T2D, fully reduced T2D, and reduced TlHg laccase with oxygen.

2+

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159

spectra of the T l H g derivative, w h i c h contains a valid type 2/type 3 trinuclear c o p p e r cluster, was investigated. F r o m F i g u r e 2 I B , the fully r e d u c e d t r i n u c l e a r c o p p e r c l u s t e r site r a p i d l y r e a c t s w i t h 0 e l i m i n a t i n g t h e 8 9 8 4 - e V p e a k . T h u s , t h e t r i n u c l e a r c o p p e r c l u s t e r is t h e m i n i m u m s t r u c t u r a l u n i t r e q u i r e d f o r 0 r e d u c t i o n (95). 2

2

B e c a u s e t h e m e r c u r i c i o n i n T l H g l a c c a s e is r e d o x i n a c t i v e , t h i s d e r i v a t i v e has o n e less e l e c t r o n e q u i v a l e n t a v a i l a b l e f o r 0 reduction t h a n n a t i v e l a c c a s e . T h i s p r o p e r t y e n a b l e d us t o s t a b i l i z e a n o x y g e n intermediate i n T l H g laccase. A c o m b i n a t i o n of l o w - t e m p e r a t u r e M C D a n d X A S has d e m o n s t r a t e d t h a t t w o c o p p e r s o f t h e t r i n u c l e a r c l u s t e r a r e o x i d i z e d i n t h i s i n t e r m e d i a t e (41). T h u s , t w o e l e c t r o n s h a v e b e e n transferred to d i o x y g e n a n d this species c o r r e s p o n d s to a p e r o x i d e l e v e l i n t e r m e d i a t e t h a t c a n b e c o m p a r e d t o t h e p e r o x o - b i n u c l e a r c u p r i c sites i n o x y h e m o c y a n i n a n d o x y t y r o s i n a s e . A s is c l e a r f r o m F i g u r e 2 2 A , t h e p e r o x i d e i n t e r m e d i a t e i n l a c c a s e has a s t r i k i n g l y d i f f e r e n t c h a r g e - t r a n s f e r s p e c t r u m from that of o x y h e m o c y a n i n a n d oxytyrosinase. T h i s requires a different geometric a n d electronic structure for this p e r o x y - t r i n u c l e a r c o p p e r c l u s t e r site (41). D e t a i l e d s p e c t r a l s t u d i e s o n t h i s i n t e r m e d i a t e are presently u n d e r w a y (Shin, W . ; C o l e , J . L . ; R o o t , D . E . ; S o l o m o n , Ε. I . , u n p u b l i s h e d r e s u l t s ) . H o w e v e r , at t h i s p o i n t o u r d a t a i n d i c a t e t h a t it c o r r e s p o n d s t o a h y d r o p e r o x i d e b o u n d e n d - o n t o o n e o f t h e c o p p e r s o f a n o x i d i z e d t y p e 3 c e n t e r a n d also l i k e l y b r i d g i n g t o a r e d u c e d t y p e 2 c o p p e r c e n t e r ( F i g u r e 2 2 B ) . M e s s e r s c h m i d t et a l . h a v e s i n c e o b t a i n e d a crystal structure of a low-affinity p e r o x i d e - b o u n d adduct of ascorbate o x i d a s e t h a t is also d e s c r i b e d as h a v i n g h y d r o p e r o x i d e e n d - o n b o u n d t o o n e o f t h e t y p e 3 c o p p e r s . ( T h i s p e r o x i d e a d d u c t has t h e t h r e e c o p p e r s of the trinuclear cluster u n b r i d g e d and therefore u n c o u p l e d i n contrast to the o x y g e n i n t e r m e d i a t e o f T l H g laccase.) T h e o x y g e n i n t e r m e d i a t e of T l H g laccase indicates the m e c h a n i s t i c r e l e v a n c e of a e n d - o n hydroperoxide-bound form of the protein. This difference i n perox i d e b i n d i n g r e l a t i v e to h e m o c y a n i n a n d t y r o s i n a s e a p p e a r s t o p l a y a k e y r o l e i n s t a b i l i z i n g t h e p e r o x i d e i n t e r m e d i a t e a n d p r o m o t i n g its i r r e v e r s i b l e f u r t h e r r e d u c t i o n t o w a t e r at t h e t r i n u c l e a r c o p p e r c l u s ter site.


2

Summary A t this point the u n i q u e spectral features associated w i t h the major classes o f a c t i v e sites i n c o p p e r p r o t e i n s a r e r e a s o n a b l y w e l l u n d e r s t o o d a n d d e f i n e a c t i v e site e l e c t r o n i c s t r u c t u r e s t h a t 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 t h e i r r e a c t i v i t i e s i n b i o l o g y . F o r t h e b l u e c o p p e r sites, w e d e t e r m i n e d that the u n i q u e spectral features d e r i v e f r o m a ground-state w a v e f u n c t i o n t h a t has a h i g h a n i s o t r o p i c c o v a l e n c y i n v o l v i n g t h e t h i o l a t e l i g a n d . T h i s c o v a l e n c y p r o v i d e s a v e r y efficient superexchange p a t h w a y


160


Charge Transfer Spectra

4000

«

π« 1 1 peroxy intermediate laccase


oxyhemocyanin and oxytyrosinase

300

Β

400

500 600 700 Wavelength (nm)

800

Structural M o d e l s for O x y - l n t e r m e d i a t e s

Coupled Binuclear Copper

Trinuclear Copper Cluster T2

Cu \

1 +

/

HOO''

/

\ Oxyhemocyanin Oxytyrosinase

T3

\

/

4

N

Cu + 2

\

ο Η

/

4

Cu

2 +

\

Peroxy Intermediate Laccase

Figure 22. Comparison of oxygen intermediates. A: Electronic absorption spectra of the peroxy-intermediate in laccase versus oxyhemocyanin and oxytyrosinase. B: Proposed structural differences between peroxide binding in oxyhemocyanin and oxytyrosinase relative to the end-on bound hydro peroxide intermediate at the trinuclear copper cluster in laccase.

for long-range e l e c t r o n transfer. F o r the c o u p l e d b i n u c l e a r c o p p e r active sites, w e h a v e s e e n t h a t t h e u n i q u e s p e c t r a l f e a t u r e s o f t h e o x y site c o r r e s p o n d to a n e w b r i d g i n g p e r o x i d e e l e c t r o n i c structure that has v e r y s t r o n g σ - d o n o r a n d 7r-acceptor p r o p e r t i e s . T h e s e p r o p e r t i e s a p p e a r to m a k e significant contributions t o the reversible b i n d i n g a n d activation o f d i o x y g e n b y t h e s e a c t i v e sites. I n t h e m u l t i c o p p e r o x i d a s e s , o u r s p e c -


5.

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161

tral studies determined that the type 3 center is fundamentally different from the coupled binuclear copper site in hemocyanin and tyrosinase, that it is part of a trinuclear copper cluster, and that this trinuclear copper cluster is the structure required for 0 reduction. We have now characterized a peroxide level intermediate at this trinuclear copper cluster site that is strikingly different from the peroxide bound in oxy hemocyanin and oxytyrosinase in that it is bound end-on as hydroper oxide. Our spectral studies presently underway should provide important insight into the geometric and electronic structure differences that are indicated by these spectral differences and their contribution to differ ences in biological function. Downloaded by PURDUE UNIV on April 9, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/ba-1995-0246.ch005

2

Acknowledgments This research was supported by the National Science Foundation (CHE9217628) for the blue copper studies and by the National Institutes of Health (DK-31450) for the coupled binuclear and multicopper oxidase studies. Edward I. Solomon expresses his sincere appreciation to all his students and collaborators who are listed as co-authors in the references for their commitment and contributions to this science.

References 1. Solomon, E.I.;Penfield, K. W.; Wilcox, D. E. Struct. Bonding 1983, 53, 1-57. 2. Solomon, Ε. I.; Baldwin, M. J.; Lowery, M. D. Chem. Rev. 1992, 92, 521542.

3. Solomon, E. I.; Lowery, M. D. Science (Washington, D.C.) 1993, 259, 15751581.

4. Solomon, Ε. I.; Lowery, M. D. In The Chemistry of Copper and Zinc Triads; Welch, A. J.; Chapman, S. K., Eds.; The Royal Society of Chemistry: Cam bridge, England, 1993; pp 12-29. 5. Solomon, Ε. I.; Hemming, B. L.; Root, D. E. In Bioinorganic Chemistry of Copper; Karlin K. D.; Tyeklár, Z., Eds.; Chapman & Hall: New York, 1992; pp 3-20. 6. Solomon, Ε. I. Comments Inorg. Chem. 1984, 3, 225-320. 7. Ballhausen, C. J. Introduction to Ligand Field Theory; McGraw-Hill: New York, 1962. 8. McClure, D. S. Electronic Spectra of Molecules and Ions in Crystals; Aca demic: New York, 1959. 9. Griffith, J. S. The Theory of Transition Metal Ions; Cambridge University Press: London, 1964. 10. Sugano, S.; Tanabe, Y.; Kamimura, H. Multiplets of Transition Metal Ions in Crystals; Academic: New York, 1970. 11. Figgis, Β. N. Introduction to Ligand Fields; Interscience: New York, 1967. 12. Hitchman, Μ. Α.; Cassidy, P. J. Inorg. Chem. 1979, 18, 1745-1754. 13. Johnson, Κ. H. Adv. Quantum Chem. 1973, 7, 143-185. 14. Johnson, Κ. H.; Norman, J. G., Jr.; Connolly, J. W. D. In Computational Methods


162

15. 16. 17. 18. 19.


20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

MECHANISTIC BIOINORGANIC CHEMISTRY

for Large Molecules and Localized States in Solids; Herman, F.; McLea A. D.; Nesbet, R. K., Eds.; Plenum: New York, 1973; pp 161-201. Connolly, J. W. D. In Semiempirical Methods of Electronic Structure Cal culations, Part A: Techniques; Segal, G. Α., Ed.; Plenum: New York, 1977. Rosch, Ν. In Electrons in Finite and Infinite Structures; Phariseu,P.;Scheire, L., Eds.; Wiley: New York, 1977. Slater, J. C. The Calculation of Molecular Orbitals; John Wiley & Sons: New York, 1979;p104. Gewirth, Α. Α.; Cohen, S. L.; Schugar, H. J.; Solomon, Ε. I. Inorg. Chem. 1987, 26, 1133-1146. Solomon, Ε. I.; Gewirth, Α. Α.; Cohen, S. L. In Understanding Molecular Properties; Avery, J.; Dahl, J. P.; Hansen, A. E., Eds.; D. Reidel: Dordrecht, Netherlands, 1987; pp 27-68. Didziulis, S. V.; Cohen, S. L.; Gewirth, Α. Α.; Solomon, Ε. I. J. Am. Chem. Soc. 1988, 110, 250-268. Bencini, Α.; Gatteschi, D. J. Am. Chem. Soc. 1983, 105, 5535-5541. Chow, C.; Chang, K.; Willett, R. D. J. Chem. Phys. 1973, 59, 2629-2640. Solomon, Ε. I.; Lowery, M. D.; LaCroix, L. B.; Root, D. E. In Methods in Enzymology; Riordan, J. F.; Vallee, B. L., Eds.; 1993; Vol. 226, Part C; pp 1-33. Desjardins, S. R.; Penfield, Κ. W.; Cohen, S. L.; Musselman, R. L.; Solomon, E. I. J. Am. Chem. Soc. 1983, 105, 4590-4603. Mulliken, R. S.; Rieke, C. Α.; Orloff, D.; Orloff, H. J. Chem. Phys. 1949, 17, 1248-1267. Solomon, E. I.; Hare, J. W.; Gray, H. B. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 1389-1392. Guss, J. M.; Freeman, H. C. J. Mol. Biol. 1983, 169, 521-563. Lowery, M. D.; Guckert, J. Α.; Gebhard, M. S.; Solomon, Ε. I. J. Am. Chem. Soc. 1993, 115, 3012-3013. Penfield, K. W.; Gay, R. R.; Himmelwright, R. S.; Eickman, N. C.; Norris, V. Α.; Freeman, H. C.; Solomon, Ε. I. J. Am. Chem. Soc. 1981,103,43824388. Bates, C. Α.; Moore, W. S.; Standley, K. J.; Stevens, Κ. W. H. Proc. Phys. Soc. 1962, 79, 73. Sharnoff, M. J. Chem. Phys. 1965, 42, 3383-3395. Roberts, J. E.; Brown, T. G.; Hoffman, B. M.; Peisach, J. J. Am. Chem. Soc. 1980, 102, 825-829. Gewirth, Α. Α.; Solomon, E. I. J. Am. Chem. Soc. 1988, 110, 3811-3819. Shadle, S. E.; Penner-Hahn, J. E.; Schugar, H. J.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 1993, 115, 767-776. Scott, R. Α.; Hahn, J. E.; Doniach, S.; Freeman, H. C.; Hodgson, K. O. J. Am. Chem. Soc. 1982, 104, 5364-5369. Penfield, Κ. W.; Gewirth, Α. Α.; Solomon, Ε. I. J. Am. Chem. Soc. 1985, 107, 4519-4529. Norman, J. G. J. Mol. Phys. 1976, 31, 1191-1198. George, S. J.; Lowery, M. D.; Solomon, Ε. I.; Cramer, S. P. J. Am. Chem. Soc. 1993, 115, 2968-2969. Hughey, J. L., IV; Fawcett, T. G.; Rudich, S. M.; Lalancette, R. Α.; Potenza, J. Α.; Schugar, H. J. J. Am. Chem. Soc. 1979, 101, 2617-2623. Messerschmidt, Α.; Ladenstein, R.; Huber, R.; Bolognesi, M.; Avigliano, L.; Petruzzelli, R.; Rossi, Α.; Finazzi-Agro, A. J. Mol. Biol. 1992, 224, 179-205.


5. 41.

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Cole, J. L.; Ballou, D. P.; Solomon, Ε. I. J. Am. Chem. Soc. 1991, 113,

8544-

8546.

42. Jolly, R. L., Jr.; Evans, L. H.; Makino, N.; Mason, H. S.J.Biol. Chem. 1974, 249, 335.

43. Himmelwright, R. S.; Eickman, N. C.; LuBien, C. D.; Lerch, K.; Solomon, Ε. I.J.Am. Chem. Soc. 1980, 102, 7339-7344.

44. Woolery, G. L.; Powers, L.; Winkler, M.; Solomon, Ε. I.; Spiro, T. G.J.Am. Chem. Soc. 1984,106, 86-92. 45. Woolery, G. L.; Powers, L.; Winkler, M.; Solomon, Ε.I.;Lerch, K.; Spiro, T. G. Biochim. Biophys. Acta 1984, 788, 155-161. 46. Freedman, T. B.; Loehr, J. S.; Loehr, T. M. J. Am. Chem. Soc. 1976, 98, 2809-2815.

Larrabee, J. Α.; Spiro, T. G. J. Am. Chem. Soc. 1980, 102, 4217-4223. 48. Eickman, N. C.; Solomon, Ε. I.; Larrabee, J. Α.; Spiro, T. G.; Lerch, K. Downloaded by PURDUE UNIV on April 9, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/ba-1995-0246.ch005

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for review July 19, 1993. ACCEPTED revised manuscript May 10, 1994.


Electronic Structures of Active Sites in Copper Proteins and Their

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