Structure and Electron Transfer Reactions of Blue Copper Proteins

8 Structure and Electron Transfer Reactions of

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Blue Copper Proteins H A R R Y B. GRAY, C A T H E R I N E L . C O Y L E , D A V I D M . D O O L E Y , P A U L A J. G R U N T H A N E R , J E F F R E Y W . H A R E , R O B E R T A. H O L W E R D A , JAMES V. M c A R D L E , D A V I D R. M c M I L L I N , JILL R A W L I N G S , R O B E R T C. R O S E N B E R G , N. SAILASUTÁ, E D W A R D I. S O L O M O N , P. J. S T E P H E N S , SCOT W H E R L A N D , and JAMES A. W U R Z B A C H

Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, CA 91125 Complete assignments of the electronic spectra of stellacyanin, plastocyanin, and azurin have been made. Bands attributable to d-d transitions have been located in the near-infrared region for the first time, and their positions are consistent with a distorted tetrahedral geometry for the blue copper center. The kinetics of the electron transfer reactions of stellacyanin, azurin, and plastocyanin with Fe(EDTA) and Co(phen) have been studied. Kinetic parameters indicate that reduction of azurin and plastocyanin by Fe(EDTA) occurs by long distance transfer to a buried blue copper center. However, the pathway for oxidation involves substantial protein rearrangement, thereby allowing contact of Co(phen) with the copper ligands. In contrast, the blue copper center of stellacyanin is equally accessible in solution to redox agents. 2-

3+ 3

2-

3+ 3

H P h e r e has b e e n m u c h recent interest i n t h e m e c h a n i s m of e l e c t r o n transfer i n v o l v i n g b l u e c o p p e r p r o t e i n s a n d n o n p h y s i o l o g i c a l as w e l l as p h y s i o l o g i c a l r e d o x agents ( 1 ).

Q u e s t i o n s s u c h as h o w f a r a p a r t t h e

t w o r e d o x centers are at the instant of e l e c t r o n transfer, w h e t h e r t h e r e are m u l t i p l e p a t h w a y s , w h e t h e r t h e r e is a h i g h degree of specificity, a n d , i f so, w h a t is its o r i g i n , are b e i n g a s k e d . E x p e r i m e n t s i n o u r l a b o r a t o r y a n d elsewhere have p r o v i d e d enough information on the spectroscopic p r o p erties a n d the k i n e t i c s of e l e c t r o n transfer reactions of b l u e c o p p e r p r o teins to m a k e serious s t r u c t u r a l a n d m e c h a n i s t i c discussions p o s s i b l e . T h e b l u e c o p p e r p r o t e i n s w h i c h w e discuss h e r e are b e a n p l a s t o c y a n i n (Phaseolus vulgaris; m o l w t 10,700 (2);

Ε 350 m V ( 3 ) ), a z u r i n (Pseudo-

145

Raymond; Bioinorganic Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1977.

146

BIOINORGANIC

CHEMISTRY

II

monas aeruginosa;

m o l w t 13,900 (4); Ε 330 m V (5) ) , a n d s t e l l a c y a n i n

(Rhus vernicifera;

m o l w t 20,000 (6); Ε 184 m V (7) ). A l l these proteins

are t h o u g h t to h a v e e l e c t r o n transfer f u n c t i o n s , a l t h o u g h a p h y s i o l o g i c a l p a r t n e r is n o t k n o w n f o r s t e l l a c y a n i n . P l a s t o c y a n i n accepts a n e l e c t r o n f r o m c y t o c h r o m e / i n t h e p h o t o s y n t h e t i c c h a i n of green leaves, a n d a z u r i n is b e l i e v e d to b e t h e p h y s i o l o g i c a l o x i d a n t f o r f e r r o c y t o c h r o m e

c 551 i n

Pseudomonas aeruginosa a n d r e l a t e d b a c t e r i a . T h e b l u e c o p p e r proteins


as a class a r e p a r t i c u l a r l y a t t r a c t i v e f o r systematic k i n e t i c studies, as b a s i c a l l y t h e same c h r o m o p h o r e potentials.

spans a f a i r l y large r a n g e of r e d u c t i o n

T h e p o t e n t i a l that is a d o p t e d a p p a r e n t l y d e p e n d s

on

finer

details of t h e b l u e c o p p e r e n v i r o n m e n t , a l l o w i n g a n o p p o r t u n i t y f o r correlations of e l e c t r o n transfer r e a c t i v i t y w i t h s u c h s t r u c t u r a l differences. Structural

Considerations

X - r a y c r y s t a l structure analysis has n o t b e e n c o m p l e t e d to date f o r a n y b l u e ( o r t y p e 1) c o p p e r p r o t e i n . T h e p r o b a b i l i t y that t h e c o p p e r c o o r d i n a t i o n e n v i r o n m e n t is h i g h l y u n u s u a l , h o w e v e r ,

has l o n g

been

r e c o g n i z e d , as a result of v a r i o u s spectroscopic studies (8, 9). A t y p i c a l b l u e c o p p e r p r o t e i n is c h a r a c t e r i z e d b y a n intense e l e c t r o n i c a b s o r p t i o n b a n d system, w h i c h peaks at a b o u t 600 n m (c 3* 2 χ 10 ), as w e l l as b y 3

a n e x t r e m e l y s m a l l Α μ E P R s p e c t r a l p a r a m e t e r . N e i t h e r of these s p e c t r a l p r o p e r t i e s has b e e n Cu(II)

complexes.

d u p l i c a t e d satisfactorily i n l o w m o l e c u l a r A s square p l a n a r C u ( I I )

weight

centers, i n p a r t i c u l a r , ex

h i b i t o p t i c a l a n d E P R s p e c t r a that are m u c h different f r o m those o b s e r v e d for b l u e proteins, most m o d e l s h a v e f e a t u r e d geometries b a s e d o n t e t r a h e d r a l or five c o o r d i n a t i o n .

T w o different explanations of t h e intense

600-nm a b s o r p t i o n h a v e b e e n p r o p o s e d . f r o m one o r m o r e

a l l o w e d d-d

O n e treats t h e b a n d as a r i s i n g

transitions i n a

non-centrosymmetric

center, a n d t h e other attributes t h e s t r o n g a b s o r p t i o n to a charge transfer process, p r o b a b l y of t h e l i g a n d - t o - m e t a l ( L M C T ) t y p e (10, 11). Spectroscopic

studies of C o ( I I )

d e r i v a t i v e s of s t e l l a c y a n i n , p l a s t o

c y a n i n , a n d a z u r i n h a v e e s t a b l i s h e d that t h e charge transfer i n t e r p r e t a t i o n is p r e f e r r e d (10, 11).

Intense b a n d s ( e ^ 2 χ 10 ) that a p p e a r t o 3

b e analogous to t h e 600-nm system of b l u e proteins are o b s e r v e d

between

300 a n d 350 n m i n t h e C o ( I I ) d e r i v a t i v e s . T h e shift i n b a n d p o s i t i o n of a b o u t 16 k K [ C u ( I I ) < < C o ( I I ) ] accords w e l l w i t h e x p e c t a t i o n f o r a n LMCT MCD

transition. T h e visible a n d near-infrared absorption, C D , a n d spectra of C o ( I I )

d e r i v a t i v e s of s t e l l a c y a n i n , p l a s t o c y a n i n , a n d

a z u r i n h a v e b e e n i n t e r p r e t e d (12) successfully i n terms of t h e d-d t r a n sitions e x p e c t e d f o r d i s t o r t e d t e t r a h e d r a l m e t a l centers ( T a b l e I ) . A v e r age l i g a n d field parameters a r e t h e same f o r a l l three C o ( I I )

proteins

(Dq = 490, Β = 730 c m ) , w h i c h s t r o n g l y suggests t h a t t h e d o n o r a t o m - 1


8.

147

Blue Copper Proteins

GRAY E T A L .

set of a b l u e site does n o t v a r y f r o m case to case, e v e n t h o u g h t h e p o t e n t i a l does. I n t e r e s t i n g l y , t h e s p l i t t i n g of t h e Γ ι state is smallest f o r t h e 4

C o (II)

d e r i v a t i v e of s t e l l a c y a n i n , w h i c h is t h e p r o t e i n w h o s e n a t i v e

f o r m exhibits t h e lowest p o t e n t i a l . Table I .


Protein

Energies of the Ligand Field States in C o ( I I ) Protein Derivatives"

T

4

Stellacyanin Azurin Plastocyanin

T> ( F)

4

2

5000

6800 6600 6900

b b

Avg.

4

9700 10,150 10,000

8250 8375 8450

E n e r g i e s i n c m " ; g r o u n d state (zero) is A N o t observed.

a

4

1

b

T

4

( F)

Avg.

4

t

15,500 15,700 15,200

18,500 19,200 19,700

17,000 17,400 17,400

(12).

2

N e w absorptions a n d C D s p e c t r a l features a t t r i b u t a b l e to d-d t r a n s i tions h a v e b e e n o b s e r v e d

i n the near-infrared region for stellacyanin,

p l a s t o c y a n i n , a n d a z u r i n ( T a b l e I I ) . T h e s e d-d transitions h a v e a n a l y z e d (13) successfully i n terms of a s l i g h t l y g e o m e t r y f o r b l u e c o p p e r centers. energies (W) B

2

2

I n s u c h a geometry

been

tetrahedral

the electronic

of t h e l i g a n d field states of C u ( I I ) increase a c c o r d i n g t o T h e v a l u e of t h e a n g l e (β)

< E < B i < Αχ (14). 2

flattened

2

2

z-axis a n d t h e m e t a l - l i g a n d b o n d s (β = 90° at t h e D

4

h

between the

l i m i t ) for stella

c y a n i n w a s fixed at 60° i n o r d e r to g i v e reasonable values f o r A W ( A i — 2

i n t h e t e t r a h e d r a l a n d s q u a r e p l a n a r l i m i t i n g geometries.

B)

2

2

AW( E -B) 2

2

= 5250 a n d A W ( B 2

2

444 c m " ) , values of A W ( A 1

cm"

2

at t h e D

1

4

h

and T

X

-

2

2

2

1

and B

become A i 2

2

tions, B 2

2

2

g

2

andB ,

g

and B

-» E

lg

2

l g

lg

2

respectively, i n D

4 h

T h e energy

— B i ) , as Αχ 2

i g

2

g

. T h e other D

4

h

transi

B , are p r e d i c t e d at 24,890 a n d 15,550 c m " , 1

2 g

respectively. A l l the calculated D 20%,

Taking

(Ds = 765, Dt

( £ = 54.74°) l i m i t s , r e s p e c t i v e l y .

d

s e p a r a t i o n of 22,800 c m " is c o r r e c t l y d e n o t e d A W ( A 2

1

are c a l c u l a t e d to b e 22,800 a n d 6915

B)

2

= 8750 c m

-B)

X

4

h

values s h o u l d b e r e d u c e d b y a b o u t

a l l o w i n g f o r t h e slight increase ( ~ 0 . 1 A ) i n m e t a l - l i g a n d b o n d

lengths that is e x p e c t e d

to a c c o m p a n y

a change

f r o m t e t r a h e d r a l to

square p l a n a r c o o r d i n a t i o n . T h e a d j u s t e d values for t h e D

4

h

d-d t r a n s i

t i o n energies are i n reasonable agreement w i t h t h e o b s e r v e d b a n d p o s i tions

i n square

l i g a n d s (14).

planar C u ( I I )

complexes

containing

nitrogen-donor

I n s h a r p contrast, l i g a n d field parameters d e r i v e d f r o m β

values a f e w degrees b e l o w o r a b o v e 60° g i v e a b s u r d e n e r g y in the D

4

limit.

h

F o r e x a m p l e , AW( A 2

lg

—B) 2

lg

differences

is c a l c u l a t e d t o b e

58,800 c m " f r o m β = 57° parameters ( D s = 2240, Dt = 505 c m " ) a n d 1

1

5050 c m " f r o m β = 70° ones (Ds = 77, Dt = 1

320 c m " ) . 1

T h e d e r i v e d l i g a n d field parameters (β = 6 0 ° , Ds = 765, Dt = 444 c m " ) p r e d i c t A i to b e 11,540 c m " a b o v e t h e B g r o u n d state f o r s t e l l a 1

cyanin.

2

1

2

2

A n a b s o r p t i o n b a n d a n d C D m a x i m u m are o b s e r v e d near this

American Chemical Society Library 1155Bioinorganic 16th St. Chemistry—II N. W. Raymond; Advances in Chemistry; American Chemical Society: Washington, DC, 1977. Washington, D. C. 20036

148

BIOINORGANIC C H E M I S T R Y


Table II. Protein

ν (cm' )

Stellacyanin

5250 8750 11,470 13,040 16,580 17,840 22,570 5500 10,300 H ,940 13,540 16,600 18,140 21,540 23,640 10,300 12,940 15,940 17,770 20,840

Absorption and C D Spectral AefM"

1

Plastocyanin

Azurin

cm' ) 1

1

0.45 -0.35 2.4 -5.0 3.6 0.75 -7.35 0.125 -0.165 ~ 1.5 -3.78 4.08 0.4 -1.32 1.26 -0.475 -3.6 3.96 0.72 -1.11

" F r o m R e f . 13. A b s o r p t i o n for each b l u e p r o t e i n are t a k e n f r o m r e s o l u t i o n of the 35 Κ n e a r - i n f r a r e d a n d v i s i b l e a b s o r p t i o n s p e c t r u m .

energy.

II

a

Gaussian

T h e r e s o l v e d a b s o r p t i o n b a n d has a m o l a r e x t i n c t i o n coefficient

of 565, w h i c h is a p p r o x i m a t e l y five times l a r g e r t h a n t h a t o b s e r v e d the m a x i m u m at 8750 c m ' .

It is e x p e c t e d t h a t the B - » A

1

2

2

2

for

transition

X

s h o u l d b e m o r e intense, as i t is e l e c t r i c - d i p o l e a l l o w e d , w h e r e a s B - » B i 2

2

2

is not. E x c i t e d d - l e v e l energies for p l a s t o c y a n i n a n d a z u r i n are v e r y s i m i l a r . T a k i n g A W ( £ — B ) — 5500, A W ( B x — B ) = 2

2

2

2

2

2

10,300 c m " , a n d β — 1

6 0 ° for b o t h p r o t e i n s , Ds a n d Dt are c a l c u l a t e d to b e 764 a n d 508 c m " , 1

respectively, a n d the B 2

2

2

Αχ t r a n s i t i o n is p r e d i c t e d to b e at 12,520 c m " . 1

P l a s t o c y a n i n exhibits a G a u s s i a n - r e s o l v e d p e a k at 11,940 c m "

1

(Table II),

w h i c h m a y b e a t t r i b u t e d to B - » A i . I n a z u r i n , t h e r e l a t i v e l y intense 2

b a n d at ~ 13,000 c m

- 1

2

2

p r o b a b l y o v e r l a p s extensively w i t h

absorption

o w i n g to B - » Αχ. 2

2

2

R e s e a r c h a i m e d at i d e n t i f y i n g the l i g a n d s c o m p r i s i n g the

flattened

t e t r a h e d r a l b l u e c o p p e r center has b e e n p a r t i c u l a r l y intense i n the case of p l a s t o c y a n i n . D i r e c t e v i d e n c e for a s u l f u r l i g a n d has c o m e f r o m x - r a y photoelectron spectral ( X P S ) experiments on bean plastocyanin, where a l a r g e shift of the S2p core e n e r g y of the s i n g l e cysteine ( C y s - 8 5 )

residue

i n the p r o t e i n u p o n m e t a l i n c o r p o r a t i o n ( 164.5, a p o ; 169.8, n a t i v e ; 168.8 e V , C o ( I I ) derivative) was observed

(15).

T h e two histidines i n s p i n

a c h p l a s t o c y a n i n e x h i b i t pfC values b e l o w 5 i n N M R t i t r a t i o n e x p e r i m e n t s ,


8.

149


GRAY E T A L .

D a t a for Blue Copper Proteins" efM'

1

cm' )


~ ~

100 100 565 341 3549 1542 942 ~ 100 200 1162 1289 4364 1163 300 ~ 100 82 686 3798 504 185 6

Assignment

Ύ

1

B -* E β -* βι

~ ~

0.018 0.014 0.017 0.059 0.0041 0.0019 0.031 0.005 0.0033 0.0052 0.012 0.0037 0.0014 0.018 ~ 0.050 0.023 0.021 0.0042 0.0057 0.024

2

2

2

2

2

2

7rS —> d 2 . aS—* d 2. 2 x

x

y

2

y

b 2

2

£ -+ £ 2

2

B -» ^i 7rS —> d 2 2

2

X

. y2

d 2. 2

aS-^

x

y

b

ΤΤΝ*^ B^B 7rS —>

2

2

2

d 2.y2 X

t

d 2 _ y2 X

—> CÎ 2 . y2 b X

πΝ*·^

d 2.y x

2

Not assigned.

s u g g e s t i n g t h a t t h e y are c o o r d i n a t e d to c o p p e r ( 16).

I t is r e a s o n a b l e to

assume, t h e r e f o r e , t h a t t h e a n a l o g o u s t w o residues i n the b e a n p r o t e i n , H i s - 3 8 a n d H i s - 8 8 , are also l i g a n d s (13).

T h e f o u r t h l i g a n d i n the p r o

p o s e d d o n o r set f o r b e a n p l a s t o c y a n i n has b e e n i d e n t i f i e d i n extensive i n f r a r e d s p e c t r a l studies (17).

These experiments have revealed that a

short s e c t i o n of α-helix i n a p o p l a s t o c y a n i n is s t r o n g l y p e r t u r b e d u p o n metal [ C u ( I I ) or C o ( I I ) ] incorporation, thereby i m p l i c a t i n g a backbone p e p t i d e n i t r o g e n or o x y g e n as a l i g a n d .

T h e p r e f e r e n c e of c o p p e r

for

n i t r o g e n d o n o r s , as w e l l as e v i d e n c e f r o m c h a r g e t r a n s f e r s p e c t r a

(vide

i n f r a ) , favors c o o r d i n a t i o n b y a d e p r o t o n a t e d p e p t i d e n i t r o g e n

(N*).

C o n s i d e r a t i o n of the b e a n p l a s t o c y a n i n s e q u e n c e places t h e α-helix, a n d t h e r e f o r e the b a c k b o n e p e p t i d e n i t r o g e n , a f e w residues a b o v e H i s - 3 8 (14). A f l a t t e n e d t e t r a h e d r a l C u N N * S m o d e l is also r e a s o n a b l e f o r a z u r i n . 2

Recent X P S experiments have s h o w n copper

(18)

t h a t a s u l f u r is b o u n d

i n azurin, a n d the electronic spectroscopic

C u ( I I ) and Co (II)

p r o p e r t i e s of

to

both

f o r m s of t h e p r o t e i n are c l o s e l y s i m i l a r to those of

a n a l o g o u s b e a n p l a s t o c y a n i n d e r i v a t i v e s . I t is p r o b a b l e t h a t t h e n e a r t e t r a h e d r a l b i n d i n g site i n e a c h b l u e p r o t e i n is r a t h e r r i g i d , as l i g a n d field

s t a b i l i z a t i o n factors

strongly favor

structure for four-coordinate

Cu(II).

a square

planar geometrical

T h e site-structure rigidity


built

150

BIOINORGANIC CHEMISTRY-

-II

b y t h e p r o t e i n m u s t o v e r c o m e the e l e c t r o n i c s t a b i l i z a t i o n e n e r g y

associ

ated w i t h a Jahn—Teller distortion t o w a r d square planar C u ( I I )

geome

t r y , t h e r e b y c o n t r i b u t i n g to t h e r e l a t i v e i n s t a b i l i t y of the o x i d i z e d state of

the system.

T h u s the r e l a t i v e l y h i g h r e d u c t i o n p o t e n t i a l s of

blue

c o p p e r p r o t e i n s m a y b e a t t r i b u t a b l e , at least i n p a r t , to e l e c t r o n i c factors associated w i t h the r i g i d l y c o n s t r a i n e d ,

flattened

tetrahedral C u N N * S 2

site s t r u c t u r e .


S t r o n g e v i d e n c e for cysteine s u l f u r c o o r d i n a t i o n i n s t e l l a c y a n i n has b e e n o b t a i n e d (18)

i n X P S experiments.

T h i o e t h e r c o o r d i n a t i o n is r u l e d

o u t i n this case, as the p r o t e i n does n o t possess a n y m e t h i o n i n e (6).

It

is p r o b a b l e t h a t the o t h e r l i g a n d s are s i m i l a r , b u t n o t n e c e s s a r i l y i d e n t i c a l , to those of b e a n p l a s t o c y a n i n . A n i n t e r p r e t a t i o n of the intense a b s o r p t i o n b a n d s o b s e r v e d at a b o u t 13,000, 16,000, a n d 22,000 c m " s e n t e d (13).

1

i n b l u e c o p p e r p r o t e i n s has b e e n p r e

T h e analysis is b a s e d o n the a s s u m p t i o n t h a t the

of t h e h i g h e s t o c c u p i e d

l i g a n d orbitals i n a C u N N * S 2

a c c o r d i n g to TTS > aS > ττΝ* > ?rN.

C h a r g e transfer e x c i t e d states

r i v e d f r o m transitions of the t y p e π-> netic-dipole allowed.

energies

u n i t decrease de

d 2_ 2 are b o t h e l e c t r i c - a n d m a g X

y

T h e t r a n s i t i o n a S - » i Z 2 _ , o n t h e other h a n d , is X

y2

o n l y e l e c t r i c - d i p o l e a l l o w e d . T h e r o t a t i o n a l s t r e n g t h (R)

of a C D b a n d

is r e l a t e d to t h e e l e c t r i c d i p o l e

dipole

(/^i)

a n d the m a g n e t i c

(^

m&g

)

moments b y :

w h e r e ψ a n d ψ are the i n i t i a l a n d final states, r e s p e c t i v e l y . {

£

The rota

t i o n a l s t r e n g t h m a y also b e d e t e r m i n e d f r o m the e x p e r i m e n t a l q u a n t i t y Ac, or ci — c , a c c o r d i n g to E q u a t i o n 2: r

(2) F u r t h e r , t h e d i p o l e s t r e n g t h ( D ) is r e l a t e d to t h e m o l a r e x t i n c t i o n coeffi cient c b y :

(3) and 4 R / D « mated

(19)

γ, the K u h n the

integralf

anisotropy

factor.

Moscowitz

(e/v)dv as l n 2 V^°8°/v°,

has

approxi

w h e r e e° is the

m a x i m u m v a l u e of c, δ° is t h e h a l f - w i d t h at h a l f - m a x i m u m , a n d v° is the f r e q u e n c y of c°. A s s u m i n g t h a t δ° a n d v° are t h e same f o r

corresponding

a b s o r p t i o n a n d C D b a n d s , y m a y b e c a l c u l a t e d f r o m E q u a t i o n 4:


8.

151


GRAY E T A L .

y =

(4)

|Ac/c|

B a n d s associated w i t h m a g n e t i c - d i p o l e a l l o w e d , ττ c h a r g e transfer t r a n s i tions a r e e x p e c t e d t o h a v e m u c h l a r g e r y v a l u e s t h a n those a t t r i b u t a b l e to aS—»

as t h e i n t e n s i t y - g i v i n g m e c h a n i s m i n t h e latter case is

d 2. 2, x

y

p u r e l y e l e c t r i c d i p o l e i n o r i g i n . V a l u e s of γ , Ac, a n d c f o r t h e e l e c t r o n i c s p e c t r a l features of s t e l l a c y a n i n , p l a s t o c y a n i n , a n d a z u r i n a r e l i s t e d i n T a b l e I I . T h e results c l e a r l y i n d i c a t e t h a t t h e b a n d s a t ~ 13,000 a n d ~ 22,000 c m " b e a s s i g n e d t o π c h a r g e transfer, as t h e y h a v e r e l a t i v e l y


1

l a r g e v a l u e s of y. Specific assignments are d 2. 2 at 22,000 c m ' .

ΤΓΝ*

x

y

TTS - »

T h e 16,000-cm"

1

1

p l a s t o c y a n i n ( 4.6 Χ 10 ) > a z u r i n 5

1

( 2.2 χ 10" ) .

Correction

2

for electrostatic c h a r g e effects does not alter the r e a c t i v i t y o r d e r .

By

e s t i m a t i n g t h e charges o n the proteins at p H 7 f r o m sequence d a t a , elec t r o s t a t i c s - c o r r e c t e d self-exchange rate constants ( f c n c u l a t e d (23)

c o r r

) have been cal

as f o l l o w s ( M " s e c " ) : s t e l l a c y a n i n (2.7 Χ 1

1

c y a n i n (1.8 Χ 1 0 ) > a z u r i n (1.1 χ 1

10 ) 5

>

plasto

10" ). 2

U n f o r t u n a t e l y , e x p e r i m e n t a l ku v a l u e s are not a v a i l a b l e f o r a n y one of the three b l u e p r o t e i n s .

It has b e e n e s t a b l i s h e d , h o w e v e r , t h a t the

self-exchange rates for a z u r i n (24) o n the N M R t i m e scale at 2 5 ° .

a n d p l a s t o c y a n i n ( 2 5 ) are b o t h s l o w It is not l i k e l y that the p r e d i c t e d

ku

v a l u e s f r o m the F e ( E D T A ) " reactions w i l l c o r r e s p o n d to the m e a s u r e d 2

self-exchange rate constants. I n d e e d , agreement b e t w e e n c a l c u l a t e d a n d measured k

lx

values is o n l y to b e e x p e c t e d i f the a c t i v a t i o n r e q u i r e m e n t s

of b o t h partners i n a cross r e a c t i o n are e x a c t l y t h e same as those u s e d i n t h e i r respective self-exchanges.

Access to a b u r i e d , or at best p a r t i a l l y

exposed, outer-sphere redox center s h o u l d v a r y s u b s t a n t i a l l y f o r different reactants, a n d i t is l i k e l y t h a t c a l c u l a t e d ku v a l u e s w i l l s p a n a w i d e r a n g e i n s u c h cases. B a s e d o n the F e ( E D T A ) " results, the b l u e c o p p e r center i n s t e l l a 2

c y a n i n appears to b e m u c h m o r e accessible t h a n t h a t s i t u a t e d i n either a z u r i n or p l a s t o c y a n i n .

T h u s it s h o u l d b e p r o f i t a b l e to c o m p a r e

the

e l e c t r o n transfer reactivities of these three p r o t e i n s w i t h a v a r i e t y of r e d o x agents. K i n e t i c studies of the o x i d a t i o n of the three b l u e p r o t e i n s by Co(phen)

3

3 +

have been made

(26),

a n d the results together

with

those for other r e d o x agents are set out i n T a b l e I V . T h e electrostatic corrections to the p r e d i c t e d ku values are m o d e s t b o t h for the l a r g e c h a r g e o n p l a s t o c y a n i n a n d the s m a l l one o n a z u r i n , as the p r o t e i n selfe x c h a n g e a n d the cross r e a c t i o n w o r k terms compensate.

T h e reactivity


8.

153


GRAY E T A L .

of t h e s t e l l a c y a n i n r e d o x center as c a l c u l a t e d f r o m t h e cross r e a c t i o n with Co(phen)

3

3 +

is m u c h greater t h a n t h e c o r r e s p o n d i n g

fcn

for

corr

a z u r i n o r p l a s t o c y a n i n , i n a c c o r d w i t h o u r e a r l i e r finding b a s e d o n k

12

[ F e ( E D T A ) " ] values. T h e rather good agreement between 2

based on F e ( E D T A ) " a n d C o ( p h e n ) 2

3

3 +

fcn

values

corr

provides strong evidence that

t h e r e d o x center i n s t e l l a c y a n i n is accessible to reactants i n s o l u t i o n . S u c h g o o d agreement b e t w e e n ku

values contrasts m a r k e d l y w i t h t h e

corr

situation

i n azurin

a n d plastocyanin, where

fcn [Fe(EDTA) "]

Structure and Electron Transfer Reactions of Blue Copper Proteins

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