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
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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
Raymond; Bioinorganic Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1977.
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 .
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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
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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 ,
Raymond; Bioinorganic Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1977.
8.
149
Blue Copper Proteins
GRAY E T A L .
D a t a for Blue Copper Proteins" efM'
1
cm' )
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~ ~
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
Raymond; Bioinorganic Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1977.
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 .
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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:
Raymond; Bioinorganic Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1977.
8.
151
Blue Copper Proteins
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
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/ba-1977-0162.ch008
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
Raymond; Bioinorganic Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1977.
8.
153
Blue Copper Proteins
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) "]