Long-Range Electron Transfer Within Mixed-Metal Hemoglobin Hybrids

13 Long-Range Electron Transfer Within Mixed-Metal Hemoglobin Downloaded by COLUMBIA UNIV on March 20, 2013 | http://pubs.acs.org Publication Date: May 5, 1991 | doi: 10.1021/ba-1991-0228.ch013

Hybrids Michael J. Natan, Wade W. Baxter, Debasish K u i l a , D a v i d J. Gingrich, Gregory S. M a r t i n , and B r i a n M. Hoffman Department of Chemistry, Northwestern University, Evanston, IL 60208-3113

Studies of long-range electron transfer (ET) within mixed-metal hemoglobin (Hb) hybrids [MP, Fe (L)P, where M is Mg or Zn; Ρ is protoporphyrin IX; and L is H 0, imidazole, N -, F-, or CN-] are discussed. Because the structure of Hb is crystallographically known, ET occurs between redox centers held at known distance and ori entation. The ET energetics are easily manipulated through variation of M and L. In these systems, cyclic ET is initiated through photoproduction of the strong reductant (MP). ET quenching yields the charge-separated intermediate, [(MP) , Fe (L)P], which returns to the ground state by thermal ET. Direct spectroscopic observation of [(MP) , Fe (L)P] confirms the cyclic ET scheme. Comparison of rate constants for photoinitiated and thermally activated ET within various [MP, Fe (L)P] hybrids indicates that ET is direct and not "gated" by either protein conformational changes or ligand loss. 3+

2

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E L E C T R O N - T R A N S F E R R E A C T I O N S A R E C E N T R A L to b i o l o g y a n d c h e m i s t r y

(J, 2), b u t o n l y r e c e n t l y have techniques b e e n d e v e l o p e d to s t u d y l o n g range i n t e r p r o t e i n e l e c t r o n transfer ( E T ) (3, 4) w i t h o u t t h e c o m p l i c a t i o n of second-order processes t h r o u g h use of m o d i f i e d proteins that h o l d an e l e c t r o n d o n o r - a c c e p t o r redox p a i r at fixed distance. I n one a p p r o a c h , several groups have d e v e l o p e d t e c h n i q u e s for s t u d y i n g E T w i t h i n p r o t e i n s m o d i f i e d b y covalent attachment of redox-active i n o r g a n i c complexes to surface a m i n o acid residues (5-11). F o r e x a m p l e , [ ( L ) R u ] , w h e n b o u n d to a h i s t i d i n e r e s i d u e o n the outside of proteins s u c h as c y t o c h r o m e c or m y o g l o b i n , c a n 5

2 +

0065-2393/91/0228-0201$06.00/0 © 1991 American Chemical Society

In Electron Transfer in Inorganic, Organic, and Biological Systems; Bolton, J., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1991.

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E T IN INORGANIC, ORGANIC, A N D BIOLOGICAL SYSTEMS

exchange an e l e c t r o n w i t h a m e t a l - c o n t a i n i n g redox c e n t e r located o n the i n s i d e of the p r o t e i n . I n parallel w i t h M c L e n d o n (3), w e focused o n studies of long-range E T w i t h i n p r o t e i n - p r o t e i n complexes, such as the p h y s i o l o g i c a l l y i m p o r t a n t c o m p l e x of c y t o c h r o m e c peroxidase w i t h c y t o c h r o m e c (12-14) or the [ α β ] c o m p l e x of the h e m o g l o b i n t e t r a m e r (15-17). O u r a p p r o a c h involves r e p l a c i n g the h e m e (FeP) of one p r o t e i n p a r t n e r w i t h a c l o s e d - s h e l l p o r p h y r i n M P ( M is Z n or M g ; Ρ is p r o t o p o r p h y r i n IX) a n d s t u d y i n g E T b e t w e e n the M P a n d F e P groups (12-17). R e v e r s i b l e E T w i t h i n such m e t a l - s u b s t i t u t e d E T complexes (Scheme I) is i n i t i a t e d b y laser flash p h o t o p r o d u c t i o n of the

Downloaded by COLUMBIA UNIV on March 20, 2013 | http://pubs.acs.org Publication Date: May 5, 1991 | doi: 10.1021/ba-1991-0228.ch013

1 ?

2

A* [ (MP),Fe *(L)P] 3

3

I [(MPr,Fe *(L)P] 2

[MP,Fe (L)P] 2+

c Scheme I. slowly d e c a y i n g ( M P ) t r i p l e t state (A*). T h e ( M P ) is a good reductant a n d can r e d u c e the f e r r i h e m e p a r t n e r ( F e P ) b y long-range E T w i t h a p h o toinitiated E T rate constant fc (eq 1). 3

3

3 +

t

3

(MP) + F e

3 +

P - ^ » (MP)

+

+ Fe

2 +

P

(1)

T h e r e s u l t i n g charge-separated i n t e r m e d i a t e , [ ( M P ) , F e P ] (I), r e turns to the g r o u n d state b y t h e r m a l E T from F e P to the cation radical ( M P ) (eq 2) w i t h rate constant k . +

2 +

2 +

+

h

(MP)

+

+ F e

2

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P - ^ MP + Fe

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P

(2)

I n o u r studies w e u s e d transient absorption a n d e m i s s i o n t e c h n i q u e s to m o n i t o r A * a n d I, t h e r e b y a l l o w i n g us to measure b o t h k a n d k . T h e k e y benefit to s t u d y i n g long-range E T processes w i t h i n h e m o g l o b i n h y b r i d s (Hb) is that, u n d e r the conditions of o u r e x p e r i m e n t s , the h e m o g l o b i n tetramers i n s o l u t i o n adopt d e o x y - H b (T-state) g e o m e t r y w i t h a crystallographically t

h


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k n o w n structure. T h u s , e l e c t r o n transfer occurs b e t w e e n redox centers h e l d at k n o w n distance a n d o r i e n t a t i o n .

Preparation, Structure, and Characterization of Mixed-Metal Hemoglobin Hybrids P r e p a r a t i o n of m i x e d - m e t a l h e m o g l o b i n h y b r i d s is a c h i e v e d b y separation of [2α, 2 β ] h e m o g l o b i n i n t o its constituent a a n d β chains, f o l l o w e d b y d e m e t a l a t i o n of one of the chains, metalation w i t h M P , a n d c h a i n r e c o m b i n a t i o n , to y i e l d the t e t r a m e r i c [2α(ΜΡ), 2 β ( Ρ β Ρ ) ] or [ 2 α ( Ρ β Ρ ) , 2 β ( Μ Ρ ) ] species (18). T h u s , M P —» F e P E T m i g h t i n p r i n c i p l e o c c u r b e t w e e n α - β ! or α ! ~ β subunits. H o w e v e r , the distance b e t w e e n a a n d β h e m e s is m o r e than 10 A greater t h a n that b e t w e e n a a n d β · T h i s extra distance is e x p e c t e d a n d i n d e e d is f o u n d to r e d u c e E T rates b y several orders of m a g n i t u d e . H e n c e , for a l l p r a c t i c a l purposes w e may treat the [ 2 α , 2 β ] t e t r a m e r i n t e r m s of two i n d e p e n d e n t [ α β ] E T complexes. 3 +

3+


χ

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If the g e o m e t r y of m i x e d - m e t a l h e m o g l o b i n h y b r i d s is fixed i n the k n o w n structure of T-state (deoxy) H b , m e t a l r e p l a c e m e n t s h o u l d not p e r t u r b that structure. T h e first structural issue that m u s t b e c o n s i d e r e d is l o c a l : D o e s substitution o f Z n or M g cause significant p e r t u r b a t i o n s ? T h e s t r u c t u r e o f M g H b , i n w h i c h a l l four prosthetic groups are M g P , has r e c e n t l y b e e n crystallographically d e t e r m i n e d at 2 . 2 - A r e s o l u t i o n (19). W i t h the a t o m i c m o d e l of deoxy H b as the starting p o i n t i n a least-squares r e f i n e m e n t , o n l y t r i v i a l l y s m a l l structural differences w e r e n o t e d . T h e r e f o r e , r e p l a c e m e n t o f Fe with M g i n h e m o g l o b i n does not significantly alter the s t r u c t u r e . 2 +

2 +

T h e second structural issue is global: Is the q u a t e r n a r y structure of a m i x e d - m e t a l h y b r i d significantly different from that of T-state H b ? A g a i n the answer is no, as i n d i c a t e d b y an X - r a y structure of the [ a ( F e C O ) ^ ( M n ) ] h y b r i d (20). T h u s , the distances a n d g e o m e t r i c relationships of the h e m e groups i n v o l v e d i n E T w i t h i n the [ α β ] E T c o m p l e x are p r e s e r v e d i n the m e t a l - s u b s t i t u t e d species. W i t h this corroborative structural data, it is pos sible to discuss structure o f the [ α β ] c o m p l e x w i t h h i g h p r e c i s i o n . ΐ 5

ΐ 5

2

2

A s a n e x a m p l e w e c o n s i d e r e d the [ F e , M ] h y b r i d s , w h e r e M is M g or Z n . I n the [ α β ] E T c o m p l e x ( F i g u r e 1), the β ( Μ Ρ ) a n d a ( F e P ) are r o u g h l y p a r a l l e l , w i t h distances of 25 Â b e t w e e n metals a n d about 17 À e d g e to-edge (21). T h i s s t r u c t u r a l l y d e f i n e d b u t c h e m i c a l l y m a n i p u l a b l e system offers m a n y avenues for study. R e c e n t l y w e focused o n the effects of c h a n g i n g E T energetics. O n e means to do this is to vary the c l o s e d - s h e l l M P . T h e ( M g P ) - M g P r e d u c t i o n p o t e n t i a l is about 100 m V l o w e r t h a n the Z n P - Z n P r e d u c t i o n p o t e n t i a l (15, 16). C o n s e q u e n t l y , the free energy change, - A G , for p h o t o i n i t i a t e d A * —» I process is - 1 . 0 e V for [ ( Z n P ) , F e P ] , a n d - 1 . 1 e V for [ ( M g P ) , F e P ] . F o r the I A E T , - A G is - 0 . 8 e V for [(ZnP) , F e P ] a n d - 0 . 7 e V for [(MgP) , F e P ] (15, 16). 1 ?

2

2

x

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3 +

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H e m e l i g a n d v a r i a t i o n provides an e v e n m o r e effective means of a l t e r i n g



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Figure 1. a-Carbon backbone of an [α β ] ET complex within [2a(Fe(CO)P, 2β(ΜηΡ)]. (Reproduced with permission from ref 20. Copyright 1986 Aca demic Press.) Ϊ9

2

the energetics for E T w i t h o u t c h a n g i n g the structure o f the E T c o m p l e x . A t n e u t r a l p H , the F e P i n m e t h e m o g l o b i n has H 0 i n the distal c o o r d i n a t i o n site, w i t h the r e m a i n i n g five sites taken b y the nitrogens of Ρ a n d o f the p r o x i m a l h i s t i d i n e (22). T h e c o o r d i n a t e d H 0 can be r e p l a c e d b y o t h e r ligands L , b o t h n e u t r a l ( L = L ° is imidazole) a n d a n i o n i c ( L = X " is C N ~ , F " , or N ~ ) . A s the F e P - F e P redox p o t e n t i a l depends o n L , the d r i v i n g force for E T changes c o r r e s p o n d i n g l y . X - r a y crystallographic m e a s u r e m e n t s of l i g a n d e d h e m o g l o b i n s show n e g l i g i b l e changes u p o n l i g a n d v a r i a t i o n , a n d thus the h e m e g e o m e t r y is r e t a i n e d (23). 3 +

2

2

3

3 +

2 +

T h e c o m b i n a t i o n o f m e t a l - l i g a n d v a r i a t i o n i n these h y b r i d s not o n l y allows alteration of the E T energetics b u t also p r o v i d e s a means to study m e c h a n i s t i c questions. It c o u l d b e u s e d to d e t e r m i n e w h e t h e r E T is d i r e c t or i n v o l v e s a h o p p i n g m e c h a n i s m and w h e t h e r E T is " g a t e d " (24-27) b y linkage to p r o t e i n conformational change or h e m e l i g a n d dissociation.


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Kinetics by Triplet Decay R e v e r s i b l e E T w i t h i n M g - a n d Z n - s u b s t i t u t e d h e m o g l o b i n h y b r i d s is i n i t i a t e d b y flash p h o t o p r o d u c t i o n of the l o n g - l i v e d t r i p l e t state ( M P ) . F i g u r e 2 shows the progress curves for t r i p l e t decay i n [ M g , F e ] , [ M g , F e ( H 0 ) ] , [ Z n , F e ] , a n d [ Z n , F e ( H 0 ) ] , as m o n i t o r e d b y ( M P ) - M P absorbance dif ference spectra. T h e data are s h o w n n o r m a l i z e d to u n i t t r i p l e t p o p u l a t i o n . T h e t r i p l e t decay for b o t h r e d u c e d h y b r i d s , [ M g , F e ] a n d [ Z n , F e ] , is e x p o n e n t i a l o v e r five half-lives. T h e rate constant for this i n t r i n s i c t r i p l e t decay, Jt , is 2 0 ( ± 2 ) s" at 25 °C w h e n M is M g a n d 5 3 ( ± 5 ) s" at 25 °C w h e n M is Z n . I d e n t i c a l rate constants are o b t a i n e d b y f o l l o w i n g triplet-state emission. 3

2 +

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d

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T r i p l e t decay i n [ M g , F e ( H 0 ) ] a n d [ Z n , F e ( H 0 ) ] m o n i t o r e d at λ = 415 n m , the F e P isosbestic p o i n t , o r at 475 n m , w h e r e c o n t r i b u t i o n s from the charge-separated i n t e r m e d i a t e are m i n i m a l , r e m a i n s e x p o n e n t i a l . H o w e v e r , the decay rate i n the o x i d i z e d h y b r i d s , fe , is i n c r e a s e d to 5 5 ( ± 5 ) s" w h e n M is M g a n d to 1 3 8 ( ± 7 ) s" w h e n M is Z n . T w o a d d i t i o n a l q u e n c h i n g processes can c o n t r i b u t e to deactivation of ( M P ) w h e n the i r o n - c o n t a i n i n g c h a i n of the h y b r i d is o x i d i z e d to the F e P state: E T q u e n c h i n g as i n e q 1 (with rate constant k ) a n d F o r s t e r e n e r g y transfer (with rate constant k ). E T q u e n c h i n g is not possible i n the F e P h y b r i d , a n d F o r s t e r e n e r g y transfer also is u n i m p o r t a n t because spectral overlap is m i n i m a l (17). T h e 3 +

3 +

2

2

3 + / 2 +

p

1

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3 +

t

0

2 +

Tine

(ns)

Figure 2. Normalized triplet-decay curves for [M(P), Fe(P)] hybrids. For a given M (M is Zn or Mg), the arrow is directed from the curve for the Fe P state toward that for the Fe P state. 2+

3+


206


net rate of t r i p l e t disappearance i n o x i d i z e d h y b r i d s is thus the s u m of three terms: fc = fc + k + k . T h e difference i n t r i p l e t - d e c a y rate constants for the o x i d i z e d a n d r e d u c e d h y b r i d s gives the q u e n c h i n g rate constant, k k - k = k + fc , w h i c h is thus an u p p e r b o u n d to k . S u b t r a c t i o n y i e l d s k = 8 5 ( ± 1 0 ) s for [ZnP, F e ( H 0 ) P ] a n d k = 3 5 ( ± 5 ) s" for [MgP, F e ( H 0 ) P ] . p

q

=

t

d

p

d

t

0

t

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q

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q

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2

B y d e f i n i t i o n , the i n t r i n s i c t r i p l e t - d e c a y rate constant, k , is i n d e p e n d e n t of h e m e l i g a t i o n . T h e r e f o r e , differences i n k i n e t i c progress curves for [ ( M P ) , F e ( L ) P ] reflect i n e q u i v a l e n t values of k for the various ligands. T h e data for M = Z n clearly fall into two classes: H y b r i d s w i t h f e r r i h e m e c o o r d i n a t e d to the n e u t r a l ligands H 0 a n d i m i d a z o l e give k ~ 80 s" , b u t those w i t h b o u n d anionic ligands give dramatically r e d u c e d values [ 3 ( ± 2 ) s" < k < 1 2 ( ± 3 ) s" ]. T h e data for [ M g P , F e ( L ) P ] h y b r i d s show a s i m i l a r g r o u p i n g . d

3

3 +

q


1

q

2

1

1

q

3 +

I n o u r i n i t i a l studies, w h i c h w e r e solely l i m i t e d to m e a s u r e m e n t s of t r i p l e t q u e n c h i n g , w e c o n s i d e r e d w h e t h e r energy transfer c o u l d be c o n t r i b u t i n g to fc . F o r s t e r energy transfer w o u l d be p r o p o r t i o n a l to spectral overlap b e t w e e n the ( M P ) e m i s s i o n s p e c t r u m a n d the F e ( L ) P absorption spec t r u m . T h u s , a lack of correlation b e t w e e n the ligated h e m e o p t i c a l spectra a n d the o b s e r v e d k i n d i c a t e d that for L = L° = H 0 (and i m i d a z o l e ) , most, i f not a l l , t r i p l e t q u e n c h i n g is associated w i t h E T (17). H o w e v e r , w i t h the s m a l l e r rate constants for t r i p l e t q u e n c h i n g b y the a n i o n - l i g a t e d h e m e s , it was b y no means clear w h e t h e r E T was the p r e d o m i n a n t q u e n c h i n g m e c h a n i s m . T h a t is, a small value for k w o u l d have m i n i m a l c o n s e q u e n c e for L = L ° , b u t c o u l d account for m u c h or a l l of k i n the case w h e r e L = X ~ . T h u s , d i r e c t observation of the charge-separated i n t e r m e d i a t e I , i n a d d i t i o n to y i e l d i n g fc , the rate constant for the t h e r m a l process, is r e q u i r e d to c o n f i r m the v e r y existence of long-range E T i n cases w h e r e k is small. q

3

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q

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0

q

b

q

Direct Observation of Charge-Separated ET Intermediates T h e t i m e course of the charge-separated i n t e r m e d i a t e I can be m e a s u r e d i n a flash photolysis e x p e r i m e n t that monitors the I - A transient absorbance difference at a g r o u n d s t a t e - t r i p l e t state isosbestic p o i n t (e.g., λ = 432 n m w h e n M is M g a n d 435 n m w h e n M is Zn). W e have o b s e r v e d this i n t e r mediate for the [ M P , F e P ] h y b r i d s w h e n M is M g or Z n ; representative k i n e t i c progress curves are s h o w n i n F i g u r e 3 (15). I n a k i n e t i c s c h e m e that i n c l u d e s eqs 1 a n d 2 as the o n l y E T processes, w h e n fe > fc , as is the case h e r e , I appears exponentially w i t h rate constant k a n d disappears c o m p l e t e l y 3 +

b

p

h

i n an e x p o n e n t i a l fall w i t h rate constant fc . H o w e v e r , the o c c u r r e n c e of a persistent absorbance change ( Δ Α „ ) for the [ M , F e ] h y b r i d s r e q u i r e s an e x t e n d e d k i n e t i c m o d e l (Scheme I). I n this m o d e l , ( M P ) is r e d u c e d not o n l y b y F e P w i t h rate constant k (regen e r a t i n g the [ M P , F e P ] state), b u t also b y an a s - y e t - u n i d e n t i f i e d a m i n o a c i d p

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Figure 3. Normalized kinetic progress curves at 5 °C for ET intermediate (I) plus photoproduct (C) (see Scheme I) formed upon flash photolysis of the mixed-metal Hb hybrids: [$(MgP), a(Fe P)] (\ = 432 nm); [β(ΖηΡ), a(Fe P)] (λ = 435 nm). Solid lines are nonlinear least-squares fits to the equations in ref. 15. For[Mg, Fe], k = 155(±15) s" , k = 47(±5) s' , andk = 20(±5) s^for [Zn, Fe], k = 350(±35) s , k = 122(±10) s , and k = 40(±8) s- . Buffer: 0.01 M KP pH 7.0. 3+

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r e s i d u e X a n d / o r solution i m p u r i t i e s w i t h rate constant fc , l e a d i n g to [ M P , F e P ] (eq 3): m

2 +

[(MP ), Fe +

2 +

P] + X - ^ +

[MP, Fe

2 +

P] + X

(3)

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S o l u t i o n of the k i n e t i c equations i m p l i c i t i n S c h e m e I indicates that the m a g n i t u d e of àA is p r o p o r t i o n a l to fe , a n d that I appears e x p o n e n t i a l l y w i t h rate constant k = k + k . F i g u r e 3 shows that the k i n e t i c progress curves for I for the Z n - a n d M g - s u b s t i t u t e d h y b r i d s are w e l l - d e s c r i b e d b y n o n l i n e a r least-squares fits to these k i n e t i c equations (15). T h e data i n F i g u r e 3 show that the t i m e course of the i n t e r m e d i a t e [ ( M P ) , F e P ] (I) strongly depends o n M . A t 5 °C w h e n M is M g , k = 155(±15) s , fc = 4 7 ( ± 5 ) s , a n d k = 2 0 ( ± 5 ) s" ; w h e n M is Z n , k = 3 5 0 ( ± 3 5 ) s , k = 1 1 2 ( ± 1 0 ) s" , a n d k = 4 0 ( ± 8 ) s" . D i r e c t observation of I , the charge-separated i n t e r m e d i a t e , has v e r i f i e d the occurrence of long-range E T w i t h i n [ ( M P ) , F e ( L ) P ] for b o t h M a n d all L . F i g u r e 4 shows a c o m p a r i s o n of the k i n e t i c progress curves o b t a i n e d for [β(ΖηΡ), a ( F e ( H 0 ) P ] a n d [β(ΖηΡ), a ( F e ( C N ) P ] (16). T h e l o n g - t i m e exponential fall for the latter, k = 6 5 ( ± 8 ) s" , is i n agreement w i t h that œ

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0 0

40

20 Tine (ns)

Figure 4. Kinetic progress curves as in Figure 3 at 435 nm for [β(ΖηΡ), a(Fe (L)P)]. Experimental points and nonlinear least-squares fits for L = H 0 and L = CN~ are shown, with absorbance changes normalized to a zerotime triplet concentration (A *) = 10~ M. For [$(ZnP), 0L(Fe (H O)P)], k = 345(±45) s- and k = 134(±15) s" ; for [β(ΖηΡ), a(Fe (CN~)P)], k = 240(±30) s- and k = 65(±8) s" . Buffer: 0.01 M KP pH 7.0. 3+

2

6

0

1

b

3+

2

p

1

b

2

p

2

3+

h

o b s e r v e d i n t r i p l e t - d e c a y data. A b s o r b a n c e changes r e s u l t i n g from f o r m a t i o n o f t h e charge-separated i n t e r m e d i a t e I are p r o p o r t i o n a l to the rate constant k . T h u s k can b e calculated i n d e p e n d e n t l y of any o t h e r c o n t r i b u t i o n s to t

t

triplet-state q u e n c h i n g i f the q u a n t u m y i e l d for the f o r m a t i o n of I can b e determined. W i t h this p r o c e d u r e , analysis o f the r e l a t i v e l y large absorbance changes o b s e r v e d w i t h t h e i n t e r m e d i a t e i n the M = Z n , L = fc (H 0) Zn

t

k

= 90(±30) s

2

3 +

2

t

H 0 . O n the o t h e r h a n d , k

=

(CN")P],

but analysis of absorbance

changes associated

d

a(Fe

H Q h y b r i d gives

a n d thus confirms the p r e v i o u s assignment fc =

for L =

- k

p

_ 1

[β(ΖηΡ)

+

q

2

14(±4) s

1

for [β(ΖηΡ),

, a ( F e ( C N " ) P ] gives an e v e n s m a l l e r v a l u e , J f c ^ i C N " ) = 2 +

with 6(±3)

s" . T h u s , r e p l a c e m e n t of H 0 b y C N " i n the h e m e c o o r d i n a t i o n sphere 1

2

reduces k b y o v e r a n o r d e r of m a g n i t u d e . Q u a n t i t a t i o n o f I w h e n M is M g t

o r Z n , a n d w i t h a l l the ligands s t u d i e d so far, gives t h e

fc (L) M

t

shown i n

F i g u r e 5. R e p l a c e m e n t of H 0 w i t h another n e u t r a l l i g a n d , i m i d a z o l e , does 2

not significantly alter k . R e p l a c e m e n t w i t h o t h e r anions ( N ~ , F~) affects k t

3

t

i n the same fashion as C N " , a 10-fold r e d u c t i o n i n rate. T h e effect o f a n i o n b i n d i n g o n fc is not n e a r l y as great. T h e data i n b

F i g u r e 5 show that, for b o t h metals, a less than 5 0 % r e d u c t i o n i n t h e r m a l l y activated E T rate constant is o b s e r v e d b e t w e e n h y b r i d s c o n t a i n i n g n e u t r a l a n d a n i o n i c ligands.



13.

NATAN ET AL.


209

Hybrids

Figure 5. Ligand dependence of rate constants for photoinitiated ET (k , light, speckled) and thermal ET (kb, dark, crosshatched) within the hybrids (A) [ZnP, Fe +(L)P] and (B) [MgP, Fe (L)P]. When M is Zn, the data refer to 1methylimidazole rather than imidazole. t

3

3+

Mechanistic Aspects of Electron Transfer within [MP, Fe +(L)P] 3

T h e single most i m p o r t a n t mechanistic q u e s t i o n c o n c e r n i n g long-range E T i n h e m o g l o b i n h y b r i d s is w h e t h e r the E T reactions u n d e r consideration are single-step events o r m u l t i p l e - s t e p processes w i t h one or m o r e r e a l i n t e r m e d i a t e states (such as one w i t h an o x i d i z e d or r e d u c e d a m i n o a c i d residue). A second k e y issue i n m e c h a n i s t i c studies of long-range e l e c t r o n transfer i n proteins is the r o l e of gating. I f p h o t o i n i t i a t e d or t h e r m a l E T r e q u i r e s a


210


p r o t e i n conformational change, t h e n fe , the o b s e r v e d rate constant, m a y actually b e m e a s u r i n g a conformational rate rather than an E T rate (24-27). O n e aspect of this issue involves the fate of the h e m e l i g a n d . R e d u c t i o n of F e ( H 0 ) P p r o m p t l y y i e l d s the u n l i g a n d e d ferroheme F e P , b u t the fate of a n i o n i c ligands u p o n r e d u c t i o n of F e ( X " ) P is not clear. I n some cases i n v o l v i n g exogenous reductants, it has b e e n s h o w n that l i g a n d dissociation is a p r e r e q u i s i t e to r e d u c t i o n (28, 29). T h u s , the p o s s i b i l i t y of " l i g a n d g a t i n g " m u s t also be c o n s i d e r e d . T h e ability to alter the redox potentials of b o t h M P a n d F e ( L ) P allows us to address these questions d i r e c t l y , a n d o u r data show that k a n d k represent rate constants for d i r e c t , u n g a t e d e l e c t r o n transfer b e t w e e n the M P a n d F e P . obs

3 +

2 +

2

3 +


t

h

P h o t o i n i t i a t e d E T is easily p r o v e d to be direct. I n d i r e c t h o p p i n g of an e l e c t r o n f r o m ( M P ) to F e ( L ) P w o u l d c o r r e s p o n d to oxidative t r i p l e t q u e n c h i n g b y an a m i n o a c i d , w i t h subsequent r e d u c t i o n of F e P b y the a m i n o a c i d a n i o n . H o w e v e r , any such q u e n c h i n g of ( M P ) w o u l d o c c u r e q u a l l y i n the r e d u c e d ( F e P ) h y b r i d s because this process does not i n v o l v e the F e P . C o n s e q u e n t l y , E T b y this m e c h a n i s m w o u l d not give rise to an increase i n t r i p l e t decay, a n d the increased t r i p l e t q u e n c h i n g i n the F e P h y b r i d s m u s t be associated w i t h a d i r e c t E T process. 3

3 +

3 +

3

2 +

3 +

O u r data also indicate that the I —> A E T process is direct. I f i t w e r e not, t h e n there m u s t exist an a m i n o a c i d , y, that mediates e l e c t r o n flow f r o m F e P to ( M P ) v i a an i n t e r n a l E T . T h i s is e q u i v a l e n t to p o s t u l a t i n g a t h e r m o d y n a m i c a l l y accessible discrete i n t e r m e d i a t e , [ ( M P ) , y , ( F e P ) ] , w h i c h w o u l d decay b y a second E T process back to the g r o u n d state. I f the ( M P ) - » y E T process w e r e r a p i d a n d F e P - > y E T w e r e rate l i m i t i n g , c h a n g i n g M w o u l d not affect the o b s e r v e d rate constant a n d fc would e q u a l fc . I f a r a t e - l i m i t i n g ( M P ) —» y E T w e r e s u c c e e d e d b y r a p i d F e P —» y E T , c h a n g i n g the h e m e l i g a n d L w o u l d not affect the E T rate a n d fc (H 0) would equal fc (CN). However, fc (H 0) Φ fc (H 0) Φ fc (CN~). W e c o n c l u d e that a two-step e l e c t r o n h o p p i n g m e c h a n i s m does not o b t a i n a n d that k describes d i r e c t F e P -> ( M P ) E T . 2 +

+

2 +

+

+

2 +

+

Mg

b

Zn

+

b

2 +

+

Zn

b

Zn

2

Mg

b

b

Zn

2

b

2

Zn

b

2 +

h

+

C o m p a r i s o n of rates for the [ M , F e ] h y b r i d s w h e r e M is M g a n d Z n p r o v i d e s a test of w h e t h e r E T is gated (i.e., c o n t r o l l e d b y a slow confor mational transformation) to an " E T - a c t i v e " state i n w h i c h E T is p r e s u m e d to b e r a p i d . T h e rate of such a conformational transformation w o u l d not change because of the alteration i n d r i v i n g force caused b y the Z n - M g s w i t c h . Because k is i n d e e d different w h e n M is M g a n d Z n , it cannot represent a rate constant for conformational i n t e r c o n v e r s i o n . t

W h a t is the fate of the h e m e - l i g a n d , L , d u r i n g the E T cycle of S c h e m e I? W h e n L is H 0 , r e d u c t i o n of the a q u o - b o u n d h e m e y i e l d s the fivecoordinate f e r r o h e m e , F e P . F o r C N " , l i g a n d dissociation from F e ( C N " ) P is slow; this fact indicates that the I —> A process involves reoxidation of the C N - b o u n d species. T h e data i n F i g u r e 5 show that N ~ a n d F " also r e m a i n b o u n d o n the E T t i m e scale. I f E T - i n d u c e d l i g a n d loss w e r e r a p i d c o m p a r e d 2

2 +

2 +

3


13.

NATAN ET AL.

Mixed-Metal

Hemoglobin

Hybrids

211

to the I —» A E T process, one w o u l d p r e d i c t that for a g i v e n m e t a l , fc (X ) = fc (H 0). T h i s p r e d i c t i o n is contrary to observation w i t h b o t h metals. M

b

M

b

2

T h e data also show that for a g i v e n a n i o n , fc (X~) < k (X-). This d e p e n d e n c e o n m e t a l i o n indicates that fc (X~) cannot r e p r e s e n t a ratel i m i t i n g l i g a n d dissociation f r o m the F e ( X ~ ) P p a r t n e r of I, f o l l o w e d b y fast E T (i.e., l i g a n d gating of I —» A ) . T h u s the variation i n the t h e r m a l E T rate, fc (L), as a f u n c t i o n of l i g a n d a n d m e t a l indicates that a l l a n i o n i c ligands r e m a i n b o u n d d u r i n g the e n t i r e E T cycle of S c h e m e I. I n contrast, the e x t r e m e l y slow r e d u c t i o n of ligated f e r r i m y o g l o b i n [ M b ( L ) ] b y exogenous S 0 ~ r e q u i r e s anionic dissociation for most ligands, notably fluoride (28, 29). T a k e n together, these data suggest a difference i n the m e c h a n i s m s for r e d u c t i o n b y exogenous d i t h i o n i t e a n d b y an i n t e r n a l ( M P ) . Mg

Zn

h

b

M

b

2 +

M

b

3 +


2

4

2

3

In s u m m a r y , the simple variation of ligands and metals w i t h i n [ M P , F e ( L ) P ] allows a heretofore u n p a r a l l e l e d v i e w of the m e c h a n i s t i c aspects of long-range E T b e t w e e n proteins. 3 +

Conclusions and Prospectus M e t a l - s u b s t i t u t e d h e m o g l o b i n h y b r i d s , [ M P , F e ( H 0 ) P ] are w e l l - s u i t e d to the study of long-range E T w i t h i n p r o t e i n complexes. B o t h p h o t o i n i t i a t e d a n d t h e r m a l l y activated E T can be s t u d i e d b y flash excitation of Z n - or M g s u b s t i t u t e d complexes. D i r e c t spectroscopic observation of the charge-sep arated i n t e r m e d i a t e , [(MP) , F e P ] , u n a m b i g u o u s l y demonstrates p h o t o i n i t i a t e d E T , a n d the t i m e course of this E T p r o d u c t indicates the p r e s e n c e of t h e r m a l E T . R e p l a c e m e n t of the c o o r d i n a t e d H 0 b y a n i o n i c ligands ( C N ~ , F ~ , o r N ~ ) i n the f e r r i h e m e s u b u n i t dramatically lowers the p h o t o i n i t i a t e d rate constant, k , b u t has a r e l a t i v e l y m i n o r effect o n the t h e r m a l rate, k . 3 +

+

2

2 +

2

3

t

h

Because m e t a l substitution a n d l i g a n d variation can b e effected w i t h o u t structural p e r t u r b a t i o n of the E T complex, such changes can be u s e d to p r o b e m e c h a n i s t i c aspects o f E T . T h e data show that b o t h p h o t o i n i t i a t e d a n d t h e r m a l E T are direct processes. F u r t h e r m o r e , E T is not gated e i t h e r b y p r o t e i n conformational changes or b y l i g a n d o n - o f f processes. T h e sta b i l i t y of h e m o g l o b i n tetramers i n cryosolvent, c o u p l e d w i t h the absence of gating i n these systems, has a l l o w e d observation of long-range E T at t e m peratures near 77 Κ (30), w h e r e q u a n t u m m e c h a n i c a l t u n n e l i n g is operative. T h e ease w i t h w h i c h E T can be s t u d i e d i n m i x e d - m e t a l h e m o g l o b i n h y b r i d s suggests that this system w i l l be of value i n addressing several l o n g standing p r o b l e m s i n this field. F o r example, m i x e d - m e t a l m u t a n t h e m o globins, i n w h i c h a m i n o acids b e t w e e n p o r p h y r i n s have b e e n c h a n g e d f r o m aliphatic to aromatic a n d vice versa, are b e i n g used to assess the role of E T pathways a n d of hole superexchange i n long-range e l e c t r o n transfer. W e are e x t e n d i n g o u r studies of E T w i t h i n [ M P , F e ( L ) P ] to l i q u i d h e l i u m (4 K ) temperatures. F i n a l l y , a m o r e c o m p l e t e p i c t u r e of the role of energetics i n long-range E T is b e i n g r e a l i z e d t h r o u g h e x p a n d e d m e t a l s u b s t i t u t i o n a n d 3 +


212

ET IN INORGANIC, ORGANIC, A N D BIOLOGICAL SYSTEMS

l i g a n d v a r i a t i o n . B y a l t e r i n g the p r o t e i n e n v i r o n m e n t , the solvent, the t e m p e r a t u r e , a n d the E T sites themselves, w e h o p e to greatly a d d to the u n d e r s t a n d i n g o f this i m p o r t a n t biological process.

Acknowledgment


T h i s research was s u p p o r t e d b y N a t i o n a l Institutes of H e a l t h G r a n t s H L 13531 a n d H L 40453, a n d b y N a t i o n a l Science F o u n d a t i o n G r a n t D M B 8907559 to B r i a n M . H o f f m a n , a n d b y N a t i o n a l Institutes o f H e a l t h N a t i o n a l R e s e a r c h S e r v i c e A w a r d postdoctoral fellowship H L 0 7 5 3 1 to M i c h a e l J . Natan.

Β.

Β.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. M. 14. 15. 16. M. 17. 18. 19. 20. 21. 22.

Gray, H. B.; Malmström, B. G . Biochemistry 1989, 28, 7499. Marcus, R. Α.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265. M c L e n d o n , G . Acc. Chem. Res. 1988, 21, 160. Mayo, S. L.; Ellis, W. R., Jr.; Crutchley, R. J.; Gray, Η. B. Science (Washington, D.C.) 1986, 233, 948. Bowler, Β. E.; Meade, T. J.; Mayo, S. L.; Richards, J. H.; Gray, Η. B. J. Am. Chem. Soc. 1989, 111, 8757. Meade, T. J.; Gray, Η. B.; Winkler, J. R. J. Am. Chem. Soc. 1989, 111, 4353. Elias, H.; Chou, M. H.; Winkler, J. R. J. Am. Chem. Soc. 1988, 110, 429. Cowan, J. Α.; Upmacis, R. Κ.; Beratan, D. Ν.; Onuehic, J. Ν.; Gray, Η. Β. Ann. Ν.Ύ. Acad. Sci. 1988, 550, 68. Axup, A. W.; Albin, M.; Mayo, S. L.; Crutchley, R. J.; Gray, Η. Β. J. Am. Chem. Soc. 1988, 110, 435. Jackman, M. P.; McGinnis, J.; Powls, R.; Salmon, G. Α.; Sykes, A. G. J. Am. Chem. Soc. 1988, 110, 5880. Osvath, P.; Salmon, G. Α.; Sykes, A. G. J. Am. Chem. Soc. 1988, 110, 7114. Nocek, J. M.; Liang, N.; Wallin, S. Α.; Mauk, A. G.; Hoflman, Β. M. J. Am. Chem. Soc. 1990, 112, 1623. Liang, N.; Mauk, A. G.; Pielak, G. J.; Johnson, J. Α.; Smith, M.; Hoflman, Science (Washington, D.C.) 1988, 240, 311. Liang, N.; Pielak, G. J.; Mauk, A. G.; Smith, M.; Hoflman, Β. M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 1249. Natan, M. J.; Hoffman, Β. M. J. Am. Chem. Soc. 1989, 111, 6468. Natan, M. J.; Kuila, D.; Baxter, W. W.; King, B. C.; Hawkridge, F. M.; Hoffman, J. Am. Chem. Soc. 1990, 112, 4081. McGourty, J. M.; Peterson-Kennedy, S. E.; Ruo, W. Y.; Hoffman, Β. M. Biochemistry 1987, 26, 8302. Gingrich, D. J.; Hoffman, Β. M., unpublished results. Kuila, D.; Natan, M. J.; Rogers, P.; Gingrich, D. J.; Baxter, W.; Arnone, Α.; Hoffman, Β. M. J. Am. Chem. Soc., unpublished results. Arnone, Α.; Rogers, P.; Blough, Ν. V.; McGourty, J. L.; Hoffman, Β. M. J. Mol. Biol. 1986, 188, 693. Gingrich, D. J.; Nocek, J. M.; Natan, M. J.; Hoffman, Β. M. J. Am. Chem. Soc. 1987, 109, 7533. Antonini, E.; Brunori, M. Hemoglobin and Myoglobin in Their Reactions with Ligands; North Holland Publishing Co.: Amsterdam, Netherlands, 1971.


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Hemoglobin

Hybrids

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23. Moffat, J. K.; Deatherage, J . F.; Seybert, D . W. Science (Washington, D.C.) 1979, 206, 1035. 24. Hoffman, B. M.; Ratner, M. R. J. Am. Chem. Soc. 1987, 109, 6237. 25. Hoffman, Β. M.; Ratner, Μ. Α.; Wallin, S. A . In Electron Transfer in Biology and the Solid State; Johnson, M. K.; King, R. B.; Kurtz, D. M., J r . ; Kutal, C.; Norton, M. L.; Seott, R. Α., E d s . ; Advances in Chemistry Series 226; American Chemical Society: Washington, D C , 1990; pp 125-146. 26. Brunschwig, B. S.; Sutin, N. J. Am. Chem. Soc. 1989, 111, 7454. 27. M c L e n d o n , G . ; Pardue, K . ; Bak, P. J. Am. Chem. Soc. 1987, 109, 7540. 28. Cox, R. P.; Holloway, M. R. Eur. J. Biochem. 1977, 74, 575. 29. Olivas, E.; deWaal, D . J. Α.; Wilkins, R. G. J. Biol Chem. 1977, 252, 4038. 30. Kuila, D.; Baxter, W. W.; Natan, M. J.; Hoffman, Β. M. J. Phys. Chem. 1991, 95, 1. R E C E I V E D for review April 27, 1990. A C C E P T E D revised manuscript August 17, 1990.


Long-Range Electron Transfer Within Mixed-Metal Hemoglobin Hybrids

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