Utility of Proton Hyperfine Shifts in Elucidating the Electronic Structure

27 Utility of Proton Hyperfine Shifts in Elucidating the Electronic Structure of Horseradish Peroxidase Compounds I and II and Their Model Compounds Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch027

L. LATOS-GRAZYNSKI, ALAN L. BALCH, and GERD N. LA MAR University of California, Department of Chemistry, Davis, CA 95616

This chapter reviews proton ΝMR spectroscopic studies of the electronic and molecular structure of horseradish peroxidase (HRP) in its reactive forms. The resting state is characterized by a rigid heme pocket with the iron bound to an axial imidazole with intact N -H and a quantum mechanical admixture S =3/3,5/2spin state for the metal. HRP Compound II is characterized by very small hyperfine shifts in its proton NMR spectrum, and a complete assignment of the spectrum awaits further de velopments. The proton NMR spectrum of HRP Com pound I is unlike that of any previously characterized iron porphyrin, and supports the (FeO) /porphyrin π cation radical model. The heme environment of HRP Compound I is rigid, with evidence that the heme vinyl groups are locked in two in-plane positions. The spectra of various states of HRP are compared with those of relevant model compounds. Data obtained with carbene complexes of iron porphyrins suggest that the oxidizing equivalents in an iron-oxo complex may be stored by inserting an (formal) oxygen atom into an iron-nitrogen bond. Models for (FeO) porphyrin complexes are available through the reaction of N-methylimidazole with peroxo-bridged iron porphyrin dimers. 1

2+

2+

| " n e 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 c o n s t i t u t e s t h e t e r m i n a l s t e p i n the respiratory electron transport c h a i n . H o w e v e r , d i o x y g e n m e t a b o l i s m at o t h e r stages c a n a l s o r e s u l t i n t h e f o r m a t i o n o f t h e p a r t i a l l y 0065-2393/82/0201-0661$06.00/0 © 1982 A m e r i c a n C h e m i c a l Society Kadish; Electrochemical and Spectrochemical Studies of Biological Redox Components Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

662

BIOLOGICAL REDOX COMPONENTS

Downloaded by CORNELL UNIV on May 18, 2017 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch027

r e d u c e d species, hydrogen peroxide a n d superoxide. These species h a v e p o t e n t i a l t o x i c effects. T h e c a t a l a s e s a n d s u p e r o x i d e d i s m u t a s e s are e x i s t i n g e n z y m e s t h a t are c a p a b l e o f d e s t r o y i n g t h e s e s p e c i e s . A n other group o f enzymes, the peroxidases, c o u p l e the destruction o f h y d r o g e n p e r o x i d e w i t h the oxidation o f organic substrates. H o r s e r a d i s h p e r o x i d a s e ( H R P ) , a h e m e e n z y m e , is a r e a d i l y a v a i l a b l e r e p r e s e n t a t i v e o f t h i s g r o u p o f p e r o x i d a s e s a n d has r e c e i v e d c o n s i d e r a b l e s t u d y ( I , 2 ) . A n u m b e r o f d i f f e r e n t o x i d a t i o n states o f t h e p r o s t h e t i c g r o u p h a v e b e e n d e t e c t e d . T h e s e o x i d a t i o n states are s h o w n i n S c h e m e I, a l o n g w i t h the reaction p a t h w a y s that i n t e r c o n n e c t t h e m ( J , 2). T h e p r o d u c t i v e catalytic c y c l e i n v o l v e s : the resting e n z y m e , w h i c h contains iron(III); the green C o m p o u n d I ( H R P I); a n d the r e d C o m p o u n d II ( H R P II). T h e p h y s i o l o g i c a l significance o f the other o x i d a t i o n states s h o w n i n S c h e m e I is less c l e a r . M a n y d e t a i l s o f t h e m o l e c u l a r a n d e l e c t r o n i c s t r u c t u r e o f t h e H R P states r e m a i n u n r e solved, despite extensive studies u s i n g a w i d e variety o f physicoc h e m i c a l t e c h n i q u e s . S o m e o f these p o i n t s i n c l u d e the l o c a t i o n o f the o x i d i z i n g equivalents i n H R P I a n d II, a n d the role o f axial i m i d a z o l e deprotonation i n s t a b i l i z i n g these forms.

NMR Spectroscopy of Heme and Hemoproteins P r o t o n N M R h y p e r f i n e shifts c a n p r o v i d e , i n p r i n c i p l e , a w e a l t h o f e l e c t r o n i c a n d structural information about the paramagnetic center i n h e m o p r o t e i n s i n v a r i o u s o x i d a t i o n a n d s p i n states ( 3 , 4). T h e u n p a i r e d s p i n o f the h e m i n perturbs the proton resonances o f the prosthetic g r o u p , c o o r d i n a t e d l i g a n d s , a n d some o f the a m i n o a c i d s i n the n e i g h b o r h o o d o f the h e m e iron. T h i s perturbation permits the resolution o f m a n y p e a k s t h a t are s h i f t e d a w a y f r o m t h e g e n e r a l l y p o o r l y r e s o l v e d diamagnetic envelope [the region 0-10 ppm from sodium 3-(trimethylsilyl)-l-propanesulfonate ( D S S ) ] (3, 5). These hyperfine s h i f t e d r e s o n a n c e s are e x t r e m e l y s e n s i t i v e to t h e o x i d a t i o n , l i g a t i o n , a n d s p i n states o f t h e s y s t e m , a n d are i n f l u e n c e d b y t h e h e m e a p o p r o t e i n i n t e r a c t i o n t h a t c a n affect t h e o r i e n t a t i o n o f p o r p h y r i n b o u n d substituents ( p a r t i c u l a r l y v i n y l groups) a n d the p l a n e o f the axial imidazole ligands (3-23). E a c h p r e v i o u s l y c h a r a c t e r i z e d s p i n , l i g a t i o n , a n d o x i d a t i o n state ("state") o f i r o n p o r p h y r i n s has a characteristic s p e c t r a l pattern. I n m o s t cases, t h e h y p e r f i n e s h i f t p a t t e r n s c a n b e r a t i o n a l i z e d o n t h e b a s i s o f i n t e r a c t i o n o f a v a i l a b l e , s p i n - c o n t a i n i n g rf-orbitals o f i r o n a n d the a p p r o p r i a t e p o r p h y r i n a n d a x i a l l i g a n d orbitals. A n extensive re v i e w d e s c r i b e s t h e v a r i o u s d e r e a l i z a t i o n p a t h w a y s (4). W e u s e d c h a r a c t e r i s t i c a v e r a g e s h i f t v a l u e s ( T a b l e I) for w e l l - c h a r a c t e r i z e d states o f i r o n p o r p h y r i n s to assess t h e l i k e l i h o o d t h a t h y p e r f i n e s h i f t p a t t e r n s a c c o u n t for t h e p r o p e r t i e s o f t h e r e a c t i v e f o r m s o f H R P .

Kadish; Electrochemical and Spectrochemical Studies of Biological Redox Components Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


27.

LATOS-GRAZYNSKi

ET AL.

AH

Horseradish

2

Peroxidase

A H + H

Compounds

663

+

Scheme I. The productive catalytic cycle involves Native HRP, HRP I, and HRP II. The overall reaction catalyzed is H 0 + H A —> 2H Ο + A. 2

2

2

2

T w o i m p o r t a n t factors r e l e v a n t to o u r i n t e r p r e t a t i o n s have e m e r g e d from our previous studies on b o t h m o d e l s a n d more r e a d i l y c h a r a c t e r i z e d h e m o p r o t e i n s (i.e., m y o g l o b i n , a n d i n s e c t h e m o g l o b i n ) . F i r s t , the r e l a t i v e hyperfine shift o f b o t h p y r r o l e - b o u n d protons a n d m e t h y l g r o u p s o f the h e m e core m u s t b e o b s e r v e d to c h a r a c t e r i z e the h e m e state. T h i s r e s t r i c t i o n r e q u i r e s o b s e r v a t i o n s to b e m a d e o n i s o structural systems containing protoporphyrin (A, R = vinyl) a n d deut e r o p o r p h y r i n ( A , R = h y d r o g e n ) . U n a m b i g u o u s a s s i g n m e n t o f res o n a n c e s to p a r t i c u l a r p r o t o n s n e c e s s i t a t e s t h e u s e o f s t e r e o s p e c i f i c a l l y d e u t e r a t e d p o r p h y r i n s . S e c o n d , for a g i v e n state, m o d e l s a n d 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 a v e r a g e h y p e r f i n e s h i f t for a p a r t i c u l a r f u n c t i o n a l g r o u p (4). H o w e v e r , p r o t e i n - b o u n d h e m e i n v a r i a b l y e x h i b i t s a l a r g e , i n - p l a n e a n i s o t r o p y t h a t is m a n i f e s t e d b y a l a r g e s p r e a d o f h y p e r f i n e shifts i n a p a r t i c u l a r f u n c t i o n a l g r o u p . M o s t i m p o r t a n t l y , i n t h e l o w s p i n i r o n ( I I I ) state t h e p a t t e r n o f t h e m e t h y l h y p e r f i n e shifts c a n b e u s e d to u n i q u e l y d e t e r m i n e t h e o r i e n t a t i o n o f t h e a x i a l h i s t i d y l imidazole. A n o t h e r s p e c t r a l p r o p e r t y o f i n t e r e s t for t h e s e p a r a m a g n e t i c h e m o p r o t e i n s is t h e l i n e w i d t h , w h o s e r e l a t i v e v a l u e s r e f l e c t a n i n verse, sixth d e p e n d e n c e on the distance o f the p r o t o n i n v o l v e d from the p a r a m a g n e t i c center (7). A n a l y s i s o f l i n e w i d t h s b e c o m e s p a r t i c u l a r l y i m p o r t a n t i n cases w h e r e t w o p a r a m a g n e t i c c e n t e r s h a v e b e e n p r o p o s e d to e x i s t . T h e spectroscopic p r o b e f o u n d most useful i n c h a r a c t e r i z i n g the a x i a l h i s t i d i n e a n d t h e i r o n - i m i d a z o l e b o n d i n g is t h e r e s o n a n c e o f t h e



664

exchangeable i m i d a z o l e N r - H (B). A g a i n , the resonance position o f t h i s N i - H s i g n a l f a l l s i n t h e s a m e s p e c t r a l w i n d o w for i d e n t i c a l states o f m o d e l s a n d proteins. H e n c e , the detection o f this proton resonance e l i m i n a t e s t h e presence o f a d e p r o t o n a t e d i m i d a z o l e . H o w e v e r , shift d i f f e r e n c e s b e t w e e n m o d e l s a n d p r o t e i n s m a y a l l o w for a n i n t e r p r e t a tion o f the extent o f i m i d a z o l a t e character i m p o s e d on the axial l i g a n d b y hydrogen b o n d i n g to a protein-acceptor residue.


Proton NMR Studies of HRP R e s t i n g S t a t e . T h e r e s t i n g state c o n t a i n s i r o n ( I I I ) w i t h o n e a x i a l i m i d a z o l e l i g a n d ( 2 3 - 3 4 ) . A l t h o u g h p r o t o n N M R s p e c t r o s c o p y has not r e s o l v e d t h e q u e s t i o n (31-34) o f w h a t l i g a n d , i f a n y , o c c u p i e s t h e r e m a i n i n g a x i a l s i t e , i t has c o n t r i b u t e d v a l u a b l e i n f o r m a t i o n a b o u t o t h e r s i g n i f i c a n t 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 i n c l u d e t h e s p i n state o f t h e i r o n , t h e r i g i d i t y o f t h e h e m e c r e v i c e , a n d t h e state o f i m i d a z o l e d e p r o tonation. A s s h o w n later, the q u e s t i o n o f i m i d a z o l e N i - H protonation/ d e p r o t o n a t i o n is s i g n i f i c a n t for a l l f o r m s o f H R R T h e s p e c t r u m o f rest i n g H R P , a l o n g w i t h r e s o n a n c e a s s i g n m e n t s , is s h o w n i n F i g u r e 1. T h e r e d u c e d m a g n e t i c s u s c e p t i b i l i t y o f r e s t i n g state H R P , a l o n g w i t h i t s e l e c t r o n s p i n r e s o n a n c e ( E S R ) s p e c t r u m , l e d to t h e s u g g e s t i o n t h a t t h e iron was present i n a n u n u s u a l q u a n t u m m e c h a n i c a l a d m i x e d s p i n state w i t h f a n d f c h a r a c t e r (36-40). C o m p a r i s o n o f t h e p r o t o n N M R spectra o f native a n d deuteroheme reconstituted H R P confirms the m i x e d s p i n state b e c a u s e t h e o b s e r v e d s h i f t p a t t e r n i s m o s t s i m i l a r t o t h e s h i f t p a t t e r n s o f m o d e l c o m p o u n d s t h a t a l s o h a v e t h e a d m i x e d f-I state (11). T h e t e m p e r a t u r e d e p e n d e n c e o f the resonances o f the side chains r e f l e c t s a r i g i d h e m e p o c k e t . T h i s finding c o n t r a s t s w i t h o b s e r v a t i o n s m a d e o n m y o g l o b i n (JO, 41). T h e h i g h l y b u r i e d n a t u r e o f t h e h e m e e n v i r o n m e n t is d e m o n s t r a t e d further b y t h e e x t r e m e l y s l o w rate o f e x c h a n g e o f the Ν χ - H o f the p r o x i m a l h i s t i d i n e . D e t e c t i o n o f a b r o a d , b u t e x c h a n g e a b l e , p r o t o n at + 1 0 0 p p m , w h i c h is e s s e n t i a l l y t h e s a m e l o c a t i o n as f o u n d i n m y o g l o b i n , e n s u r e s t h a t t h e a x i a l i m i d a z o l e l i g a n d is p r e s e n t w i t h t h e N ^ H i n t a c t . M o r e o v e r , t h e i n s e n s i t i v i t y o f t h e N i - H resonance position to substrate b o n d i n g disproves the p r e v i o u s c o n t e n t i o n (42) t h a t s t a b i l i z a t i o n o f t h e s u b s t r a t e / p r o t e i n b i n d i n g i n volves h y d r o g e n b o n d i n g to the p r o x i m a l h i s t i d i n e N i - H . A d d i t i o n o f s o d i u m cyanide to resting H R P gives a hexacoordi n a t e d f o r m , w h i c h , a l t h o u g h p h y s i o l o g i c a l l y i r r e l e v a n t , is p a r t i c u l a r l y u s e f u l for s p e c t r o s c o p i c s t r u c t u r e d e t e r m i n a t i o n . T h e C u r i e b e h a v i o r o f the resonances o f the h e m e p e r i p h e r a l substituents a n d the observa t i o n o f a N i - H s i g n a l are s i m i l a r to t h o s e f e a t u r e s i n m e t c y a n o m y o g l o b i n . T h i s r e s u l t offers f u r t h e r c o n f i r m a t i o n o f t h e r i g i d n a t u r e o f t h e


LATOS-GRAZYNSKi ET AL.

27.

Horseradish

Peroxidase

Compounds

665


cd

100

80

60

40

Shift, In ppm from DSS Figure deutero 8-CH ; H > (?), 5-CH ; 3

a

8

3

20

0

-20

—*>

1. Proton NMR spectra (360 MHz) of: A, native HRP; and B, HRP in H 0, pH 7, 2 5 ° C . Assignments [11] A: a, NjH; b, c, 5-CH ; d, 3-CH ; e, 1-CH ; f, vinyl; g-l, vinyl, propionic acid proximal histidyl Η >^?); and m,n, vinyl Η . Β: a, NjH; b, c, 1-CH ; d, 8-CH ; e, 3-CH ; and h-m, propionic acid Η > (?), proximal histidyl Η . Spectra from Ref 3 5 . 2

2

3

3

3

β

3

β

3

3

α

8

β8

heme pocket a n d the presence o f an essentially neutral axial i m i d a z o l e . D e u t e r i u m l a b e l i n g studies establish that t h e axial i m i d a z o l e ' s p l a n e is o r i e n t e d a l o n g t h e N - F e - N a x i s o f P y r r o l e s 1 a n d 3 . S i g n i f i c a n t l y , d e u t e r o h e m e r e c o n s t i t u t i o n o f H R P r e v e a l s a 180° rota tion o f the p o r p h y r i n ( h e m e disorder), w h i c h m a y a c c o u n t for t h e dif ferential stabilities o f C o m p o u n d I formed from differently substituted p o r p h y r i n s ( v i d a infra). 2 +

H R P C o m p o u n d I I . H R P II contains a n ( F e O ) unit w i t h a trip l e t g r o u n d state a n d a n a x i a l h i s t i d y l i m i d a z o l e ( 3 7 , 38, 43-47). T h e proton N M R s p e c t r u m o f this form o b t a i n e d from d - d i a c e t y l d e u t e r o h e m e ( A , R = H , C O C D g r o u p s i n t h e 6- a n d 7 - p o s i t i o n s i n p l a c e o f t h e p r o p i o n i c a c i d s i d e c h a i n s ) r e c o n s t i t u t e d H R P is s h o w n i n F i g u r e 2. S i m i l a r spectra w e r e r e c o r d e d for n a t i v e H R P a n d for ferryl m y o g l o b i n , the green species obtained b y treating m e t m y o g l o b i n w i t h e x c e s s h y d r o g e n p e r o x i d e (49-51). I n a l l cases, t h e p a r a m a g n e t i c shifts a r e a m o n g t h e l o w e s t r e c o r d e d f o r a n y h e m e p r o t e i n . T h e p e a k s i n t h e 1 7 - 1 4 - p p m r e g i o n t e n t a t i v e l y h a v e b e e n as6

3




666

Figure 2. Proton NMR spectrum (360 MHz) of à^diacetyl deutero Compound II (15°C). Spectrum taken from Reference 48.

HRP

s i g n e d to h e m e m e t h y l g r o u p s ( 4 9 - 5 5 ) . H o w e v e r , t h e a c t u a l a s s i g n ment only can be m a d e w h e n suitable d e u t e r i u m l a b e l i n g studies, n o w i n progress, are c o m p l e t e d . T h e l o w s t a b i l i t y o f the H R P I I form has h a m p e r e d this w o r k . M o d e l c o m p o u n d s , N - M e l m P F e O ( N - M e l m is N - m e t h y 1 i m i d a z o l e , Ρ is a s y n t h e t i c p o r p h y r i n d i a n i o n ) r e c e n t l y w e r e o b t a i n e d . T h e i r p r o p e r t i e s are d i s c u s s e d i n a l a t e r s e c t i o n . H R P C o m p o u n d I.

T h e electronic a n d m o l e c u l a r structures o f

H R P I have b e e n the subjects o f extensive investigation. N u m e r o u s different

electronic structures

oxidized

species. These

have been

structures

for t h i s

highly

include: simple iron(V),

proposed

meso-

p o r p h y r i n a t t a c k e d i r o n ( I V ) , a n d i r o n ( I V ) w i t h a free r a d i c a l t h a t m a y b e b a s e d on an a m i n o a c i d s i d e c h a i n or o n the p o r p h y r i n ( p o r p h y r i n 7r-cation r a d i c a l ) . M a g n e t i c s u s c e p t i b i l i t y s t u d i e s (38)

i n d i c a t e that

H R P I has three u n p a i r e d electrons, a l t h o u g h data f r o m spectroscopy

(43,44, 52)

Môssbauer

i n d i c a t e that the i r o n e n v i r o n m e n t s i n H R P I

a n d H R P I I are v e r y s i m i l a r . T h e E S R , E N D O R

(52-54),

and elec

t r o n i c s p e c t r a (55, 56) o f H R P I a l l are consistent w i t h the p r e s e n c e o f a p o r p h y r i n cation r a d i c a l . H o w e v e r , i n the related species, C o m p o u n d I o f c y t o c h r o m e c peroxidase, the presence o f an a m i n o acid-based radical was detected b y E S R spectroscopy (57, 58). Initial proton N M R studies

(49-51)

i n d i c a t e d that h i g h s p i n

F e ( I V ) w a s present i n H R P I. T h i s interpretation w a s b a s e d o n the a p p a r e n t s i m i l a r i t i e s o f t h e h e m e m e t h y l shifts i n r e s t i n g H R P a n d H R P I , a n d h e n c e t h e p r e s e n c e o f a n a m i n o a c i d - b a s e d free r a d i c a l w a s r e q u i r e d . T h e use o f isotope l a b e l i n g a n d d e u t e r o h e m e later r e v e a l e d

(19,35) t h a t

substitution

the h y p e r f i n e shift pattern for H R P I (whose

p r o t o n N M R s p e c t r u m is s h o w n i n F i g u r e 3) is i n c o n s i s t e n t w i t h t h e p r e s e n c e o f h i g h s p i n i r o n ( I V ) , b u t l i k e w i s e d i d n o t c o r r e s p o n d to a n y


Horseradish Peroxidase Compounds




667


668

o f t h e p r e v i o u s l y c h a r a c t e r i z e d i r o n p o r p h y r i n states g i v e n i n T a b l e I . M o r e o v e r , q u a n t i t a t i v e analysis o f the l i n e w i d t h s o f the i n d i v i d u a l l y a s s i g n e d p e r i p h e r a l g r o u p s e l i m i n a t e d a n e a r b y a m i n o a c i d - b a s e d free r a d i c a l , a n d s u p p o r t e d t h e c a t i o n r a d i c a l f o r m u l a t i o n for t h e s e c o n d o x i d i z i n g e q u i v a l e n t . I n c o n t r a s t to o p t i c a l s p e c t r o s c o p y at l o w t e m perature, the h e m e m e t h y l l i n e w i d t h s i n the p r o t o n N M R spectra sup p o r t i d e n t i c a l o r b i t a l g r o u n d states for H R P I a n d d e u t e r o H R P I . T h e N M R s h i f t s h a v e n o t e n a b l e d i d e n t i f i c a t i o n o f t h e o r b i t a l g r o u n d state. T h e o r e t i c a l c a l c u l a t i o n s for t h e p r o p o s e d a state i n d i c a t e t h a t the s p i n d e n s i t y is l o c a l i z e d at t h e p y r r o l e n i t r o g e n a n d raeso-carbons ( 5 9 ) . T h e i n a b i l i t y to d e t e c t raeso-proton r e s o n a n c e s is i n d i r e c t s u p p o r t for t h e a state. P l a n n e d e x p e r i m e n t s to s e a r c h for t h e raeso-resonance by H - N M R spectroscopy w i t h raeso-deuterated H R P may provide direct support.


lu

lu

2

T h e p r o t o n N M R s p e c t r u m o f H R P I p r o v i d e s conformation o f the steric constraints i m p o s e d o n the m o t i o n o f the h e m e p e r i p h e r a l sub s t i t u e n t s b y t h e p r o t e i n . T h u s , t h e r e s o n a n c e s i d e n t i f i e d as t h e v i n y l α - p r o t o n s , i n fact, r e p r e s e n t t w o e n v i r o n m e n t s t h a t i n t e r c o n v e r t s l o w l y o n t h e N M R t i m e s c a l e a n d h a v e b e e n i n t e r p r e t e d as r e p r e s e n t i n g i n - p l a n e v i n y l g r o u p s that differ b y 180° i n rotational p o s i t i o n . A ra t i o n a l i z a t i o n o f t h e u n u s u a l c o n f o r m a t i o n i n v o l v e s a p o t e n t i a l r o l e for the v i n y l groups i n e x t e n d i n g the conjugated π - s y s t e m o f the p o r p h y r i n , t h e r e b y s t a b i l i z i n g t h e h i g h l y o x i d i z e d s p e c i e s . N a t i v e H R P I is m o r e stable than d e u t e r o p o r p h y r i n reconstituted H R P I. T h i s differen t i a l s t a b i l i t y m a y b e a t t r i b u t e d to t h e r o l e o f t h e v i n y l g r o u p s a n d t h e i r o r i e n t a t i o n i n s t a b i l i z i n g t h i s o x i d i z e d state.

Table I.

Typical Porphyrin Hyperfine Shifts of Iron Complexes and Hemoproteins (25°C, ppm) Porphyrin Functional Group

Oxidation State Fe(II)

Spin

Fe(III)

1 2 1/2 3/2 5/2

Fe(IV)

5/2" 1 2

a

c

a

CH

3

45 8 13 60 45 65

H

Il

-3 42 -20 0 65 55 -5 70

32 5 4

0-3 2-3 -8

35 38

-4 -9

a

Reference

a

60

15 9, 16 6, 8, 14 11, 12 10, 13 18 17

— C H , Η attached to peripheral pyrrole carbons. H«, Η vinyl protons. Pentacoordinated. Hexacoordinated. 3

6

β

c d


27.

LATOS-GRAZYNSKi ET AL. Ferrous [Fe(II)] H R P .

Horseradish Peroxidase Compounds T h e proton N M R s p e c t r u m o f the

669

Fe(II)

form o f H R P r e s e m b l e s that o f d e o x y m y o g l o b i n (59). I n particular, the Ni-H

proton

is r e s o l v e d , a n d

f a l l s i n a r e g i o n s i m i l a r to t h a t o f

d e o x y m y o g l o b i n . A protein conformational change m o d u l a t e d b y an other

titratable

paramagnetic

residue

induces

a significant change

shift. I n c o n j u n c t i o n

i n the

w i t h resonance R a m a n

t h e s e r e s u l t s s h o w t h a t t h e state o f h y d r o g e n b o n d i n g o f t h e p r o t o n is i n v o l v e d i n t h i s p r o c e s s ( 5 9 ,

Ni-H studies, Ni-H

60).


Models of Oxidizing Enzymes Carbene Complexes of Iron Porphyrins.

T h e vinylidene carbene

[ V C = : C = C ( p - C H C l ) 2 ] complexes of iron porphyrins, P F e ( V C ) , and 6

4

t h e i r o x i d a t i o n p r o d u c t s , P ( V C ) F e C l , p o s s i b l y m a y s e r v e as m o d e l s for H R P II a n d H R P I (61, 62). T h i s c l a i m w a s b a s e d o n s i m i l a r i t i e s i n the o p t i c a l spectra o f the m o d e l c o m p o u n d s a n d the e n z y m e

intermedi

ates, as w e l l as t h e m a g n e t i c s u s c e p t i b i l i t y o f P ( V C ) F e C l , w h i c h i n d i c a t e s a n S = f s p i n state. P r o t o n N M R spectra o f P F e ( V C ) alone a n d i n the presence o f pyridine

or

N-methylimidazole

indicate

that

this

molecule

is

d i a m a g n e t i c (S = 0), a n d h e n c e is i n a d e q u a t e as a m o d e l for H R P I I . T h e s e p r o t o n N M R spectra are, h o w e v e r , f u l l y consistent w i t h

the

existence of a c o m p l e x containing a normal iron p o r p h y r i n unit a n d a c a r b e n e l i g a n d a x i a l l y b o u n d to i r o n . T h e p r o t o n N M R s p e c t r a o f P ( V C ) F e C l e x h i b i t e d r e m a r k a b l e fea t u r e s (63).

C h a r a c t e r i s t i c s p e c t r a l patterns are p r e s e n t e d i n F i g u r e 4.

T h e peak assignments were verified b y selective deuteration. T h e ob servation o f four p y r r o l e p r o t o n resonances, t h r e e w i t h h y p e r f i n e shifts o f - 3 0 to - 5 0 p p m a n d a f o u r t h w i t h a s h i f t o f 12 p p m , is p a r t i c u l a r l y s t r i k i n g . T h i s observation, c o u p l e d w i t h the analysis o f the spectra o f the analogous c o m p l e x o b t a i n e d from d e u t e r o h e m i n

(which clearly

s h o w s 16 p o r p h y r i n m e t h y l r e s o n a n c e s ) i n d i c a t e s t h a t t h e p o r p h y r i n no longer retains

its f o u r f o l d s y m m e t r y .

Indeed,

the

carbene

inserted into an i r o n - n i t r o g e n b o n d on oxidation, a n d the

has

reversible

p r o c e s s i n t e r c o n v e r t i n g P F e ( V C ) a n d P ( V C ) F e C l is s h o w n i n E q u a t i o n 1. T h u s ,

four

unique

products

are

analogous

to

Structure

2

for

d e u t e r o h e m i n a n d h e n c e , t h e 16 m e t h y l r e s o n a n c e s . T h e s t r u c t u r e o f T P P ( V C ) F e C l was d e t e r m i n e d b y x-ray c r y s t a l l o g r a p h y

(64).

T h e s e o b s e r v a t i o n s r a i s e n e w p o s s i b i l i t i e s for t h e s t r u c t u r e o f t h e reactive forms o f o x i d i z i n g h e m e e n z y m e s . B e c a u s e the e n d - c a r b o n o f t h e c a r b e n e is i s o e l e c t r o n i c w i t h a n o x y g e n a t o m , c o m p l e x e s c o n t a i n i n g the F e 0

2

+

and F e O

s +

u n i t s m a y c h o o s e to s t o r e t h e i r o x i d i z i n g

equivalents b y inserting an oxygen atom into an iron-nitrogen bond. C o n s e q u e n t l y , Structure 3 p o s s i b l y m a y be i n v o l v e d i n the c h e m i c a l




670

t r a n s f o r m a t i o n s o f o x o - i r o n c o m p l e x e s . S u c h a s t r u c t u r e for t h e a c t i v e h y d r o x y l a t i n g f o r m o f c y t o c h r o m e P - 4 5 0 w o u l d a p p e a r to b e a n i n triguing possibility. Ferryl (FeO) C o m p l e x e s of Iron Porphyrins. T w o groups of iron(IV) porphyrin complexes were examined. Electrochemical oxida tion o f iron(III) c o m p l e x e s P F e X a n d P F e O F e P p r o d u c e the i r o n ( I V ) species P F e X a n d P F e O F e P , a n d the m i x e d valence species PFeOFeP (17, 5 5 , 5 6 ) . O n l y P F e X is r e l e v a n t as a m o d e l for a monomeric heme enzyme. 2 +

+

2 +

+

+

A n o t h e r g r o u p o f i r o n ( I V ) p o r p h y r i n s is a v a i l a b l e v i a E q u a t i o n 2 ( 1 8 ) , w h e r e Β m a y b e p y r i d i n e or N - m e t h y l i m i d a z o l e . T h e s e c o m p l e x e s are 2PFe(II) + 0

2

-> P F e ( I I I ) O O F e ( I I I ) P -4 2 B P F e ( I V ) 0

(2)

s t a b l e i n s o l u t i o n at l o w t e m p e r a t u r e ( - 8 0 ° to ~ - 2 0 ° C ) a n d w e r e characterized b y their proton N M R a n d electronic spectra. T h e i r m a g n e t i c s u s c e p t i b i l i t i e s a n d the C u r i e b e h a v i o r o f the hyperfine shifts i n d i c a t e t h a t t h e c o m p l e x e s h a v e a t r i p l e t g r o u n d state. T h e


27.

LATOS-GRAZYNSKI ET A L .

ability

of BPFeO

Horseradish

to transfer

Peroxidase

its o x y g e n

Compounds

atom

671

quantitatively to

t r i p h e n y l p h o s p h i n e a n d form t r i p h e n y l p h o s p h i n e o x i d e verifies the 2

1

existence o f the FeO " " u n i t i n this r e a c t i v e i n t e r m e d i a t e (65). F u r t h e r w o r k c o m p a r i n g t h e c h e m i c a l shifts o f i d e n t i c a l f u n c t i o n a l g r o u p s i n t h e s e m o d e l c o m p o u n d s w i t h t h o s e o b s e r v e d for H R P I I a r e n e c e s s a r y . P r e l i m i n a r y s t u d i e s o n ( B ) O E P F e O ( O E P is t h e d i a n i o n o f o c t a e t h y l p o r p h y r i n ) i n d i c a t e t h a t t h e meso-proton r e s o n a n c e a p p e a r s at 14 p p m d o w n f i e l d (extrapolated to 2 5 ° C ) from tetramethylsilane ( T M S ) . T h i s assignment suggests that the d o w n f i e l d hyperfine shifted resonances o f H R P II actually may b e

raeso-protons

r a t h e r t h a n t h e h e m e m e t h y l s , as


o r i g i n a l l y a s s i g n e d (49-51). F u r t h e r s t u d i e s o f t h i s p r o b l e m a r e i n progress.

J

J

I

I

I

50

I

I

I

0

Figure 4. Proton NMR spectra DPDME(VC)FeCl in CDCl /25°C role; and m, methyl, propionic ?

I

I

ppm

I

L

-50

(360 MHz) of: I , TPP(VC)FeCl and 2 , (VC, vinylidene; Ph, phenyl; p, pyracid H > ). Spectra taken from Reference 63. a

s



672 Acknowledgments

We thank our collaborators Κ. M . Smith, J. deRopp, D . H . Chin, and R. J. Cheng for their assistance, and the National Institutes of Health (HL-16087, GM-26226), and the U C D N M R Facility for support.


Literature

Cited

1. Dunford, H. B.; Stillman, J. S. Coord. Chem. Rev. 1976, 19, 182-251. 2. Hewson, W. D.; Mayer, L. P. In "The Porphyrins"; Dolphin, D., Ed.; Academic: New York, 1979; Vol. 7, pp. 295-332. 3. La Mar, G. N. In "Biological Application of Magnetic Resonance"; Shul man, R. G., Ed.; Academic: New York, 1979; pp. 61-157. 4. La Mar, G. N.; Walker, F. A. In "The Porphyrins"; Dolphin, D., Ed.; Academic: New York, 1979; pp. 61-157. 5. Morrow, J. S.; Gurd, F. R. N.; Crit. C. R. C. Rev. Biochem. 1975, 3, 221-287. 6. Mayer, Α.; Ugame, S.; Shulman, R. G.; Uamane, T.; Cavaleiro, J. A. S.; Rocha-Gonsalves, A. M. d'A.; Venner, G. W.; Smith, K.M.;J.Mol. Biol. 1974, 86, 749-756. 7. Swift, T. J. In "NMR of Paramagnetic Molecules"; La Mar, G. N.; Hor rocks, Jr., W. D.; Holm, R. H., Eds.; Academic: New York, 1973, pp. 53-83. 8. La Mar, G. N.; Budd, D. L.; Viscio, D. B.; Smith, K. M.; Langry, K. C. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 5755-5759. 9. La Mar, G. N.; Budd, D. L.; Sick, H.; Gersonde, K., Biochim. Biophys. Acta 1978, 537, 270-283. 10. La Mar, G. N.; Budd, D. L.; Smith, Κ. M.; Langry, K. C.J.Am. Chem. Soc. 1980, 102, 1822-1827. 11. La Mar, G. N.; deRopp, J. S.; Smith, K. M.; Langry, K. C. J. Biol. Chem. 1980, 255, 6646-6652. 12. Goff, H.; Shimomura, E. J. Am. Chem. Soc. 1980, 102, 31-37. 13. Budd, D. L.; La Mar, G. N.; Smith, K. M.; Nayyir-Mazhir, R.J.Am. Chem. Soc. 1979, 101, 6091-6096. 14. La Mar, G. N.; Viscio, D. B.; Smith, K. M.; Caughey, W. S.; Smith, M. L. J. Am. Chem. Soc. 1978, 100, 8085-8092. 15. Goff, H.; La Mar, G. N.; Reed, C. A. J. Am. Chem. Soc. 1977, 99, 36413646. 16. Goff, H.; La Mar, G. N.J.Am. Chem. Soc. 1977, 99, 6599-6606. 17. Phillipi, Μ. Α.; Goff, H. J. Am. Chem. Soc. 1979, 101, 7641-7643. 18. D.-H. Chin; Blach, A. L.; La Mar, G. N. J. Am. Chem. Soc. 1980, 102, 1446-1448. 19. La Mar, G. N.; de Ropp, J. S.; Smith, Κ. M.; Langry, K. C. J. Biol. Chem. 1981, 256, 237-243. 20. Williams, R.J.P.; Wright, P. E.; Mazza, G.; Ricard,J.R. Biochim. Biophys. Acta 1975, 412, 127-147. 21. Morishima, I.; Ogawa, S. J. Biol. Chem. 1979, 254, 2814-2820. 22. Izuha, T.; Ogawa, S.; Inubushi, T.; Yonezawa, T.; Morishima, I. FEBS Lett. 1976, 64, 156-158. 23. Morishima, I.; Ogawa, S.; Inubushi, T.; Yonezawa, T.; Izuha, T. Biochemistry 1977, 16, 5109-5115. 24. George, P.; Lyster, R. L. Proc. Nat. Acad. Sci. U.S.A. 1974, 44. 25. Yamada, H.; Yamazaki, I. Arch. Biochem. Biophys. Acta 1974, 165, 728738. 26. Richard,J.;Marne, G.; Williams, R.J.P. Eur.J.Chem. Biochem. 1972, 28, 566-578.



27.


Horseradish Peroxidase Compounds

673

27. Morrison, R.; Schonbaum, G. R. Annu. Rev. Biochem. 1976, 45, 867-888. 28. Phelps, C.; Forlani, L.; Antonini, E . Biochem.J.1971, 124, 605-614. 29. Yonetani, T.; Yamamoto, H.; Erman, J. E.; Leigh, Jr., J. S.; Reed, G. H. J. Biol. Chem. 1972, 247, 2447-2455. 30. La Mar, G. N.; de Ropp,J.S. Biochem. Biophys. Res. Commun. 1979, 90, 36-41. 31. Lanir, Α.; Schejter, A. Biochem. Biophys. Res. Commun. 1975, 62, 199203. 32. Vak-Pavlovic, S.; Siterer, Y. Biochem. Biophys. Res. Commun. 1977, 79, 885-891. 33. Spiro, T. G.; Strong, J. D.; Stein, P.J.Am. Chem. Soc. 1979, 101, 26482655. 34. Rakshit, G., Spiro, T. G. Biochemistry 1974, 13, 5317-5323. 35. La Mar, G. N.; deRopp, J. S. J. Am. Chem. Soc. 1980, 102, 395-397. 36. Leigh, J. S.; Maltempo, M. M.; Ohlsson, P. I.; Paul, K. G. FEBS Lett. 1975, 51, 304-308. 37. Maltempo, M. M.; Ohlsson, P. I.; Paul, K. G.; Petersson, L.; Ehrenberg, A. Biochemistry 1979, 18, 2935-2941. 38. Theorell, H.; Ehrenberg, A. Arch. Biochem. Biophys. 1952, 41, 442-461. 39. Schonbaum, G. R. J. Biol. Chem. 1973, 248, 502-511. 40. Tamura, M. Biochim. Biophys. Acta 1971, 243, 249-258. 41. La Mar, G. N.; Viscio, D. B.; Gersonde, K.; Sick, H. Biochemistry 1978, 17, 361-367. 42. Schejter, Α.; Lanir, Α.; Epstein, N. Arch. Biochem. Biophys. 1976, 174, 36-44. 43. Maeda, Y.; Morita, Y. Biochem. Biophys. Res. Commun. 1967, 29, 680685. 44. Moss, T. -H.; Ehrenberg, Α.; Bearden, A. J. Biochemistry 1969, 8, 41594162. 45. Mauk, M. R.; Girotti, A. W. Biochemistry 1974, 13, 1757-1763. 46. Mayer, L. P.; Doubek, D. L.; Silverstein, R. M.; Margis,J.H.; Martin,J.C. J. Am. Chem. Soc. 1972, 94, 4364-4366. 47. Loew, G. H.; Kert, C. H.; Jhelmeland, L. M.; Kirchner, R. F.J.Am. Chem. Soc. 1977, 99, 3534-3536. 48. deRopp, J. S. Ph.D. Thesis, University of California, Davis, 1981. 49. Morishima, I.; Ogawa, S. J. Am. Chem. Soc. 1978, 100, 7125-7127. 50. Morishima, I.; Ogawa, S. Biochem. Biophys. Res. Commun. 1978, 83, 946-953. 51. Morishima, I.; Ogawa, S. Biochemistry 1978, 17, 4384-4388. 52. Schulz, C. E.; Devaney, P. W.; Winkler, H.; Debrunner, P. G.; Doan, N.; Chiang, R.; Rutter, R.; Hager, L. P. FEBS Lett. 1979, 103, 102-105. 53. Aasa, R.; Vanngard, T.; Dunford, H. B. Biochim. Biophys. Acta 1975, 391, 259-264. 54. Roberts, J. E.; Hoffman, B. H.; Rutter, R.; Hager, L. P.J.Biol. Chem. 1980, 256, 2118-2121. 55. Dolphin, D.; Felton, R. H.; Borg, D. C.; Fajer,J.J.Am. Chem. Soc. 1970, 92, 743-745. 56. Dolphin, D.; Forman, Α.; Borg, D. C.; Fajer,J.;Felton, R. H. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 614-618. 57. Yonetani, T.; Schleyer, M.; Ehrenberg, A. J. Biol. Chem. 1966, 241, 3240-3247. 58. Hottman, B. M.; Roberts, J. G.; Brown, T. G.; Kang, C. H.; Margoliash, E . Proc. Natl. Acad. Sci. U.S.A. 1978, 76, 6132-6136. 59. La Mar, G. N.; deRopp, J. S. J. Am. Chem. Soc. in press. 60. Teroka, J.; Kitagawa, T. Biochem. Biophys. Res. Comm. 1980, 93, 694700. 61. Mansuy, D.; Longe, M.; Chottard, J. C. J. Am. Chem. Soc. 1979, 101, 6437-6438. 62. Mansuy, D.; Pure Appl. Chem. 1980, 52, 681-690.


674


63. Latos-Grazynski, L.; Cheng, R.-J.; La Mar, G. N . ; Blach, A. L .J.Am. Chem. Soc. 1981, 102, 4270-4272. 64. Cheng, R.-J; Olmstead, M. M.; Balch, A. L., unpublished data. 65. Chin, D.-H.; La Mar, G. N.; Balch, A. L.J.Am. Chem. Soc. 1980, 102, 5945-5947. 66. Tamura, M.; Hori, H. Biochim. Biophys. Acta 1972, 289, 20-29. 67. Hanson, L. K.; Chang, G. K.; Davis, M. S.; Fajer, J. J. Am. Chem. Soc. 1981, 103, 663-670.


RECEIVED for review June 12, 1981. A C C E P T E D December 7, 1981.


Utility of Proton Hyperfine Shifts in Elucidating the Electronic Structure

Recommend Documents