14 Electrochemistry of Protoporphyrin IX Compared to Synthetic Models 1
KAREY L. H O L L A N D and JOSEPH JORDAN
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Pennsylvania State University, Department of Chemistry, University Park, PA 16802
Normal pulse polarography and cyclic voltammetry of the free base protoporphyrin IX (PP) yielded well -defined reduction waves in aqueous solutions in the presence of tetramethylammonium hydroxide. Elec troreduction occurred in three discrete steps, which were each coulometrically identified as two-electron transfers. Each step was characterized as an EC'-type mechanism, with proton transfer as the rate-determining reaction. All reduction sites on PP were methine bridges yielding, successively, phlorin, porphomethene, and porphyrinogen structures. Remarkably, these products are entirely analogous to the electroreduction products of porphyrin c and differ significantly from the chlorin-type products obtained from Collman's picket fence porphyrin. The picket fence porphyrin is a vital component of a synthetic myoglobin model, but undergoes electroreduction at different acceptor sites, viz., at pyrrolic double bonds. h e e l e c t r o n t r a n s f e r k i n e t i c s o f c y t o c h r o m e c a r e r e m a r k a b l y fast: t h e rate c o n s t a n t o f c e l l u l a r r e s p i r a t i o n i s o n t h e o r d e r o f 1 0 t o 1 0 L / m o l · s ( I ) a n d t h e rate o f t h e cross r e a c t i o n 7
9
f e r r o c y t o c h r o m e c 4- F e ( C N ) i ~ = f e r r i c y t o c h r o m e c + F e ( C N ) e ~ (1) 7
is c o m p a r a b l e (k = 1 0 L / m o l · s) (2, 3). O n t h e o t h e r h a n d , t h e a n a l o gous reaction o f h e m o g l o b i n : hemoglobin + F e ( C N ) i " = methemoglobin + Fe(CN)J"
(2)
1
Current address : International Business Machines, Inc., General Technology Divi sion, Essex Junction, VT 05452 0065-2393/82/0201-0313$06.00/0 © 1982 A m e r i c a n C h e m i c a l Society In Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.
BIOLOGICAL REDOX COMPONENTS
314
is t h r e e o r d e r s o f m a g n i t u d e s l o w e r (4). T h e v i e w is w i d e s p r e a d t h a t t h e e l e c t r o n transfer, w h i c h c o n v e r t s t h e c e n t r a l i r o n a t o m f r o m t h e d i v a l e n t to t h e t r i v a l e n t ( f o r m a l ) o x i d a t i o n state v i a R e a c t i o n s 1 a n d 2 , occurs t h r o u g h the plane o f the equatorial p o r p h y r i n l i g a n d s o f the p r o s t h e t i c g r o u p s (5). I n t h i s c o n t e x t , t h e e l e c t r o o x i d a t i o n - r e d u c t i o n p r o p e r t i e s o f p o r p h y r i n s are o f o b v i o u s r e l e v a n c e . I n a p r e v i o u s s t u d y (6, 7), w e r e p o r t e d that the e l e c t r o r e d u c t i o n o f p o r p h y r i n c p r o c e e d e d v i a a n E C m e c h a n i s m i n v o l v i n g a t w o - e l e c t r o n transfer f o l l o w e d b y p r o t o n a t i o n . T h e p r o d u c t w a s a p h l o r i n , I , t h a t is, t h e r e d u c t i v e a t t a c k
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o c c u r r e d at a m e t h i n e b r i d g e . A t m o r e n e g a t i v e p o t e n t i a l s , t h e s a m e electroreductive
pattern
recurred
i m p l i c a t i n g successive
methine
bridges yielding, i n turn, a porphomethene, II, and a porphyrinogen, I I I . T h i s t y p e o f b e h a v i o r , t h a t is, e l e c t r o r e d u c t i v e a t t a c k at m e t h i n e b r i d g e p o s i t i o n s , is c o m m o n t o n u m e r o u s p o r p h y r i n s (8-12) a n d w a s i n f e r r e d ( 9 ) , b y a n a l o g y , for p r o t o p o r p h y r i n I X (the p r o s t h e t i c g r o u p o f h e m o g l o b i n a n d m y o g l o b i n ) . O n the other h a n d , i n c h e m i c a l r e d u c t i o n s o f p o r p h y r i n s (13) t h e i n i t i a l e l e c t r o n a c c e p t o r s i t e is a p y r r o l e d o u b l e b o n d ( r a t h e r t h a n a m e t h i n e b r i d g e ) , a n d t h e p r o d u c t is a c h l o rin,
I V , (rather t h a n a p h l o r i n ) . R e m a r k a b l y , recent studies (12,
r e v e a l e d t h a t C o l l m a n ' s p i c k e t f e n c e p o r p h y r i n (15)
I (phlorin)
III (porphyrinogen)
Π
14)
[tetra(a,a,a,a-o-
(porphomethene)
I V (chlorin)
In Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.
HOLLAND A ND JORDAN
14.
Protoporphyrin IX
315
porphin p r o t o p o r p h y r i n : 1,3,5,8 C H 3 ; 2 , 4 C H = C H ; 6,7 Downloaded by UNIV OF SYDNEY on September 24, 2015 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch014
2
CH CH COOH 2
2
p o r p h y r i n c: 1,3,5,8 C H ; 2,4 C H ( C H 3 ) S C H C H ( N H ) C O O H ; 6,7 3
2
2
CH CH COOH 2
2
h e m a t o p o r p h y r i n : 1,3,5,8 C H 3 ; 2,4 C H C H O H ; 6,7 2
CH CH COOH
2
2
2
p i c k e t fence p o r p h y r i n : t e t r a ( a , a , a , a - o - p i v a l a m i d o p h e n y l ) p o r p h i n p i v a l a m i d o p h e n y l ) p o r p h i n ] , w h i c h is t h e
equatorial ligand of iron
i n a synthetic m y o g l o b i n m o d e l , was e x c e p t i o n a l i n that it y i e l d e d a c h l o r i n b y electroreduction (via an E C E m e c h a n i s m d e s c r i b e d else where). This unexpected
finding
suggests that the
electroreduction
of p r o t o p o r p h y r i n m a y p r o c e e d b y a m e c h a n i s m analogous to that of the
p i c k e t fence
porphyrin (yielding a chlorin). Alternatively,
protoporphyrin may produce
a phlorin in a manner
s i m i l a r to
t y p i c a l behavior o f most other porphyrins. S u r p r i s i n g l y , no
the
defini
t i v e v o l t a m m e t r i c i n v e s t i g a t i o n o f t h e p r o t o p o r p h y r i n free b a s e h a s b e e n r e p o r t e d i n a q u e o u s s o l u t i o n to d a t e , a l t h o u g h t h e a q u e o u s p r o t o p o r p h y r i n d i a c i d w a s t h e s u b j e c t o f o n e p a p e r (16). T h e k i n e t i c s a n d m e c h a n i s m s o f e l e c t r o r e d u c t i o n o f t h e a q u e o u s p r o t o p o r p h y r i n free b a s e , as r e v e a l e d t h r o u g h p o l a r o g r a p h y , c y c l i c v o l t a m m e t r y ,
and
c o u l o m e t r y , are r e p o r t e d a n d d i s c u s s e d i n t h i s c h a p t e r .
Experimental G e n e r a l . A l l potentials reported i n this chapter were referred to the aqueous saturated c a l o m e l reference electrode, whose potential is +0.2412 V vs. the normal hydrogen electrode ( N H E ) at 25°C. T h e sign convention of the International U n i o n of Pure a n d A p p l i e d C h e m i s t r y ( I U P A C ) is used through out, that is, the more anodic (oxidizing) a potential, the more positive the assignment. A l l current values reported were corrected for residual current. U n c o m p e n s a t e d I R drops were n e g l i g i b l e under the experimental conditions. N u m e r i c a l assignments for electrochemical rate parameters are "apparent v a l ues," that is, they were not corrected for d o u b l e layer effects (even though these may have been appreciable due to specific adsorption). P r e c i s i o n is ex pressed as the standard deviation of the mean [s/(n) ]. 112
In Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.
BIOLOGICAL REDOX COMPONENTS
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C h e m i c a l s . P r o t o p o r p h y r i n I X d i s o d i u m salt, obtained from I C N B i o c h e m i c a l , was converted to the free a c i d b y ether extraction (17) a n d crystal l i z e d b y solvent evaporation. P u r i t y was ascertained b y spectrophotometry a n d b y elemental analysis (Schwartzkopf Analytical). T e t r a m e t h y l a m m o n i u m h y d r o x i d e ( T M A H ) ( 1 M ) was purchased from Southwestern A n a l y t i c a l C h e m icals, Inc. A l l water was d e i o n i z e d and t r i p l y d i s t i l l e d . Instrumentation. T h e reference half-cell was an aqueous saturated c a l o m e l electrode ( S C E ) . T h e w o r k i n g electrode u s e d for pulse polarography was a conventional d r o p p i n g mercury electrode ( D M E ) , e q u i p p e d w i t h a drop knocker M o d e l 172 [ s u p p l i e d b y E G & G Princeton A p p l i e d Research C o r p . (PAR)]. A stirred mercury p o o l was e m p l o y e d for coulometry a n d a K e m u l a ' s h a n g i n g drop m e r c u r y electrode ( H D M E ) ( M e t r o h m M o d e l B M 5 - 0 3 , distrib uted b y B r i n k m a n n Instruments Inc.) was e m p l o y e d for c y c l i c voltammetry. A u x i l i a r y electrodes were mercury pools for polarography a n d c y c l i c voltam metry a n d p l a t i n u m gauze for coulometry. C y c l i c voltammetry a n d polarog raphy were performed i n a 15-ml water-jacketed c e l l ; water c i r c u l a t i n g through the water jacket was thermostated at (25.0 ± 0.1)°C. N o r m a l pulse polarography (drop time, 0.5 s; pulse w i d t h , 57 ms; current s a m p l i n g t i m e , 17 ms, mercury flow rate, 2.3 mg/s) was performed on P A R M o d e l s 170 a n d 174 E l e c t r o c h e m i c a l Systems. T h e P A R M o d e l 170 also was u t i l i z e d for c y c l i c voltammetry, a n d data acquisition at h i g h scan rates was e x p e d i t e d b y a m i n i c o m p u t e r . C o u l o m e t r i c experiments were carried out on both the P A R M o d e l 170 and a W e n k i n g potentiostat ( s u p p l i e d b y B r i n k m a n n Instruments, Inc.) i n c o m b i n a t i o n w i t h a P A R M o d e l 379 coulometer and a P A R 175 univer sal programmer. V i s i b l e spectra were obtained on a G a r y M o d e l 118 (Varian Associates) as w e l l as a H e w l e t t - P a c k a r d M o d e l 8 4 5 0 A Spectrophotometer (Hewlett-Packard Co.).
Results P r o t o p o r p h y r i n I X ( P P ) is a n a m p h o t e r i c m o l e c u l e , s o l u b l e o n l y i n a c i d i c m e d i a (as t h e d i c a t i o n ) o r b a s i c m e d i a ( w h e r e t h e p r o p i o n a t e groups are not protonated). T o s t u d y the e l e c t r o r e d u c t i o n o f the pro t o p o r p h y r i n free b a s e i n a n a q u e o u s e n v i r o n m e n t , a s t r o n g a l k a l i (0.5 M T M A H ) w a s e m p l o y e d as t h e s u p p o r t i n g e l e c t r o l y t e . T h e s t a b i l i t y of P P i n aqueous T M A H was ascertained spectrophotometrically: P P w a s s t a b l e for at l e a s t 2 0 d a y s . Polarography. Conventional polarography (using drop times of 2 - 4 s) o f P P r e v e a l e d t h r e e r e d u c t i o n w a v e s , o n l y t h e m o s t p o s i t i v e (anodic) b e i n g w e l l - d e f i n e d . O n the other h a n d , n o r m a l p u l s e p o l a r o g r a p h y u s i n g d r o p s k n o c k e d o f f at t = 0.5 s, p r o d u c e d t h r e e w e l l defined waves, p r e s u m a b l y because adsorption was m i n i m i z e d . T h e w a v e s are s h o w n i n F i g u r e 1 a n d a r e d e s i g n a t e d as W a v e s Χ , Ύ, a n d Z . T h e l i m i t i n g currents o f W a v e s X , Y , a n d Ζ w e r e p r o p o r t i o n a l to c o n c e n t r a t i o n i n t h e r a n g e o f 1 0 " t o 10~ M P P a n d iJC ( w h e r e id is t h e d i f f u s i o n l i m i t e d c u r r e n t a n d C is t h e b u l k c o n c e n t r a t i o n ) w a s a p p r o x i m a t e l y 1.5 μ Α / m m o l / L . S t u d i e s o f t h e l i m i t i n g c u r r e n t s as a f u n c t i o n o f m e r c u r y c o l u m n h e i g h t (h, a p p r o p r i a t e l y c o r r e c t e d for b a c k 4
2
In Electrochemical and Spectrochemical Studies of Biological Redox Components; Kadish, K.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.
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14.
HOLLAND A ND JORDAN
317 Protoporphyrin
IX
Potential ( V o l t ) Figure 1. Normal pulse polarograms of 9.1 x 10^* M protoporphyrin. Supporting electrolytes: pH 13.3, 0.5 m aqueous TMAH; pH 12.4, mix ture of TMAH + TMACl. Curves shifted arbitrarily along ordinate axis. p r e s s u r e ) s u b s t a n t i a t e d d i f f u s i o n as t h e p r e d o m i n a n t c o n t r o l m e c h a n i s m . A s l i g h t effect o f a d s o r p t i o n w a s a p p a r e n t : t h e s l o p e o f t h e l o g l o g p l o t o f id vs. h w a s 0 . 5 5 , w h i c h c o m p a r e d t o 0 . 5 0 for p u r e d i f f u s i o n c o n t r o l . T o assess w h e t h e r N e r n s t i a n r e v e r s i b i l i t y p r e v a i l e d a n d t o estimate the n u m b e r o f F a r a d a y s transferred p e r m o l e o f P P (i.e., the η - v a l u e ) , t h e n o r m a l p u l s e p o l a r o g r a m s w e r e s u b j e c t e d to c o n v e n t i o n a l w a v e a n a l y s i s . L o g [i/(id ~ 0 ] w a s p l o t t e d v s . p o t e n t i a l i n a c c o r dance w i t h the equation
T h e r e l e v a n t p l o t s for W a v e s X a n d Y are s h o w n i n F i g u r e 2 , w h e r e
s
(
s
I
o
p
e
)
-dlog[
