UBC Theses and Dissertations

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UBC Theses and Dissertations

Peroxidatic & spectrophotometric characteristics of a 65-residue heme peptide derivative from cytochrome… Chang, Edwin 1986

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PEROXIDATIC & SPECTROPHOTOMETRIC CHARACTERISTICS OF A 65-RESIDUE HEME PEPTIDE DERIVATIVE FROM CYTOCHROME c_ By EDWIN CHANG B . S c , B r o c k U n i v e r s i t y , 1984 A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF -THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Depar tmen t o f B i o c h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e * i _ s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A u g u s t 1986 (c) Edwin Chang, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 )E-6 (3/81) A1£XBA£X 1-65H, a 65 amino a c i d r e s i d u e l o n g , h e m e - c o n t a i n i n g p e p t i d e , was p roduced by i n c u b a t i n g h o r s e h e a r t cy toch rome c_ i n a s o l u t i o n c o n t a i n i n g t w o - h u n d r e d f o l d excess o f CNBr. A c o m b i n a t i o n o f CO, CN , I m i d a z o l e , SCN and pH s p e c t r a l t i t r a t i o n s t u d i e s have d e m o n s t r a t e d t h a t t h e heme b i n d i n g s i t e o f 1-65H i s more exposed t o w a r d s t h e e x t e r n a l e n v i r o n m e n t . A consequence o f t h i s i n c r e a s e d exposu re i s t h e 88 t o 8 7 5 - f o l d a m p l i f i c a t i o n o f t h e p e r o x i d a s e a c t i v i t y i n 1-65H o v e r h o r s e h e a r t cy toch rome c_. The b i n d i n g o f exogenous l i g a n d s t o 1-65H i n h i b i t e d t h e p e r o x i d a s e a c t i v i t y t h u s d e m o n s t r a t i n g t h a t a n e c e s s a r y c o n d i t i o n f o r p e r o x i d a s e a c t i v i t y i s i n c r e a s e d a c c e s s i b i l i t y o f t he heme m o i e t y t o ^ 2 ^ 2 ' However t h e p e r o x i d a s e a c t i v i t y o f Horse R a d i s h P e r o x i d a s e (HRP) i s a p p r o x i m a t e l y t h r e e o r d e r s o f m a g n i t u d e g r e a t e r t h a n r a t e s f o r 1-65H i n d i c a t i n g t h a t heme exposu re i s n o t s u f f i c i e n t f o r f u l l p e r o x i d a s e f u n c t i o n . A second consequence o f g r e a t e r heme e x p o s u r e i s t h e ease i n wh ich exogenous l i g a n d s b i n d t o t h e heme g roup o f 1-65H when compared t o hhc_. The r e l a t i v e i n c r e a s e i n exogenous l i g a n d b i n d i n g has l e d t o a number o f i n t e r e s t i n g f i n d i n g s . Of s p e c i a l n o t e i s t h e d i s c o v e r y t h a t h o r s e h e a r t cy toch rome £. has a r e d o x - s t a t e dependent a f f i n i t y f o r CN b u t t h a t 1-65H d i s p l a y e d no such p r o p e r t y . As a r e s u l t , i t i s p roposed t h a t t h e n a t u r e o f t h e p r o t e i n - b o u n d a x i a l l i g a n d s p l a y a v i t a l r o l e i n d e t e r m i n i n g t h e r e d o x -s t a t e dependency o f heme a c c e s s i b i l i t y . Based on exogenous l i g a n d s u b s t i t u t i o n s t u d i e s on 1-65H, i t i s a l s o p roposed t h a t t h e t y p e o f a x i a l l i g a n d s i n f l u e n c e s t h e n a t u r e o f t h e S o r e t abso rbance b a n d . I n p a r t i c u l a r , e l e c t r o n - w i t h d r a w i n g a x i a l l i g a n d s w i l l r e d u c e t h e i n t e n s i t y o f t h e S o r e t abso rbance peak w h i l e e l e c t r o n - d o n a t i n g l i g a n d s w i l l i n c r e a s e t h e i n t e n s i t y o f t h e S o r e t abso rbance p e a k . ( i i ) TABLE Q£ CONTENTS ABSTRACT ( i i ) TABLE OF CONTENTS ( i i i ) L IST OF TABLES ( v ) L IST OF FIGURES ( v i ) ABBREVIATIONS USED ( v i i i ) ACKNOWLEDGEMENTS . . . ( i x ) INTRODUCTION O u t l i n e 1 I : T r u n c a t e d P e p t i d e s o f Horse H e a r t Cytochrome c_ (A) F r a g m e n t a t i o n o f Cytochrome c_ . . . . 6 (B) Fragment C o m p l e m e n t a t i o n S t u d i e s o f Cyt c_ 11 (C) S e m i s y n t h e s i s o f M o d i f i e d Cytochrome £. 15 I I : S t e r i c and E l e c t r o n i c C o n t r i b u t i o n s o f A x i a l L i g a n d s (A) S t e r i c F a c t o r s i n f l u e n c i n g Heme R e a c t i v i t y 17 (B) E l e c t r o n i c E f f e c t s o f A x i a l L i g a n d s 23 I I I : T h e s i s I n v e s t i g a t i o n : F u n c t i o n a l P r o p e r t i e s o f 1-65H 27 MATERIALS A M METHODS I : Reagents and Chemica ls 30 I I : I s o l a t i o n and P u r i f i c a t i o n o f 1-65H 31 I I I : P r o t o c o l f o r t h e P e r o x i d a s e Assay 34 I V : L i g a n d B i n d i n g and L i g a n d S u b s t i t u t i o n S t u d i e s 34 V: pH T i t r a t i o n o f 1-65H . . 3 5 V I : S p e c t r o p h o t o m e t r y I n s t r u m e n t s Used . . . . 3 5 ( i i i ) V I I : H - N u c l e a r M a g n e t i c Resonance S p e c t r o s c o p y 36 RESULTS I : G e n e r a l C h a r a c t e r i s t i c s o f Cyt c_ 37 I I : L i g a n d B i n d i n g S t u d i e s o f 1-65H (A) CN _ A d d i t i o n 46 (B ) I m i d a z o l e A d d i t i o n 52 (C) SCN" A d d i t i o n 52 I I I : L i g a n d S u b s t i t u t i o n S t u d i e s f o r 1-65H 53 V: ^H-NMR S p e c t r a o f f r e e and cyano complexes o f o x i d i z e d 1-65H . . . . . 5 5 I V : P e r o x i d a s e Assay R e s u l t s 59 DISCUSSION I : Heme A c c e s s i b i l i t y o f 1-65H 64 I I : L i g a n d B i n d i n g E x p e r i m e n t s 65 I I I : L i g a n d S u b s t i t u t i o n E x p e r i m e n t s 67 I V : F u n c t i o n a l P r o p e r t i e s o f 1-65H . . . . 69 V: Summary . . . . 7 3 REFERENCES 74 APPENDIX I 78 ( i v ) L I S T Of TABLES TABLE I ; Amino a c i d c o m p o s i t i o n o f p u r i f i e d 1-65H. p. 39 TABLE I I : Lambda maxima and E x t i n c t i o n c o e f f i c i e n t maxima f o r v a r i o u s s p e c i e s o f 1 -65H. p . 49 TABLE I I I : Summary o f R e s u l t 6 f o r L i g a n d S u b s t i t u t i o n E x p e r i m e n t s , p . 57 TABLE I V ; C o m p a r a t i v e P e r o x i d a s e Rates o f 1 -65H, Horse Rad ish P e r o x i d a s e and Horse H e a r t Cytochrome £. a t pH 's 3 , 7 , and 1 0 . p . 60 ( v ) LIST OF FIGURES INTRODUCTION F i g . 1. D iag ram o f P r o t o p o r p h y r i n IX; t h e p r o s t h e t i c g roup f o r heme p r o t e i n s , p . 3 F i g . 2. Amino A c i d Sequence o f Horse H e a r t Cy tochrome p . 4 F i g . 3. R ibbon D iagram o f Horse H e a r t Cytochrome c_. p . 5 F i g . 4. B a l l and S t i c k Model o f a " P i c k e t Fence P r o p h y r i n " . p . 19 F i g . 5A. B i n d i n g Geormet ry o f 0„ and CO t o model Heme compounds, p . 20 £ ig j . 5B. D i s t o r t i o n o f L i g a n d B i n d i n g Geometry by t h e d i s t a l h i s t i d i n e s o f Hb. p . 20 F i g . 6. S y n t h e s i s pathway f o r " P i c k e t Pocke t P o r p h y r i n s " , p. 25 Eigj . ZJ. N a t u r e o f e l e c t r o n w i t h d r a w i n g and e l e c t r o n d o n a t i n g a x i a l l i g a n d s . p . 26 MATERIALS AND METHODS F i g . 8. F low D iag ram showing t h e i s o l a t i o n and p u r i f i c a t i o n o f 1-65H. p. 33 F i g . 9_t G-75 Ge l F i l t r a t i o n p r o f i l e f o r hhc_ t r e a t e d w i t h CNBr. p . 38 RESULTS F i g . 10. Soret absorbance as a function of 1-65H concentration, p . 40 F i g . 11. Reduced and oxidzed spectra of 1-65H at pH 7.0 . p. 41 F i g . 12. Absorption spectra of oxidized 1-65H at pH 3.0 and 7.0. p. 43 F i g . 13• pH t i t r a t i o n plot for the acid t r ans i t ion of 1-65H. p. 44 F i g . 14. Absorption spectra of the reduced C0-1-65H complex, p. 45 F i g . 15. Absorption spectra of the reduced and oxidized cyano-l-65H complex ( F i g . 15A and 15B), the oxidized imidazole-l-65H complex ( F i g . 15C and 15D) and the oxidized SCN-1-65H complex ( F i g . 15E and 15F). p. 48 (v i ) F i g . 16. ( a ) CN ( b ) I m i d a z o l e ( c ) SCN t i t r a t i o n p l o t s for 1-65H. p. 51 F i g . 1 7 . R e p r e s e n t a t i v e t r a c i n g o f a L i g a n d S u b s t i t u t i o n E x p e r i m e n t ( + S C N + I m i d a z o l e ) . p . 56 F i g . 1 8 . ^H-NMR s p e c t r a o f t h e f r e e and cyano fo rms o f 1-65H. p . 58 F i g . 1 9 . E f f e c t s o f ( a ) t e m p e r a t u r e , ( b ) Gdn.HCl c o n c e n t r a t i o n , ( c ) L i g a n d a d d i t i o n on t h e p e r o x i d a s e a c t i v i t y o f 1-65H. p . 62 ( v i i ) ABBREVIATIONS 1-65H - A h e m e - c o n t a i n i n g , 65 amino a c i d r e s i d u e l o n g f r a g m e n t o f h o r s e h e a r t cy toch rome c_. 1-80H - A h e m e - c o n t a i n i n g , 65 amino a c i d r e s i d u e l o n g f r a g m e n t o f h o r s e h e a r t cy toch rome c_. hhc_ - Horse h e a r t cy toch rome c_. HRP - Horse R a d i s h P e r o x i d a s e . Hb - H e m o g l o b i n . Mb - M y o g l o b i n . ( v i i i ) Acknowledgements I b e l i e v e I owe my d e a r e s t d e b t s t o L i n d s a y E l t i s and An thony L i m , who have p r o v i d e d i n v a l u a b l e i n s i g h t s , s u g g e s t i o n s , and c r i t i c i s m s . D r . B h a v i n i P a t e l has t a u g h t me a w e a l t h o f c h e m i c a l know ledge so i t i s o n l y a p p r o p r i a t e t h a t I acknowledge D r . P a t e l . I a l s o t h a n k D r . A . Gran t Mauk and D r . M a r c i a Mauk f o r b e i n g s t r i c t and c o n s c i e n t i o u s s u p e r v i s o r s . As f o r t h e p e o p l e o u t s i d e o f t h e Mauk l a b , I acknowledge D r . G e o f f r e y R. Moore and D r . C o l i n T i l c o c k f o r h e l p i n g me w i t h my NMR s p e c t r a . F i n a l l y , I acknowledge Gordon L o u i e f o r p r o v i d i n g me w i t h a f i g u r e showing the b a l l and s t i c k model o f t u n a cy toch rome c_ ( F i g . 3 ) . ( i x ) INTRODUCTION Heme p r o t e i n s can be c l a s s i f i e d i n t o t h r e e f u n c t i o n a l g r o u p s : 1 . Those t h a t f u n c t i o n i n t r a n s p o r t o f l i g a n d s such as 0^ o r CO2 ( e . g . m y o g l o b i n o r h e m o g l o b i n ) . 2 . Those t h a t f u n c t i o n i n e l e c t r o n t r a n s -f e r ( e . g . cy toch rome c_). 3. Those t h a t f u n c t i o n i n a c a t a l y t i c c a p a c i t y ( e . g . h o r s e r a d i s h p e r o x i -d a s e , c a t a l a s e o r c y t o c h r o m e P - 4 5 0 ) . D e s p i t e t h i s f u n c t i o n a l d i v e r s i t y , a l l t h r e e c l a s s e s share a common p r o s t h e t i c g r o u p , p ro toheme I X . D i f f e r e n t heme p r o t e i n s may have d i f f e r e n t heme p e r i p h e r a l s u b s t i t u e n t s t h a t may l e a d t o s i g n i f i c a n t a l t e r a t i o n s i n f u n c t i o n a l p a r a m e t e r s ( e . g . Re id e t a l . 1 9 8 4 , 1 9 8 6 ) . U s u a l l y , such m o d i f i c a t i o n s p roduce a q u a n t i t a t i v e a l t e r a t i o n i n t h e heme p r o t e i n ' s o r i g i n a l f u n c t i o n . For a c o n v e r s i o n f r o m one f u n c t i o n a l t y p e t o a n o t h e r , a l t e r a t i o n o f t h e heme b i n d i n g s i t e i s r e q u i r e d . L a r g e f r a g m e n t s o f cy toch rome £. have been used t o a d d r e s s i s s u e s o f t h i s l a t t e r t y p e by p e r m i t t i n g c o n s i d e r a t i o n o f t h e f o l l o w i n g t y p e s o f q u e s t i o n s : What a r e t h e minimum c o n d i t i o n s r e q u i r e d f o r t h e c o m p l e t e o r n e a r c o m p l e t e r e s t o r a t i o n o f o r i g i n a l a c t i v i t y ( i n t h e case o f h o r s e h e a r t cy toch rome c_, o r i g i n a l a c t i v i t y w o u l d be e l e c t r o n t r a n s f e r ) ? How much p e r t u r b a t i o n i s n e c e s s a r y t o c o m p l e t e a t r a n s f o r m a t i o n o f f u n c t i o n ? Cytochrome c_ has been a m a j o r f o c u s o f t h e s e s t u d i e s f o r a v a r i e t y o f r e a s o n s . I t i s s m a l l (MW=12,500) , m o n o m e r i c , s o l u b l e and s t a b l e . These c h a r a c t e r i s t i c s make cy toch rome c_ c o n v e n i e n t t o i s o l a t e and t o a n a l y z e . The 1 heme g roup ( F i g . 1) i s c o v a l e n t l y l i n k e d t o t h e p r o t e i n v i a two t h i o e t h e r l i n k a g e s ( F i g . 2) and l o c a t e d i n a h y d r o p h o b i c p r o t e i n e n v i r o n m e n t ( F i g . 3 ) . The c o v a l e n t a t t a c h m e n t o f t he heme g roup t o t h e p o l y p e p t i d e backbone p r e v e n t s t h e l o s s o f t h e p r o s t h e t i c g r o u p d u r i n g an i s o l a t i o n o r m o d i f i c a t i o n p r o c e d u r e . F i n a l l y , t h e f u n c t i o n a l c h a r a c t e r i s t i c s o f cy toch rome c_ a r e r e l a t i v e l y s i m p l e w i t h no a l l o s t e r i c i n t e r a c t i o n s and no m a j o r c o n f o r m a t i o n a l changes t o c o n s i d e r . The m o d i f i c a t i o n s t u d i e s r e p o r t e d f o r cy toch rome c_ can be c l a s s i f i e d i n t o t h r e e a r e a s : 1 . Chemica l m o d i f i c a t i o n o f s p e c i f i c amino a c i d r e s i d u e s on cy toch rome 2 . I s o l a t i o n and c h a r a c t e r i z a t i o n o f cy toch rome c_ m u t a n t s . 3 . I s o l a t i o n and c h a r a c t e r i z a t i o n o f t r u n c a t e d , h e m e - c o n t a i n i n g p e p t i d e s o f cy toch rome £.. The f i r s t two t y p e s o f s t u d i e s w i l l n o t be d i s c u s s e d as a comprehens ive t r e a t m e n t o f t h e s e t o p i c s i s beyond t h e scope o f t h i s t h e s i s . S e v e r a l r e c e n t and comprehens ive r e v i e w s have appeared on t h e s u b j e c t o f s p e c i f i c m o d i f i c a t i o n i n cy toch rome c_. ( F e r g u s o n - M i l l e r e_L a l . . 1979 ; M a r g o l i a s h , 1 9 8 2 ) . D e t a i l e d r e p o r t s a r e a l s o a v a i l a b l e c o n c e r n i n g cy toch rome £. m u t a n t s o b t a i n e d by c l a s s i c a l m u t a g e n i c t e c h n i q u e s ( e . g . Hampsey £_£. a l . . 1986) and by s i t e - d i r e c t e d m u t a g e n e s i s ( Z o l l e r and S m i t h , 1 9 8 3 ; P i e l a k e i . a_Lu, 1 9 8 5 ) . The m a j o r f i n d i n g s f r o m t h e t h i r d c l a s s o f s t u d i e s w i l l be s u r v e y e d a l o n g w i t h work c o n c e r n i n g model heme compounds and the i n f l u e n c e o f a x i a l l i g a n d s on b o t h p h y s i o l o g i c a l and model heme compounds. 2 N I H 2 CH2 CHt H2C H,C \ \ C02H C02H FIG 11 Heme £.: The prosthetic group for cytochrome c_. 3 10 NH 2 - G ly - Asp - V a l - G lu - Lys - G l y - Lys - Lys - l i e - Phe 20 V a l - G lu - Lys - CYS - A l a - G in - CYS - HIS. - Thr - Va l 30 Glu - Lys - G l y - G l y - Lys - H i s - Lys - Thr - G l y - Pro 40 Asn - Leu - H i s - G ly - Leu - Phe - G l y - A r g - Lys - Thr 50 G l y - G in - A l a - Pro - G l y - Phe - Thr - T y r - Thr - Asp 60 A l a - Asn - Lys - Asn - Lys - G l y - l i e - Thr - T rp - Lys 70 Glu - G lu - Thr - Leu - Met - G lu - T y r - Leu - G lu - Asn 80 Pro - Lys - Lys - T y r - H e - Pro - G l y - Thr - Lys - HEX 90 H e - Phe - A l a - G l y - H e - Lys - Lys - Lys - Thr - G lu 100 A r g - G lu - Asp - Leu - H e - A l a - T y r - Leu - Lys - Lys A l a - Thr - Asn - G lu - COOH Eigj. 2. Amino a c i d sequence f o r h o r s e h e a r t cy toch rome £.. Res idues s e r v i n g as a x i a l l i g a n d s ( H i s - 1 8 , M e t - 8 0 ) a r e d e s i g n a t e d by u n d e r l i n e d , c a p i t a l l e t t e r s . The c y s t i n e r e s i d u e s t h a t f o r m t h i o e t h e r l i n k a g e s t o t h e heme g roup ( C y s - 1 4 , C y s - 1 7 ) a r e d e n o t e d by c a p i t a l , b o l d -f a c e d l e t t e r s . 4 EI£ 3_i Diagram shoving the arrangement of the polypeptide backbone of cytochrome £.; the positions of the axial ligands (Met-80, His-18) and of Cya-14 and Cys-17 are labelled accordingly. The portion of the polypeptide backbone which is retained after CNBr treatment is shown in black. The non-solid portion of the backbone is the part that is cleaved off. Diagram courtesy of Gordon Louie. 5 J j . TRUNCATED PEPTIDES QL £ H C A l F r a g m e n t a t i o n S t u d i e s cJL c y t o c h r o m e c f a ) P h y s i c a l S t u d i e s o f Cytochrome c Heme P e p t i d e Fragments H i s t o r i c a l l y , t h r e e t e c h n i q u e s f o r t h e c l e a v a g e o f cy toch rome c_ have been d e v e l o p e d : 1. T r y p s i n d i g e s t s cy toch rome £. t o p roduce e i -t h e r a 3 8 - r e s i d u e , h e m e - c o n t a i n i n g f r a g m e n t ( 1 - 3 8 H ) o r a h e m e - c o n t a i n i n g u n d e c a p e p t i d e (11 -21H) (Fanger and H a r b u r y , 1 9 6 5 ) . 2 . P e p s i n d i g e s t s o f cy toch rome c_ t o p roduce a h e m e - c o n t a i n i n g f r a g m e n t w h i c h i s e i g h t r e s i d u e s l o n g ( 1 4 - 2 1 H ) ( H a r b u r y and L o a c h , 1 9 6 0 ) . 3 . Cyanogen b rom ide (CNBr) c l e a v e s cy toch rome c_ a t m e t h i o n i n e r e s i d u e s and l e a v e s a heme-con-t a i n i n g f r a g m e n t w h i c h i s 65 r e s i d u e s i n l e n g t h ( 1 - 6 5 H ) ( C o r r a d i n and H a r b u r y , 1 9 7 0 ) . Numerous reports have appeared that assess the f u n c t i o n a l p r o p e r t i e s of heme peptides prepared from cytochrome c_. Most studies have employed e i t h e r 11-21H or 14-21H as these two peptides are reasonably close to free hemes and at the same time are f a i r l y s o l u b l e i n aqueous s o l u t i o n s . These p r o t e i n fragments of t e n have been used as model compounds f o r cytochrome P-450 and p e r o x i d a s e - l i k e r e a c t i o n s . Wilson and Janson (1977) examined the e l e c t r o n i c spectra of 11-21H as a f u n c t i o n of pH and found i t to have three pH-dependent s p e c t r a l t r a n s i t i o n s w i t h pKa's at 7.6, 5.8 and 3.4. These values were concluded to 6 c o r r e s p o n d t o t h e d i s s o c i a t i o n f r o m t h e heme i r o n o f t h e p r o t o n a t e d ^ - N H ^ g roup i n l y s i n e - 1 3 , t h e O^-Nr^ g r o u p i n v a l i n e - 1 1 o r v a l i n e - 2 0 , and an u n i d e n t i f i e d i m i d a z o l e g roup r e s p e c t i v e l y . By way o f c o n t r a s t , h o r s e h e a r t cy toch rome c_ m a i n t a i n s a s t a b l e s t r u c t u r e f r o m pH 4 t o pH 12 (Moore and W i l l i a m s , 1 9 8 0 ) . These t i t r a t i o n d a t a f o r 11-21H a r e c o n s i s t e n t w i t h a more exposed heme p o c k e t as e x p e c t e d f r o m t h e 90% l o s s o f p e p t i d e backbone r e s u l t i n g f r o m e n z y m a t i c c l e a v a g e . A n o t h e r use o f heme p e p t i d e s has been t o assess t h e r o l e o f heme e n v i r o n m e n t h y d r o p h o b i c i t y on t h e r e d u c t i o n p o t e n t i a l o f t h e heme c e n t r e . F o l l o w i n g t h e s u g g e s t i o n by Kassner ( 1 9 7 2 ) t h a t t h e m i d - p o i n t p o t e n t i a l o f a heme g roup i s a measure o f heme e x p o s u r e , a p o s i t i v e m i d - p o i n t p o t e n t i a l w o u l d mean t h a t a heme s p e c i e s r e s i d e s i n a h y d r o p h o b i c e n v i r o n m e n t w h i l e a n e g a t i v e m i d - p o i n t p o t e n t i a l w o u l d d e s c r i b e an exposed heme. As the f o r m a l c h a r g e on t h e heme c e n t r e s o f f e r r i c and f e r r o u s cy toch rome c_ a re +1 and 0 r e s p e c t i v e l y , i n c r e a s e d e n v i r o n m e n t a l h y d r o p h o b i c i t y s h o u l d enhance the s t a b i l i t y o f t h e r e d u c e d s t a t e . E l e c t r o c h e m i c a l work by W i l g u s e_t a l . ( 1 9 7 8 ) has shown t h a t t h e m i d - p o i n t p o t e n t i a l o f 14-21H ( - 2 0 8 mV) i s more n e g a t i v e t h a n t h a t o f h o r s e h e a r t cy toch rome c. (+265 mV) o r 1-65H ( -108 mV) . Kazmi e_L. a l . - ( 1 9 8 5 ) however r e p o r t a m i d - p o i n t p o t e n t i a l o f 0 mV a t pH 7 .0 f o r 1 4 - 2 1 H . T h i s d i s c r e p a n c y may have a r i s e n f r o m t h e d i f f e r e n c e s i n t h e e l e c t r o c h e m i c a l t e c h n i q u e s used by t h e two g r o u p s . The W i l g u s e_L a l . g roup employed t h e t e c h n i q u e o f s p e c t r o e l e c t r o c h e m i s t r y w h i l e Kazmi eJL a l . used p u l s e d p o l a r o g r a p h y . I n e i t h e r c a s e , t h e m i d - p o i n t p o t e n t i a l o f 14-21H has been shown t o be more n e g a t i v e t h a n t h a t o f h o r s e h e a r t cy toch rome c_. I f 11-21H i s h i g h - s p i n l i k e 14-21H t h e n one wou ld s u s p e c t t h a t t he m i d -p o i n t p o t e n t i a l s must be s i m i l i a r as i t i s d i f f i c u l t t o see how t h r e e e x t r a amino a c i d r e s i d u e s c o u l d a l t e r t h e h y d r o p h o b i c i t y o f t h e heme p o c k e t . However , f r o m Mossbauer s p e c t r a o f 1 1 - 2 1 H , P e t e r s o n fit. a l . ( 1 9 8 0 ) have shown 7 t h a t 11-21H p r e p a r a t i o n s have a low s p i n ( I ) and a h i g h s p i n ( I I ) component . P e t e r s o n e_£ a l . c o n s i d e r I I t o be a n o n a p e p t i d e i m p u r i t y a r i s i n g f r o m t h e a c t i o n o f a p e p s i n c o n t a m i n a n t i n t h e t r y p s i n used t o make 1 1 - 2 1 H . O t h e r g roups r e p o r t a p r e d o m i n a n t h i g h s p i n component f o r 11-21H ( e . g . Baumgar tner e t a l . . 1974) a l t h o u g h t h e r e l a t i v e r a t i o o f I t o I I seems t o v a r y f r o m p r e p a r a t i o n t o p r e p a r a t i o n . The a m b i g u i t y i n d e t e r m i n i n g t h e s p i n s t a t e o f 11-21H i s an i m p o r t a n t p r o b l e m because t h e m i d - p o i n t p o t e n t i a l i s d e f i n a t e l y i n f l u e n c e d by t h e l i g a t i o n number and t h e s p i n s t a t e o f t h e heme p r o t e i n (Moore and W i l l i a m s , 1 9 7 7 ) . I f 11-21H i s h i g h s p i n , t h e n i t s m i d - p o i n t p o t e n t i a l i s most l i k e l y s i m i l i a r t o 1 4 - 2 1 H . I f 11-21H i s low s p i n , t h e n the m i d - p o i n t p o t e n t i a l i s most l i k e l y more p o s i t i v e t h a n t h a t o f 1 4 - 2 1 H . One c o m p l i c a t i n g p r o p e r t y o f 14-21H and p o s s i b l y 11-21H i s t h a t even t h o u g h t h e s e f r a g m e n t s a r e s o l u b l e i n aqueous s o l u t i o n s , t h e y do have a t e n d e n c y t o a g g r e g a t e . U r r y (1967) f i r s t a r r i v e d a t t h i s c o n c l u s i o n f r o m t h e M a g n e t i c C i r c u l a r D i c h r o i s m (MCD) s p e c t r a o f 11-21H a t d i f f e r e n t m i c r o m o l a r c o n c e n t r a t i o n s . U r r y s u g g e s t e d t h a t t h e a g g r e g a t i o n mechanism stemmed f r o m t h e head t o t a i l a l i g n m e n t o f t h e heme p l a n e s as opposed t o t h e c a r d s t a c k i n g " m a n n e r o f a g g r e g a t i o n p r e v a l e n t i n a g g r e g a t e d f r e e hemes ( M a r g a l i t e_£ a l . . 1 9 8 3 ) . I t w o u l d seem t h a t t he heme n o n a - and u n d e c a p e p t i d e s possess t h e same d i s a d v a n t a g e as f r e e hemes; n a m e l y , t h e p r o p e n s i t y t o a g g r e g a t e . The degree and t h e mechanism o f a g g r e g a t i o n , h o w e v e r , do d i f f e r c o n s i d e r a b l y . ( b ) : P e r o x i d a t i c Functions o_f. C_y_£. c_ Fragments One p r o p e r t y o f 11-21H and 14-21H t h a t has been r e c o g n i z e d f o r y e a r s has been t h e i r enhanced p e r o x i d a s e a c t i v i t i e s ( d e c o m p o s i t i o n o f ^ 2 ^ 2 * n t 0 8 has been t h e i r enhanced p e r o x i d a s e a c t i v i t i e s ( d e c o m p o s i t i o n o f i n t o ^ 0 and C^) r e l a t i v e t o n a t i v e cy toch rome c_. U s i n g an I ^ C ^ / m e s i d i n e assay sys tem a t pH 4 . 9 , P a l e u s , E h r e n b e r g and Tuppy ( 1 9 5 5 ) f o u n d 11-21H t o have a 2 0 - f o l d g r e a t e r p e r o x i d a s e a c t i v i t y t h a n h o r s e h e a r t cy tochrome c_. Presumab ly t h e o p e n i n g up o f t h e heme p o c k e t and change i n a x i a l l i g a t i o n t h a t o c c u r s on c h a n g i n g hhc_ t o 11-21H i n c r e a s e s p e r o x i d a s e a c t i v i t y a l t h o u g h t h e r a t e s s t i l l a r e f a r b e l o w t h e l e v e l s o f h o r s e r a d i s h p e r o x i d a s e (HRP) a c t i v i t y . C l o r e £ £ a l . ( 1 9 8 1 ) has e x t e n d e d t h e s e p e r o x i d a s e s t u d i e s by e x a m i n i n g t h e p e r o x i d a s e mechanism o f 1 1 - 2 1 H . U s i n g s t o p - f l o w t e c h n i q u e s , C l o r e e_t a l . e s t a b l i s h e d t h e number o f i n t e r m e d i a t e s fo rmed d u r i n g the p e r o x i d a s e r e a c t i o n . For pH 7 . 0 c o n d i t i o n s , t h e y p roposed c a t a l y s i s o c c u r s by a f o u r - s t e p mechan ism: 1 . ( 1 1 - 2 1 H ) 3 + + H 2 0 2 — I c — H c — I I I C 2 . I I I C + H 2 0 2 - I V C w h i l e a t h r e e - s t e p mechanism o c c u r s a t pH 1 0 . 4 : 1 . ( 1 1 - 2 1 H ) 3 + +. H 2 0 2 — I D — I I I D 2 . I I I D + H 2 0 2 - I V D where I , 1^ I I , 11^, I I I C . I l l , I V , and I \ L a r e i n t e r m e d i a t e s o f ( 1 1 - 2 1 H ) d u r i n g t h e p e r -o x i d a s e r e a c t i o n s . The r a t e c o n s t a n t s , e s p e c i a l l y t h o s e o f t h e f i r s t c o m m i t t i n g s t e p s (1^ , and I p ) , a r e 4 t o 5 o r d e r s o f m a g n i t u d e s m a l l e r t h a n a n a l o g o u s r a t e c o n s t a n t s f o r h o r s e r a d i s h p e r o x i d a s e , m y o g l o b i n and c a t a l a s e . E v i d e n t l y i n c r e a s e d heme exposu re a l o n e i s n o t s u f f i c i e n t t o r e a c h r a t e s comparab le t o HRP. C l o r e e_£ a l . p e r f o r m e d t h e i r e x p e r i m e n t s a t 1.63 uM p e p t i d e c o n c e n t r a t i o n s , 9 and Pa leus e_£ a l . p e r f o r m e d t h e i r s a t 4 . 9 uM p e p t i d e c o n c e n t r a t i o n s , so i t i s p o s s i b l e t h a t d i f f e r e n t degrees o f a g g r e g a t i o n a f f e c t e d t h e number o f * * 2 ^ 2 ~ a c c e s S ^ k ^ e heme g r o u p s . A n o t h e r f a c t o r i n f l u e n c i n g p e r o x i d a s e r a t e s i s t h e s p i n s t a t e o f 1 1 - 2 1 H . A l t h o u g h t h e s p i n c h a r a c t e r o f 11-21H i s e q u i v o c a l , s p i n s t a t e i s a l s o e x p e c t e d t o i n f l u e n c e t h e p e r o x i d a s e a c t i v i -t i e s o f 11-21H and o f heme p r o t e i n s and p e p t i d e s i n g e n e r a l . A n o t h e r f a c t o r t h a t must be c o n s i d e r e d i s t h e r o l e o f r e s i d u e s i n t h e b i n d i n g s i t e i n s t a b i l i z i n g p e r o x i d e i n t e r m e d i a t e s . For e x a m p l e , i t has been p o s t u l a t e d t h a t t h e g u a n i d i n i u m g r o u p o f A r g 48 i n Cytochrome c_ P e r o x i d a s e fo rms a h y d r o g e n bond w i t h t h e d e v e l o p i n g n e g a t i v e -charge on the oxygen m o l e c u l e o f a p e r o x i d e s p e c i e s ( P o u l o s and F i n z e l , 1 9 8 4 ) . The f o r m a t i o n o f t he a n i o n i c p e r o x i d e i s a r e s u l t o f p r o t o n t r a n s f e r t o t h e a x i a l l i g a n d H is 5 2 . Thus t h e r e a s o n s f o r d e f i c i e n t p e r o x i d a s e a c t i v i t y i n 11-21H i s t h e l a c k o f a p r o t e i n t e r t i a r y s t r u c t u r e t h a t i s d e s i g n e d t o p r e v e n t a g g r e g a t i o n and t o o r i e n t t h e a p p r o p r i a t e r e s i d u e s a round t h e heme b i n d i n g s i t e . However Frew and Jones ( 1 9 8 3 ) have r e p o r t e d c u r i o u s f i n d i n g s c o n c e r n i n g t h e p e r o x i d a s e a c t i v i t y o f c h l o r o d e u t e r o f e r r i h e m e (DFH) . U s i n g an ^ 2 ^ 2 ^ a n ^ ^ ^ n e a s s a y s y s t e m , t h e y f o u n d t h e p e r o x i d a s e r a t e o f DFH was o f t h e same o r d e r o f m a g n i t u d e as t h e p e r o x i d a s e o f HRP. Oddly enough , a l t h o u g h Frew and Jones r e c o g n i z e d t h a t t h e i r DFH may be a g g r e g a t i n g under t h e i r p e r o x i d a s e assay c o n d i t i o n s , t h e y c l a i m t h a t " n e i t h e r t h e p r e s e n t d a t a , n o r e a r l i e r s t u d i e s o f r e d u c t i o n o f t h e i n t e r m e d i a t e w i t h i o d i d e show c o m p l e x i t i e s t h a t m i g h t sugges t a r e a c t i v i t y d i s c r i m i n a t i o n between monomer ic and a g g r e g a t e d s p e c i e s . " Chance £ t a l . ( 1 9 8 6 ) have r e p o r t e d r e l e v a n t o b s e r v a t i o n s c o n c e r n i n g compound ES o f CCP ( c y t o c h r o m e c. p e r o x i d a s e ) , compound I o f HRP (Horse R a d i s h P e r o x i d a s e ) and o x y f e r r y l - m y o g l o b i n . U s i n g t h e t e c h n i q u e o f Ex tended X - r a y A b s o r p t i o n F i n e S t r u c t u r e (EXAFS) s p e c t r o s c o p y , o b t a i n e d the i r o n -10 above t h r e e compounds. They f o u n d t h a t t h e i r o n - l i g a n d and N p ~ N p d i s t a n c e s f o r CCP and f o r HRP were v i r t u a l l y i d e n t i c a l b u t t h a t t h e N -N d i s t a n c e s P P f o r o x y f e r r y l - m y o g l o b i n were c o n s i d e r a b l y l o n g e r t h a n t h e N^-N d i s t a n c e s f o r CCP or f o r HRP. They c o n c l u d e d f r o m t h e s e d i s t a n c e v a l u e s t h a t t h e s t a b i l i t y and a c t i v i t y o f t h e p e r o x i d a s e i n t e r m e d i a t e s a r e v e r y dependent upon, t he e l e c t r o n i c o r b i t a l d i s t r i b u t i o n o f t h e heme g r o u p s . M o d i f i c a t i o n o f t h e heme o r b i t a l s and c o n s e q u e n t l y t h e p e r o x i d a s e a c t i v i t y o f 11-21H i s most l i k e l y i n f l u e n c e d by h e m e - l i g a n d i n t e r a c t i o n s . I t seems r e a s o n a b l e t h e n t o p r o p o s e t h a t t h e a p p r o p r i a t e h e m e - p r o t e i n i n t e r a c t i o n r e q u i r e d t o f o r c e the i r o n - p o r p h y r i n c e n t e r i n t o t h e c o r r e c t c o n f o r m a t i o n f o r t h e p u r p o s e o f maximum p e r o x i d a s e a c t i v i t y i s s i m p l y n o t p r e s e n t i n t h e t r u n c a t e d p e p t i d e s . A t p r e s e n t , t h e p o s s i b i l i t y i s d i f f i c u l t t o add ress by e x p e r i m e n t . £ i Fragment C o m p l e m e n t a t i o n S t u d i e s o_f_ £y_L c_ The s t r a t e g y o f c o m p l e m e n t a t i o n i n v o l v e s t h e m i x i n g o f f r a g m e n t s A + B d e r i v e d f r o m a g i v e n p r o t e i n and a l l o w i n g them t o f o r m a f u n c t i o n a l n o n -c o v a l e n t complex i n s o l u t i o n . C o m p l e m e n t a t i o n work was f i r s t r e p o r t e d f o r RNase ( R i c h a r d s , 1958) and s u b s e q u e n t l y f o r S t a p h y l o c o c c u s n u c l e a s e ( T a n i u c h i e_L a l . . 1967). Fragment c o m p l e m e n t a t i o n s t u d i e s on cy toch rome c_ f o l l o w a c o n s i s t e n t s t r a t e g y : a h e m e - c o n t a i n i n g f r a g m e n t and a non-heme c o n t a i n i n g f r a g m e n t a r e i s o l a t e d and t h e n a r e r e c o m b i n e d , and t h e complex i s c h a r a c t e r i z e d f o r f u n c t i o n . The f i r s t a u t h o r s t o r e p o r t t h i s a p p r o a c h as a p p l i e d t o cy toch rome c_ were C o r r a d i n and H a r b u r y (1970). These a u t h o r s t r e a t e d h o r s e h e a r t cy toch rome c_ w i t h CNBr and o b t a i n e d a 65 amino a c i d r e s i d u e , heme-c o n t a i n i n g f r a g m e n t d e r i v e d f r o m t h e amino t e r m i n u s o f t h e n a t i v e p r o t e i n 11 and a 38 amino a c i d r e s i d u e , non-heme fragment derived from the carboxy end of the nati v e p r o t e i n . These fragments were designated 1-65H and 66-104 r e s p e c t i v e l y . Mixing these two fragments produced a complex w i t h a f u n c t i o n a l a c t i v i t y c l o s e to that of unmodified cytochrome c_; however, these authors f a i l e d to r e a l i z e that the aerobic c o n d i t i o n s of the complementation process caused the two fragments to be c o v a l e n t l y l i n k e d at residue 66. Lat e r complementation experiments i n anaerobic c o n d i t i o n s produced a non-covalent complex of 1-65H w i t h 66-104 that d i d not r e t a i n cytochrome c. a c t i v i t y (Corradin and Harbury, 1974). As the Methionine-65 residue i s i n the middle of an a l p h a - h e l i c a l region of cytochrome c_, CNBr-induced cleavage of cytochrome c. probably disrupted the t e r t i a r y s t r u c t u r e of the h e l i x to such an extent that non-covalent complementation i s not s u f f i c i e n t to r e e s t a b l i s h the nati v e conformation. These r e s u l t s were an e a r l y i n d i c a t i o n of the requirement of a p r e c i s e t e r t i a r y arrangement f o r the recovery of f u l l f u n c t i o n a l a c t i v i t y . Other heme peptides have a l s o been studied i n t h i s manner. Wilgus e_L a l . (1978) examined the p r o p e r t i e s of s e v e r a l complexes composed e i t h e r of , 1-38H, 11-21H, 14-21H or 1-65H and se v e r a l overlapping and non-overlapping non-heme co n t a i n i n g fragments. These authors concluded that the p a r t i a l or f u l l r e s t o r a t i o n of e l e c t r o n t r a n s f e r a c t i v i t y and of mid-point p o t e n t i a l required the complementation of overlapping heme-containing and non-heme containing fragments. These f i n d i n g s only a p p l i e d to complexes that contained e i t h e r 1-38H, 1-65H or 1-80H. Overlapping complexes having the heme-containing fragments 11-21H (e.g. (11-21H):(1-104)) or 14-21H (e.g. (14-21H):(1-104)) produced no r e s t o r a t i o n of o r i g i n a l f u n c t i o n . Both 11-21H and 14-21H are missing the conserved amino a c i d residue Phe-10, which may be an i n d i c a t i o n of the importance of conserved residues and of conserved 12 be an i n d i c a t i o n o f the i m p o r t a n c e o f c o n s e r v e d r e s i d u e s and o f conse rved p e p t i d e sequences i n d e t e r m i n i n g t h e v i a b i l i t y o f an a c t i v e s i t e . W i l g u s £_£. a l . sugges t t h a t c o m p l e m e n t a t i o n r e s t o r e s a c t i v i t y by r e s t o r i n g t h e h y d r o p h o b i c p o c k e t a round t h e heme p r o s t h e t i c g r o u p . T h i s p o s s i b i l i t y i s c o n s i s t e n t w i t h t he argument t h a t t h e m i d - p o i n t p o t e n t i a l , a c r i t i c a l p a r a m e t e r i n e l e c t r o n t r a n s f e r a c t i v i t y , i s l a r g e l y d e t e r m i n e d by t h e d i e l e c t r i c c o n s t a n t o f t h e heme e n v i r o n m e n t ( K a s s n e r , 1972 ; S t e l l w a g e n , 1 9 7 8 ) . W i l g u s £_£. a l . r e g a r d t h i s f a c t o r as b e i n g more c r i t i c a l t o heme c e n t r e r e a c t i v i t y t h a n t h e i d e n t i t y o f t h e a x i a l l i g a n d s . A s l i g h t l y d i f f e r e n t v i e w i s t a k e n by Hantgan and T a n i u c h i ( 1 9 7 7 ) and by P a r r , Hantgan and T a n i u c h i ( 1 9 7 8 ) . These a u t h o r s f o u n d t h a t t h e y c o u l d make a c t i v e complexes w i t h ( 1 - 5 3 H ) : ( 3 9 - 1 0 4 ) , ( 1 - 5 3 H ) : ( 4 0 - 1 0 4 ) , ( 1 - 5 3 H ) : ( 5 4 -104) and most i m p o r t a n t l y , ( 1 - 2 5 H ) : ( 3 9 - 1 0 4 ) , ( 1 - 3 8 H ) : ( 5 6 - 1 0 4 ) . The a c t i v i t y o f t h e s e complexes was m o n i t o r e d by a cy toch rome a s s a y , and t h e degree of f o l d i n g was m o n i t o r e d t h e q u e n c h i n g o f T r p - 5 9 f l u o r e s c e n c e . F u n c t i o n a l a c t i v i t y i n t h e l a t t e r two complexes l e d t h e T a n i u c h i g roup t o sugges t t h a t o n l y c e r t a i n p o r t i o n s o f t h e p e p t i d e backbone a r e r e q u i r e d f o r a c t i v i t y . Of g r e a t e r i n t e r e s t i s t h e deve lopmen t on t h e i r i d e a s i n t h e dynamics o f p r o t e i n f o l d i n g and u n f o l d i n g (Han tgan and T a n i u c h i , 1978 ; T a n i u c h i , 1 9 7 3 ) . The T a n i u c h i g r o u p has r e p o r t e d e x t e n s i v e i n v e s t i g a t i o n s i n t o t h e exchange o f 1 4 C - l a b e l l e d ( 5 4 - 1 0 4 ) f r o m (1 -53H): (54 -104) w h i c h i s i n c u b a t e d i n an medium o f c o l d ( 54 -104) . They measured t h e e n t h a l p i c ( 4 H ) , e n t r o p i c (4S) and f r e e e n e r g y (4G) changes o f t h e e q u i l i b r i u m p r o c e s s and f o u n d t h a t A S i n c r e a s e s w i t h i n c r e a s i n g t e m p e r a t u r e b u t t h a t A H r e m a i n s r e l a t i v e l y c o n s t a n t . An i n c r e a s e i n A S , a measure o f d i s o r d e r i n t h e c o m p l e x , must mean a g r e a t e r exchange between t h e o r d e r e d and d i s o r d e r e d f o r m o f t h e c o m p l e x . Thus t h e complex i s n o t s t a t i c b u t i s i n a s o r t o f " b r e a t h i n g " mode. A n o t h e r i n t e r e s t i n g f i n d i n g i s t h a t t h e dependence ofA S on 13 t e m p e r a t u r e i s l e s s p ronounced i n t h e reduced f o r m t h a n i n t h e o x i d i z e d f o r m . I t i s known t h a t t h e a x i a l l i g a n d M e t h i o n i n e - 8 0 b i n d s more s t r o n g l y t o t h e reduced heme i r o n t h a n t o t h e o x i d i z e d heme i r o n ( H a r b u r y e_£. a l j _ , 1 9 6 5 ) . The i m p l i c a t i o n i s t h a t t h e a x i a l l i g a n d s i n c y t o c h r o m e £. may p l a y a r o l e i n d e t e r m i n i n g t h e heme e n v i r o n m e n t by r e s t r i c t i n g the movement o f t h e p o l y p e p t i d e b a c k b o n e . T a n i u c h i * s g roup has ex tended t h e i r k i n e t i c and thermodynamic s t u d i e s o f t he c o m p l e m e n t a t i o n p r o c e s s . For e x a m p l e . P a r r and T a n i u c h i (1981) s t u d i e d t h e r a t e o f a s s o c i a t i o n o f ( 1 - 2 5 H ) : ( 2 3 - 1 0 4 ) , ( 1 - 3 8 H ) : ( 5 6 - 1 0 4 ) , and ( 1 - 5 3 H ) : ( 5 4 - 1 0 4 ) i n t h e p r e s e n c e and i n t h e absence o f i m i d a z o l e . I n t h e abscence o f i m i d a z o l e , a p r o d u c t i v e complex was formed i n each c a s e . The k i n e t i c s o f t h i s c o m p l e x a t i o n p r o c e s s c o n s i s t e d o f an i n i t i a l s e c o n d - o r d e r s t e p m o n i t o r e d by f l u o r e s c e n c e q u e n c h i n g f o l l o w e d by a f i r s t - o r d e r s t e p m o n i t o r e d by abso rbance c h a n g e s . I n t h e p r e s e n c e o f i m i d a z o l e (10 mM o r 50 mM) no abso rbance change c o u l d be d e t e c t e d a l t h o u g h f l u o r e s c e n c e q u e n c h i n g was s t i l l o b s e r v e d . The complex fo rmed was n o n - p r o d u c t i v e . P a r r and T a n i u c h i i n t e r p r e t e d t h e s e r e s u l t s i n te rms o f a t w o - s t e p c o m p l e x a t i o n m o d e l . The i n i t i a l s t e p c o n s i s t s o f t h e comp lementa ry f o l d i n g o f heme-c o n t a i n i n g and n o n - h e m e - c o n t a i n i n g f r a g m e n t s i n t o an o r d e r e d s t a t e . The second s t e p e n t a i l s t h e l i g a t i o n o f M e t - 8 0 o n t o t h e heme. I n t h e p resence o f i m i d a z o l e , t h e c o o r d i n a t i o n s i t e f o r M e t - 8 0 i s p r o b a b l y b l o c k e d , and a n o n - p r o d u c t i v e p e p t i d e - p e p t i d e complex i s f o r m e d . For p r o t e i n s , A H r e f l e c t s t h e p o l a r i t y o f t h e a c t i v e s i t e r e g i o n , A S a measure o f t h e s t a t e o f o r d e r i n t h e p e p t i d e - p e p t i d e c o m p l e x . The thermodynamic p a r a m e t e r s o f t h e c o m p l e x a t i o n p r o c e s s were such t h a t d u r i n g the i n i t i a l s t e p , t h e r e was a l a r g e e n t h a l p i c ( ^ H ) c o n t r i b u t i o n t o f r e e energy ( ^ G) b u t a s m a l l e n t r o p i c (AS) component . D u r i n g t h e second s t e p , 14 o n e . P a r r and T a n i u c h i have shown t h a t t h e i n i t i a l c o m p l e x a t i o n s t e p se rves t o e n v e l o p e the heme g roup i n t o a more h y d r o p h o b i c e n v i r o n m e n t and t h a t t h e second s t e p e n t a i l s t h e r e d u c t i o n i n p o l y p e p t i d e m o b i l i t y v i a M e t -80 l i g a t i o n t o t h e heme g r o u p . Subsequent work f r o m t h i s l a b o r a t o r y has shown t h a t c e r t a i n r e s i d u e s i n cy toch rome c_ f o r m a n e t w o r k o f i n t r a - a t o m i c i n t e r a c t i o n s t h a t s t a b i l i z e t h e t e r t i a r y s t r u c t u r e o f t h e p r o t e i n ( J u i l l e r a t and T a n i u c h i * 1 9 8 6 ) . Chemica l a l t e r a t i o n o f Leu -32 t o N o r v a l i n e o r H e d e s t a b i l i z e d t h e ( 1 - 2 5 H ) : ( 2 8 - 3 8 ) : ( 3 9 - 1 0 4 ) complex by a f a c t o r o f 4 0 0 . I n a d d i t i o n , t h e i m p r e s s i v e s e r i e s o f y e a s t i s o - l - c y t o c h r o m e c_ m u t a n t s t h a t Hampsey £_£. a l ' . ( 1 9 8 6 ) r e p o r t a l l m a n i f e s t t h e i r m u t a t i o n s as a dec rease i n s t a b i l i t y . P r o t e i n s t a b i l i z a t i o n by i n t e r - a t o m i c i n t e r a c t i o n may s e r v e t o i n c r e a s e t h e l i g a n d s t r e n g t h o f M e t h i o n i n e - 8 0 o r H i s t i c T i n e - 1 8 as s u g g e s t e d by t h e f i n d i n g t h a t t h e b i n d i n g a f f i n i t y o f t h e heme g roup f o r m e t h i o n i n e i s f o u r o r d e r s o f m a g n i t u d e h i g h e r i n t h e ( 1 - 3 8 H ) : ( 1 - 1 0 4 ) complex t h a n j u s t t h e a d d i t i o n o f exogenous N - a c e t y l m e t h i o n i n e ( W i l g u s e_£. a l . . 1 9 7 8 ) . P r e s u m a b l y , t h e c o m b i n a t i o n o f o v e r l a p p i n g f r a g m e n t s r e s t o r e s t h e n e t w o r k o f i n t e r - a t o m i c i n t e r a c t i o n s a n d , t h i s i n t e r a c t i o n s e r v e s t o o r i e n t t h e a x i a l l i g a n d s t o t h e i r f a v o u r e d b i n d i n g p o s i t i o n s . C_L S e m i s y n t h e s i B aL c_y_L £. D e r i v a t i v e s The s e m i s y n t h e t i c a p p r o a c h i n v o l v e s t h e i s o l a t i o n o f c l e a v e d p r o t e i n f r a g m e n t s , m o d i f y i n g one o f them and t h e n c o v a l e n t l y r e c o n s t i t u t i n g t h e two p o l y p e p t i d e c h a i n s t o p r o d u c e a s i n g l e p o l y p e p t i d e c h a i n o f t h e same l e n g t h as t h e n a t i v e p r o t e i n . The p r o d u c t i o n o f t h e p e p t i d e f r a g m e n t s i n v o l v e s b o t h c h e m i c a l (CNBr) and e n z y m a t i c ( t r y p s i n , p e p s i n ) t e c h n i q u e s (see S e c t i o n I A ) . One a d v a n t a g e o f i n i t i a l l y p r o d u c i n g p e p t i d e f r a g m e n t s o f t h e n a t i v e 15 p r o t e i n i s t h e a b i l i t y t o m o d i f y amino a c i d r e s i d u e s t h a t a r e n o t a c c e s s i b l e i n t h e n a t i v e p r o t e i n . A second advan tage i s t h a t t h e s p e c i f i c i t y o f a m o d i f i c a t i o n p r o t o c o l i s enhanced ow ing t o t h e l o s s o f c o m p e t i n g r e s i d u e s t h a t were p r e s e n t i n t h e n a t i v e p r o t e i n . W e s t e r h u i s e_L a l • ( 1 9 8 2 ) made m a j o r c o n t r i b u t i o n s t o w a r d s t h e deve lopment o f t h i s t e c h n i q u e as a p p l i e d t o c y t o c h r o m e c_. The M e r r i f i e l d method ( M i t c h e l l e t a l . . 1 9 7 6 ) , a t e c h n i q u e f o r t h e s o l i d phase s y n t h e s i s o f p o l y p e p t i d e s , f r e q u e n t l y has been used t o p r o d u c e s e m i s y n t h e t i c f r a g m e n t s ( B a r s t o w £_£. a l . . 1977 ; V e l o s o £ i a_Lt.» 1 9 8 4 ) . For e x a m p l e , t e n K o r t e n a a r QL a l . . ( 1 9 8 5 ) used t h i s method t o p roduce a 6 6 -104 cy toch rome c_ f r a g m e n t whose e v o l u t i o n a r i l y c o n s e r v e d Phe-82 r e s i d u e was a l t e r e d t o L e u - 8 2 . C o v a l e n t r e c o m b i n a t i o n o f t h e m o d i f i e d 6 6 - 1 0 4 f r a g m e n t w i t h 1-65H p roduced a complex whose m i d - p o i n t p o t e n t i a l was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f t h e n a t i v e p r o t e i n . These r e s u l t s a r e i n t e r e s t i n g i n l i g h t o f t he work by P i e l a k £_£. a l . ( 1 9 8 5 ) who showed t h a t h y d r o p h o b i c amino a c i d r e s i d u e s a t p o s i t i o n 82 r e t a i n e d t h e p o t e n t i a l o f t h e w i l d - t y p e p r o t e i n w h i l e s u b s t i t u t i o n o f s m a l l o r h y d r o p h o b i c r e s i d u e s ( G l y o r S e r ) a t t h i s p o s i t i o n p roduced a s i g n i f i c a n t d r o p i n t h e p o t e n t i a l . F u r t h e r work on t h e m o d i f i c a t i o n o f amino a c i d r e s i d u e s i n cy tochrome £. by s e m i s y n t h e t i c methods has been r e p o r t e d by W a l l a c e and Rose (1983) and by W a l l a c e ( 1 9 8 4 ) . The m a j o r f i n d i n g a r i s i n g f r o m t h e i r work i s t h a t t h e m o d i f i c a t i o n o f t h e e v o l u t i o n a r i l y c o n s e r v e d A r g - 3 8 r e s i d u e r e s u l t s i n t h e l o s s o f e l e c t r o n t r a n s f e r a c t i v i t y b u t m o d i f i c a t i o n a t t h e A r g - 9 1 p o s i t i o n had no such e f f e c t . 16 H i STERIC AND. ELECTRONIC CONTRIBUTIONS BJ_ AXIAL LIGANPS Based on t h e k i n e t i c s o f h o r s e h e a r t cy toch rome c. r e d u c t i o n by d i t h i o n i t e a t l e a s t two pathways i n w h i c h r e d u c i n g e q u i v a l e n t s may e n t e r and l e a v e cy toch rome £. have been i d e n t i f i e d ( C r e u t z and S u t i n , 1 9 7 3 ) . The " a d j a c e n t * * pa thway i n v o l v e s t h e r u p t u r e o f t h e F e ( l l l ) - M e t h i o n i n e - 8 0 bond and the subsequen t t r a n s f e r o f e l e c t r o n s f r o m d i t h i o n i t e t o F e . The r a t e c o n s t a n t ( k ^ ) f o r t h i s pathway i s a p p r o x i m a t e l y 30s The " r e m o t e " p a t h w a y , w h i c h i n v o l v e s t h e t r a n s f e r o f r e d u c i n g e q u i v a l e n t s t o t he p e r i p h e r a l edge o f t h e heme g r o u p , has a r a t e c o n s t a n t ( k ^ ) o f abou t 4 - 1 - 1 1 .17x10 M s . A x i a l l i g a n d s can be r e g a r d e d as i n f l u e n c i n g t h e c h a r a c t e r and t h e s i t e o f r e a c t i v i t y o f heme g roups t h r o u g h s t e r i c h i n d r a n c e o f t h e a x i a l c o o r d i n a t i o n p o s i t i o n a n d / o r t h r o u g h e l e c t r o n i c p e r t u r b a t i o n o f p o r p h y r i n o r b i t a l s . ( a ) : S t e r i c F a c t o r s I n f l u e n c i n g Heme R e a c t i v i t y As i s f r e q u e n t l y t h e case i n t h e s t u d y o f heme p r o t e i n f u n c t i o n , t h e most d e t a i l e d mode ls f o r t h e r o l e o f s t e r i c f a c t o r s i n heme p r o t e i n f u n c t i o n have been d e v e l o p e d f o r h e m o g l o b i n . T h i s l i t e r a t u r e w i l l be c o n s i d e r e d b r i e f l y h e r e i n s o f a r as a s p e c t s o f t h i s work may se rve t o be u s e f u l by a n a l o g y i n u n d e r s t a n d i n g t h e b e h a v i o u r o f cy toch rome c_ and i t s heme p e p t i d e s . P e r u t z ( 1 9 7 0 , 1 9 7 2 ) was t h e f i r s t t o p r o p o s e a r o l e f o r p r o t e i n a x i a l l i g a n d s i n i n f l u e n c i n g t h e a f f i n i t y o f h e m o g l o b i n f o r exogenous l i g a n d s . Based on h i s s p e c t r o p h o m e t r i c and c r y s t a l l o g r a p h i c work o f o x y - and d e o x y -Hb, P e r u t z i n t r o d u c e d t h e " t e n s i o n a t t h e heme" m o d e l . The f e a t u r e s o f h i s model a r e as f o l l o w s : 17 ( 1 ) The i n i t i a l t r a n s i t i o n f r o m oxy-Hb t o deoxy-Hb c o n s i s t s o f t h e l o s s o f t h e 0 „ l i g a n d f r o m Hb. ( 2 ) T h i s l o s s t r i g g e r s a change i n t h e t e r t i a r y s t r u c t u r e o f deoxy-Hb r e s u l t i n g i n t he move-ment o f t h e p r o x i m a l H i s t i d i n e l i g a n d away f r o m t h e p o r p h y r i n r i n g : t h e " t e n s i o n a t t h e heme**. ( 3 ) T e n s i o n a t t h e heme i n d u c e s a movement o f t h e i r o n atom out o f t h e p l a n e o f t h e r i n g . ( 4 ) The t e n s i o n f o r c e s t h e deoxy-Hb q u a t e r n a r y s t r u c t u r e f r o m t h e R o x y - f o r m i n t o t h e T d e o x y - f o r m . T h i s model c o n t a i n s one c l e a r p r e d i c t i o n : t h e deoxy T - s t a t e o f Hb s h o u l d have a v e r y d i f f e r e n t s e t o f F e - N ^ l e n g t h s t h a n t h e R - s t a t e . EXAFS d a t a ( E i s e n b e r g e r , 1978) f o r Hb, Hb-C^ and v a r i o u s model p o r p h y r i n compounds have shown t h a t t h e F e - ^ and F e - L i g a n d d i s t a n c e s a r e i d e n t i c a l w i t h i n e x p e r i m e n t a l e r r o r f o r t he o x y - and d e o x y - s p e c i e s . F e r m i ' s ( 1 9 7 5 ) c r y s t a l l o g r a p h i c d a t a have a l s o shown no d i f f e r e n c e i n Fe~Np d i s t a n c e s . A m o d i f i e d v i e w has been p o s t u l a t e d by Co l lman and c o - w o r k e r s . C o l l m a n ' s emphasis i s n o t on t h e p r o x i m a l h i s t i d i n e b u t on s t e r i c h i n d r a n c e s p roduced by the d i s t a l h i s t i d i n e o f Hb w h i c h i s n o t bound t o t h e heme i r o n . T h i s v i e w stems f r o m Co l lman e_L a L t 1 s ( 1 9 7 9 ) work on p i c k e t f e n c e p o r p h y r i n s . P i c k e t f e n c e p o r p h y r i n a r e m o d i f i e d fo rms o f t e t r a p h e n y l -p o r p h y r i n (TPP) t h a t possess a s u b s t i t u t e d p h e n y l g roup c o v a l e n t l y a t t a c h e d t o each meso b r i d g e o f t h e p o r p h y r i n r i n g (see F i g . 4 ) . Two t y p e s o f P i c k e t Fence P o r p h y r i n s were s y n t h e s i z e d . I n one t y p e 1 , 2 - d i m e t h y l i m i d a z o l e ( 1 , 2 d i M e I m i d ) a c t s as a n o n c o v a l e n t l y a t t a c h e d p r o x i m a l l i g a n d ( F i g . 4 A ) . I n t h e o t h e r , an a c y l i m i d a z o l e g roup i s c o v a l e n t l y a t t a c h e d t o one o f t h e p h e n y l r i n g s and a r r a n g e d so t h a t t h e i m i d a z o l e i s a s s o c i a t e d w i t h t h e Fe i n t he heme r i n g ( F i g . 4 B ) . I n each c a s e , t h e s i x t h a x i a l l i g a n d c o o r d i n a t i o n 18 FIG 4: Five coordinate picket fence porphyrins. (A) meso-tetra(°^,^.^»°(-o-pivalamidophenyl)porphyrinate(l,2-dimethy1 imidazole) (B) meso-tri( ,3-o-pivalamidophenyl)-/!-o-5-(l-imidazolyl)varleramidophenylporphyr-inate. Figures from Collman ££. a l . (1979). 19 (A) 0 I Fe 0 I c I Fe ( B ) • H i s 1 - / ^ ' 0 Fe 0 ' Vol) x^  V 0 / i His / p v - ' / (Vol) Fe X > i'HisN' S V C Fe 0 'Vol x^ V "t i l ted" bent £I£ i i . Ligand binding geometry of 0- and CO i n (A) f i v e coordinate model i r o n ( I I ) complexes (B) the Beta chain of hemoblobin. Figures from Collman e_L aJu. (1979) 20 p o s i t i o n rema ins u n o c c u p i e d . Compar ison o f t h e 0^ and CO b i n d i n g c h a r a c t e r -i s t i c s o f t h e s e mode l compounds d e m o n s t r a t e d t h a t f o r a b e n t l i g a n d l i k e O ^ i t h e r e vas l i t t l e d i f f e r e n c e i n b i n d i n g a f f i n i t i e s be tween Hb and the p i c k e t f e n c e p o r p h y r i n s w h i l e t h e a f f i n i t y f o r CO was much l o w e r i n t h e case o f Hb t h a n f o r t h e model compounds. CO i s n o r m a l l y a l i n e a r l i g a n d , b u t i t has 63 67 been s u g g e s t e d t h a t s t e r i c h i n d r a n c e s by b e t a - H i s and b e t a - V a l o f Hb f o r c e s t h e CO i n t o a " b e n t " o r a " t i l t e d * * c o n f i g u r a t i o n (see F i g . 5 B ) . As a c o n s e q u e n c e , t h e r e a r e p e r t u r b a t i o n s i n t h e e l e c t r o n i c o r b i t a l i n t e r a c t i o n s between CO and Fe w h i c h reduce t h e a f f i n i t y o f Hb f o r CO. As t h e r e i s no d i s t a l l i g a n d i n t h e p i c k e t f e n c e p o r p h y r i n s , t h e r e i s no s t e r i c h i n d r a n c e and hence no r e d u c t i o n i n CO a f f i n i t y . I f t h i s model i s c o r r e c t , then t h e t e r t i a r y s t r a i n i s n o t m a n i f e s t e d i n t h e d i s s o c i a t e d Hb s p e c i e s (as i t i s i n t h e P e r u t z m o d e l ) b u t i n t h e l i g a t e d s p e c i e s . There i s some e v i d e n c e t o show t h a t s t r a i n can o c c u r i n t h e l i g a t e d f o r m o f h e m o g l o b i n . For example i n t h e p r e s e n c e o f t h e a x i a l l i g a n d NO and t h e a l l o s t e r i c m o d u l a t o r i n o s i t o l h e x a p h o s p h a t e , t h e p r o x i m a l H i s l i g a n d o f Hb i s c l e a v e d f r o m t h e h e m e - i r o n ( M a x w e l l and C a u g h l e y , 1976 ; Szabo and P e r u t z , 1 9 7 6 ) . C l e a r l y , i n t h i s case i t i s t h e l i g a t e d , T s t a t e o f Hb w h i c h e x p e r i e n c e s s u f f i c i e n t s t r a i n t o p roduce a change i n t h e a x i a l l i g a t i o n . C o l l m a n ' s r e s u l t s a r e i n c o n t r a s t t o t h o s e o f T r a y l o r and B e r z i n i s ( 1 9 8 0 ) , who have s y n t h e s i z e d mode l p o r p h y r i n compounds s i m i l i a r t o C o l l m a n ' s i n t h e sense t h a t an a c y l - i m i d a z o l e i s c o v a l e n t l y a t t a c h e d t o t h e heme and i s a x i a l l y l i g a t e d t o t h e heme i r o n . These compounds possess no p h e n y l s u b s t i t u e n t s so t h e heme g r o u p e x p e r i e n c e s a more p o l a r e n v i r o n m e n t . I f Co l lman and c o w o r k e r s a r e c o r r e c t t h e n t h e CO s h o u l d b i n d such a compound w i t h a g r e a t e r a f f i n i t y t h a n Hb. However T r a y l o r and B e r z i n i s f i n d t h a t t h e b i n d i n g a f f i n i t i e s o f CO f o r t h e R and T fo rms o f Hb a r e no d i f f e r e n t f r o m t h e b i n d i n g a f f i n i t i e s o f t h e i r p o r p h y r i n d e r i v a t i v e s . The r e s u l t s i n d i c a t e t h a t e i t h e r C O i s l i n e a r i n t h e R s t a t e o f H b o r s t e r i c h i n d r a n c e i s n o t a s s i g n i f i c a n t a f a c t o r i n i n f l u e n c i n g b i n d i n g a f f i n i t y a s w a s o r i g i n a l l y t h o u g h t . S u b s e q u e n t w o r k b y C o l l m a n ' s g r o u p (1981) e x t e n d e d t h e p i c k e t -f e n c e p o r p h y r i n c o n c e p t b y m a k i n g " p i c k e t p o c k e t " p o r p h y r i n s w h e r e t h e i r o n i s b u r i e d i n a t r u e c r e v i c e i n a m a n n e r a n a l o g o u s t o H b ( s e e F i g . 6 ) . T h e a f f i n i t i e s f o r 0^ a n d C O w e r e m e a s u r e d a n d c o m p a r e d w i t h n o r m a l H b . P r e d i c t a b l y , t h e a f f i n i t y c o n s t a n t s a r e s i m i l i a r t o t h o s e o f H b b u t n o t t o H b - Z u r i c h w h e r e a m u t a t i o n o f t h e d i s t a l h i s t i d i n e t o a n a r g i n i n e r e s i d u e e l i m i n a t e s a n y s t e r i c h i n d r a n c e . T h u s , t h e s t e r i c a l l y h i n d e r e d p i c k e t -p o c k e t p o r p h y r i n s d o p o s s e s s a d e c r e a s e d a f f i n i t y f o r C O . O n e e x p l a n a t i o n o f t h e d i s c r e p a n t f i n d i n g s m a y b e t h e d i f f e r e n t s o l v e n t s y s t e m s u s e d b y t h e d i f f e r e n t r e s e a r c h g r o u p s . B i n d i n g a f f i n i t i e s f o r C O a n d 0^ d o c h a n g e w i t h s o l v e n t p o l a r i t y a n d t h e s o l v e n t s y s t e m s f o r h e m e p r o t e i n s a r e d i f f e r e n t f r o m t h o s e o f t h e p o r p h y r i n m o d e l s ( C h a n g a n d T r a y l o r , 1975). C o l l m a n ' s m o d e l s a r e a m o r e a c c u r a t e r e p r e s e n t a t i o n o f t h e H b h e m e c r e v i c e b e c a u s e h e d o e s t a k e c a r e t o m a k e t h e F e - p o r p h y r i n c r e v i c e a s h y d r o p h o b i c a s p o s s i b l e . F u r t h e r m o r e , R e s o n a n c e R a m a n s t u d i e s o n e l e p h a n t m y o g l o b i n ( w h e r e t h e d i s t a l a x i a l l i g a n d i s g l u t a m i n e i n s t e a d o f h i s t i d i n e ) ( K e u r e _ L a l . . 1985) a n d l i g a n d b i n d i n g s t u d i e s o n c y t o c h r o m e £ . o c t a p e p t i d e s ( S m i t h a n d M c L e n d o n , 1980) h a v e i n d i c a t e d t h a t s t e r i c h i n d r a n c e d o e s p l a y a p a r t i n i n f l u e n c i n g a x i a l l i g a n d a f f i n i t i e s . B a s e d o n t h e a b o v e r e s u l t s a n d h i s o w n c r y s t a l l o g r a p h i c s t u d i e s o f v a r i o u s H b d e r i v a t i v e s , M o f f a t ( M o f f a t fit a l , . , 1979; M o f f a t , 1980-1981) h a s p r o p o s e d a n a l t e r n a t i v e s c h e m e f o r l i g a n d b i n d i n g t o h e m e p r o t e i n s . T h i s s c h e m e i s b a s e d o n d a t a f r o m H b , b u t t h e b a s i c p r i n c i p l e s m a y b e a p p l i c a b l e t o h e m e p r o t e i n s i n g e n e r a l : 1. S t e r i c h i n d r a n c e t h r o u g h t h e d i s t a l a x i a l l i g a n d contributes to a lower 0^ a f f i n i t y in the T s truc-ture of Hb. 2. Ster ic hindrance is less pronounced in the R-state. 3. S ter ic hindrance on l inear ligands (e .g . CO) is more pronounced than for bent ligands (e .g . O j ) . A. Ster ic hindrance is manifested largely on the k i -net ics and thermodynamics of ligand associa t ion . 5. Dissocia t ion of ligands i s under e lectronic not s t e r i c con t ro l . 6. Cooperativity of subunits in Hb is different for l inear or bent l igands. Whatever model turns out to be the most useful , i t is clear that ax ia l ligands have a pronounced effect on the functional heme center. (b): Elec t ronic Effects oJL A x i a l Ljgands The studies into the electronic effects of a x i a l ligands on metal porphyrins have focused on the manner in which a x i a l ligands influence the reduction potent ia l of heme proteins and of model heme compounds and the manner in which a x i a l ligands d is t r ibutes porphyrin o rb i t a l s and influences the s i t e of electron transfer . Studies by Moore and Williams (1977) and by Marchon e_L a l . (1982) on heme model compounds have shown that electron withdrawing ligands (e .g. Tetrahydrothiophene) tend to raise reduction potentials while electron donating ligands (e .g . N-methyl imidazole) tend to lower reduction po ten t ia l s . The data below are representative of resul ts obtained for some relevant model compounds ( a l l potentials stated in this thesis are in r e l a t i on to the normal hydrogen electrode) . COMPOUND REDUCTION POTENTIAL REFERENCE 1. (NMelmid).(TPP) 2. (NMelmid)TC IM)(TPP) 3. (THT) (TPP) -19 mV +18 mV +230 mV a a a 23 4 . 5. 6. 7. Mesoheme B i s h i s t i d i n e mesoheme H i s / M e t mesoheme B i s m e t h i o n i n e mesoheme -158 mV -220 mV -110 mV +20 mV b b b b where NMelm - N - m e t h y l - i m i d a z o l e C_Im - a 5 c a r b o n a r y l i m i d a z o l e TPP - T e t r a p h e n y l p o r p h y r i n THT - T e t r a h y d r o t h i o p h e n e R e f e r e n c e s : a . Marchon e £ a l * . (1982) b . Moore and W i l l i a m s (1978) The l i g a n d s e t o f cy toch rome £. i s H i s / M e t , and i t s r e d u c t i o n p o t e n t i a l i s +265 mV. As can be seen f r o m t h e above d a t a , t h e o n l y compound t h a t approaches such a v a l u e i s Compound 3, w h i c h has two e l e c t r o n - w i t h d r a w i n g l i g a n d s . Compound 7, w h i c h a l s o has two e l e c t r o n w i t h d r a w i n g l i g a n d s has a m i d - p o i n t p o t e n t i a l o f o n l y +20 mV. C l e a r l y , a x i a l l i g a n d s a r e n o t t h e s o l e d e t e r m i n a n t s o f m i d - p o i n t p o t e n t i a l f o r heme p r o t e i n s . Marchon e_£ aJLt's TPP compounds have a more h y d r o p h o b i c e n v i r o n m e n t (due t o t h e p h e n y l s u b s t i t u e n t s ) t h a n Moore and W i l l i a m ' s heme g roups and have h i g h e r r e d o x p o t e n t i a l s , so i t i s most l i k e l y t h a t t h e m i s s i n g f a c t o r i s t h e h y d r o p h o b i c i t y o f t h e heme p o c k e t . G r e a t e r h y d r o p h o b i c i t y u s u a l l y means a d e c r e a s e i n t h e d i e l e c t r i c c o n s t a n t o f t h e heme e n v i r o n m e n t w h i c h w o u l d r e s u l t i n g r e a t e r l i g a h d - i r o n a t t r a c t i o n . G r e a t e r l i g a n d s t r e n g t h ensues w h i c h means an i n c r e a s e i n t h e r e d u c t i o n p o t e n t i a l . There i s some e v i d e n c e f r o m model heme p e p t i d e s t o s u p p o r t t h e s e c o n t e n t i o n s ( B l u m e n t h a l and K a s s n e r , 1979; Huang and K a s s n e r , 1981; J a i n and K a s s n e r , 1984) . Moore and W i l l i a m s c l a i m t h a t a c o n t r a c t i o n o f t h e Fe-S bond i n cy toch rome £. (and i n d i c a t i o n o f i n c r e a s e d l i g a n d s t r e n g t h ) by abou t 0.1 angs t roms l e a d s t o an i n c r e a s e i n t h e r e d o x p o t e n t i a l by abou t 400 mV! A t p r e s e n t , t h i s s t a t e m e n t canno t be s u b j e c t e d t o e x p e r i m e n t a l assessment d i r e c t l y . 24 HG_ 6_L R e a c t i o n pa thway f o r t h e p r o d u c t i o n o f " p i c k e t - p o c k e t " p o r p h y r i n s . From C o l l m a n e t a l . ( 1 9 8 1 ) . 25 (A) (B) FIG 7_i. Schematic diagrams showing the direction of electron flow when the heme is ligated to (A) an electron donating ligand and (B) an elec-tron withdrawing ligand. From Ferguson-Miller ££. a l . (1979). 26 I n t h e case o f one heme p r o t e i n , I k e d a - S a i t o and P r i n c e ( 1 9 8 5 ) have shown t h a t a t pH 4 . 3 a C I complex o f m y e l o p e r o x i d a s e has a r e d u c t i o n p o t e n -t i a l o f +143 mV compared t o a v a l u e o f +21 mV f o r t h e n a t i v e p r o t e i n . T h i s r e s u l t d e m o n s t r a t e s t h a t l i g a n d s can i n f l u e n c e t h e r e d u c t i o n p o t e n t i a l s i g n i f i c a n t l y . Heme l i g a n d s w i l l n o t o n l y h e l p d e t e r m i n e t h e m i d - p o i n t p o t e n t i a l o f a heme g r o u p , t h e y may a l s o i n f l u e n c e t h e s i t e o f e l e c t r o n t r a n s f e r . S t u d i e s on t h e e f f e c t s o f a x i a l and e q u a t o r i a l l i g a n d s on t h e e l e c t r o n t r a n s f e r mechanisms o f model heme systems ( C o c o l i o s and K a d i s h , 1 9 8 5 ) have shown t h a t e l e c t r o n - w i t h d r a w i n g l i g a n d s ( F i g . 7B) t e n d t o f a v o u r r i n g - c e n t e r e d 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 b u t e l e c t r o n - d o n a t i n g l i g a n d s t e n d t o promote m e t a l -c e n t r e d 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 . These r e s u l t s a r e r e a s o n a b l e because l i g a n d s w h i c h w i t h d r a w e l e c t r o n s f r o m t h e p o r p h y r i n r i n g w i l l make the heme e l e c t r o n - d e f i c i e n t e l e c t r o p h i l l i e . L i g a n d s t h a t d o n a t e e l e c t r o n s i n t o t h e heme r i n g ( F i g . 7A) w i l l t e n d t o make i t e l e c t r o n - r i c h n u c l e o p h i l l i c . T h u s , t h e movement o f e l e c t r o n s i n and o u t o f t h e heme w i l l t e n d t o be m e t a l -c e n t r e d . H I THESIS INVESTIGATION; FUNCTIONAL PROPERTIES OF CYTOCHROME C PEPTIDE. 1 -65H C l e a r l y , much e f f o r t has gone i n t o s t u d y i n g t h e f u n c t i o n a l p r o p e r t i e s o f heme p e p t i d e s d e r i v e d f r o m c y t o c h r o m e £. and o f mode l heme compounds i n an e f f o r t t o d e l i n e a t e t h e r o l e o f v a r i o u s a s p e c t s o f t h e heme e n v i r o n m e n t i n r e g u l a t i n g t h e a c t i v i t y o f t h e heme c e n t e r . W h i l e t h e emphasis o f t h i s work has been on s m a l l heme p e p t i d e s and model heme compounds, r e l a t i v e l y l i t t l e a t t e n t i o n has been d i r e c t e d t o w a r d t h e l a r g e r heme p e p t i d e s o b t a i n e d by CNBr c l e a v a g e . 27 P a r t i a l c h a r a c t e r i z a t i o n o f 1-65H was i n i t i a t e d by B a b u l , McGowan and S t e l l w a g e n ( 1 9 7 1 ) . T h i s p e p t i d e has many f e a t u r e s i n common w i t h cy tochrome c_ i n t h a t 1-65H i s s m a l l (MW 7760 D a l t o n s ) , w a t e r - s o l u b l e , s t a b l e and easy t o i s o l a t e . Hydrodynamic a n a l y s i s was c o n s i s t e n t w i t h a m o l e c u l a r w e i g h t o f 7520 D a l t o n s w h i c h i n d i c a t e s t h a t t h i s p e p t i d e does n o t a g g r e g a t e a p p r e c i a b l y i n s o l u t i o n . T h i s f i n d i n g i s c o r r o b o r a t e d by t h e i n v a r i a n c e o f s o l u t i o n v i s c o s i t y w i t h 1-65H c o n c e n t r a t i o n o v e r t h e range o f 1 mg/ml (340 uM) t o 7 mg/ml (902 u M ) . Removal o f 38 r e s i d u e s f r o m t h e c a r b o x y l t e r m i n a l end o f c y t o c h r o m e c. w o u l d seem l i k e l y t o make t h e heme g r o u p more a c c e s s i b l e t o t h e e x t e r n a l e n v i r o n m e n t and weaken t h e a x i a l l i g a n d s i n r e l a t i o n t o t h a t o f h o r s e h e a r t cy toch rome c_. Two d e s i r a b l e consequences emerge f r o m t h e s e p r o p e r t i e s : F i r s t , t h e r e w i l l be a r e l a t i v e l y f a c i l e t r a n s i t i o n f r o m a 6 -c o o r d i n a t e l o w - s p i n heme s p e c i e s t o a 5 - c o o r d i n a t e h i g h - s p i n s p e c i e s ; and s e c o n d , t h e r e i s t h e p o t e n t i a l o p p o r t u n i t y f o r t h e s u b s t i t u t i o n o f t h e n a t u r a l a x i a l l i g a n d s w i t h s e l e c t e d exogenous l i g a n d s . As 1-65H l a c k s M e t - 8 0 , w h i c h i s one o f t h e a x i a l l i g a n d s i n t h e n a t i v e p r o t e i n , some e f f o r t has gone i n t o a s s e s s i n g t h e l i g a t i o n s t a t u s o f t h e heme i r o n i n t h i s p e p t i d e . C a r b o x y m e t h y l a t i o n o f t h e h i s t i d i n e r e s i d u e s o f 1-65H has e s t a b l i s h e d the a x i a l l i g a n d c o u p l e f o r t h e heme Fe t o be H i s / H i s . The c h e m i c a l m o d i f i c a t i o n o f t h e h i s t i d i n e r e s i d u e s p roduced a c o n v e r s i o n o f low s p i n 1-65H t o a h i g h s p i n s p e c i e s i n d i c a t i v e o f s p e c i f i c m o d i f i c a t i o n t o t h e a x i a l l i g a n d s . Chemica l m o d i f i c a t i o n o f r e s i d u e s t h a t m i g h t be c a n d i d a t e s f o r a x i a l l i g a n d s , such as l y s i n e , d i d n o t p r o d u c e a change i n t h e s p i n s t a t e . The l i g a n d c o u p l e o f h o r s e h e a r t cy toch rome c_, by c o n t r a s t , i s H i s / M e t . Because 1-65H i s s o l u b l e , amenable t o l i g a n d s u b s t i t u t i o n , and monomer ic i n s o l u t i o n , i t has been employed i n t h e work o u t l i n e d i n t h i s t h e s i s t o a d d r e s s t h e f o l l o w i n g q u e s t i o n s : I s i n c r e a s e d heme e x p o s u r e s u f f i c i e n t and 28 n e c e s s a r y t o i n d u c e p e r o x i d a s e a c t i v i t y ? What a r e t h e e f f e c t s o f a x i a l l i g a t i o n on t h e p e r o x i d a s e a c t i v i t y o f 1-65H and what a r e t h e e f f e c t s o f a x i a l l i g a t i o n on t h e e l e c t r o n i c d i s t r i b u t i o n and hence t h e s p e c t r a o f t he heme g r o u p i n 1-65H? These q u e s t i o n s a r e r e a l l y s u b - q u e s t i o n s o f a more g e n e r a l q u e s t i o n : What i s t h e r o l e o f p r o t e i n o r g a n i z a t i o n i n d e t e r m i n i n g heme c e n t r e r e a c t i v i t y ? 29 MATERIALS AND METHODS I i REAGENTS AND CHEMICALS The f o l l o w i n g i s a c o m p i l a t i o n o f a l l t h e r e a g e n t s , c h e m i c a l s and enzymes u s e d , t h e i r c h e m i c a l g rade and t h e i r s u p p l y company. £ a k £ x Chemica l Company. P h i l l i p s b u r g . N . J . B o r i c a c i d (ACS g r a d e ) ; KCN (ACS g r a d e ) EM C h e m i c a l s . Toronto 98% F o r m i c A c i d (ACS g r a d e ) ; Na„HP0, and NaH PO, ( a n a l y t i c a l g r a d e ) 32% H 2 0 2 B i o r a d . R ichmond. C a l i f o r n i a G l y c i n e ( E l e c t r o p h o r e s i s g r a d e ) Eas tman-Kodak . R o c h e s t e r . N.Y. o - D i a n i s i d i n e ( T e c h n i c a l g r a d e ) M a t h e s o n . Coleman £ B e l l . Norwood. QhJP Cyanogen b rom ide ( r e a g e n t g r a d e ) ; NaSCN (ACS g r a d e ) ; I m i d a z o l e ( r e c r y s t a l l i z e d f r o m benzene) Pharmac ia F i n e C h e m i c a l s . U p p s a l a , Sweden G-75 Sephadex Sigma. Chemica l Company. S t , L o u j g , MQ_ Horse H e a r t Cy tochrome c. (Type V I ) ; Horse R a d i s h P e r o x i d a s e (Type 30 I L L ISOLATION QL 1-65H A m o d i f i e d p r o c e d u r e based on t h e p r o t o c o l s o f Babu l si. a l . ( 1 9 7 2 ) and o f C o r r a d i n and H a r b u r y ( 1 9 7 0 ) was used t o p u r i f y t h e p e p t i d e f r a g m e n t 1 -65H. A f l o w c h a r t o f t h e i s o l a t i o n p r o c e d u r e i s shown i n F i g . 8 . A p p r o x i m a t e l y 100 mg. o f h o r s e h e a r t cy toch rome £. (Type V I , Sigma) was d i s s o l v e d i n 10 ml o f 70% f o r m i c a c i d . Cyanogen b rom ide (CNBr) was added t o t h e s o l u t i o n i n two h u n d r e d - f o l d excess o v e r cy toch rome c_ and a l l o w e d t o r e a c t f o r 24 h o u r s a t room t e m p e r a t u r e ( H a r b u r y and C o r r a d i n , 1 9 7 0 ) . The r e a c t i o n was s t o p p e d by a d d i n g 10 vo lumes o f d o u b l y d i s t i l l e d w a t e r . The d i l u t e d s o l u t i o n was d i v i d e d e v e n l y i n t o t e n 13x100 mm Kimax t e s t t u b e s ; t h e t u b e s were p l a c e d i n a Savant Speed Vac C o n c e n t r a t o r , and the s o l u t i o n was e v a p o r a t e d t o d r y n e s s . The d r i e d sample was t h e n r e d i s s o l v e d i n 7% f o r m i c a c i d and e l u t e d o v e r a Sephadex G-75 ( F i n e ) column ( 2 . 5 x 9 2 . 5 cm) a t 4 °C and i n 7% f o r m i c a c i d . The ave rage f l o w r a t e was 25 m l / h r and 3 ml f r a c t i o n s o f t h e e l u a n t were c o l l e c t e d o v e r a t o t a l volume o f a p p r o x i m a t e l y 270 ml ( F i g . 9 ) . For each f r a c t i o n , abso rbance r e a d i n g s a t 280 nm, 407, nm and 495 nm were r e c o r d e d . A l l f r a c t i o n s w i t h an ^ 2 8 0 ^ 4 9 5 r a t i o o f 2 . 2 8 + 0 . 1 (Han tgan and T a n i u c h i , 1977) were p o o l e d and c o n c e n t r a t e d by u l t r a f i l t r a t i o n t h r o u g h a s t i r r i n g , W p r e s s u r i z e d Amicon Model 12 o r 52 c e l l w h i c h was f i t t e d w i t h a YM-5 membrane. The c r u d e , c o n c e n t r a t e d 1-65H was r e a p p l i e d o n t o a s m a l l G-75 ( 2 . 5 x 1 5 cm) column w h i c h had been e q u i l l i b r a t e d w i t h 7% f o r m i c a c i d . F r a c t i o n s w i t h A ^ g g / A ^ j . r a t i o s o f 2 . 2 8 + 0 .05 were p o o l e d and c o n c e n t r a t e d as d e s c r i b e d a b o v e . Amino a c i d a n a l y s i s c o n f i r m e d t h a t t h e second i s o l a t e c o n t a i n e d p u r e 1-65H (see Re-s u l t s , T a b l e I ) . The a v e r a g e y i e l d o f 1-65H was 20% based on a m o l a r b a s i s . 31 FIG 8 : F l o w c h a r t showing t h e i s o l a t i o n p r o t o c o l f o r 1 -65H. 100 mg. HORSE HEART £ + 10 ml 70% HCOOH pH 1.6 + 100-fold XS CNBR/MET grp. WAIT 24 hr. (room temp.) + 10 vol. H20 (100 ml) EVAPORATE TO DRYNESS i DISSOLVE IN 7% HCOOH (2-3 ml) ELUTE AT 2 0-24 ml/hr. 24 hr. COLLECT 3 ml FRACTIONS EQUILIBRATE G-75 (2.5 x 98 cm.) in 7% HCOOH pH 1.6 PURE? 1 NC i YES ELUTE THRU G-75 (1.5 x 20 cm.) i n 7% HCOOH pH 1.6 CONCENTRATE STORE AT -70 C 3 3 I l l i PEROXIDASE ASSAY The H 2 0 2 / o - d i a n i s i d i n e assay sys tem ( W o r t h i n g t o n Enzyme M a n u a l , 1972) was used t o examine t h e p e r o x i d a t i c a c t i v i t y o f 1 -65H, hhc. and h o r s e r a d i s h p e r o x i d a s e (HRP) . The f o l l o w i n g s t o c k s o l u t i o n s were r e q u i r e d : 1% o -d i a n i s i d i n e s o l u t i o n ( i n MeOH), 0.3% H ^ . N a P i pH 7 . 9 1 = 0 . 1 , NaGly pH 3 . 0 1 = 0 . 1 , NaBor pH 1 0 . 0 1 = 0 . 1 and e i t h e r 1 mg/ml o f 1 -65H, 1 mg/ml o f h o r s e h e a r t cy toch rome c. o r 0 . 0 1 mg/ml o r HRP. The f i n a l c o n c e n t r a t i o n o f each component was 0.003% ^ 2 ° 2 * ^ .008% o - d i a n i s i d i n e and e i t h e r 0 . 0 0 1 m g / m l , 0 . 0 1 mg/ml hhc. o r 0 . 0 1 mg/ml HRP i n a 1 ml v o l u m e . The r e a g e n t s o - d i a n i s i d i n e and ^ 2 ^ 2 w e r e a ^ d e d t o t n e c u v e t t e f i r s t , and t h e s o l u t i o n a l l o w e d t o e q u i l i b r a t e t o t he d e s i r e d t e m p e r a t u r e . The assay was t h e n i n i t i a t e d by t h e a d d i t i o n o f t h e a p p r o p r i a t e vo lume o f p r o t e i n . P e r o x i d a s e r a t e s were c a l c u l a t e d f r o m t h e r a t e o f o - d i a n i s i d i n e o x i d a t i o n as m o n i t o r e d a t 460 nm f r o m t h e f o l l o w i n g r e l a t i o n s h i p : Rate o f o - d i a n i s i d i n e o x i d a t i o n = A . , - / m i n 460 nm 1 1 . 3 x (mg enzyme/ m l . s o l u t i o n ) H i LIGAND BINDING AND LIGAND SUBSTITUTION STUDIES L i g a n d b i n d i n g s t u d i e s i n v o l v e d t h e t i t r a t i o n o f 1-65H w i t h t h e l i g a n d i n q u e s t i o n and t h e r e c o r d i n g o f t h e a b s o r p t i o n s p e c t r u m subsequen t t o each a d d i t i o n . For each l i g a n d , t h e K^^-value was o b t a i n e d ( i . e . , t h e r a t i o o f L i g a n d t o 1-65H r e q u i r e d f o r h a l f max ima l change i n a b s o r b a n c e ) . L i g a n d s u b s t i t u t i o n e x p e r i m e n t s i n v o l v e d r e c o r d i n g t h e s p e c t r a l changes t h a t o c c u r 34 upon t h e r e p l a c e m e n t o f one l i g a n d by a n o t h e r . The l i g a n d s used w e r e : KCN, NaSCN, I m i d a z o l e and N - a c e t y l M e t h i o n i n e . N - a c e t y l m e t h i o n i n e was employed o n l y f o r t h e l i g a n d s u b s t i t u t i o n s t u d i e s . For most l i g a n d s , s t o c k s o l u t i o n s were p r e p a r e d i n t h e same b u f f e r as t h a t o f t h e p r o t e i n s o l u t i o n . For SCN , t i t r a t i o n e x p e r i m e n t s were p e r f o r m e d by a d d i n g s o l i d samples o f t h e l i g a n d . W i t h the e x c e p t i o n o f i m i d a z o l e , a d d i t i o n s o f each l i g a n d d i d n o t a l t e r t h e pH o f t h e s o l u t i o n by more t h a n 0 . 2 pH u n i t s . A d d i t i o n s o f i m i d a z o l e t h a t r e s u l t i n a f i n a l c o n c e n t r a t i o n o f more t h a n 15 mM have been known t o a l k a l i n i z e a c i d (pH 3 . 0 ) b u f f e r s t o above pH 8 . 0 . A l l l i g a n d s u b s t i t u t i o n and l i g a n d b i n d i n g e x p e r i m e n t s were p e r f o r m e d a t T=25 °C and i n e i t h e r N a P / p H 7 . 0 ( 1 = 0 . 1 ) o r NaGly pH 3 . 0 ( 1 = 0 . 1 ) . Hantgan and T a n i u c h i ( 1 9 7 7 ) e s t a b l i s h e d t h e e x t i n c t i o n c o e f f i c i e n t s f o r o x i d i z e d 1-65H t o be 1 . 2 7 + 0 . 0 1 x l 0 5 M _ 1 c m ~ 1 a t 406nm and 9 . 9 x l 0 3 M _ 1 c m " 1 a t 526 nm. The e x t i n c t i o n c o e f f i c i e n t s o f reduced and l i g a n d - c o m p l e x e d 1-65H were d e t e r m i n e d by c o m p a r i n g t h e abso rbance peaks t o the c o r r e s p o n d i n g peaks i n t h e s p e c t r a o f o x i d i z e d 1-65H and a d j u s t i n g t h e e x t i n c t i o n c o e f f i c i e n t v a l u e s a c c o r d i n g l y . i i p £ TITRATIONS A l l pH t i t r a t i o n s were p e r f o r m e d i n s o l u t i o n s o f NaP^ pH 7 . 0 ( 0 . 5 mM) w i t h a f i n a l 1-65H c o n c e n t r a t i o n o f 12 .4 uM. The pH was a d j u s t e d by t h e a d d i t i o n o f a p p r o p r i a t e vo lumes o f 1 N HC1 a n d / o r 2 N NaOH. The pH o f each a d d i t i o n was measured w i t h a c o m b i n a t i o n e l e c t r o d e ( R a d i o m e t e r , GK2321C). Y U . SPECTROPHOTOMETRIC INSTRUMENTS Two s p e c t r o p h o t o m e t e r s were used f o r t h i s i n v e s t i g a t i o n . Hard c o p i e s 35 s e t i n e i t h e r t h e TIME SCAN o r t h e OPT. PROG Dl mode. A b s o r p t i o n s p e c t r a were r e c o r d e d on t h e Cary 219 s p e c t r o p h o t o m e t e r w h i c h was equ ipped w i t h a p rogrammable i n t e r f a c e a c e s s o r y t h a t a l l o w e d a b s o r p t i o n s p e c t r a t o be d i g i t i z e d and d i s p l a y e d on a N o r t h S t a r m i c r o c o m p u t e r , s t o r e d on d i s k e t t e o r p l o t t e d on p a p e r . The g r a p h i c s package c o n t r o l l i n g t h e i n t e r f a c e o p e r a t i o n was SPECTRO (Rev . 4 . 0 8 ) by OLIS (On L i n e I n s t r u m e n t Sys tems , J e f f e r s o n GA) . B o t h t h e Cary and t h e Shimadzu i n t r u m e n t s were f i t t e d w i t h w a t e r j a c k e t e d sample h o l d e r s t h a t were m a i n t a i n e d a t c o n s t a n t t e m p e r a t u r e ( + 0 . 1 °C) w i t h a t h e r m o s t a t t e d c i r c u l a t i n g w a t e r b a t h . V I I : ^H-NMR EXPERIMENTS '''H-NMR s p e c t r a o f f r e e and c y a n o - f o r m s o f 1-65H were r e c o r d e d on a 200 MHz B r u k e r WP-200 s p e c t r o m e t e r . A p p r o x i m a t e l y 1.5 uM o f t h e heme p e p t i d e was o x i d i z e d by t h e a d d i t i o n o f excess d i p i c o l i n a t o c o b a l t a t e ( I II ) ( N H ^ C o ^ i p i c ^ ) i n NaP^ b u f f e r p H = 7 . 0 . The sample was p r e p a r e d f o r NMR a n a l y s i s by c e n t r i f u g a l u l t r a c e n t r i f u g a t i o n (Amicon C e n t r i c o n - 1 0 ) . The s o l u t i o n was exchanged and excess o x i d a n t was removed by w a s h i n g f o u r t i m e s w i t h d e u t e r a t e d NaP^ b u f f e r pD=7.1 ( 1 = 0 . 1 ) . C y a n o - l - 6 5 H was p roduced by a d d i n g a few c r y s t a l s o f KCN i n t o t h e NMR t u b e t h a t c o n t a i n e d t h e o x i d i z e d 1-65H. 36 R E S U L T S I i GENERAL CHARACTERISTICS P_E 1-65H A r e p r e s e n t a t i v e G-75 g e l f i l t r a t i o n p r o f i l e of hhc_ f o l l o w i n g treatment w i t h CNBr i s shown i n F i g . 9. F r a c t i o n s from peak " c " were c o l l e c t e d , concentrated and p u r i f i e d according to the p u r i f i c a t i o n p r o t o c o l o u t l i n e d i n the M a t e r i a l s and Methods s e c t i o n . R-values ^ 2 8 0 ^ 4 9 5 r a t ^ o s ^ f o r the p u r i f i e d 1-65H was experimentally e s t a b l i s h e d to be 2.32 + 0.02. The amino a c i d a n a l y s i s of 1-65H (Table I) agreed w i t h the expected amino a c i d composition. These r e s u l t s confirm the i d e n t i t y of the i s o l a t e to be pure 1-65H. Most of the experiments presented i n t h i s t h e s i s required a 1-65H concentration which was f a r less than 340 uM so the dependence of Soret absorbance on 1-65H concentration was examined. Most experiments required a concentration of 1-65H f a r less than 10 uM. The r e s u l t s ( F i g . 10) revealed a l i n e a r dependence from 1 uM to 9 uM i n d i c a t i n g that 1-65H does not aggregate over that concentration range. Absorption spectra of o x i d i z e d and d i t h i o n i t e reduced 1-65H are shown i n F i g . 11. As f o r a l l heme p r o t e i n s , the spectrum of 1-65H can be considered as having two p r i c i p a l spectrophotometric regions: the Soret region ( F i g . 11A) from 350-450 nm and the v i s i b l e region ( F i g . 11B) from 450 to 700 nm. The spectrum of o x i d i z e d 1-65H displayed a Soret maximum of 407 nm (E =1.27xl0 5M _ 1cm~ 1) and a v i s i b l e maximum of 526 nm (E = 9.9xl0 3M _ 1 max max cm The Soret band of reduced 1-65H has a maximum at 417 nm (E = max 1.30xl0 5M~ 1cm~ 1) w i t h the v i s i b l e maxima at 521 nm (E = 1.09xl0 4M _ 1cm _ 1) max and 550 nm (E = 1.76xl0**M *cm The spectrum of reduced 1-65H i s max s i m i l i a r to the spectrum of reduced horse heart cytochrome £.. Reduced horse 37 FIG 9: Elution profile of horse heart cytochrome c_ which has been digested with a 200-fold excess of CNBr. Conditions are as described in Materials and Methods. The identities of the separated components are (a) unreacted cytochrome c. (b) 1-80H (c) 1-65H (d) 81-104 (e) 66-80. 38 TABLE I ; Amino A c i d C o m p o s i t i o n o f 1-65H Number o f amino a c i d s / R e s i d u e Res idue O b t a i n e d E x p e c t e d ASP 5 .0 5 THR 6 . 5 7 SER 0 .2 0 GLU 7 . 0 7 PRO 2.2. 2 GLY 9 .5 10 ALA 3 . 1 3 VAL 2.7 3 MET 0 . 0 0 ILE 1.8 2 LEU 3 . 0 3 TYR 1.1 1 HIS 2 . 9 3 LYS 10 .7 11 ARG 1.1 1 Amino a c i d a n a l y s i s samples were p r e p a r e d by i n c u b a t i n g a p p r o x i m a t e l y 10 nmoles o f 1-65H i n 6N HC1 a t 110 °C f o r 24 h o u r s . The samples were t h e n e v a p o r a t e d on a Savant Speed-Vac and t h e n d i s s o l v e d i n s t a n d a r d 0 . 2 N N a C i t r a t e pH 2 . 2 . The equ ipment used was t h e Durrum Model D-500 Amino A c i d A n a l y s e r . The o b s e r v e d v a l u e s have an u n c e r t a i n t y o f + 5%. 39 40 ID O 2 < CD DC O W m < .6 .5 .4 .3 ,2 1 0 - (A) - (B) j O X I D I Z E D A l \ R E D U C E D \ / i — 1 . 1 1 R E D U C E D 1 i 1 / / I i \ i " 'l' / / I 1 — \ \ 1 \ I i \ X \ \ \ \ \ \ 1 l \ O X I D I Z E D 1 1 I 1 1 1 1 1 1 1 1 . 1 .08 .06 04 .02 360 380 400 420 440 450 500 550 600 650 700 W A V E L E N G T H £I£ H i (A) Soret and (B) Visible absorption spectra of oxidized and dithionite-reduced 1-65H (1=0.1 NaPi pH 7.0. T=25 ° C . ) 41 h e a r t cy toch rome £. possesses a b s o r p t i o n maxima a t 416 nm ( 1 . 2 9 x 1 0 M cm ) , 3 - 1 - 1 3 - 1 - 1 521 nm ( 1 5 . 9 x 1 0 M cm ) and 550 nm ( 2 7 . 7 x 1 0 M cm ) r e s p e c t i v e l y ( M a r g o l i a s h £ t a j j . , 1 9 5 9 ) . The s p e c t r u m o f o x i d i z e d h o r s e h e a r t cy toch rome £. was c o n s i d e r a b l y d i f f e r e n t f r o m t h a t o f 1-65H b o t h i n te rms o f a b s o r p t i o n maxima and e x t i n c t i o n c o e f f i c i e n t s . 1-65H had a S o r e t peak a t 410 nm ( 1 . 0 6 x l O ^ M ^cm * ) and a v i s i b l e peak a t 526 nm ( 1 1 . 2 x l 0 3 M *cm * ) w h i l e h o r s e h e a r t cy toch rome c had maxima a t 409 nm ( 1 . 0 6 x l O ^ M ^cm * ) and 528 nm ( 1 1 . 2 x l 0 3 M " 1 c m ~ 1 ) . There a r e s e v e r a l i n d i c a t i o n s t h a t t h e b i n d i n g s t r e n g t h s o f t h e a x i a l l i g a n d s t o t h e heme i r o n o f 1-65H a r e weaker t h a n t h e b i n d i n g s t r e n g t h s o f t h e a x i a l l i g a n d s t o h o r s e h e a r t cy toch rome c_. One manner i n a s s e s s i n g t h e b i n d i n g s t r e n g t h s was i n m o n i t o r i n g t h e pH-dependen t s p i n s t a t e o f 1-65H. The t r a n s i t i o n f r o m a low s p i n s p e c i e s t o a h i g h s p i n s p e c i e s i s a r e s u l t o f t h e p r o t o n a t i o n o f a t l e a s t one o f t h e a x i a l l i g a n d s and i t s subsequent d i s s o c i a t i o n f r o m t h e heme m o i e t y . pH t i t r a t i o n s s h o u l d p r o v i d e some i n d i c a t i o n s as t o how weak o r s t r o n g an a x i a l l i g a n d appears t o b e . A n a l y s i s o f t h e pH dependent a b s o r p t i o n s p e c t r a o f 1-65H shows t h a t t h e p r o t e i n i s h i g h s p i n i n a c i d c o n d i t i o n s and low s p i n i n n e u t r a l o r b a s i c c o n d i t i o n s ( F i g . 12A and F i g . 1 2 B ) . T i t r a t i o n p l o t s o f S o r e t absorbance v s . pH f o r 1-65H f r o m pH 6 . 8 t o pH 1.8 ( F i g . 13) show a pK o f a round 3 . 4 . s The pK f l f o r an e q u i v a l e n t t i t r a t i o n t o h o r s e h e a r t cy toch rome c_ i s 2 .5 ( B a b u l and S t e l l w a g e n , 1 9 7 2 ) . A n o t h e r method f o r e x a m i n i n g t h e b i n d i n g s t r e n g t h s o f t h e a x i a l l i g a n d s t o 1-65H i s t o s t u d y t h e CO b i n d i n g p r o p e r t i e s o f 1 -65H. I t was found t h a t CO b i n d s q u i t e r e a d i l y t o 1-65H ( F i g . 1 4 ) . By way o f c o n t r a s t , n o n -deamida ted fo rms o f h o r s e h e a r t cy toch rome £. w i l l n o t b i n d t o CO (Tsou C. L . , 1 9 5 1 ; F l a t m a r k T . , 1 9 6 6 ) . I t has been shown t h a t t h e b i n d i n g s t r e n g t h o f t h e m e t h i o n i n e a x i a l l i g a n d i s a f u n c t i o n o f t h e number o f r e s i d u e s 42 UJ o < CD DC O CO m < - (A) u pH 3 - (B) A IS i • / • -; i • / n p H 7 i pH 3 - 1 / V \ p H 7 \ - • v \\ /' / A ^ ^ ^ ^ ^ ^ ^ ^ \ ^ ^^^^^ \ \ i i i i i i i i i i i i i i .06 .05 .04 .03 .02 ,01 0 300 320 340 360 380 400 420 440 450 500 550 600 650 700 WAVELENGTH E I £ 121. (A ) S o r e t and (B) v i s i b l e a b s o r p t i o n s p e c t r a o f 3 .4 uM 1-65H i n e i t h e r u=0.1 NaGly pH 3.0 o r u=0.1 NaP^^ pH 7 . 0 . T=25 ° C . 43 FIG 13: Plot shoving the change in 395 nm absorbance of 12.4 uM 1-65H as a function of pH. Starting conditions vere 0.5 mM NaP. pH 7.0, T= 25 °C, Titration of 1-65H sample vas as described in tfee Materials and Methods section. 44 FIG 14; (A) Soret and (B) visible absorption spectra of the oxidized, dithionite-reduced and the reduced + CO forms of 1-65H. Experimental conditions were 4.3 uM 1-65H in u=0.1 NaP. pH 7.0, T=25 °C. Reduced 1-65H was bubbled with CO for about 30 seconds. 45 i n v o l v e d i n f o r m i n g t h e h y d r o p h o b i c p o c k e t o f t h e heme b i n d i n g s i t e ( W i l g u s e t a l . . 1 9 7 8 ) . I n 1 -65H, a number o f t h e s e t y p e s o f r e s i d u e s a r e l o s t and hence t h e l i g a n d b i n d i n g s t r e n g t h s a r e w e a k e r . The CO r e s u l t s , a l o n g w i t h t h e pH t i t r a t i o n d a t a a r e s t r o n g e v i d e n c e t h a t wou ld c o n f i r m t h e dec rease i n t h e b i n d i n g a f f i n i t i e s o f t h e a x i a l l i g a n d s i n 1-65H r e l a t i v e t o cy tochrome £.» H i LTGANp BINDING STUDIES Q£ 1-65H l A l E H " A d d i t i o n The s p e c t a o f reduced and o x i d i z e d c y a n o - l - 6 5 H a r e shown i n F i g . 15A and F i g . 15B. A d d i t i o n o f CN t o e i t h e r r e d u c e d o r o x i d i z e d s p e c i e s o f 1 -65H p roduced a r e d s h i f t ( i . e . a s h i f t o f an abso rbance peak towards h i g h e r w a v e l e n g t h s ) i n b o t h t h e S o r e t and t h e v i s i b l e r e g i o n s . The o x i d i z e d CN complex has a So re t maximum a t 411 nm ( 9 . 7 2 x l O ^ M ^cm and a v i s i b l e 3 - 1 - 1 abso rbance maxima a t 530 nm ( 8 . 6 x 1 0 M cm ) . D i t h i o n i t e - r e d u c e d c y a n o - l - 6 5 H had a S o r e t maximum a t 421 nm ( 1 . 2 2 x l O ^ M *cm * ) and v i s i b l e maxima a t 526 nm (1 . S l x l O ^ ^ c m " 1 ) and 555 nm ( 1 . 4 4 x l 0 4 M ~ 1 c m " 1 ) . The c y a n i d e complex i s s i g n i f i c a n t l y more s t a b l e t h a n t h a t o f n a t i v e hhc.. W i t h 1 -65H, t h e c y a n o - c o m p l e x r e m a i n s i n t a c t t h r o u g h s e v e r a l c y c l e s o f t h e r e d o x s t a t e . By way o f c o n t r a s t , t h e hhc. complex i s v e r y s t a b l e i n t h e o x i d i z e d f o r m b u t r e l a t i v e l y u n s t a b l e i n t h e r e d u c e d f o r m . R e d u c t i o n o f t h e o x i d i z e d c y a n o - c o m p l e x o f h o r s e h e a r t cy toch rome c. w i t h d i t h i o n i t e causes r e v e r s i o n o f t h e p r o t e i n t o i t s u n l i g a t e d f o r m w i t h i n t w e n t y m i n u t e s ( B r o o k s , Chanady and N i c h o l l s , 1 9 8 2 ) . The s t r e n g t h o f CN as an a x i a l l i g a n d was m o n i t o r e d by e x a m i n i n g t h e change i n t h e S o r e t maximum as a f u n c t i o n o f l i g a n d c o n c e n t r a t i o n . The CN 46 W A V E L E N G T H ( n m ) *1 FIG 15! (A) Soret and (B) visible absorption spectra of the reduced and reduced-cyano forms of 1-65H. 4.33 uM 1-65H in 1 ml u=0.1 NaP. pH 7.0, T=25 °C. Final concentration of CN~ was 600 uM. 1 FIG 15; (C) Soret and (D) visible absorption spectra of 3.4 uM 1-65H in the abscence and presence of 35 mM Imidazole. 1-65H in 1 ml u=0.1 NaGly pH 3.0, T=25 °C. FIG 15; (E) Soret and (F) visible absorption spectra of 4.05 uM 1-65H in the abscence and presence of 327 mM SCN. 1-65H in 1 ml u=0.1 NaGly pH 3.0. T=25 °C. 48 TABLE I I : S p e c t r a l Data f o r v a r i o u s d e r i v a t i v e s o f 1-65H [ W a v e l e n g t h ( E x t i n c t i o n C o e f f i c i e n t ) ] CONDITIONS S o r e t V i s i b l e o x i d i z e d , pH 3 . 0 395 nm ( 2 . 1 1 x 1 0 ) 495 nm ( 9 . 7 x 1 0 ) 621 nm ( 3 . 9 6 x 1 0 ) pH 7 . 0 , o x i d i z e d o x i d i z e d + CN 407 nm ( 1 . 2 7 x l 0 p 411 nm ( 9 . 7 2 x 1 0 ) 526 nm ( 9 . 9 x 1 0 , ) 530 nm ( 8 . 6 x 1 0 ) pH 7 . 0 , reduced reduced + CN 417 nm ( 1 . 3 0 x 1 0 ) 421 nm ( 1 . 2 2 x l 0 5 ) 521 nm ( 1 . 0 9 x 1 0 ^ ) 550 nm ( 1 . 7 6 x 1 0 7 ) 526 nm ( 1 . 3 1 x 1 0 ? ) 555 nm ( 1 . 4 4 x 1 0 ) pH 7 . 0 , d i t h + CO 414 nm ( 1 . 8 5 x 1 0 ) 528 nm ( 1 . 0 4 x 1 0 , ) 558 nm ( 8 . 1 4 x 1 0 ) pH 3 . 0 , o x i d i z e d + i m i d 407 nm ( 1 . 2 2 x 1 0 ) (35 mM) 526 nm (9.95x10,) 557 nm (7.10x10 ) pH 3 . 0 , o x i d i z e d + SCN (327 mM) 399 nm (1.19x10 ) 492 nm (9.85x10,) 589 nm (2.96x10,) 635 nm (2.52x10,) 623 nm (2.96x10 ) 49 FIG 1 6 : Changes i n t h e S o r e t abso rbance o f 1-65H as a f u n c t i o n o f (A) [CN ] /C1-65H] (B ) [ l m i d a z o l e ] / [ l - 6 5 H ] (C) C S C N ] / [ 1 - 6 5 H ] . Excep t f o r C N _ . a l l t i t r a t i o n s were p e r f o r m e d 1 = 0 . 1 NaGly pH 3 . 0 . For CN 1 = 0 . 1 NaP. pH 7 . 0 was u s e d . A d d i t i o n p r o c e d u r e s : (A ) A l i q u o t a d d i t i o n s o f s t o c k 20 mM KCN (B) a l i q u o t a d d i t i o n s o f s t o c k 1M I m i d a z o l e (C) m i l l i g r a m a d d i t i o n s o f s o l i d NaSCN. 50 ABSORBANCE at 395 nm ABSORBANCE at 395 nm ABSORBANCE at 407 nm t_n • en ' —2 ' . . . . . . * jjfc. j*-. • c_n (_n (_n c n t_n *-o cn oo en o~> ~-o co co rsj -t*~ cn co t_n no t i t r a t i o n p l o t i s shown i n F i g . 16A and t h e v a * u e a e r i v e c ^ f r o m t h e t i t r a t i o n d a t a was d e t e r m i n e d t o be 2 8 . i l l I m i d a z o l e T i t r a t i o n I m i d a z o l e had l i t t l e e f f e c t on t h e 1-65H s p e c t r a a t pH 7 . 0 . Pronounced changes i n a b s o r p t i o n s p e c t r a c o u l d o n l y be seen a t low pH ( 1 = 0 . 1 NaGly pH 3 . 0 ) . S a t u r a t i o n o f h i g h s p i n ( a c i d i c ) 1-65H by i m i d a z o l e y i e l d e d a low s p i n s p e c i e s whose s p e c t r u m i s v e r y s i m i l i a r i n appearance t o t h e n a t i v e 1 -65H a t pH 7 . 0 ( F i g . 15C and F i g . 1 5 D ) . I n t h e p r e s e n c e o f 30 mM i m i d a z o l e , 1-65H possessed a S o r e t band a t 407 nm ( 1 . 2 2 x l 0 ^ M ^cm ^ ) and a v i s i b l e peak 3 - 1 - 1 a t 526 nm ( 9 . 9 5 x 1 0 M cm ) . These v a l u e s a r e s i m i l i a r t o t h o s e o f n a t i v e 1 -65H. P r e s u m a b l y , t h e s i m i l a r i t y be tween i m i d a z o l e and h i s t i d i n e i n c h e m i c a l s t r u c t u r e a c c o u n t s f o r t h e l a c k o f d i f f e r e n c e i n t h e n a t i v e and i m i d a z o l e - c o m p l e x s p e c t r a . Subsequent t i t r a t i o n p l o t s ( F i g . 16B) c o n f i r m e d t h a t i m i d a z o l e i s an i n t e r m e d i a t e s t r e n g t h l i g a n d w i t h a v a l u e o f 5 5 0 0 . These r e s u l t s s h o u l d be r e g a r d e d w i t h c a u t i o n because pH measurements o f t h e i m i d a z o l e - p e p t i d e complex showed t h a t by t h e end o f t h e t i t r a t i o n e x p e r i m e n t s , t he pH i n c r e a s e d f r o m an i n i t i a l v a l u e o f 3 . 0 t o a f i n a l v a l u e o f 6 . 1 . Thus i t i s u n c e r t a i n w h e t h e r o r n o t t h e s p e c t r a o f F i g . 8C and F i g . 8D a r e a c t u a l l i g a n d complexes o r m e r e l y low s p i n n a t i v e 1-65H. However , t h e p e r o x i d a s e assay r e s u l t s f o r t h e complex ( v i d e i n f r a S e c t i o n V) does i n d i c a t e t h a t t he i m i d a z o l e l i g a n d i s c a p a b l e o f a s s o c i a t i n g w i t h t h e heme on 1 -65H. i£l ££N_~ T i t r a t i o n T i t r a t i o n o f 1-65H by SCN a t pH 7 . 0 p r o d u c e d m i n i m a l a l t e r a t i o n s i n t h e s p e c t r u m o f 1 -65H. The a d d i t i o n o f 327 mM SCN~ t o 4 . 0 5 uM 1-65H produced a r e d s h i f t o f t he S o r e t band f r o m 395 nm ( 2 . 1 1 x 1 0 M cm ) t o 399 nm ( 1 . 1 9 x l 0 5 M _ 1 c m " 1 ) (see F i g . 1 5 E ) . F i g . 15F shows t h a t SCN~ a d d i t i o n p roduces a m ixed s p i n s p e c i e s i . e . a s p e c i e s w i t h s p e c t r o p h o t o m e t r i c f e a t u r e s t h a t a r e composed o f h i g h s p i n and low s p i n p e a k s . F u r t h e r a d d i t i o n s o f SCN p r o d u c e d no s h i f t s i n t h e ,X o f t h e v i s i b l e r e g i o n max a l t h o u g h t h e r e i s a s m a l l d rop i n a b s o r b a n c e . T i t r a t i o n p l o t s o f 1-65H by SCN have e s t a b l i s h e d t h e K ^ 2 v a l u e t o be a round 12000 ( F i g . 1 6 C ) . I l l ; LIGAND SUBSTITUTION STUDIES O F 1-6 5H The d i f f e r e n c e i n t h e So re t s p e c t r a be tween cyano-hhc. and c y a n o - l - 6 5 H p r o v i d e t h e b a s i s f o r t he l i g a n d s u b s t i t u t i o n e x p e r i m e n t s . A d d i t i o n o f CN t o reduced or o x i d i z e d hhc_ p roduces a r e d s h i f t i n t h e S o r e t and an i n c r e a s e i n abso rbance ( B r o o k s e_t a l . . 1 9 8 2 ) . I n c o n t r a s t , t h e a d d i t i o n o f CN t o 1 -65H p roduced a r e d s h i f t and a d e c r e a s e i n absorbance a t t h e S o r e t r e g i o n . Two h y p o t h e s e s were d e v e l o p e d t o e x p l a i n t h e s e o b s e r v a t i o n s . I n i t i a l l y , i t was p roposed t h a t t h e g r e a t e r b i n d i n g s t r e n g t h s o f t h e a x i a l l i g a n d s i n h o r s e h e a r t c y t o c h r o m e c_ , p l u s t h e p r e s e n c e o f a more e n c l o s e d heme s i t e imposed s t e r i c d i s t o r t i o n s t o t h e b i n d i n g geomet ry o f any exogenous a x i a l l i g a n d s . P r e s u m a b l y , t h i s d i s t o r t i o n f a c t o r i s n o t as p ronounced 1-65H and may a c c o u n t f o r t h e d i f f e r e n c e i n S o r e t i n t e n s i t y be tween t h e two c h r o m o p h o r e s . I f t h i s h y p o t h e s i s i s c o r r e c t t h e n heme p r o t e i n s w i t h r e l a t i v e l y l e s s s t e r i c h i n d r a n c e ( e . g . Mb) s h o u l d p roduce s p e c t r a c l o s e t o t h o s e o f t h e c y a n o - c o m p l e x o f 1 -65H. On t h e o t h e r h a n d , t h o s e p r o t e i n s w i t h s t r o n g a x i a l l i g a n d s ( e . g . c y t . b_^) s h o u l d have a b s o r p t i o n s p e c t r a o f t h e c y a n o - c o m p l e x w h i c h i s s i m i l i a r t o t h e s p e c t r a o f t h e c y a n o - c o m p l e x o f ho rse h e a r t cy toch rome c_. However t h e cyano -comp lexes o f b o t h b_^  and Mb d i s p l a y e d a b s o r p t i o n s p e c t r a t h a t showed a l o s s i n S o r e t abso rbance r e l a t i v e 53 t o t he c o r r e s p o n d i n g uncomplexed f o r m s . An a l t e r n a t i v e e x p l a n a t i o n a r i s e s f r o m t h e n a t u r e o f t h e a x i a l l i g a n d s . I n h o r s e h e a r t cy toch rome c_, one o f t h e a x i a l l i g a n d s i s e l e c t r o n w i t h d r a w i n g ( H i s ) w h i l e t h e o t h e r i s e l e c t r o n d o n a t i n g ( M e t ) . I n 1-65H; h o w e v e r , b o t h a x i a l l i g a n d s a r e e l e c t r o n d o n a t i n g H i s l i g a n d s To i n v e s t i g a t e t h e i n f l u e n c e o f e l e c t r o n - d o n a t i n g and e l e c t r o n - w i t h d r a w i n g t y p e l i g a n d s on t h e S o r e t abso rbance s p e c t r a , l i g a n d s u b s t i t u t i o n were d e s i g n e d t h a t w o u l d m imic t h e s w i t c h f r o m t h e a x i a l l i g a n d s o f ho rse h e a r t c y t o c h r o m e c. t o t he a x i a l l i g a n d s o f 1 -65H. The p r o t o c o l employed was : 1 . T i t r a t i o n o f 1-65H w i t h e l e c t r o n - w i t h d r a w i n g l i g a n d s . 2 . Replacement o f t h e f i r s t l i g a n d w i t h a second l i g a n d w h i c h i s e l e c t r o n - d o n a t i n g . I f t h e e l e c t r o n d o n a t i n g / w i t h d r a w i n g p r o p e r t i e s o f t h e a x i a l l i g a n d s a r e r e s p n s i b l e f o r t h e s p e c t r o s c o p i c o b s e r v a t i o n s a b o v e , t h e n t h e r e p l a c e m e n t o f t h e f i r s t l i g a n d by t h e second s h o u l d p r o d u c e an i n c r e a s e i n t h e So re t a b s o r b a n c e . The l i g a n d c o u p l e s w h i c h were used w e r e : ( i ) + N - A c e t y l - M e t h i o n i n e + I m i d a z o l e ( i i ) + N - A c e t y l - M e t h i o n i n e + CN~ ( i i i ) + SCN + I m i d a z o l e ( i v ) + SCN + CN* F i g u r e 17 i s a r e p r e s e n t a t i v e example o f t h e s p e c t r o p h o t o m e t r i c changes t h a t a r e the r e s u l t o f t h e s e l i g a n d s u b s t i t u t i o n e x p e r i m e n t s . These e x p e r i m e n t s were p e r f o r m e d on b o t h 1-65H and Mb t o e s t a b l i s h w h e t h e r o r n o t t h e 54 prediction could be extended to proteins other than 1-65H. The results of the ligand substitution experiments are summarized in Table III. For 1-65H, experiments ( i ) , ( i i ) and (iv) showed an increase in the Soret absorbance t o t he v a l u e s r e p o r t e d f o r t h e i m i d a z o l e o r cyano -comp lexes o f 1 -65H. However , t h e r e s u l t s were l e s s c o n c l u s i v e f o r Mb. I n t h i s c a s e , t h e i n t e n s i t y o f t h e So re t i n c r e a s e d f o r t h e + Met + I m i d e x p e r i m e n t b u t t h e Met+CN~, SCN+Imid, SCN+CN~ e x p e r i m e n t s a l l showed a l o s s i n S o r e t a b s o r b a n c e . The d i s c r e p a n c i e s i n t h e SCN e x p e r i m e n t s may stem f r o m t h e f a c t t h a t a l l t h e SCN e x p e r i m e n t s w i t h Mb were r u n a t pH 7.0, t h e e x p e r i m e n t s f o r 1-65H were r u n a t pH 3.0. U s u a l l y , t h e g r e a t e s t s p e c t r o s c o p i c changes f o r SCN and i m i d a z o l e b i n d i n g were o b s e r v e d a t pH 3.0. However , t h e heme g r o u p o f Mb i s n o t c o v a l e n t l y bound t o t h e p r o t e i n and d i s s o c i a t e s f r o m i t a t low pH so a l l e x p e r i m e n t s f o r Mb were p e r f o r m e d a t pH 7.0. H-NMR spectra of the ligand-free and cyano forms of oxidized (and hence, paramagnetic) 1-65H in the region from 10 ppm to 40 ppm are shown in Fig. 18. This portion of the spectrum contains resonances attributable to heme substituent groups that are shifted away from the main protein resonances by the proximity of the paramagnetic iron atom (La Mar e_L a l . . 1980). For the free form of 1-65H, this portion of the spectrum is composed of very broad and nearly featureless resonances. But for the cyano form of 1-65H, this region exhibits a series of well resolved resonances. The results in Fig. 18 would indicate that the native axial ligands are so weakly bound that the frequency of association and dissociation of these axial ligands on the NMR with respect to the f i r s t axial ligand. The X max values were a l l identical 1 55 FIG 17; Representative tracing of a (+SCN+Imidazole) ligand substitution experiment, (a) Soret absorption spectrum of 2.8 uM oxidized 1-65H in 1 ml u=0.1 NaGly pH 3.0, T=25 °C (b) +387 mM SCN (c) +53.12 mM Imidazole. 56 1ABL£ I I I I Data Summary o f t h e L i g a n d S u b s t i t u t i o n E x p e r i m e n t s 1-65H M y o g l o b i n L i g a n d A d d i t i o n (mM) max ^ m a x L i g a n d A d d i t i o n (mM) max X max Met ( 8 . 5 ) + I m i d ( 1 7 . 8 ) +17.8 408 Met ( 1 4 2 ) + I m i d ( 2 3 5 ) +10.5 414 Met ( 8 . 5 ) + CN~ ( 2 ) +0.4 421 Met ( 8 . 1 ) + CN" ( 2 ) -15.6 420 SCN (387 ) + I m i d ( 2 9 6 ) +17.2 407 SCN ( 2 0 9 ) + I m i d ( 2 7 0 ) -18.8 413 SCN ( 4 2 0 ) + CN~ ( 4 ) ND ND SCN ( 3 6 0 ) + CN" ( 4 ) -6.9 421 [ 1 - 6 5 H ] = 4 - 5 uM [Mb] = 2 . 4 - 3 uM For Mb, a l l l i g a n d s u b s t i t u t i o n e x p e r i m e n t s were done i n u = 0 . 1 NaP. pH 6 . 0 . For 1 -65H, a l l e x p e r i m e n t s i n v o l v i n g m e t h i o n i n e were done i n u=0.1 NaP. pH 7 . 0 b u f f e r w h i l e a l l e x p e r i m e n t s i n v o l v i n g SCN were done i n u = 0 . 1 NaGly pH 3 . 0 b u f f e r . 57 I _ ! 1 I 1 1 40 30 20 10 0 -10 CHEMICAL SHIFT (ppm) FIG 1 8 : H-NMR s p e c t r a o f t h e f r e e and cyano f o r m s o f 1.3 mM o x i d i z e d 1-65H i n d e u t e r a t e d NaP. p D = 7 . 1 . 58 t i m e - s c a l e i s s u f f i c i e n t t o b r o a d e n t h e p a r a m a g n e t i c a l l y - s h i f t e d heme resonances beyond d e t e c t i o n . The r e p l a c e m e n t o f t h e h i s t i d y l l i g a n d w i t h c y a n i d e e l i m i n a t e s t h i s r a p i d exchange t o p roduce t h e w e l l r e s o l v e d resonances shown f o r c y a n o - l - 6 5 H . Y-i P e r o x i d a s e Assays The p e r o x i d a s e a c t i v i t y o f 1-65H was i n v e s t i g a t e d t o assess t h e degree t o w h i c h heme e x p o s u r e and a c c e s s i b i l i t y t o 1*^2 ^ e a ( ^ t o i n c r e a s e d p e r o x i d a s e a c t i v i t y . As t h e heme g roup o f 1-65H i s p r o b a b l y more exposed t o t h e s o l v e n t t h a n t h a t o f hhc_, t h e measured p e r o x i d a s e a c t i v i t i e s f o r 1 -65H, hhc_ and HRP were compared . The r e s u l t s o f t h e s e c o m p a r a t i v e s t u d i e s a r e summarized i n T a b l e I V . A t a l l v a l u e s o f pH, 1-65H d i s p l a y e d an enhanced p e r o x i d a s e a c t i v i t y o v e r hhc_. The i n c r e a s e was anywhere f r o m 8 8 -f o l d (pH 3 . 0 ) t o 8 7 5 - f o l d (pH 7 . 0 ) . On t h e o t h e r h a n d , t h e r a t e s observed f o r HRP a re g r e a t e r t h a n t h o s e f o r 1-65H by n e a r l y t h r e e o r d e r s o f m a g n i t u d e . I n t e r e s t i n g l y , a t low pH where 1-65H s h o u l d be p r e d o m i n a n t l y h i g h s p i n , no i n c r e a s e o f a c t i v i t y was o b s e r v e d . I n s t e a d , t h e a c i d i f i c a t i o n l e a d t o a d e c r e a s e i n p e r o x i d a s e a c t i v i t y . However , a c i d i f i c a t i o n d i d p roduce i n c r e a s e d r a t e s f o r hhc. and HRP. The a b s o r p t i o n s p e c t r a o f o - d i a n i s i d i n e a t pH 3 . 0 i s d i f f e r e n t f r o m t h e s p e c t r a a t pH 7 . 0 so t h e d i s c r e p a n c y c o u l d be e x p l a i n e d by t h e r e l a t i v e i n s t a b i l i t y o f o - d i a n i s i d i n e o r even ^ 0 2 u n d e r a c i d c o n d i t i o n s . The i n c r e a s e d a c t i v i t y o f HRP and h o r s e h e a r t cy tochrome £. u n d e r a c i d i c c o n d i t i o n s makes t h e c o n t e n t i o n d o u b t f u l . The p e r o x i d a s e a c t i v i t y o f 1-65H a l s o seemed t o be dependent on t e m p e r a t u r e and G u a n i d i n e , H C 1 (Gdn .HC l ) c o n c e n t r a t i o n . I t was found t h a t an i n c r e a s e i n t e m p e r a t u r e l e d t o an i n c r e a s e i n p e r o x i d a s e a c i t i v i t y ( F i g . 1 9 A ) . 59 TABLE I V : C o m p a r a t i v e P e r o x i d a s e A c i t i v i t i e s o f Horse R a d i s h P e r o x i d a s e (HRP) , Horse h e a r t cy toch rome c_ (hhc.) and 1-65H a t pH 3 , 7 , and 10 S p e c i e s P e r o x i d a s e Rate (umole H202 /min mg p r o t e i n ) pH 3 . 0 pH 7 . 0 pH 1 0 . 0 HRP 2411 + 282 1706 + 286 2 9 + 1 . 4 1-65H 4 . 4 + 0 . 8 7 . 0 + 2 .6 1.03 + 0 .14 hhc. 0 .05 + 0 . 0 1 * 0 .008 + 0 . 0 0 1 0 .002 + 0 . 0 0 1 0 . 2 4 + 0 . 0 1 * Slow phase o f p e r o x i d a s e r e a c t i o n * * F a s t phase o f p e r o x i d a s e r e a c t i o n P e r o x i d a s e r e a g e n t s - 0.003% H ^ 0.008% o - d i a n i s i d i n e u = 0 . 1 NaGly (pH 3 . 0 ) o r NaP. (pH 7 . 0 ) o r NaBor (pH 1 0 . 0 ) A l l s o l u t i o n s made up t o 1 m l . Sample vo lumes added - 10 u l 1-65H (1 m g / m l ) o r 100 u l hhc. (1 m g / m l ) o r 1 u l HRP ( 0 . 0 1 m g / m l ) 60 FIG 1 9 ; The e f f e c t s o f (A ) T e m p e r a t u r e (B ) [ G d n . H C l ] and (C) L i g a n d a d d i t i o n on t h e p e r o x i d a s e a c t i v i t y o f 1 -65H. 61 Gdn.HCl a d d i t i o n s p roduced s i m i l i a r r e s u l t s ; t h e g r e a t e r t h e Gdn.HCl c o n -c e n t r a t i o n , t h e g r e a t e r t h e p e r o x i d a s e a c t i v i t y ( F i g . 1 9 B ) . Gdn.HCl i s a known d e n a t u r a n t and i n c r e a s i n g Gdn.HCl c o n c e n t r a t i o n does push 1-65H i n t o t h e h i g h s p i n s t a t e . Thus t h e t e m p e r a t u r e dependence and Gdn.HCl e x p e r i m e n t s seem t o show some c o r r e l a t i o n be tween s p i n s t a t e and p e r o x i d a s e a c t i v i t y . I n p a r t i c u l a r , as 1-65H i s g a i n i n g i n h i g h s p i n c h a r a c t e r , t h e r e i s an i n c r e a s e i n t h e p e r o x i d a s e a c i t i v i t y . T h i s i s i n d i r e c t c o n t r a d i c -t i o n t o t h e pH dependent p e r o x i d a s e assays f o r 1 -65H. The e f f e c t o f a d d i n g exogenous a x i a l l i g a n d s t o 1-65H p e r o x i d a s e a c t i v i t y i s shown i n F i g . 19C. I n each c a s e , an i n c r e a s e i n t h e l i g a n d t o 1-65H r a t i o l e a d s t o an i n h i b i t i o n o f p e r o x i d a s e a c t i v i t y . P r e d i c t a b l y , t h e s t r o n g e r t h e l i g a n d f i e l d s t r e n g t h o f t h e g roup a d d e d , t h e l o w e r t h e l i g a n d t o 1-65H r a t i o needed t o m a x i m a l l y i n h i b i t t h e p e r o x i d a s e a c t i v i t y . T h u s , CN has t h e l o w e s t [ l i g a n d ] / [ l - 6 5 H ] r a t i o w h i l e SCN has t h e h i g h e s t [ l i g a n d ] / [ l - 6 5 H ] r a t i o . A s u r p r i s i n g and n o t v e r y e x p l a i n a b l e r e s u l t i s t h e c o n n e c t i o n be tween l i g a n d s t r e n g t h and t h e e x t e n t o f p e r o x i d a s e i n h i b i t i o n . I n p a r t i c u l a r , SCN w i t h an K^^~va^-ue ° f 12000 p roduced a max ima l i n h i b i t i o n o f p e r o x i d a s e a c t i v i t y o f a p p r o x i m a t e l y 50% w h i l e i m i d a z o l e and CN w i t h K j y 2 v a l u e s ° f 5800 and 28 r e s p e c t i v e l y p roduced i n h i b i t i o n s o f up t o 90%. C o n t r o l e x p e r i m e n t s d e m o n s t r a t e d t h a t H 2 O 2 d i d n o t r e a c t w i t h SCN , I m i d a z o l e , and CN . 63 DISCUSSION Heme A c c e s s i b i l i t y o j . 1-65H Three l i n e s o f e v i d e n c e i n d i c a t e t h a t t h e heme group o f 1-65H i s more exposed t o the e x t e r n a l e n v i r o n m e n t t h a n t h a t o f cy toch rome c_: ( i ) CO w i l l b i n d t o t h e heme g roup o f 1-65H w h i l e i t w i l l n o t b i n d t o t h e heme g roup o f cy toch rome c_. ( i i ) pH t i t r a t i o n d a t a showed an a c i d t r a n s i t i o n w i t h a pK h i g h e r t h a n t h a t o f h o r s e h e a r t cy tochchrome £.. Si ( i i i ) SCN , I m i d a z o l e and CN~ w i l l b i n d r e a d i l y t o 1-65H as when compared w i t h t h e i r b i n d i n g c h a r a c t e r i s t i c s t o h o r s e h e a r t cy toch rome c_. For an exogenous l i g a n d t o b i n d t o t h e Fe group on the heme, i t must t r a v e r s e two e n e r g y b a r r i e r s . One b a r r i e r i n v o l v e s p e n e t r a t i o n o f t h e w a t e r - p r o t e i n i n t e r f a c e and o f t r a v e r s i n g t h e h y d r o p h o b i c e n v i r o n m e n t o f t h e heme p o c k e t ( A n s a r i £ i a l . . 1986; K y t e and D o o l i t t l e , 1982). The second b a r r i e r i n v o l v e s r u p t u r i n g t h e h e m e - a x i a l l i g a n d bond ( C r e u t z and S u t i n , 1973). For Met-80 o f h o r s e h e a r t cy toch rome c_, l i g a n d b i n d i n g s t r e n g t h i s r e l a t e d t o t h e p r e s e n c e o r absence o f t h e p o l y p e p t i d e backbone w h i c h b r a c k e t s t h e p r o t e i n l i g a n d ( W i l g u s si. a l . . 1978). The p r o d u c t i o n o f 1-65H i n v o l v e s t h e e l i m i n a t i o n o f a 39 amino a c i d r e s i d u e f r a g m e n t f r o m t h e c a r b o x y t e r m i n u s o f h o r s e h e a r t cy toch rome c. w h i c h r e s u l t s i n a weaken ing o f h e m e - l i g a n d i n t e r a c t i o n as r e s i d u e s i n v o l v e d i n h y d r o p h o b i c and i n t e r a t o m i c i n t e r a c t i o n s ( J u i l l e r a t and T a n i u c h i , 1985) have been e l i m i n a t e d . T h i s i s t h e r e a s o n why CO and o t h e r exogenous l i g a n d s a r e a b l e t o b i n d t o 1-65H; 64 t h e e n e r g y r e q u i r e d t o r u p t u r e t h e p r o t e i n l i g a n d - h e m e bond i s n o t as g r e a t as i n h o r s e h e a r t cy toch rome c_. I n a d d i t i o n , t h e heme s i t e o f 1-65H i s more exposed (and l e s s h y d r o p h o b i c ) t h a n t h e e q u i v a l e n t s i t e i n cy tochrome £. w h i c h means l e s s e n e r g y i s r e q u i r e d t o p e n e t r a t e t h e s i t e . A consequence i s t h a t H + i o n s more c a p a b l e o f p e n e t r a t i n g t h e heme p o c k e t , p r o t o n a t e t h e a x i a l l i g a n d ( s ) and d i s s o c i a t e i t f r o m t h e heme i r o n . As a r e s u l t , t h e pK o f t h e a c i d t r a n s i t i o n i s i n c r e a s e d because o f t h e weakened b i n d i n g s t r e n g t h o f t h e a x i a l l i g a n d and because o f t h e i n c r e a s e d a c c e s s i b i l i t y o f t h e heme b i n d i n g s i t e . The m i d - p o i n t p o t e n t i a l has been s u g g e s t e d t o be an i n d i c a t o r o f heme e x p o s u r e ( S t e l l w a g e n , 1 9 7 8 ) . L i t e r a t u r e v a l u e s f o r t h e m i d - p o i n t p o t e n t i a l o f 1-65H have been r e p o r t e d t o be -108 mV w h i c h i s f a r be low t h e o p e r a t i o n a l m i d -p o i n t p o t e n t i a l o f cy toch rome c_ (+265 mV) ( W i l g u s ££. a l . . 1971) and w h i c h i s c o n s i s t e n t w i t h a more h y d r o p h i l l i c heme e n v i r o n m e n t f o r 1-65H t h a n i n h o r s e h e a r t cy toch rome c_. LIGAND ADDITION The changes i n X and i n e x t i n c t i o n c o e f f i c i e n t s obse rved i n t h e max p r e s e n t s t u d y a r e summar ized i n T a b l e I I I o f t h e R e s u l t s s e c t i o n . There a r e s e v e r a l a s p e c t s o f t h e s e l i g a n d b i n d i n g s t u d i e s t h a t a r e w o r t h y o f m e n t i o n . The f i r s t o f t h e s e i s t h e r e c u r r e n t o b s e r v a t i o n o f a red s h i f t i n t h e S o r e t whenever CN , I m i d a z o l e o r SCN i s a d d e d . A p o s s i b l e e x p l a n a t i o n c o u l d stem f r o m t h e phenomenom o f e l e c t r o n r e p u l s i o n . The p r o c e s s o f l i g a n d b i n d i n g i n t r o d u c e s more e l e c t r o n d e n s i t y i n t o t h e heme a r e a . R e p u l s i v e f o r c e s i n d u c e t h e e l e c t r o n s t o become more d e l o c a l i z e d . As a r e s u l t , e l e c t r o n i c o r b i t a l s become more d i f f u s e . D i f f u s e o r b i t a l s mean t h a t l e s s e n e r g y i s r e q u i r e d t o p r o d u c e e l e c t r o n i c t r a n s i t i o n s , hence the red s h i f t i n 65 t h e S o r e t . The amount o f d e r e a l i z a t i o n may be a f u n c t i o n o f l i g a n d s t r e n g t h as t h e amount o f r e d s h i f t i s CN >Imid>SCN . The s t r o n g e r t h e a x i a l l i g a n d , t h e g r e a t e r t h e e l e c t r o n i c i n t e r a a c t i o n s , hence t h e g r e a t e r t e n d e n c y f o r t h e e l e c t r o n s t o become d e l o c a l i z e d . The end r e s u l t i s a red s h i f t i n t h e S o r e t t h a t i s f u n c t i o n o f t he l i g a n d s t r e n g t h i n t h e l i g a n d -p r o t e i n c o m p l e x . I t i s a p p a r e n t t h a t a x i a l l i g a n d s a r e i m p o r t a n t i n d e t e r m i n i n g t h e f u n c t i o n a l c h a r a c t e r i s t i c s o f a heme p r o t e i n . I n g e n e r a l , t he change f r o m a h i g h s p i n s p e c i e s t o a low s p i n s p e c i e s can be r e g a r d e d as r e s u l t i n g f r o m b i n d i n g o f an a x i a l l i g a n d t o an open c o o r d i n a t i o n p o s i t i o n on the heme ( B a b u l and S t e l l w a g e n , 1972) a l t h o u g h h i g h - s p i n s i x - c o o r d i n a t e i r o n heme systems a r e w e l l known ( e . g . f l u o r o m e t M b , aquometMb) . The change i n e l e c t r o n i c s p e c t r u m t h a t r e s u l t s f r o m an i n c r e a s e i n pH o r f r o m l i g a t i o n o f a cyano or an i m i d a z o l a t e g roup i s e s s e n t i a l l y t he same because i n each c a s e , t h e s p i n s t a t e changes f r o m h i g h t o l o w . There a r e d i f f e r e n c e s i n abso rbance maxima and i n e x t i n c t i o n c o e f f i c i e n t s , i n t h e s e two e x a m p l e s , but t h e r e i s one b a s i c t r e n d : a r e d s h i f t i n t h e S o r e t , a l o s s o f t h e h i g h s p i n marke r band a t a p p r o x i m a t e l y 620 nm and t h e appearance o f peaks i n t h e v i s i b l e r e g i o n on c o n v e r s i o n t o t h e l o w - s p i n f o r m . I n d e e d t h e p resence and t h e t y p e o f a x i a l l i g a n d may be more i m p o r t a n t i n d e t e r m i n i n g t h e n a t u r e o f t h e a b s o r p t i o n s p e c t r a t h a n p r o t e i n c o n f o r m a t i o n . For i n s t a n c e , t h e r e i s t h e g r e a t s i m i l a r i t y i n s p e c t r a be tween t h e n a t i v e 1-65H and the i m i d a z o l a t e f o r m . T h i s i s e x p e c t e d as i m i d a z o l e i s v e r y s i m i l i a r t o h i s t i d i n e , and t h e l i g a t i o n o f i m i d a z o l e t o h i g h s p i n 1-65H r e s u l t s i n a complex w i t h t h e same a x i a l l i g a n d s as t h e b i s - i m i d a z o l e ( t w o h i s t i d i n e r e s i d u e s ) complex o f n a t i v e 1 -65H. A l s o t h e reduced f o r m o f 1-65H i s v e r y s i m i l i a r t o t h e r e d u c e d f o r m o f c y t o c h r o m e £. i n t h e absence o f exogenous l i g a n d s w h i c h s u g g e s t s t h a t t h e p r e s e n c e o f an a x i a l l i g a n d i s t h e m a j o r d e t e r m i n a n t t o 66 the c h a r a c t e r i s t i c s o f t he a b s o r p t i o n s p e c t r a . The c o n c l u s i o n t h a t t h e a x i a l l i g a n d s a r e t h e ma in m o d u l a t o r s o f heme s p e c t r a wou ld e x p l a i n the pH 7 . 0 s p e c t r a o f t h e i m i d a z o l a t e and SCN complexes o f 1 -65H. A t pH 7 . 0 , n e i t h e r t he a d d i t i o n o f i m i d a z o l e n o r t h e a d d i t i o n o f SCN p r o d u c e d a d r a m a t i c change i n t h e abso rbance s p e c t r u m o f 1 -65H. T h i s was n o t t r u e a t pH 3 . 0 c o n d i t i o n s . One p o s s i b l e e x p l a n a t i o n i s t h a t t h e two l i g a n d s a r e n o t c a p a b l e o f b i n d i n g t o 1-65H a t pH 7 . 0 . T h i s p o s s i b i l i t y i s u n l i k e l y i n l i g h t o f t h e d a t a showing t h e e f f e c t s o f l i g a n d a d d i t i o n on p e r o x i d a s e a c t i v i t y ( F i g . 19C) and i n l i g h t o f t h e s m a l l s p e c t r a l changes upon l i g a n d a d d i t i o n . An a l t e r n a t i v e e x p l a n a t i o n i s t h a t b o t h SCN and i m i d a z o l e p r o v i d e an F e - l i g a n d i n t e r a c t i o n t h a t i s c h e m i c a l l y s i m i l i a r t o t he n a t i v e h i s t i d i n e l i g a n d s o f f r e e 1 -65H. I n o t h e r w o r d s , b o t h i m i d a z o l e and SCN a c t as e l e c t r o n d o n a t i n g l i g a n d s . C e r t a i n l y , i m i d a z o l e i s an e l e c t r o n d o n a t i n g l i g a n d a l t h o u g h t h e a s s i g n m e n t o f l i g a n d t y p e t o SCN i s more ambiguous as w i l l be d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n . LIGAND SUBSTITUTION EXPERIMENTS I t was s u g g e s t e d above t h a t e l e c t r o n w i t h d r a w i n g g roups had t h e e f f e c t o f d e c r e a s i n g t h e e x t i n c t i o n c o e f f i c i e n t i n t h e S o r e t w h i l e t h e e l e c t r o n d o n a t i n g g roups had t h e e f f e c t o f i n c r e a s i n g t h e S o r e t e x t i n c t i o n c o e f f i c i e n t s w i t h r e s p e c t t o t h e f o r m e r s e t o f a x i a l l i g a n d s . One e x p l a n a t i o n o f t h i s phenomenom r e l a t e s t o t h e i n f l u e n c e o f t h e s e l i g a n d s on t h e p o p u l a t i o n o f t h e heme e l e c t r o n i c o r b i t a l s . E l e c t r o n w i t h d r a w i n g l i g a n d s , as t h e i r name s u g g e s t s , w i t h d r a w e l e c t r o n s f r o m t h e p i - o r b i t a l s o f t h e heme g r o u p . C o n s e q u e n t l y , t h e e l e c t r o n p o p u l a t i o n o f t he heme i s r e d u c e d meaning a d e c r e a s e d l i k e l i h o o d o f p r o d u c i n g t h e c o r r e c t e l e c t r o n i c t r a n s i t i o n s i n t h e S o r e t . E l e c t r o n d o n a t i n g l i g a n d s have t h e a b i l i t y t o f e e d e l e c t r o n s i n t o t h e heme g r o u p t h u s p r o d u c i n g an e l e c t r o n r i c h p r o s t h e t i c g roup and r e n d e r i t more l i k e l y t o p r o d u c e t h e e l e c t r o n i c t r a n s i t i o n s i n t h e S o r e t . As shown i n t h e R e s u l t s s e c t i o n and i n T a b l e I I I , w h i l e t h e r e s u l t s f o t h e l i g a n d s u b s t i t u t i o n e x p e r i m e n t s f o r 1-65H c o n f i r m e d t h e h y p o t h e s i s , t h e e x p e r i m e n t s f o r Mb were i n c o n s i s t e n t w i t h t h i s t r e n d . The d i s c r e p a n t r e s u l t s may stem f r o m the p H - i n f l u e n c e d b i n d i n g p r o p e r t i e s o f SCN i n one s e t o f exper iment ' s and f r o m t h e d i f f e r e n c e i n e l e c t r o n i c d i s t r i b u t i o n f o r l i n e a r and s t e r i c a l l y - b e n t CN i n t h e o t h e r s e t o f e x p e r i m e n t s . The pH c o n d i t i o n s a r e s i g n i f i c a n t i n t h a t t h e SCN e x p e r i m e n t s f o r Mb were r u n a t pH 7.0 w h i l e t h e SCN e x p e r i m e n t s f o r 1-65H were r u n a t pH 3 .0. The d i f f e r e n c e i n pH c o n d i t i o n s may be s i g n i f i c a n t as t h e n a t u r e o f SCN b i n d i n g may be pH d e p e n d e n t . Under a c i d i c c o n d i t i o n s , b i n d i n g o c c u r s t h r o u g h t h e s u l f u r atom t o p r o v i d e an e l e c t r o n - w i t h d r a w i n g l i g a n d w h i l e u n d e r b a s i c o r n e u t r a l c o n d i t i o n s , b i n d i n g o c c u r s p r i m a r i l y t h r o u g h t h e n i t r o g e n atom t o p r o v i d e an e l e c t r o n - d o n a t i n g l i g n d . As a r e s u l t , SCN i s c a p a b l e o f b e h a v i n g i n an a m b i v a l e n t , pH-dependent manner . The r e s u l t s o b s e r v e d on CN b i n d i n g t o metMb may be a t t r i b u t a b l e t o t h p o t e n t i a l o f t h e d i s t a l h i s t i d i n e t o i n d u c e s t e r i c d i s t o r t i o n s o f o t h e r w i s e l i n e a r exogenous a x i a l l i g a n d s . As CN n o r m a l l y i s a l i n e a r l i g a n d , t h e p roposed d i s t o r t i o n c o u l d p o s s i b l y a l t e r t h e e l e c t r o n - d o n a t i n g c h a r a c t e r o f c y a n i d e . I m i d a z o l e i s n o t a f f e c t e d i n t h i s manner because o n l y c y a n i d e possesses b o t h s i g m a - e l e c t r o n o r b i t a l s t h a t a r e e l e c t r o n - d o n a t i n g and p i -back b o n d i n g o r b i t a l s t h a t a r e e l e c t r o n - w i t h d r a w i n g . I t may be p o s s i b l e t h a t t h e p r o p o s e d d i s t o r t i o n i n h i b i t s t h e s igma b o n d i n g c h a r a c t e r i s t i c s o f CN~. 68 I n a d d i t i o n , H-NMR s p e c t r a o f f r e e and cyano complexes o f o x i d i z e d 1 -65H showed t h a t t he heme m e t h y l peaks o n l y became sha rp and d i s t i n c t i n t h e p r e s e n c e o f c y a n i d e . These f i n d i n g s w o u l d i n d i c a t e t h a t t h e n a t i v e a x i a l l i g a n d s a r e so w e a k l y bound t h a t t h e r e i s f r e q u e n t a s s o c i a t i o n , d i s s o c i a t i o n o f t h e a x i a l l i g a n d s such t h a t t h e heme m e t h y l peaks a r e b r o a d e n e d . The s e n s i t i v i t y o f t h e nmr s p e c t r a i n r e l a t i o n t o t h e e l e c t r o n i c s p e c t r a i n t h i s r e g a r d i l l u s t r a t e s t h e comp lementa ry n a t u r e o f t h e two t e c h n i q u e s and t h e d i f f e r e n c e i n t h e t i m e - s c a l e on w h i c h t h e two t e c h n i q u e s o p e r a t e . Func t i ona l P r o p e r t i e s QL 1-65H P e r o x i d a s e r a t e f i g u r e s f r o m T a b l e I V can be i n t e r p r e t e d i n te rms o f a r e l a t i o n s h i p be tween heme-exposure and p e r o x i d a s e a c t i v i t y . The p e r o x i -dase a c t i v i t y o f 1-65H has been f o u n d t o be a m p l i f i e d f r o m 8 8 - t o 8 7 5 - f o l d o v e r t he c o m p a r a t i v e p e r o x i d a s e r a t e s f o r h o r s e h e a r t cy toch rome c_. However , i f one examines T a b l e I V , i t i s a p p a r e n t t h a t t h e r a t e s f o r 1-65H a r e f a r be low t h e r a t e s o b s e r v e d f o r a t r u e p e r o x i d a s e (Horse Rad ish P e r o x i d a s e ) . I n c r e a s e d heme exposu re i s a n e c e s s a r y b u t n o t a s u f f i c i e n t c o n d i t i o n f o r f u l l y f u n c t i o n a l p e r o x i d a s e a c t i v i t y . C l e a r l y , t h e e lements r e q u i r e d f o r t h e maximum p o s s i b l e e x p r e s s i o n o f p e r o x i d a s e a c t i v i t y a r e m i s s i n g . The p r o s t h e t i c g roup o f 1-65H n o t o n l y has a more exposed a x i a l c o o r d i n a t i o n p o s i t i o n b u t t h e p e r i p h e r a l heme edge i s more exposed as w e l l . I t i 6 known t h a t ^ C ^ and o t h e r p e r o x i d e - l i k e compounds a r e c a p a b l e o f d e g r a d i n g heme i n t o b i l i r u b i n by a t t a c k i n g t h e m e t h i n e b r i d g e s o f t h e heme edge ( S t r y e r , 1 9 7 5 ) . I n a d d i t i o n , a number o f r e l a t e d heme p r o t e i n s ( c y c l o o x y g e n a s e , c y t o c h r o m e P-450) a r e s u s c e p t i b l e t o s e l f c a t a l y z e d 69 d e s t r u c t i o n f r o m t h e p e r o x i d e r a d i c a l i n t e r m e d i a t e s t h a t a r e formed d u r i n g p r o t e i n t u r n o v e r ( S m i t h and B o r g e a t , 1 9 8 5 ) . I n t h e second c a s e , t he m e t h i n e b r i d g e s a r e a l s o t h e s i t e s o f a t t a c k . Thus t h e p e r i p h e r a l heme edge must be p r o t e c t e d f o r t h e p r o t e i n t o d i s p l a y f u l l p e r o x i d a s e a c t i v i t y . The heme edge i n CCP and t o some e x t e n t i n c y t o c h r o m e £. i s p r o t e c t e d by t h e p r o t e i n ' s t e r t i a r y s t r u c t u r e ( P o u l o s and F i n z e l , 1 9 8 4 ) , b u t t he l i g a n d - b i n d i n g and pH t i t r a t i o n s t u d i e s on 1-65H s u g g e s t s t h a t t h i s mode o f p r o t e c t i o n i s n o t p r e s e n t i n t h e p e p t i d e . P r o t e i n c o n f o r m a t i o n and a x i a l l i g a n d s a r e t h u s r e s p o s i b l e f o r e i t h e r t h e s t e r i c a c c e s s i b i l i t y ( o r s t e r i c h i n d r a n c e ) o f t h e p r o s t h e t i c g roup f r o m exogenous s u b s t r a t e s . C o m p e t i t i o n f o r H jC^ be tween t h e p e r i p h e r a l edge o f t h e heme and t h e i r o n c e n t r e c o u l d e x p l a i n t h e pH dependent changes i n t h e p e r o x i d a s e a c t i v i t y o f 1 -65H. I n p a r t i c u l a r , i t was f o u n d t h a t a dec rease i n pH i n c r e a s e d t h e p e r o x i d a s e a c t i v i t i e s o f h o r s e h e a r t cy toch rome c_ and HRP b u t d e c r e a s e d the r a t e f o r 1-65H ( T a b l e I V ) . As 1-65H i s h i g h s p i n a t pH 3 . 0 , t h e heme g roup s h o u l d be more a c c e s s i b l e . B u t , i t may be p o s s i b l e t h a t t h e r e i s a p o i n t where f u r t h e r d e n a t u r a t i o n w i l l o n l y a m p l i f y t he pathway f o r t h e p e r i p h e r a l a t t a c k o f t h e heme by ^ 2 ^ 2 ' ^ n e r e * n e f f e c t , a range i n t h e degree o f d e n a t u r a t i o n i n w h i c h one can a m p l i f y p e r o x i d a s e a c t i v i t y . Below t h i s r a n g e , t h e r a t e s a r e supp ressed as t h e heme i s n o t s u f f i c i e n t l y e x p o s e d . Above t h i s r a n g e , p e r i p h e r a l a t t a c k p r e d o m i n a t e s . There i s some e v i d e n c e t o s u p p o r t t h i s h y p o t h e s i s . The p e r o x i d a s e a c t i v i t y o f 1-65H and t o some e x t e n t h o r s e h e a r t cy toch rome c. was t e s t e d u n d e r c o n d i t i o n s t h a t w o u l d i n d u c e even g r e a t e r heme e x p o s u r e by e x a m i n i n g t h e e f f e c t o f Gdn.HCl and t e m p e r a t u r e on p e r o x i d a s e a c t i v i t y . The i n c r e a s e i n Gdn.HCl c o n c e n t r a t i o n , a known p r o t e i n d e n a t u r a n t , had the u n u s u a l e f f e c t o f i n c r e a s i n g t h e p e r o x i d a s e a c t i v i t y o f b o t h 1-65H and cy tochrome c_ ( F i g . 1 9 B ) . But f o r 1 -65H, t h e p e r o x i d a s e a c t i v i t y s t a r t s t o d e c l i n e a t 6M 70 Gdn.HCl w h i c h may be a t t r i b u t e d t o t h e excedance o f t h e range o f d e n a t u r a t i o n f o r 1 -65H. No such d e c l i n e o c c u r s f o r hhc. as t h e range f o r p e p t i d e d e n a t u r a t i o n has n o t been e x c e e d e d . An i n c r e a s e i n t h e assay t e m p e r a t u r e a l s o i n c r e a s e d t h e p e r o x i d a s e a c t i v i t y o f 1-65H ( F i g . 1 9 A ) . An i n c r e a s e i n t e m p e r a t u r e w o u l d i n d u c e g r e a t e r random m o t i o n on a p r o t e i n hence t h e n e t e f f e c t i s p a r t i a l d e n a t u r a t i o n . The f a c t t h a t hea t i s c a p a b l e o f p r o d u c i n g a g r e a t e r p e r o x i d a s e a c t i v i t y i n 1-65H shows t h a t t h e e f f e c t o f i n c r e a s e d p e r o x i d a s e a c t i v i t y i s n o t a f u n c t i o n o f some c h e m i c a l m o d i f i c a t i o n o f 1-65H on t h e p a r t o f Gdn.HCl b u t o f t h e a c t u a l d e n a t u r i n g e f f e c t o f t h e compound. The p o s s i b i l i t y t h a t t he i n c r e a s e i n p e r o x i d a s e a c t i v i t y o v e r hhc. c o u l d be a t t r i b u t e d t o t h e a l t e r a t i o n f r o m t h e H i s / M e t c o u p l e o f hhc. t o t h e H i s / H i s c o u p l e o f 1-65H was e x a m i n e d . The a x i a l l i g a n d s CN , I m i d a z o l e and SCN a r e c a p a b l e o f i n h i b i t i n g t h e p e r o x i d a s e a c t i v i t y o f 1-65H. As i m i d a z o l e i s v e r y s i m i l i a r i n c h e m i c a l s t r u c t u r e t o h i s t i d i n e i i t wou ld seem t h a t t h e change i n i n one a x i a l l i g a n d f r o m m e t h i o n i n e t o h i s t i d i n e i s no t c a p a b l e o f p r o m o t i n g p e r o x i d a s e a c t i v i t y . I n h i b i t i o n o f t h e p e r o x i d a s e a c t i v i t y by i m i d a z o l e a l s o w o u l d i n d i c a t e t h a t t h e l i g a n d i s b i n d i n g o n t o the heme g roup and t h a t t h e h i g h t o low s p i n t r a n s i t i o n o f t he s p e c t r u m o f 1-65H upon i m i d a z o l e a d d i t i o n i s n o t due t o t h e e f f e c t o f a l k a l i n i z a t i o n ( F i g . 8C and F i g . 8 D ) . There was r e p o r t e d a d i f f e r e n c e i n t h e % max ima l i n h i b i t i o n o f p e r o x i d a s e a c t i v i t y o f t h e SCN l i g a n d as opposed t o t h e CN o r i m i d a z o l a t e l i g a n d ( F i g . 1 9 C ) . The d i f f e r e n c e p r o b a b l y stems f r o m t h e f a c t t h a t SCN i s a weak l i g a n d ( K ^ 2 = i 2 0 0 0 ) w h i l e i m i d a z o l e and CN a r e i n t e r m e d i a t e ( K j y 2 = 5 5 0 0 ) and s t r o n g - f i e l d (K^^=28) l i g a n d s r e s p e c t i v e l y . I f one v i e w s t h e K j y 2 v a i u e s a s a r a t i o o f l i g a n d d i s s o c i a t i o n r a t e s t o l i g a n d 71 a s s o c i a t i o n r a t e s (K. . =k r r / k ) ( F e r s h t , 1977) t h e n a weak l i g a n d such as 1/2 o f t on SCN s h o u l d have a r e l a t i v e l y f a s t k c c r a t e and a r e l a t i v e l y s low k r a t e . J o f f on The consequence o f t h i s i s t h a t t h e second a x i a l c o o r d i n a t i o n p o s i t i o n o f 1 -65H i s a c t u a l l y empty f o r a s i g n i f i c a n t p e r i o d o f t i m e - enough t i m e f o r **2^2 t o k i n d a n ^ f o r c e a p e r o x i d a t i c r e a c t i o n . T h i s i s a t l e a s t a t e s t a b l e h y p o t h e s i s f o r t h e t i m e r e q u i r e d f o r ^ 2 ^ 2 t C > k^ n <* must be a f u n c t i o n o f t h e H 2 ^ 2 ^ ~ ^ H r a t i o . So i f one i n c r e a s e s t h e c o n c e n t r a t i 0 1 1 *-n t n e p e r o x i d a s e assay o f i m i d a z o l a t e and c y a n o - complexes o f 1 -65H, t h e n t h e m a x i m a l i n h i b i t i o n o f p e r o x i d a t i c a c t i v i t y s h o u l d d e c r e a s e t o t h e l e v e l s o f t h e SCN-1-65H e x p e r i m e n t s i f n o t l o w e r . M o f f a t i n 1979 used t h e c o n c e p t o f s t e r i c h i n d r a n c e t o e x p l a i n t h e r e l a t i v e b i n d i n g a f f i n i t i e s o f 0^ and CO t o h e m o g l o b i n (see I n t r o d u c t i o n ) . The c y a n i d e b i n d i n g s t u d i e s o f 1-65H have shown t h a t t h e M o f f a t model i s amenable t o m o d i f i c a t i o n s . The enhanced s t a b i l i t y o f t h e reduced c y a n o -complex o f 1-65H compared t o t h a t o f t h e reduced c y a n o - c o m p l e x o f h o r s e h e a r t cy toch rome c. can be e x p l a i n e d by t h e i n c r e a s e d a f f i n i t y f o r reduced hemes on t h e p a r t o f M e t h i o n i n e . M e t - 8 0 i s p r e s e n t i n h o r s e h e a r t c y t o c h r o m e £. thus i t i s a v a i l a b l e t o compete f o r t h e same c o o r d i n a t i o n p o s i t i o n as c y a n i d e . T h i s i s n o t t r u e f o r 1-65H w h i c h most l i k e l y has a b i s - i m i d a z o l a t e a x i a l l i g a n d p a i r . We c o n c l u d e t h a t a x i a l l i g a n d s p r o v i d e s t e r i c h i n d r a n c e s n o t o n l y t o t h e a s s o c i a t i o n o f exogenous l i g a n d s b u t t h e y a l s o i n f l u e n c e t h e d i s s o c i a t i o n r a t e o f t h e exogenous l i g a n d s . T h i s c o n c l u s i o n i s i n c o n t r a s t t o M o f f a t ' s model f o r h e m o g l o b i n i n w h i c h d i s s o c i a t i v e p r o c e s s e s a r e w h o l l y u n d e r " e l e c t r o n i c c o n t r o l " . M o f f a t d e s i g n e d h i s t h e o r y t o e x p l a i n t h e l i g a n d b i n d i n g k i n e t i c s o f h i g h - s p i n compounds such as h e m o g l o b i n o r m y o g l o b i n so t h e d i s c r e p a n c y may r e f l e c t a g e n e r a l d i f f e r e n c e between oxygen b i n d i n g p r o t e i n s and c_-type c y t o c h r o m e s . 72 Summary pH t i t r a t i o n , l i g a n d b i n d i n g and r e d u c t i o n p o t e n t i a l d a t a i n d i c a t e t h a t t h e heme g roup o f 1-65H r e s i d e s i n a more h y d r o p h i l i c e n v i r o n m e n t t h a n t h e p r o s t h e t i c g r o u p o f hhc.. The consequence o f i n c r e a s e d heme e x p o s u r e i s t h e g r e a t e r ease i n w h i c h l i g a n d s can be l i g a t e d t o t h e i r o n c e n t e r . T h i s p r o p e r t y f a c i l i t a t e s l i g a n d s u b s t i t u t i o n s t u d i e s on 1-65H such t h a t we were a b l e t o t e s t t h e h y p o t h e s i s t h a t e l e c t r o n - d o n a t i n g l i g a n d s have a t e n d e n c y t o i n c r e a s e t h e S o r e t abso rbance band w h i l e e l e c t r o n - w i t h d r a w i n g l i g a n d s w i l l d e c r e a s e t h e S o r e t p e a k . For 1 -65H, t h e r e s u l t s w o u l d i n d i c a t e t h a t t h i s h y p o t h e s i s was t r u e a l t h o u g h t h e r e s u l t s f o r Mb a r e more a m b i g u o u s . The d i s c r e p a n c i e s f o r t h e Mb case may stem f r o m t h e ( p o s t u l a t e d ) pH-dependent b i n d i n g c h a r a c t e r i s t i c s o f SCN and t h e s t e r i c a l l y i n f l u e n c e d c h a r a c t e r i s t i c s o f CN . I n c r e a s e d heme exposu re a l s o p roduced an i n c r e a s e i n p e r o x i d a s e a c t i v i t y . T h i s i n c r e a s e was a m p l i f i e d f u r t h e r by t h e a d d i t i o n o f a d e n a t u r a n t , G d n . H C l , and an i n c r e a s e i n t e m p e r a t u r e . The r a t e dec reased upon a c i d i f i c a t i o n o f t he assay medium t h u s s u g g e s t i n g t h a t t h e r e i s a range o f d e n a t u r a t i o n i n w h i c h a m p l i f i c a t i o n w i l l o c c u r . T h i s r e s u l t i n d i c a t e s t h a t o n l y s e l e c t i v e e x p o s u r e o f t h e heme m o i e t y w i l l i n c r e a s e t h e a c t i v i t y . The i n h i b i t i o n o f p e r o x i d a s e a c t i v i t y by t h e a d d i t i o n o f exogenous a x i a l l i g a n d s shows t h a t a v i t a l component f o r p e r o x i d a s e a c t i v i t y i s t h e a v a i l a b i l i t y o f an open a x i a l c o o r d i n a t i o n p o s i t i o n . 73 REFERENCES Ansari, A., Dilorio, E. E., Dlott, D. D., Frauenfelder, Iben, I. E. T., Langer, P., Roder, H., Sauke, T. B., and Shyamsunder, E. (1986) Biochemistry 21, 3139-3146. Babul, J., and Stellwagen, E. (1972) Biochem JLI, 1195-1200. Babul, J., McGowan, E. B., and Stellwagen, E. (1972) Arch. Biochem. Biophys. 1M. 141-147. Baumgartner, C. P., Sellers M., Nassif, R., and May, L. (1974) Eur. J. Biochem. 4j>., 625-629. Blumenthal, D. C , and Kassner, R. J. (1979) J. Biol. Chem. 254. 9617-9620. Brooks, S. P. J., Chanady, G. A., and Nicholls P. (1982) Can. J . Biochem. £0_, 763-770. Bryant, C , and Stellwagen, E. (1985) J. Biol. Chem. 2M>. 332-336. Chance, M., Powers, L., Poulos, T., and Chance B. (1986) Biochemistry 21, 1259-1265. Chance, M., Powers, L., and Chance, B. (1986) Biochemistry 21, 1266-1270. Clore, G. M., Hollaway, M. R., Orengo, C , Peterson, J., and Wilson, M. T. (1981) Inorg. Chim. Acta 143-148. Cocolios, P., and Kadish, K. M. (1985) Isr. J. Chem. 21, 138-147. Collman, J. P., Brauman, J. P., Collins, T. I., Iverson, B., and Sessler, J.L. (1981) J. Am. Chem. Soc. 1£2, 2450-2452. Collman, J. P., Brauman, J. I., and Doxsee, K. M. (1979) Proc. Natl. Acad. Sci. USA ]£, 6035-6039. Corradin, H., and .Harbury H. A. (1970) Biochim. Biophys. Acta 221. 489-496. Corradin, G.» and Harbury, H. A. (1974) Biochem. Biophys. Res. Commun. 4100-4106. Creutz, C , and Sutin, N. (1973) 1Q., 1701-1703. Dawson, A., and Wood, E. J. (1983) Biochem. J. 201, 519-526. Fanger, M. W., and Harbury, H. A. (1965) Biochemistry 4_, 2541-2545. Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1979) in The Porphyrins VIIB (Dolphin D. ed.) pp. 149-240, Academic Press Inc., New York. 74 F e r m i , G. ( 1 9 7 5 ) J . M o l . B i o l . 21, 2 3 7 - 2 5 6 . F e r s h t , A . ( 1 9 7 7 ) i n Enzyme S t r u c t u r e and. Mechanism p p . 8 4 - 9 9 , W. H. Freeman and C o . , New Y o r k . F l a t m a r k , T . ( 1 9 6 6 ) A c t a Chem. Scand. 20., 1 4 7 0 - 1 4 7 5 . F l a t m a r k , T . ( 1 9 6 6 ) A c t a Chem. Scand. 20_. 1 4 7 6 - 1 4 8 6 . F rew, J . E . , and J o n e s , P. ( 1 9 8 3 ) J . I n o r g . B i o c h e m . H , 3 3 - 3 9 . Hampsey, D. M . , Goutam, D . , and Sherman, F. ( 1 9 8 6 ) J . B i o l . Chem. 2 6 1 ( 7 ) . 3 2 5 9 - 3 2 7 1 . H a n t g a n , R. R . , and T a n i u c h i , H. ( 1 9 7 8 ) J . B i o l . Chem. 2 5 3 ( 1 5 ) . 5373-5 3 8 0 . H a n t g a n , R. R . , and T a n i u c h i , H. ( 1 9 7 7 ) J . B i o l Chem. 2 5 2 ( 4 ) . 1367-1 3 7 4 . H a r b u r y , H. A . , C r o n i n , J . R . , F a n g e r , M. M . , H e t t i n g e r , T . P . , M u r p h y , A. J . , M y e r , Y . P . , and V i n o g r a d o v S. ( 1 9 6 5 ) B i o c h e m i s t r y 5 4 . 1 6 5 8 - 1 6 6 4 . H a r b u r y , H. A . , and L o a c h , P. A . ( 1 9 6 0 ) J . B i o l . Chem. 2 2 1 . 3 6 4 0 -3 6 4 5 . Huang, Y a - P i n g , and K a s s n e r , R. J . ( 1 9 8 1 ) J . B i o l . Chem. 2J>6_, 5327-5331 . I k e d a - S a i t o , M . , and P r i n c e , R. C. ( 1 9 8 5 ) J . B i o l . Chem. 26_Q_, 8 3 0 1 -8 3 0 5 . J a i n , A . , and K a s s n e r , R. J . ( 1 9 8 4 ) J . B i o l . Chem. 2Ji£, 1 0 3 0 9 - 1 0 3 1 4 . J u i l l e r a t , M. A . , and T a n i u c h i , H. ( 1 9 8 6 ) J . B i o l . Chem. 2 6 1 . 2697-2 7 1 1 . K a s s n e r , R. J . ( 1 9 7 2 ) P r o c . N a t l . A c a d . S c i . USA fi£, 2 2 6 3 - 2 2 6 7 . K a z m i , S. A . , M i l l s , M. A . , P i t l u k , Z . W. , and S c o t t , R. A . ( 1 9 8 5 ) J . I n o r g . B iochem. 2 4 , 9 - 1 2 . K e u r , E. A . , N a i - T e n g Y u , B a r t n i c k i , D. E . , and M i z u k a m i , H. ( 1 9 8 5 ) J . B i o l . Chem. 2&0_, 8 3 6 0 - 8 3 6 5 . t e n K o r t e n a a r , P. B. W . , Adams, P. J . H. M . , and T e s s e r , G. I . ( 1 9 8 5 ) P r o c . N a t l . A c a d . S c i . USA £ 2 . 8 2 7 9 - 8 2 8 3 . K y t e , J . , and D o o l i t t l e , R. F. ( 1 9 8 2 ) J . m o l . B i o l . 1 5 1 . 1 0 5 - 1 3 2 . La Mar , G. N . , Budd , D. L . , S m i t h , K. M . , and L a n g r y , K. C. ( 1 9 8 0 ) J . Am. Chem. Soc . 10_2_. 1 8 2 2 - 1 8 2 7 . M a r c h o n , J . C , M a s h i k o , T . and Reed, C. A . ( 1 9 8 2 ) i n E l e c t r o n T r a n s f e r and Oxygen U t i l i z a t i o n ( C h i e n Ho e d . ) p p . 6 7 - 7 2 . , E l s e v i e r N o r t h H o l l a n d I n c . , New Y o r k . M a r g o l i a s h , E. ( 1 9 8 2 ) i n E l e c t r o n Transport flM oxygen U t i l i z a t i o n ( C h i e n Ho e d . ) p p . 3 - 1 5 . , E l s e v i e r N o r t h H o l l a n d I n c . , New Y o r k . M a r g o l i a s h , E . , F r o h w i r t , N . , and W i e n e r , E. ( 1 9 5 9 ) B iochem. J . 7 1 . 5 5 9 - 5 7 2 . M a x w e l l , J . C , and Caughey, W. S. ( 1 9 7 6 ) B i o c h e m i s t r y 15_, 3 8 8 - 3 9 6 . M i t c h e l l , R. A . , E r i c k s o n , B. W. , Ryabsev , M. N . , Hodges , R. S . , and M e r r i f i e l d , R. B. ( 1 9 7 6 ) J . Am. Chem. Soc. 9_8_, 7 3 5 7 - 7 3 6 2 . M o f f a t , K . , D e a t h e r a g e , J . F . , and S e y b e r t , D. W. ( 1 9 7 9 ) Sc ience 2 0 6 . 1 0 3 5 - 1 0 4 2 . M o f f a t , K. ( 1 9 8 0 ) T e x . Rep. B i o l . Med. 4_0_» 1 9 2 - 1 9 8 . M o o r e , G. R . , and W i l l i a m s , R. J . P. ( 1 9 7 7 ) FEBS L e t t . 12., 2 2 9 - 2 3 2 . Moore , G. R, and W i l l i a m s , R. J . P. (1986) E u r . J . B iochem. 1Q3_, 5 1 3 -5 2 1 . P a r r , G. R . , H a n t g a n , R. R . , and T a n i u c h i , H. ( 1 9 7 8 ) J . B i o l . Chem. 2 5 3 . 5 3 8 1 - 5 3 8 8 . P a r r , G. R . , and T a n i u c h i ; H. ( 1 9 8 2 ) J . B i o l . Chem. 251, 10103-1 0 1 1 1 . P a r r , G. R . , and T a n i u c h i , H. ( 1 9 8 1 ) J . B i o l . Chem. 2J& , 1 2 5 - 1 3 2 . P e r u t z , M. B. ( 1 9 7 2 ) N a t u r e 221, 4 9 5 - 4 9 9 . P e t e r s o n , J . , S i l v e r , J . , and W i l s o n , M. T . ( 1 9 8 0 ) J . I n o r g . B iochem. i i . 7 5 - 8 2 . P i e l a k , ' G. J . , Mauk, A . G . , and S m i t h , M. ( 1 9 8 5 ) N a t u r e 3 1 1 . 1 5 2 - 1 5 4 . P o u l o s , T . L . , a n d , F i n z e l B. C. ( 1 9 8 4 ) i n P e p t i d e and P r o t e i n Reviews (Hearn M. T . W . , e d . ) p p . 1 1 5 - 1 7 1 , M a r c e l Dekker I n c . , New Y o r k . R e i d , L . S . , L i m A . R . , and Mauk A . G. (1986) s u b m i t t e d t o J . Am. Chem. Soc. R e i d , L . S . , Mauk, M. R . , and Mauk, A . G. ( 1 9 8 4 ) J . Am. Chem. Soc. 1 0 6 . 2 1 8 2 - 2 1 8 5 . R i c h a r d s , F . M. ( 1 9 5 8 ) P r o c . N a t l . A c a d . S c i . USA 4A, 1 6 2 - 1 6 8 . Shoemaker, D. P . , G a r l a n d , C. W . , S t e i n f e l d , J . I . , and N i b l e r , J . W. ( 1 9 8 1 ) i n E x p e r i m e n t s i n P h y s i c a l C h e m i s t r y . 4 t h . e d . , p p . 3 5 8 - 3 6 9 , M c G r a w - H i l l Book C o . , T o r o n t o . S m i t h , W. L . , and B o r g e a t , P. ( 1 9 8 5 ) "The E i c o s a n o i d s " i n B i n r h e m i s t r v oJL L i p i d s and Membranes ( D . E. Vance and J . E. Vance e d s . ) p p . 3 3 6 - 3 3 7 , Benjamin/Cummings P u b l i s h i n g Co. I n c . , C a l i f o r n i a . S m i t h , M. C , and McLendon, G. ( 1 9 8 0 ) J . Am. Chem. Soc. 1 0 2 . 5 6 6 6 -5 6 7 0 . S t e l l w a g e n , E. ( 1 9 7 8 ) N a t u r e 2 7 5 . 7 3 - 7 4 . S t r y e r , L . ( 1 9 7 5 ) i n B i o c h e m i s t r y , p p . 5 2 4 - 5 2 5 , W. H. Freeman and C o . , San F r a n c i s c o . Szabo, A . , and P e r u t z , M. F. ( 1 9 7 6 ) B i o c h e m i s t r y L i . 4 4 2 7 - 4 4 2 8 . Tamura , M . , A s a k u r a , T . , and Y o n e t a n i , T . ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 211, 4 6 7 - 4 7 9 . T a n i u c h i , H. ( 1 9 7 3 ) J . B i o l . Chem. 2 4 J L , 5 1 6 4 - 5 1 7 4 . T a n i u c h i , H . , A n f i n s e n , C. B . , and S o d j a , A . (1967) P r o c . N a t l . A c a d . S c i . USA 5JL, 1 2 3 5 - 1 2 3 9 . T r a y l o r , T . G . , and B e r z i n i s , A . P. ( 1 9 8 0 ) P r o c . N a t l . A c a d . S c i . USA 21, 3 1 7 1 - 3 1 7 5 . T s o u , C. L . ( 1 9 5 1 ) B iochem. J . £ £ , 3 6 7 - 3 7 4 . U r r y , D. W. ( 1 9 6 7 ) J . Am. Chem. Soc. £ 2 . 4 1 9 0 - 4 1 9 6 . V e l o s o , D . , J u i l l e r a t , M . , and T a n i u c h i , H. ( 1 9 8 4 ) J . B i o l . Chem. 211, 6 0 6 7 - 6 0 7 3 . W a l l a c e , C. J . A . ( 1 9 8 4 ) B iochem. J . 211, 5 9 5 - 5 9 9 . W a l l a c e , C. J . A . , and Rose, K. ( 1 9 8 3 ) B iochem. J . 211, 6 5 1 - 6 5 8 . W e s t e r h u i s , L . W. , T e s s e r , G. I . , and N i v a r d , R. J . F. ( 1 9 8 2 ) I n t . J . P e p t i d e - R e s . L i . 2 9 0 - 2 9 9 . W i l g u s , H . , R a n w e i l l e r , J . S . , W i l s o n , G. S . , and S t e l l w a g e n , E. ( 1 9 7 8 ) J . B i o l . Chem. 211, 3 2 6 5 - 3 2 7 2 . The W o r t h i n g t o n Enzyme Manual ( 1 9 7 2 ) p p . 4 2 - 4 5 , W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n , New J e r s e y USA. Z o l l e r , M. J . , and S m i t h , M. ( 1 9 8 3 ) Me th E n z y m o l . 1 0 0 . 4 6 8 - 5 0 0 . 77 A p p e n d i x I : N o m e n c l a t u r e o f Cytochrome c. P e p t i d e F ragments P e p t i d e f r a g m e n t s o f cy toch rome c_ a r e i d e n t i f i e d by t h e i r l e n g t h and by t h e p r e s e n c e o r abscence o f a c o v a l e n t l y l i n k e d heme g r o u p . T h u s , t h e d e s i g n a t i o n : 1-65H d e n o t e s a f r a g m e n t t h a t r e t a i n s r e s i d u e s 1 t o 65 f r o m h o r s e h e a r t c y t o c h r o m e c_. The **H~ s i g n i f i e s a heme g roup t h a t i s s t i l l c o v a l e n t l y bound t o t h e p e p t i d e f r a g m e n t . Complexes o f p e p t i d e f r a g m e n t s a r e t i t l e d by t h e f o l l o w i n g f o r m a t : ( 1 - 6 5 H ) : ( 6 6 - 1 0 4 ) The i d e n t i t y o f 1-65H has been m e n t i o n e d . 6 6 - 1 0 4 i s t h e n o m e n c l a t u r e f o r a non-heme c o n t a i n i n g f r a g m e n t o f cy toch rome £. c o m p r i s i n g o f r e s i d u e s 66 t o 104 o f t he o r i g i n a l p r o t e i n . The s e m i c o l o n i n d i c a t e s a n o n - c o v a l e n t complex between 1-65H and 6 6 - 1 0 4 . A h y p h e n , w o u l d i n d i c a t e a c o v a l e n t complex between the two f r a g m e n t s . 78 

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