UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on carbon monoxide and dioxygen binding to cytochrome P-450cam 1984

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STUDIES ON CARBON MONOXIDE AND DIOXYGEN BINDING TO CYTOCHROME P-450cam. By NIMAL RAJAPAKSE B . S c , U n i v e r s i t y of S r i Lanka, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard. THE UNIVERSITY OF BRITISH COLUMBIA December 1984 © Nimal Rajapakse,1984 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. N. RAJAPAKSE Department of Chemistry The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 2nd January 1985. DE-6 (3/81) i i ABSTRACT I n t e r e s t has r e m a i n e d v e r y i n t e n s e d u r i n g t h e l a s t two d e c a d e s on t h e h e m e - c o n t a i n i n g monooxygenase sy s t e m , c y t o c h r o m e P-450. The P-450 h e m o p r o t e i n s a r e w i d e l y d i s t r i b u t e d i n n a t u r e and engage i n o x y g e n a t i o n of a wide v a r i e t y of s u b s t r a t e s a c c o r d i n g t o t h e r e a c t i o n , R-H + 0 2 + 2H ++ 2e~ > R-OH + H 2 0 where R-H r e p r e s e n t s an u n a c t i v a t e d c a r b o n - h y d r o g e n bond. I n v e s t i g a t i o n s on b i n d i n g of s m a l l gas m o l e c u l e s s u c h as CO and 0 2 t o t h e P-450 enzymes a r e i m p o r t a n t n o t o n l y i n u n d e r s t a n d i n g v a r i o u s a s p e c t s of m o n o o x y g e n a t i o n but a l s o i n d e v e l o p i n g p r o t e i n - f r e e model s y s t e m s t h a t can mimic t h e c a t a l y t i c p r o p e r t i e s of P-450. T h i s t h e s i s d e s c r i b e s gas b i n d i n g s t u d i e s c a r r i e d o u t on c y t o c h r o m e P-450cam. A p r o c e d u r e i s g i v e n f o r g r o w i n g t h e b a c t e r i u m Pseudomonas p u t i d a s t r a i n 786 from w h i c h s o l u b l e , camphor h y d r o x y l a t i n g P-450 enzyme i s i s o l a t e d and p u r i f i e d . The b i n d i n g o f CO t o t h e s t o i c h i o m e t r i c a l l y r e d u c e d s u b s t r a t e - f r e e enzyme a t d i f f e r e n t t e m p e r a t u r e s was s t u d i e d u s i n g a s t a n d a r d s p e c t r o p h o t o m e t r i c p r o c e d u r e . From t h e s e e x p e r i m e n t a l d a t a , t h e thermodynamic p a r a m e t e r s A H 0 and AS 0 were c a l c u l a t e d f o r t h e r e a c t i o n , ( P - 4 5 0 ) F e ( I I ) + CO ^ ^ ( P - 4 5 0 ) F e ( 1 1 ) - C 0 . A t t e m p t s t o d e t e r m i n e s u c h thermodynamic p a r a m e t e r s f o r t h e b i n d i n g of d i o x y g e n t o t h e s u b s t r a t e - b o u n d P-450 enzyme were not s u c c e s s f u l . i i i On c o m p a r i s o n of t h e d e t e r m i n e d thermodynamic p a r a m e t e r s f o r t h e s u b s t r a t e - f r e e s y s t e m w i t h t h e l i t e r a t u r e v a l u e s f o r s u b s t r a t e - b o u n d enzyme, h e m o g l o b i n , m y o g l o b i n and P-450 model s y s t e m s , i t i s c o n c l u d e d t h a t the s u b s t r a t e m o l e c u l e was bonded i n t h e immediate v i c i n i t y of t h e a c t i v e - s i t e t h e r e b y l o w e r i n g t h e CO a f f i n i t y t o t h e s u b s t r a t e - b o u n d s y s t e m . i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS X ACKNOWLEDGEMENTS x i i CHAPTER I INTRODUCTION 1 References 6 CHAPTER II LITERATURE REVIEW 7 11.1 B r i e f H i s t o r y of Cytochrome P-450 . 8 11.2 Nature and Cytochrome P-450 15 11.3 S t r u c t u r a l C o n s i d e r a t i o n s 17 11.4 The Mechanism of C a t a l y s i s 20 11.4.1 Types of O x i d a t i o n Reactions 20 11.4.2 The C a t a l y t i c C y c l e 24 11.4.2.1 Binding of Substrate 24 11.4.2.2 F i r s t Reduction 25 11.4.2.3 Bind i n g of Dioxygen 26 11.4.2.4 Second Reduction 27 11.4.2.5 S p l i t t i n g of Oxygen-Oxygen Bond ... 27 11.4.2.6 O x i d a t i o n of Su b s t r a t e 28 11.4.2.7 D i s s o c i a t i o n of Product 29 11.5 E l e c t r o n i c Spectroscopy 31 11.6 I n t e r a c t i o n of Cytochrome P-450 with Carbon Monoxide and Dioxygen 34 References 43 V CHAPTER I I I EXPERIMENTAL PROCEDURES 49 111.1 Growth of B a c t e r i a 50 I I I . 1.1 G e n e r a l I n f o r m a t i o n 50 I I I . 1 . 2 Media 52 I I I . 1 . 2 . 1 M i n i m a l Agar 52 1 1 1 . 1.2. 2 L - B r o t h 54 I I I . 1 .2.3 500 mL Shake F l a s k 54 I I I . 1 . 2 . 4 14 L F e r m e n t e r 56 I I I . 1.3 Growth P r o c e d u r e 57 111 . 2 I s o l a t i o n and P u r i f i c a t i o n of Cytochrome P-450 63 111.2.1 G e n e r a l I n f o r m a t i o n 63 111.2.2 M a t e r i a l s 63 111.2.3 B u f f e r S o l u t i o n s 64 111.2.4 Column C h r o m a t o g r a p h y 68 111.2.5 I s o l a t i o n of C y t o c h r o m e P-450 69 111.2.5.1 C e l l - F r e e E x t r a c t 69 111.2.5.2 S e p a r a t i o n o f C y t o c h r o m e P-450 .... 69 111.2.5.3 Ammonium S u l p h a t e F r a c t i o n a t i o n ... 70 111.2.6 G e l F i l t r a t i o n C h r o m a t o g r a p h y 72 111.2.6.1 P u r i f i c a t i o n of C y t o c h r o m e P-450 .. 72 111.2.6.2 Removal of Camphor S u b s t r a t e 72 111.2.7 S p e c t r a l A n a l y s i s 74 1 1 1 . 3 D e t e r m i n a t i o n of E q u i l i b r i u m C o n s t a n t a t D i f f e r e n t T e m p e r a t u r e s f o r t h e R e a c t i o n of C a r b o n Monoxide w i t h S u b s t r a t e - F r e e C y t o c h r o m e P-450 ... 77 v i 111.3.1 G e n e r a l I n f o r m a t i o n 77 111.3.2 P r o c e d u r e 77 I I I . 4 D e t e r m i n a t i o n of E q u i l i b r i u m C o n s t a n t f o r t h e R e a c t i o n of D i o x y g e n w i t h S u b s t r a t e - B o u n d Cytochrome P-450 .. 80 I I I . 4.1 G e n e r a l I n f o r m a t i o n 80 I I I .4.2 P r o c e d u r e 80 R e f e r e n c e s 83 CHAPTER IV RESULTS AND DISCUSSION 84 IV. 1 Growth of Pseudomonas p u t i d a S t r a i n 786 85 IV.2 P r o p e r t i e s of Cytochrome P-450cam . 87 IV.2 S u b s t r a t e - B o u n d Cytochrome P-450cam 87 IV.2.2 S u b s t r a t e - F r e e Cytochrome P-450cam 91 IV.2.3 P u r i t y and S t a b i l i t y of Enzyme P r e p a r a t i o n s 91 IV.3 D e t e r m i n a t i o n of E q u i l i b r i u m C o n s t a n t a t D i f f e r e n t T e m p e r a t u r e s f o r t h e R e a c t i o n o f C a r b o n Monoxide w i t h S u b s t r a t e - F r e e Cytochrome P-450cam 97 IV.3.1 A c q u i s i t i o n and T r e a t m e n t of D a t a . 97 IV.4 C o m p a r i s o n o f M e a s u r e d A H 0 and A S 0 V a l u e s t o Those of O t h e r H e m o p r o t e i n s and Model Systems .... 106 R e f e r e n c e s 111 A p p e n d i c e s 113 v i i L I S T OF TABLES T a b l e Page 11.1 K i n e t i c D a t a f o r CO B i n d i n g t o Cytochrome P-450 and R e l a t e d Systems 37 11.2 E q u i l i b r i u m D a t a f o r CO b i n d i n g t o C y t o c h r o m e P-450 and R e l a t e d Systems 38 1 1 . 3 E q u i l i b r i u m and K i n e t i c D a t a f o r R e v e r s i b l e D i o x y g e n B i n d i n g t o S u b s t r a t e - B o u n d C y t o c h r o m e P-450 and Mb 42 111.1 Volumes of B u f f e r S o l u t i o n s R e q u i r e d f o r a T y p i c a l I s o l a t i o n and P u r i f i c a t i o n of C y t o c hrome P-450 66 111.2 Q u a n t i t i e s of R e a g e n t s R e q u i r e d f o r a T y p i c a l I s o l a t i o n and P u r i f i c a t i o n o f C ytochrome P-450 67 IV.1 A b s o r b a n c e D a t a f o r S u b s t r a t e - B o u n d C y t o c h r o m e P-450cam 89 IV.2 A b s o r b a n c e D a t a f o r S u b s t r a t e - F r e e C y t o c h r o m e P-450cam 9 3 IV.3 The E q u i l i b r i u m C o n s t a n t V a l u e s f o r CO B i n d i n g t o S u b s t r a t e - F r e e C y t o c h r o m e P-450cam 102 IV.4 K ^ , A H 0 and A S 0 V a l u e s f o r CO B i n d i n g t o Some H e m o p r o t e i n s and M o d e l s of C y t o c h r o m e P-450 1 05 IV.5 H a l f - l i v e s o f A u t o x i d a t i o n R e a c t i o n of S u b s t r a t e - B o u n d C y t o chrome P-450cam 110 v i i i L I S T OF FIGURES F i g u r e Page I . 1 The S t r u c t u r e o f t h e Heme U n i t 3 I I . 1 The F i r s t P u b l i s h e d R e c o r d of t h e C a r b o n Monoxide D i f f e r e n c e S p e c t r u m of Cy t o c h r o m e P-450 8 11.2 The C a r b o n Monoxide D i f f e r e n c e S p e c t r a o f Cy t o c h r o m e P-450 and P-420 9 11 . 3 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 i n B a c t e r i a l S y s t e m L e a d i n g t o Camphor H y d r o x y l a t i o n . ... 12 11.4 The S u b s t r a t e I n d u c e d S p e c t r a l Changes f o r C y t o c h rome P-450cam 13 11. 5 B e n z o p y r e n e and i t s C a r c i n o g e n i c M e t a b o l i t e 7 , 8 - D i o l - 9 , 1 0 - e p o x i d e 16 11. 6 The C a t a l y t i c C y c l e o f Cy t o c h r o m e P-450. ... 23 11.7 H y p o t h e t i c a l Scheme f o r t h e B i n d i n g of S u b s t r a t e s t o Cy t o c h r o m e P450 25 11.8 'Proposed Mechanism f o r P-450 M e d i a t e d H y d r o x y l a t i o n R e a c t i o n 29 11 . 9 Heme E n v i r o n m e n t s of M y o g l o b i n , H e m o g l o b i n and P e r o x i d a s e 35 11.10 V a l e n c e Bond S t r u c t u r e s o f t h e CO-Complex of C y t o c h r o m e P-450 39 111.1 O D 6 6 0 and pH P r o f i l e s f o r t h e B a c t e r i a l Growth i n t h e F i r s t S e t o f S h a k e - F l a s k s .... 58 111. 2 O D 6 6 0 and pH P r o f i l e s f o r t h e B a c t e r i a l G r owth i n t h e Second S e t o f S h a k e - F l a s k s ... 60 ix 111.3 O D 6 6 0 and pH P r o f i l e s f o r t h e B a c t e r i a l Growth i n t h e 14 L F e r m e n t e r s 61 111.4 The C e l l - t o n o m e t e r 75 111.5 The 10 cm P a t h - l e n g t h C e l l ( S l u s h B a t h ) 81 IV.1 The A b s o r p t i o n S p e c t r a of V a r i o u s S t a t e s of S u b s t r a t e - B o u n d C y t o c h r o m e P-450cam 88 IV.2 The A b s o r p t i o n S p e c t r a o f V a r i o u s S t a t e s of S u b s t r a t e - F r e e C y t o c h r o m e P-450cam 92 IV.3 The A b s o r p t i o n S p e c t r a of O x i d i z e d S t a t e s o f S u b s t r a t e - F r e e and S u b s t r a t e - B o u n d C y t o c h r o m e P-450cam 94 IV.4 The A b s o r p t i o n S p e c t r a o f P-420 S p e c i e s Formed i n S u b s t r a t e - F r e e Cytochrome P-450cam S o l u t i o n 96 IV. 5 S p e c t r a l Changes O b s e r v e d f o r t h e P 1 / 2 E x p e r i m e n t no. 5 98 IV.6 A T y p i c a l H i l l L o g / L o g P l o t 100 IV.7 T h e v a n ' t H o f f P l o t 103 X L I S T OF ABBREVIATIONS The f o l l o w i n g a b b r e v i a t i o n s and common names a r e us e d i n t h i s t h e s i s a l o n g w i t h t h e s t a n d a r d a b b r e v i a t i o n s u s e d commonly i n c h e m i c a l l i t e r a t u r e . A A 0 Ace B u f f e r P B u f f e r P-100 B u f f e r T B u f f e r T-50 B u f f e r T-100 B u f f e r T-600 BME o cam o r camphor DNase 1 DTT EXAFS ENDOR EPR ESR A H 0 A b s o r b a n c e O b s e r v e d a b s o r b a n c e a t t h e b e g i n n i n g of an e x p e r i m e n t O b s e r v e d a b s o r b a n c e a t t h e end o f an e x p e r i m e n t 50 mM p h o s p h a t e b u f f e r , pH 7.4 50 mM p h o s p h a t e b u f f e r , pH 7.4, 100 mM KC1 50 mM T r i s b u f f e r , pH 7.4, 50 mM T r i s b u f f e r , pH 7.4, 50 mM KC1 50 mM T r i s b u f f e r , pH 7.4, 100 mM KC1 50 mM T r i s b u f f e r , pH 7.4, 600 mM KC1 B - m e r c a p t o e t h a n o l D e g r e e s C e n t i g r a d e D-(+)-camphor D e o x y r i b o n u c l e a s e 1 D i t h i o t h r e i t o l E x t e n d e d 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 s p e c t r o s c o p y E l e c t r o n n u c l e a r d o u b l e r e s o n a n c e E l e c t r o n p a r a m a g n e t i c r e s o n a n c e E l e c t r o n s p i n r e s o n a n c e S t a n d a r d e n t h a l p y change x i Hb H e m o g l o b i n max Maximum Mb M y o g l o b i n NADH Reduced n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e NADPH Reduced n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e NMR N u c l e a r m a g n e t i c r e s o n a n c e P-450 C y t o c h r o m e P-450 P-450cam Camphor h y d r o x y l a t i n g P-450 P-450LM L i v e r - m i c r o s o m a l P-450 RNase A R i b o n u c l e a s e A RR Resonance Raman SDS Sodium d o d e c y l s u l p h a t e Ago Standard entropy change x i i ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my g r a t i t u d e t o P r o f e s s o r s D . H . D o l p h i n and B.R.James f o r t h e i r g u i d a n c e and encourag e m e n t t h r o u g h o u t t h e c o u r s e of t h i s work. My t h a n k s a r e a l s o due t o M e s s r s . Simon A l b o n , G a r y H e w i t t and L a l i t h T a l a g a l a f o r t h e i r h e l p . F i n a l l y , I w i s h t o e x p r e s s my d e e p e s t a p p r e c i a t i o n t o my f a m i l y f o r t h e i r l o v e and u n d e r s t a n d i n g . CHAPTER 2 I . I n t r o d u c t i o n The h e m o p r o t e i n s w h i c h m e d i a t e t r a n s p o r t a t i o n , s t o r a g e and a c t i v a t i o n o f d i o x y g e n a r e v i t a l f o r most l i v i n g o r g a n i s m s . F o r example, i n mammals, d i o x y g e n i s t r a n s p o r t e d by h e m o g l o b i n , s t o r e d by m y o g l o b i n and r e d u c e d t o water i n t h e t e r m i n a l s t e p of r e s p i r a t i o n by c y t o c h r o m e c o x i d a s e . A l s o , d e c o m p o s i t i o n and f u r t h e r a c t i v a t i o n o f h y d r o g e n p e r o x i d e formed i n b i o l o g i c a l s y s t e m s a r e u n d e r t a k e n by c a t a l a s e and p e r o x i d a s e w h i l e a number o f o t h e r h e m o p r o t e i n s f u n c t i o n a s mono- and d i o x y g e n a s e s . Amongst a l l t h e s e h e m o p r o t e i n s , c y t o c h r o m e P-450 (P- 450) i s c o n s i d e r e d t o be one of t h e most u n u s u a l and i n t r i g u i n g . The i n t e n s e l e v e l o f r e s e a r c h c o n d u c t e d d u r i n g t h e l a s t 25 y e a r s s i n c e i t s d i s c o v e r y 1 - 5 r e f l e c t s , i n p a r t , t h e i m p o r t a n c e of c y t o c h r o m e P-450 as a d r u g - m e t a b o l i z i n g enzyme, i t s c e n t r a l r o l e i n c h e m i c a l c a r c i n o g e n e s i s , and i t s b r o a d s u b s t r a t e s p e c i f i c i t y . The P-450 m o l e c u l e c o n s i s t s o f a s i n g l e p r o t e i n c h a i n w i t h a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 45,000 , j o i n e d t o an i r o n p r o t o p o r p h y r i n m o i e t y ( F i g u r e 1.1) v i a a t h i o l a t e (-S~ ) l i g a n d d e r i v e d f r o m a c y s t e i n e r e s i d u e . The P-450 h e m o p r o t e i n s g e n e r a l l y c a t a l y z e t h e s p l i t t i n g of m o l e c u l a r d i o x y g e n and i n s e r t i o n of one oxygen atom i n t o an u n a c t i v a t e d C-H bond t o f o r m an a l c o h o l , t h e o t h e r atom of oxygen b e i n g c o n v e r t e d t o a m o l e c u l e of w a t e r ( R e a c t i o n 1 . 1 ) . RH + 0 2 + 2H ++ 2e" > ROH + H 2 0 (1.1) C H , H ca C H 2 C O O H  C H 2 C 0 0 H The Structure of the Heme Unit. 4 T h e r e f o r e , c y t o c h r o m e P-450's a r e c o l l e c t i v e l y known as monooxygenases, w h i c h a r e a c l a s s of enzymes t h a t i n c o r p o r a t e one atom of oxygen from m o l e c u l a r oxygen i n t o a s u b s t r a t e . The a b i l i t y of P-450 t o c a r r y out t h i s h y d r o x y l a t i o n r e a c t i o n under p h y s i o l o g i c a l c o n d i t i o n s w i t h h i g h s t e r e o - and r e g i o s p e c i f i c i t y i s i n f a c t r e m a r k a b l e , when compared t o t h e f o r c i n g r e a c t i o n c o n d i t i o n s and t h e number of d i f f e r e n t p r o d u c t s t h a t r e s u l t , i f t h e same h y d r o x y l a t i o n r e a c t i o n were t o be c a r r i e d out i n t h e l a b o r a t o r y u s i n g non e n z y m a t i c , c h e m i c a l r e a g e n t s . 6 T h r o u g h o u t t h e b r i e f h i s t o r y of c y t o c h r o m e P-450, numerous a t t e m p t s t o s o l u b i l i z e and p u r i f y t h e membrane- bound enzyme t h a t i s p r e s e n t i n mammalian s y s t e m s r e s u l t e d i n t h e i s o l a t i o n o f an ' i n a c t i v e ' f o r m o f enzyme. 7 However, t h e d i s c o v e r y o f s o l u b l e P-450 i n t h e b a c t e r i a l s t r a i n Peudomonas p u t i d a grown on D-( + )-camphor l e d t o t h e i s o l a t i o n o f p u r e enzyme. T h e r e f o r e , i n t h e p r e s e n t s t u d y , s o l u b l e camphor monooxygenase s y s t e m , P-450cam, has been u s e d as a b i o l o g i c a l model f o r mammalian P-450. R e a c t i o n s of o r g a n i c compounds w i t h d i o x y g e n i n t h e g r o u n d s t a t e a r e r e s t r i c t e d by s p i n c o n s e r v a t i o n . 8 T h i s may be overcome by f o r m a t i o n o f m e t a l - d i o x y g e n c o m p l e x e s w h i c h i n t u r n r e a c t w i t h t h e o r g a n i c s u b s t r a t e . However, t h e same 5 m e t a l c o m p l e x e s a l s o r e a c t d i r e c t l y w i t h a l k y l h y d r o p e r o x i d e s t o g e n e r a t e c h a i n - i n i t i a t i n g r a d i c a l s . 8 S i n c e a l k y l h y d r o p e r o x i d e s a r e u b i q u i t o u s i n most h y d r o c a r b o n m i x t u r e s , t h e m e t a l - h y d r o p e r o x i d e i n t e r a c t i o n s t e n d t o mask any r e a c t i o n v i a 'oxygen a c t i v a t i o n ' . T h e r e f o r e i t i s of immense i m p o r t a n c e t o d e v e l o p c a t a l y t i c s y s t e m s t h a t a r e c a p a b l e of p r o m o t i n g s e l e c t i v e o x i d a t i o n s , p r e f e r a b l y u s i n g 0 2 under m i l d r e a c t i o n c o n d i t i o n s . The s t u d y o f c y t o c h r o m e P-450 i s t h u s of g r e a t i m p o r t a n c e not o n l y i n w i d e n i n g our knowledge of t h i s r e m a r k a b l e b i o l o g i c a l s y s t e m but a l s o i n d e v e l o p i n g p r o t e i n - f r e e c a t a l y t i c s y s t e m s t h a t would mimic t h e 0 2 a c t i v a t i o n c a p a b i l i t y of t h e s e enzymes; s u c h s y s t e m s b e a r an u n l i m i t e d i n d u s t r i a l p o t e n t i a l . 9 I n a d d i t i o n t o m a j o r unanswered q u e s t i o n s a b o u t t h e mechanism o f P-450 c a t a l y t i c f u n c t i o n , q u e s t i o n s c o n c e r n i n g t h e i n t e r a c t i o n s o f t h e s u b s t r a t e s w i t h t h e a c t i v e - s i t e o f t h e enzyme r e m a i n e q u a l l y c h a l l e n g i n g . In t h e p r e s e n t s t u d y , t h e a t t e n t i o n was f o c u s e d t o g a i n some i n s i g h t i n t o a c t i v e s i t e - s u b s t r a t e i n t e r a c t i o n s by s t u d y i n g t h e CO and 0 2 b i n d i n g t o P-450cam. The t h e s i s b e g i n s w i t h an e x t e n s i v e l i t e r a t u r e s u r v e y i n C h a p t e r I I , t h a t i n t r o d u c e s v a r i o u s a s p e c t s o f m o n o o x y g e n a t i o n m e d i a t e d by P-450. C h a p t e r I I I d e s c r i b e s t h e d e t a i l s of e x p e r i m e n t a l p r o c e d u r e s . In C h a p t e r IV, t h e r e s u l t s o f e x p e r i m e n t s a r e d i s c u s s e d i n c o m p a r i s o n t o o t h e r known h e m o p r o t e i n s and model s y s t e m s , and some c o n c l u s i o n s a r e drawn. 6 REFERENCES 1. D . Y . C o o p e r , 0 . R o s e n t h a l , R . S n y d e r , C . W i l n e r , E d s . , C y t o c h r o m e P-450 and b 5 S t r u c t u r e , F u n c t i o n and I n t e r a c t i o n , Plenum, New Y o r k , 1974. 2. D . W . C o o p e r , H . A . S a l h a n i c k , E d s . , M u l t i e n z y m e Systems i n E n d o c h r i n o l o g y : P r o g r e s s i n P u r i f i c a t i o n and Methods of I n v e s t i g a t i o n , New York A c a d . S c i . , New Y o r k , 1973. 3. T . E . K i n g , H . S . M a s o n , M . M o r r i s o n , E d s . , O x i d a s e s and R e l a t e d Redox S y s t e m s , U n i v e r s i t y P a r k P r e s s , B a l t i m o r e , M a r y l a n d , 1973. 4. R . W . E s t a b r o o k , J . R . G i l l e t t e , K . C . L e i b m a n , E d s . , M i c r o s o m e s and D r u g O x i d a t i o n s , W i l l i a m s and W i l k i n s , B a l t i m o r e , M a r y l a n d , 1972. 5. M . J . C o o n , R . E . W h i t e , M e t a l Ion A c t i v a t i o n o f D i o x y g e n , ( E d . T . G . S p i r o ) , W i l e y I n t e r s c i e n c e , New Y o r k , 1980, pp73- 123. 6. J.H.Groves,W.Kroper,T.E.Nemo, R.S.Myers, J . M o l . C a t a l . , 7, 169 ( 1 9 8 0 ) . 7. B . W . G r i f f i n , J . A . P e t e r s o n , R . W . E s t a b r o o k , The P o r p h y r i n s , ( E d . D . D o l p h i n ) , Academic P r e s s , New Y o r k , V I I I , 1979, p.335. 8. R . A . S h e l d o n , J . K . K o c h i , M e t a l C a t a l y z e d O x i d a t i o n s o f O r g a n i c Compounds, Academic P r e s s , New Y o r k , 1981, Ch.8. 9. B.R.James,The P o r p h y r i n s , ( E d . D . D o l p h i n ) , A c a d e m i c P r e s s , New Y o r k ; V, 1979, p.205. CHAPTER II LITERATURE REVIEW 8 II . 1 B r i e f H i s t o r y of Cytochrome P-450 The p r e s e n c e i n r a t l i v e r m i c r o s o m e s of a pigment w i t h a p e c u l i a r c a r b o n monoxide b i n d i n g s p e c t r u m was f i r s t n o t e d by W i l l i a m s i n 1955 1 w h i l e s t u d y i n g t h e o x i d a t i o n - r e d u c t i o n k i n e t i c s of microsome-bound c y t o c h r o m e b 5 . T h i s n o v e l o b s e r v a t i o n was not p u b l i s h e d u n t i l 1958, a t w h i c h t i m e K l i n g e n b e r g 1 f i r s t r e p o r t e d t h e c a r b o n m o n o x i d e - d i f f e r e n c e s p e c t r u m o f t h i s m i c r o s o m a l p i g m e n t ( F i g u r e I I . 1 ) . 450 * - i 1 1 1 1 20 10 — 0 T -10 -20 600 400 440 480 520 560 Wavelength (nro) Figure I I . 1 The f i r s t published record of the carbon mono- x i d e - d i f f e r e n c e spectrum of cytochrome P-450. (from r e f . 1 ) . A l t h o u g h t h e b i n d i n g o f c a r b o n monoxide s t r o n g l y s u g g e s t e d t h e p r e s e n c e of a heavy m e t a l i o n i n t h e chromophore o f t h e p i g m e n t , t h e e l e c t r o n i c a b s o r p t i o n s p e c t r u m showed no r e s e m b l a n c e t o any known c o l o u r e d m e t a l l o p r o t e i n i n c l u d i n g h e m o p r o t e i n s . In 1962, Omura and S a t o 2 p r e s e n t e d c o n c l u s i v e s p e c t r a l e v i d e n c e f o r t h e h e m o p r o t e i n n a t u r e of t h e pig m e n t and c o n f i r m e d t h a t t h e pigment was a new b - t y p e c y t o c h r o m e . They a l s o p r o p o s e d a 9 ' t e n t a t i v e ' name "P-450" w h i c h meant "a pigment which a b s o r b s a t 450 nm". The p r e s e n c e of a- and | 3 - bands, i n a d d i t i o n t o i n t e n s e S o r e t bands i n t h e c a r b o n m o n o x i d e - d i f f e r e n c e s p e c t r u m o f t h e r e d u c e d pigment ( F i g u r e I I . 2 ) , c l e a r l y r e v e a l e d t h e h e m o p r o t e i n n a t u r e of t h e new p i g m e n t . I t a l s o 0.4 0.3 02 S 0.1 | 0.0 < -0.1 -0.2 400 450 500 550 600 650 Wavelength (nm) Figure II.2 The carbon monoxide-difference spectra of cytochrome P-450 (A) and P-420 (B).(from r e f . l ) . was o b s e r v e d t h a t t r e a t m e n t of t h e pigment w i t h d e t e r g e n t s c o n v e r t e d i t q u a n t i t a t i v e l y i n t o a s p e c t r a l l y d i s t i n c t s o l u b i l i z e d form, w i t h a p r o m i n e n t peak a t 420 nm i n t h e C O - d i f f e r e n c e s p e c t r u m . S i n c e t h e e s t a b l i s h m e n t of t h e h e m o p r o t e i n n a t u r e of "P-450", t h e a t t e n t i o n of many b i o c h e m i s t s who were engaged i n t h e s t u d y o f p h y s i c o c h e m i c a l p r o p e r t i e s of h e m o p r o t e i n s became f o c u s e d on t h i s new b - t y p e c y t o c h r o m e . U t i l i z i n g t h e p h o t o c h e m i c a l a c t i o n s p e c t r u m t e c h n i q u e , i n 1963, E s t a b r o o k e t a l . 3 d e m o n s t r a t e d f o r t h e f i r s t t i m e t h e p a r t i c i p a t i o n 10 o f c y t o c h r o m e P-450 i n a s t e r o i d C - 2 1 - h y d r o x y l a t i o n r e a c t i o n c a t a l y z e d by t h e a d r e n a l c o r t e x m i c r o s o m e s ( R e a c t i o n I I . 1 ) . The p h o t o c h e m i c a l a c t i o n s p e c t r u m f o r t h e l i g h t r e v e r s a l of t h e C O - i n h i b i t e d a c t i v e monooxygenase enzyme p r e s e n t i n t h e s e m i c r o s o m e s showed an i n t e n s e a b s o r p t i o n peak a t 450 nm t h a t was i d e n t i c a l t o t h e r e d u c e d C O - d i f f e r e n c e s p e c t r u m of t h e l i v e r m i c r o s o m e s . In a d d i t i o n , E s t a b r o o k e t a l . " were a b l e t o d e m o n s t r a t e t h e p a r t i c i p a t i o n of c y t o c h r o m e P-450 i n many o t h e r m i x e d f u n c t i o n o x i d a s e r e a c t i o n s i n c l u d i n g h y d r o x y l a t i o n s and o x i d a t i v e d e a l k y l a t i o n o f d r u g s c a t a l y z e d by l i v e r m i c r o s o m e s , c o n f i r m i n g t h e p h y s i o l o g i c a l f u n c t i o n o f c y t o c h r o m e P-450 t o be m o n o o x y g e n a t i o n . As t h e n a t u r e and f u n c t i o n o f c y t o c h r o m e P-450 was e l u c i d a t e d , i t was a l s o shown t h a t t h i s enzyme c o u l d be i n d u c e d by t r e a t i n g a n i m a l s w i t h v a r i o u s d r u g s . 5 ' 6 A l t h o u g h c y t o c h r o m e P-450 was i n i t i a l l y t h o u g h t t o be 1 1 p r e s e n t o n l y i n m i c r o s o m e s of a n i m a l t i s s u e , 7 i n a m a t t e r of a f i v e - y e a r p e r i o d f r o m t h e o r i g i n a l d i s c o v e r y i n m i c r o s o m e s , i t was shown t o be p r e s e n t i n a l m o s t a l l forms of l i f e . 8 In f a c t , c y t o c h r o m e P-450 i s d i s t r i b u t e d i n n a t u r e from p r i m i t i v e b a c t e r i a t o h i g h l y d e v e l o p e d mammals, and p a r t i c i p a t e s i n d i v e r s i f i e d m e t a b o l i c r e a c t i o n s as t h e oxygen a c t i v a t i n g component of monooxygenase s y s t e m s . In 1967, A p p l e b y 9 p r e s e n t e d t h e f i r s t c o n v i n c i n g e v i d e n c e f o r t h e p r e s e n c e o f P-450 i n a b a c t e r i a l s p e c i e s . The c y t o c h r o m e P-450 f o u n d i n t h e b a c t e r i u m R h i z o b i u m j a p o n i c u m was not bound t o membranous c e l l p a r t i c l e s and h ence c o u l d be p a r t i a l l y p u r i f i e d by c o n v e n t i o n a l p r o c e d u r e s . 9 S h o r t l y a f t e r w a r d s t h e camphor h y d r o x y l a t i n g c y t o c h r o m e P-450 was i s o l a t e d f r o m b a c t e r i u m Pseudomonas p u t i d a 1 0 and p u r i f i e d t o h o m o g e n i e t y , 1 1 and c r y s t a l l i z e d . 1 2 T h i s s o u r c e of t h e enzyme has p r o v i d e d t h e b e s t m a t e r i a l f o r s t u d y i n g t h e c h e m i c a l and p h y s i c o c h e m i c a l p r o p e r t i e s - o f t h i s c l a s s o f e n z y m e s . 8 S i n c e t h e e s t a b l i s h m e n t of t h e p h y s i o l o g i c a l f u n c t i o n , t h e n e x t c h a l l e n g e was t o d e t e r m i n e t h e mechanism o f e l e c t r o n s u p p l y t o c y t o c h r o m e P-450. The d i f f i c u l t y i n s o l u b i l i z i n g t h e l i v e r m i c r o s o m a l P-450 s y s t e m , w h i c h was v e r y t i g h t l y a s s o c i a t e d w i t h t h e membranes, c a u s e d r e s e a r c h e r s t o l o o k f o r a more s o l u b l e form o f P-450. The components of t h e 1 1 - / 3 - h y d r o x y l a s e s y s t e m o f a d r e n a l c o r t e x m i t o c h o n d r i a were f i n a l l y s e p a r a t e d by i o n i c t r e a t m e n t 1 3 - 1 " i n t o a P - 4 5 0 - c o n t a i n i n g membrane f r a g m e n t and a s o l u b l e 12 NADH-P-450 r e d u c t a s e s y s t e m , w h i c h was l a t e r s e p a r a t e d by c h r o m a t o g r a p h y i n t o an i r o n - s u l f u r p r o t e i n and a f l a v o p r o t e i n . A l l of t h e s e components were r e q u i r e d t o r e c o n s t i t u t e t h e 1 1 - / ^ - h y d r o x y l a s e a c t i v i t y of t h e sys t e m , and t h e r o l e o f i r o n - s u l f u r p r o t e i n was f o u n d t o be t h e t r a n s f e r r i n g of e l e c t r o n s from N A D P H - l i n k e d f l a v o p r o t e i n t o c y t o c h r o m e P-450. In 1968, c o n s t i t u e n t s o f t h e camphor h y d r o x y l a t i n g s y s t e m were s e p a r a t e d and i d e n t i f i e d as an i r o n - s u l f u r p r o t e i n , a f l a v o p r o t e i n a nd c y t o c h r o m e P-450 ( F i g u r e II .3) . 1 5 NAD+ NADH +H+ REDUCTASE C F A D>red REDUCTASE (FAD) PUTIDAREDOXIN ( F e 3 + ) PUTIDAREDOXIN (Fe2 +) P-450cam(SH) ( F e 2 + ) P-450cam(SH) ( F e 3 + ) 0 2 SOH +H20 Figure II.3 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 i n b a c t e r i a l system leading to camphor (SH) hydroxylation. A m a j o r b r e a k t h r o u g h t o w a r d s t h e u n d e r s t a n d i n g of t h e r e a c t i o n c y c l e o f c y t o c h r o m e P-450 o c c u r r e d when N a r a s i m h u l u e t a l . 1 6 , i n 1965, o b s e r v e d t h e " s u b s t r a t e b i n d i n g s p e c t r u m " of t h e C - 2 1 - h y d r o x y l a s e s y s t e m o f a d r e n a l c o r t e x m i c r o s o m e s . When t h e s u b s t r a t e , 17- h y d r o x y p r o g e s t e r o n e , was added t o a s u s p e n s i o n o f a d r e n a l c o r t e x m i c r o s o m e s , t h e S o r e t peak was o b s e r v e d t o s h i f t t o 13 a s h o r t e r w a v e l e n g t h , and t h e s p e c t r u m r e t u r n e d t o t h e o r i g i n a l p o s i t i o n upon t h e a d d i t i o n of NADPH. The s u b s t r a t e - i n d u c e d s p e c t r a l change f o r t h e s o l u b l e c y t o c h r o m e P-450 from Pseudomonas p u t i d a was o b s e r v e d 1 7 a l s o and t h e a s s o c i a t i o n c o n s t a n t f o r camphor b i n d i n g was c a l c u l a t e d t o be (0.47±0. 07) 1 0 6W 1 ( F i g u r e I I . 4 ) . 1 8 0 5 s B *! 01 0 350 4 0 0 4 5 0 9 0 0 5 5 0 6 0 0 6 5 0 7 C 0 W A V E L E N G T H <i>o0 Figure II.4 The substrate-induced spectral changes for cytochrome P-450cam. F i n a l camphor concentrati- ons (WM) were, 0 ( ) ; 2 ( 0 ) ; and 6 ( 0 ) ; and 20.5 ( A ),(fromref. 18). The a s s o c i a t i o n r e a c t i o n between P-450 and camphor c h a n g e s t h e i r o n ( I I I ) f r o m a low t o h i g h s p i n form a s d e t e c t e d f r o m EPR e x p e r i m e n t s , 1 9 and t h i s i n t u r n r e s u l t s i n a s i g n i f i c a n t change i n t h e red o x p o t e n t i a l of t h e heme i r o n . 2 0 Thus, t h e f i r s t s t e p i n t h e r e a c t i o n of t h i s r e m a r k a b l e b i o c a t a l y s t s y s t e m became w e l l u n d e r s t o o d . The m i c r o s o m a l f r a c t i o n s were shown t o r e a c t w i t h a wide v a r i e t y of s u b s t r a t e s u s i n g t h e p h o t o c h e m i c a l a c t i o n s p e c t r u m t e c h n i q u e , 3 t h i s b e i n g u n u s u a l b e h a v i o r f o r a s i n g l e enzyme s p e c i e s . A l s o , i t was o b s e r v e d t h a t t h e 14 c y t o c h r o m e P-450 from m i c r o s o m e s of a d r e n a l c o r t e x and l i v e r had e s s e n t i a l l y t h e same C O - d i f f e r e n c e s p e c t r u m , a l t h o u g h t h e s u b s t r a t e s were c h e m i c a l l y v e r y d i f f e r e n t . 2 1 However, t h e e x p l a n a t i o n o f t h i s o b s e r v a t i o n was d e l a y e d u n t i l l a t e r s e p a r a t i o n and p u r i f i c a t i o n of m i c r o s o m a l c y t o c h r o m e P-450. In 1975, i t was shown t h a t r a b b i t l i v e r m i c r o s o m a l P-450 (P-450LM) p u r i f i e d t o e l e c t r o p h o r e t i c h o m o g e n i e t y c o n t a i n e d a s many as s i x forms of P- 4 5 0 L M s , 2 2 - 2 5 d e s i g n a t e d a c c o r d i n g t o d e c r e a s i n g m o b i l i t y i n S D S - g e l e l e c t r o p h o r e s i s a s P-450LM,, P-450LM 2 and so o n . 2 6 Some of t h e s e m u l t i p l e forms ( i s o z y m e s ) have been c h a r a c t e r i z e d 2 7 and f o u n d t o c o n t a i n d i f f e r e n t C- and N- t e r m i n a l r e s i d u e s and o t h e r s i g n i f i c a n t s t r u c t u r a l d i f f e r e n c e s . 15 11.2 N a t u r e and C y t o c hrome P-450 A l l human b e i n g s and most o t h e r a n i m a l s on e a r t h a r e c h r o n i c a l l y e x p o s e d t o numerous n o n - n u t r i e n t d i e t a r y c h e m i c a l s , t o d r u g s u s e d i n t r e a t m e n t o r p r e v e n t i o n of d i s e a s e , and t o an e v e r i n c r e a s i n g number of man-made e n v i r o n m e n t a l p o l l u t a n t s . In o r d e r t o s u s t a i n l i f e i n t h i s p o t e n t i a l l y h a z a r d o u s s u r r o u n d i n g s , n a t u r e has p l a y e d a r e m a r k a b l e r o l e i n d e s i g n i n g and a c t i v a t i n g r e l a t i v e l y e f f i c i e n t ways of d e t o x i f i c a t i o n and e x c r e t i o n of t h e s e ' d e a d l y ' x e n o b i o t i c s . S i n c e i t s d i s c o v e r y i n 1958, many for m s o f c y t o c h r o m e P-450 have been shown t o o c c u r i n a l m o s t a l l forms of l i f e , f u n c t i o n i n g a s a h i g h l y v e r s a t i l e o x y g e n a t i n g c a t a l y s t i n d i v e r s i f i e d b i o c h e m i c a l r e a c t i o n s . C y t o c h r o m e P-450 i s known t o be p r e s e n t i n y e a s t s , 2 8 f u n g i , 9 p l a n t s , 2 9 b a c t e r i a , 3 0 - 3 1 i n s e c t s , 3 2 f i s h , b i r d s , r e p t i l e s , a m p h i b i a n s , 3 3 • 3 * and mammals. 3 5 In mammals, c y t o c h r o m e P-450s a r e f o u n d i n m i c r o s o m e s o f t h e l i v e r , k i d n e y , s m a l l - i n t e s t i n e , l u n g , a d r e n a l c o r t e x , s k i n , t e s t i s , p l a c e n t a and s e v e r a l o t h e r t i s s u e s , 8 and a r e c a p a b l e o f m e t a b o l i s i n g d r u g s , f o o d a d d i t i v e s , a n a e s t h e t i c s , p e t r o l e u m p r o d u c t s , p e s t i c i d e s and c a r c i n o g e n s . 2 7 In t h i s c o n t e x t , i t i s c o n c e i v a b l e t h a t t h e b a s i c f u n c t i o n of P-450 monooxygenase s y s t e m i s t o c o n v e r t t h e l y p o p h y l i c x e n o b i o t i c compounds t o more p o l a r m e t a b o l i t e s w h i c h i n t u r n can be e x c r e t e d from t h e body. However, t h e s i t u a t i o n i s by no means s i m p l e , s i n c e a number o f c a s e s have been r e p o r t e d i n w h i c h x e n o b i o t i c s a r e 16 c o n v e r t e d t o more h a z a r d o u s m e t a b o l i t e s . 3 6 ' 3 7 F o r example, h a l o g e n a t e d b e n z e n e s a r e c o n v e r t e d t o t h e i r a r e n e o x i d e s , w h i c h b i n d t o c e l l u l a r m a c r o m o l e c u l e s c a u s i n g c e l l damage, and p o l y c y c l i c h y d r o c a r b o n s a r e c o n v e r t e d t o t r u e c a r c i n o g e n s ( s e e F i g u r e I I . 5 ) . 3 8 Hence, i t a p p e a r s t h a t Figure I I .5 Benzopyrene (A) and i t s carcinogenic metabolite 7,8-diol-9,10-epoxide (B). n a t u r e has f a i l e d t o a c e r t a i n e x t e n t i n d e s i g n i n g t h e s e enzyme s y s t e m s by a l l o w i n g a w i d e r s u b s t r a t e s p e c i f i c i t y and t h u s c a u s i n g t h e b i o t r a n s f o r m a t i o n o f h a r m l e s s c h e m i c a l s t o p o t e n t i a l c a r c i n o g e n s . The wide d i s t r i b u t i o n of c y t o c h r o m e P - 4 5 0 - c o n t a i n i n g monooxygenase s y s t e m s from mammals t o b a c t e r i a r e p r e s e n t s an i n t e r e s t i n g example of how t h i s enzyme s y s t e m e v o l v e d when t h e s u r r o u n d i n g s were c h a n g e d . A l l components of t h e b a c t e r i a l monooxygenase s y s t e m e x i s t i n a ' p r i m i t i v e ' s o l u b l e form and t h e more d e v e l o p e d m i t o c h o n d r i a l s y s t e m c o n s i s t s o f a s o l u b l e r e d u c t a s e component and a membrane- bound P-450; i n t h e m i c r o s o m e s , a l l components a r e t i g h t l y membrane-bound and hence have a c q u i r e d enough h y d r o p h o b i c i t y t o become i n c o r p o r a t e d i n t o biomembranes. 17 11.3 S t r u c t u r a l C o n s i d e r a t i o n s I t i s of immense i m p o r t a n c e t o e l u c i d a t e t h e s t r u c t u r e of t h e c y t o c h r o m e P-450 enzymes i n o r d e r t o f u l l y u n d e r s t a n d t h e mechanisms of o x y g e n a t i o n and d i o x y g e n r e d u c t i o n so t h a t t h e b i o l o g i c a l r e a c t i o n s c a n be m i m i c k e d . In t h i s d i s c u s s i o n of t h e s t r u c t u r a l a s p e c t s o f c y t o c h r o m e P-450, a t t e n t i o n w i l l be f o c u s e d m a i n l y on t h e b a c t e r i a l P- 450cam enzyme, s i n c e i t i s t h e most s t u d i e d and most u n d e r s t o o d . The heme p r o s t h e t i c g r o u p i n P-450 i s bound t o t h e p o l y p e p t i d e c h a i n v i a an a c i d l a b i l e , non c o v a l e n t l i n k a g e and hence t h e s y s t e m i s c l a s s i f i e d as a b - t y p e c y t o c h r o m e . 3 9 The c y t o c h r o m e P-450 i s composed o f a s i n g l e p o l y p e p t i d e c h a i n and a s i n g l e m o l e c u l e o f p r o t o p o r p h y r i n a t o i r o n ( 1 1 1 ) p e r m o l e c u l e o f p r o t e i n . 3 9 ' " 0 The immediate e n v i r o n m e n t of t h e heme g r o u p a t t r a c t e d t h e a t t e n t i o n of many i n v e s t i g a t o r s i n l a t e 1960's b e c a u s e of t h e a b n o r m a l s p e c t r a l c h a r a c t e r i s t i c s o b s e r v e d i n s u b s t r a t e b i n d i n g and C O - c o m p l e x a t i o n . S i n c e t h e n , enough d i r e c t and c o m p e l l i n g e v i d e n c e has been g a t h e r e d t o i n d i c a t e t h e p r e s e n c e o f a s u l f u r atom i n t h e f i r s t c o o r d i n a t i o n s p h e r e o f t h e h e m e - i r o n , and t h e i r o n s u l f u r d i s t a n c e has been shown t o be c o n s i s t e n t w i t h t h i o l a t e (RS~) l i g a t i o n i n a l l f o u r s t a t e s of t h e enzyme." 1 In 1968, M i y a k e and G a l o r " 2 were a b l e t o show u s i n g EPR e x p e r i m e n t s t h a t t h e s u b s t r a t e - f r e e f e r r i c enzyme i s p r e d o m i n a n t l y i n low s p i n ( S = l / 2 ) form. T h i s o b s e r v a t i o n was s i m i l a r l y f o u n d 18 i n a l k y l m e r c a p t i d e c o m p l e x e s o f f e r r i c h e m o g l o b i n or f e r r i c m y o g l o b i n . * 3 ' * * The model s t u d i e s o f Chang and D o l p h i n " 5 and o t h e r s * 6 • " 7 were a b l e t o mimic t h e s p e c t r u m of c a r b o n y l a t e d P-450 u s i n g s i m p l e i r o n ( I I ) - p o r p h y r i n s h a v i n g m e r c a p t i d e i o n and CO l i g a t i o n . A l s o t h e models showed a h i g h e n e r g y , low i n t e n s i t y S o r e t band a r o u n d 360 nm i n t h e CO-complex w h i c h was n o t o b s e r v e d i n t h e c a s e of t h e enzyme CO-complex, due t o t h e use of e x c e s s sodium d i t h i o n i t e r e d u c t a n t w h i c h o b s c u r e d t h e a b s o r p t i o n s below 380 nm. However, s t o i c h i o m e t r i c a l l y r e d u c e d enzyme, when c o m p l e x e d w i t h CO, gave t h e s e c o n d S o r e t peak a t 365 nm. The o c c u r r e n c e of s p l i t S o r e t bands, o r " h y p e r s p e c t r a " , a r e c h a r a c t e r i s t i c o f i r o n p o r p h y r i n s c o o r d i n a t e d by l i g a n d s w h i c h can d o n a t e h i g h e l e c t r o n d e n s i t y . * 8 The measurement of c h a n g e s i n t h e p r o t o n c o n c e n t r a t i o n d u r i n g t h e b i n d i n g o f CO,0 2 and e x c h a n g e of 0 2 by CO, i n d i c a t e d t h a t t h e Fe-S l i n k a g e r e m a i n e d i n t a c t d u r i n g t h e r e a c t i o n s . 4 9 R e c e n t d i r e c t e v i d e n c e f r o m R esonance Raman (RR) s p e c t r o s c o p y 5 0 and e x t e n d e d 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 ( E X A F S ) * 1 s p e c t r o s c o p y has c o n f i r m e d t h i o l a t e l i g a t i o n t o h e m e - i r o n . As i n t h e c a s e of o t h e r low s p i n f e r r i c h e m o p r o t e i n s , t h e s u b s t r a t e - f r e e c y t o c h r o m e P-450 i s b e l i e v e d t o have a s i x t h l i g a n d c o o r d i n a t e d . A n i t r o g e n o r oxygen b a s e d r e s i d u e h a s been p r o p o s e d as t h e b e s t c a n d i d a t e . 5 0 A l t h o u g h t h e p r o b l e m s t i l l r e m a i n s a f t e r e x t e n s i v e i n v e s t i g a t i o n s , a w a ter l i g a n d has been i m p l i c a t e d from t h e NMR r e l a x a t i o n 19 r a t e o f water p r o t o n s 5 1 and e l e c t r o n n u c l e a r d o u b l e r e s o n a n c e (ENDOR) 5 2 s t u d i e s . The amino a c i d s e quence of c y t o c h r o m e P-450cam c o n s i s t s o f 412 amino a c i d r e s i d u e s wi^th a c a l c u l a t e d m o l e c u l a r w e i g h t of 46,820 i n c l u d i n g heme. Of t h e s e r e s i d u e s , 210 a r e h y d r o p h o b i c , 98 a r e p o l a r n e u t r a l , and 104 a r e i o n i c (54 a c i d i c and 50 b a s i c ) amino a c i d s . 5 3 T h e r e a r e e i g h t c y s t e i n e r e s i d u e s w i t h f o u r i n t h e N H 2 - t e r m i n a l h a l f and f o u r i n t h e C O O H - t e r m i n a l h a l f , and a l l o f them a r e s i t u a t e d i n t h e i n t e r i o r of t h e p o l y p e p t i d e c h a i n . 5 3 The above d a t a p r e d i c t t h a t t h e c o n f o r m a t i o n o f t h e P- 450cam m o l e c u l e c o n s i s t s of 46% a - h e l i x , 16% 0- p l e t e d s h e e t , 21 /3-turns and 38% random c o i l s t r u c t u r e . 5 3 Based on t h e n a t u r e and t h e number of amino a c i d r e s i d u e s p r e s e n t , i t i s c o n c e i v a b l e t h a t t h e p r o s t h e t i c g r o u p o f t h e enzyme i s s u r r o u n d e d m a i n l y by h y d r o p h o b i c , n o n - a r o m a t i c amino a c i d s . The h y d r o p h o b i c p o c k e t t h u s formed would have a c a v i t y l a r g e enough f o r s u b s t r a t e b i n d i n g and w o u l d a l s o p r o h i b i t any amino a c i d g r o u p b e i n g c l o s e t o t h e s i t e o f f o r m a t i o n of t h e e x t r e m e l y p o w e r f u l o x i d i z i n g a g e n t . The t h r e e d i m e n s i o n a l shape o f P-450 m o l e c u l e , w h i c h i s under i n v e s t i g a t i o n i n t h e l a b o r a t o r y of I . C . G u n s a l u s , has been d e s c r i b e d a s a "donut w i t h a t a i l " . 5 " 20 11.4 The Mechanism of C a t a l y s i s 11. 4 .1 T y p e s o f O x i d a t i o n R e a c t i o n s A l t h o u g h t h e major i n t e r e s t l i e s i n c y t o c h r o m e P-450 enzymes t h a t a c t i v a t e r a t h e r i n e r t C-H bonds, t h e d i v e r s i t y of s u b s t r a t e s and t h e v a r i e t y o f t r a n s f o r m a t i o n s t h a t t h e enzyme c a n e x e c u t e d e s e r v e s a b r i e f d i s c u s s i o n . The m e c h a n i s t i c c y c l e w i l l be d e s c r i b e d w i t h p a r t i c u l a r r e f e r e n c e t o C-H h y d r o x y l a t i n g s y s t e m s , a l t h o u g h w i t h m i n o r m o d i f i c a t i o n s i t can be a p p l i e d t o o t h e r t y p e s of r e a c t i o n s as w e l l . G u e n g e r i c h and M a c D o n a l d 5 5 c l a s s i f y t h e o x i d a t i v e r e a c t i o n s o f c y t o c h r o m e P-450 i n t o s i x c a t e g o r i e s . ( 1 ) . C a r b o n h y d r o x y l a t i o n : The f o r m a t i o n of an a l c o h o l a t a m e t h y l , m e t h y l e n e or m e t h i n e p o s i t i o n . E g . ( 2 ) . H e t e r o a t o m r e l e a s e ; The o x i d a t i v e c l e a v a g e o f t h e h e t e r o a t o m i c p o r t i o n of a m o l e c u l e . The m o l e c u l e i s h y d r o x y l a t e d a d j a c e n t t o t h e h e t e r o a t o m and t h e g e m i n a l h y d r o x y h e t e r o a t o m i n t e r m e d i a t e t h u s f o r m e d l o s e s t h e h e t e r o a t o m t o form a c a r b o n y l compound. Eg. C H 3 C H 2 C H 2 C H 2 B r > CH 3CH 2CH 2CHBr(OH) > CH 3CH 2CH 2CHO 21 (3) . H e t e r o a t o m o x y q e n a t i o n : C o n v e r s i o n of a h e t e r o a t o m - c o n t a i n i n g s u b s t r a t e t o i t s c o r r e s p o n d i n g h e t e r o a t o m o x i d e . E g . Me 3N > Me 3N=0 (4) . E p o x i d a t i o n : F o r m a t i o n of an o x i r a n e d e r i v a t i v e f r o m o l e f i n s and a r o m a t i c compounds. Eg. (5) . O x i d a t i v e g r o u p t r a n s f e r : 1,2-Carbon s h i f t o f a g r o u p w i t h c o n c o m i t a n t i n c o r p o r a t i o n o f oxygen as a c a r b o n y l a t C-1 p o s i t i o n . E g . C1 2C=CHC1 > C C I 3 C H O (6) . O l e f i n i c s u i c i d e d e s t r u c t i o n ; I n a c t i v a t i o n of t h e heme o f c y t o c h r o m e P-450 by an enzyme p r o d u c t o r an enzyme i n t e r m e d i a t e . The common f e a t u r e i n a l l of t h e s e r e a c t i o n s i s t h a t one atom of oxygen, from t h e r e d u c t i v e s c i s s i o n of d i o x y g e n , i s b e i n g i n s e r t e d i n t o t h e s u b s t r a t e w h i l e t h e o t h e r i s r e d u c e d t o a w ater m o l e c u l e . A f t e r c o n s i d e r a t i o n 22 of t h e o b s e r v e d s t o i c h i o m e t r y , t h e r e s u l t s o f l a b e l l e d - oxygen e x p e r i m e n t s , t h e r e g i o s e l e c t i v i t y of t h e h y d r o x y l a t i o n , and t h e number o f e l e c t r o n s r e q u i r e d from t h e r e d u c t a s e , a scheme has been c o n s t r u c t e d t o i l l u s t r a t e t h e v a r i o u s s t e p s of t h e o x y g e n a s e r e a c t i o n c y c l e ( F i g u r e I I . 6 ) . 5 6 Figure 11.6 The P-450 c a t a l y t i c c y c l e . 24 I I . 4 . 2 The C a t a l y t i c C y c l e As i n any e n z y m a t i c c a t a l y s i s , t h e f i r s t s t e p i n t h e r e a c t i o n c y c l e i s t h e f o r m a t i o n of an e n z y m e - s u b s t r a t e c omplex. T h i s i s f o l l o w e d by r e d u c t i o n o f i r o n ( I I I ) t o i r o n ( I I ) w h i c h a l l o w s f o r d i o x y g e n b i n d i n g . A s e c o n d r e d u c t i o n , t h a t i n d u c e s t h e s p l i t t i n g of d i o x y g e n , forms t h e " a c t i v e oxygen complex" and a m o l e c u l e o f w a t e r . Then t h e ' a c t i v e oxygen' i s i n s e r t e d i n t o a c a r b o n - h y d r o g e n bond, f o r m i n g t h e a l c o h o l p r o d u c t , w h i c h i n t u r n i s r e l e a s e d a s t h e enzyme i s r e t u r n e d t o t h e o r i g i n a l f e r r i c s t a t e . I I . 4 . 2 . 1 B i n d i n g of S u b s t r a t e The a s s o c i a t e d change i n t h e c o n f o r m a t i o n due t o t h e b i n d i n g o f t h e s u b s t r a t e t o t h e enzyme c a u s e s t h e s p i n s t a t e of t h e i r o n ( I I I ) t o s h i f t f r o m low s p i n ( S = l / 2 ) , t o h i g h s p i n , ( S = 5 / 2 ) . 1 9 T h i s change has been o b s e r v e d by EPR s p e c t r o s c o p y 1 9 and by t h e measurement o f t h e m a g n e t i c s u s c e p t i b i l i t y . 1 8 The b i n d i n g p r o c e s s p r o c e e d s a t a h i g h r a t e w i t h a s e c o n d o r d e r r a t e c o n s t a n t o f 3 . 7x 106M"1 s"1 a t 8 ° . 5 7 The e q u i l i b r i u m c o n s t a n t i n t h e p r e s e n c e of p o t a s s i u m i o n s has been e s t i m a t e d t o be 4 . 7 x 1 0 5 M ~ 1 . 1 8 The main b o n d i n g f o r c e s a r e b e l i e v e d t o be h y d r o p h o b i c i n n a t u r e , a l t h o u g h t h e r e may be some d i p o l e i n t e r a c t i o n s between t h e s u b s t r a t e and t h e enzyme. T h i s t y p e of b o n d i n g i s l a r g e l y r e s p o n s i b l e f o r t h e o b s e r v e d i n c r e a s e i n e n t r o p y on t r a n s f e r o f t h e l i p o p h i l i c s u b s t r a t e from t h e aqueous phase i n t o 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 a c t i v e c e n t e r . 5 8 25 A l t h o u g h t h e p r e c i s e l o c a t i o n o f t h e s u b s t r a t e i s s t i l l unknown, i n h i b i t i o n e x p e r i m e n t s show t h e b o n d i n g s i t e of camphor t o be i n t h e immediate v i c i n i t y of t h e oxygen b o n d i n g s i t e . 5 9 The p r o c e s s of s u b s t r a t e b i n d i n g c o u l d p r o c e e d as i n d i c a t e d i n F i g u r e I I . 7 . The most s i g n i f i c a n t change upon s u b s t r a t e b i n d i n g i s a s h i f t i n r e d o x p o t e n t i a l of t h e heme i r o n . The s u b s t r a t e - f r e e c y t o c h r o m e (low s p i n ) has a r e d u c t i o n p o t e n t i a l (E°) of -270 mV w h i l e t h a t of s u b s t r a t e - b o u n d enzyme ( h i g h s p i n ) i s -170 mV. 2 0 S i n c e t h e E° of p u t i d a r e d o x i n i s -240 mV, t h e s p i n change i n P-450 f a c i l i t a t e s r e d u c t i o n by p u t i d a r e d o x i n , t h e r e b y e n a b l i n g t h e s u c c e s s i v e b i n d i n g o f d i o x y g e n . 11.4.2.2 F i r s t R e d u c t i o n The n e x t s t e p i n t h e r e a c t i o n c y c l e i s t h e t r a n s f e r o f an e l e c t r o n t o t h e c y t o c h r o m e . D e s p i t e t h e c o n s i d e r a b l e e f f o r t d e v o t e d t o s t u d y t h e n a t u r e o f t h i s r e a c t i o n , some q u e s t i o n s s t i l l r e m a i n unanswered. S t o p p e d - f l o w s p e c t r o p h o t o m e t r i c s t u d i e s 6 0 i n d i c a t e t h i s r e d u c t i o n t o be Figure II.7 Hypothetical scheme f o r the binding of substrates to cytochrome P-450. (from r e f . 58). 26 f i r s t o r d e r b i p h a s i c , b ut no r a t i o n a l e f o r t h i s b e h a v i o r has been p r e s e n t e d . The r a t e c o n s t a n t s c a l c u l a t e d 6 0 i n d i c a t e t h i s s t e p i s not r a t e - l i m i t i n g . A l t h o u g h t h e e x a c t mechanism of e l e c t r o n c h a n n e l i n g f r o m p u t i d a r e d o x i n t o t h e i r o n ( l l l ) c e n t e r i s n o t known, t h e p u t i d a r e d o x i n i s t h o u g h t t o b i n d t o a h i g h l y p o s i t i v e l y c h a r g e d r e g i o n of t h e P-450 p r o t e i n , between t h e 340 and 412 p o s i t i o n s from t h e NH 2 t e r m i n a l end. I I . 4 . 2 . 3 B i n d i n g o f D i o x y g e n The a s s o c i a t i o n r e a c t i o n o f t h e r e d u c e d s u b s t r a t e - bound enzyme w i t h d i o x y g e n , t o form t h e f e r r o u s - d i o x y g e n complex, has been w e l l e s t a b l i s h e d , owing t o t h e f a c t t h a t t h e complex i s f a i r l y s t a b l e a t low t e m p e r a t u r e s . 2 7 , 6 1 The f o r m a t i o n of o x y - c y t o c h r o m e P-450cam, as d e t e r m i n e d from t h e s t o p p e d - f l o w t e c h n i q u e , i s f i r s t - o r d e r w i t h r e s p e c t t.o b o t h d i o x y g e n and t h e r e d u c e d enzyme, t h e s e c o n d - o r d e r r a t e c o n s t a n t b e i n g 7.7x10 s M"1 s'1 a t 4° and pH 7 . 4 . 6 2 The d i o x y g e n complex t h u s formed s l o w l y d e c a y s even a t low t e m p e r a t u r e s t o g i v e t h e o x i d i z e d enzyme. T h i s p r o c e s s , known as t h e " a u t o x i d a t i o n " , and t h e s t r u c t u r e and s t a b i l i t y of d i o x y g e n a d d u c t , w i l l be d i s c u s s e d i n d e t a i l i n S e c t i o n I I . 6 . The s u b s t r a t e - f r e e e n z y m e - d i o x y g e n complex u n d e r g o e s t h e a u t o x i d a t i o n r e a c t i o n 1 0 2 t i m e s f a s t e r t h a n t h e s u b s t r a t e - b o u n d e n z y m e - d i o x y g e n c o m p l e x . 3 0 T h i s i n d i c a t e s t h a t t h e o x y - c y t o c h r o m e P-450cam for m e d i n t h e p r e s e n c e of camphor i s not a s i m p l e a d d u c t of d i o x y g e n but a t e r n a r y complex of d i o x y g e n , e n z y m e and camphor. 27 11.4.2.4 Second R e d u c t i o n The a b i l i t y t o b y - p a s s t h e s e c o n d , t h i r d and f o u r t h s t e p s ( F i g u r e I I . 6 ) o f t h e e n z y m a t i c c y c l e by a d d i n g h y d r o g e n p e r o x i d e t o o x i d i z e d enzyme, and s t i l l o b t a i n t h e h y d r o x y l a t e d s u b s t r a t e w i t h o u t any e x t e r n a l e l e c t r o n s u p p l y , s u g g e s t s t h a t t h e n a t u r e of t h e t w o - e l e c t r o n r e d u c e d enzyme-oxo complex t o be F e ( I I I ) - 0 2 ~ 2 i n n a t u r e . 6 3 In f a c t , t h e model s t u d i e s i n d i c a t e t h a t t h e s t r u c t u r e of t h i s s p e c i e s may be h i g h - s p i n f e r r i c j i 2 - p e r o x i d e . 6 * - 6 5 U n f o r t u n a t e l y , t h e model F e ( I I I ) - p e r o x o c o m p l e x e s , p r o d u c e d e i t h e r s y n t h e t i c a l l y 6 4 o r e l e c t r o c h e m i c a l l y , 6 5 do not e x i b i t t h e o x i d i z i n g power o f t h e enzyme s y s t e m p o s s i b l y b e c a u s e of t h e a b s e n c e of t h i o l a t e l i g a n d . 6 5 The r a t e o f t h e s e c o n d r e d u c t i o n a g r e e s w e l l w i t h o b s e r v e d t u r n - o v e r number f o r camphor h y d r o x y l a t i o n , and h e n c e a p p e a r s t o be t h e r a t e d e t e r m i n i n g s t e p i n t h e c a t a l y t i c c y c l e . 6 6 , 6 7 11. 4 . 2. 5 S ' p l i t t i n g of t h e Oxygen-Oxygen Bond The s t e p , where t h e bond between t h e two oxygen atoms i s c l e a v e d h e t e r o l y t i c a l l y , i s l e a s t u n d e r s t o o d of t h e e n t i r e m e c h a n i s t i c scheme and has been t h e s u b j e c t o f much s p e c u l a t i o n . The c o n v e r s i o n o f t h e i r o n - p e r o x i d e complex t o t h e " a c t i v e o x y g e n " i n t e r m e d i a t e i n v o l v e s p r o t o n a t i o n , f o l l o w e d by h e t e r o l y t i c c l e a v a g e o f t h e 0-0 bond w i t h t h e p r o d u c t i o n of a m o l e c u l e o f w a t e r . The r e m a i n i n g oxygen atom s t a y s c o o r d i n a t e d t o t h e h e m e - i r o n . The o v e r a l l c h a r g e on t h i s two-atom u n i t , n e g l e c t i n g t h e c o n t r i b u t i o n s of t h e 28 p o r p h y r i n and t h i o l a t e , becomes +3, l e a d i n g t o d e s i g n a t i o n s s u c h as [ F e ( l I I ) - 0 ] 3 + , o r [ Fe ( I V ) - 0 - ] 3 + , or [ Fe (V) - 0 2 " ] 3 + . E v i d e n c e has been g a t h e r e d t o d e m o n s t r a t e t h e o c c u r r e n c e of t h i s " o x e n o i d " t y p e i n t e r m e d i a t e by u s i n g f e r r i c p o r p h y r i n s w i t h s i n g l e oxygen atom d o n o r s l i k e i o d o s y l b e n z e n e , C 6 H 5 I - 0 6 8 and p e r a c i d s 2 7 > 6 9 t o e f f e c t h y d r o x y l a t i o n s s i m i l a r t o t h a t of t h e n a t i v e P-450,NADPH,0 2 s y s t e m . I t has been s u g g e s t e d t h a t t h e p r e s e n c e o f t h i o l a t e l i g a n d may f a c i l i t a t e t h e s p l i t t i n g of t h e p e r o x i d e m o i e t y by w eakening i t t h r o u g h c h a r g e r e p u l s i o n by an e l e c t r o n - r i c h i r o n a t o m . 7 0 But no d e f i n i t i v e e x p l a n a t i o n has been g i v e n t o t h e f a c t t h a t t h i o l a t e i s n o t a r e q u i r e d l i g a n d f o r c a t a l a s e and p e r o x i d a s e , w h i c h undergo s i m i l a r p e r o x y - i r o n i n t e r m e d i a t e s i n t h e i r r e a c t i o n c y c l e s . 11.4.2.6 O x i d a t i o n of S u b s t r a t e S i n c e c y t o c h r o m e P-450 i s t h e o n l y h e m o p r o t e i n c a p a b l e o f h y d r o x y l a t i n g an a l k a n e a t an u n a c t i v a t e d C-H bond, t h e e l e c t r o p h i - l i c i r o n - o x e n o i d s p e c i e s and i t s r e a c t i o n w i t h s u b s t r a t e a r e e s p e c i a l l y i n t e r e s t i n g . I f t h e c l e a v a g e of a C-H bond i s a n e c e s s a r y s t e p i n t h e h y d r o x y l a t i o n r e a c t i o n , t h e n a s u b s t r a t e f r e e - r a d i c a l w o uld be t h e l o w e s t e n e r g y i n t e r m e d i a t e t h a t c o u l d e x i s t i n t h e s t r o n g l y h y d r o p h o b i c a c t i v e s i t e ; any i o n i c s p e c i e s w ould be e n e r g e t i c a l l y u n f a v o u r a b l e . I n d e e d , i s o t o p e e f f e c t s , and r e g i o - and s t e r e o - s e l e c t i v i t y o f t h e s e r e a c t i o n s , a l l p o i n t t o w a r d s s u c h a m e c h a n i s m . 2 7 - 7 1 ' 7 2 Two e l e g a n t e x amples, t h a t a c c o u n t f o r t h e e x i s t e n c e of an 29 u n c h a r g e d s u b s t r a t e i n t e r m e d i a t e have been d i s c u s s e d by G r o v e s e t a l . 7 3 and D o l p h i n e t a l . 7 4 A p l a u s i b l e mechanism of f o r m a t i o n and h y d r o x y l a t i o n of t h e s u b s t r a t e r a d i c a l i s o u t l i n e d i n F i g u r e I I . 8 . 7 1 > R : c r , H < > V Z H • 9 : ROH + Fe(lll)P F«(lW)P Figure II.8 Proposed mechanism f o r the formation and hydroxylation of substrate (RH) r a d i c a l . The a b s t r a c t i o n o f a h y d r o g e n atom from s u b s t r a t e RH by t h e a c t i v e i r o n - o x e n e complex forms an F e ( I V ) h y d r o x i d e s p e c i e s and a s u b s t r a t e f r e e - r a d i c a l , R*; t h i s i n t u r n a c c e p t s t h e i r o n - b o u n d h y d r o x y r a d i c a l t o f o r m R-OH and r e g e n e r a t e s F e ( l I I ) enzyme r e a d y f o r t h e n e x t r e a c t i o n c y c l e . 11.4.2.7 D i s s o c i a t i o n of P r o d u c t The h y d r o x y l a t e d p r o d u c t a p p a r e n t l y d i s s o c i a t e s b e f o r e a new c y c l e b e g i n s , s i n c e d i o l s a r e n o t o b s e r v e d i n a l k a n e h y d r o x y l a t i o n s . The h y d r o x y l g r o u p of t h e p r o d u c t a l c o h o l may be c o o r d i n a t e d t o i r o n , b ut t h i s w ould make t h e r e d u c t i o n of t h e i r o n ( I I I ) c e n t e r more d i f f i c u l t and a l s o , even i f t h e i r o n i s r e d u c e d , would p r o h i b i t 0 2 b i n d i n g . • « Fe(llf )p+* A 30 T h e r m o d y n a m i c a l l y , t h e b i n d i n g of a p o l a r m o l e c u l e w i l l be l e s s f a v o u r a b l e and a n o t h e r s u b s t r a t e m o l e c u l e t h a t i s i n abundance i n t h e s u r r o u n d i n g s w i l l be p r e f e r r e d ; t h e h i g h s p i n F e ( 1 1 1 ) - s u b s t r a t e complex formed y i e l d s t h e way f o r a n o t h e r r e a c t i o n c y c l e . 31 11.5 E l e c t r o n i c S p e c t r o s c o p y E l e c t r o n i c a b s o r p t i o n s p e c t r o s c o p y has been u s e d e x t e n s i v e l y t o s t u d y t h e c y t o c h r o m e P-450 s y s t e m i n r e l a t i o n t o m o l e c u l a r s t r u c t u r e , geometry, e l e c t r o n i c c o n f i g u r a t i o n and o x i d a t i o n s t a t e . O t h e r s p e c t r o s c o p i c t e c h n i q u e s s u c h as e l e c t r o n p a r a m a g n e t i c r e s o n a n c e (EPR), M o s s b a u e r , c i r c u l a r d i c h r o i s m (CD), 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), n u c l e a r m a g n e t i c r e s o n a n c e (NMR) and r e s o n a n c e Raman (RR) have been u s e d t o c o n f i r m u n a m b i g u o u s l y t h e r e s u l t s o b t a i n e d from e l e c t r o n i c s p e c t r o s c o p y . A l s o , heavy r e l i a n c e has been p l a c e d on c o m p a r i s o n s w i t h o t h e r known h e m o p r o t e i n s and low m o l e c u l a r w e i g h t i r o n - p o r p h y r i n c o m p l e x e s . The P-450 h e m o p r o t e i n s from a l l known s o u r c e s a r e r e m a r k a b l y s i m i l a r i n e l e c t r o n i c a b s o r p t i o n s p e c t r a l p r o p e r t i e s . 2 7 The s u b s t r a t e - f r e e , f e r r i c ( " r e s t i n g " ) s t a t e of t h e enzyme i s t y p i c a l of low s p i n f e r r i c h e m e p r o t e i n s , w i t h a S o r e t band a t 416 nm and w e l l d e f i n e d a and /3 b a n d s . Upon s u b s t r a t e b i n d i n g , t h e h e x a c o o r d i n a t e low s p i n f o r m c h a n g e s t o p e n t a c o o r d i n a t e h i g h s p i n f o r m w i t h a s h i f t o f t h e S o r e t band t o 391 nm and d i s a p p e a r a n c e o f d i s t i n c t bands i n 500-600 nm r e g i o n ; however, a new low e n e r g y , l o w i n t e n s i t y band a t 643 nm i s f o r m e d . On r e d u c i n g t h e s u b s t r a t e - f r e e s p e c i e s i n t h e a b s e n c e o f p o t e n t i a l l i g a n d s s u c h as CO o r 0 2 , t h e S o r e t band moves t o 408 nm and t h e a and 0 bands c o l l a p s e f o r m i n g a new peak a t 542 nm. However, t h e most s t r i k i n g f e a t u r e i s o b s e r v e d when t h e r e d u c e d 32 enzyme i s e x p o s e d t o a CO a t m o s p h e r e when a s h a r p , i n t e n s e peak a p p e a r s a t 446 nm, an u n u s u a l l y h i g h w a v e l e n g t h f o r a h e m o p r o t e i n . The low e n e r g y band of t h e CO complex i s o b s e r v e d a t 540 nm w i t h about th e same i n t e n s i t y as t h e peak of t h e r e d u c e d s p e c i e s a t 542 nm. The s p e c t r u m of t h e d i o x y g e n complex i s c o m p r i s e d of a S o r e t band a t 418 nm and a low e n e r g y band a t 552 nm. The s m a l l s e p a r a t i o n between S o r e t p e a k s of t h e r e d u c e d (408 nm) and 0 2 - c o m p l e x (418 nm) c r e a t e d d i f f i c u l t i e s i n s t u d y i n g t h e d i o x y g e n b i n d i n g as a f u n c t i o n of gas p r e s s u r e i n e f f o r t s t o d e t e r m i n e t h e e q u i l i b r i u m c o n s t a n t (see C h a p t e r IV). Normal p o r p h y r i n s p e c t r a show t h r e e s e t s of bands c l a s s i f i e d a c c o r d i n g t o o r b i t a l symmetry c o n s i d e r a t i o n s . The bands a p p e a r i n t h e v i s i b l e , S o r e t and n e a r UV r e g i o n s . " 8 T h e s e p e a k s a r e due t o t h e w e l l c h a r a c t e r i z e d 7 r - 7 r * t r a n s i t i o n s . But some p o r p h y r i n - m e t a l c o m p l e x e s show " h y p e r " s p e c t r a , w i t h s t r o n g e x t r a bands i n t h e 300-800 nm r e g i o n , a' common s p e c t r a l p a t t e r n e x i b i t e d by a number o f h y p e r p o r p h y r i n s b e a r i n g two S o r e t bands ( s p l i t S o r e t ) , one i n t h e 380 nm r e g i o n and t h e o t h e r i n t h e 440-480 nm r e g i o n . The CO and 0 2 c o m p l e x e s o f r e d u c e d P-450 s p e c i e s a l s o e x i b i t t h i s f e a t u r e . As d e m o n s t r a t e d by t h e model s t u d i e s " 5 - " 7 t h e p r e s e n c e of t h i o l a t e i n an 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 i s r e q u i r e d t o mimic t h e P-450 s p e c t r u m , i n c l u d i n g t h e s p l i t S o r e t b a n d s . Hansen e t a l . " B u s i n g i t e r a t i v e e x t e n d e d H u c k e l (IEH) c a l c u l a t i o n s were a b l e t o show t h a t t h e s p l i t S o r e t bands a r i s e from t h e 33 s t r o n g i n t e r a c t i o n of c h a r g e t r a n s f e r t r a n s i t i o n s ( f r o m t h i o l a t e p- o r b i t a l s t o p o r p h y r i n 7 r * - o r b i t a l s ) w i t h t h e p o r p h y r i n TT-VT* t r a n s i t i o n s . 34 11.6 I n t e r a c t i o n of C y t o c h r o m e P-450 w i t h C a r b o n Monoxide and D i o x y g e n The o b s e r v a t i o n t h a t t h e a d d i t i o n of c a r b o n monoxide t o sodium d i t h i o n i t e - t r e a t e d l i v e r m i c r o s o m e s r e s u l t e d i n arv u n u s u a l S o r e t peak l e d t o t h e d i s c o v e r y of c y t o c h r o m e P-450 (see S e c t i o n I I . 1) . A l s o , t h e b i o l o g i c a l f u n c t i o n o f t h i s monooxygenase enzyme was d e t e r m i n e d by a s t u d y of i n h i b i t i o n of C - 2 1 - h y d r o x y l a s e r e a c t i o n of a d r e n a l c o r t e x m i c r o s o m e s v i a p h o t o c h e m i c a l a c t i o n s p e c t r u m o f t h e CO c o m p l e x . 3 S i n c e t h e s e f i n d i n g s , many s t u d i e s on CO and 0 2 b i n d i n g t o r e d u c e d , n a t i v e and r e c o n s t i t u t e d P-450 enzyme and t o i t s model compounds have been r e p o r t e d . 6 1 - 7 5 - 8 0 The i n f o r m a t i o n g a t h e r e d f r o m s u c h s t u d i e s w i l l be d i s c u s s e d and compared t o t h e o t h e r h e m e - c o n t a i n i n g b i o m o l e c u l e s s u c h as h e m o g l o b i n (Hb), and m y o g l o b i n (Mb), w h i c h have been t h o r o u g h l y s t u d i e d and a r e w e l l u n d e r s t o o d . A l t h o u g h Hb and Mb c o n t a i n t h e same p r o s t h e t i c g r o u p as P-450, p r o t o h e m i n , t h e y d i f f e r i n many ways i n t h e i r s t r u c t u r e and p r o p e r t i e s . The d i f f e r e n c e i n a x i a l l i g a t i o n ( s e e F i g u r e I I . 9 ) must p l a y an i m p o r t a n t r o l e . 8 1 I t i s i n t e r e s t i n g t o n o t e t h a t Hb and Mb do n o t u n d e r g o a change i n t h e o x i d a t i o n s t a t e o f t h e p r o s t h e t i c g r o u p s i n any s t a g e of t h e i r n o r m a l b i o l o g i c a l f u n c t i o n s . In t h e c a s e of P-450 and Mb, where o n l y one heme u n i t p e r m o l e c u l e i n t e r a c t s , t h e r e a c t i o n of CO w i t h t h e r e d u c e d heme u n i t i s r e p r e s e n t e d by r e a c t i o n I I . 2 , F i g u r e I I . 9 Heme environments of myoglobin ( A ) , hemoglobin (B), and p e r o x i d a s e (C). (from r e f . 8 1 ) . 36 F e ( I I ) + C O ^ ^ F e ( I I ) - C O , K c o = 1 o n / ' b f f < J I « 2 ) where F e ( I I ) i s t h e r e d u c e d heme u n i t , K c o i s t h e e q u i l i b r i u m c o n s t a n t , and k Q n and k D f f a r e a s s o c i a t i o n and d i s s o c i a t i o n r a t e c o n s t a n t s , r e s p e c t i v e l y . The l i g a n d a s s o c i a t i o n r e a c t i o n i s an o v e r a l l s e c o n d o r d e r p r o c e s s , w h i l e t h e d i s s o c i a t i o n r e a c t i o n i s f i r s t o r d e r . 8 2 . 8 3 The k i n e t i c and e q u i l i b r i u m d a t a f o r CO b i n d i n g t o P-450 and r e l a t e d s y s t e m s a r e g i v e n i n T a b l e s I I . 1 and I I . 2 , r e s p e c t i v e l y . 37 T a b l e I I . 1 K i n e t i c D a t a f o r CO B i n d i n g t o Cytochrome P-450 and R e l a t e d S y s t e m s . System Method PH T°C k o n (M-'s- 1 ) k o f f ( s - 1 ) Ref . P-450cam SF 7.4 4 5. 1x10 s 2.3 77 ( c a m . - f r e e ) FP 7.4 5 2.7x10 s 1 .2 82 1 5 4.7x10 6 25 8.4x10 6 P-450cam SF 7.4 4 3.8x10" 77 (cam.-bound) SF 7.0 20 3.6x10 s 2.8 84 SF 7.0 24 2.2x10 s 1 .7 75 FP 7.0 5 8x1 0" 82 15 1.3x10 5 25 2.3x10 5 FP 7.3 12 3.5x10" 85 M i c r o s o m a l FP 7.4 4 3.4x10 5 0.068 13 P-450 SF 7.5 4 4.5x10 s 0.63 88 H o r s e Mb FP 7.0 20 5x1 0 5 0.02 83 FP 7.0 25 3.8x10 s 86 P-450 M o d e l FP 23 1 . 1x10 s 18 87 S F = s t o p p e d - f l o w , F P = f l a s h p h o t o l y s i s 38 T a b l e I I . 2 E q u i l i b r i u m D a t a f o r CO B i n d i n g t o C y t o c h r o m e P-450 and R e l a t e d S y s t e m s . System PH T°C K C 0 ( M - 1 ) A H 0 ( k c a l /mol) As 0 ( c a l /mol .deg) R e f . P-450cam 7.4 4 2.2x10 6 77 ( c a m . - f r e e ) 7.0 24 1 .7X10 5 75 P-450cam 7.4 4 2.6x10 5 77 (cam.-bound) 7.0 12 2.6x10 s -12 -17 75 7.0 24 1 . 3 x 1 0 5 75 P-450 Model 23 1.1x10" -16 -35 87 H o r s e Mb 7.0 20 2.9x10 7 -12.6 -7.4 83 39 The CO and 0 2 c o m p l e x e s of P-450, Hb and Mb a r e d i a m a g n e t i c . 8 9 The m e t a l - c a r b o n b o n d - o r d e r d e r i v e d f r o m t h e r e l a t i o n s h i p w i t h t h e C-0 s t r e t c h i n g f r e q u e n c y i n d i c a t e s t h a t c a r b o n monoxide i s i n v o l v e d i n b o t h a and u — t y p e m o l e c u l a r o r b i t a l s of t h e c o m p l e x . 8 3 The s p - h y b r i d i z e d l o n e p a i r of t h e c a r b o n atom i s i n v o l v e d i n 0=C—> Fe t y p e x b o n d i n g , w h i l e t h e two pit o r b i t a l s o v e r l a p w i t h two dir o r b i t a l s of i r o n . The P a u l i n g v a l e n c e bond s t r u c t u r e s of t h e CO complex a r e g i v e n i n F i g u r e 11.10. N N N N . \ / -\/ P r o t e i n F e « — C s Q : P r o t e i n F e = C = 0 N N N N I H Figure 11.10 Pauling valence bond s t r u c t u r e s of carbon monoxide complex of cytochrome P-450. A l t h o u g h CO i s t h o u g h t t o b i n d i n a l i n e a r f a s h i o n t o t r a n s i t i o n m e t a l s , i t c a n be a t t a c h e d a t an a n g l e i n a heme p r o t e i n t o m i n i m i z e t h e s t e r i c h i n d r a n c e . In t h e c h i r o n o m o u s h e m o g l o b i n CO complex t h e Fe-C-0 a n g l e i s 1 4 5 ± 1 5 0 . 8 3 T h i s o b s e r v a t i o n p r o v i d e s a b a s i s f o r t h e e x p l a n a t i o n o f t h e l o w e r a f f i n i t y of s u b s t r a t e - b o u n d P-450 t o w a r d s CO, compared t o t h e s u b s t r a t e - f r e e s y s t e m (see T a b l e I I . 2 ) . The camphor m o l e c u l e bound i n t h e v i c i n i t y of t h e heme m o i e t y 7 7 i s c o n s i d e r e d n o t o n l y t o r e s t r i c t t h e a c c e s s o f t h e CO m o l e c u l e t o t h e heme u n i t , b u t a l s o t o have a d e s t a b i l i z i n g e f f e c t on t h e heme-CO complex once i t i s f o r m e d . However, a t 2 4 ° , t h e e q u i l i b r i u m c o n s t a n t v a l u e f o r t h e s u b s t r a t e - f r e e s y s t e m , r e p o r t e d by D o l p h i n e t a l 7 5 , 40 a p p r o a c h e s t h a t f o r t h e s u b s t r a t e - b o u n d s y s t e m . In o r d e r t o u n d e r s t a n d b e t t e r t h e e f f e c t of t h e s u b s t r a t e on CO b i n d i n g t o P-450, t h e n e c e s s i t y o f more s t u d i e s has been m e n t i o n e d . 8 7 ' 9 0 More d e t a i l e d i n t e r p r e t a t i o n of t h e s u b s t r a t e e f f e c t on CO b i n d i n g can be p r o v i d e d by e v a l u a t i o n of t h e e n t h a l p y and e n t r o p y c o n t r i b u t i o n s t o t h e b i n d i n g c o n s t a n t v a l u e s a t d i f f e r e n t t e m p e r a t u r e s . The b i n d i n g of CO t o s u b s t r a t e - b o u n d P-450 i s a b o u t 200 t i m e s weaker t h a n t h a t f o r Mb ( s e e T a b l e I I . 2 ) . T h i s d i f f e r e n c e i n a f f i n i t y t o w a r d s l i g a n d b i n d i n g p r o b a b l y r e f l e c t s t h e e f f e c t of t h e p r o x i m a l l i g a n d , i m i d a z o l e i n Mb, and t h i o l a t e i n P-450. The t h i o l a t e a n i o n c o o r d i n a t e d t o heme may p r o d u c e an e l e c t r o n - r i c h i r o n atom, t h e r e b y l o w e r i n g t h e a f f i n i t y f o r t h e s i x t h l i g a n d . 9 1 S i n c e i m i d a z o l e i s a weaker a-donor t h a n t h i o l a t e , t h i s c o u l d 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 b i n d i n g c o n s t a n t s . The thermodynamic p a r a m e t e r s , c a l c u l a t e d from t h e . l i m i t e d d a t a a v a i l a b l e 7 5 f o r t h e s u b s t r a t e - b o u n d s y s t e m ( T a b l e I I . 2 ) , i n d i c a t e t h a t t h e AH 0 v a l u e i s c o m p a r a b l e i n m a g n i t u d e t o t h a t of Mb (-12 k c a l / m o l f o r P-450; -12.6 k c a l / m o l f o r Mb). B i n d i n g of a gas m o l e c u l e t o a m e t a l c e n t r e would be u n f a v o u r a b l e i n e n t r o p y t e r m s due t o t h e l o s s of t r a n s l a t i o n a l and r o t a t i o n a l m o t i o n o f t h e gas m o l e c u l e . But t h e e n t r o p y of b i n d i n g of CO t o P-450 i s more u n f a v o u r a b l e (AS=-17 c a l / m o l . d e g . ) t h a n f o r Mb; (AS°=-7.4 c a l / m o l . d e g . ) . Such d i f f e r e n c e s i n e n t r o p y c o n t r i b u t i o n s have been r a t i o n a l i z e d , b a s e d on c i s - and t r a n s - e f f e c t s 41 and s t e r i c e f f e c t s due t o t h e p r e s e n c e o f p r o t e i n as w e l l as t h e s u b s t r a t e . 7 5 - 7 7 The AH 0 t e r m f o r t h e p r o t e i n f r e e model s y s t e m (-16 k c a l / m o l ) i s more f a v o u r a b l e , but t h e e n t r o p y c o n t r i b u t i o n i s not so (-35 c a l / m o l . d e g . ) . The k i n e t i c and e q u i l i b r i u m d a t a a v a i l a b l e t o d a t e f o r d i o x y g e n b i n d i n g t o c y t o c h r o m e P-450 a r e n o t as e x t e n s i v e as t h o s e f o r CO b i n d i n g t o P-450, o r 0 2 b i n d i n g t o Mb. The p r i n c i p a l r e a s o n f o r t h i s i s t h e i n s t a b i l i t y o f t h e P-450- 0 2 complex a t a m b ient t e m p e r a t u r e s ; i n t h e c a s e of s u b s t r a t e - b o u n d enzyme a t room t e m p e r a t u r e and pH 7.0 t h e a u t o x i d a t i o n ( R e a c t i o n I I . 3 ) o c c u r s w i t h a h a l f - l i f e < 1 m i n u t e , 9 2 w h i l e t h e r a t e o f a u t o x i d a t i o n o f t h e d i o x y g e n complex of t h e s u b s t r a t e - f r e e enzyme i s a t l e a s t 100 t i m e s f a s t e r . 7 5 P - 4 5 0 F e ( l I ) - 0 2 + H+ > P - 4 5 0 F e ( I I I ) + H0 2 ( I I . 3 ) The b i n d i n g of d i o x y g e n t o s u b s t r a t e bound P-450 i s a t l e a s t 6 t i m e s weaker t h a n t o Mb ( T a b l e I I . 3 ) . T h i s r e s u l t s m a i n l y f r om a much s l o w e r o n - r a t e i n t h e P-450 s y s t e m ; t h i s c o u l d r e s u l t f r o m a g e o m e t r i c a l l y r e s t r i c t e d c o o r d i n a t i o n s i t e due t o t h e p r e s e n c e of t h e s u b s t r a t e , 7 5 a l t h o u g h t h e e l e c t r o n i c e f f e c t s due t o t h e s i x t h l i g a n d may a l s o p l a y a r o l e . 42 T a b l e I I . 3 E q u i l i b r i u m and K i n e t i c D a t a f o r R e v e r s i b l e 0 2 B i n d i n g t o S u b s t r a t e - B o u n d Cytochrome P - 4 5 0 7 5 and Mb. 9 3 System P 1/2 (mmHg) B i n d i n g C o n s t a n t (M- 1) ^on (M- 1s-M k o f f ( S - 1 ) P-450 H o r s e Mb 2.5 (0°C) •52(20°C) 2.2x10 5 (0°C) 1.3x10 s (20°C) 7.7x10 s (4°C) 1 .4x 1 0 7 (20°C) 3.5 (4°C) 10. (20°C) 4 3 REFERENCES 1 . M . K l i n g e n b e r g , A r c h . Biochem. B i o p h y s . , 7J5 , 3 7 6 ( 1 9 5 8 ) . 2 . T.Omura, R.Sato, J . B i o l . C h e m , 2 3 7 , 1 3 7 5 ( 1 9 6 2 ) . 3 . 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P e t e r s o n , R.W.Estabrook, The P o r p h y r i n s , ( E d . D . D o l p h i n ) , V I I , Aca d e m i c P r e s s , New Y o r k , 1979, p.333. 85. C . B o n f i l s , J . L . S a l d a n a , P.Debey, P . M a u r e l , C . B a l n e y , P.Dousou, B i o c h i m i e , 6±, , 6 8 1 ( 1 9 7 9 ) . 86. B . B . H a s i n o f f , B i o c h e m i s t r y , J_3, 3111 ( 1 9 7 4 ) . 87. C.K.Chang, D . D o l p h i n , P r o c . N a t l . A c a d . S c i . , U.S.A., 73, 3338(1976) 88. P.Debey, G.Hui Bon Hoa, P.Dousou, FEBS L e t t . , 32, 227(1973) 89. L . P a u l i n g , C . D . C o r r y e l l , P r o c . N a t l . A c a d . S c i . , U.S.A., 22, 1 5 9 ( 1 9 3 6 ) . 90. S . A l b o n , M . S c . T h e s i s , D e p t . C h e m i s t r y , U.B.C., ( I 9 8 3 ) p p . 7 4 , 79. 91. D . D o l p h i n , B.R.James, C.H.Welborn, R e f . 25, p.183. 92. S . G . S l i g a r , J . D . L i p s c o m b , P.G.Debruner, I . C . G u n s a l u s , Biochem. B i o p h y s .Res .Commun. , 6J_, 290( 1974). 48 B.R.James, The P o r p h y r i n s , ( E d . D . D o l p h i n ) , Academic P r e s s , New Y o r k , V, 1978, p.205. CHAPTER I I I EXPERIMENTAL PROCEDURES 50 111,1 Growth o f B a c t e r i a 111.1 . 1 G e n e r a l I n f o r m a t i o n The b a c t e r i a Pseudomonas p u t i d a s t r a i n PpG 786 (ATTC 29607) d e r i v e d f r o m s t r a i n PpG 1 (ATTC 17453) by G u n s a l u s and Wagner 1 was u s e d t h r o u g h o u t t h e s e s t u d i e s t o p r o d u c e c y t o c h r o m e P-450. The p a r e n t s t r a i n PpG 1 was o r i g i n a l l y i s o l a t e d f r o m s o i l by e n r i c h m e n t on D-(+)-camphor. The s t r a i n was k e p t a l i v e by b i - w e e k l y t r a n s f e r t o m i n i m a l a g a r p l a t e s c o n t a i n i n g D-(+)-camphor as c a r b o n and e n e r g y s o u r c e . The f o u r - s t a g e g r o w th p r o c e d u r e f o r t h e mass p r o d u c t i o n of b a c t e r i a as d e s c r i b e d by G u n s a l u s and Wagner 1 was u s e d w i t h s e v e r a l m i n o r m o d i f i c a t i o n s . T h i s g r o w t h p r o c e d u r e i s d e s i g n e d t o p r o d u c e maximum amounts of c y t o c h r o m e P-450 and o t h e r monooxygenase components a t t h e e x p e n s e of camphor and l i q u i d media and has a r e l a t i v e l y s h o r t p e r i o d of g r o w th w i t h a g e n e r a t i o n t i m e of a b o u t t h r e e hour's. The s t r a i n PpG 786 has t h e a b i l i t y t o r e l e a s e much of t h e h y d r o x y l a s e p r o t e i n s by f r e e z e - t h a w a u t o l y s i s t o c i r c u m v e n t more t e d i o u s c e l l b r e a k a g e t e c h n i q u e s . A s i n g l e f r e e z e - t h a w c y c l e t y p i c a l l y l i b e r a t e s a b o u t one h a l f of t h e c y t o c h r o m e P-450 c o n t e n t as s o l u b l e enzyme i n t h e c e l l - f r e e e x t r a c t , b a s e d on t h e t o t a l amount of enzyme i n t h e whole c e l l s u s p e n s i o n as d e t e r m i n e d f r o m t h e f e r r o u s c a r b o n monoxide d i f f e r e n c e s p e c t r u m . The f o u r s t a g e g r o w th p r o c e d u r e , 1. M i n i m a l a g a r s t a g e 51 2. L - B r o t h s t a g e 3. 500 mL Shake f l a s k s t a g e 4. 14 L F e r m e n t e r s t a g e t y p i c a l l y y i e l d e d a b o u t 500 g of b a c t e r i a b a s e d on t h e wet w e i g h t i n a b o u t 96 h o u r s o f e l a p s e d t i m e . A l l i n o c u l a t i o n s e x c e p t f o r 14 L f e r m e n t e r s were c a r r i e d o u t i n a l a m i n a r f l o w c l e a n - a i r s t a t i o n t o m i n i m i z e t h e p o s s i b i l i t y o f c o n t a m i n a t i o n . T h r e e a g a r p l a t e s were i n o c u l a t e d e a c h t i m e and t h e w i r e l o o p u s e d t o t r a n s f e r t h e b a c t e r i a between p l a t e s was h e a t e d u n t i l r e d h o t b e f o r e e a c h use, i n t h e f l a m e of a Bunsen b u r n e r . The L - b r o t h and t h e shake f l a s k s were i n o c u l a t e d and i n c u b a t e d i n a p l a t f o r m s h a k e r t h e r m o s t a t e d t o 3 0 ° . T h r e e 14 L f e r m e n t e r s ( L a b r o f e r m ; New B r u n s w i c k S c i e n t i f i c , N .J, U.S.A) were us e d i n t h e f i n a l s t a g e of g r o w t h . The g r o w th medium was made up t o 12 L and s t i r r e d a t 300 rpm w i t h 15 L/min r a t e o f a e r a t i o n . The t e m p e r a t u r e was m a i n t a i n e d a t 3 0 ° . The r a t e of g r o w t h o f b a c t e r i a was d e t e r m i n e d t u r b i d o m e t r i c a l l y by m e a s u r i n g t h e o p t i c a l d e n s i t y a t 660 nm u s i n g a B a u s c h and Lomb s p e c t r o m e t e r model S p e c t r o n i c 20. D u r i n g t h e g r o w t h o f b a c t e r i a i n 500 mL shake f l a s k and 14 L f e r m e n t e r s t a g e s , t h e f l u c t u a t i o n o f pH was m o n i t o r e d u s i n g a F i s h e r Accumet model 210 pH-meter e q u i p p e d w i t h a g l a s s p H - e l e c t r o d e . When th e o p t i c a l d e n s i t y of t h e medium r e a c h e d a maximum v a l u e , t h e b a c t e r i a were c o l l e c t e d u s i n g a c o n t i n u o u s f l o w c e n t r i f u g e ( C a r l P a d b e r g , S c h n e l l - Z e n t r i f u g e , M odel LE) a t 37, 000 rpm. The b a c t e r i a were t h e n s t o r e d under d r y - i c e 52 t e m p e r a t u r e i n a Dewar i n 500 g b a t c h e s . I l l . 1 .2 M e d i a M e d i a f o r a l l f o u r s t a g e s were p r e p a r e d a c c o r d i n g t o G u n s a l u s and Wagner 1 u s i n g r e a g e n t g r a d e c h e m i c a l s and f r e s h l y d i s t i l l e d w a t e r . A l l s o l u t i o n s were s t e r i l i z e d a t 121° under p r e s s u r i z e d steam u s i n g an a u t o c l a v e ( A m e r i c a n S t e r i l i z e r Co. Model AS-DTT616GE). S o l u t i o n s o f volumes l e s s t h a n 200 mL r e q u i r e d 20 min a u t o c l a v i n g w h i l e 14 L f e r m e n t e r s r e q u i r e d 45 min t o e n s u r e c o m p l e t e s t e r i l i z a t i o n . The s t e r i l i z e d s o l u t i o n s were u s e d as e a r l y as p o s s i b l e t o a v o i d r e - c o n t a m i n a t i o n . 111 . 1 . 2.1 M i n i m a l Agar In o r d e r t o s t o r e t h e b a c t e r i a a l i v e , m i n i m a l a g a r p l a t e s were p r e p a r e d u s i n g t h e f o l l o w i n g i n g r e d i e n t s . K 2HPO a 3.5-g K H 2 P O « 1.0 g N a C l 0.06 g M g S O « . 7 H 2 0 0.05 g ( N H a ) 2 S O „ 0.5 g D-( + )-camphor 0.68 g B a c t o a g a r 7.5 g Water 540 mL The s a l t s , camphor and a g a r were d i s s o l v e d i n w a t e r , s t e r i l i z e d , c o o l e d , combined and p o u r e d i n t o d i s p o s a b l e p e t r i d i s h e s o b t a i n e d from C a n l a b . The m i n i m a l a g a r d i s h e s 53 were s t o r e d a t 5 ° , t h r e e of w h i c h were u s e d e v e r y two weeks t o i n o c u l a t e w i t h t h e b a c t e r i a . 54 I I I . 1 . 2 . 2 L - B r o t h A t t h e i n i t i a l s t a g e o f l a r g e s c a l e g r o w t h of b a c t e r i a , a v e r y r i c h medium c a l l e d L - b r o t h 2 was u s e d t o f a c i l i t a t e t h e r a p i d m u l t i p l i c a t i o n o f t h e o r g a n i s m . The f o l l o w i n g i n g r e d i e n t s were mixed, p o u r e d i n t o f i v e 50 mL E r l e n m e y e r f l a s k s and s t e r i l i z e d . B a c t o t r i p t o n e 1.0 g Y e a s t e x t r a c t 0.5 g N a C l 0.5 g G l u c o s e 0.1 g Water 1 00 mL T h i s s o l u t i o n was b r o u g h t t o pH 7.0 by a d d i n g 1M NaOH d r o p w i s e b e f o r e t h e s t e r i l i z a t i o n . I l l . 1 .2.3. 500 mL Shake F l a s k At t h i s s t a g e of g r o w t h a phosphate-ammonium (PA) b u f f e r s o l u t i o n was u s e d t o m a i n t a i n t h e d e s i r e d pH l e v e l . E s s e n t i a l m i n e r a l s a l t s were s u p p l i e d f r o m a h u n d r e d - f o l d s t r o n g s t o c k s o l u t i o n (100-X s a l t s ) and a c a r b o n and e n e r g y s o u r c e was a l s o a d d e d t o t h e medium. In o r d e r t o m a x i m i z e t h e p r o d u c t i o n o f c y t o c h r o m e P-450 c o n t e n t , t h e 500 mL shake f l a s k s t a g e was p e r f o r m e d t w i c e , f i r s t w i t h g l u t a m i c a c i d as t h e c a r b o n and e n e r g y s o u r c e and s e c o n d l y w i t h camphor as t h e o n l y c a r b o n and e n e r g y s o u r c e . In e a c h c a s e 1 L o f medium was p r e p a r e d u s i n g t h e f o l l o w i n g i n g r e d i e n t s and d i v i d e d i n t o f i v e 500 mL E r l e n m e y e r f l a s k s and 55 s t e r i l i z e d . PA b u f f e r 1 L 100-X s a l t s 16 mL G l u t a m a t e 2.9 g o r Camphor 2.5 g PA b u f f e r and 100-X s a l t s were p r e p a r e d by m i x i n g f o l l o w i n g i n g r e d i e n t s . PA b u f f e r : K 2HPO, 10.7 g K H 2 P O « 3.1 g N H « C 1 4.0 g Water 1 L 100-X s a l t s : MgSO,.7H 20 19.5 g MnSO,.H 20 5.0 g F e S O « . 7 H 2 0 5.0 g C a C l 2 . 2 H 2 0 0.3 g L - A s c o r b i c a c i d 1.0 g Water 1 L 56 I I I . 1.2.4 14 L F e r m e n t e r The f i n a l s t a g e o f growth was c a r r i e d o u t i n a somewhat s i m i l a r medium t o t h e 500 mL shake f l a s k s t a g e , but i t was e s s e n t i a l t o keep t h e medium a l w a y s s a t u r a t e d w i t h camphor d u r i n g t h e whole p e r i o d of g r o w t h . The f o l l o w i n g c h e m i c a l s were added i n e a c h f e r m e n t e r . K 2HPO f l 128.2 g K H 2 P 0 „ 36.3 g NH,C1 46.8 g 100-X s a l t s 120 mL B a c t o y e a s t e x t r a c t 3.0 g A n t i f o a m 1 .0 mL Camphor i n DMF (3M) 30 mL Water 1 1 .6 L 57 111.1.3 Growth P r o c e d u r e F o u r of t h e f i v e E r l e n m e y e r f l a s k s , e a c h c o n t a i n i n g 20 mL o f L - b r o t h medium, were i n o c u l a t e d w i t h t h e b a c t e r i a and shaken a t 30° f o r 10 h o u r s . The f i f t h f l a s k s e r v e d as a r e f e r e n c e . At t h e end of t h e 10 hour p e r i o d i t was n o t e d t h a t t h e f l a s k s t h a t were i n o c u l a t e d t u r n e d t u r b i d , w h i l e t h e r e f e r e n c e f l a s k r e m a i n e d c l e a r . On s w i r l i n g t h e i n o c u l a t e d f l a s k s , c l o u d s o f b a c t e r i a c o u l d be s e e n . The t h i r d s t a g e of growth was s t a r t e d by i n o c u l a t i n g e a c h of t h e f i r s t s e t of f o u r 500 mL shake f l a s k s w i t h 5 mL of L - b r o t h c u l t u r e . The f i f t h f l a s k s e r v e d a s a r e f e r e n c e . E a c h of t h e s e f l a s k s c o n t a i n e d 200 mL o f b a s a l PA b u f f e r , 3.2 mL of 100-X s a l t s and 10 mM g l u t a m a t e a s t h e c a r b o n and e n e r g y s o u r c e . A l l f i v e f l a s k s were shaken a t 30° u n t i l t h e o p t i c a l d e n s i t y a t 660 nm ( O D 6 6 0 ) r e a c h e d 0.3. At t h i s t i m e th e i n d u c t i o n of camphor 5-monooxygenase s y s t e m was i n i t i a t e d by t h e a d d i t i o n o f s t o c k camphor s o l u t i o n (3M i n DMF) t o a f i n a l c o n c e n t r a t i o n of 5 mM. The O D 6 6 o a n d t h e pH of one o f t h e i n o c u l a t e d f l a s k s and t h e r e f e r e n c e f l a s k were t a k e n e v e r y hour i n o r d e r t o m o n i t o r t h e g r o w t h and th e m e t a b o l i s m of camphor. A d d i t i o n of camphor was c o n t i n u e d a t O D 6 6 0 0.7, 1.0 and once e v e r y t h r e e h o u r s t h e r e u p o n u n t i l t h e growth r e a c h e s i t s l a t e - l o g r i t h m i c p h ase (see F i g u r e I I I . l ) . At t h i s t i m e t h e s e c o n d s e t .of shake f l a s k s w i t h camphor as t h e o n l y c a r b o n and e n e r g y s o u r c e was i n o c u l a t e d w i t h 10 mL e a c h o f t h i s s o l u t i o n . F o r t h i s i n o c u l a t i o n t h e c u l t u r e medium was u s e d o n l y from t h e 58 59 t h r e e shake f l a s k s t h a t were l e f t u n d i s t u r b e d w h i l e s h a k i n g a t 3 0 ° . The f l a s k t h a t was u s e d t o w i t h d r a w a l i q u o t s f o r pH and o p t i c a l d e n s i t y measurements was n e v e r u s e d as an i n o c u l u m , i n o r d e r t o a v o i d any p o s s i b i l i t y of c o n t a m i n a t i o n . The f l a s k s were h e a t e d a r o u n d t h e neck b e f o r e o p e n i n g , by r o t a t i n g on t h e f l a m e of a Bunsen b u r n e r whenever t h e y were opened e i t h e r f o r a d d i t i o n o f camphor or f o r w i t h d r a w i n g a l i q u o t s . The n e e d l e of t h e 1 mL d i s p o s a b l e s y r i n g e t h a t was u s e d t o measure t h e camphor s o l u t i o n was h e a t e d t o r e d h o t b e f o r e u s e . P r e - s t e r i l i z e d 2 mL P a s t u r e p i p e t t e s were us e d t o w i t h d r a w a l i q u o t s from t h e f l a s k s . The i n i t i a l r a t e of g r o w t h i n t h e p r e s e n c e of camphor as t h e o n l y c a r b o n and e n e r g y s o u r c e was s m a l l e r compared t o t h a t i n t h e p r e s e n c e o f g l u t a m a t e ( s e e F i g u r e I I I . 2 ) , b u t t h e o p t i c a l d e n s i t y r e a c h e d s i m i l a r v a l u e s a f t e r a b o u t 24 h o u r s . When t h e g r o w t h r e a c h e d i t s l a t e - l o g a r i t h m i c p hase ( a b o u t 14 h o u r s ) , t h e c o n t e n t s o f t h e t h r e e u n d i s t u r b e d f l a s k s were u s e d t o i n o c u l a t e 14 L f e r m e n t e r s . The b a c t e r i a were a l l o w e d t o grow under c o n t i n u o u s a g i t a t i o n and a e r a t i o n ( s e e S e c t i o n I I I . 1.1) u n t i l t h e OD 6 6 0 r e a c h e d 0.6 ( s e e F i g u r e I I I . 3 ) , a t w h i c h t i m e t h e f e e d i n g w i t h camphor was s t a r t e d . A s o l u t i o n o f camphor i n DMF (3M) as w e l l as s o l i d camphor was u s e d a t a r a t e of 15 mL/h or 6.7 g/h, r e s p e c t i v e l y . The use o f a s o l u t i o n o f camphor was p r e f e r r e d t o w a r d s l a t e s t a g e s of g r o w th s i n c e t h e s o l i d camphor t e n d e d t o c l o g t h e c o n t i n u o u s f l o w c e n t r i f u g e . However , t h e use of s o l i d camphor h e l p e d t o r e d u c e t h e T i i 1 1 1 1 1 1 1 1 1 r inoculum for the 14 L fermenters Time (h) Figure I I I . 2 O.IXggn and pH p r o f i l e s for the b a c t e r i a l growth i n the second set of shake-flasks. 61 to in to to r-' to tn to to to' I I i 1 r i 1 1 1 r readjusted to 7.4 ' ' I L J L to l _ o O — CO 3 r d harvest J I I L J I I L 0.0 16.0 32.0 48.0 64.0 80.0 96.0 Time Ch) Figure III.3 O.UggQ and pH p r o f i l e s for the b a c t e r i a l growth i n the 14 L fermenters. 112.0 6 2 amount o f DMF added t o t h e m e d i a . I t i s l i k e l y t h a t an e x c e s s of DMF may i n t e r f e r e w i t l r t h e g r o w t h . The c e n t r i f u g a t i o n was c a r r i e d out when t h e O D 6 6 0 r e a c h e d a b o u t 1 . 2 ( a b o u t 2 4 h o u r s ) and a b o u t 1 5 0 g of b a c t e r i a had c o l l e c t e d . The growth was c o n t i n u e d u n t i l t h e o p t i c a l d e n s i t y a g a i n r e a c h e d 1 . 2 ( a b o u t 24 h o u r s from l a s t h a r v e s t ) , a t w h i c h t i m e t h e c e n t r i f u g a t i o n was c a r r i e d o ut a g a i n . The pH o f t h e medium d r o p s below 6 . 0 a t t h i s t i m e and enough p o t a s s i u m h y d r o g e n p h o s p h a t e ( K 2 H P O i , ) was added t o t h e medium t o b r i n g t h e pH v a l u e t o 7 . 4 . A n o t h e r 120 mL of 1 0 0-X s a l t s was added a l s o i n o r d e r t o r e p l e n i s h t h e medium. The f i n a l c e n t r i f u g a t i o n was c a r r i e d o ut a g a i n when t h e o p t i c a l d e n s i t y r e a c h e d 1 . 2 . The t o t a l wet w e i g h t of th e b a c t e r i a c o l l e c t e d was 4 5 0 - 5 0 0 g. The t o t a l g r o w t h p r o c e d u r e t a k e s a b o u t 5 . 5 d a y s . The p a s t e of b a c t e r i a was s t o r e d under d r y - i c e ( - 7 8 ° ) f o r a t l e a s t 10 d a y s b e f o r e b e i n g u s e d t o i s o l a t e c y t o c h r o m e P - 4 5 0 . 63 I I I .2 I s o l a t i o n and P u r i f i c a t i o n of C y t o c h r o m e P-450 1 1 1.2.1 G e n e r a l I n f o r m a t i o n The f r e e z e - t h a w a u t o l i z e d c e l l s o f b a c t e r i a Pseudomonas p u t i d a s t r a i n 786 (see S e c t i o n I I I . 1 . 3 ) were u s e d t o i s o l a t e c y t o c h r o m e P-450. The method of G u n s a l u s and Wagner 1 was u s e d w i t h some minor m o d i f i c a t i o n s . B a s i c a l l y , t h e i s o l a t i o n p r o c e d u r e i n v o l v e s r e m o v a l of c e l l d e b r i s by c e n t r i f u g a t i o n o f s t i r r e d c e l l s u s p e n s i o n i n a b u f f e r s o l u t i o n , and s e p a r a t i n g c y t o c h r o m e P-450 from o t h e r monooxygenase components by a t w o - s t e p a n i o n exchange column c h r o m a t o g r a p h y p r o c e d u r e . The p u r i f i c a t i o n o f i s o l a t e d c r u d e c y t o c h r o m e P-450 was done by a t w o - s t e p g e l f i l t r a t i o n column c h r o m a t o g r a p h y p r o c e d u r e . Once i s o l a t e d and p u r i f i e d , P-450 samples can be s t o r e d f r o z e n i n l i q u i d n i t r o g e n (-196°) or can be k e p t as a l i q u i d i n t h e r e f r i g e r a t o r ( 5 ° ) . The camphor s u b s t r a t e was removed by a n o t h e r t w o - s t e p g e l f i l t r a t i o n c h r o m a t o g r a p h i c p r o c e d u r e . 1 1 1.2.2 M a t e r i a l s The c h e m i c a l s and s o u r c e s were a s f o l l o w s . D i e t h y l a m i n o m e t h y l c e l l u l o s e (DE 52) from Whatman C h e m i c a l Co.; Sephadex G-10 and G-100 from P h a r m a c i a F i n e C h e m i c a l s ; Enzyme g r a d e ammonium s u l p h a t e from Schwarz/Mann C h e m i c a l Co.; 0 - m e r c a p t o e t h a n o l (0ME), d e o x y r i b o n u c l e a s e 1 (DNase) i s o l a t e d f r o m b o v i n e p a n c r e a s e , r i b o n u c l e a s e - A (RNase) i s o l a t e d f r o m b o v i n e p a n c r e a s e , d i t h i o t h r e i t o l (DTT) and 64 t.r i s - ( h y d r o x y m e t h y l )aminomethane ( T r i z m a Base) f r o m Sigma C h e m i c a l Co.; p o t a s s i u m c h l o r i d e from A m e r i c a n S c i e n t i f i c C h e m i c a l Co.. I l l .2.3. B u f f e r S o l u t i o n s A l l b u f f e r s o l u t i o n s were p r e p a r e d f r o m g l a s s - d i s t i l l e d w a t e r , d e a e r a t e d by r e p e a t e d a s p i r a t i o n f o l l o w e d by f l u s h i n g w i t h a r g o n . The a r g o n was p a s s e d t h r o u g h a column of c o p p e r c a t a l y s t p e l l e t s (BASF C h e m i c a l Co.) h e a t e d t o 50° t o remove t r a c e d i o x y g e n , f o l l o w e d by d r y i n g t h r o u g h p h o s p h o r u s p e n t o x i d e and I n d i c a t i n g D r i e r i t e (Hammond D r i e r i t e C o . ) . The T r i s . C I b u f f e r s o l u t i o n s were p r e p a r e d f r o m a 1.0M s t o c k T r i z m a Base ( s e e S e c t i o n I I I . 2 . 2 . ) s o l u t i o n by d i l u t i n g t o 50 mM w i t h d i s t i l l e d w a t e r f o l l o w e d by a d d i t i o n o f 6M H C l t o o b t a i n a pH of 7.4. F o u r b u f f e r s o l u t i o n s (4 L o f ea c h ) were p r e p a r e d w i t h d i f f e r e n t i o n i c s t r e n g t h s by a d d i n g 0, 50, 100 and 600 mM KC1 t o 50 mM T r i s . C I b u f f e r . F o r s i m p l i f i e d n o m e n c l a t u r e t h e s e b u f f e r s a r e s p e c i f i e d a s T, T-50, T-100, T-600, r e s p e c t i v e l y . P r i o r t o u s e , a l l t h e b u f f e r s o l u t i o n s were made 10 mM w i t h 0ME . The p h o s p h a t e b u f f e r s o l u t i o n s were p r e p a r e d by m i x i n g a p p r o p r i a t e volumes of 1 M K 2 H P O i , and 1 M K H 2 P O « s t o c k s o l u t i o n s t o o b t a i n a pH v a l u e of 7.4, when d i l u t e d t o a 50 mM s o l u t i o n . The 50 mM p h o s p h a t e b u f f e r s o l u t i o n (P- b u f f e r ) was made 100 mM w i t h KC1 t o p r o d u c e b u f f e r P-100. S o l i d camphor was added t o a p p r o p r i a t e b u f f e r s t h a t were s t i r r e d f o r a b o u t 6 h o u r s t o o b t a i n a s a t u r a t e d (8 mM) 65 s o l u t i o n . The a p p r o x i m a t e v o l u m e s of b u f f e r s o l u t i o n s u s e d i n a t y p i c a l p r e p a r a t i o n of c y t o c h r o m e P-450 from a b o u t 400 g o f b a c t e r i a a r e l i s t e d i n T a b l e 111. 1 . The q u a n t i t i e s o f r e a g e n t s u s e d a r e l i s t e d i n T a b l e I I I . 2 . 6 6 T a b l e I I I . 1 Volumes of B u f f e r S o l u t i o n s R e q u i r e d f o r a T y p i c a l I s o l a t i o n and P u r i f i c a t i o n of C y t ochrome P-450. S t e p B u f f e r Volume C e l l - F r e e E x t r a c t T 0.6 L A n i o n Exchange C h r o m a t o g r a p h y (DE52) (a) E q u i l i b r a t i o n o f column A T 4.0 L (b) E q u i l i b r a t i o n o f column B T-50 4.0 L (c) S e p a r a t i o n of p r o t e i n components T-1 00 4.0 L T-600 4.0 L G e l - F i l t r a t i o n (Sephadex G-100) (a) Column A T-1 00 1 .0 L - (1OOmM DTT) (b) Column B P-1 00 1 .0 L (8 mM Cam.) 67 T a b l e I I I . 2 Q u a n t i t i e s o f R e a g e n t s R e q u i r e d f o r a T y p i c a l I s o l a t i o n and P u r i f i c a t i o n of Cytochrome P-450. Reagent Q u a n t i t y T r i z m a b a s e , 1M 800 mL KH 2P0 4, 1M 20 mL K 2HP0 4, 1M 35 mL KC1 250 g ( N H 4 ) 2 S0 4 100 g NH 40H, 15M 2 mL B ME 1 0 mL DTT 0.2 g DNase 1 .0 mg RNase 1 .0 mg 68 111.2. 4 Column C h r o m a t o g r a p h y S i n c e t h e s u c c e s s o f i s o l a t i o n and p u r i f i c a t i o n o f c y t o c h r o m e P-450 v e r y much depend on t h e p r e c i s i o n e x e r c i s e d i n t h e p r e p a r a t i o n of c h r o m a t o g r a p h i c r e s i n s and o p e r a t i o n o f t h e c h r o m a t o g r a p h y c o l u m n s , t h e m a n u f a c t u r e r ' s r e c o m m e n d a t i o n s were f o l l o w e d w i t h s p e c i a l c a r e . The c o l u m n s f o r DE-52 a n i o n exchange c h r o m a t o g r a p h y were m a n u f a c t u r e d by B i o - R a d L a b o r a t o r i e s (Model B i o - R e x ) and t h e c o l u m n s f o r Sephadex g e l f i l t r a t i o n column c h r o m a t o g r a p h y were c o n s t r u c t e d i n t h e m e c h a n i c a l shop o f t h e C h e m i s t r y D e p a r t m e n t , U.B.C. The ends of t h e DE-52 colu m n s were f i t t e d w i t h p o r o u s p o l y e t h y l e n e and 100 mesh n y l o n i n s e r t s . The ends of Sephadex columns c o n t a i n e d o n l y 100 mesh n y l o n i n s e r t s . B o t h DE-52 c o l u m n s were e q u i l i b r a t e d i n t h e d e s c e n d i n g f l o w mode u s i n g a p e r i s t a l t i c pump ( B u c h l e r I n s t r u m e n t s ) t o a c h i e v e c o n s t a n t f l o w r a t e s . The Sephadex columns were e q u i l i b r a t e d u nder g r a v i t y f e e d . The enzyme s a m p l e s were c o l l e c t e d w i t h a LKB Broma, 7000 U l t r a r a c f r a c t i o n c o l l e c t o r , and t h e enzyme samp l e s were c o n c e n t r a t e d u s i n g an Amicon u l t r a f i l t r a t i o n a p p a r a t u s f i t t e d w i t h a YM-10 D i a f l o w u l t r a f i l t r a t i o n membrane (10, 000 m o l e c u l a r w e i g h t c u t - o f f ) . 69 111.2.5. I s o l a t i o n of C y t o c hrome P-450 I I I .2.5.1. C e l l - F r e e E x t r a c t About 400 g of b a c t e r i a , w h i c h had been f r o z e n f o r a t l e a s t two weeks i n d r y - i c e , were thawed a t room t e m p e r a t u r e and made i n t o a creamy p a s t e w i t h 320 mL of T - b u f f e r . T h i s s u s p e n s i o n was t h e n s t i r r e d i n t h e c o l d room (5°) f o r 8 h o u r s a t w h i c h t i m e 2 mg e a c h of DNase 1 and RNase A were added, and t h e s t i r r i n g c o n t i n u e d o v e r n i g h t ( c a . 12 h) a t 5 ° . A n o t h e r 120 mL of T - b u f f e r were t h e n added and t h e m i x t u r e c e n t r i f u g e d a t 10,000 rpm f o r 15 m i n u t e s i n a S o r v a l l RC-2 r e f r i g e r a t e d c e n t r i f u g e e q u i p p e d w i t h a GSA r o t o r . T h i s removed most o f t h e c e l l d e b r i s from t h e enzyme e x t r a c t . The r e s u l t a n t c l o u d y l i q u i d was a g a i n c e n t r i f u g e d on a SS-34 r o t o r a t 15,000 rpm f o r 30 m i n u t e s t o o b t a i n a c l e a r p i n k c o l o u r e d s o l u t i o n . T h i s c e l l - f r e e e x t r a c t was k e p t on i c e a t a l l t i m e s under an a r g o n a t m o s p h e r e . I l l .2.5.2.' S e p a r a t i o n o f C y t o c h r o m e P-450 S e p a r a t i o n o f c y t o c h r o m e P-450 f r o m t h e o t h e r monooxygenase components was c a r r i e d o u t a t 2 5 ° , u n l e s s o t h e r w i s e s p e c i f i e d , u s i n g a t w o - s t e p a n i o n exchange column c h r o m a t o g r a p h i c p r o c e d u r e . The c e l l - f r e e e x t r a c t d e r i v e d from a b o u t 400 g of b a c t e r i a was a p p l i e d i n t h e d e s c e n d i n g mode a t a f l o w r a t e of 180 mL/h t o a DE-52 a n i o n exchange column (150 g, 5x15 cm) p r e v i o u s l y e q u i l i b r a t e d w i t h b u f f e r T ( s e e T a b l e I I 1.1). T h i s column was t h e n washed w i t h b u f f e r T-100 f o r 0.5h and t h e e f f l u e n t d i s c a r d e d . 70 S u b sequent e f f l u e n t from t h i s column was t h e n a p p l i e d i n t h e a s c e n d i n g mode t o a s e c o n d DE-52 a n i o n exchange column (300 g, 5x30 cm) e q u i l i b r a t e d w i t h b u f f e r T-50. The e l u t i o n w i t h b u f f e r T-100 c o n t i n u e d u n t i l t h e t h r e e enzyme bands were w e l l s e p a r a t e d . At t h i s t i m e t h e y e l l o w p u t i d a r e d o x i n r e d u c t a s e band was r u n n i n g i n f r o n t of t h e p i n k c y t o c h r o m e P-450 band w h i c h was f o l l o w e d by t h e brown p u t i d a r e d o x i n band. At t h i s p o i n t a l i n e a r s a l t g r a d i e n t from T-100 t o T- 600 was s t a r t e d i n o r d e r t o f u r t h e r s e p a r a t e y e l l o w and p i n k bands from t h e brown band. The f i r s t DE-52 column was d i s c o n n e c t e d when t h e y e l l o w and p i n k bands e n t e r e d t h e s e c o n d c o l u m n , and t h e e l u t i o n o f s e c o n d column was c o n t i n u e d . The y e l l o w band w h i c h e l u t e d f i r s t was d i s c a r d e d and t h e p i n k c y t o c h r o m e P-450 band was p a s s e d t h r o u g h a 6 m c o i l of s m a l l d i a m e t e r t u b i n g immersed i n i c e p r i o r t o c o l l e c t i o n a t 5 ° . The t o t a l t i m e r e q u i r e d f o r r u n n i n g t h e two columns was a b o u t 14 h o u r s . 111.2.5.3. Ammoniurn S u l p h a t e F r a c t i o n a t i o n The c r u d e enzyme s o l u t i o n f r o m t h e s e c o n d DE-52 column was made 1mM w i t h D-(+)-camphor and f r a c t i o n a t e d w i t h ammonium s u l p h a t e (enzyme g r a d e ) by a d d i n g 200 g/L (36% s a t u r a t i o n ) o v e r a p e r i o d of 30 m i n u t e s . The m i x t u r e was kept on i c e a t a l l t i m e s under an a r g o n a t m o s p h e r e w i t h g e n t l e s t i r r i n g . To m a i n t a i n a c o n s t a n t pH v a l u e , 1 mL of c o n c e n t r a t e d N H „ O H was s l o w l y added t o t h e m i x t u r e f o r e a c h 30 g of (NHjJzSO,, added. The m i x t u r e was s t i r r e d f o r 71 a n o t h e r 30 m i n u t e s and c e n t r i f u g e d a t 10, 000 rpm f o r 10 m i n u t e s ; t h e r e s u l t i n g w h i t e a p o - c y t o c h r o m e P-450 p r e c i p i t a t e was d i s c a r d e d . The s u p e r n a t a n t was p r e c i p i t a t e d by a d d i n g 140 g/L of ( N H „ ) 2 S O » ( 6 0% s a t u r a t i o n ) u s i n g t h e same p r o c e d u r e . The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and t h e y e l l o w s u p e r n a t a n t d i s c a r d e d . The p r e c i p i t a t e was r e s u s p e n d e d i n a minimum volume of b u f f e r p-100 c o n t a i n i n g 8 mM camphor. T h i s c r u d e enzyme s o l u t i o n was s t o r e d under a r g o n a t 5° i n a S c h l e n k t u b e u n t i l p u r i f i e d by g e l f i l t r a t i o n c h r o m a t o g r a p h y . 72 I I I . 2 . 6 . G e l - F i l t r a t i o n C h r o m a t o g r a p h y 111.2.6.1. P u r i f i c a t i o n of C y t ochrome P-450 The c r u d e enzyme samples were made 50 mM w i t h DTT and i n c u b a t e d f o r 30 m i n u t e s a t room t e m p e r a t u r e p r i o r t o p u r i f i c a t i o n by two Sephadex G-100 ( p a r t i c l e s i z e 40-120 m i c r o n ) columns e q u i l i b r a t e d w i t h T-100 (10 mM DTT) and P- 100 (8 mM camphor) b u f f e r s , r e s p e c t i v e l y . A l l enzyme sa m p l e s were c o n c e n t r a t e d t o 1-2 mL ( c a . 0.5 mM) a l i q u o t s by u l t r a - f i l t r a t i o n b e f o r e a p p l y i n g t o t h e c o l u m n s . The enzyme s o l u t i o n from 400 g of b a c t e r i a was d i v i d e d i n t o two p o r t i o n s and p u r i f i e d s e p a r a t e l y i n o r d e r t o a v o i d o v e r l o a d i n g t h e c o l u m n s . B o t h columns were e q u i l i b r a t e d and run a t 5° under g r a v i t y f l o w w i t h a h e a d - p r e s s u r e l e s s t h a n 200 mm. The f r a c t i o n s from t h e f i r s t column were s e l e c t e d by i n s p e c t i n g t h e c o l o u r d e n s i t y so t h a t a b o u t 60-70% of t h e p i n k enzyme e l u t e was i n c l u d e d ; a f t e r b e i n g c o n c e n t r a t e d t o a b o u t 2 mL t h o s e f r a c t i o n s were t h e n a p p l i e d t-o t h e s e c o n d c o l u m n . The m i d d l e f r a c t i o n s o f t h i s column c o n t a i n e d t h e most p u r e enzyme samples w h i c h were s p e c t r o s c o p i c a l l y a n a l y z e d . The u s u a l y i e l d c a l c u l a t e d from a m o l e c u l a r w e i g h t o f 45,000 D a l t o n s amounted t o a b o u t 135 mg of p r o t e i n ( c a . 3 mL of 1 mM s o l u t i o n ) i s o l a t e d f r o m 400 g o f b a c t e r i a . 111.2.6.2. Removal o f Camphor S u b s t r a t e The camphor s u b s t r a t e was removed by a n o t h e r t w o - s t e p g e l - f i l t r a t i o n column c h r o m a t o g r a p h i c p r o c e d u r e u s i n g Sephadex G-10 a t 5 ° . P u r e c y t o c h r o m e P-450 s o l u t i o n s were 73 made 50 mM w i t h DTT and i n c u b a t e d a t room t e m p e r a t u r e f o r 30 m i n u t e s . Camphor was t h e n removed by g e l - f i l t r a t i o n on a Sephadex G-10 column (1.Ox 20 cm) e q u i l i b r a t e d w i t h T-100 b u f f e r c o n t a i n i n g 1OmM DTT. O n l y about 1.0 mL of enzyme s o l u t i o n was u s e d f o r e a c h r u n and t h e h e a d - p r e s s u r e was a d j u s t e d so t h a t t h e column r a n o v e r a 24 hour p e r i o d . The e f f l u e n t enzyme s o l u t i o n was t h e n c h r o m a t o g r a p h e d on a s e c o n d Sephadex G-10 column e q u i l i b r a t e d w i t h b u f f e r P-100 i n o r d e r t o e f f e c t a b u f f e r e x c h a n g e and t o remove t h e e x c e s s t h i o l r e a g e n t . D u r i n g t h e r e m o v a l o f camphor, s p e c i a l c a r e was t a k e n t o a v o i d t h e g l a s s w a r e and ' t u b i n g w h i c h came i n c o n t a c t w i t h camphor, as t h i s sometimes l e d t o t h e i s o l a t i o n o f p a r t l y s u b s t r a t e - b o u n d enzyme. The enzyme sam p l e s p r e p a r e d i n t h i s manner d e m o n s t r a t e d no a p p a r e n t r e s i d u a l bound camphor, as i n d i c a t e d by t h e a b s e n c e o f an o p t i c a l a b s o r p t i o n s h o u l d e r a t 391 nm i n t h e p r e s e n c e o f 50 mM p o t a s s i u m i o n ( s e e S e c t i o n I I I . 2 . 7 . ) . 74 111.2.7 . S p e c t r a l A n a l y s i s The p u r i t y of c y t o c h r o m e P-450 samples can be e s t i m a t e d c o n v e n i e n t l y by c o r r e l a 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 o f t h e e l e c t r o n i c a b s o r p t i o n maximum of t h e heme p r o t e i n w i t h t h e e l e c t r o p h o r e t i c m o b i l i t y i n sodium d o d e c y l s u l p h a t e (SDS) g e l s . 1 The a n a l y t i c a l SDS-tube g e l e l e c t r o p h o r e s i s by t h e i n t e g r a t i o n of d e n s i o m e t r i c r e c o r d i n g s f r om an a u t o m a t i c g e l s c a n n e r u s i n g t h e method of Weber and O s b o r n , 3 combined w i t h t h e o p t i c a l a b s o r p t i o n s p e c t r a l d a t a of t h e heme p r o t e i n , i n d i c a t e t h a t c r y s t a l l i n e c y t o c h r o m e P-450 ( g r e a t e r t h a n 99% homogeneous by t h e SDS-tube g e l e l e c t r o p h o r e s i s ) i s c h a r a c t e r i z e d by an o p t i c a l a b s o r p t i o n r a t i o of t h e peak a t 391 nm t o t h e peak a t 280 nm o f 1.63. 1 P u r i f i e d but non c r y s t a l l i z e d s amples g r e a t e r t h a n 95% homogeneous have an a b s o r p t i o n r a t i o , A 3 9 1 : A 2 so o r 1.45. E l e c t r o n i c 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 from 250- 700 nm i n a C a r y s p e c t r o p h o t o m e t e r model 17D a t room t e m p e r a t u r e , a s p e c i a l c e l l t o n o m e t e r ( F i g u r e I I I . 4 ) d e s i g n e d t o h a n d l e enzyme s o l u t i o n s a n a e r o b i c a l l y b e i n g u s e d . The c e l l t o n o m e t e r c o n s i s t e d o f a c l e a r r e c t a n g u l a r q u a r t z c e l l (1x1x5 c m ) a t t a c h e d t o a s i d e a r m r e s e r v o i r (10 mL) t h r o u g h g l a s s t u b i n g (20 cm) c o n t a i n i n g a s i l i c o n r u b b e r septum f i t t i n g . A h i g h - vacuum O - r i n g j o i n t ( K o n t e s G l a s s Co.) w i t h a B-14 s i d e a r m s o c k e t was u t i l i z e d f o r a t t a c h m e n t t o t h e vacuum-gas h a n d l i n g l i n e . The t o t a l volume of t h e c e l l t o n o m e t e r was 60 mL. 1 cm q u a r t z c e l l g u r e I I I . 4 The c e l l - t o n o m e t e r . 76 P r e v i o u s l y degassed. P-100 b u f f e r (4.8 mL) was t r a n s f e r r e d t o t h e s i d e a r m r e s e r v o i r and r e p e a t e d l y f l u s h e d w i t h a r g o n and e v a c u a t e d t h r e e t i m e s . S t o c k enzyme s o l u t i o n ( c a . 0 . 2 mL) was added t o t h e r e s e r v o i r , mixed by s w i r l i n g and t h e n t r a n s f e r r e d t o t h e c e l l c a v i t y by t i l t i n g t h e t o n o m e t e r . The o p t i c a l a b s o r p t i o n s p e c t r u m of t h e f e r r i c s t a t e of t h e enzyme was t h e n r e c o r d e d . S t o i c h i o m e t r i c r e d u c t i o n o f t h e enzyme t o t h e f e r r o u s s t a t e was c a r r i e d o u t by a d d i n g s m a l l amounts of a sodium d i t h i o n i t e s o l u t i o n ( c a . 4 mg/mL i n d e g a s s e d b u f f e r P-100) t h r o u g h t h e r u b b e r septum u s i n g a s y r i n g e , u n t i l a s m a l l a b s o r p t i o n peak a p p e a r e d a t 314 nm due t o e x c e s s d i t h i o n i t e . Any e x c e s s sodium d i t h i o n i t e was removed by a d d i n g s m a l l amounts o f d i o x y g e n v i a a g a s - t i g h t s y r i n g e ; t h e o p t i c a l s p e c t r u m o f r e d u c e d enzyme was t h e n r e c o r d e d . The c a r b o n monoxide complex o f t h e r e d u c e d enzyme was c o n v e n i e n t l y f o r m e d by g e n t l y b u b b l i n g t h e gas t h r o u g h t h e s o l u t i o n f o r 30 s e c o n d s a t room t e m p e r a t u r e . 77 111.3 D e t e r m i n a t i o n of t h e E q u i l i b r i u m C o n s t a n t f o r t h e R e a c t i o n o f C a r b o n Monoxide w i t h S u b s t r a t e - f r e e C y t o c h r o m e P-450 a t P i f f e r e n t T e m p e r a t u r e s 111 . 3 . 1 G e n e r a l I n f o r m a t i o n The e q u i l i b r i u m c o n s t a n t f o r t h e r e a c t i o n of c a r b o n monoxide w i t h r e d u c e d s u b s t r a t e - f r e e c y t o c h r o m e P-450 a t a g i v e n t e m p e r a t u r e can be measured c o n v e n i e n t l y by o b s e r v i n g t h e e l e c t r o n i c a b s o r p t i o n s p e c t r a l c h a n g e s as a f u n c t i o n of gas p r e s s u r e . The p r o c e d u r e f o r m e a s u r i n g t h e s p e c t r a l c h a n g e s as a f u n c t i o n o f gas p r e s s u r e and d e t e r m i n i n g t h e e q u i l i b r i u m c o n s t a n t v i a H i l l l o g / l o g p l o t s has been d e s c r i b e d p r e v i o u s l y . " • 5 In o r d e r t o a l l o w f o r t h e a d d i t i o n of s m a l l p a r t i a l p r e s s u r e s of CO gas ( c a.0.8 mm Hg), a vacuum-gas h a n d l i n g a p p a r a t u s was c o n s t r u c t e d w i t h an i n c o r p o r a t e d d i - n - b u t y l p h t h a l a t e (DBT) manometer i n s t e a d of t h e u s u a l m e r c u r y manometer.. P a r t i a l p r e s s u r e s of CO i n mm DBT were c o n v e r t e d t o mm Hg f o r d a t a a n a l y s i s u s i n g t h e f o r m u l a , mm DBT 7 . 7 3 X 1 0 ~ 2 = mm Hg, d e r i v e d u s i n g d e n s i t y d a t a . 111 . 3.2 P r o c e d u r e A l l b u f f e r s o l u t i o n s were f r e e z e - t h a w d e g a s s e d and a r g o n - s a t u r a t e d b e f o r e u s e . About 4.8 mL o f c a m p h o r - f r e e P- 100 b u f f e r was added t o t h e o p t i c a l c e l l t o n o m e t e r and t h e s y s t e m e v a c u a t e d and r e f i l l e d w i t h a r g o n t h r e e t i m e s . The c e l l was t h e n t r a n s f e r r e d t o t h e t h e r m o s t a t e d c e l l compartment o f t h e C a r y 17D and t h e b a s e - l i n e s p e c t r u m 78 r e c o r d e d from 250-700 nm. F o r t h e r m o s t a t i n g , two Haake model FK c o n s t a n t t e m p e r a t u r e b a t h s were u t i l i z e d , one c o n n e c t e d t o t h e s p e c i a l c e l l compartment of t h e s p e c t r o p h o t o m e t e r and t h e o t h e r a t t h e CO-gas h a n d l i n g l i n e . B e f o r e t h e b e g i n i n g of e a c h e x p e r i m e n t , t h e c o n s t a n t t e m p e r a t u r e b a t h s were b r o u g h t t o t h e r e q u i r e d t e m p e r a t u r e (±0.2°) and a l l o w e d t o e q u i l i b r a t e a t t h e t e m p e r a t u r e f o r a t l e a s t 30 m i n u t e s . The c e l l t o n o m e t e r was a g a i n a t t a c h e d t o t h e a r g o n l i n e and a b o u t 0.2 mL of s t o c k s u b s t r a t e - f r e e enzyme s o l u t i o n added a n e r o b i c a l l y w i t h g e n t l e s w i r l i n g . At a l l t i m e s , c a r e was t a k e n not t o form b u b b l e s i n t h e enzyme s o l u t i o n as t h i s c a n l e a d t o d e n a t u r a t i o n o f 'the p r o t e i n . Then t h e c e l l t o n o m e t e r was a g a i n p l a c e d i n t h e t h e r m o s t a t e d c e l l compartment and t h e s p e c t r u m o f o x i d i z e d s u b s t r a t e - f r e e enzyme r e c o r d e d . The s t o i c h i o m e t r i c r e d u c t i o n of t h e enzyme was t h e n c a r r i e d o u t as d e s c r i b e d i n S e c t i o n I I I . 2 . 7 and t h e s p e c t r u m r e c o r d e d . The c e l l t o n o m e t e r ' was t h e n t a k e n t o t h e t h e CO-gas h a n d l i n g l i n e and t h e r m o s t a t e d t o t h e d e s i r e d t e m p e r a t u r e by immersion i n a Dewar f u l l o f water c o n t a i n i n g a s p i r a l c o p p e r c o i l a t t a c h e d t o t h e w a t e r c i r c u l a t i o n p o r t o f t h e c o n s t a n t t e m p e r a t u r e b a t h . Two t h e r m o m e t e r s were u s e d t o measure t h e t e m p e r a t u r e ( ± 0 . 2 ° ) , one a t t h e C O - l i n e and t h e o t h e r a t t h e s p e c t r o p h o t o m e t e r . T h e c e l l t o n o m e t e r was t h e n s l o w l y e v a c u a t e d t o remove t h e a r g o n a t m o s p h e r e and t h e s y s t e m a l l o w e d t o e q u i l i b r a t e ( >5 m i n u t e s ) b e f o r e r e a d i n g t h e v a p o u r p r e s s u r e o f t h e enzyme s o l u t i o n . T h i s p r o c e d u r e was 79 r e p e a t e d t h r e e t i m e s . The v a l u e o f v a p o u r p r e s s u r e o b t a i n e d was c o n v e r t e d t o mm Hg and c h e c k e d w i t h p u b l i s h e d d a t a 6 f o r a c c u r a c y , i n o r d e r t o make s u r e t h a t t h e r e were no l e a k s i n t h e a p p a r a t u s . Then a s m a l l amount o f CO gas was c a r e f u l l y a d m i t t e d t o t h e s y s t e m and a l l o w e d t o e q u i l i b r a t e (3 m i n u t e s ) . The p r e s s u r e was r e c o r d e d and t h e c e l l t o n o m e t e r was c l o s e d , removed from t h e gas h a n d l i n g l i n e , s l o w l y s w i r l e d and t a k e n t o t h e s p e c t r o p h o t o m e t e r and a l l o w e d t o e q u i l i b r a t e f o r a n o t h e r 3 m i n u t e s p r i o r t o t h e s p e c t r u m of p a r t i a l l y c a r b o n y l a t e d enzyme b e i n g r e c o r d e d . Once t h e s p e c t r u m was r e c o r d e d , t h e c e l l was t a k e n back t o t h e CO- l i n e and t h e p r e s s u r e was r e a d j u s t e d u s i n g t h e same p r o c e d u r e as a b o v e . At l e a s t f i v e d i f f e r e n t C O - p r e s s u r e s were added and t h e s p e c t r a r e c o r d e d i n e a c h c a s e . The f i n a l s pectrum.was r e c o r d e d under 1 a t m o s p h e r e of CO. T h i s g e n e r a l p r o c e d u r e was f o l l o w e d a t f o u r d i f f e r e n t t e m p e r a t u r e s i n o r d e r t o i n v e s t i g a t e t h e t e m p e r a t u r e d e p endence of t h e e q u i l i b r i u m c o n s t a n t . 80 111.4 D e t e r m i n a t i o n of E q u i 1 i b r ium C o n s t a n t f o r t h e R e a c t i o n o f D i o x y g e n w i t h S u b s t r a t e - b o u n d c y t o c h r o m e P450cam 111.4.1 G e n e r a l I n f o r m a t i o n The e q u i l i b r i u m c o n s t a n t f o r t h e r e a c t i o n o f d i o x y g e n w i t h s u b s t r a t e - b o u n d P-450 c a n be e s t i m a t e d by an e x p e r i m e n t a l p r o c e d u r e s i m i l a r t o t h a t u s e d f o r t h e d e t e r m i n a t i o n of CO b i n d i n g c o n s t a n t , d i s c u s s e d i n t h e p r e v i o u s s e c t i o n . In o r d e r t o e q u i l i b r a t e t h e s y s t e m a t s u b - z e r o t e m p e r a t u r e s a s l u s h b a t h ( F i g u r e I I I . 5 ) was u s e d . T h e r e f o r e , t h e use of c o n s t a n t t e m p e r a t u r e b a t h s as w e l l as t h e s p e c i a l c e l l compartment was not n e c e s s a r y . The b u f f e r s o l u t i o n u s e d was 50% d i e t h y l e n e g l y c o l i n P - b u f f e r . 111 .4 .2 P r o c e d u r e The c e l l - c a v i t y o f t h e s l u s h b a t h was f i l l e d t o 2/3 o f i t s volume ( 30 mL ) w i t h t h e b u f f e r s o l u t i o n and t h e b a s e - l i n e s p e c t r u m was r e c o r d e d i n t h e C a r y s p c t r o p h o t o m e t e r as d e s c r i b e d _ i n S e c t i o n I I I . 3 . 2 . S t o c k s u b s t r a t e - b o u n d enzyme s o l u t i o n was t h e n added t o a f i n a l c o n c e n t r a t i o n o f a b o u t 2 1CT6 M and t h e s p e c t r u m r e c o r d e d . Then, t h e s t o i c h i o m e t r i c r e d u c t i o n was c a r r i e d out as g i v e n i n S e c t i o n I I I . 3 . 2 . The c e l l was t h e n c o o l e d t o t h e d e s i r e d t e m p e r a t u r e (± 1°) u s i n g t h e s u i t a b l e s l u s h 7 from t h e l i s t b elow. S l u s h T e m p e r a t u r e B e n z o n i t r i l e / l i q u i d N 2 -13° C a r b o n t e t r a c h l o r i d e / l i q u i d N 2 -23° o - X y l e n e / l i q u i d N 2 -29° 81 Serum cap To vacuum l i n e Dewar fl a s k Constant temperature bath Optical c e l l Quartz windows Figure i n . 5 1 0 cm path-length o p t i c a l c e l l (Slush Bath). 82 The t e m p e r a t u r e was m a i n t a i n e d c o n s t a n t by t h e a d d i t i o n of s m a l l amounts o f l i q u i d N 2 p e r i o d i c a l l y . S m a l l p a r t i a l p r e s s u r e s of 0 2 were t h e n added u s i n g a low p r e s s u r e gas h a n d l i n g l i n e a s d e s c r i b e d i n S e c t i o n I I I . 3 . 2 , and t h e c o r r e s p o n d i n g s p e c t r a were r e c o r d e d . A t t e m p t s were made t o r e c o r d t h e s p e c t r u m o f t h e f u l l y f ormed o x y g e n a t e d s p e c i e s under 1 a t m o s p h e r e of d i o x y g e n ( s e e S e c t i o n I V . 4 ) . 83 REFERENCES 1. I.C.Gunsalus,G.C.Wagner,Methods E n z y m o l . , 5 2 , 1 6 6 ( 1 9 7 8 ) . 2. E.S.Lenox, V i r o l o g y , J_, 1 9 0 ( 1 9 5 5 ) . 3. K.Weber, M.Osborn, J . B i o l . Chem., 244, 4 4 0 6 ( 1 9 6 9 ) . 4. D . D o l p h i n , B.R.James, H.C.Welborn, Biochem. B i o p h y s . Res. Commun., 88, 4 1 5 ( 1 9 7 9 ) . 5. D.V.Stynes,B.R.James,J.Am.Chem.Soc.,96,2733(1974). 6. Handbook o f C h e m i s t r y and P h y s i c s , 4 1 s t Edn, C h e m i c a l Rubber P u b l i s h i n g Co., C l e v e l a n d ( 1 9 5 9 - 6 0 ) p . 2 3 2 6 . 7. R.E.Randeau, J . Chem. Eng. D a t a , 11 , 124( 1966). CHAPTER IV RESULTS AND DISCUSSION 85 IV.1 G rowth.of Pseudomonas p u t i d a S t r a i n 786 The monooxygenase enzyme s y s t e m p r e s e n t i n t h e b a c t e r i u m Pseudomonas p u t i d a has p r o v i d e d a d e f i n i t i v e b i o l o g i c a l model f o r e x t e n s i v e b i o c h e m i c a l and p h y s i c a l s t u d i e s r e l e v a n t t o t h e c y t o c h r o m e P-450 m e d i a t e d h y d r o x y l a t i o n r e a c t i o n s o c c u r r i n g i n mammalian s y s t e m s . 1 In t h e p r e s e n t s t u d y , t h e Pseudomonas p u t i d a s t r a i n 786 was u s e d t o i s o l a t e t h e camphor h y d r o x y l a t i n g monooxygenase component, c y t o c h r o m e P-450cam. The s t r a i n 786 p o s s e s s e s s e v e r a l a d v a n t a g e s o v e r t h e p a r e n t s t r a i n PpG 1 w h i c h was o r i g i n a l l y i s o l a t e d from s o i l by e n r i c h m e n t on D-(+)- camphor. 1 Among t h e s e a d v a n t a g e s a r e t h e s h o r t e r g e n e r a t i o n t i m e , r e s i s t a n c e t o l y s i s by b a c t e r i o p h a g e , and t h e a b i l i t y t o r e l e a s e t h e h y d r o x y l a s e p r o t e i n s by f r e e z e - t h a w a u t o l y s i s , t h e r e b y p r o v i d i n g e a s y a c c e s s t o t h e P-450 enzyme. In o r d e r f o r t h e b a c t e r i a t o r e t a i n t h e a b i l i t y t o m e t a b o l i z e camphor, and hence t o p r o d u c e t h e P-450 enzyme, i t was n e c e s s a r y t o m a i n t a i n t h e s t r a i n i n m i n i m a l a g a r p l a t e s c o n t a i n i n g camphor as t h e o n l y c a r b o n s o u r c e . F a i l u r e t o do so r e s u l t s i n t h e b a c t e r i u m l o s i n g t h e a b i l i t y t o m u l t i p l y i n camphor s a t u r a t e d m e d i a , w i t h s u b s e q u e n t l o n g l a g - p h a s e s i n t h e growth c u r v e s . 2 I t was o b s e r v e d t h a t t h e pH t r e n d s o f t h e c u l t u r e media depend on t h e b a s i c c a r b o n and e n e r g y s o u r c e p r o v i d e d f o r t h e g r o w t h . In t h e c a s e of L - b r o t h and 500 mL shake f l a s k s t a g e s , where g l u c o s e and g l u t a m i c a c i d were u s e d , 86 r e s p e c t i v e l y , t h e pH of t h e media i n c r e a s e d , w h i l e t h e g r o w t h on camphor r e s u l t e d i n a d e c r e a s e o f pH. The pH d e c r e a s e d u r i n g camphor m e t a b o l i s m i s known t o be due t o t h e a c c u m u l a t i o n of a c i d i c m e t a b o l i t e s , 3 " w h i l e no c l e a r e x p l a n a t i o n has been g i v e n f o r t h e i n c r e a s e i n pH d u r i n g t h e m e t a b o l i s m of t h e o t h e r o r g a n i c s u b s t r a t e s . The b a s i c d i f f i c u l t y e n c o u n t e r e d i n t h e g r o w t h of t h e b a c t e r i a i s t h e ' f oaming o u t ' , d e s p i t e t h e use of a n t i f o a m i n g a g e n t s , a t t h e l a t e r p a r t of t h e 14 L f e r m e n t e r s t a g e . The foam c a u s e d t h e e x i t a i r f i l t e r s t o c l o g and t h e a i r s u p p l y t o t h e c u l t u r e medium was t h u s c u t o f f by t h e b u i l d - u p o f h i g h p r e s s u r e i n t h e f e r m e n t e r s . T h i s p r o c e s s , w h i c h u s u a l l y happened d u r i n g o v e r n i g h t g r o w t h , r e s u l t e d i n t h e d e a t h o f b a c t e r i a l c e l l s ; t h e use of an e x t e r n a l c o n t a i n e r , c o n n e c t e d t o t h e f e r m e n t e r s v i a r u b b e r t u b i n g ( w i t h a i r f i l t e r s r emoved), i n o r d e r t o c o l l e c t t h e foam ( w h i c h c o n t a i n e d p i n k b a c t e r i a l c e l l s ) s e r v e d as an a l t e r n a t i v - e , but t h i s a l s o r e s u l t e d i n t h e d e a t h of a c o n s i d e r a b l e f r a c t i o n of t h e c e l l s due t o l a c k o f a e r a t i o n and a g i t a t i o n . The d u r a t i o n of t h e g r o w t h p r o c e d u r e was f o u n d t o be a t l e a s t t w i c e as l o n g as t h a t g i v e n i n t h e l i t e r a t u r e p r o c e d u r e , 1 a l t h o u g h a l l t h e i n s t r u c t i o n s were f o l l o w e d c l o s e l y . As a c o n s e q u e n c e o f t h e l e n g t h y g r o w t h p e r i o d , t h e amount o f enzyme i s o l a t e d was g e n e r a l l y s m a l l e r t h a n t h a t r e p o r t e d . 1 87 IV.2 P r o p e r t i e s o f Cytochrome P-450cam IV.2.1 S u b s t r a t e - B o u n d Cytochrome P~450cam The a b s o r p t i o n s p e c t r a o f t h e v a r i o u s s t a t e s of p u r e , s u b s t r a t e - b o u n d c y t o c h r o m e P-450cam ( S e c t i o n s I I . 2 . 5 and I I . 2 . 6 . 1 ) a r e g i v e n i n F i g u r e I V . 1 . The w a v e l e n g t h s of t h e a b s o r p t i o n maxima ( Amax), as w e l l as t h e c o r r e s p o n d i n g m i l l i m o l a r e x t i n c t i o n c o e f f i c i e n t s ( e mM) a r e p r e s e n t e d t o g e t h e r w i t h l i t e r a t u r e d a t a i n T a b l e I V . 1 . The o x i d i z e d , s u b s t r a t e - b o u n d enzyme a b s o r b s a t 280 nm ( p r o t e i n p e a k ) , 391 nm ( S o r e t p e a k ) , and 510, 542, and 643 nm ( v i s i b l e p e a k s ) . The c a l c u l a t e d EmM v a l u e s a g r e e r e a s o n a b l y w e l l w i t h t h e p u b l i s h e d d a t a , e x c e p t f o r t h e a b s o r p t i o n a t 280 nm, w h i c h i s due t o a r o m a t i c g r o u p s i n t h e p r o t e i n c h a i n . The i n t e n s i t y of t h e 280 nm peak v a r i e d s l i g h t l y (± 5 % ) , i n d i f f e r e n t enzyme p r e p a r a t i o n s , due t o t h e p r e s e n c e o f s m a l l amounts of a p o - c y t o c h r o m e P-450. On r e d u c i n g t h e o x i d i z e d s t a t e w i t h s t o i c h i o m e t r i c amounts of sodium d i t h i o n i t e (^max 314 nm), t h e c o l o u r of enzyme s o l u t i o n c h a n g e s from l i g h t brown t o l i g h t p i n k w i t h g e n e r a t i o n o f a b s o r p t i o n p e a k s a t 408 and 544 nm; t h e p r o t e i n a b s o r p t i o n r e m a i n s unchanged as e x p e c t e d . The CO- complex of t h e r e d u c e d enzyme, formed by b u b b l i n g CO t h r o u g h t h e enzyme s o l u t i o n f o r a few s e c o n d s , has a g o l d e n y e l l o w c o l o u r w i t h a b s o r p t i o n s a t 365 and 445 nm ( n e a r uv and low e n e r g y bands of a s p l i t S o r e t ) and 550 nm. The d i s c r e p a n c y i n v a l u e s f o r t h e 365 nm peak ( T a b l e IV.1) i s due t o t h e i n t e r f e r e n c e of e x c e s s d i t h i o n i t e i n t h e 1.0 0) o c (0 M O 5 o x i d i z e d reduced reduced-CO D i t h i o n i t e a b s o r p t i o n oo 300 400 —I 500 6 0 0 W a v e l e n g t h ( n m ) 7 0 0 o o oo F i g u r e IV.1 Various s t a t e s of pure, substrate-bound cytochrome P-450cam. 89 T a b l e IV.1 A b s o r b a n c e D a t a f o r S u b s t r a t e - B o u n d Cytochrome P-450cam. S t a t e (a) E x p t . P r e p . (b) L i t . P r e p . (c) L i t . P r e p . X max emM X max emM X max emM O x i d i z e d 280 81.2 - - 280 63.3 391 1 02 391 101 391 102 510 12.2 510 - 510 13.0 542 10.9 540 - 540 11.2 643 4.2 645 - 645 5.4 Reduced 408 80.7 408 83 408 86.5 544 14.5 542 15.2 542 16.0 Reduced-CO 365 51.1 - - 364 60.8 445 1 20 446 1 19 446 120 550 13.8 550 13.0 550 14 (a) D e t e r m i n e d i n P-100 b u f f e r , pH 7.4, 8 mM camphor a t 2 3 + 2 ° ; e mM c a l c u l a t e d a s s u m i n g emM=120 f o r reduced-CO s p e c i e s a t 445 nm. (b) From r e f . 5 (c) From r e f . 1 90 l i t e r a t u r e p r e p a r a t i o n ; and t h e s m a l l e r e v a l u e o b t a i n e d i n t h e p r e s e n t work i s c o n s i d e r e d more a c c u r a t e . 91 IV.2.2 S u b s t r a t e - F r e e C y t o c h r o m e P~450cam The a b s o r p t i o n s p e c t r a of t h e v a r i o u s s t a t e s o f p u r e , s u b s t r a t e - f r e e c y t o c h r o m e P-450cam ( S e c t i o n I I . 2 . 6 . 2 ) a r e shown i n F i g u r e IV.2; t h e xmax and emM v a l u e s f o r t h e e x p e r i m e n t a l p r e p a r a t i o n a r e g i v e n w i t h t h e l i t e r a t u r e d a t a i n T a b l e I V .2. E x a m i n a t i o n of t h e s p e c t r a l c h a r a c t e r i s t i c s of v a r i o u s s t a t e s o f t h e s u b s t r a t e - b o u n d and s u b s t r a t e - f r e e enzyme ( T a b l e s IV.1 and IV.2) r e v e a l s t h a t b o t h forms have v e r y s i m i l a r a b s o r p t i o n s p e c t r a i n t h e r e d u c e d and reduced-CO s t a t e s . However, t h e o x i d i z e d s t a t e s of t h e forms have r e m a r k a b l y d i f f e r e n t s p e c t r a l p r o p e r t i e s ( F i g u r e I V . 3 ) . The b i n d i n g of camphor t o s u b s t r a t e - f r e e enzyme i s s t r o n g and o c c u r s r a p i d l y ( S e c t i o n I I . 4 . 2 . 1 ) . B e c a u s e of t h e s i m i l i a r i t i e s i n s p e c t r a o f r e d u c e d s t a t e s (and reduced-CO s t a t e s ) o f s u b s t r a t e - f r e e and s u b s t r a t e - b o u n d enzymes, i t was e s s e n t i a l t o make s u r e t h a t no t r a c e camphor was p r e s e n t i-n t h e b u f f e r s o r g l a s s w a r e u s e d i n t h e s t u d i e s o f s u b s t r a t e - f r e e enzyme. IV.2.3 P u r i t y and S t a b i l i t y o f t h e Enzyme P r e p e r a t i o n s The i s o l a t e d and p u r i f i e d ( S e c t i o n s I I . 2 . 5 and 11.2.6.1) s u b s t r a t e - b o u n d enzyme samples were a t l e a s t 80% homogeneous, 1 h a v i n g an a b s o r p t i o n r a t i o A 3 9 1 : A 2 B 0 = 1.14. The a b s o r p t i o n r a t i o f o r some of t h e l i t e r a t u r e p r e p a r a t i o n s was 1.00 5 , 1.13 2 , 1.37 6 , 1.45 ( c r y s t a l l i n e s a m p l e ) 1 . A l l s u b s t r a t e - f r e e enzyme sa m p l e s were of t h e same D i t h i o n i t e absorption Figure IV.2 Various states of pure, substrate-free cytochrome P-450cam. 93 T a b l e IV.2 A b s o r b a n c e D a t a f o r S u b s t r a t e - F r e e Cytochrome P-450cam. S t a t e (a) Expt P r e p . (b) L i t P r e p . ( c ) L i t . P r e p . X max emM X max emM X max emM O x i d i z e d 278 107 - - 280 68.3 360 41.0 - - 360 36.7 416 117.9 418 1 04 417 1 15 535 9.7 535 10.3 535 11.6 568 9.3 570 10.4 569 11.9 R e d uced 408 78.9 408 69.0 408 76.7 540 12.5 540 13.5 540 15.1 Reduced-CO 365 445 120 447 104 447 120 550 12.5 550 12. 550 14.3 (a) D e t e r m i n e d i n b u f f e r P-100, pH 7.4, no camphor added, a t 2 3 ± 2 ° ; emM c a l c u l a t e d a s s u m i n g emM=120 f o r reduced-CO s p e c i e s a t 445 nm. (b) From r e f . 5 (c) From r e f . 1  95 o r d e r o f p u r i t y a s t h e s u b s t r a t e - b o u n d enzyme, from w h i c h t h e y were p r e p a r e d . B u t , s t o r a g e a t 5° under a r g o n f o r more t h a n 48 h o u r s c a u s e d t h e f o r m a t i o n of d e t e c t a b l e amounts of P-420 s p e c i e s ( d e n a t u r e d P-450) as seen i n t h e a b s o r p t i o n s p e c t r u m ( F i g u r e I V . 4 ) . T h e r e f o r e , s u b s t r a t e - f r e e enzyme s a m p l e s f o r t h e e q u i l i b r i u m l i g a n d b i n d i n g s t u d i e s were p r e p a r e d on a d a i l y b a s i s . The s u b s t r a t e - b o u n d enzyme, once p u r i f i e d , c o u l d be s t o r e d a t 5° f o r o v e r 6 months w i t h o u t t h e f o r m a t i o n of any P-420 s p e c i e s . The s u b s t r a t e - b o u n d enzyme c o u l d be s t o r e d i n d e f i n i t e l y a t l i q u i d n i t r o g e n t e m p e r a t u r e w i t h o u t d e t e c t a b l e l o s s of p u r i t y . F i g u r e IV.4 P-420 species formed i n s u b s t r a t e - f r e e cytochrome P-450cam s o l u t i 97 IV.3 D e t e r m i n a t i o n of t h e E q u i l i b r i u m C o n s t a n t a t P i f f e r e n t T e m p e r a t u r e s f o r t h e B i n d i n g of C a r b o n Monoxide by S u b s t r a t e - F r e e Cytochrome P-450cam IV.3.1 A c q u i s i t i o n and T r e a t m e n t of P a t a E q u i l i b r i u m c o n s t a n t v a l u e s f o r t h e b i n d i n g of CO w i t h s u b s t r a t e - f r e e c y t o c h r o m e P-450 were e s t i m a t e d f r o m t h e e x p e r i m e n t a l d a t a o b t a i n e d as g i v e n i n S e c t i o n I I I . 3 . At e a c h t e m p e r a t u r e , a t l e a s t t h r e e d i f f e r e n t d e t e r m i n a t i o n s were c a r r i e d out t o g i v e a mean v a l u e f o r t h e e q u i l i b r i u m c o n s t a n t s . The s p e c t r a l c h a n g e s o b s e r v e d f o r e x p e r i m e n t no.5 ( T a b l e IV.3) a r e g i v e n i n F i g u r e I V . 5 . A t a f i x e d enzyme c o n c e n t r a t i o n (7-8 10 " 6 M), t h e CO p r e s s u r e was v a r i e d from 0.4-760 mm Hg, r e s u l t i n g i n a d e c r e a s e of t h e 408 nm peak ( r e d u c e d ) and an i n c r e a s e i n 445 nm peak ( C O - c o m p l e x e d ) . I s o s b e s t i c p o i n t s were o b s e r v e d a t 373, 430, 466 and 583 nm. Raw d a t a from a l l t h e e x p e r i m e n t s a r e g i v e n i n A p p e n d i x ( i ) , a l o n g w i t h t h e p r o c e s s e d d a t a . The d e g r e e of f o r m a t i o n of t h e CO-complex o f t h e s u b s t r a t e - f r e e c y t o c h r o m e P-450cam ( R e a c t i o n I I . 2 ) can be d e t e r m i n e d u s i n g t h e f a m i l i a r H i l l l o g / l o g p l o t w h i c h y e i l d s t h e P w 2 and hence K v a l u e . J CO The r a t i o of t h e F e ( I I ) - C O t o t h e d e c a r b o n y l a t e d complex p r e s e n t a t a g i v e n CO p r e s s u r e ( P c o ^ c a n D e e x p r e s s e d as (A-A 0 ) / ( A a o - A ) , where, A = a b s o r b a n c e o f F e ( I l ) - C O a t a known P C Q A Q = a b s o r b a n c e of F e d I) enzyme when P c o = 0 F i g u r e IV.5 S p e c t r a l changes o b s e r v e d f o r t h e P. / 0 e x p e r i m e n t no. 5. 99 A ^ a b s o r b a n c e o f f u l l y f ormed F e ( I I ) - C O C l e a r l y , A-A 0 a. [ F e ( I I ) - C O ] , and A^-A o c [ F e ( I I ) ] . S i n c e , = [ F e ( I I ) - C O ] / [ F e ( I I ) ] [ C O ] , t h e f o l l o w i n g r e l a t i o n s h i p s a r e d e r i v e d . Log K C Q = L o g ( A - A 0 ) / ( A O D - A ) - Log [CO] Log ( A - A 0 ) / ( A o o - A ) = L o g Pco+ Log K' where, K'= x.K and x= t h e s o l u b i l i t y of CO i n w a t e r e x p r e s s e d i n M/(mm Hg) a t t h e r e a c t i o n t e m p e r a t u r e . T h e r e f o r e , t h e p l o t o f l o g ( A - A 0 ) / ( A ^ - A ) v s . l o g P C O s h o u l d g i v e a s t r a i g h t l i n e w i t h a t h e o r e t i c a l g r a d i e n t e q u a l t o u n i t y f o r t h e s i m p l e e q u i l i b r i u m ; and when A-A 0 =Aoo_A o r , l o g (A-A 0 ) / ( A^-A) =0 , t h e c o r r e s p o n d i n g PQ-, v a l u e w o u l d be t h e P , y 2 . The K C O ( M - 1 ) v a l u e s c a n be c a l c u l a t e d u s i n g t h e e q u a t i o n , K c o = 1 / X . P 1 / 2 The l o g ( A - A 0 ) / ( A r o - A ) v a l u e s were c a l c u l a t e d from t h e c o r r e s p o n d i n g a b s o r b a n c e v a l u e s a t 445 nm. P̂ -, v a l u e s were c o n v e r t e d f r o m mm DBT t o mm Hg a s d e s c r i b e d i n S e c t i o n I I I . 3 . 1 . The d a t a were p l o t t e d a s i n F i g u r e IV.6 u s i n g t h e computer p r o g r a m " a c e : g r a p h " a v a i l a b l e from t h e computer f a c i l i t y a t U.B.C. The g r a d i e n t s of a l l t h e p l o t s were i n t h e r a n g e 0.90-1.15; i n c e r t a i n i n s t a n c e s , r e m o v a l o f an e x p e r i m e n t a l d a t a p o i n t ( c o n s i d e r e d t o c o n t a i n a l a r g e e r r o r ) , a p p l y i n g t h e c o n d i t i o n t h a t a t l e a s t f o u r o t h e r d a t a p o i n t s r e m a i n e d , r e s u l t e d i n a much more s a t i s f a c t o r y 100 101 c o r r e l a t i o n of d a t a , a c c o r d i n g t o l e a s t - s q u a r e s l i n e a r r e g r e s s i o n a n a l y s i s . The p l o t s o b t a i n e d f o r a l l P,^ e x p e r i m e n t s a r e g i v e n i n A p p e n d i x ( i i ) . E a c h P 1 / 2 v a l u e c a l c u l a t e d from t h e s e p l o t s w r e c o r d e d and t h e mean v a l u e c a l c u l a t e d ( T a b l e I V . 3 ) . The P,^ v a l u e s were c o n v e r t e d t o e q u i l i b r i u m c o n s t a n t ( K c o , M _ 1 ) v a l u e s u s i n g p u b l i s h e d d a t a 7 f o r t h e s o l u b i l i t y of CO i n water a t v a r i o u s t e m p e r a t u r e s . An i m p o r t a n t a s p e c t of t h e s t u d y o f r e a c t i o n s o f h e m o p r o t e i n s w i t h l i g a n d s i s t h e d e t e r m i n a t i o n of t h e thermodynamic p a r a m e t e r s A H 0 and A S 0 . In t h e c a s e o f b i o l o g i c a l s y s t e m s , t h e d e t e r m i n a t i o n of s u c h p a r a m e t e r s has u s u a l l y been v i a v a n ' t H o f f p l o t s , 8 a l t h o u g h h i g h l y s e n s i t i v e m i c r o c a l o r i m e t r i c t e c h n i q u e s a r e a l s o a v a i l a b l e . 9 The v a n ' t H o f f p l o t , ( l o g K C Q v s . 1/T ; F i g u r e IV.7) was o b t a i n e d u s i n g t h e computer p r o g r a m m e n t i o n e d a b o v e , and t h e thermodynamic p a r a m e t e r s were c a l c u l a t e d . The t hermodynamic r e l a t i o n s h i p , Log K = - ( A H ° / 2 . 3 0 3 R ) . 1 / T + A S ° / 2 . 3 0 3 R , a l l o w s f o r a p l o t o f l o g K v s . 1/T t o g i v e & H ° f r o m t h e g r a d i e n t and A S 0 from t h e y - i n t e r c e p t . Good l i n e a r i t y i s o b s e r v e d i n t h e s e p l o t s owing t o t h e f a c t t h a t t h e measurements a r e done i n a narrow t e m p e r a t u r e r a n g e 8 ( u s u a l l y 0°-40°) b e c a u s e o f t h e i n s t a b i l i t y of p r o t e i n s a t h i g h e r t e m p e r a t u r e s . The A H 0 v a l u e f o r t h e CO b i n d i n g t o r e d u c e d s u b s t r a t e - f r e e c y t o c h r o m e P-450cam was e s t i m a t e d t o be -18±1 k c a l / m o l 102 T a b l e IV.3 The E q u i l i b r i u m C o n s t a n t V a l u e s f o r CO B i n d i n g t o S u b s t r a t e - F r e e C y t o c hrome P-450cam. T ( ° C ) # Pimm Hg) 1/2 Mean P̂  K C Q (M* ) Mean Lo g K C O l / T x l C T 3 4.0 1 0.372 1 .55X10 6 2 0.423 0.398 1.37X10 6 1.46x10 s 6.1644 3.608 3 0.398 1 .45x1 0 6 12.0 4 1 .38 5.07x10 5 5 1 .32 1 .40 5.30X10 5 5.00x10 s 5.6990 3.507 6 1 .57 4.63x10 s 18.0 7 2.63 2.95x10 s 8 2.69 2.78 2.88x10 s 2.80x10 s 5.4472 3.434 9 3.02 2.57x10 s 24.0 10 4.68 1.57x10 s 1 1 4.47 4.59 1.65x10 s 1.60x10 s 5.2041 3.365 1 2 4.62 1.59x10 s 103 1 0 3.36 3.4 3.44 3.4B 3.52 3.56 3.6 3.64 1/T*10 F i g u r e IV.7 The van't Hoff p l o t . 104 The AS 0 v a l u e was -37±4 c a l / m o l . d e g . ( s t a n d a r d s t a t e o f 1 M). T a b l e IV.4 summarizes p u b l i s h e d thermodynamic d a t a f o r t h e CO b i n d i n g t o s u b s t r a t e - b o u n d c y t o c h r o m e P-450cam, h o r s e Mb, sheep Hb ( p e r mole o f heme), and t o some model compounds. G i v e n a l s o a r e t h e v a l u e s d e t e r m i n e d i n t h i s s t u d y f o r s u b s t r a t e - f r e e c y t o c h r o m e P450cam. 105 T a b l e IV.4 K c o , AH 0 and AS 0 V a l u e s f o r CO B i n d i n g t o Some H e m o p r o t e i n s and M o d e l s o f Cytochrome P-450. System K c o , 2 5 ° AH 0 AS 0 Ref . (M- 1 ) ( k c a l / m o l ) ( c a l / m o l . d e g . ) P-450cam 1.3x10 5 -12 -17 1 1 (sub.-bound) H o r s e Mb 2.5x10 7 -12.6 -7.4 8 Sheep Hb - -16 - 8 Human Hb - -17.7 - 9 M o d e l * (a) 1.3x10' -16 -35 1 0 (b) 2.8x10 3 -19 -48 1 0 P-450cam 1.3x10 5 -18±1 - 3 7 ± 4 t h i s ( s u b . - f r e e ) work * F e ( I I ) P p ( I X ) D M E - B u S complex (a) i n a p o l a r medium ( N , N - d i m e t h y l a c e t a m i d e ) (b) i n a n o n - p o l a r medium ( t o l u e n e ) 106 IV.4 C o m p a r i s o n of M e a s u r e d AH° and AS 0 V a l u e s t o t h o s e of O t h e r H e m o p r o t e i n s and Model Systems S t r u c t u r a l a n a l y s e s of c a r b o n y l a t e d h e m o p r o t e i n s r e v e a l t h a t t h e CO u n i t i s bent a n d / o r t i l t e d from t h e p e r p e n d i c u l a r t o t h e p o r p h y r i n p l a n e , due t o i n t e r a c t i o n s w i t h t h e d i s t a l r e s i d u e s . 1 2 However, i n t h e p r o t e i n - f r e e model s y s t e m s , a s e x p e c t e d , t h e Fe-C-0 u n i t i s l i n e a r and n o r m a l t o p o r p h y r i n p l a n e . 1 3 I t has been p r o p o s e d 1 * " 1 7 t h a t i n h e m o p r o t e i n s , t h e CO a f f i n i t i e s a r e l o w e r e d s i g n i f i c a n t l y due t o t h i s ' d i s t a l - s i d e s t e r i c e f f e c t ' w h i c h c a u s e s t h e Fe-C-0 u n i t t o be b e n t . R e s u l t s from t h e p r e s e n t s t u d y show t h a t t h e b i n d i n g of CO t o s u b s t r a t e - f r e e c y t o c h r o m e P-450cam i s a t l e a s t 5 k c a l / m o l more f a v o u r a b l e i n terms of AH 0 compared t o t h e p u b l i s h e d v a l u e 1 1 f o r t h e s u b s t r a t e - b o u n d s y s t e m ( T a b l e I V . 4 ) . The r e v e r s e t r e n d o b s e r v e d i n t e r m s of AS 0 i s c o n s i d e r e d t o be due m a i n l y t o d i f f e r e n c e s i n c o n f o r m a t i o n a l c h a n g e s i n t h e p r o t e i n c h a i n . 1 8 As p r o p o s e d by Lumry and R a j e n d e r , 1 8 t h e r e e x i s t a s p e c i f i c l i n e a r r e l a t i o n s h i p between t h e e n t r o p y change and e n t h a l p y change w i t h i n a v a r i e t y o f p r o t e i n r e a c t i o n s t e r med ' e n t h a l p y - e n t r o p y c o m p e n s a t i o n phenomena'. I t has been d e m o n s t r a t e d t h a t t h e ' c o m p e n s a t i o n e f f e c t ' p r o d u c e s p a r a l l e l e n t h a l p y and e n t r o p y c h a n g e s so t h a t t h e n e t change i n t h e f r e e - e n e r g y o f t h e s y s t e m under i n v e s t i g a t i o n i s r e l a t i v e l y m i n o r . A l t h o u g h t h i s p r i n c i p l e i s not r i g o r o u s l y e s t l i s h e d , t h e f e e l i n g i s t h a t e x p a n s i o n s and 107 c o n t r a c t i o n s of t h e p r o t e i n r e s u l t i n c h a n g e s i n t h e ' f r e e volume' of w a t e r , which c o n t r i b u t e s t o t h e e n t r o p y change of t h e s y s t e m . F o r example, th e e f f e c t o f v a r y i n g e t h a n o l c o n c e n t r a t i o n s on t h e r e v e r s i b l e t h e r m a l u n f o l d i n g t r a n s i t i o n of r i b o n u c l e a s e A a t v a r i o u s t e m p e r a t u r e s i s t o p r o d u c e p a r a l l e l AH 0 and A S 0 changes t h a t g i v e r i s e t o t h e same A ( A H 0 ) / A ( A S 0 ) v a l u e a t e a c h t e m p e r a t u r e . A l s o s i m i l a r c o m p e n s a t i o n e f f e c t s have been n o t e d i n r e a c t i o n s of h e m o g l o b i n and m y o g l o b i n w i t h l i g a n d s s u c h as F~, N 3, CN~, S C N - . 1 8 The c o m p e n s a t i o n h y p o t h e s i s a p p e a r s t o a p p l y t o t h e r e s u l t s o f t h e p r e s e n t s t u d y ; t h e l a r g e n e g a t i v e e n t r o p y c o n t r i b u t i o n o b s e r v e d i n t h e c a s e of CO b i n d i n g t o s u b s t r a t e - f r e e P-450cam compensates f o r t h e l a r g e e n t h a l p y change o b s e r v e d , t h e o v e r a l l p r o c e s s r e s u l t i n g i n no change i n t h e AG 0 of t h e r e a c t i o n , compared t o t h e s u b s t r a t e - b o u n d s y s t e m . Q u a l i t a t i v e l y , t h e o b s e r v e d l e s s - f a v o u r a b l e A H 0 f o r t h e b i n d i n g of CO t o s u b s t r a t e - b o u n d enzyme, compared t o t h e s u b s t r a t e - f r e e s y s t e m , may be due m a i n l y t o t h e p r e s e n c e o f t h e s u b s t r a t e m o l e c u l e s i t t i n g c l o s e t o t h e a c t i v e - s i t e , p e r h a p s c a u s i n g t h e CO m o l e c u l e t o b i n d i n a more a n g u l a r f a s h i o n . In f a c t , s u c h a h y p o t h e s i s i n d i r e c t l y s u p p o r t s t h e o b s e r v a t i o n s by P e t e r s o n e t a l . 1 9 i n t h e i r i n h i b i t i o n e x p e r i m e n t s where t h e y c o n c l u d e t h a t t h e b o n d i n g s i t e of t h e s u b s t r a t e must l i e i n t h e immediate v i c i n i t y of t h e heme i r o n . 108 The r e l a t i v e l y l a r g e r CO b i n d i n g c o n s t a n t t o m y o g l o b i n ( T a b l e IV.4) may r e s u l t from t h e weaker 6"-donor p r o p e r t y of t h e f i f t h l i g a n d , i m i d a z o l e , compared t o t h e t h i o l a t e l i g a n d i n P-450. The A H 0 term f o r t h e CO b i n d i n g t o sheep Hb i s more f a v o u r a b l e t h a n t h a t f o r s u b s t r a t e - b o u n d P- 450cam o r Mb, and i n d e e d i t i s s i m i l a r t o t h e A H ° d e t e r m i n e d f o r t h e s u b s t r a t e - f r e e P-450cam. However, t h e e n t r o p y t e r m f o r t h e s h e e p Hb s y s t e m i s not a v a i l a b l e f o r c o m p a r i s o n . The r e l a t i v e l y s m a l l e n t r o p y change i n t h e Mb s y s t e m may r e f l e c t t h a t o n l y m i n o r c o n f o r m a t i o n a l c h a n g e s o c c u r i n t h e p r o t e i n c h a i n upon CO b i n d i n g compared t o t h e s u b s t r a t e - b o u n d P-450 s y s t e m ; t h i s r e s u l t s i n a much h i g h e r b i n d i n g c o n s t a n t . The b i n d i n g of CO t o t h e model s y s t e m ( T a b l e IV.4) i s e q u a l l y f a v o u r a b l e i n t e r m s of A H 0 a s t h e b i n d i n g o f CO t o s u b s t r a t e - f r e e P-450cam. T h e r e f o r e , i t i s c o n c e i v a b l e t h a t t h e Fe-C-0 u n i t i s l i n e a r ( o r a l m o s t so) i n t h e s u b s t r a t e - f r e e P-450-CO c o m p l e x . T h e r e a r e no thermodynamic d a t a a v a i l a b l e on 0 2 b i n d i n g t o P-450 s y s t e m . In t h e p r e s e n t work, a l a r g e number of a t t e m p t s were made t o d e t e r m i n e t h e P ^ v a l u e s , a t t e m p e r a t u r e s r a n g i n g from 0° t o - 3 0 ° , f o r t h e s u b s t r a t e - bound s y s t e m u s i n g t h e p r o c e d u r e g i v e n i n S e c t i o n I I I . 4 . None o f t h e s e e x p e r i m e n t s were s u c c e s s f u l b e c a u s e o f s e v e r a l d i f f i c u l t i e s t h a t were n o t r e s o l v e d . At t e m p e r a t u r e s near 0 ° , t h e enzyme-0 2 complex was formed, but a u t o x i d i z e d ( a c c o r d i n g t o r e a c t i o n I I . 3 , t o g i v e t h e F e ( I I I ) enzyme) d u r i n g t h e t i m e of t h e e x p e r i m e n t . The o n l y 109 r e p o r t e d P,/2 v a l u e f o r 0 2 b i n d i n g t o P-450 enzyme a t 0° was d e t e r m i n e d u s i n g t h e same c o n v e n t i o n a l c e l l t o n o m e t e r method u s e d i n t h e p r e s e n t s t u d y t o d e t e r m i n e t h e CO b i n d i n g c o n s t a n t s . I t s h o u l d be n o t e d t h a t t h e same r e p o r t g i v e s t h e h a l f l i f e of a u t o x i d a t i o n a t 0° as 75 m i n u t e s . 1 1 However, t h e p u b l i s h e d 1 1 . 2 0 , 2 1 , 2 2 r a t e s of a u t o x i d a t i o n o f t h e s u b s t r a t e - b o u n d P-450cam-dioxygen complex ( T a b l e IV.5) a r e r a t h e r i n c o n s i s t e n t ; t h e r a t e s d e t e r m i n e d i n t h e p r e s e n t work were h i g h e r t h a n most o f t h e r e p o r t e d v a l u e s [ T a b l e IV.5; Raw D a t a i n A p p e n d i x ( i i i ) ] . A t l o w e r t e m p e r a t u r e s (-10° tO - 3 0 ° ) t h e a u t o x i d a t i o n r e a c t i o n was i n d e e d p r e v e n t e d , but t h e c o - s o l v e n t s y s t e m u s e d ( e t h y l e n e g l y c o l / w a t e r ) became more v i s c o u s , t h u s c a u s i n g s m a l l b u b b l e s t o f o r m when t h e s l u s h - b a t h was s h a k e n t o mix t h e a d d e d g a s . T h e s e b u b b l e s f l o a t e d t o t h e s u r f a c e o v e r a l o n g p e r i o d of t i m e ( c a . 1 h ) , and a s i n g l e b u b b l e r i s i n g t o t h e s u r f a c e d u r i n g t h e r e c o r d i n g o f a s p e c t r u m a l t e r e d a b s o r p t i o n c o n s i d e r a b l y s u c h t h a t t h e i s o s b e s t i c p o i n t s were not o b s e r v e d . A f t e r many u n s u c c e s s f u l a t t e m p t s i t was d e c i d e d t h a t t h e c u r r e n t a p p r o a c h was n o t s u i t a b l e f o r d e t e r m i n a t i o n of t h e P 1 / 2 v a l u e (and A H 0 , AS 0 v a l u e s ) f o r t h e b i n d i n g o f d i o x y g e n t o s u b s t r a t e - b o u n d c y t o c h r o m e P-450cam. 110 T a b l e IV.5 H a l f - l i v e s o f A u t o x i d a t i o n R e a c t i o n of D i o x y g e n Complex of S u b s t r a t e - B o u n d Cytochrome P-450cam. pH T (°C) H a l f - l i f e (min.) Ref . 7.0 0.0 75 1 1 7.4 4.0 45 20 7.4 4.0 1 15 21 7.4 2.0 21 22 7.4 4.0 18 22 7.4 10.0 9.2 22 7.4 0.0 20 t h i s work 7.4 10.0 6.7 t h i s work U n f o r t u n a t e l y , no e q u i l i b r i u m or k i n e t i c d a t a a r e a v a i l a b l e on P-450 model s y s t e m s t h a t do n o t use a v a s t e x c e s s of a ' n o n - i n n o c e n t ' l i g a n d . 2 3 M e r c a p t a n - t a i 1 p o r p h y r i n s , p r e p a r e d r e c e n t l y by C o l l m a n e t a l . 2 3 by c o v a l e n t l y a t t a c h i n g a t h i o l g r o u p t o t h e p o r p h y r i n so t h a t t h e t h i o l c a n s e r v e as a b u i l t - i n a x i a l l i g a n d , a p p e a r t o be good c a n d i d a t e s f o r s t u d i e s on CO v s . 0 2 b i n d i n g f o r c o m p a r i s o n w i t h P-450 s y s t e m s i n t h e c o n t e x t o f s t e r i c c o n s i d e r a t i o n s . I l l REFERENCES 1. I . C . G u n s a l u s , G.C.Wagner, Methods E n z y m o l . , 5_2, 166 ( 1 9 7 8 ) . 2. S . 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S e s l e r , R . M . M o r r i s , Q.H.Gibson, J . Am. Chem. S o c , 1 0 5 ( 1 0 ) , 3 0 5 2 ( 1 9 8 3 ) . 112 1 6 . P.W T u c k e r , S . E . P h i l l i p s , M . F . P e r u t z , R.Houtchens, W.H.Caughey, P r o c . N a t l . A c a d . S c i . , U.S.A., 7 5 , 1 0 7 6 ( 1 9 7 8 ) . 1 7 . W . J . W a l l a c e , J . A . V o l p e , J . C . M a x w e l l , W.S.Caughey, S . C h a r a c h e , Biochem. B i o p h y s . Res. Commun., 6 8 , 1 3 7 9 ( 1 9 7 6 ) . 1 8 . R.Lumry, S . R a j e n d e r , B i o p o l y m e r s , 9 ( 1 0 ) , 1 1 2 5 ( 1 9 7 0 ) . 1 9 . J . A . P e t e r s o n , V . U l l r i c h , A . H i l d e r b r a n d t , A r c h . Biochem. B i o p h y s . , J_45, 5 3 1 ( 1 9 7 1 ) . 2 0 . R.W.Estaboook, J . B a r o n , J . A . P e t e r s o n , Y . I s h i m u r a , B i o c h e m . J . , 1 2 1 , 3 ( 1 9 7 1 ) . 2 1 . J . A . P e t e r s o n , Y . I s h i m u r a , B . W . G r i f f i n , A r c h . Biochem. B i o p h y s . , J_49, 1 9 7 ( 1 9 7 2 ) . 2 2 . L . E i n s t e i n , P.Debey, P.Dousou, Biochem. B i o p h y s . Res. Commun., 7_7, 1 3 7 7 ( 1 9 7 7 ) . 2 3 . J . P . C o l l m a n , S.E.Groh, H e m o g l o b i n and Oxygen B i n d i n g , ( E d . C h i e n Ho), E l s e v i e r B i o c h e m i c a l , New Y o r k , 1 9 8 2 p . 3 7 . 113 A P P E N D I C E S Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 4 ± Q±2° C Vapour Pressure of Water: 79 mm DBT Experiment No. 1 Raw Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) P rco (mm DBT) A445nm 1 - 76 - 0.177 2 5 80 4 0.327 3 10 89 13 0.416 4 20 97 21 0.443 5 40 111 35 0.467 6 100 183 107 0.492 Buffer: P-100 PH: 7.4 Processed Data Pco (mm Hg) A-A 0 A.-A A-A 0 A— -A Log A _ A o A.-A L°8 P ™ CO 0.309 0.150 0.180 0.833 -0.07918 -0.5098 1.01 0.239 0.091 2.626 0.4194 -0.002120 1.62 0.266 0.064 4.156 0.6187 0.2104 2.71 0.290 0.040 7.250 0.8603 0.4323 8.27 0.315 0.015 21.00 1.322 0.9176 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 4f0.2°C Vapour Pressure of Water:79 Experiment No. 2 Raw Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) P rco (mm DBT) A445nm 1 - 76 - 0.205 2 5 80 4 0.385 3 10 85 9 0.475 4 15 91 15 0.490 5 25 98 22 0.548 6 - - 0.063 Buffer: P _ 1 0 0 P H : 7.4 Processed Data Pco (mm Hg) A-A 0 A.-A A-A 0 A<« -A Log A _ A o A..-A Log P CO 0.309 0.189 0.245 0.735 -0,1339 -.5100 0,696 0.270 8.155 1.742 0.2410 -0.1600 1.16 0,285 -.140 2.036 0.3087 0.0643 (?) 1.70 0,343 0.082 4,183 0.6215 0.2306 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 4+0.2°C Buffer: P-100 Vapour Pressure of Water: 79 mm DBT pH: 7 . 4 Experiment No. 3 Raw Data Processed Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) P rco (mm DBT) A445nm 1 - 74 - 0.150 2 5 79 5 0.350 3 20 95 21 0.485 4 40 l i t 0.520 5 - - 0.560 Pco (mm Hg) A-A o A-A QD A-A o A- -A Log A - A o Log P CO 0.387 0.200 0.210 0.9524 -0.0212 -0.4129 1.62 0.335 0.0750 4.466 0.6499 0.2104 3.48 0.370 0.0400 9.250 0.9661 0.5414 Appendix (1) contM. Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 12+0.2°C Buffer: P-100 Vapour Pressure of Water: 135 mm DBT pH: 7.4 Experiment No.4 Raw Data Processed Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) p RCO (mm DBT) A445nm Pco (mm Hg) A-A o A.-A A-A o A<» -A Log A _ A o A.-A Log P CO 1 - 135 - 0.195 2 5 140 5 0.285 0.387 0.090 0.355 0.254 -0.5960 -0.4129 3 10 164 29 0.480 2.24 0.285 0.160 1.78 0.2507 0.3506 4 30 215 80 0.565 6.18 0.370 0.075 4.93 0.6931 0.7913 5 50 405 270 0.610 20.9 0.415 0.030 13.8 1.141 1.320 6 oo - - 0.640 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 1 2 + Q 2 o c Buffer: p _ l 0 0 Vapour Pressure of Water: 135 mmDET 7.4 Experiment No. 5 Raw Data Processed Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) P rco (mm DBT) A445nm Pco (mm tt^) A-A 0 A.-A A-A 0 Ao -A Log A _ A o A.-A Log P CO 1 - 134 - 0.222 2 5 138 4 0.260 ? 0.310 0.038 0.600 0.0633 -1.198 -0.5100? 3 10 141 7 0.397 0.541 0.175 0.463 0.378 -0.4225 -0.2668 4 20 163 29 0.610 2.24 0.388 0.250 1.15 0.1909 0.3506 5 50 196 62 0.727 4.79 0.505 0.133 3.79 0.5794 0.6806 6 150 290 156 0.787 12.1 0.565 0.073 7.74 0.8887 1.081 7 OO - - 0.860 00 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 6 Raw Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) p rco (mm DBT) A445nm 1 _ 135 - 0.190 2 10 153 18 0.372 3 20 163 28 0.430 4 40 188 53 0.478 5 60 246 111 0.530 6 CO - - 0.582 Buffer: pH: Processed Data p c o (mm Hg) A-A o A-A GD A-A o A— -A Log A Ao A^-A Log P CO 1.39 0.182 0.210 0,867 -0.0621 0.1435 2.16 0.240 0.152 1.58 0.198^ 0.3353 4.10 0.288 0.104 2.77 0.4424 0.6125 8.58 0.340 0.052 6.54 0.8155 0.9335 Appendix (i) (cont'd) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 18± 0.2°C Buffer: P-100 Vapour Pressure of Water: 200 mm DBT pH: 7.4 Experiment No. 7 Raw Data Processed Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) p (mm DBT) A445nm (mm Hg) A-A o A.-A A-A o A<» -A Log A _ A o A.-A Log P CO 1 0 192 - 0.220 2 10 200 8 0.325 0.618 0.105 0.445 0.236 -0.6270 -0.2087 3 20 219 27 0.470 2.09 0.250 0.300 0.833 -0.0792 0.3195 4 40 268 76 0.595 5.88 0.375 0.175 2.14 0.3310 0.7690 5 80 313 121 0.635 9.35 0.415 0.135 3.07 0.4877 0.9710 6 120 371 179 0.685 13.8 0.465 0.085 5.47 0.7380 1.141 7 oO - - 0.770 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 8 Raw Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) p rco (mm DBT) A445nm 1 0 203 - 0.165 2 10 212 9 0.252 3 20 313 110 0.482 4 40 403 200 0.528 5 80 429 226 0.565 ? 6 oo - - 0.585 Buffer: pH: Processed Data Pco (mm Hg) A-A o A.-A A-A o A- -A Log A _ A o A„-A Log P CO 0.696 0.0870 0.333 0.261 -0.5829 -0.1576 8.50 0.317 0.103 3.08 0.4882 0.9295 15.5 0.363 0.057 6.37 0.8040 1.189 17.5 0.400 0.020 20.0 1.301 1.242 Appendix (1) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 9 Raw Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) p rco (mm DBT) A445nm 1 0 205 - 0.225 2 10 216 11 0.310 3 20 221 16 0.348 4 40 239 34 0.400 5 CO - - 0.605 Buffer: pH: Processed Data Pco (mm Hg) A-A 0 A.-A A-A o A- -A Log A _ A o A.-A Log P CO 0.850 0.085 0.295 0.288 -0.5404 -0.07040 1.24 0.123 0.257 0.479 -0.3200 0.09230 2.63 0.175 0.205 0.854 -0.06872 0.4197 Appendix (i) (cont'd) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: 24±0.2° C Buffer: P_ioo Vapour Pressure of Water: 290 mm DBT pH: 7.4 Experiment No.IQ Raw Data Processed Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) p rco (mm DBT) A445nm Pco (mm Hg) A-A o A-A CD A-A o A«j -A Log A _ A o A^-A Log P CO 1 0 250 - 0.290 2 15 266 16 0.326 1.23 0.036 0.500 0.0720 -1.1427 -0.1118 3 25 275 25 0.390 1.91 o . i e o 0.436 0.229 -0.6395 0.2810 4 40 286 36 0.490 2.82 0.200 0.336 0.595 -0.2253 0.4502 5 65 318 68 0.600 5.25 0.310 0.226 1.37 0.1373 0.7201 6 100 360 110 0.652 8.51 0.362 0.174 2.08 0.3182 0.9299 7 - - 0.826 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 12 Raw Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) p 'co (mm DBT) A445nm 1 0 263 - 0.190 2 10 355 102 0.415 3 30 454 191 0.455 4 80 533 270 0.480 5 - 0.540 Buffer: pH: Processed Data Pco (mm Hg) A-A o A.-A A-A o A A -A Log A - A o A»-A Log P CO 7.88 0.225 0.125 1.80 0.2553 0.8968 14.81 0.265 0.085 3.12 0.4938 1.170 20.9 0.290 0.060 4.83 0.4842 1.320 Appendix (i) Equilibrium Data for CO Binding to Substrate-free Cytochrome P-450cam. Temperature: Vapour Pressure of Water: Experiment No. 11 Raw Data Species P CO wanted (mm DBT) p t o t a l (mm DBT) p cco (mm DBT) A445nm 1 0 272 - 0.220 2 5 277 5 0.260 3 10 281 9 0.293 4 20 290 18 0.340 5 50 356 84 0.543 6 80 387 115 0.590 7 - - 0.770 Buffer: PH: Processed Data Pco (mm Hg) A-A o A-A OD A-A o Aaa -A Log A - A o Log P CO 0.387 0.040 0.510 0.0784 -1.1055 -0.4129 0.696 0.073 0.477 0.153 -0.8152 -0.1577 1.39 0.120 0.430 0.279 -0.5543 0.1435 6.49 0.323 0.227 1.42 0.1532 0.8125 8.89 0.370 0.180 2.06 0.3129 0.9489 Appendix ( i i ) Appendix ( i i ) (cont'd) -r~n—1—1—r -,—T 1 1 1 r — i " r -0 2 0? OS • • • 1 1 1 L 10 u i' 2 2 -0.3 - 0 1 i.< log Pco r 6 Experiment No. 4_ — ~~ The H i l l l o g / l o g p l o t s obtained f o r the CO-binding to s u b s t r a t e - f r e e cytochrome P-450cam a t 12°C. Appendix ( i i ) (cont'd) The H i l l log/log plots obtained for the CO-binding to substrate-free cytochrome P-450cam at 18°C. ro oo Appendix (ii) (cont'd) i«« Experiment No. 10 11 — The H i l l log/log plots obtained for the CO-binding to substrate-free cytochrome P-450cam at 24°C. Appendix ( i i i ) Determination of rate of autoxidation of the dioxygen compl of reduced, substrate-bound cytochrome P-450cam. Raw Data Expt. No. : 1 Temperature : 0 ± 0.2 °C Buffer : P-100 A =1.68 A Aoo-A -In (Aoc-A) Time (min.) 1.28 0.396 0.9263 1.0 1.45 0.230 1.4697 15 1.50 0.176 1.7323 20 1.55 0.130 2.0402 30 1.58 0.100 2.3025 36 1.62 0.0560 2.8824 55 Expt. No. : 2 Temperature : 0 ± 0.2°C Buffer : P-100 ^=1.09 A A^-A -In(A^-A) Time (Min; 0.790 0.306 1.1841 1.0 0.856 0.240 1.4271 7.0 0.920 0.176 1.7372 16 0.996 0.100 2.30 6 30 1.05 0.0460 3.0790 54 Expt. NO. : 3 Temperature : 10 + 0.2°C Buffer : P-100 A~=1.63 A Aoo-A -In (Aoo-A) Time (Min.) 1.38 0.254 1.3704 1.0 1.48 0.146 1.9241 5.0 1.53 0.100 2.3026 9.0 1.57 0.0600 2.8134 14 1.61 0.0200 3.9120 24

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