Cytochrome P-450âIts Role in Oxygen Activation for Drug Metabolism

1 Cytochrome P-450—Its Role in Oxygen Activation for Drug Metabolism R. W. ESTABROOK and J. WERRINGLOER

Downloaded by 80.82.77.83 on March 26, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch001

Department of Biochemistry, University of Texas Health Science Center, Dallas, TX 75235

At t h i s time many of our colleagues are directing their a t t e n t i o n (1,2) to e v a l u a t i n g the e x i s t e n c e of oxygen on the p l a n e t Mars - an effort d i c t a t e d by the d e s i r e to l e a r n whether any life form, as we know it, e x i s t s on that p l a n e t . What is it t h a t conf e r s on oxygen s p e c i a l p r o p e r t i e s t h a t make it so critical f o r the f u n c t i o n i n g of cellular metabolism. Much has been written on the "fitness of oxygen" (3) p o i n t i n g to the capability to metabolically reduce atmospheric oxygen to hydrogen peroxide or water by two or f o u r e l e c t r o n t r a n s f e r processes, r e s p e c t i v e l y . More important to the t o p i c of this symposium is the ability to enzymatically " a c t i v a t e " molecular oxygen, p e r m i t t i n g the i n c o r p o r a t i o n of one atom of oxygen i n t o an organic s u b s t r a t e molecule concomitant w i t h the r e d u c t i o n of the other atom of oxygen to water. The enzyme systems r e s p o n s i b l e f o r c a t a l y z i n g these types of r e a c t i o n s (Figure 1) are termed mixed-function oxidases, hydroxylases or oxygenases. In many i n s t a n c e s , the i n t r o d u c t i o n of a h y d r o x y l group to the hydrophobic s u b s t r a t e molecule provides a site f o r subsequent conjugation w i t h h y d r o p h i l i c compounds thereby i n c r e a s i n g the solubility of the product f o r its t r a n s p o r t and e x c r e t i o n from the organism. C e n t r a l to the f u n c t i o n i n g of many mixed-function o x i d a t i o n r e a c t i o n s i s a f a m i l y of hemoproteins g e n e r a l l y classified as cytochromes P-450. The present symposium is directeded to f u r t h e r our understanding of how t h i s unique hemoprotein p a r t i c i p a t e s in "oxygen activation" and " s u b s t r a t e h y d r o x y l a t i o n " . Studies w i t h the perfused r a t l i v e r by Thurman and Scholz (4j 5^) as w e l l as S i e s , et a l . (6,7) i n d i c a t e t h a t approximately 60 percent of c e l l u l a r r e s p i r a t i o n by t h i s organ i s s e n s i t i v e to i n h i b i t i o n by Antimycin A - a chemical w e l l recognized as a powerf u l i n h i b i t o r of the m i t o c h o n d r i a l r e s p i r a t o r y c h a i n . Of the r e maining 40 percent of c e l l u l a r r e s p i r a t i o n approximately h a l f i s s e n s i t i v e to the i n h i b i t o r sodium cyanide. A s i g n i f i c a n t p o r t i o n

Supported i n p a r t by a r e s e a r c h grant from the N a t i o n a l I n s t i t u t e s of Health (NIGMS - 16488).

1 Jerina; Drug Metabolism Concepts ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


2

DRUG M E T A B O L I S M CONCEPTS

of the antimycin A i n s e n s i t i v e r e s p i r a t i o n of the l i v e r c e l l i s presumed to occur by o x i d a t i v e enzymes a s s o c i a t e d w i t h the endoplasmic r e t i c u l u m , i . e . the microsomal f r a c t i o n . A c o n s i d e r a t i o n of microsomal 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 (Figure 2) r e v e a l s the f u n c t i o n i n g of a t l e a s t three f l a v o p r o t e i n s , two hemoproteins, and an i r o n c o n t a i n i n g p r o t e i n . The f l a v o p r o t e i n s f u n c t i o n as reduced p y r i d i n e n u c l e o t i d e dehydrogenases and they p a r t i c i p a t e i n the t r a n s f e r of reducing e q u i v a l e n t s to cytochrome 1>5 from NADH, v i a the f l a v o p r o t e i n ( f p i ) , NADH-cytochrome b$ reductase, or from NADPH by (fp2>, NADPH-cytochrome P-450 reductase ( 8 ) . Reduced cytochrome b$ may i n t e r a c t w i t h a r e c e n t l y i s o l a t e d i r o n cont a i n i n g p r o t e i n (9) i n the cyanide s e n s i t i v e d e s a t u r a t i o n of f a t t y acyl-Coenzyme A compounds (10,11). The reduced f l a v o p r o t e i n , f p 2 , can a l s o undergo o x i d a t i o n by f e r r i c cytochrome P-450 i n r e a c t i o n s to be described below, o r , by an as y e t p o o r l y understood r e a c t i o n , t h i s reduced f l a v o p r o t e i n has been p o s t u l a t e d (12,13) to r e a c t w i t h molecular oxygen g i v i n g r i s e to a superoxide anion f o r the i n i t i a t i o n of l i p i d p e r o x i d a t i o n or heme degradation. A t h i r d f l a v o p r o t e i n , f p 3 , p a r t i c i p a t e s i n the o x i d a t i o n of t e r t i a r y amines g i v i n g r i s e to N-oxides as described by Z i e g l e r et a l . (14,15). Of i n t e r e s t are recent r e s u l t s reported by Z i e g l e r et a l . (16) t h a t t h i s f l a v o p r o t e i n , f p 3 , may a l s o f u n c t i o n i n an oxygen dependent o x i d a t i o n of s u l f h y d r y l groups f o r the format i o n of d i s u l f i d e bonds. Cytochrome P-450 has many i n t e r e s t i n g p r o p e r t i e s that serve as a challenge to the biochemist concerned w i t h understanding the f u n c t i o n of t h i s hemoprotein as i t p a r t i c i p a t e s i n a broad spectrum of o x i d a t i v e r e a c t i o n s . I t s n a t u r a l environment i n most mammalian t i s s u e s i s the membrane s t r u c t u r e c a l l e d the endoplasmic r e t i c u l u m ; t h e r e f o r e , considerable i n t e r e s t i s d i r e c t e d to understanding the i n f l u e n c e imposed by a presumed r e s t r i c t e d m o b i l i t y of p r o t e i n molecules i n a m i l e u of l i p i d as t h i s pigment i n t e r a c t s w i t h oxygen, s u b s t r a t e molecules, and the f l a v o p r o t e i n e l e c t r o n donor. F u r t h e r , i t r e q u i r e s the s k i l l and perseverance of groups such as those l e d by Coon e t a l . (17-19) as w e l l as Lu and L e v i n (20-22) to i s o l a t e and p u r i f y v a r i o u s forms of t h i s hemoprotein f o r p h y s i c a l and chemical c h a r a c t e r i z a t i o n . Such s t u d i e s have revealed that the f a m i l y of cytochromes P-450 have molecular weights i n the range of 46,000 to 52,000 and d i s p l a y a great p r o p e n s i t y to aggregate because of t h e i r hydrophobic p r o p e r t i e s . F u r t h e r , cytochrome P-450 i s r e a d i l y i n d u c i b l e upon treatment of an animal w i t h v a r i o u s drugs or p o l y c y c l i c hydrocarbons. Although t h i s property i s p o o r l y understood i t appears to be r e s p o n s i b l e i n p a r t f o r the long observed phenomenon of drug t o l e r a n c e (23«24). As w i l l be discussed by Dr. Coon during t h i s symposium (25), cytochrome P-450 can e x i s t i n m u l t i p l e forms w i t h an ever i n c r e a s i n g r o s t e r of new p r o t e i n s i d e n t i f i e d as p u r i f i c a t i o n methodology becomes more s o p h i s t i c a t e d and w i d e l y used.

Jerina; Drug Metabolism Concepts ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


1.

ESTABROOK A N D WERRINGLOER

Cytochrome

P-450 in Oxygen Activation

3

I n i t i a l i n t e r e s t i n microsomal mixed-function o x i d a t i o n r e a c t i o n s occured i n the 1950's; such s t u d i e s were focused on three g e n e r a l areas of b i o m e d i c a l importance. Brodie and h i s c o l l a b o r a t o r s (26,27) recognized the p o t e n t i a l r o l e of h y d r o x y l a t i o n r e a c t i o n s i n the o x i d a t i v e t r a n s f o r m a t i o n of many drugs - hence t h i s r e a c t i o n system was looked upon as a general mechanism f o r d e t o x i f i c a t i o n of f o r e i g n chemicals. In c o n t r a s t , the M i l l e r s and t h e i r colleagues (28,29) were concerned w i t h the o x i d a t i v e conversion of p r e c a r c i n o g e n i c chemicals, such as p o l y c y c l i c hydrocarbons, to carcinogens - r e a c t i o n s of great p o t e n t i a l harm to the maintenance of the v i a b i l i t y of the organism. A t h i r d group, the s t e r o i d e n d o c r i n o l o g i s t s , were a c t i v e l y studying (30, 31) the o x i d a t i v e processes r e s p o n s i b l e f o r the enzymatic conv e r s i o n of c h o l e s t e r o l to g l u c o c o r t i c o i d s and m i n e r a l o c o r t i c o i d s products of c r i t i c a l importance f o r the maintenance of homeos t a s i s of the organism. We now know t h a t the general f a m i l y of hemoproteins, cytochromes P-450, p l a y p i v i t o l r o l e s i n d i r e c t i n g the o p e r a t i o n of each of these, as w e l l as other s i m i l a r r e a c t i o n s . Thus t h i s c l a s s of hemoproteins has a d u a l i t y of f u n c t i o n (Figure 3) jL.e_., i t may serve as e i t h e r a panacea or a plague f o r the c e l l . Proposed C y c l i c F u n c t i o n of Cytochrome P-450. How do we c u r r e n t l y v i s u a l i z e the f u n c t i o n of t h i s unique hemoprotein, cytochrome P-450? One p o s t u l a t e d scheme (32) i l l u s t r a t i n g the c y c l i c p a t t e r n of r e d u c t i o n and oxygenation of cytochrome P-450 as i t i n t e r a c t s w i t h s u b s t r a t e molecules, e l e c t r o n donors, and oxygen i s shown i n F i g u r e 4. B r i e f l y , these r e a c t i o n s may be summarized as f o l l o w s : ^ A. The f e r r i c hemoprotein (Fe'") can i n t e r a c t w i t h a molec u l e of s u b s t r a t e (R) r e s u l t i n g i n a complex (Fe ^»R) analogous to an enzyme - s u b s t r a t e complex. This i n t e r a c t i o n can be d i r e c t l y measured s i n c e the s u b s t r a t e molecule appears to be c l o s e l y a s s o c i a t e d w i t h the heme moiety of cytochrome P-450 r e s u l t i n g i n a s p e c t r a l p e r t u r b a t i o n measurable by e i t h e r o p t i c a l absorbance spectrophotometry or e l e c t r o n paramagnetic resonance spectrometry (33-36). The o r i e n t a t i o n of the s u b s t r a t e molecule as i t s i t s i n p r o x i m i t y of the heme i r o n , the presumed b i n d i n g s i t e f o r oxygen, remains a conjecture although the r o l e of s t e e r i n g groups on the s u b s t r a t e molecule (37) or the i n f l u e n c e of the hydrophobic environment o f the heme 738) may be considered as p o s s i b l e d i r e c t ing f o r c e s . B. The s u b s t r a t e complex of f e r r i c - cytochrome P-450 (Fe 3*R) undergoes r e d u c t i o n to a f e r r o u s cytochrome P-450 s u b s t r a t e complex (Fe ^»R) by e l e c t r o n s o r i g i n a t i n g from NADPH and t r a n s f e r r e d by the f l a v o p r o t e i n (fp2)> NADPH-cytochrome P-450 r e ductase. The q u e s t i o n of a stimultaneous two e l e c t r o n t r a n s f e r to cytochrome P-450, as suggested by Coon et a l . (39,40), or the e x i s t e n c e of two d i s c r e t e one e l e c t r o n donating s t e p s , as demons t r a t e d f o r the p u r i f i e d b a c t e r i a l cytochrome P-450 by Tyson e t +

+

+


DRUG M E T A B O L I S M

4

CONCEPTS

oxygenase 2 e ~ + Substrate + 0

Product +

2 M

(hydrophobic)

F

a

H 0 2

(hydroxylated)

Figure 1. General representation of microsomal-catalyzed oxygenase or mixed-function oxidation reactions

Conjugation i t Excretion

F A T T Y ACID DESATURATION


,NADH fp

—•fpi

>b /

3

/

N A D P H — * ffPp p2a, ^ f

-

•* X ,

x

2

V...

•

5

x

P-450

*0

2

MIXED FUNCTION OXIDATION

\

LIPID LI PI PEROXIDATION

N-OXIDATION

Figure 2. Schematic of microsomal electron transport reactions

0 ,e~ — • 2

A.

Figure 3. The duality of cytochrome P-450-catalyzed reactions

A c t i v e Drug

Inactive

Drug

0 ,e 2

B.

Precarcinogen

•

Carcinogen

CO

I 02

Figure 4. Schematic of the proposed cyclic function of cytochrome P-450. The substrate molecule is designated R. The valence state of the heme iron of cytochrome P-450 is indicated.


CO

1.


Cytochrome


a l . (41) as w e l l as Peterson (42), remains as a p o i n t o f uncert a i n t y . F o r the present d i s c u s s i o n the scheme presented i s based on the premise t h a t two separate e l e c t r o n donating r e a c t i o n s o c cur. C. Reduced cytochrome P-450 ( F e * R ) can r e a c t w i t h carbon monoxide t o form a d e r i v a t i v e which i s r e a d i l y i d e n t i f i a b l e s p e c t r o p h o t o m e t r i c a l l y by an absorbance band maximum a t about 450 nm - hence the o r i g i n (43) o f the name cytochrome P-450. A l t e r n a t i v e l y , reduced cytochrome P-450 can r e a c t w i t h oxygen t o form a complex termed (44) oxycytochrome P-450 (Fe 2»02*R). Knowledge o f the chemistry o f oxycytochrome P-450 should provide the needed c l u e t o evaluate the f i r s t step o f "oxygen a c t i v a t i o n " f o r h y d r o x y l a t i o n r e a c t i o n s ; t h e r e f o r e , much e f f o r t has been d i r e c t e d to understanding the parameters t h a t i n f l u e n c e the g e n e r a t i o n and subsequent u t i l i z a t i o n o f t h i s i n t e r m e d i a t e i n cytochrome P-450 c a t a l y z e d r e a c t i o n s . D. Oxycytochrome P-450 (Fe 2»02*R) can presumably d i s s o c i a t e to g i v e a superoxide a n i o n (02~) concomitant w i t h the r e g e n e r a t i o n of the f e r r i c hemoprotein. The r e s u l t a n t hydrogen p e r o x i d e formed by d i s m u t a t i o n o f the superoxide a n i o n may be a measure o f t h i s a b o r t i v e "uncoupling" (45,46) o f cytochrome P-450 f u n c t i o n , .i.e.., a r e a c t i o n which d i v e r t s the t e r n a r y complex o f oxygen, hemoprot e i n , and s u b s t r a t e from i t s r o l e i n oxygen a c t i v a t i o n and subsequent s u b s t r a t e h y d r o x y l a t i o n . A l t e r n a t i v e l y , the complex o f oxycytochrome P-450 may undergo f u r t h e r r e d u c t i o n t o form the equival e n t o f a p e r o x i d e a n i o n d e r i v a t i v e o f the s u b s t r a t e bound hemoprot e i n . Studies (47-50) w i t h the membrane bound cytochrome P-450 of l i v e r microsomes suggests t h a t a d o n a t i o n o f a proposed second e l e c t r o n occurs v i a cytochrome b$ (51). T h i s c o n c l u s i o n i s the b a s i s f o r e x p l a i n i n g the s y n e r g i s t i c T e f f e c t observed d u r i n g the concomitant o x i d a t i o n o f NADPH and NADH by t h i s system (52). E. The proposed p e r o x i d e a n i o n complex o f cytochrome P-450 may undergo p r o t o n a t i o n and d i s s o c i a t e as hydrogen peroxide o r i t may rearrange t o form an oxene d e r i v a t i v e (53) concomitant w i t h the r e l e a s e of water. The e x i s t e n c e o f oxygen as an oxenoid species i s c o n j e c t u r e although s t u d i e s w i t h o r g a n i c p e r o x i d e supported h y d r o x y l a t i o n r e a c t i o n s (54-58) mediated by cytochrome P-450 demonstrate the e x i s t e n c e o f an EPR species s i m i l a r t o Complex I of peroxidase as an i n t e r m e d i a t e i n the r e a c t i o n (59). C l e a r l y , more r e s e a r c h i s needed t o f i r m l y e s t a b l i s h the presence of t h i s form o f oxygen as " a c t i v e oxygen". F. L e a s t understood i s the mechanism o f d i s s o c i a t i o n o f the hydroxylated product and the r e s t o r a t i o n o f the low s p i n form of f e r r i c cytochrome P-450. I n some i n s t a n c e s (60) epoxide i n t e r mediates e x i s t , such as occurs i n those r e a c t i o n s i n v o l v i n g p o l y c y c l i c hydrocarbons (61). I n other c a s e s , product adducts r e s u l t (62-65) which impede the f u r t h e r f u n c t i o n o f cytochrome P-450 as i t p a r t i c i p a t e s i n s u b s t r a t e h y d r o x y l a t i o n r e a c t i o n s . +2

+


5

+



6


Hydrogen Peroxide Formation, The generation o f hydrogen peroxide during NADPH o x i d a t i o n by l i v e r microsomes has been r e cognized f o r a number o f years (66,67). L i t t l e a t t e n t i o n was p a i d to t h i s phenomenon - i n l a r g e p a r t because o f the a s s o c i a t i o n of c a t a l a s e w i t h the microsomal f r a c t i o n r e s u l t i n g i n the breakdown o f hydrogen peroxide as r a p i d l y as i t was formed. Recent s t u d i e s (54-58) on the p e r o x i d a t i c f u n c t i o n o f cytochrome P-450 as w e l l as t h e coupled r e a c t i o n o f hydrogen peroxide w i t h c a t a l a s e f o r the o x i d a t i o n o f a l c o h o l s (68-70) during NADPH o x i d a t i o n by l i v e r microsomes has s t i m u l a t e d a f u r t h e r c o n s i d e r a t i o n o f the r e a c t i o n s i n v o l v e d i n hydrogen peroxide formation. I n a d d i t i o n , an understanding o f the mechanism o f hydrogen peroxide generation may provide a needed c l u e f o r b e t t e r exami n i n g the p r o p e r t i e s o f " a c t i v a t e d oxygen" proposed t o be a necessary intermediate i n cytochrome P-450 c a t a l y z e d r e a c t i o n s (71). As i l l u s t r a t e d i n F i g u r e 5, the a d d i t i o n o f NADPH t o a suspension o f r a t l i v e r microsomes incubated i n the presence o f sodium a z i d e t o i n h i b i t a d v e n t i t i o u s c a t a l a s e , r e s u l t s i n a 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 oxygen. Approximately 50 percent o f the oxygen reduced can be accounted f o r as hydrogen peroxide generated during the r e a c t i o n * Repeated a d d i t i o n s of NADPH r e s u l t s i n t h e stepwise formation of equal i n c r e ments o f hydrogen peroxide i n d i c a t i n g the a b i l i t y t o a d d i t i v e l y form t h i s product. The f a i l u r e t o observe a s t o i c h i o m e t r i c amount o f hydrogen peroxide formed t o the amount o f oxygen reduced o r NADPH o x i d i z e d has been a t t r i b u t e d (72) t o the p r e sence o f "endogenous s u b s t r a t e s " a s s o c i a t e d w i t h the microsomal f r a c t i o n which can undergo mixed f u n c t i o n o x i d a t i o n r e a c t i o n s . The nature of the proposed "endogenous s u b s t r a t e s " remains t o be b e t t e r d e f i n e d . A number o f p o s s i b i l i t i e s e x i s t t o e x p l a i n the source o f hydrogen peroxide. As proposed i n the scheme presented i n F i g u r e 4 hydrogen peroxide may a r i s e e i t h e r from d i s s o c i a t i o n o f oxycytochrome P-450 o r from the two e l e c t r o n reduced form o f the t e r n a r y complex o f oxygen, s u b s t r a t e , and cytochrome P-450. A l t e r n a t i v e l y one must consider t h a t hydrogen peroxide format i o n may n o t i n v o l v e cytochrome P-450, such as by the autox i d a t i o n o f reduced f l a v o p r o t e i n s . I n order t o f i r m l y e s t a b l i s h the r o l e o f cytochrome P-450 i n hydrogen peroxide gene r a t i o n a s e r i e s o f experiments were c a r r i e d out t o examine the i n f l u e n c e o f i n h i b i t o r s o f cytochrome P-450 f u n c t i o n , such as carbon monoxide o r metyrapone, as w e l l as determine the i n f l u e n c e o f temperature, pH, and the e f f e c t o f v a r i o u s l e v e l s o f NADPH on the r e a c t i o n . I n each i n s t a n c e comparat i v e s t u d i e s were c a r r i e d out t o determine the changes observed i n the r a t e o f N-demethylation o f ethylmorphine - a s u b s t r a t e recognized t o undergo o x i d a t i v e t r a n s f o r m a t i o n c a t a l y z e d by cytochrome P-450.


1.

ESTABROOK A N D W E R R I N G L O E R

Cytochrome


7

n moles/min/mg

9.3

if ^no

u u n MM n U g


2

73liMNADPHQuid. 72 M Og utillztd M

min. 2 mg

Pb

liver m i c r o t o m e t / m l

Figure 5. The formation of hydrogen peroxide during NADPH oxidation. Liver microsomes from phenobarbital-treated rats were incubated at 2 mg of protein per ml in a reaction medium containing 50mM tris-chloride buffer, pH 7.5, 150mM KCl, 10 mM MgCl and ImM NaN . At times equal to 0 and 10 min, aliquots of NADPH were added to initiate the reaction. In the experiment shown in dashes, NADPH was added only at 0 time. Samples were removed at the points indicated and after addition to trichloroacetic acid the concentration of hydrogen peroxide formed was determined colorometrically with potassium thiocyanate and ferrous ammonium sulfate (S3). Oxygen use was measured in a comparable series of experiments polarographically, and NADPH oxidation was determined spectrophotometrically. 2>

s

y



8


CONCEPTS

Incubation o f l i v e r microsomes i n a v e s s e l designed t o m a i n t a i n f i x e d r a t i o s o f carbon monoxide t o oxygen r e s u l t s i n an e q u i v a l e n t i n c r e a s e i n i n h i b i t i o n o f both hydrogen peroxide formation and ethylmorphine N-demethylation as the concentrat i o n o f carbon monoxide i n c r e a s e s r e l a t i v e t o oxygen as shown i n F i g u r e 6. The o b s e r v a t i o n t h a t hydrogen peroxide formation i s i n h i b i t e d by carbon monoxide i s strong presumptive evidence f o r the r o l e o f cytochrome P-450 i n t h i s r e a c t i o n (73). The f a c t t h a t t h e extent o f i n h i b i t i o n i s e f f e c t i v e l y i d e n t i c a l t o t h a t observed f o r the metabolism o f ethylmorphine r e i n f o r c e s t h i s c o n c l u s i o n . However, t o date no photochemical a c t i o n spectrum s t u d i e s (74,75) have been c a r r i e d out t o c o n f i r m the r o l e o f c y t o chrome P-450 i n the formation o f hydrogen peroxide a s has been done f o r other s u b s t r a t e s o x i d a t i v e l y metabolized by t h i s hemop r o t e i n . I t i s o f i n t e r e s t t o note t h a t t h e degree o f carbon monoxide i n h i b i t i o n i s independent o f the presence o f NADH where a s y n e r g i s t i c (47-50) e f f e c t on product formation d u r i n g t h e NADPH supported r e a c t i o n may occur (see below). Studies w i t h t h e i n h i b i t o r metyrapone ( 2 - m e t h y l - l , 2 - b i s ( 3 p y r i d y l ) - l - p r o p a n o n e ) are more e q u i v o c a l s i n c e hydrogen peroxide formation i s maximally i n h i b i t e d about 50 percent by t h i s compound whereas the metabolism o f ethylmorphine i s g r e a t e r than 90 percent i n h i b i t e d (Figure 7 ) . One p o s s i b l e e x p l a n a t i o n f o r t h i s d i f f e r e n c e r e l a t e s t o the o b s e r v a t i o n t h a t o n l y 50 percent o f t h e cytochrome P-450 a s s o c i a t e d w i t h l i v e r microsomes from p h e n o b a r b i t a l t r e a t e d r a t s r e a c t s w i t h metyrapone (76) t o form a s p e c t r a l l y i d e n t i f i a b l e complex. This would imply t h a t a l l forms o f c y t o chrome P-450 present i n l i v e r microsomes can p a r t i c i p a t e i n hydrogen peroxide formation whereas a unique metyrapone b i n d i n g form f u n c t i o n s i n ethylmorphine N-demethylation r e a c t i o n s . C l e a r l y more experiments w i l l be r e q u i r e d t o support t h i s hypothesis. An examination o f the i n f l u e n c e o f v a r y i n g suboptimal steady s t a t e l e v e l s o f NADPH shows (Figure 8) t h a t the apparent Km's f o r NADPH r e q u i r e d t o support the generation o f hydrogen peroxide and t h e N-demethylation o f ethylmorphine a r e i d e n t i c a l . L i k e w i s e s t u d i e s on the i n f l u e n c e o f temperature (Figure 9) on the r a t e s o f t h e two r e a c t i o n s shows an e q u i v a l e n t c a l c u l a t e d energy o f a c t i v a t i o n o f approximately 20 k i l o c a l o r i e s per degree. Thus a wide range o f d i f f e r e n t f a c t o r s have been examined t o e s t a b l i s h the s i m i l a r i t y o f r e a c t i o n s i n which c y t o chrome P-450 serves as the pigment i n t e r a c t i n g w i t h oxygen f o r the formation o f hydrogen peroxide. The q u e s t i o n remains unanswered, however, as t o the mechanism o f hydrogen peroxide gene r a t i o n , i..je. by d i s m u t a t i o n o f t h e superoxide anion d i s s o c i a t i n g from oxycytochrome P-450 o r by p r o t o n a t i o n of the proposed two e l e c t r o n reduced s t a t e e q u i v a l e n t t o the peroxide anion complex of cytochrome P-450.



Cytochrome

PDnn. IPT PRODUCT

IOC

HCHO

u n "2 2

• • • A

U

>


§

AnniTinMC ADDITIONS o o A

80

P-450 in Oxygen

EM EM EM

CONTROL , ACTIVITY - i , -i)

( n m o l e s

m i n

NADPH NADPH NADPH+ NADH

13.3 10.7 23.5

EM

NADPH NADPH NADPH+ NADH NADPH+ NADH

9.2 10.0 10.2 10.0

1

1

EM

Activation

m q

60-

o 8

4 0

O

20

i

1

1

1

1

1

>

RATIO

C

%

Figure 6. The inhibition by carbon monoxide of the generation of hydrogen peroxide and the N-demethylation of ethyl morphine. A series of experiments was carried out as described in Figure 5 in a reaction vessel designed to permit equilibration with various gas mixtures of carbon monoxide, oxygen, and nitrogen. The concentration of oxygen was maintained at 20% for all experiments. The reaction mixture was supplemented by adding 5mM sodium isocitrate, 0.5 units of isocitrate dehydrogenase per ml, 2mM of 5'AMP, and 5/AM rotenone. The reaction was initiated by adding 200fiM NADPH and 200[xM NADPH where indicated. Samples were removed every 30 sec for the initial 5 min of the reaction and analyzed for hydrogen peroxide (S3) or formaldehyde (84;.


10


CONCEPTS

EM(HCHO)

1412.

10-

H

E


I

2°2

8-

Figure 7. The effect of metyrapone 6on the rate of hydrogen peroxide for-1 e mation as compared with the rate of 4'N-demethylation of ethylmorphine. Liver microsomes from phenobarbital< treated rats were incubated at 1 me of 2protein per ml in the reaction medium described in Figure 5 supplemented with an NADPH generating system (cf. Figure 6). Varying concentrations of metyrapone were added as indicated. The initial rates of product formation were determined as described in Figure 6.

0 .02

.1

.5 2

0 .02

METYRAPONE

.1

.5

2

(mM)

80-

Figure 8. The influence of varying g 60steady state concentrations of NADPH ^> on the rate of formation of hydrogen ^ peroxide and the N-demethylation of § ethylmorphine. 2 40Liver microsomes from phenobarbitaU &. treated rats were incubated in a reaction medium as described in Figure 5 supplemented with 5mM sodium isocitrate and 2 0 0.5 units of isocitrate dehydrogenase per ml. Where indicated, 5mM ethylmorphine was present. Varying concentrations of NADPH were added to initiate the reaction, and the rate of hydrogen peroxide or formaldehyde formed was determined as described in Figure 6.

• X

// if il §[

In m I HCHO o-«o

• ' ' • | 5

| 10 >)M

NADPH


H 0 2

I 15

2


1. E S T A B R O O K A N D W E R R I N G L O E R

Cytochrome


11

The I n f l u e n c e of Substrates o f Cytochrome P-450 on t h e Generation o f Hydrogen Peroxide. Presumably oxycytochrome P-450 serves a t a p i v i t o l p o i n t i n the c y c l i c f u n c t i o n o f cytochrome P-450 ( c f . F i g u r e 4 ) . T h i s t e r n a r y complex o f oxygen, s u b s t r a t e and cytochrome P-450 can undergo d i s s o c i a t i o n t o g i v e r i s e t o hydrogen peroxide r e g e n e r a t i n g t h e complex o f f e r r i c cytochrome P-450 w i t h s u b s t r a t e o r i t can proceed, by a mechanism as y e t unknown, t o a c t i v a t e oxygen f o r i n s e r t i o n i n t o the s u b s t r a t e r e s u l t i n g i n the formation o f a hydroxylated product and t h e low s p i n uncomplexed form o f f e r r i c cytochrome P-450. Therefore i t was o f i n t e r e s t t o c a r r y out experiments t o evaluate the i n f l u ence o f v a r i o u s s u b s t r a t e s o f cytochrome P-450, known t o undergo enzymatic h y d r o x y l a t i o n , and t o determine how such s u b s t r a t e s i n f l u e n c e t h e r a t e o f hydrogen peroxide formation. As i l l u s t r a ted i n F i g u r e 10, the presence o f ethylmorphine markedly a t tenuated t h e extent of hydrogen peroxide formed when a l i m i t i n g amount o f NADPH i s added t o i n i t i a t e the r e a c t i o n ( c f . F i g u r e 5 ) . In a d d i t i o n a s m a l l b u t r e p r o d u c i b l e i n h i b i t i o n o f t h e i n i t i a l r a t e o f hydrogen peroxide formation was observed. The decrease i n the extent o f hydrogen peroxide formation i n the presence o f ethylmorphine can be a t t r i b u t e d i n l a r g e p a r t t o t h e s t i m u l a t i o n of NADPH o x i d a t i o n observed i n t h e presence o f t h i s s u b s t r a t e , I.e. a n e a r l y a d d i t i v e e f f e c t o f NADPH o x i d a t i o n occurs when a Ndemethylation r e a c t i o n f u n c t i o n s concomitant w i t h hydrogen p e r oxide formation. Of i n t e r e s t i s the o b s e r v a t i o n t h a t a slow but p e r c e p t i b l e r a t e o f N-demethylation o f ethylmorphine cont i n u e s a f t e r the t o t a l o x i d a t i o n o f NADPH. T h i s slow r e a c t i o n ( o c c u r r i n g a f t e r 4 minutes i n t h e experiments shown i n F i g u r e 10) appears r e l a t e d t o a s t i m u l a t i o n i n the r a t e o f u t i l i z a t i o n of hydrogen peroxide i n the presence o f sodium a z i d e . As d i s cussed below, the a b i l i t y t o support the o x i d a t i v e metabolism o f a v a r i e t y o f s u b s t r a t e s by hydrogen peroxide, i n the absence o f reducing e q u i v a l e n t s generated from NADPH, d i r e c t l y demonstrates the p e r o x i d a t i c f u n c t i o n o f cytochrome P-450. A f u r t h e r s e r i e s o f experiments were c a r r i e d o u t t o examine the e f f e c t o f other s u b s t r a t e s on t h e r a t e o f formation o f hydrogen peroxide as summarized i n F i g u r e 11. I n t h i s case NADPH c o n c e n t r a t i o n was maintained by the a d d i t i o n o f sodium i s o c i t r a t e and i s o c i t r a t e dehydrogenase. I n agreement w i t h t h e experimental r e s u l t s d e s c r i b e d i n F i g u r e 10, the presence o f ethylmorphine r e s u l t e d i n approximately a 20 percent i n h i b i t i o n o f the r a t e o f hydrogen peroxide f o r m a t i o n . I n c o n t r a s t , s u b s t r a t e s o f c y t o chrome P-450 such as benzphetamine and h e x o b a r b i t a l caused a marked s t i m u l a t i o n i n the r a t e o f hydrogen peroxide f o r m a t i o n . U l l r i c h and D i e h l (45), Hildebrandt e t a l (46) and Werringloer e t a l (70) have d e s c r i b e d t h e a b i l i t y o f v a r i o u s compounds t o serve as "uncouplers" o f cytochrome P-450 f u n c t i o n , chemicals which s t i m u l a t e the r a t e o f NADPH o x i d a t i o n and oxygen u t i l i z a t i o n w i t h out a corresponding i n c r e a s e i n the r a t e o f s u b s t r a t e h y d r o x y l a t i o n . There appear t o be two c l a s s e s o f such "uncouplers"; those


12


CONCEPTS

100 50 •X.

V o Z> Q O

or o_

V

HCHO

1

1

10 5

20 Kcal udegree" jimole" X

H 0 2

2

21 Kcal xdegree" jtmole"

1


1

Figure 9. The effect of varying temperatures on the rate of hydrogen peroxide formation and the rate of Ndemetnylation of ethylmorphine. A series of experiments similar to those described in Figure 6 were carried out at the temperatures indicated.

27°

37° 3.2

33

17°

3.4

3.5, 3.6 J-.IO 5

80—EM 60-

40-

Figure 10. The influence of ethylmorphine on the rate and extent of hydrogen peroxide formation in the presence of a limiting concentration of NADPH. A series of experiments similar to those described in Figure 5 were carried out in the presence or absence of 5mM ethylmorphine or ImM sodium azide. The amount of hydrogen peroxide or formaldehyde formed * were determined as described in Figure 6. An NADPH generating system was omitted during this series of experiments. The reactions were initiated by addition of 145uM NADPH.

20•

+EM

+ Az 80-Az 60-

40-

20-

10 TIME (minutes)


1.


Cytochrome


13


compounds t h a t r e s u l t i n an i n c r e a s e d r a t e o f f o r m a t i o n o f hydrogen peroxide where t h e i n c r e a s e d r a t e o f u t i l i z a t i o n o f oxygen i s accompanied by an e q u i v a l e n t i n c r e a s e i n the r a t e o f NADPH o x i d a t i o n (such as h e x o b a r b i t a l o r benzphetamine) and a second type o f "uncoupling" where non-metabolized s u b s t r a t e s such as p e r f l u o r i nated, a l i p h a t i c f l u o r o c a r b o n s (45) o r compounds l i k e halothane (Werringloer, J and Estabrook, R.W., unpublished r e s u l t s ) r e s u l t i n a f a i l u r e t o see any a d d i t i o n a l hydrogen peroxide formed upon s t i m u l a t i o n o f oxygen u t i l i z a t i o n , l*e., two moles o f NADPH a r e u t i l i z e d f o r each a d d i t i o n a l mole o f oxygen reduced o f f s e t t i n g the s t o i c h i o m e t r y o f 1 mole o f oxygen u t i l i z e d p e r mole o f NADPH o x i d i z e d . The d e t a i l s o f how "uncouplers" d i v e r t the f u n c t i o n o f cytochrome P-450 i n a presumed a b o r t i v e r e a c t i o n r e s u l t i n g i n t h e d i s s i p a t i o n o f the oxycytochrome P-450 t e r n a r y complex remains as a c h a l l e n g e f o r f u r t h e r experimental examination. E l e c t r o n paramagnetic resonance s t u d i e s . The above d i s c u s s i o n centers on the p o t e n t i a l r o l e o f a superoxide anion as an i n t e r m e d i a t e i n the formation o f hydrogen peroxide d u r i n g NADPH o x i d a t i o n by l i v e r microsomes. Since the superoxide anion i s a f r e e r a d i c a l i t should be d e t e c t a b l e by e l e c t r o n p a r a magnetic resonance spectroscopy (77). When l i v e r microsomes are incubated i n an oxygen s a t u r a t e d b u f f e r i n the presence of sodium a z i d e and a sample i s r a p i d l y f r o z e n i n l i q u i d n i t r o g e n soon a f t e r i n i t i a t i o n o f the r e a c t i o n by the a d d i t i o n of NADPH, examination by EPR spectroscopy r e v e a l s t h e f o r m a t i o n of a s i g n a l a t about g • 2.0 as shown i n F i g u r e 12. The gene r a t i o n o f t h i s new EPR s i g n a l , however, does n o t appear t o be r e l a t e d t o hydrogen peroxide f o r m a t i o n o r t h e g e n e r a t i o n o f a f r e e r a d i c a l o f the superoxide anion type. The same EPR s i g n a l a t g » 2.0 i s obtained i f NADH i s employed r a t h e r than NADPH and t h e s i g n a l i s u n a l t e r e d i f sodium a z i d e i s omitted from the medium when reduced p y r i d i n e n u c l e o t i d e i s added. F u r t h e r the s i g n a l remains when the f r o z e n sample of microsomes i s warmed from the temperature o f l i q u i d n i t r o g e n t o -10° and i t s power s a t u r a t i o n c h a r a c t e r i s t i c s resemble those d e s c r i b e d f o r a s i m i l a r f r e e r a d i c a l s i g n a l d e s c r i b e d by Iyanagi and Mason (78) which they a t t r i b u t e d t o a f l a v i n f r e e r a d i c a l generated during t h e f u n c t i o n o f the microsomal f l a v o p r o t e i n , NADPH-cytochrome c reductase. Thus no p o s i t i v e evidence f o r the presence o f the superoxide anion c o u l d be obtained by examining l i v e r microsomes by EPR spectroscopy. A number o f exp l a n a t i o n s c o u l d be o f f e r e d t o r a t i o n a l i z e t h i s n e g a t i v e r e s u l t , such as the presence of an a c t i v e superoxide dismutase a s s o c i a t e d w i t h the microsomal f r a c t i o n ; evenso, t h i s approach to d e f i n e the presence o f the superoxide anion does remain t o be f u r t h e r e x p l o r e d . NADH Synergism. A number o f years ago (47,48) i t was r e cognized t h a t NADH o x i d a t i o n by l i v e r microsomes concomitant



20-


E

16 Figure 11. The "uncoupling effect" of various sub-c strates on the rate of gen- E eration of hydrogen peroxide during NADPH 12oxidation by rat liver microsomes. A series of experiments were carried out using liver micro- 8somes from phenobarbitaU CM treated rats incubated in I a reaction mixture as described in Figure 7 containing an o 4NADPH generating system.£ c Where indicated 5mM ethylmorphine (EM), ImM benzphetamine (BPh), or 2mM hexobarbital (Hx) were added to the reaction mixture.

E M

B

P

h

Hx

ImM

-

AZIDE

NADPH

+ NADPH

2600

2800

3000

3200

3400 H

(Oersted)

Figure 12. Changes in the electron paramagnetic resonance signals of liver microsomes associated with the initiation of NADPH oxidation by liver microsomes. Liver microsomes from phenobarbital-treated rats were suspended at 10 mg protein per ml in an oxygenated reaction medium containing 50mM tris-chbride buffer, pH 7.5, 150mU KCl, lOmU MgCL, and ImM sodium azide. An aliquot was removed, placed in a calibrated EPR tube, and rapidly frozen in liquid nitrogen (upper curve). NADPH (final concentration, 200fiM) was then added to the suspension and an aliquot removed and frozen within 15 sec (lower curve). First derivative spectra were obtained with an E-4 Varian EPR with the samples maintained at the temperature of liquid nitrogen.



1.

ESTABROOK AND WERRINGLOER

Cytochrome


15

w i t h t h e o x i d a t i o n o f l i m i t i n g c o n c e n t r a t i o n s o f NADPH r e s u l t e d i n a marked enhancement i n the o x i d a t i v e metabolism o f s u b s t r a t e s of cytochrome P-450 such as aminopyrine and ethylmorphine. As shown i n F i g u r e 13 the i n i t i a l r a t e o f N-demethylation o f e t h y l morphine by r a t l i v e r microsomes i s n e a r l y doubled when NADH i s added i n the presence o f NADPH. T h i s s t i m u l a t i o n o f a c t i v i t y i s r e f l e c t e d not o n l y i n the r a t e but a l s o t h e extent o f product formed. T h i s observed e f f e c t o f NADH g r e a t l y exceeds an a d d i t i v e e f f e c t o f the a c t i o n of the two forms o f reduced p y r i d i n e n u c l e o t i d e s s i n c e the N-demethylation of ethylmorphine i s r e l a t i v e l y slow i n the presence of NADH a l o n e . This type o f experiment t o gether w i t h measurements on changes i n the extent of steady s t a t e r e d u c t i o n o f cytochrome b$ (51) have served as one o f the foundat i o n s f o r the development of the c y c l i c scheme o f cytochrome P-450 f u n c t i o n as d e s c r i b e d i n F i g u r e 4. I t i s proposed t h a t NADH serves t o donate, v i a the f l a v o p r o t e i n reductase and c y t o chrome b^, the e l e c t r o n r e q u i r e d t o reduce oxycytochrome P-450 to an i n t e r m e d i a t e w i t h an o x i d a t i o n s t a t e e q u i v a l e n t t o a p e r oxide a n i o n form. Of i n t e r e s t was the q u e s t i o n whether a s i m i l a r NADH synergism would be observed d u r i n g t h e g e n e r a t i o n o f hydrogen p e r o x i d e a s s o c i a t e d w i t h the o x i d a t i o n o f NADPH. I n t h i s way i t may be p o s s i b l e t o g a i n some i n s i g h t i n t o which form o f the oxygen comp l e x o f cytochrome P-450 serves as t h e source o f hydrogen p e r oxide. As shown i n F i g u r e 14, NADH does not have a s i g n i f i c a n t syne r g i s t i c a f f e c t on the g e n e r a t i o n o f hydrogen p e r o x i d e d u r i n g NADPH o x i d a t i o n . A s m a l l i n c r e a s e i n the i n i t i a l r a t e o f hydrogen peroxide f o r m a t i o n i s observed i n the presence o f NADH and NADPH, r e l a t i v e t o the r a t e observed when NADPH alone i s used as the donor o f r e d u c i n g e q u i v a l e n t s , b u t t h i s i n c r e a s e i s a p p r o x i mately equal t o the r a t e observed when NADH i s used t o support the r e a c t i o n . The extent o f hydrogen peroxide f o r m a t i o n observed does i n c r e a s e measurably b u t t h i s may be a t t r i b u t e d i n l a r g e p a r t to a " s p a r i n g e f f e c t " of r e d u c i n g e q u i v a l e n t s from NADPH used t o support the mixed f u n c t i o n o x i d a t i o n o f "endogenous s u b s t r a t e s " as d e s c r i b e d i n an e a r l i e r s e c t i o n o f t h i s paper. On the b a s i s o f these r e s u l t s i t i s concluded t h a t the same type o f NADH synerg i s t i c e f f e c t c h a r a c t e r i s t i c o f h y d r o x y l a t i o n r e a c t i o n s mediated by cytochrome P-450 does not f u n c t i o n f o r the g e n e r a t i o n o f hydrogen p e r o x i d e . T h i s o b s e r v a t i o n may r e s u l t from an a l t e r a t i o n o f the r o l e f o r a needed second e l e c t r o n t o form the p e r o x i d e a n i o n form o f cytochrome P-450 o r from t h e dominant r o l e o f t h e d i s s o c i a t i o n o f oxycytochrome P-450 g i v i n g r i s e t o the superoxide a n i o n . The l a t t e r h y p o t h e s i s would p r e c l u d e the need f o r donation o f a second e l e c t r o n i n the f o r m a t i o n o f hydrogen p e r o x i d e . The p e r o x i d a t i c f u n c t i o n o f cytochrome P-450. Hrycay and O'Brien (54-56) have d e s c r i b e d experiments which demonstrate t h e



CONCEPTS

72pM NAOPH + 96JIM NADH

A" 4-


4-

2

4

6 8 TIME (minutes)

10

12

Figure 13.

The synergistic effect of NADH on the NADPH-dependent N-demethylation of ethylmorphine. Liver microsomes from phenoharhital-treated rats were incubated at 1 mg of protein per ml in the presence of 5mM ethylmorphine. NADPH and NADH were added in the concentrations indicated to initiate the reaction. Samples were removed and the amount of formaldehyde formed determined by the Nash reagent (84). Sodium azide and an NADPH generating system were omitted from the reaction medium.



1.


Cytochrome


17

TIME (minutes)

Figure 14. The effect of varying concentrations of NADH on the rate and extent of formation of hydrogen peroxide associated with the oxidation of a limiting concentration of NADPH by liver microsomes. A series of experiments similar to those described in Figure 5 were carried out in the presence of NADPH and NADH as indicated.



18


CONCEPTS

a b i l i t y o f cytochrome P-450 o f l i v e r microsomes t o serve as a peroxidase. About the same t i m e , Kadlubar e t a l (57) demonstrated t h e a b i l i t y o f o r g a n i c hydroperoxides t o support N-demethylat i o n r e a c t i o n s when l i v e r microsomes a r e added t o the r e a c t i o n mixture. R e c e n t l y c o n s i d e r a b l e i n t e r e s t has centered on the mechanism o f these peroxide c a t a l y z e d r e a c t i o n s as they i n v o l v e cytochrome P-450 s i n c e such r e a c t i o n s may p r o v i d e a d i f f e r e n t means of e v a l u a t i n g the presence and nature o f " a c t i v e oxygen". When l i v e r microsomes i n t e r a c t w i t h ethylmorphine and hydrogen p e r o x i d e , i n the presence o f sodium a z i d e t o i n h i b i t contamina t i n g c a t a l a s e , one observes (Figure 15) a s t o i c h i o m e t r i c u t i l i z a t i o n o f hydrogen p e r o x i d e concomitant w i t h the f o r m a t i o n o f formaldehyde - the product o f N-demethylation o f ethylmorphine (58). T h i s r e a c t i o n i s n o t dependent on the presence o f oxygen and i s not i n h i b i t e d by carbon monoxide. R e l a t i v e l y h i g h l e v e l s o f hydrogen p e r o x i d e a r e r e q u i r e d t o o b t a i n maximal r a t e s o f the Ndemethylation r e a c t i o n and an apparent Km o f approximately 20 mM f o r hydrogen p e r o x i d e has been determined (58). Under o p t i m a l cond i t i o n s r a t e s o f N-demethylation a r e observed i n the presence o f hydrogen peroxide which a r e g r e a t e r than 50 f o l d the r a t e o b t a i n e d when NADPH supports the o x i d a t i v e t r a n s f o r m a t i o n of ethylmorphine. Both o p t i c a l (Figure 16) and e l e c t r o n paramagnetic resonance (Figure 17) spectroscopy s t u d i e s (59) r e v e a l e d changes i n the o x i d a t i o n p r o p e r t i e s of microsomal cytochrome P-450 upon a d d i t i o n o f o r g a n i c hydroperoxides. T r a n s i e n t changes i n the o p t i c a l s p e c t r a were observed upon a d d i t i o n o f cumene hydroperoxide and these s p e c t r a l changes d i f f e r e d from those r e p o r t e d (44) f o r oxycytochrome P-450 observed d u r i n g the a e r o b i c steady s t a t e o x i d a t i o n o f NADPH by l i v e r microsomes. F u r t h e r , e l e c t r o n paramagnetic resonance s t u d i e s (Figure 17) r e v e a l e d the f o r m a t i o n of EPR s i g n a l s i n the a r e a o f g * 2.0 d i f f e r e n t from those d e s c r i b e d i n F i g u r e 12. Indeed, t h e unique nature o f the EPR s i g n a l s observed a t about g • 2.0 when cumene hydroperoxide r e a c t s w i t h microsomal cytochrome P-450 resemble t h e s i g n a l s observed when hydrogen peroxide r e a c t s w i t h metmyoglobin o r cytochrome c peroxidase (79, 80). From these r e s u l t s i t was concluded (59) t h a t cytochrome P-450 may o b t a i n h i g h e r v a l e n c e s t a t e s o f the heme i r o n i n a manner analogous t o t h a t proposed by Yamazaki e t a l (81) f o r p e r o x i dases. T h i s would suggest t h a t the e q u i v a l e n t o f aiT^oxene" form of oxygen might be formed d u r i n g t h e a c t i v a t i o n o f oxygen by cytochrome P-450 as i t f u n c t i o n s i n h y d r o x y l a t i o n r e a c t i o n s . I t i s o f i n t e r e s t t o note t h a t the EPR s i g n a l observed when p e r o x i des i n t e r a c t w i t h l i v e r microsomal cytochrome P-450 i s v e r y s i m i l a r t o t h a t r e p o r t e d by Vanneste e t a l (82) f o r the complex o f oxygen w i t h reduced cytochrome P-450 o b t a i n e d i n r a p i d f r e e z e quenching experiments. Concluding Remarks. The experimental r e s u l t s d e s c r i b e d i n the preceeding s e c t i o n s have been presented t o p r o v i d e some background as t o the present s t a t u s of our understanding o f " a c t i v e


1.


R A ITI IUOH -C H -Q RM 2

2

3

too

!{

g g 4

.

A

2

2

u t i l i 2 e
,

3.3

nmoles

J HCH0

o ? g .. liberated

It

^ £ ^

(plus

I A '

xmin '

40-

J*

s

20

i i ,

ft X " 4

8

PB microsomes/ml,

5mM

ETHYLMORPHINE) H 0 * 1 utilized I 65 2

2mg

19

* i|

b e r a t e d

0.4 °*

,

• • 8

Cytochrome

12 I mM

2

16 20 TIME (minutes) Azide :

A o a

aerob ARGON CARBON MONOXIDE

Figure 15.

The hydrogen peroxide-dependent N-demethylation of ethylmorphine as catalyzed by liver microsomes. Liver microsomes from phenobarbital-treated rats were diluted to 2 mg protein per ml in a reaction mixture similar to that described in Figure 5. Where indicated, 5mM ethylmorphine was added. The reaction was initiated by adding lOOuM. hydrogen peroxide. Special reaction vessels were used to permit equilibration with various gas mixtures and to permit sampling the reaction at the times indicated. The changes in the concentrations of hydrogen peroxide used or formaldehyde formed were determined colorometrically.


20


Z

1

41


-^%Sh

442-500nm -0.005

^^^^^^

I

\vj W V422

-0.06-

400

579

^

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|j

—0.005

g

--OJOIO

8
iM P-450) 50>iM CumtntOOH

450

500

550

600

WAVELENGTH (not)

Figure 16.

Spectrophotometric measurement of changes occurring during the oxidation of cumene hydroperoxide by rat liver microsomes. Liver microsomes from phenobarbital-treated animals were diluted to a protein concentration of 3 mg per ml in a reaction mixture containing 50mM tris-chloride buffer, pH 7.5, 150mM KCl, and 5mM MgCl . After recording a baseline of equal light absorbance, 50fiM cumene hydroperoxide was added to the contents of the sample cuvette, and the change of absorbance with time was determined by repetitive scanning at 2 nm per sec. (Insert) The kinetics of formation and decay of the absorbance at 442 nm at different concentrations of cumene hydroperoxide. 2




1.

Cytochrome


21

Biochemical and Biophysical Research Communications

Figure 17. Changes in the EPR spectra associated with the interaction of cumene hydroperoxide and liver microsomes. Experiments were carried out using liver microsomes from phenobarbital-treated rabbits. (A) no additions; (B) after adding 1.5mM cumene hydroperoxide; (C) after adding 46mM cyclohexane followed by cumene hydroperoxide; (D) 50/AM metmyoglobin mixed with 1.5mM cumene hydroperoxide; (E) a baseline obtained in the absence of liver microsomes

HO

Oxonium ion

+.p..

H0 2

fOxene

> e

2 Oxygen w

—

H

+

H0

H 0

2

2

Perhydroxyl radical

H+

HO*

2

Hydrogen peroxide

pK *4.5

7*

pK *ll.8

0

0

+

H +H0 Superoxide ion

2

Hydroperoxide Hy ion

I +

H +0

2

Peroxide ion

Hydroxy I radical

^ H M+

2

0 Water

I PK 7 S

0

H++OH" Hydroxyl ion

Figure 18. The possible various states of oxygen during its stepwise reduction to water



22


CONCEPTS

oxygen" formed d u r i n g cytochrome P-450 c a t a l y z e d r e a c t i o n s . Oxygen may e x i s t i n a number o f o x i d a t i o n s t a t e s as i l l u s t r a t e d i n F i g u r e 18. As yet i t i s not p o s s i b l e t o a s s i g n a s p e c i f i c f u n c t i o n i n h y d r o x y l a t i o n r e a c t i o n s f o r the superoxide a n i o n , peroxide a n i o n , oxene o r t h e i r protonated forms. The a b i l i t y to observe the formation of hydrogen peroxide during NADPH o x i d a t i o n by l i v e r microsomes and to a s s i g n a r o l e f o r cytochrome P-450 i n the generation of hydrogen peroxide lends credence to the scheme p r o posed i n F i g u r e 4. F u r t h e r the experimental observations obtained during the p e r o x i d a t i c f u n c t i o n of cytochrome P-450 a l l p o i n t to the c e n t r a l r o l e f o r a peroxide anion complex of t h i s pigment p l a y i n g a c e n t r a l and p i v i t o l r o l e i n the a c t i v a t i o n of oxygen. New approaches and more experiments w i l l be r e q u i r e d to b e t t e r e s t a b l i s h the v a l i d i t y of the c u r r e n t hypotheses on the proposed intermediates formed during cytochrome P-450 f u n c t i o n . Oxygen i s c e n t r a l to the maintalliances of the l i f e of higher organisms as we now understand i t . In a d d i t i o n to r e a c t i o n s i n the c e l l where oxygen i s reduced to water concomitant w i t h the c o n s e r v a t i o n of energy i n the form of ATP, as c a t a l y z e d by the m i t o c h o n d r i a l r e s p i r a t o r y c h a i n , oxygen p l a y s a key r o l e i n the s y n t h e s i s and degradation of a wide d i v e r s i t y of n a t u r a l compounds, such as s t e r o i d s , as w e l l as f o r e i g n chemical agents. For these l a t t e r r e a c t i o n s cytochrome P-450 p l a y s a c r i t i c a l r o l e s e r v i n g t o a c t i v a t e oxygen f o r i n t e r a c t i o n w i t h these organic s u b s t r a t e s . Undoubtably the f u t u r e w i l l provide many new s u r p r i s e s as we delve deeper to g a i n a f u l l e r understanding of t h i s important enzyme system.

Literature Cited 1.

2.

3.

4. 5. 6. 7.

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Cytochrome P-450 in Oxygen Activation

23

8. Masters, B.S.S., Baron, J., T a y l o r , W.E., Isaacson, E . I . , and L o S p a l l u t o , J., J. Biol. Chem. (1971) 246, 4143-4150. 9. S t r i t t m a t t e r , P., Spatz, L., Corcoran, D., Rogers, M.J., Setlow, B., and R e d l i n , R., Proc. Nat'l. Acad.Sci.(USA) (1974) 71, 4565-4569. 10. Holloway, P.W., Biochemistry (1971) 10, 1556-1560. 11. Oshino, N., Imai, Y., and Sato, R., J. Biochem. (Tokyo) (1971) 69, 155-168. 12. K e l l o g g , E.W. and Fridovich, I . , J. Biol. Chem. (1975) 250, 8812-8817. 13. Masters, B.S.S. and Schacter, B.A., Annals o f Clinical Research (1976), Vol. 8, s u p p l . 17, 18-27. 14. Z i e g l e r , D.M. and Mitchell C.H., A r c h i v e s Biochem. Biophys. (1972) 150, 116-125. 15. Kadlubar, F.F. and Ziegler, D.M., A r c h i v e s Biochem. Biophys. (1974) 162, 83-92. 16. Z i e g l e r , D.M., Hyslop, R.M., and Poulsen, L.L., HoppeS e y l e r ' s Z. Physiol. Chem. (1976) 357, 1067. 17. Coon, M.J. and Lu, A.Y.H., in "Microsomes and Drug O x i d a t i o n s " , e d i t e d by Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., F o u t s , J.R., and Mannering, G.J., pgs. 151166, Academic P r e s s , New York (1969). 18. van der Hoeven, T.A., and Coon, M.J., J. Biol. Chem. (1974) 249, 6302-6310. 19. Haugen, D.A., van der Hoeven, T.A., and Coon, M.J., J. Biol. Chem. (1975) 250, 3567-3570. 20. L u , A.Y.H. and L e v i n , W., Biochem. Biophys. Res. Comm. (1972) 46, 1334-1339. 21. L u , A.Y.H., L e v i n , W., and Kuntzman, R., Biochem. Biophys. Res. Comm. (1974) 60, 266-272. 22. Ryan, D., L u , A.Y.H., West, S., and L e v i n , W., J. Biol. Chem. 250, 2157-2163. 23. Remmer, H. and Merker, H.J., Annals o f the New York Acad. Sci. (1965) 123, 79-97. 24. Conney, A.H., Pharmacol. Rev. (1967) 19, 317-366. 25. Coon, M.J., V e r m i l i o n , J.L., Vatsis, K.P., French, J.S., Dean, W.L. and Haugen, D.A., T h i s volume. 26. B r o d i e , B.B., Science (1955) 121, 603. 27. Cooper, J.R. and B r o d i e , B.B., J. Pharmacol. exp. Ther. (1955) 114, 409-417. 28. Conney, A.H., Miller, E.C., and Miller, J.A., J. Biol. Chem. (1957) 228, 753-766. 29. Miller, E.C., Miller, J.A., Brown, R.R., and MacDonald, J.C., Cancer Res. (1958) 18, 469-477. 30. M u e l l e r , G.C. and Rumney, G., J. Amer. Chem. Soc. (1957) 79, 1004-1005. 31. Ryan, K.J. and Engel, L.L., J. Biol. Chem. (1957) 225, 103114. 32. Estabrook, R.W., Schenkman, J.B., Cammer, W., Remmer, H., Cooper, D.Y., Narasimhulu, S., and Rosenthal, O. in


24

33.

34. 35. 36.


37. 38.

39. 40. 41. 42. 43. 44.

45. 46. 47. 48. 49. 50. 51. 52.


CONCEPTS

"Biological and Chemical Aspects o f Oxygenases" e d i t e d by K. Bloch and O. H a y a i s h i , pgs. 153-170, Maruzen Co. Ltd., Tokyo (1966). Remmer, H., Schenkman, J. B., Estabrook, R.W., Sasame, H., Gillette, J., Narasimhulu, S., Cooper, D.Y., and R o s e n t h a l , 0., Molec. Pharmacol. (1966) 2, 187-190. Schenkman, J.B., Remmer, H., and Estabrook, R.W., Molec. Pharmacol. (1967) 3, 113-123. Cammer, W., Schenkman, J.B., and Estabrook, R.W., Biochem. Biophys. Res. Comm. (1968) 23, 264-268. Estabrook, R.W., Baron, J., Peterson, J. and Ishimura, Y. in "Biological Hydroxylation Mechanisms", e d i t e d by Boyd, G.S. and S m e l l i e , R.M.S., pgs. 159-186, Academic P r e s s , London (1972). Estabrook, R.W., M a r t i n e z - Z e d i l l o , G., Young S., Peterson, J.A., and McCarhty, J., J. S t e r o i d Biochem. (1975) 6, 419-425. Narasimhulu, S., in "Proceedings o f the T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug O x i d a t i o n s " , e d i t e d by Ullrich, V., Roots, I., H i l d e b r a n d t , A.G., Estabrook, R.W., and Conney, A., Pergamon P r e s s , Oxford, in press (1977). B a l l o u , D.P., Veeger, C., van der Hoeven, T.A., and Coon, M.J., FEBS L e t t e r s (1974) 38, 337-340. Guengerich, F.P., Ballou, D.P., and Coon, M.J., J. Biol. Chem. (1975) 250, 7405-7414. Tyson, C.A., Lipscomb, J.D. and Gunsalus, I.C., J. Biol. Chem. (1972) 247, 5777-5784. P e t e r s o n , J.A., A r c h i v e s Biochem. Biophys. (1971) 144, 678693. Omura, T. and Sato, R., J. Biol. Chem. (1964) 239, 2370-2378. Estabrook, R.W., H i l d e b r a n d t , A.G., Baron, J., N e t t e r , K . J . , and Leibman, K., Biochem. Biophys. Res. Comm. (1971) 42, 132139. Ullrich, V. and Diehl, H., Eur. J. Biochem. (1971) 20, 509412. H i l d e b r a n d t , A.G., Speck, M., and Roots, I., Biochem. Biophys. Res. Commun. (1093) 54, 968-975. Cohen, B.S. and Estabrook, R.W., Arch. Biochem. Biophys. (1971) 143, 46-53. Cohen, B.S. and Estabrook, R.W., Arch. Biochem. Biophys. (1971) 143, 54-65. C o r r e i a , M.A. and Mannering, G., M o l . Pharmacol. (1973) 9, 455-469. C o r r e i a , M.A. and Mannering, G.J., M o l . Pharmacol. (1973) 9, 470-485. H i l d e b r a n d t , A. and Estabrook, R.W., A r c h i v e s Biochem. Biophys. (1971) 143, 66-79. Peterson, J.A., Ishimura, Y., Baron, J., and Estabrook, R.W., in "Oxidases and R e l a t e d Redox Systems" e d i t e d by K i n g , T.E., Mason, H.S., and M o r r i s o n , M., pgs. 565-581, U n i v e r s i t y Park P r e s s , B a l t i m o r e , Md. (1973).



1.


Cytochrome P-450 in Oxygen Activation

25

53. Ullrich, V. and Staudinger, Hj., in 'Microsomes and Drug O x i d a t i o n s " edited by Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., Fouts, J.R., and Mannering, G.J., pg. 199-217, Academic P r e s s , New York (1969). 54. Hrycay, E.G. and O'Brien, P.J., A r c h i v e s Biochem. Biophys. (1972) 153, 480-494. 55. Hrycay, E.G. and O'Brien, P.J., A r c h i v e s Biochem. Biophys. (1973), 157, 7-22. 56. Hrycay, E.G. and O'Brien, P.J., Archives Biochem. Biophys. (1974), 160, 230-245. 57. Kadlubar, F.F., Morton, K.C., and Ziegler, D.M., Biochem. Biophys. Res. Comm. (1973) 54, 1255-1261. 58. W e r r i n g l o e r , J. in "Proceedings o f the T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug O x i d a t i o n s " , e d i t e d by Ullrich, V., Roots, I., H i l d e b r a n d t , A.G., Estabrook, R.W., and Conney, A., Pergamon P r e s s , Oxford, in press (1977). 59. Rahimtula, A.D., O'Brien, P.J., Hrycay, E.G., Peterson, J.A., and Estabrook, R.W., Biochem. Biophys. Res. Commun. (1974) 60, 695-702. 60. Thakker, D.R., Y a g i , H., L u , A.Y.H., L e v i n , W., Conney, A.H., and J e r i n a , D.M., Proc. N a t ' l . Acad. Sci. (USA), (1976) 73, 3381-3385. 61. H e i d e l b e r g e r , C., Ann. Rev. Biochemistry (1975) 44, 79-121. 62. P h i l p o t , R.M. and Hodgson, E., Mol. Pharm. (1972) 8, 204-214. 63. F r a n k l i n , M., X e n o b i o t i c a (1971) 1, 581-591. 64. Schenkman, J.B., Wilson, B.J., and Cinti, D.L., Biochem. Pharmacol. (1972) 21, 2373-2383. 65. W e r r i n g l o e r , J. and Estabrook, R.W., Life Sciences (1973) 13, 1319-1330. 66. Gillette, J.R., B r o d i e , B.B., and LaDu, B.N., J. Pharm. Exp. Therap. (1957) 119, 532-540. 67. Orme-Johnson, W.H. and Z i e g l e r , D.M., Biochem. Biophys. Res. Comm. (1965) 21, 78-85. 68. Oshino, N., Oshino, R., and Chance, B., Biochem. J. (1973) 131, 555-563. 69. I s s e l b a c h e r , K.J. and C a r t e r , E.A., Biochem. Biophys. Res. Comm. (1970) 39, 530-537. 70. W e r r i n g l o e r , J., Chacos, N., Estabrook, R.W., Roots, I., and H i l d e b r a n d t , A.G. in " A l c o h o l and Aldehyde M e t a b o l i z i n g Systems" e d i t e d by Thurman, R.G., W i l l i a m s o n , J.R., D r o t t , H. and Chance, B., Academic P r e s s , New York (1977) in p r e s s . 71. Estabrook, R.W. and W e r r i n g l o e r , J. in "Proceedings o f the T h i r d I n t e r n a t i o n a l Symposium on Microsomes and Drug O x i d a t i o n s " e d i t e d by Ullrich, V., Roots, I., H i l d e b r a n d t , A.G., Estabrook, R.W. and Conney, A., Pergamon P r e s s , Oxford, in press (1977). 72. W e r r i n g l o e r , J. and Estabrook, R.W., in p r e p a r a t i o n . 73. Estabrook, R.W., F r a n k l i n , M.R., and H i l d e b r a n d t , A.G., Annals New York Acad. Sci. (1970) 174, 218-232.


26 74. 75. 76. 77. 78. 79. 80.


81.

82. 83. 84.


CONCEPTS

Estabrook, R.W., Cooper, D.Y. and Rosenthal, O., Biochem. Zeit. (1963) 338, 741-755. Cooper, D.Y., L e v i n , S., Narasimhulu, S., Rosenthal, O. and Estabrook, R.W., Science (1965) 147, 400-402. W e r r i n g l o e r , J. and Estabrook, R.W., A r c h i v e s Biochem. Biophys. (1975) 167, 270-286. Knowles, P.F., Gibson, J.F., P i c k , F.M. and Bray, R.C. Biochem. J. (1969) 111, 53-58. I y a n a g i , T. and Mason, H.S., Biochemistry (1973) 12, 22972308. K i n g , N.K. and Winfield, M.P., J. Biol. Chem. (1963) 238, 1520-1528. Yonetani, T. and Schleyer, H., J. Biol. Chem. (1967) 242, 1974-1979. Yamazaki, I., Nakajima, R., M i y o s h i , K., Makino, R., and Tamura, M., in "Oxidases and R e l a t e d Redox Systems" e d i t e d by K i n g , T.E., Mason, H.S., and M o r r i s o n , M., Vol. 1, pg. 407-418, U n i v e r s i t y Park P r e s s , B a l t i m o r e , Md. (1973). Vanneste, M.Y., Vanneste, W.H., and Mason, H.S., Biochem. Biophys. A c t a (1972) 267, 268-274. H i l d e b r a n d t , A.G. and Roots, I., A r c h i v e s Biochem. Biophys. (1975) 171, 385-397. Nash, T., Biochem. J. (1953) 55, 416-421.


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