Oxidation of Organic Compounds - ACS Publications

74 Reaction Mechanism of FAD-Containing

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Monooxygenases SHOZO YAMAMOTO, YOSHITAKA MAKI, TERUKO NAKAZAWA, YASUMICHI KAJITA, HIROSHI TAKEDA, MITSUHIRO NOZAKI, and OSAMU HAYAISHI Department of M e d i c a l Chemistry, Kyoto University Faculty of Medicine, Kyoto, Japan

Imidazoleacetate and L-lysine monooxygenases from pseudomonads were obtained in crystalline form. Both enzymes were established to contain FAD probably as a sole cofactor. Available evidence indicates that the reaction catalyzed by these enzymes involves the reduction of FAD, the monooxygenation of substrate, and the reoxidation of reduced FAD. The mechanism of activating molecular oxygen by these FAD-containing monooxygenases is discussed.

TiJ~onooxygenase

( m i x e d f u n c t i o n oxidase) catalyzes t h e i n c o r p o r a t i o n

of one a t o m of m o l e c u l a r o x y g e n i n t o substrate a n d t h e r e d u c t i o n of t h e other a t o m to w a t e r . F o r t h e r e d u c t i o n of t h e o x y g e n a t o m , some monooxygenases

r e q u i r e a n exogenous r e d u c t a n t s u c h as r e d u c e d p y r i

dine nucleotide

a n d ascorbate,

h y d r o g e n atoms of substrate.

whereas

others

consume

endogenous

T h u s , the m o n o o x y g e n a s e r e a c t i o n i n v o l v e s

both the oxidation (dehydrogenation)

of a h y d r o g e n d o n o r a n d t h e

o x y g e n a t i o n o f substrate. T o c l a r i f y t h e r e a c t i o n m e c h a n i s m of monooxygenase, w e h a v e re c e n t l y p u r i f i e d t w o monooxygenases acetate m o n o o x y g e n a s e

f r o m pseudomonads.

(5) a n d L-lysine monooxygenase

(10)

Imidazole were ob

t a i n e d i n c r y s t a l l i n e f o r m , a n d b o t h w e r e s h o w n to b e flavoproteins. T h e f o r m e r requires a n exogenous h y d r o g e n d o n o r , b u t t h e latter u t i l i z e s t h e h y d r o g e n atoms of L - l y s i n e . This, p a p e r describes some of t h e results of o u r investigations of t h e t w o F A D ( f l a v i n a d e n i n e d i n u c l e o t i d e ) - c o n t a i n i n g monooxygenases.

We

also discuss the m e c h a n i s m of a c t i v a t i o n of m o l e c u l a r o x y g e n b y these enzymes.

177 In Oxidation of Organic Compounds; Mayo, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

178

OXIDATION O F ORGANIC COMPOUNDS

III

Imidazole acetate Monooxygenase Imidazoleacetate

monooxygenase

w a s p u r i f i e d a b o u t 250-fold f r o m

a cell-free extract of a p s e u d o m o n a d a n d w a s o b t a i n e d i n c r y s t a l l i n e f o r m (Figure 1).

T h e specific a c t i v i t y of t h e c r y s t a l l i n e e n z y m e w a s 25.0

/xmoles/min./mg. p r o t e i n , a n d o n t h e basis of a m o l e c u l a r w e i g h t of


90,000 its m o l e c u l a r a c t i v i t y w a s estimated t o b e 2000 at 2 4 ° C .

Figure

1.

Crystalline imidazoleacetate monooxygenase

T h e e n z y m e catalyzes t h e i n c o r p o r a t i o n of one a t o m of o x y g e n i n t o imidazoleacetate,

a n d t h e a c c u m u l a t i o n of i m i d a z o l o n e a c e t a t e

served.

T h e r e a c t i o n p r o d u c t w a s unstable a n d d e c o m p o s e d

ously.

NADH

(nicotinamide

adenine

c o n s u m e d as a h y d r o g e n d o n o r . either b y t h e s p e c t r o p h o t o m e t r i c HC=C—CH COOH

V

I NH

hydrate)

was

T h e e n z y m e assay w a s c a r r i e d o u t measurement

of N A D H o x i d a t i o n o r

HO*—C=C—CH COOH

2

I N

dinucletotide

was ob spontane

2

+ 0% + N A D H + H * 2

I I N NH

+ NAD* + H O * a

w

C H

C H

b y t h e p o l a r o g r a p h i c d e t e r m i n a t i o n of o x y g e n c o n s u m p t i o n .

K

m

values

f o r i m i d a z o l e a c e t a t e , N A D H , a n d o x y g e n w e r e estimated to b e 0.3, 0.01, a n d 0.02 m M , respectively. T h e c r y s t a l l i n e e n z y m e w a s y e l l o w , a n d its a b s o r p t i o n s p e c t r u m is presented i n F i g u r e 2. T h e prosthetic g r o u p w a s i d e n t i f i e d as F A D , one m o l e of w h i c h w a s e s t i m a t e d

p e r m o l e of the e n z y m e p r o t e i n .

In Oxidation of Organic Compounds; Mayo, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

The

74.

YAMAMOTO

ET AL.

FAD-Containing

179

Monooxygenases

1.0


250

350

450

Wavelength, m/i Figure 2. Absorption spectrum of zoleacetate monooxygenase. Protein, for visible region and 0.045% for violet region enzyme

c a t a l y z e d the

anaerobic flavine

r e d u c t i o n of

several

dyes

imida0.45% ultra-

with

NADH

under

conditions; 2,6-dichlorophenolindophenol, F A D , F M N (ribo-

5 ' - p h o s p h a t e ) , a n d f e r r i c y a n i d e . I n the absence of i m i d a z o l e a c e

tate the e n z y m e c o u l d o x i d i z e N A D H presence.

K

m

at o n l y 0 . 5 %

of the rate i n its

values of the N A D H oxidase a c t i v i t y for N A D H a n d o x y g e n

w e r e e s t i m a t e d to b e 0.3 a n d 0.02 mM,

respectively.

A n a l y s e s of the e n z y m e f a i l e d to detect significant amounts of i r o n and

c o p p e r ( T a b l e I ) , a n d most m e t a l c h e l a t i n g agents tested d i d not

s h o w significant i n h i b i t i o n of the e n z y m e a c t i v i t y T a b l e I.

(11).

Estimations of Iron a n d Copper of Imidazoleacetate a n d L - L y s i n e Monooxygenases, /miole Enzyme

Iron"

Copper

Imidazoleacetate monooxygenase (0.120)

0.001

0.000

L - L y s i n e monooxygenase (0.103)

0.000

0.003

a

Dry ashing of enzyme protein was carried out. Iron was estimated by o-phenanthroline method and copper by sodium diethyldithiocarbamate or dithizone method. a

Reduction

of

the

enzyme-bound F A D was

s o d i u m d i t h i o n i t e at p H 10.5

(Figure 3).

observed

on adding

F A D r e d u c t i o n w a s also o b

s e r v e d o n a d d i n g N A D H b o t h i n the presence of i m i d a z o l e a c e t a t e a n d i n its a b s e n c e ( F i g u r e 3 ) .

A n a b s o r p t i o n s p e c t r u m characteristic

of

the

s e m i q u i n o i d f o r m of F A D a p p e a r e d w h e n the e n z y m e w a s h a l f r e d u c e d w i t h s o d i u m d i t h i o n i t e b u t not w i t h N A D H . W h e n a n e q u i m o l a r a m o u n t of N A D H w a s a n a e r o b i c a l l y a d d e d to the e n z y m e i n the presence of i m i d a z o l e a c e t a t e , the r e d u c t i o n of F A D


180

OXIDATION O F ORGANIC COMPOUNDS

III

w a s c o m p l e t e a n d i m m e d i a t e . A i r w a s t h e n i n t r o d u c e d to r e o x i d i z e F A D , a n d t h e a m o u n t of i m i d a z o l e a c e t a t e w a s m e a s u r e d .

O n adding N A D H

( 4 5 m/*moles), F A D (44 m ^ m o l e s ) w a s r e d u c e d , a n d o n the r e o x i d a t i o n


of F A D , i m i d a z o l e a c e t a t e (42 m/xmoles) w a s c o n s u m e d .

_ i

i

400

'^1

i

500

400

500

Wavelength, m/i Figure 3. Reduction of imidazoleacetate monooxygenase with sodium dithionite at pH 10.5 (left) and with NADH (right)

L-Lysine Monooxygenase L - L y s i n e monooxygenase f r o m a p s e u d o m o n a d w a s p u r i f i e d a p p r o x i m a t e l y 100-fold, a n d t h e e n z y m e w a s c r y s t a l l i z e d (10)

(Figure 4).

Its

specific a c t i v i t y w a s 10.5 /mioles/min./mg. p r o t e i n . T h e m o l e c u l a r w e i g h t w a s e s t i m a t e d to b e 191,000 a n d the m o l e c u l a r a c t i v i t y w a s c a l c u l a t e d to b e 2082 at 3 4 ° C . T h e e n z y m e catalyzes the i n c o r p o r a t i o n of one a t o m of o x y g e n i n t o L - l y s i n e , a n d 8 - a m i n o n o r v a l e r a m i d e is f o r m e d c o n c o m i t a n t l y w i t h the e v o l u t i o n of c a r b o n d i o x i d e .

E n z y m e a c t i v i t y w a s estimated

b y the

p o l a r o g r a p h i c d e t e r m i n a t i o n of o x y g e n c o n s u m p t i o n . T h e c o n c e n t r a t i o n c u r v e of L - l y s i n e w a s s i g m o i d a l , a n d 0.18 m M L - l y s i n e w a s r e q u i r e d f o r the h a l f m a x i m a l v e l o c i t y of the e n z y m e . CH NH 2

(CH ) 2

3

2

+ 0*

CH—NH COOH

CH NH

2

2

2

(CH ) 2

3

C—NH

2

+ CO, +

H 0* 2

2

O*


74.

FAD-Containing

YAMAMOTO ET AL.

The

enzyme

was

also

s h o w n to

181

Monooxygenases contain

FAD.

The

absorption

s p e c t r u m is s h o w n i n F i g u r e 5. T w o moles of F A D w e r e estimated p e r m o l e of e n z y m e p r o t e i n .

T h e e n z y m e c a t a l y z e d the r e d u c t i o n of

d i c h l o r o p h e n o l i n d o p h e n o l i n the presence of p h e n a z i n e

A n a l y s e s of the e n z y m e s h o w e d n o i r o n , c o p p e r , or other present i n significant quantities ( T a b l e I ) .


tested w e r e not i n h i b i t o r y to the e n z y m e

Figure

4.

I 350

metal

M o s t m e t a l c h e l a t i n g agents (11).

Crystalline L-lysine genase

0 L-J 250

2,6-

methosulfate.

monooxy-

I 450

I

Wavelength, m/i Figure 5. Absorption spectrum of ^-lysine monooxygenase. Protein, 0.62% for visible region and 0.048% for ultraviolet region


182

OXIDATION OF ORGANIC

COMPOUNDS

III

The enzyme-bound F A D could be reduced b y adding sodium d i t h i o n i t e . F i g u r e 6 presents the s p e c t r a l c h a n g e of F A D at p H 7.0, a n d a n a b s o r p t i o n of s e m i q u i n o i d of F A D a p p e a r e d d u r i n g the r e d u c t i o n . W h e n the e n z y m e w a s i n c u b a t e d w i t h L - l y s i n e u n d e r the a n a e r o b i c

conditions,

F A D w a s r e d u c e d at a rate w h i c h d e p e n d e d o n the c o n c e n t r a t i o n L-lysine.

of

W i t h 0.1 m M L - l y s i n e the r e d u c t i o n o c c u r r e d v e r y s l o w l y as

p r e s e n t e d i n F i g u r e 6.


•

A A

\

o

o 0.5 o

1

1

400

500

i * ^ ^

500

400

Wavelength, m/i Figure 6. Reduction of L-lysine monooxygenase with sodium dithionite at pH 7.0 (left) and with L-lysine at 0.1 mM. Numbers indicate the incubation time in hours (right) A s t o i c h i o m e t r i c a m o u n t (0.1 m M ) e a c h of e n z y m e - b o u n d F A D a n d 4 1

C-u-L-lysine

(u =

uniformly labelled)

was incubated

anaerobically

u n t i l F A D w a s f u l l y r e d u c e d . A f t e r d e p r o t e i n i z a t i o n the r e a c t i o n m i x t u r e w a s s u b j e c t e d to h i g h voltage p a p e r electrophoresis tography.

a n d paper chroma

M o s t of the r a d i o a c t i v i t y a p p e a r e d at t h e area c o r r e s p o n d i n g

to p i p e r i d i n e 2 - c a r b o x y l i c a c i d (a-keto-c-aminocaproic

acid)

(Figure 7),

a n d a significant a m o u n t of c a r b o n d i o x i d e w a s n o t detected.

W h e n the

r e a c t i o n m i x t u r e w a s aerated to r e o x i d i z e F A D a n d t h e n d e p r o t e i n i z e d , the r a d i o a c t i v i t y w a s also f o u n d at the p o s i t i o n of p i p e r i d i n e 2 - c a r b o x y l i c a c i d .

Discussion By

i n c o r p o r a t i n g one a t o m

monooxygenases

of m o l e c u l a r

oxygen

into

substrate,

c a t a l y z e a v a r i e t y of r e a c t i o n s — n a m e l y , h y d r o x y l a t i o n ,

dealkylation, epoxide a n d N - o x i d e formation, desaturation, aromatization of steroid, a n d a c i d a m i d e f o r m a t i o n .

T h e p a r t i c i p a t i o n of a

cofactor has b e e n r e p o r t e d f o r each t y p e of monooxygenase

reaction.


flavin

74.

FAD-Containing

YAMAMOTO ETA L .

183

Monooxygenases

A n e l e c t r o n transport system is associated w i t h several m o n o o x y genase reactions.

T h e steroid l l / ? - h y d r o x y l a t i n g system of a d r e n a l m i t o

c h o n d r i a consists of a

flavoprotein,

non-heme iron protein a n d homopro-

t e i n ( 3 ) . F a t t y a c y l A C P desaturase f r o m E u g l e n a r e q u i r e s a a n d n o n - h e m e i r o n p r o t e i n (6). as a

flavoprotein

ever,

flavoprotein

I n these m o n o o x y g e n a t i o n systems F A D

is a c o m p o n e n t of a n e l e c t r o n transport system.

imidazoleacetate

and L-lysine

monooxygenases,

How

together

with

s a l i c y l a t e a n d p - h y d r o x y b e n z o a t e h y d r o x y l a s e s , a p p e a r to c o n t a i n F A D as a sole cofactor since i r o n a n d c o p p e r w e r e n o t d e t e c t e d i n significant q u a n t i t i e s i n these e n z y m e s (11,

12).


W h e n F A D of i m i d a z o l e a c e t a t e m o n o o x y g e n a s e w a s f u l l y r e d u c e d w i t h N A D H i n the presence of i m i d a z o l e a c e t a t e a n d r e o x i d i z e d b y aera t i o n , a n e q u i m o l a r a m o u n t of i m i d a z o l e a c e t a t e w a s c o n s u m e d p r o b a b l y to f o r m i m i d a z o l o n e a c e t a t e . A n analogous result has b e e n o b t a i n e d w i t h s a l i c y l a t e h y d r o x y l a s e (9, 13).

T h e s e findings i n d i c a t e that t h e r e d u c e d

f o r m of e n z y m e - b o u n d F A D serves as a h y d r o g e n d o n o r i n the h y d r o x y l a t i o n of substrate a n d reduces o n e a t o m of o x y g e n to w a t e r . W h e n a s i m i l a r experiment was carried out w i t h L-lysine monooxygenase a n d C - L - l y s i n e , 1 4

the r a d i o a c t i v i t y w a s R CH

R FAD

2

I CH—NH

R

R

I

CH | C=NH 2

or

CH 2

C—NH

1

2

COOH

COOH

COOH

2

CH

2

+ C—NH

ca

2

2

II O 8-Aminonorvaleramide

I H 0 2

Lysine

0

R CH NHo

2

+ c=o

"

COOH R:

H N—(CH ) — 2

2

3

a-Keto-eaminocaproate

Piperidine 2-carboxylate

f o u n d at t h e area c o r r e s p o n d i n g to p i p e r i d i n e 2 - c a r b o x y l i c a c i d b u t l i t t l e at the p o s i t i o n of 8 - a m i n o n o r v a l e r a m i d e . U n d e r t h e e x p e r i m e n t a l conditions, F A D was reduced very slowly, a n d a rather long incubation t i m e w a s r e q u i r e d f o r its f u l l r e d u c t i o n . If a n i n t e r m e d i a t e f o r m e d b y the d e h y d r o g e n a t i o n of L - l y s i n e is u n s t a b l e a n d r e a d i l y h y d r o l y z e d , after the l o n g i n c u b a t i o n t i m e t h e i n t e r m e d i a t e m a y b e h y d r o l y z e d t o p i p e r i d i n e 2 - c a r b o x y l i c a c i d , w h i c h is n o t susceptible to m o n o o x y g e n a t i o n .


184

OXIDATION O F ORGANIC

COMPOUNDS—HI

c.p.m. -6000

-4000

+

-2000

r

, , - T H


O

8-Aminonorvaleramide

CD

O

L-Lysine

8-Aminovalerate

O

Piperidine 2 carboxylate

Figure 7. Formation of piperidine 2-carboxylic acid. The enzymebound FAD (0.1 mM) and C-u-L-lysine (0.1 mM, 1,000,000 c.p.m.) were incubated anaerobically for 7 hours. After deproteinization by adding perchloric acid, an aliquot of the reaction mixture was subjected to high voltage paper electrophoresis (2000 volts) at pH 3.4 for 1 hour n

Although

t h e i d e n t i f i c a t i o n of the

1 4

C-labeled product

as p i p e r i d i n e

2 - c a r b o x y l i c a c i d has n o t b e e n e s t a b l i s h e d a n d the m e c h a n i s m of its f o r m a t i o n has n o t b e e n c l e a r l y e l u c i d a t e d , t h e f o r m a t i o n of t h e a-keto a c i d may

suggest

the i n t e r m e d i a t e

formation

of d e h y d r o g e n a t e d

L-lysine

c o n c o m i t a n t w i t h t h e r e d u c t i o n of F A D . T h e f u n c t i o n of m e t a l i o n i n a c t i v a t i n g m o l e c u l a r o x y g e n has l o n g b e e n i n v e s t i g a t e d a n d discussed.

S e v e r a l dioxygenases h a v e b e e n estab

l i s h e d to c o n t a i n i r o n w h i c h has b e e n p o s t u l a t e d to m e d i a t e the a c t i v a t i o n of m o l e c u l a r o x y g e n (7, 8). t a i n e d i n phenolase

(2)

T h i s is also the case w i t h t h e c o p p e r c o n

a n d d o p a m i n e /3-hydroxylase (1).

A s described

a b o v e , h o w e v e r , i r o n a n d c o p p e r a p p e a r to b e absent i n some m o n o oxygenases, a n d F A D seems to b e a sole c o f a c t o r of these

enzymes.

T h e r e f o r e , i n t h e absence of m e t a l i o n , c o n s i d e r a t i o n of t h e m e c h a n i s m of a c t i v a t i n g m o l e c u l a r o x y g e n suggests a n a d d i t i o n a l f u n c t i o n of r e d u c e d flavin

i n a d d i t i o n to its a c t i o n as a n e l e c t r o n carrier.

T h e spontaneous

o x i d a t i o n of r e d u c e d flavin l e a d i n g to a v e r y reactive h y d r o p e r o x i d e has been reported b y M a g e r a n d Berends

(4).

T h i s h y d r o p e r o x i d e is s u p

p o s e d to arise b y a r e a c t i o n b e t w e e n a s e m i q u i n o i d f o r m of flavin a n d molecular oxygen.

W h e t h e r or not such a hydroperoxide participates i n

the e n z y m a t i c a c t i v a t i o n of m o l e c u l a r o x y g e n w i l l r e q u i r e e v i d e n c e o b t a i n e d f r o m experiments w i t h F A D - c o n t a i n i n g monooxygenases. T h e s e m i q u i n o i d f o r m of F A D w a s o b s e r v e d

spectrophotometrically

w h e n t h e enzymes w e r e r e d u c e d w i t h s o d i u m d i t h i o n i t e . H o w e v e r , s u c h a species c o u l d n o t b e d e t e c t e d o n r e d u c t i o n w i t h substrate, a n d i t has


74.

YAMAMOTO ET AL.

FAD-Containing

Monooxygenases

185

not b e e n established w h e t h e r a s e m i q u i n o n e of F A D is i n v o l v e d i n the catalyses of these e n z y m e s .

Acknowledgment T h i s i n v e s t i g a t i o n has b e e n s u p p o r t e d i n p a r t b y P u b l i c

Health

Service R e s e a r c h G r a n t s C A - 0 4 2 2 2 f r o m the N a t i o n a l C a n c e r Institute a n d A M - 1 0 3 3 3 f r o m the N a t i o n a l Institute of A r t h r i t i s a n d M e t a b o l i c Diseases, a n d b y grants f r o m the Jane C o f f i n C h i l d s M e m o r i a l F u n d f o r M e d i c a l R e s e a r c h , the S q u i b b Institute of M e d i c a l R e s e a r c h a n d the


Scientific R e s e a r c h F u n d of the M i n i s t r y of E d u c a t i o n of J a p a n .

Literature Cited

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January 8,

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Oxidation of Organic Compounds - ACS Publications

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