Ascorbic Acid: Chemistry, Metabolism, and Uses - American Chemical

10 Ascorbate Oxidase: Molecular Properties and Catalytic Activity PETER M. H. KRONECK, FRASER A. ARMSTRONG , HELLMUT MERKLE, and AUGUSTO MARCHESINI 1

Downloaded by CORNELL UNIV on May 14, 2012 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch010

2

Universität Konstanz, Fakultät für Biologie, D-7750 Konstanz, Federal Republic of Germany

Ascorbate oxidase (E.C. 1.10.3.3) of the squash C. pepo medullosa was investigated by electron paramagnetic resonance (EPR); redox titrations of the different copper sites were carried out anaerobically by following the absorbance at 610 and 330 nm, or thefluorescenceat 335 nm. The kinetics of ascorbate oxidase reduction by L-ascorbate were studied by stopped-flow and rapid-freeze techniques. The enzyme contains eight copper atoms/M , four detectable by EPR (three type 1, one type 2), and four that are EPR silent (type 3). Potentiometric titrations showed equivalence among the three type 1 copper atoms (average midpoint potential 350 mV, 25°C); the midpoint potential of type 3 copper was slightly higher than that of type 1. At 10°C, increased differences between the two copper types were observed. On reduction, a free L-ascorbate radical, which was not bound to a paramagnetic copper, was formed. Type 1 and type 3 copper were reduced at similar rates, whereas the type 2 copper reacted more slowly. r

The copper enzyme ascorbate oxidase (L-ascorbate:O oxidoreductase, E.C. 1.10.3.3) was originally discovered (1) in cabbage leaves and named "hexoxidase," and has been the subject of numerous chemical and biological investigations. The literature published prior to 1963 has been 2

1

Current address: Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX 13QR, England. Current address: Istituto Sperimentale Per La Nutrizione Délie Pian te, Sezione Periferica Operativa Di Torino, Via Ormea 47, 10125 Torino, Italy. 2

0065-2393/82 /0200-0223$07.50/0 © 1982 American Chemical Society In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

224

ASCORBIC

reviewed

(2).

properties

M o r e r e c e n t results c o n c e r n i n g s t r u c t u r a l a n d c a t a l y t i c

of the metalloenzyme

have been

summarized

r e v i e w e d i n c o n n e c t i o n w i t h c o p p e r p r o t e i n s (5-8). and Dawson

ACID

(9)

(3,4), and

I n addition, L e e

have recently p u b l i s h e d a n article s u m m a r i z i n g the

c o p p e r content a n d a c t i v i t y d a t a of 137 p u r i f i e d samples of

ascorbate

oxidase p r e p a r e d i n D a w s o n ' s l a b o r a t o r y d u r i n g 1951-1977. A s c o r b a t e oxidase b e l o n g s to t h e class of b l u e oxidases t h a t also i n c l u d e s t h e laccases a n d c e r u l o p l a s m i n ( 5 ) . A l l three p r o t e i n s c o n t a i n at least f o u r c o p p e r atoms a n d are c a p a b l e of c a t a l y z i n g t h e f o u r - e l e c t r o n r e d u c t i o n of d i o x y g e n , y i e l d i n g t w o m o l e c u l e s implies, L-ascorbate : 0


towards

2

L-ascorbic acid

oxidoreductase (vitamin C ) .

of w a t e r .

A s its n a m e

d i s p l a y s its greatest

specificity

T h e substrate is o x i d i z e d to

L - d e h y d r o a s c o r b a t e w i t h t h e p r o d u c t i o n of 1 m o l o f w a t e r / m o l of L - a s c o r bate o x i d i z e d .

A s w i l l b e d i s c u s s e d l a t e r i n this c h a p t e r , m a n y

r e d u c t a n t s m a y serve as e l e c t r o n donors t o ascorbate

other

oxidase, m a k i n g

this e n z y m e n o t o n l y a v a l u a b l e d i a g n o s t i c agent f o r t h e d e t e r m i n a t i o n of v i t a m i n C i n m e d i c i n e a n d f o o d c h e m i s t r y (10), b u t also a p o w e r f u l c h e m i c a l reagent, p e r f o r m i n g r e d o x reactions w i t h s t r u c t u r a l l y c o m p l i cated organic molecules

(11,12).

A s c o r b a t e oxidase, w h i c h has b e e n f o u n d o n l y i n p l a n t tissues, is g e n e r a l l y i s o l a t e d f r o m g r e e n o r y e l l o w squash.

Considerable caution

m u s t b e u s e d w h e n e v a l u a t i n g t h e ascorbate a c t i v i t y of p l a n t extracts (2).

M a n y m e t a l - c o n t a i n i n g p r o t e i n s , o r free C u

2 +

ions i n s o l u t i o n , c a n

u n d e r g o o x i d o r e d u c t i o n i n t h e presence of ascorbate, g e n e r a l l y p r o d u c i n g h y d r o g e n p e r o x i d e i n s t e a d of w a t e r . T h e r e are also reports m e n t i o n i n g a f u n g a l e n z y m e f r o m Myrothecium " a t y p i c a l ascorbate

verrucaria

(13) t h a t is t e r m e d a n

o x i d a s e " because i t is unaffected

b y inhibitors of

h e a v y m e t a l catalysis, i n contrast to t h e c o p p e r e n z y m e s

from higher

plants. W e h a v e r e p e a t e d t h e p u r i f i c a t i o n p r o c e d u r e o f W h i t e a n d K r u p k a (13) a n d subjected t h e y e l l o w - b r o w n m a t e r i a l , c o l l e c t e d after d i e t h y l a m i n o e t h y l c e l l u l o s e c h r o m a t o g r a p h y , to s p e c t r o p h o t o m e t r y a n d e l e c t r o n p a r a m a g n e t i c resonance

( E P R ) spectroscopy.

Neither a typical absorb

a n c e i n t h e 35O-1000-nm r e g i o n n o r a c h a r a c t e r i s t i c E P R s i g n a l c o u l d be detected under o x i d i z i n g a n d reducing conditions; only a very weak E P R s i g n a l at a g of a p p r o x i m a t e l y 4, i n d i c a t i v e o f n o n s p e c i f i c a l l y b o u n d iron from a denatured protein, w a s detected. A s c o r b a t e o x i d a s e is m o s t l y f o u n d i n t h e p e r i p h e r a l p a r t o f t h e p l a n t , as s h o w n i n F i g u r e 1 f o r c a u l i f l o w e r a n d a p p l e that h a d b e e n c u t t h r o u g h the m i d d l e a n d pressed o n a p i e c e of p a p e r c o a t e d w i t h a s o l u t i o n of L-ascorbate a n d the redox

dye dichloroindophenol

(14).

T h e close

a s s o c i a t i o n w i t h t h e c e l l - w a l l m a t e r i a l gives some s u p p o r t to t h e t h e o r y that t h e e n z y m e m i g h t b e i m p o r t a n t f o r p l a n t g r o w t h a n d r i p e n i n g o f

In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


10.

KRONECK

ET

AL.

225

Ascorbate Oxidase

Figure 1. Peripheral location of ascorbate oxidase in cauliflower (left) and apple (right). The fruit was cut through the middle and pressed onto a piece of filter paper soaked with a mixture of L-ascorbate and dichloroindophenol, phosphate buffer (pH 6.0).

the f r u i t (2).

B e c a u s e of the h i g h r e a c t i v i t y of r e d u c e d ascorbate oxidase

t o w a r d s d i o x y g e n , the e n z y m e has also b e e n l i n k e d to p l a n t r e s p i r a t i o n b y a n a l o g y w i t h c y t o c h r o m e oxidase i n m a m m a l i a n tissues. I n contrast to tree a n d f u n g a l laccase, w h o s e m o l e c u l a r p a r a m e t e r s a n d m e c h a n i s m s of a c t i o n h a v e b e e n t h o r o u g h l y i n v e s t i g a t e d ( 8 ) , s u c h studies h a v e b e e n r e p o r t e d for ascorbate oxidase.

few

T h i s is m a i n l y

because of t h e r e l a t i v e l y difficult i s o l a t i o n a n d p u r i f i c a t i o n p r o c e d u r e of ascorbate oxidase i n c o m p a r i s o n w i t h laccase.

F u r t h e r m o r e , this e n z y m e

appears t o b e m o r e sensitive to e n v i r o n m e n t a l factors

s u c h as i o n i c

s t r e n g t h of the buffer m e d i u m , its p H , or t h e presence

of

extraneous

m e t a l ions. C o n s e q u e n t l y , m a n y samples i s o l a t e d o v e r a l o n g p e r i o d w e r e f o u n d to b e h o m o g e n e o u s f r o m t h e s t a n d p o i n t of t h e p r o t e i n b i o c h e m i s t but appeared inhomogeneous

w i t h respect

to t h e c a t a l y t i c a l l y a c t i v e

c o p p e r sites ( 9 ) . T h i s c h a p t e r s u m m a r i z e s some r e c e n t d e v e l o p m e n t s i n the p u r i f i c a t i o n of ascorbate oxidase, t h e n u m b e r of c o p p e r atoms p e r a c t i v e m o l e c u l e , a n d the s t o i c h i o m e t r y of the different c o p p e r sites w i t h

reference

to the c l a s s i f i c a t i o n i n t r o d u c e d b y M a l k i n a n d M a l m s t r o m ( 5 ) .

Further

m o r e , p h y s i c a l p r o p e r t i e s of t h e m e t a l centers are d i s c u s s e d i n r e l a t i o n to other s i m p l e c o p p e r p r o t e i n s t h a t h a v e b e e n c h a r a c t e r i z e d i n r e c e n t years.

Finally,

k i n e t i c i n v e s t i g a t i o n s of

ascorbate

are p r e s e n t e d as s t u d i e d b y a n a e r o b i c s t o p p e d - f l o w

oxidase

reduction

and rapid-freeze

techniques.


226

ASCORBIC

ACID

Purification of the Enzyme and Copper Content A h o m o g e n e o u s s a m p l e of ascorbate oxidase w a s first p r e p a r e d i n 1951. S i n c e t h e n , t h e specific a c t i v i t y a n d the c o p p e r content of h o m o g f r o m 740 u n i t s / / x g of

copper

a n d six c o p p e r a t o m s / M to 1000 units a n d t e n to t w e l v e c o p p e r

eneous p r e p a r a t i o n s g r e a t l y i n c r e a s e d ( 9 )

atoms.

r

T h e c h r o n o l o g i c a l c o r r e l a t i o n of the specific a c t i v i t y of t h e e n z y m e as a f u n c t i o n of

copper

content

documents

i m p r e s s i v e l y t h e difficulties

associated w i t h the p r e p a r a t i o n of " p u r e " ascorbate oxidase. d r a s t i c changes observed

of these a c t i v i t y values p e r c o p p e r

b y D a w s o n a n d coworkers

m o l e c u l a r w e i g h t (M )

been

( 2 , 3 , 9 ) , other c r i t e r i a , s u c h as

or h o m o g e n e i t y a n a l y z e d b y u l t r a c e n t r i f u g a t i o n ,

r


Although

atom have

or a m i n o a c i d c o m p o s i t i o n , h a v e

r e m a i n e d r e l a t i v e l y constant.

This

e x p e r i m e n t a l fact is h y p o t h e s i z e d to result f r o m v a r y i n g degrees p r o s t h e t i c c o p p e r loss o c c u r r i n g d u r i n g the p u r i f i c a t i o n steps A procedure was developed

(15)

that yielded a 1 4 %

of

(9). recovery

of

the t o t a l e n z y m a t i c a c t i v i t y present i n the c r u d e j u i c e extract f r o m green squash.

T h e o p t i m u m specific a c t i v i t y w a s i n the r a n g e of 4025 ±

u n i t s / m g protein i n a preparation containing 0.46-0.52%

copper

50

(10-12

C u atoms/140,000 M ) . r

T h r e e other p u r i f i c a t i o n methods h a v e b e e n d e s c r i b e d i n the l i t e r a t u r e — f o r c u c u m b e r (16)

a n d for the e n z y m e f r o m green s q u a s h (17,18).

T a b l e I s u m m a r i z e s the salient m o l e c u l a r p r o p e r t i e s of ascorbate oxidase purified

according

to

the

different p r o c e d u r e s

mentioned

d e t a i l e d p u r i f i c a t i o n t a b l e is p r e s e n t e d i n References

above.

15 a n d 18,

A and

i n c l u d e s the e n z y m e y i e l d c a l c u l a t e d o n t h e basis of t o t a l p r o t e i n present i n the c r u d e juice. T h e y i e l d is 1 4 % f o r t h e m e t h o d of L e e a n d D a w s o n Table I.

Specific Activity (Units/mg

RefSource

erence

Physical and Chemical Properties

M

r

Y e l l o w squash Cucumber G r e e n squash G r e e n squash

48 16 17 15

140,000 132,000 140,000 140,000

G r e e n squash

28

140,000

G r e e n squash

17

—

G r e e n squash

18

140,000

of

Protein)

3600 3500

Cu EPR/M

T

Cu/M

r

(%)

A o/A,

— —

> 1 0.79 1.9 1.8

3 3

3800-4250

8 8 8 8-10

—

3609

8

47 ± 3

0.68

5.4-6.5 7.5-8.0

59 48

0.87

—

3930

8

45

48 ±

2

0.65

° g values (g , g , g ) obtained from computer simulations. Source: Reprinted, with permission, from Ref. 18. Copyright 1979, Springer. z

v

x


10.

Ascorbate Oxidase

KRONECK E T A L .

227

( 9 ) vs. 3 4 % f o r t h e m e t h o d of M a r c h e s i n i a n d K r o n e c k (18). N a k a m u r a et a l . (16) recover 0.093 g of p u r e e n z y m e f r o m 15 k g o f f r e s h c u c u m b e r vs. 0.015 g f r o m fresh s q u a s h b y A v i g l i a n o et a l . (17).

T h e preparations

of L e e a n d D a w s o n ( 9 ) a n d of A v i g l i a n o et a l . (17) e x h i b i t r e l a t i v e l y h i g h variations i n c o p p e r / M

r

( T a b l e I ) , r a n g i n g f r o m 5.4 t o 10 C u

a t o m s / 1 4 0 , 0 0 0 M . A constant v a l u e of 7.95 ± 0.1 C u a t o m s / 1 4 0 , 0 0 0 M r

c a n b e a c h i e v e d b y t h e m e t h o d of M a r c h e s i n i a n d K r o n e c k

r

(18).

A m o n g t h e b l u e oxidases o n l y laccase appears to e x h i b i t r e p r o d u c i b l e values f o r the c o p p e r / M r a t i o . T h u s , i t is g e n e r a l l y a g r e e d that laccase r

contains f o u r c o p p e r atoms p e r e n z y m e m o l e c u l e ( 5 ) , w h e r e a s i n c e r u l o -


p l a s m i n ( 1 9 ) a n d ascorbate oxidase t h e m e t a l is m u c h m o r e l a b i l e a n d sensitive t o w a r d s v a r i o u s agents a n d e n v i r o n m e n t a l factors.

T h i s sensi

t i v i t y t o w a r d s extraneous agents is also e v i d e n t f r o m t h e d a t a r e p o r t e d b y A v i g l i a n o et a l . ( 2 0 ) , w h o r e m o v e d v a r y i n g amounts of t y p e 2 a n d type 3 copper (EDTA)

( 5 ) b y treatment w i t h ethylenediaminetetraacetic a c i d

or a c o m b i n a t i o n of E D T A

a n d dimethylglyoxime ( D M G ) .

A c c o r d i n g to these authors, ascorbate oxidase contains o n l y five to six p r o s t h e t i c c o p p e r a t o m s / M , whereas t w o to three of t h e eight r

copper

atoms o r i g i n a l l y present are e x t r e m e l y l a b i l e . U n f o r t u n a t e l y , no precise a c t i v i t y values a r e specified f o r t h e s e v e r a l e n z y m e p r e p a r a t i o n s

con

t a i n i n g eight, six to seven

plus

(after E D T A ) , a n d five (after E D T A

D M G ) c o p p e r atoms p e r e n z y m e m o l e c u l e . I n investigations o n the s o - c a l l e d " r e a c t i o n i n a c t i v a t i o n " o f ascorbate oxidase b y h y d r o g e n p e r o x i d e (21,22), essentially n o p r o s t h e t i c

copper

b e c a m e b o u n d t o the r e s i n d u r i n g 64-copper exchange experiments o n a n A m b e r l i t e I R - 1 0 0 c o l u m n , at p H 5.6. of Ascorbate Oxidase from Different Type

A

Cu/

M

r

(nm)

(M'

Sources

1 Copper

Type 2

A„

1

cm' ) 1

g,,

— — 2

608 607 610

10,400 9,680 10,700

—

610

9,600

2.24

3

610

9,680

2.227

3

610

11,000

2.229

3

610

9,700

2.227

g j L

— 2.22 2.22

An

Cu/

M

r

g„

g

— — 2.22

— — 2.05

x

(mT)

— 2.06 2.05

— 5.0 5.4

— — 2

2.07

6.0

—

—

—

5.6

1

2.242°

2.053

19.0

5.8

1

2.248

—

18.8

5.6

1

2.242°

2.053

19.0

a

— a

(mT)

Copper

—


— — 19.5

228

ASCORBIC

ACID

T h e c o p p e r c o n t e n t a n d e n z y m e a c t i v i t y is also m a r k e d l y d e p e n d e n t o n the p H of the s o l u t i o n . E x t e n s i v e studies (23,24) s h o w a r a p i d loss of the m e t a l b e l o w

p H 4.6;

this loss is t e m p e r a t u r e d e p e n d e n t

a c c o m p a n i e d b y a n i r r e v e r s i b l e u n f o l d i n g of the p r o t e i n m o i e t y . 11, d i s s o c i a t i o n of a s u b u n i t (M

~

r

and

At p H

65,000) is o b s e r v e d b y u l t r a f i l t r a t i o n .

T h i s process is n o t a c c o m p a n i e d b y loss of c o p p e r , a n d r e s i d u a l a c t i v i t y ( - 2 5 % ) is detectable. A p a r t f r o m p r o s t h e t i c c o p p e r loss d u e

to

environmental

c o o r d i n a t i o n site s t r u c t u r a l changes t h a t are d e t e c t a b l e b y techniques can occur.


o n spectroscopic

factors,

spectroscopic

T h e s e changes are d i s c u s s e d later i n the section

properties.

Molecular Properties of the Protein T h e most extensive studies o n t h e m o l e c u l a r p r o p e r t i e s of oxidase h a v e b e e n c a r r i e d out i n D a w s o n ' s l a b o r a t o r y .

ascorbate

Sedimentation

e q u i l i b r i u m experiments h a v e c o n f i r m e d a m o l e c u l a r w e i g h t of a v a l u e of

137,000 was d e t e r m i n e d for t h e a p o p r o t e i n .

i s o l a t e d a n d p u r i f i e d a c c o r d i n g to the different m e t h o d s

140,000;

The

enzyme

(Table I)

is

h o m o g e n e o u s b y e l e c t r o p h o r e t i c m e t h o d s a n d u l t r a c e n t r i f u g a t i o n (s o,w 2

=

7.52)

i n the p H r a n g e 5.2-10.

r e p o r t e d e a r l i e r (23, 24).

A b o v e p H 11 d i s s o c i a t i o n occurs

as

W h e n exposed to s o d i u m d o d e c y l sulfate ( S D S )

or g u a n i d i n i u m c h l o r i d e , the e n z y m e dissociates into t w o e q u i v a l e n t s u b units of 65,000 M

r

a c c o m p a n i e d b y the loss of p r o s t h e t i c c o p p e r .

I n the

presence of a strong r e d u c i n g agent, s u c h as 2 - m e r c a p t o e t h y l a m i n e

(or

its c o r r e s p o n d i n g e t h a n o l d e r i v a t i v e ) , a n d S D S , t w o other s u b u n i t s are found.

T h e s e s u b u n i t s are t e r m e d c h a i n a (M

r

28,000).

(M

r

38,000) a n d c h a i n /?

O n the basis of these results ascorbate oxidase is p r o p o s e d

to be a t e t r a m e r c o m p o s e d of t w o a a n d t w o ft c h a i n s , w h e r e e a c h «/? p a i r is c o v a l e n t l y c o n n e c t e d

by

disulfide bonds

(25).

A

quaternary

structure s i m i l a r to that of the n a t i v e e n z y m e is b e l i e v e d to exist for t h e copper-free

a p o p r o t e i n , w h i c h is o b t a i n e d b y exhaustive d i a l y s i s against

c y a n i d e (26).

R e i n c o r p o r a t i o n of t h e m e t a l i n t o t h e a p o p r o t e i n

t h e p r o t e i n m o l e c u l e to d i m e r i z e , because w i t h g e l a M

r

of 285,000

(s o,w° = 2

9-79)

was obtained.

filtration

causes

techniques

Unfortunately neither

o p t i c a l n o r E P R spectra of the different p r o t e i n species

(which might

h a v e g i v e n some s t r u c t u r a l i n f o r m a t i o n a b o u t the r e c o n s t i t u t e d c o p p e r sites, p a r t i c u l a r l y w i t h respect to the r a t i o of t y p e 1 to t y p e 2 c o p p e r ) were presented. A q u a t e r n a r y structure c o n s i s t i n g of t w o i d e n t i c a l , l a c c a s e - l i k e , a c t i v e sites p e r m o l e c u l e , e a c h c o n t a i n i n g f o u r c o p p e r atoms, w a s for t h e n a t i v e e n z y m e

(27).

suggested

I n t h e e n z y m e t h e s u b u n i t s a a n d p are

a r r a n g e d i n a s y m m e t r i c a l w a y , aft/Pa,

w h e r e a s i n the a p o e n z y m e


an

10.

229

Ascorbate Oxidase

KRONECK E T A L .

a s y m m e t r i c a l g e o m e t r y , aa/fift,

is a s s u m e d .

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

the

q u a t e r n a r y s t r u c t u r e of n a t i v e ascorbate oxidase a n d its a p o p r o t e i n is somewhat

contradictory

to

reported

results

Evidence

(18,28).

was

p r o v i d e d for a n " a s y m m e t r i c " s t o i c h i o m e t r y of the t h r e e different c o p p e r classes ( 5 ) , that is, o n l y one t y p e 2 c o p p e r out of e i g h t c o p p e r atoms, a n d not t w o as p r e d i c t e d for t w o l a c c a s e - l i k e s u b u n i t s . L i k e laccase a n d c e r u l o p l a s m i n , ascorbate oxidase is a n a c i d i c p r o t e i n , w i t h a s p a r t i c a c i d a n d g l u t a m i c a c i d i n excess over h i s t i d i n e , l y s i n e , a n d a r g i n i n e . F o r t h e a m i n o a c i d c o m p o s i t i o n see the d e t a i l e d d a t a i n References 3 a n d 18.

U n l i k e laccase, ascorbate oxidase has a r e l a t i v e l y

l o w c a r b o h y d r a t e content, 2 . 4 %

vs. a p p r o x i m a t e l y 4 5 %

(29).


A s p e c i a l interest has b e e n d i r e c t e d t o w a r d s the n u m b e r a n d accessi b i l i t y of cysteine ( R S H ) s u l f u r a n d cystine ( R S S R ) s u l f u r groups, w h i c h h a v e b e e n p r o p o s e d as b i n d i n g sites for the b l u e t y p e 1 c o p p e r , a n d as p o t e n t i a l e l e c t r o n a c c e p t i n g sites (30-34).

F o r ascorbate oxidase

from

y e l l o w squash, t e n to t w e l v e cysteine residues p l u s six to e i g h t c y s t i n e residues w e r e f o u n d ; n o n e of the S H groups w a s accessible to m e r c u r i a l s (35).

T h e s e results are i n g o o d a g r e e m e n t w i t h the d a t a r e p o r t e d

for the enzyme

from

green

squash.

O n l y three to

residues w e r e d e t e r m i n e d for t h e laccases

four

(18)

half-cystine

(36).

Spectroscopic Properties and Stoichiometry of the Copper Types T h e a b s o r p t i o n s p e c t r u m of the p u r e e n z y m e i n

Optical Spectra. p h o s p h a t e buffer

( p H 7.0)

exhibits the t y p i c a l b l u e m a x i m u m at 610

n m , a s s i g n e d to the b l u e or t y p e - 1 c o p p e r , a n d the s h o u l d e r at 330 n m , assigned to the E P R - n o n d e t e c t a b l e type 3 c o p p e r ( 5 ) I).

tions b y L e e a n d D a w s o n ( 1 5 ) , is

( F i g u r e 2, T a b l e

T a b l e I also i n c l u d e s m o l e c u l a r p a r a m e t e r s f r o m p r e v i o u s p r e p a r a the

absence

of

a

a n d other authors.

distinct absorption

A notable

maximum around

r e p o r t e d b y L e e a n d D a w s o n for t h e i r p u r e s t p r e p a r a t i o n . to o u r standards (18) 6

A io/A oo = 6

5

7.00

0

=

25 =b 0.5, A O/AQ 33

d= 0.25.

10

=

nm,

According

p u r e ascorbate oxidase is c h a r a c t e r i z e d b y

optical indices A 8 o / A i 2

feature

800

0.65 d b 0.05,

the and

D e v i a t i o n s f r o m these v a l u e s i n d i c a t e t h e

loss of the t y p e 1 c o p p e r , as is also s u p p o r t e d b y the c o r r e s p o n d i n g E P R spectra.

A n increase of the a b s o r b a n c e at 500 n m is associated w i t h a

d e n a t u r a t i o n of the p r o t e i n , as o b s e r v e d hydrogen peroxide

(18).

at the e n d of t i t r a t i o n s w i t h

U p o n a d d i t i o n of a r e d u c t a n t , for

example,

L - a s c o r b a t e or r e d u c t a t e , t h e b l u e c h r o m o p h o r e a n d the a b s o r b a n c e 330 n m are b o t h b l e a c h e d . reported

for

laccase (5-8).

the

two

at

N e a r l y i d e n t i c a l e l e c t r o n i c spectra h a v e b e e n

other

multicopper

oxidases,

ceruloplasmin and

I n the latter case the o p t i c a l p r o p e r t i e s h a v e b e e n e l u c i

d a t e d i n m u c h greater d e t a i l u s i n g l o w - t e m p e r a t u r e s p e c t r o s c o p y


(37,

230

ASCORBIC

ACID

050

^025


0 1 300

, 400

, 500

, 600

• 700 X (nm)

800

1000

900

Figure 2. Absorption spectra of pure ascorbate oxidase. Enzyme 48 fxM in 0.1 M phosphate buffer (pH 7.0), 20°C, 1.0-cm cell. Key: 1, oxidized enzyme; 2, reduced enzyme, slight excess of L-ascorbate. (Reproduced, with permission, from Ref. 18. Copyright 1979, Springer.) 38).

Furthermore, based on earlier calculations (39)

for the type

1

copper protein plastocyanin, ligand-field parameters for the blue copper i n laccase h a v e b e e n d e r i v e d .

T h e s e reports

(37,38)

also i n c l u d e a

s t r u c t u r a l r e p r e s e n t a t i o n of t h e t y p e 1 c e n t e r c o m p o s e d of a

flattened

tetrahedron ( D j symmetry) w i t h t w o i m i d a z o l e side-chains, a cysteine 2 (

sulfur, a n d a f o u r t h l i g a n d

(which probably

is m e t h i o n i n e s u l f u r ) ,

b o u n d to t h e m e t a l i o n . A l t h o u g h no s u c h l o w - t e m p e r a t u r e e x p e r i m e n t s h a v e b e e n p e r f o r m e d w i t h ascorbate oxidase, one m i g h t a n t i c i p a t e s i m i l a r s t r u c t u r a l features f o r t h e b l u e t y p e 1 centers. R e m o v a l of t h e t y p e 2 c o p p e r a c c o r d i n g to a r e p o r t e d (20)

procedure

leads to significant decreases of t h e a b s o r b a n c e at 330 a n d 7 5 0 n m .

T h e s e decreases i n d i c a t e t h a t t y p e 2 c o p p e r c o n t r i b u t e s to t h e a b s o r b a n c e i n these regions. F o r f u n g a l a n d t r e e laccase, structures b a s e d o n t e t r a g o n a l six, five, o r s q u a r e - p l a n a r f o u r c o o r d i n a t i o n , as f o u n d i n s e v e r a l low-molecular-weight copper complexes, were proposed

(37).

F l u o r e s c e n c e s p e c t r a of o x i d i z e d , p a r t i a l l y r e d u c e d , a n d f u l l y r e d u c e d ascorbate oxidase are s h o w n i n F i g u r e 3. A t p H 7.0 ( p h o s p h a t e b u f f e r ) , t h e p u r e e n z y m e gives a n e x c i t a t i o n m a x i m u m at 295 n m , a n d a n e m i s s i o n m a x i m u m at 330 n m .

E a r l i e r r e p o r t e d values

(15,28)

differ slightly

f r o m o u r figures, t h a t is, 325 a n d 335 n m are q u o t e d f o r t h e e m i s s i o n m a x i m u m . R e c e n t l y , a v a l u e of 328 n m f o r ascorbate o x i d a s e f r o m g r e e n s q u a s h w a s m e a s u r e d (40).

T h i s v a l u e , i n c o n t r a s t to a l l o t h e r v a l u e s

mentioned above, was obtained on a corrected optical data the A

3 3

fluorometer.

F r o m the

o / A i o i n d e x w a s e s t i m a t e d to b e greater t h a n 6

1.0;

f u r t h e r m o r e , t h e a b s o r p t i o n m a x i m u m w a s l o c a t e d at 605 n m . F o r o u r p r e p a r a t i o n s t h e e m i s s i o n m a x i m u m w a s a l w a y s at 330 n m , i n d e p e n d e n t of w h e t h e r w e u s e d t h e p u r e s t f r a c t i o n , e n z y m e w i t h a l o w e r

copper

content, o r e n z y m e t h a t h a d b e e n t r e a t e d w i t h C h e l e x 100 o r s u b j e c t e d t o l y o p h i l i z a t i o n (18).

O b v i o u s l y , d e s p i t e s i g n i f i c a n t differences


within

In Ascorbic Acid: Chemistry, Metabolism, and Uses; Seib, P., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982. 380

(20

X(nrn)

1

2

3

Mole Ascorbate/Enzyme

4

Anaerobic reduction of ascorbate oxidase by L-ascorbate, as followed byfluorescencespectroscopy (A) and by the change in absorbance at 610 nm (B).

340

cm

t80

(A) Enzyme 4.38 fM in 0.1 M phosphate buffer, pH 7.0, Ai . = 1.3; L-ascorbic acid 1.0 mM, 10.0 fM EDTA; 20°C, 1.0-cm cell, excitation at 295 nm, slit width 6 mm. Key: 1, oxidized enzyme; 2, half-reduced enzyme, 2 equivalents of L-ascorbate enzyme; 3, fully reduced enzyme, 4 equivalents of iL-ascorbate enzyme. (B) Enzyme 36.3 fiM in 0.1 M phosphate buffer, pH 7.0; L-ascorbic acid 5.0 mM, 10.0 fiM EDTA, 20°C, 1.0-cm cell. (Reproduced, with permission, from Ref. 18. Copyright 1979, Springer.)

Figure 3.

300


to ^

232

ASCORBIC

ACID

t h e o p t i c a l o r E P R p r o p e r t i e s of t h e i n d i v i d u a l p r e p a r a t i o n s of ascorbate oxidase, t h e Upon

fluorescence

stepwise

e m i s s i o n m a x i m u m retains its 3 3 0 - n m v a l u e .

addition of

stoichiometric

amounts

of

reducing

substrates u n d e r t h e r i g o r o u s e x c l u s i o n of d i o x y g e n ( f o r t e c h n i c a l d e t a i l s , see R e f . 18), t h e i n t r i n s i c 1.5-1.75

fluorescence

e m i s s i o n at 330 n m increases b y

( F i g u r e 3 ) w i t h o u t changes

i n p e a k shape

or position.

In

c o m p a r i s o n w i t h tree laccase o r f u n g a l laccase (41,42), ascorbate oxidase reacts r a p i d l y w i t h L - a s c o r b a t e , e v e n after t h e a c c e p t a n c e o f five to six electrons p e r m o l e c u l e .

F o r the identical experiment, a

fluorescence

e n h a n c e m e n t f a c t o r of t w o f o r a s a m p l e of ascorbate oxidase p r e p a r e d a c c o r d i n g to R e f e r e n c e 17 w a s o b s e r v e d


increase p a r a l l e l to changes detected,

F u r t h e r m o r e , at i n t e r

(40).

m e d i a t e r e d u c t i o n stages a t i m e - d e p e n d e n t i n absorbance

fluorescence

decrease a n d

at 3 3 0 a n d 605 n m w a s

a n d w a s a t t r i b u t e d to a n i n t r a m o l e c u l a r e l e c t r o n

transfer

b e t w e e n t h e t y p e 1 a n d t h e t y p e 3 c o p p e r sites. A

similar relationship between

the intrinsic

fluorescence

a n d the

r e d o x state of t h e c o p p e r atoms i n l a c q u e r tree laccase w a s r e p o r t e d (43). E P R Spectra and Stoichiometry of the Copper Classes.

A m o n g the

p h y s i c a l m e t h o d s that h a v e c o n t r i b u t e d t o o u r k n o w l e d g e a b o u t s t r u c t u r a l a n d m e c h a n i s t i c aspects of c o p p e r e n z y m e s a n d p r o t e i n s , E P R spectros c o p y has p l a y e d a d o m i n a n t role over t h e past t w o decades (5,6).

At

p H 7.0 ( p h o s p h a t e b u f f e r ) , p u r e ascorbate oxidase gives t h e E P R s i g n a l ( r e c o r d e d at X - b a n d , ~

9.3 G H z ) i l l u s t r a t e d i n F i g u r e 4. T h e s i g n a l

demonstrates t h e presence o f t h e t y p e 1 a n d t y p e 2 copper, s h o w n b y t h e different h y p e r f i n e s p l i t t i n g s i n t h e g

M

r e g i o n ( T a b l e I ) . T h e spec

t r u m s h o w n i n F i g u r e 4 is n e a r l y i d e n t i c a l w i t h t h e spectra p u b l i s h e d earlier for the enzyme from cucumber

(16), a n d f o r ascorbate

isolated from green squash, cucumber, or m a r r o w squash

oxidase

(28,44).

D o u b l e i n t e g r a t i o n of t h e area u n d e r t h e first d e r i v a t i v e reveals that 48 ±

2% of t h e c h e m i c a l l y d e t e r m i n e d c o p p e r

(18) is E P R d e t e c t a b l e

i n f r o z e n s o l u t i o n . T a b l e I s u m m a r i z e s t h e e x p e r i m e n t a l g a n d A values m e a s u r e d f r o m t h e r e c o r d e d spectra. A best fit o f t h e E P R s p e c t r u m of o x i d i z e d ascorbate o x i d a s e is o b t a i n e d b y c o m p u t e r s i m u l a t i o n , u s i n g t h e h i g h - f r e q u e n c y measurements at 35 G H z (28).

T h e ratio of t y p e 1

to t y p e 2 c o p p e r is e s t i m a t e d b y d o u b l e i n t e g r a t i o n of t h e first l o w - f i e l d l i n e , w h i c h arises f r o m t h e t y p e 2 c o p p e r , at a p p r o x i m a t e l y 0.270 T

(18).

R o u g h l y 2 5 % of t h e E P R - d e t e c t a b l e c o p p e r i n ascorbate oxidase is t y p e 2, whereas

7 5 % is b l u e t y p e

1 copper.

T h i s r a t i o is c o n f i r m e d b y

c o m p u t e r analysis (18) a n d agrees w i t h e a r l i e r results (28) ( F i g u r e 4 ) . A d d i t i o n of r e d u c i n g e q u i v a l e n t s causes c o m p l e t e loss of t h e E P R s i g n a l ( F i g u r e 2 ) , w h i c h reappears r a p i d l y a n d c o m p l e t e l y u p o n r e o x i d a t i o n w i t h d i o x y g e n or f e r r i c y a n i d e . and type 2 copper technique.

S t r u c t u r a l changes

of t h e t y p e 1

sites c a n b e c o n v e n i e n t l y m o n i t o r e d b y t h e E P R

T h u s , after l y o p h i l i z a t i o n of t h e p u r e e n z y m e i n p h o s p h a t e


10.

233

Ascorbate Oxidase

KRONECK E T A L .

buffer ( p H 7.0) a s e c o n d p e a k close to the m =

— 3 / 2 l i n e of the t y p e 2

center appears i n t h e E P R s i g n a l ( F i g u r e 4 ) . to t y p e 2 c o p p e r r a t i o b e c o m e s 1.5:1 changes i n the p u r i t y i n d e x A o / A i o 3 3

6

F u r t h e r m o r e , the t y p e 1

or e v e n 1:1, from

0.65

as s h o w n

to

0.9 a n d

by

the

greater.

R e c e n t l y , s i m i l a r observations w e r e m a d e f o l l o w i n g dialysis ( a e r o b i c or a n a e r o b i c ) against acetate buffer ( p H 5.0 or 4.5)

(45).

t y p e 1 c o p p e r decreases significantly, y i e l d i n g a n e w

T h e amount EPR

of

spectrum

w i t h l a r g e r n u c l e a r hyperfine splittings Aj| ( F i g u r e 4; for f u r t h e r details,


see Ref.

IS).

© /

© A

5A ®A A y i

w

2 6i 0

i i

1 290

1

,

i 320

Magnetic flux density Figure 4A.

,

i

i 350

i

(mT)

Experimental X-band EPR spectra of ascorbate oxidase.

Enzyme 0.228 mM in 0.1 M phosphate buffer (pH 7.0), temperature 105 K, 100 kHz modulation frequency, 1.0 mT modulation amplitude, ~ 2 mW power (20 dB), 0.2 s time constant, 0.10 mT/s scan rate, 9.39148 GHz microwave frequency. Key: 1, oxidized enzyme; V as 1, but 5 X instrument sensitivity; 2, reduced enzyme, sensitivity as in 1'; 3, oxidized enzyme, lyophilized at 0°C, redissolved, AWA*w = 1.1; 4, oxidized enzyme, after treatment with Chelex 100, AWA«i* = 1.0, sensitivity in 3 and 4 as in 1'. (Reproduced, with permission, from Ref. 18. Copyright 1979, Springer.)



234

ASCORBIC

265

285

305

325

ACID

345

Magnetic flux density (mT) Figure 4B.

Simulated X-band EPR spectra of ascorbate oxidase.

Spectra were simulated with the parameters of Table 111. Lorentzian lineshape (18). Key: 1, 3 type 1 Cu, 1 type 2 Cu; 2, 2 type 1 Cu, 2 type 2 Cu. (Reproduced, with permission, from Ref. 18. Copyright 1979, Springer.)

T h e presence of a s i n g l e t y p e 2 center i n ascorbate oxidase is not consistent w i t h the p r o p o s e d c o n c e p t of a q u a t e r n a r y s t r u c t u r e c o m p o s e d of t w o i d e n t i c a l subunits afS (25).

O n the other h a n d , a l l the m u l t i c o p p e r

oxidases d e s c r i b e d i n t h e l i t e r a t u r e (5-^5) h a v e o n l y one t y p e 2 center p e r active m o l e c u l e .

A d d i t i o n a l c o p p e r w i t h t y p e 2 characteristics c a n

b e b o u n d b y the m a c r o m o l e c u l e d u r i n g i s o l a t i o n a n d p u r i f i c a t i o n

(19).

A close e x a m i n a t i o n of t h e E P R spectra p r e s e n t e d b y L e e a n d D a w s o n (9)

i n d i c a t e s t h e presence

h y p e r f i n e s p l i t t i n g s at g|,.

of so-called nonspecific

copper w i t h

A s expected, the r a t i o A O / A Q 3 S

1 0

large

is a p p r o x i

m a t e l y 1.5-2 for these p r e p a r a t i o n s ( e s t i m a t e d f r o m F i g u r e 2 i n R e f . 9 ) .


10.

KRONECK E T A L .

235

Ascorbate Oxidase

Anaerobic Reduction of Ascorbate Oxidase—Number of Redox Equivalents and Midpoint Potentials of the Copper Sites R e d u c t i o n a n d r e o x i d a t i o n of t h e c o p p e r i n ascorbate oxidase c a n b e easily f o l l o w e d b y s p e c t r o p h o t o m e t r y at 330 a n d 610 n m ,

fluorescence

emission

and E P R

at 330 n m ( d e s c r i b e d

spectroscopy

(Figure 4).

i n the previous

section),

U s i n g the anaerobic titration techniques de

s c r i b e d i n R e f e r e n c e 18, c o m p l e t e r e d u c t i o n of t h e e n z y m e is a c h i e v e d b y f o u r e q u i v a l e n t s of L - a s c o r b a t e o r r e d u c t a t e .

W i t h total reduction,

the a b s o r b a n c e at 610 n m c o m p l e t e l y disappears, w h e r e a s some r e s i d u a l a b s o r b a n c e i n t h e 3 3 0 - n m r e g i o n r e m a i n s , c a u s e d b y t h e a b s o r b a n c e of


r e d u c e d p r o t e i n a n d o x i d i z e d substrate. P o t e n t i o m e t r i c t i t r a t i o n s o f t h e t y p e 1 c o p p e r w i t h f e r r i c y a n i d e as a m e d i a t o r ( e q u a l a m o u n t s o r a t e n f o l d e x c e s s / e n z y m e g i v e a N e r n s t factor, n i o , of 1.1 a n d a m i d p o i n t 6

potential, E '

O f 6

i , of 344 m V [vs. s t a n d a r d h y d r o g e n electrode 0

2 5 ° C , p h o s p h a t e buffer

(SHE),

( p H 7.0) w i t h I — 0.1 M ] ( F i g u r e 5 ) . F o r

m a n y titrations a r e l a t i v e l y l o n g l a g phase o f u p to t h r e e electrons w a s o b s e r v e d before a n y decrease i n a b s o r b a n c e at e i t h e r 610 o r 330 n m w a s

Ed

610

vs. S H E

[mV ]

Figure 5A. Anaerobic potentiometric titration of the type-1 Cu centers in ascorbate oxidase using ferricyanide as mediator, phosphate buffer (pH 7.0), I = 0.10 M, 25.0°C, K Fe(CN) 210 fiM, enzyme 21 fiM. 3

6



236

ASCORBIC

log ( A / ( A o - A ) )

ACID

[X=610nm]

Figure 5B. Double Nernst plot of the reduction by L-ascorbate at 610 and 330 nm, phosphate buffer (pH 7.0), I = 0.20 M , 10.0°C, enzyme 28 fiM. Potentials are reported in mV vs. SHE. detected.

T h i s effect p r o b a b l y arises f r o m traces of d i o x y g e n left i n t h e

r e s t i n g e n z y m e e v e n after r i g o r o u s d e o x y g e n a t i o n

(18).

T h e results of a s i m u l t a n e o u s s p e c t r o p h o t o m e t r i c

t i t r a t i o n at 330

a n d 610 n m are d e p i c t e d i n a d o u b l e N e r n s t p l o t i n F i g u r e 5, g i v i n g a n £'0,330 v a l u e f o r t h e t y p e 3 c e n t e r t h a t is n e a r l y i d e n t i c a l t o E'o.eio of t h e type 1 copper. 30 ±

H o w e v e r , at l o w e r t e m p e r a t u r e s (e.g., 1 0 ° C ) a v a l u e of

10 m V w a s m e a s u r e d f o r the difference E'0,330 ~~ JE'ceio* A s i m i l a r

r e d o x s i t u a t i o n is f o u n d f o r tree laccase (E'

Q

m V ; E ' f o r the t y p e 3 c o p p e r , 434 m V ) . 0

for t h e t y p e 1 c o p p e r , 394

H o w e v e r , at 2 5 ° C , w h e r e t h e

difference £'0,330 ~ £'0,610 is c o n s i d e r a b l y d i m i n i s h e d i n ascorbate oxidase ( i n t h e p r e s e n c e of t h e r e d o x m e d i a t o r f e r r i c y a n i d e ) ; t h e r m o d y n a m i c c o n t r o l f o r t h e o c c u p a n c y of t h e i n d i v i d u a l c o p p e r sites is less p r o n o u n c e d . I n t e r e s t i n g l y , i n the case o f b o t h t h e t h r e e t y p e 1 centers a n d t h e f o u r t y p e 3 centers, a l i n e a r N e r n s t r e l a t i o n s h i p exists i n t h e 1 0 - 2 5 ° C


10.

t e m p e r a t u r e r a n g e , p h o s p h a t e buffer among

237

Ascorbate Oxidase

KRONECK E T A L .

the single members

( p H 7.0), indicating equivalence

of the t w o copper

classes.

T h e Nernst

coefficient r a t i o n 3 o / n i o ( F i g u r e 5 ) , w h i c h h e r e does n o t a p p r o a c h t h e 3

6

e x p e c t e d n u m b e r of 2, raises some c o n t r o v e r s i a l p o i n t s r e g a r d i n g t h e m o d e of e l e c t r o n t r a n s f e r b e t w e e n t h e t w o c h r o m o p h o r e s .

Based largely

o n p o t e n t i o m e t r i c e v i d e n c e , a n d t h e c o m p l e t e l a c k of a n E P R s i g n a l i n e i t h e r t h e f u l l y o x i d i z e d o r f u l l y r e d u c e d state (8,46), t h e t y p e 3 c o p p e r pair probably

functions

as a c o o p e r a t i v e

two-electron

acceptor

(7).

H o w e v e r , this is o p e n t o q u e s t i o n ; u n c o u p l i n g of t h e t y p e 3 c o p p e r p a i r w a s p r o p o s e d to o c c u r i n t h e m a j o r i t y of cases (47).

Double Nernst

plots f o r s p e c t r o p h o t o m e t r i c t i t r a t i o n s of tree laccase ( s u c h as F i g u r e 5 )


p r o d u c e d slopes o f 1.0-2.0, d e p e n d i n g o n t h e t i t r a n t u s e d . T i t r a n t s s u c h as h y d r o q u i n o n e o r f e r r o c y a n i d e gave slopes of 2.0, w h e r e a s R u ( N H ) 3

6

2 +

gave a slope of 1.0. A c c o r d i n g t o some reports s t r o n g r e d u c i n g agents are c a p a b l e o f u n c o u p l i n g t h e t y p e 3 c o p p e r successive

one-electron

acceptor.

existence of p s e u d o c o o p e r a t i v i t y

unit, turning i t into a

A n a l t e r n a t i v e i n t e r p r e t a t i o n is t h e between

the type 1 copper

and the

optically almost invisible type 2 center h a n d l i n g two-electron packages to t h e t y p e 3 d i m e r . B e c a u s e t h e t y p e 2 c o p p e r i n tree laccase has t h e l o w e s t p o t e n t i a l , its a b i l i t y to b e c o m e i n v o l v e d i n a p s e u d o c o o p e r a t i v e e l e c t r o n - t r a n s f e r m e c h a n i s m w o u l d increase w i t h t h e r e d u c i n g of t h e t i t r a n t , i n a g r e e m e n t w i t h e x p e r i m e n t a l

findings

power

(47).

Mechanism of Action of Ascorbate Oxidase—Kinetics of the Anaerobic Reduction by Ascorbate and Reductate F a c e d w i t h t h e p r o b l e m of e l u c i d a t i n g t h e i n d i v i d u a l roles of t h e different c o p p e r centers i n t h e b l u e oxidases, t h e researcher has n a t u rally focused

i n recent

years

o n t h e laccases

(9).

Being

isolate, b e t t e r c h a r a c t e r i z e d , a n d c o n t a i n i n g f e w e r c o p p e r ceruloplasmin

o r ascorbate

l a c q u e r tree Rhus vernicifera

oxidase,

t h e laccases

from

easier t o

atoms

than

t h e Japanese

a n d t h e f u n g u s Polyporus versicolor h a v e

b e e n t h e subject of several t r a n s i e n t k i n e t i c studies i n t h e m i l l i s e c o n d r a n g e , t h a t i s , studies u s i n g 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 y a n d r a p i d freeze E P R spectroscopy

(9,49,50).

L a c c a s e , b y a n a l o g y w i t h ascorbate oxidase, catalyzes the o x i d a t i o n of o- o r p - a r y l d i a m i n e s a n d d i p h e n o l s b y d i o x y g e n t o p r o d u c e w a t e r a n d the corresponding quinones.

S t e a d y state k i n e t i c s h a v e e s t a b l i s h e d t h a t

a " p i n g - p o n g " m e c h a n i s m is o p e r a t i v e (51).

T h i s finding i m p l i e s t h a t t h e

r e d u c t i o n o f d i o x y g e n a n d the o x i d a t i o n of t h e o r g a n i c substrate a r e separate events, r e q u i r i n g t h e e n z y m e to f u n c t i o n as a m u l t i e l e c t r o n mediator.

D u r i n g t h e c a t a l y t i c c y c l e t h e o r g a n i c substrate u n d e r g o e s a


238

ASCORBIC

r a p i d o n e - e l e c t r o n o x i d a t i o n to a free r a d i c a l , w h i c h p r o b a b l y

ACID

decays

b y n o n e n z y m a t i c d i s m u t a t i o n ( 5 2 ) , as f o u n d i n v a r i o u s e n z y m e - c a t a l y z e d o x i d a t i o n reactions ( 5 3 ) . K i n e t i c experiments u n d e r a n a e r o b i c c o n d i t i o n s h a v e l e d to t h e c o n c l u s i o n t h a t t h e t y p e 1 c o p p e r is t h e e n t r y site of electrons i n t o t h e e n z y m e (49,54).

T h e t y p e 3 c o p p e r p a i r is r e d u c e d

i n a process d e p e n d e n t u p o n t h e i n t r a m o l e c u l a r transfer of o n e e l e c t r o n from the type

1 copper.

F u r t h e r m o r e , t h e rate o f e l e c t r o n transfer

b e t w e e n t h e t y p e 1 a n d t h e t y p e 3 centers is c o n t r o l l e d b y t h e p H of the m e d i u m , p o s s i b l y v i a a t r i g g e r m e c h a n i s m i n v o l v i n g t h e t y p e 2 copper

(49).

A c o m p l e x m e c h a n i s m f o r t h e f o u r - e l e c t r o n r e d u c t i o n of d i o x y g e n


to w a t e r m u s t c o m p l e t e t h e c a t a l y t i c c y c l e .

A n y mechanism involving

f o u r c o n s e c u t i v e s i n g l e - e l e c t r o n transfers is u n l i k e l y ( 5 5 ) . F o r laccase some e x p e r i m e n t a l e v i d e n c e i n d i c a t e s that at least t w o single electrons are e x c h a n g e d , because d u r i n g d i o x y g e n r e d u c t i o n a p a r a m a g n e t i c i n t e r m e d i a t e , w h i c h decays s l o w l y b y t h e r e a c t i o n w i t h t h e r e d u c e d t y p e 2 center, is f o r m e d radical O

t

(56,57).

T h i s p a r a m a g n e t i c species, most l i k e l y t h e

, is t r a p p e d d u r i n g s i m p l e o x i d a t i o n reactions. U s i n g o x y g e n -

1 7 - i s o t o p e - e n r i c h e d d i o x y g e n , o n l y o n e p r o d u c t w a t e r m o l e c u l e is r a p i d l y released i n t o t h e s o l u t i o n , w h e r e a s t h e second o n e r e m a i n s c o o r d i n a t e d to t h e t y p e 2 c o p p e r site a n d exchanges s l o w l y i n c o m p a r i s o n to t h e first F r o m these results i t appears t h a t t h e t y p e 2 c o p p e r p l a y s a n

(56,57).

i m p o r t a n t r o l e i n b o t h t h e r e d u c t i v e a n d o x i d a t i v e f u n c t i o n i n g of t h e enzyme.

( F o r f u r t h e r d e t a i l s , see R e f s . 9 a n d 49).

A n o t h e r i n t e r e s t i n g aspect of t h e r e d u c t i o n of d i o x y g e n b y laccase arises f r o m t h e o b s e r v a t i o n t h a t t h e e n z y m e forms a stable i n t e r m e d i a t e w i t h h y d r o g e n p e r o x i d e (58,59,60).

T h i s p e r o x y c o m p o u n d of laccase

is o b t a i n e d e i t h e r b y o x i d a t i o n of t h e r e d u c e d e n z y m e w i t h h y d r o g e n p e r o x i d e o r b y d i r e c t t i t r a t i o n of t h e o x i d i z e d e n z y m e w i t h h y d r o g e n peroxide.

P r i m a r i l y o n t h e basis of a b s o r p t i o n a n d c i r c u l a r d i c h r o i s m

spectra i t w a s p r o p o s e d t h a t t h e p e r o x i d e b i n d s to t h e t y p e 3 c o p p e r p a i r . M e a n w h i l e , these i n v e s t i g a t i o n s w e r e e x t e n d e d to m a g n e t i c s u s c e p t i b i l i t y measurements (60), w h i c h g i v e f u r t h e r s u p p o r t f o r a p e r o x i d e - c o o r d i n a t e d type 3 copper couple. S o m e o f these experiments h a v e b e e n r e p e a t e d w i t h ascorbate oxidase (two type 3 copper p a i r s ) , confirming the spectrophotometric data of R e f e r e n c e 58. T w o e q u i v a l e n t s o f h y d r o g e n p e r o x i d e , t h a t i s , 1 m o l / t y p e 3 c o p p e r p a i r (18), w h e r e s p e c i f i c a l l y b o u n d to ascorbate oxidase, p r o d u c e d a n increase i n t h e a b s o r b a n c e a r o u n d 330 n m . T h e s e e x p e r i m e n t a l results, a l t h o u g h of r a t h e r p r e l i m i n a r y n a t u r e , suggest t h a t there m i g h t b e some s i m i l a r i t i e s b e t w e e n t h e laccases a n d ascorbate oxidase, w i t h respect t o t h e d i o x y g e n - r e d u c i n g sites of t h e e n z y m e .


10.

Ascorbate

KRONECK E T A L .

239

Oxidase

T h e k i n e t i c results of the a n a e r o b i c r e d u c t i o n of ascorbate

oxidase

b y L - a s c o r b a t e o r r e d u c t a t e p r e s e n t e d i n this c h a p t e r h a v e b e e n o b t a i n e d by

anaerobic

spectroscopy.

stopped-flow

spectrophotometry

a n d rapid-freeze E P R

I n v i e w of the d i o x y g e n s e n s i t i v i t y of t h e r e d u c e d e n z y m e ,

extreme care m u s t b e exercised to o b t a i n r e p r o d u c i b l e a n d statistically m e a n i n g f u l d a t a . F o r t h e a n a e r o b i c i t y of the i n s t r u m e n t , b o t h t h e m i x i n g a n d t h e o b s e r v a t i o n c h a m b e r i n c l u d i n g t h e s y r i n g e system w e r e e m b e d d e d into a t h e r m o s t a t e d , w a t e r c i r c u l a t i n g b a t h ( n o r m a l l y m a i n t a i n e d at 1 0 - 1 5 ° C ) , w h i c h w a s k e p t u n d e r a constant pressure o f p u r i f i e d a r g o n ( 1 8 ) , as d e s c r i b e d i n R e f e r e n c e 61. A s i m i l a r s y s t e m w a s c o n s t r u c t e d for t h e r a p i d - f r e e z e a p p a r a t u s , a l l o w i n g a n a e r o b i c m i x i n g

of t h e r e -


actants p r i o r to f r e e z e - q u e n c h i n g of the r e a c t i o n m i x t u r e i n a n i s o p e n t a n e c o o l i n g b a t h at a p p r o x i m a t e l y — 1 4 5 ° C

(62). T h e r e d u c t i o n of t h e t y p e

Stopped-Flow Spectrophotometry.

c o p p e r [ o b s e r v e d at 6 1 0 n m , p h o s p h a t e buffer ( p H 7 . 0 ) , 7 =

1

0.20 M ,

2 5 ° C ] was very r a p i d a n d complex, b y both L-ascorbate a n d reductate. F u r t h e r m o r e , trace amounts of d i o x y g e n

(less t h a n 1 fiM)

significant p l a t e a u regions i n t h e r e d u c t i o n profile.

produced

A d d i t i o n of s m a l l

a m o u n t s of r e d u c t a n t r e s u l t e d first i n the r e d u c t i o n of t h i s p l a t e a u p h a s e to zero a n d t h e n i n t h e p a r t i a l r e d u c t i o n o f the e n z y m e , j u d g i n g f r o m t h e t o t a l a b s o r b a n c e at 6 1 0 n m . T o decrease t h e r e d u c t i o n rate, t h e t e m p e r a t u r e w a s l o w e r e d to 1 0 ° C w i t h s i m i l a r b u t s l o w e r o p t i c a l changes. S i m i l a r observations w e r e m a d e f o r t h e change i n a b s o r b a n c e at 330 n m (Figure 6 ) . REDUCTION

KINETICS

COPPER.

OF T H E TYPE-1

r a p i d p h a s e d u r i n g w h i c h plots of l o g (A

t

yielding &init

6 1 0

first-order

rate

constants,

is

an

initial

x

fc it . in

There

— A ) v s . t i m e are l i n e a r , A

610

linear

dependence

of

o n r e d u c t a t e c o n c e n t r a t i o n is f o u n d w i t h i n t h e l i m i t of 5.2 m M

reductate.

T h e a m p l i t u d e of the i n i t i a l phase increases w i t h r e d u c t a t e

c o n c e n t r a t i o n , l e v e l i n g off to a p p r o a c h a l i m i t o f a p p r o x i m a t e l y 5 0 % of the t o t a l a b s o r b a n c e at 6 1 0 n m . A t 2 5 ° C , t h e i n i t i a l r e a c t i o n is m u c h faster, i n d i c a t i n g a c o n s i d e r a b l e a c t i v a t i o n e n t h a l p y , a n d the c o n c e n t r a t i o n r a n g e is l i m i t e d to a v a l u e e q u a l to or less t h a n 1.35 m M f o r the t i m e scale of o u r stopped-flow spectrophotometer.

A g a i n the rate is first o r d e r w i t h respect to b o t h

e n z y m e a n d substrate. T h e a m p l i t u d e of t h e i n i t i a l phase at this h i g h e r t e m p e r a t u r e also increases w i t h r e d u c t a t e c o n c e n t r a t i o n , b u t i n a m o r e p r o n o u n c e d m a n n e r . A t the highest c o n c e n t r a t i o n u s e d , this i n i t i a l r a p i d phase represents 8 0 % of t h e t o t a l r e a c t i o n a m p l i t u d e . I n t h e absence of d i o x y g e n , t h e i n i t i a l r a p i d step is f o l l o w e d d i r e c t l y by a slower reaction.

T h i s second

rate,

fc ,

enzyme concentration.

F r o m a p l o t w h i c h is linear, of l / f c

610

2

is a g a i n first o r d e r i n 2

6 1 0


v s . the


240

ASCORBIC

r e c i p r o c a l c o n c e n t r a t i o n of r e d u c t a t e , a l i m i t i n g rate of

ACID

approximately

80 s" c a n be e s t i m a t e d . T h e a b o v e - m e n t i o n e d features w e r e r e m a r k a b l y 1

reproducible

u s i n g separate

ascorbate

oxidase

stored i n l i q u i d n i t r o g e n over a 3-year p e r i o d

samples

prepared

and

(18).

T h e r e is a v e r y s l o w final stage, a c c o u n t i n g for a p p r o x i m a t e l y 4 - 5 % of the t o t a l r e a c t i o n a m p l i t u d e at 610 n m .

T h e l o w amplitude makes

q u a n t i t a t i v e e v a l u a t i o n of this stage v e r y difficult, a n d the r e p r o d u c i b i l i t y w a s not v e r y satisfactory. H o w e v e r , the rate is p r o b a b l y i n d e p e n d e n t of the r e d u c t a t e c o n c e n t r a t i o n w i t h a rate constant, k

, of less t h a n 1.0 s" .

610

3

1

I n t h e presence of d i o x y g e n , the i n i t i a l r a p i d p h a s e a n d the s l o w e r second phase are separated b y a p l a t e a u o n the stopped-flow also m e a s u r e d f o r the d e c a y of t h e absorbance

trace, as

at 330 n m ( F i g u r e 7 ) .

T h e extent of t h e i n i t i a l r a p i d phase decreases s i g n i f i c a n t l y w i t h i n c r e a s i n g dioxygen concentration.

T h e effects of d i o x y g e n

removal by


induced

10.

241

Ascorbate Oxidase

KRONECK E T A L .

e n z y m e t u r n o v e r , a n d of p a r t i a l p r e r e d u c t i o n o f t h e e n z y m e , a r e s h o w n i n F i g u r e 7. V a r i a t i o n of p H f r o m 7.0 to 6.1 r e s u l t e d i n little c h a n g e q u a l i t a t i v e l y , although measured

fcinit

610

values w e r e s l i g h t l y h i g h e r . A single r u n at

p H 7.8 c o n f i r m e d this t r e n d ( T a b l e I I ) . REDUCTION

ment

KINETICS O F T H E T Y P E

of t h e c o m p l e x

kinetic behavior

3

COPPER.

Quantitative

assess

at 330 n m is difficult.

This

difficulty p r o b a b l y is p a r t i a l l y c a u s e d b y t h e s m a l l a n d v a r i e d c o n t r i b u t i o n f r o m t h e p r o d u c t i o n a n d d e c a y of t h e substrate r a d i c a l species ( 6 3 ) , a f e a t u r e that is r e v e a l e d w h e n r u n s at at v a r i o u s w a v e l e n g t h s are compared.

T h e d e c a y r a t e of t h e r a d i c a l , p r e s u m a b l y d e c a y

by non-


e n z y m a t i c d i s m u t a t i o n , has a m a g n i t u d e s i m i l a r t o t h e r e d u c t i o n rate of t h e t y p e 3 c o p p e r .

I n t h e experiments w i t h ascorbate as substrate,

0.100

E c o CO CO

CD

g a

0.010

o (/)

Ascorbic Acid: Chemistry, Metabolism, and Uses - American Chemical

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