Bioinorganic ChemistryâII

15 Characterization of the Copper(II) Site in Galactose Oxidase R O B E R T D . B E R E M A N , M U R R A Y J. E T T I N G E R , D A N I E L J. K O S M A N , and R O B E R T J. K U R L A N D

Downloaded by UNIV LAVAL on April 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/ba-1977-0162.ch015

The Bioinorganic Graduate Research Group, Departments of Chemistry and Biochemistry, State University of New York at Buffalo, Buffalo, N Y 14214

Spectral and model studies of the Cu(II)-containing metallo protein galactose oxidase suggest that the enzymic metal coordination center is a square planar system involving two nitrogenous ligands. Model studies suggest that exogenous ligands coordinate in an equitorial position. Difference absorbance spectra with exogenous anions establish that only the 314-nm transition exhibited by the enzyme is of charge-transfer character. Difference absorbance, EPR, and fluoride relaxation are consistent with formation of a com plex with the anion ferricyanide near the copper site. In the enzyme, a histidine imidazole and tryptophan indole con tribute directly to catalysis. Spectral results imply that enzyme activity is associated with a relatively unique ge ometry of the active site Cu(II) complex.

/ ^ o p p e r is a n essential e l e m e n t t o most l i f e forms. ^

I n h u m a n s i t is t h e

t h i r d most a b u n d a n t trace e l e m e n t ; o n l y i r o n a n d z i n c a r e present

i n h i g h e r q u a n t i t y . U t i l i z a t i o n of c o p p e r u s u a l l y i n v o l v e s a p r o t e i n a c t i v e site w h i c h catalyzes a c r i t i c a l o x i d a t i o n r e a c t i o n , e.g., c y t o c h r o m e a m i n e oxidases, s u p e r o x i d e d i s m u t a s e , ferroxidases,

oxidase,

dopamines-hydrox-

ylase, a n d tyrosinase. A c c o r d i n g l y , a n i m a l s e x h i b i t u n i q u e

homeostatic

mechanisms for the absorption, distribution, utilization, a n d excretion of c o p p e r ( 1 ) . M o r e o v e r , at least t w o p o t e n t i a l l y l e t h a l i n h e r i t e d diseases of c o p p e r m e t a b o l i s m a r e k n o w n : W i l s o n ' s D i s e a s e a n d M e n k e s ' s K i n k y H a i r Syndrome ( 1 ) . C u ( I I ) sites i n p r o t e i n s h a v e b e e n classified i n t o t h r e e types b a s e d o n t h e i r s p e c t r a l p r o p e r t i e s ( 2 ) . T y p e I C u ( I I ) sites a r e c h a r a c t e r i z e d b y v e r y h i g h m o l a r a b s o r b t i v i t y values f o r t h e v i s i b l e b a n d n e a r 600 n m 263

Raymond; Bioinorganic Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1977.

264

BIOINORGANIC CHEMISTRY

(16.5kK).

II

A c c o r d i n g l y , p r o t e i n s w h i c h c o n t a i n T y p e I sites are o f t e n

r e f e r r e d to as b l u e c o p p e r proteins since t h e i r solutions are b l u e typical enzyme

at

concentrations i n a r e s e a r c h l a b o r a t o r y ( 1 0 " - 1 0 " M ). 5

4

T h e 6 0 0 - n m r e g i o n b a n d has b e e n s u g g e s t e d to arise f r o m η —» π* ( σ* ) c h a r g e transfer i n v o l v i n g a copper—cysteine studies ( 3 , 4 )

bond.

a n d recent m o d e l studies ( 5 )

Metal

replacement

t e n d to substantiate this

assignment. E l e c t r o n s p i n resonance ( E S R ) p a r a m e t e r s for these T y p e I sites are also different t h a n for s i m p l e c o p p e r complexes, p a r t i c u l a r l y i n the u n u s u a l l y l o w v a l u e for the s p i n H a m i l t o n i a n p a r a m e t e r , A is t y p i c a l l y less t h a n 100 G ( 2 ) .

z z

, which

T h e C u ( I I ) atoms i n these sites are b e

l i e v e d to b e i n a t r i g o n a l e n v i r o n m e n t ( 4 or 5 c o o r d i n a t e )

(6,7),

and


f u n c t i o n a l a c t i v i t y is associated w i t h a c h a n g e i n o x i d a t i o n state of the copper (8). T y p e I I C u ( I I ) , or l o w - b l u e c o p p e r , is less c o l o r e d at

common

r e s e a r c h concentrations. T h e s e systems h a v e r e c e i v e d less a t t e n t i o n t h a n T y p e I c o p p e r . H o w e v e r , e v e n l o w - b l u e c u p r i c c o p p e r c a n possess h i g h m o l a r a b s o r b t i v i t i e s w h e n c o m p a r e d w i t h s i m p l e c o o r d i n a t i o n complexes of C u ( I I ) . T h e C u ( I I ) sites i n s u c h proteins also y i e l d A

z z

values n o r

m a l l y greater t h a n 140 G , i.e., m o r e l i k e that of l o w m o l e c u l a r w e i g h t s q u a r e p l a n a r C u ( I I ) complexes (2, 8 ) . T h e o n l y a v a i l a b l e c r y s t a l s t r u c t u r e of a c o p p e r p r o t e i n is that of a l o w b l u e p r o t e i n b o v i n e e r y t h r o c y t e superoxide dismtuase ( 9 ) .

T h e t w o c o p p e r atoms i n this p r o t e i n are

e a c h c o o r d i n a t e d to f o u r h i s t i d i n e nitrogens i n a n a p p r o x i m a t e square planar array. F e w prototypes are a v a i l a b l e for T y p e I I I C u ( I I ) .

These

systems

are E S R i n a c t i v e , i.e., a l t h o u g h C u ( I I ) is present, n o E S R s p e c t r u m c a n be

obtained.

R e c e n t m a g n e t i c s u s c e p t i b i l i t y results i n d i c a t e that the

T y p e I I I C u ( I I ) i n R h u s laccase is a n a n t i f e r r o m a g n e t i c - c o u p l e d C u ( I I ) d i m e r (10).

L i t t l e , h o w e v e r , is k n o w n a b o u t the c o p p e r l i g a n d s or the

n a t u r e of the d i m e r i c i n t e r a c t i o n . T h e entire m e t a l a c t i v e site i n a n y m e t a l l o p r o t e i n consists of

the

m e t a l chelate p l u s a l l of the p r o t e i n groups w h i c h c o n t r i b u t e to its s p e c t r a l a n d c a t a l y t i c p r o p e r t i e s . A c o p p e r p r o t e i n has b o t h v e r y s p e c i a l a n d specific c a t a l y t i c a n d s p e c t r a l p r o p e r t i e s .

T h e p r e m i s e of o u r r e

s e a r c h is that b o t h the c a t a l y t i c a n d s p e c t r a l properties m u s t reflect the same u n i q u e characteristics of the l i g a n d s to the m e t a l , the g e o m e t r y of t h e m e t a l c o m p l e x , the p r o p e r t i e s of the a c t i v e site p r o t e i n groups, a n d the g e n e r a l p r o t e i n e n v i r o n m e n t of the a c t i v e site. O u r u l t i m a t e objective is to e l u c i d a t e the p e r t i n e n t i n t e r r e l a t i o n s h i p s a m o n g these p a r a m e t e r s . V a r i a t i o n s i n a n y or a l l of these factors m i g h t b e the u n d e r l y i n g basis for the d i s t i n g u i s h i n g s p e c t r a l a n d c h e m i c a l properties of the t w o classes of p a r a m a g n e t i c c o p p e r i n c o p p e r p r o t e i n s .


15.

Galactose

265

Copper(II) Site in Galactose Oxidase

BEREMAN ET AL.

Oxidase—Background

O u r recent interest has c e n t e r e d o n the f u n g a l e n z y m e ,

galactose

oxidase, w h i c h m a y b e the o n l y c o p p e r p r o t e i n w i t h a single n o n - b l u e Cu(II)

( i n the n o n - b l u e f a m i l y ) a n d w h i c h contains no other p r o s t h e t i c

groups.

( W e take the v i e w that E S R parameters e s t a b l i s h the f a m i l i e s

of the c u p r i c sites.

O p e r a t i o n a l l y , one g o a l of the copper—protein

re-

s e a r c h is to d e t e r m i n e p r e c i s e l y w h a t is constant a n d w h a t varies a m o n g the v a r i o u s examples of e a c h t y p e of C u ( I I ) site.) a f e w p e r t i n e n t properties of this e n z y m e . i s o l a t e d b y C o o p e r i n 1959 ( I I ) .

W e can summarize

G a l a c t o s e oxidase w a s

first

E a r l y available literature about


e n z y m e was c o n t r i b u t e d p r i m a r i l y b y H o r e c k e r a n d c o - w o r k e r s T h e e n z y m e is e l a b o r a t e d b y Dactylium p r o t e i n (13).

dendroides a n d is a n e x t r a c e l l u l a r

Its m o l e c u l a r w e i g h t has b e e n r e c e n t l y e s t a b l i s h e d i n these

laboratories to b e 68,000 ± 3,000 daltons (14). has b e e n r e p o r t e d pH =

the (12).

12 ( 1 5 ) .

T h e single C u ( I I )

diethyl dithiocarbamate

A n a m i n o a c i d analysis

T h e p r o t e i n has a n isoelectric p o i n t a r o u n d

(14).

atom can be readily removed

coordination

or b y

H S. 2

The

by

apoenzyme

is

stable, a n d r e c o n s t i t u t i o n of the e n z y m e results i n t o t a l r e a c t i v a t i o n (12, 16).

A s the n a m e i m p l i e s , the e n z y m e catalyzes the o x i d a t i o n of g a l a c -

tose b y m o l e c u l a r o x y g e n as i n d i c a t e d b e l o w : CH OH

CHO

2

HO +

H 0 2

N e a r l y a n y p r i m a r y a l c o h o l serves as a substrate w i t h the e x c e p t i o n methanol and ethanol. hexachloroiridate(IV)

F e r r i c y a n i d e (17, 1 8 ) , p o r p h y r e x i d e (18)

c a n r e p l a c e o x y g e n as o x i d a n t .

(18),

2

of and

Hexachloro-

i r i d a t e ( I V ) is c o n s u m e d to the e x c l u s i o n of o x y g e n i n a e r o b i c m i x t u r e s . W h e n hexachloroiridate(IV) and H 0 2

2

serve as o x i d a n t a n d r e d u c t a n t

r e s p e c t i v e l y , the n o r m a l r e a c t i o n , v i s - a - v i s H 0 , is r e v e r s e d , a n d o x y g e n 2

is p r o d u c e d

2

(18).

T h e first s p e c t r a l s t u d y of galactose oxidase w a s the r e p o r t of the e l e c t r o n s p i n resonance

s p e c t r u m b y B l u m b e r g et a l . ( 1 9 ) .

c e n t l y , C l e v e l a n d et a l . (20)

M o r e re-

reported a further E S R study w h i c h was

b a s e d o n a c o m p u t e r fit to the s p e c t r u m . T h e y c o n c l u d e d that f o u r n i t r o gens w e r e b o u n d to the C u ( I I ) Ligands

atom.

to Metal in Galactose Oxidase:

ESR and Model Studies

M o d e l systems m a y b e v e r y u s e f u l i n e l u c i d a t i n g the atoms l i g a n d e d to the c o p p e r . W e earlier p r o p o s e d a p s e u d o - s q u a r e p l a n a r N 0 2


2

Schiff

266

BIOINORGANIC C H E M I S T R Y

II

b a s e c o m p l e x of c o p p e r as a m o d e l for the e q u i t o r i a l c o o r d i n a t i o n of C u ( I I ) i n galactose oxidase ( 2 1 ) .

T h a t m o d e l c o m p l e x m i m i c s some of

the s p e c t r a l properties of the e n z y m i c C u ( I I ) . I n s u p p o r t of that m o d e l , b e t t e r - r e s o l v e d E S R spectra of the e n z y m e h a v e b e e n o b t a i n e d . F i g u r e 1 shows the t i m e - a v e r a g e d E S R s p e c t r u m of galactose oxidase at 100 °K. A five-line s u p e r h y p e r f i n e s p l i t t i n g o n the p a r a l l e l lines ( A

z z

component)

c a u s e d b y t w o nitrogens is c l e a r l y i n d i c a t e d . T h i s c o n c l u s i v e l y d e m o n strates that at least t w o n i t r o g e n atoms m u s t b e l i g a n d e d to the c o p p e r a t o m ; the presence

of f o u r nitrogens ( 2 0 )

appears u n l i k e l y .

(Recent

s p i n - e c h o d a t a are consistent w i t h c o o r d i n a t i o n of the C u ( I I ) b y h i s t i d i n e i m i d a z o l e n i t r o g e n atoms.

(Work

two

i n collaboration w i t h J .


P e i s a c h a n d W . M i m s ). ) H o w e v e r , the Schiff base c o m p l e x lacks the s t a b i l i t y t o w a r d s r e d u c t i o n b y C N " that characterizes the C u ( I I ) i n galactose oxidase. W h i l e the e n z y m e b i n d s a single C N " e v e n at l a r g e C N " excess ( 2 2 ) , the C u ( I I ) i n the m o d e l is r e d u c e d b y the l i g a n d . T o assess the u n d e r l y i n g s t r u c t u r a l c o m p o n e n t s w h i c h s t a b i l i z e the e n z y m i c C u ( I I ) t o w a r d s r e d u c t i o n by C N " , a

five-coordinate

m o d e l ( F i g u r e 2) h a v i n g square b i p y r a m i d a l

symmetry was prepared (23).

( T h e c o n d i t i o n s a n d system

procedures

Figure I . ESR spectrum of galactose oxidase at ~ 5 Χ Ι0~ Μ. Τ = 100°Κ, average of six scans. Obtained with Nicolet Lab-80 CAT on a Varian E-9 spectrometer. Insert shows the second and third parallel lines and the fiveline superhyperfine splitting. 4


15.

BEREMAN E T AL.

Copper(II)

Site

Cu (TAAB) + S (CH CH 0Na) 2

2

2

in Galactose

F i g u r e

2

.

267

Oxidase

Cu[Nfi]

(see

model

text)

r e p o r t e d b y B u s c h et a l . ( 2 3 ) , f o r t h e N S c o m p l e x d o n o t y i e l d t h e 4

complex reported.

M o r e likely, the complex they obtained was one i n


w h i c h o n l y one side of t h e b r i d g i n g - O - C - C - S - C - C - O - g r o u p is a t t a c h e d to the T A A B b a c k b o n e a n d the other e n d is free, p r o b a b l y as the a l c o h o l . A

detailed synthetic procedure

preparation

(24)).

for the complex

l i g a t i o n y i e l d s i o n i c b o n d s to C u ( I I I ) . synethetically more purpose

atom

T h u s a n N S system, w h i c h is 4

easily a p p r o a c h e d , w i l l p r o b a b l y serve t h e same

as a n N 0 S 2

d i s c u s s e d here is i n

I n a qualitative w a y , nitrogen a n d oxygen

2

system.

T h e utility

of t h e m o d e l

lies i n its

encompassing t w o possibly important structural features—axial ligation b y s u l f u r (14, 2 5 ) a n d a " c a g e " s h i e l d i n g o n e a x i a l p o s i t i o n f r o m exogenous l i g a n d s . T a b l e I s u m m a r i z e s t h e p e r t i n e n t E S R a n d C N " d a t a . I n fact, n o n e of these m a c r o c y c l i c C u ( I I )

complexes

b i n d C N " at a l l as

e v i d e n c e d b y b o t h o p t i c a l a b s o r b a n c e a n d E S R studies. T h u s , a l t h o u g h t h e y are stable to C N " , t h e i r s t a b i l i t y is c a u s e d b y a t o t a l l a c k of r e a c t i v i t y t o w a r d s C N " , q u i t e u n l i k e t h e e n z y m e . A l s o , as i n d i c a t e d i n T a b l e I, there is little i n d i c a t i o n of a x i a l l i g a t i o n b y either - S - o r - O - as e v i denced

b y t h e E S R parameters.

W e conclude

f r o m these d a t a t h a t

b i n d i n g of exogenous l i g a n d s r e q u i r e s access t o a n essentially e q u i t o r i a l c o o r d i n a t i o n site a n d that t h e r i g i d m a c r o c y c l e is s i m p l y too r i g i d t o a l l o w this s u b s t i t u t i o n . T h e a p p a r e n t l a c k of a x i a l c o o r d i n a t i o n b y - S m a y b e c a u s e d b y t h e t h i o e t h e r s s o m e w h a t w e a k e r affinity f o r C u ( I I ) (cf. RS~) a n d does n o t r u l e o u t m e r c a p t i d e l i g a t i o n i n t h e p r o t e i n (14, 25).

H o w e v e r , b o t h t h e m a g n i t u d e of t h e s p i n H a m i l t o n i a n p a r a m e t e r s

w h e n c o m p a r e d w i t h other systems ( 2 6 ) a n d t h e l a c k of a s u b s t a n t i a l l i n e a r electric field effect ( L E F E ) o n t h e g-values m a k e i t u n l i k e l y that s u l f u r l i g a t i o n , i f present at a l l , is e q u i t o r i a l . I n this l i m i t e d w a y , t h e N S system is g e o m e t r i c a l l y , i f n o t c h e m i c a l l y , a p p r o p r i a t e . 4

Galactose Oxidase:

Optical

Transitions

and Anion

Binding

G i v e n some n o t i o n of t h e n a t u r e of t h e e n d o g e n o u s l i g a n d s , w e c a n next ask h o w exogenous l i g a n d s p e r t u r b t h e c o p p e r a t o m a n d w h a t this c a n t e l l us a b o u t t h e e l e c t r o n i c transitions e x h i b i t e d b y t h e C u ( I I ) a t o m i n galactose oxidase.


268


Table I.

Stoichiometry of C N " Ligation to (and Redox Stability

Complex

Ligand

C u ( T A A B ) [S ( C H C H 0 ) Cu ( T A A B ) [0 ( C H C H 0 ) Cu(TAAB) [CH N(CH CJ Cu(TAAB) [CH (CH CH Cu(tren-0H)BPh ' Cu(F Ac) en Cu(Ac) en Cu(tren-NH Ph)BPh ' G a l a c t o s e oxidase 2

2

2

2

2

3

2

2

2

4

3

c


h c d

N N N N N N N N N

2 2

2

2

d

r

2

a

2

] ] 0) ] 0) ] b

e

2

2

II

4

4 4 4

Symmetry

Type S 0 N

C (pseudo) C (pseudo) C (pseudo) C (pseudo) C v (pseudo?) rhombic planar ( C ) rhombic planar (C v) C (pseudo) rhombic planar (C y) 4 V

4 V

4 V

4

4

2 2 4

2

4 V

0 0 0 N 0

3

2

2 V

2

2

3 V

2

(?)

2

Values in Gauss. See Figure 2 and Refs. 23 and 24 for explanation of terminology. tren-NH Ph-(2,2^2''-triaminotriethylamine-phenylamine)copper(II). Ph = - C H . 2

6

5

A t least five e l e c t r o n i c transitions c a n b e d e t e c t e d b y

absorbance,

difference a b s o r b a n c e , a n d c i r c u l a r d i c h r o i c ( C D ) s p e c t r a f o r

galactose

oxidase p r i o r to r e s o l u t i o n b y c o m p u t e r analysis or m a g n e t i c C D studies. T h e s e o c c u r at energies w h i c h c o r r e s p o n d to 314, 395, 500, 630, a n d about 7 7 5 n m ( T a b l e I I )

(27).

N o t e w o r t h y a m o n g these b a n d s are t h e

transitions n e a r 650, 450, a n d 800 n m since t h e y are e x h i b i t e d b y a l l copper proteins ( 2 ) .

I n a n y event, since o n l y a m a x i m u m of f o u r db-d

transitions are p e r m i t t e d , at least one of the t r a n s i t i o n s e x h i b i t e d b y galactose oxidase m u s t b e c h a r g e transfer i n n a t u r e . M o r e o v e r , a l t h o u g h the pseudo-square w o u l d not b e

p l a n a r s y s t e m w h i c h is l i k e l y i n galactose

expected

to e x h i b i t l o w e r e n e r g y

oxidase

transitions, C D

and

m a g n e t i c C D s p e c t r a i n the n e a r - i n f r a r e d r e g i o n s h o u l d b e o b t a i n e d as h a v e b e e n r e c e n t l y r e p o r t e d i n T y p e I C u ( I I ) systems

(28).

It is i n t e r e s t i n g to c o n s i d e r the effect of exogenous l i g a n d s ( w h i c h h a v e p r e v i o u s l y b e e n s h o w n to b i n d to t h e C u ( I I ) a t o m i n n e r s p h e r e b y E S R studies ( 2 2 ) ) o n the o p t i c a l s p e c t r u m of galactose oxidase. Table II.

Electronic Transitions Exhibited by Galactose Circular

(While

Oxidase"

Dichroism

Absorbance

[Θ] [(dey cm )/ dMol] 2

X(nm)

v(cm~ )

314 445 630 775

31,800 22,500 15,900 12,900

1

1

e(M~

1

cm' )

1,370 1,155 1,015 905

1

\(nm)

v(

cm' )

314 395 500 610

31,800 25,300 20,000 16,400

1

+18,900 +3,000 +1,500 -8,200

Data from Ref. 27.


15.

BEREMAN E T AL.

of)

Some 4 - and 5-Coordinate C o p p e r ( I I ) Complexes"


269

(24)

CN- Effects &zz

g^

1:1 Complex

144/2.160' 145/2.159' 179/2.171 144/2.160' 163 187.2/2.220" 211/2.186* 163/2.244* 173/2.273

no no no no ? no no ? yes

9


h

e f 0 Λ

Excess stable stable stable stable reduces reduces reduces reduces stable

Cu(II) Cu(II) Cu(II) Cu(II)

-

Cu(I) Cu(I) Cu(I) Cu(I)

(F3Ac)2en == ^A^'-ethylenebisCtrifluoroacetylacetoniminato)copper(II). (Ac)2en = N,iV'-ethylenebis(acetylacetoniminato)copper(II). Data in D M F solvent. Data in C H O H solvent, 2

5

it w a s n o t i n d i c a t e d i n o u r p r e v i o u s l y r e p o r t e d a n i o n b i n d i n g studies t h a t i n n e r c o o r d i n a t i o n sphere b i n d i n g occurs, m o r e recent s u p e r h y p e r f i n e d e t e c t i o n of exogenous l i g a n d b i n d i n g c e r t a i n l y substantiates this fact. F u r t h e r changes i n the A

z z

c o m p o n e n t are q u i t e s i m i l a r to the fine studies

of C o l e m a n et a l . (29, 30) o n a r t i f i c i a l C u ( I I ) p r o t e i n s w h e r e exogenous l i g a n d s gave s u p e r h y p e r f i n e s t r u c t u r e to the E S R s p e c t r u m . )

A z i d e , for

e x a m p l e , at a p p r o x i m a t e l y 100:1 m o l a r excess causes a v e r y l a r g e b l u e shift of t h e 7 7 5 - n m p e a k n o t s h o w n h e r e a n d t h e 630- a n d 4 4 5 - n m a b s o r b a n c e b a n d s ( F i g u r e 3 ). A b s o r b a n c e m a x i m a n e a r 380 n m w i t h a z i d e h a v e b e e n a t t r i b u t e d to c h a r g e transfer c o m p l e x e s i n o t h e r p r o t e i n s ( 3 1 ) , b u t since the shift f r o m 445 is t h e same as for the 6 3 0 - n m b a n d i n e n e r g y terms, w e suggest t h a t this 3 8 0 - n m b a n d is r e l a t e d to the 4 5 0 - n m b a n d . C y a n i d e also u n i f o r m l y b l u e shifts the three l o w e n e r g y transitions. M o s t i m p o r t a n t l y , c o m m o n a n i o n exogenous l i g a n d s affect m a i n l y the i n t e n s i t y of the 3 1 4 - n m t r a n s i t i o n , b u t n o t its energy. G e n e r a l l y , t r a n s i t i o n i n t e n sity increases are t w o to three f o l d w i t h the b i n d i n g of s u c h a n i o n s . T h e s i m p l e fact that anions affect the e n e r g y of t h e l o w e s t e n e r g y t r a n s i t i o n s s i m i l a r l y suggests t h a t these are p r i m a r i l y d-d 314-nm transition m a y be

a transition w i t h

i n c h a r a c t e r w h i l e the u n i q u e c h a r g e transfer

character. A l l anions w h i c h b i n d to the C u ( I I ) i n galactose oxidase l o w e r t h e g

z z

and A

values ( 2 2 ) .

z z

b l u e shift i n the "d-d"

T h i s is consistent w i t h ( b u t n o t r e q u i r e d f o r ) a t r a n s i t i o n s (32, 3 3 , 3 4 ) .

Fe(CN)

6

3

" is the o n l y

a n i o n a m o n g the l i m i t e d ones w e h a v e s t u d i e d w h i c h p r o d u c e s a r e d shift i n the o p t i c a l b a n d s ( F i g u r e 4 ) . of F e ( C N )

6

3

A t 1:1, 5 : 1 , or 100:1 m o l a r ratios

~ to e n z y m e the same difference a b s o r b a n c e s p e c t r u m is

o b t a i n e d , a n d i t is consistent w i t h c o m p l e x f o r m a t i o n b e t w e e n galactose oxidase a n d the a n i o n . N a m e l y , the p o s i t i v e difference peaks at 455, 830,


270


II

a n d near-650 n m i n d i c a t e r e d shifts of the c o p p e r t r a n s i t i o n s . S i m i l a r to the other anions s t u d i e d , f e r r i c y a n i d e also leads to r e l a t i v e l y l a r g e i n creases i n a b s o r b a n c e .

A n a l o g o u s to the effects of a z i d e , for e x a m p l e , the

i n t e n s i t y effects of f e r r i c y a n i d e are m o r e p r o n o u n c e d o n the 445- a n d 7 7 5 - n m transitions t h a n o n the 6 3 0 - n m b a n d .

F u r t h e r e v i d e n c e for a

F e ( C N ) ~ - p r o t e i n c o m p l e x is the o b s e r v a t i o n that r e m o v a l of the a n i o n 3

6

is v e r y difficult, e.g., b y t r e a t m e n t w i t h a n a n i o n exchange r e s i n . Fe(CN) absorbance

G

4

'

also

apparently binds

spectrum

( F i g u r e 4)

to

the p r o t e i n ; the

difference

i n d i c a t e s a s m a l l r e d shift.

Again,

i n c r e a s i n g the m o l a r r a t i o of f e r r o c y a n i d e causes n o f u r t h e r a b s o r b a n c e changes.

T h e t w o anions a d d e d at i d e n t i c a l 5:1 m o l a r ratios to e n z y m e


cause a difference s p e c t r u m consistent w i t h a c o m p e t i t i v e b i n d i n g to the p r o t e i n ; the r e s u l t a n t difference a b s o r b a n c e is that e x p e c t e d for a n e q u a l p a r t i t i o n i n g of the e n z y m e b e t w e e n the t w o anions ( F i g u r e 4 ) . S i m i l a r results are o b t a i n e d b y relaxation Fe(CN) by 60%

6

3

of

added

1 9

F " or

of

m o n i t o r i n g the C u ( I I ) - m e d i a t e d

bulk water

(35,36).

For

example,

~ decreases t h e r e l a x i v i t y of the C u ( I I ) t o w a r d s w a t e r protons while F e ( C N )

6

4

" has little effect.

A d d i t i o n of e q u a l m o l a r

a m o u n t s of these anions p r o d u c e s a 3 0 % r e d u c t i o n . Effects s u c h as these o c c u r at as l i t t l e as 1:1 m o l a r ratios

(37).

T h e s e results c o u l d b e a t t r i b u t e d to a c h a n g e i n the r e d o x state of the e n z y m e c o p p e r (17,38). Ί

1

1

2.0

1

H o w e v e r , E S R spectra at 1:1 a n d 6:1 m o l a r

Γ

ι

/ \ / χ / \

1

1

1

1

1

1—^i

1

Γ

1.5

ο

M 1.0

5h

360

400

440

480

560

600

640

Figure 3. Copper absorbance spectrum of galactose oxidase (775 nm not recorded) (-) and the enzyme in the presence of sodium azide ( ). Spectra were recorded in 5-cm cells, 0.1 M sodium phosphate buffer, pH 7.0.



15.

BEREMAN

E TA L .

271


Figure 4. Visible absorbance spectrum of galactose oxidase ( ) and the difference absorbance spectra with 5:1 molar ratios of ferricyanide ( ), ferrocyanide (- · and the two together (- · -). The scale on the right is for both solutions containing Fe(CN) ~. Spectra were recorded in 4.5-cm double compartment cells; 0.1 M sodium phosphate buffer, pH 7.0. 3

6

ratios of F e ( C N ) ~ - t o - e n z y m e i n d i c a t e l i t t l e c h a n g e i n t h e s p e c t r a l i n 6

3

tensity ( F i g u r e 5 ) . T h u s , t h e effects seen i n a b s o r b a n c e a n d n u c l e a r s p i n r e l a x a t i o n at these ratios c a n n o t o b v i o u s l y b e a t t r i b u t e d to a n o x i d a t i o n of C u ( I I ) to C u ( I I I ) , f o r e x a m p l e (17,38).

T h e fact t h a t t h e difference

a b s o r b a n c e does n o t c h a n g e e v e n u p to 1 0 0 : 1 m o l a r r a t i o suggests t h a t as f a r as t h e l i g a n d field e n e r g y of t h e e n z y m i c c o p p e r a n d t h e t r a n s i t i o n p r o b a b i l i t i e s are c o n c e r n e d , t h e a d d i t i o n of f u r t h e r F e ( C N )

6

3

" is u n i m -

p o r t a n t . T h i s is of interest since t h e C u ( I I ) E S R s i g n a l (as i n F i g u r e 5 ) d i s a p p e a r s at these h i g h ratios. T h u s , t h e effects of f e r r i c y a n i d e o n t h e o p t i c a l a b s o r b a n c e a n d s p i n characteristics of t h e C u ( I I ) a r e a p p a r e n t l y d i s t i n c t . W h e t h e r o r n o t t h e differences are r e l a t e d to differences i n t h e observation temperature used, w h i l e a possible explanation ( 3 9 ) , remains a n i n t r i g u i n g b u t u n r e s o l v e d q u e s t i o n . F u r t h e r i n f o r m a t i o n o n t h e effects of F e ( C N )

6

3

' m a y b e p r o v i d e d b y x-ray spectroscopic experiments n o w

i n progress. A t i m e - d e p e n d e n t , a p p a r e n t r e d u c t i o n of F e ( C N )

6

3

" b y both the

h o l o - a n d a p o e n z y m e s is i n d i c a t e d b y difference a b s o r b a n c e


measure-


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BIOINORGANIC

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Figure 5. Electron spin resonance spectrum of galactose oxidase (A) and galactose oxidase and Fe(CN) ~ at a 1:1 (B) and 1:6 (C) molar ratio spectra were recorded at 100°K, with a power of 20 mw (9.115 GHz) and a modulation amplitude of 2G; [galactose oxidase] = 0.5 mM. 3

6

merits, as i n F i g u r e 4. A t a 1:1 m o l a r r a t i o of f e r r i c y a n i d e - t o - e n z y m e , the difference s p e c t r u m ( F i g u r e 4) s l o w l y ( τ ι /

2

^ 7 h r ) changes to y i e l d a

single n e g a t i v e difference p e a k at 420 n m , the A

m a x

for F e ( C N )

6

3

~.

The

s o l u t i o n , too, loses its c h a r a c t e r i s t i c y e l l o w color c a u s e d b y F e ( C N ) d u r i n g this p e r i o d .

6

3

~

T h e f a c t t h a t this r e d u c t i o n is i n d e p e n d e n t of t h e

p r e s e n c e of the C u ( I I ) i n the e n z y m e a n d t h a t , w i t h t h e

holoenzyme,

t h e o n l y difference p e a k is that a t t r i b u t a b l e to the r e d u c t i o n of F e ( C N )

6

3

~

(i.e., n o c o p p e r difference peaks p r e s e n t ) i n d i c a t e s that the p r o t e i n , a n d n o t t h e C u ( I I ) , is i n v o l v e d i n this r e d o x r e a c t i o n . T h i s p r o t e i n p r o d u c t has not b e e n c h a r a c t e r i z e d . Non-Ligand

Active

Site Groups

O t h e r p r o t e i n groups w h i c h c o n t r i b u t e to the m o l e c u l a r d y n a m i c s of t h e e n t i r e c o p p e r a c t i v e site m u s t b e c o m p l e m e n t a r y to i n n e r - c o o r d i n a -


15.

BEREMAN E T A L .

273


t i o n sphere l i g a n d s to the c o p p e r a t o m . O n e of the most d r a m a t i c results w h i c h first suggested

that a t r y p t o p h a n r e s i d u e m i g h t b e w i t h i n

the

a c t i v e site locus was the n e a r - u v - C D s p e c t r u m of galactose oxidase i n t h e presence of d i h y d r o x y a c e t o n e

( w h i c h is a n excellent s u b s t r a t e )

or

the a l d e h y d e p r o d u c t of the galactose r e a c t i o n , e a c h i n the absence

of

oxygen.

B i n d i n g of a substrate or p r o d u c t has e n o r m o u s effects o n the

t r y p t o p h a n o p t i c a l a c t i v i t y i n the 2 8 5 - 3 0 0 - n m r e g i o n (40).

Furthermore,

i n c o r p o r a t i o n of c o p p e r i n t o the a p o e n z y m e causes a 2 9 % r e d u c t i o n i n tryptophan

fluorescence

(41).

A holoenzyme-apoenzyme

difference a b

sorbance s p e c t r u m also c l e a r l y shows p e r t u r b a t i o n of a t y p t o p h a n e n v i r o n m e n t b y the c o p p e r a t o m ( 2 7 ) .

W h i l e these results c o u l d reflect v e r y


i n d i r e c t i n t e r a c t i o n s , selective o x i d a t i o n of the t r y p t o p h a n s i n galactose oxidase w i t h n - b r o m o s u c c i n i m i d e

( N B S ) revealed a critical structure-

f u n c t i o n r o l e for at least one r e s i d u e (41, 42).

G a l a c t o s e oxidase is i n a c

t i v a t e d as exactly t w o of its 18 t r y p t o p h a n s are o x i d i z e d . M o r e o v e r , the i n a c t i v a t i o n profile i m p l i e s that just one of the most r e a c t i v e residues i n the e n z y m e is p r o b a b l y associated w i t h the i n a c t i v a t i o n (41).

One mani

festation of the specificity of the r e a c t i o n is the o b s e r v a t i o n that t r y p t o phan

o p t i c a l a c t i v i t y associated

affected.

with

o n l y the 2 9 0 - n m

T h e 2 9 5 - n m p e a k is unaffected ( F i g u r e 6 ) .

e x t r e m u m is

A new extremum

80

60

θ

W 20

0

-20 -40 250

270

290

310

Wavelength ,nm Figure 6. Near-uv CD spectrum of galactose oxidase in 0.1 M sodium phosphate buffer, pH 7.0 ( ) and the NBS-modified enzyme; 2 trp equivalents oxidized (- · ·). Spectra were recorded in a 1-cm cell.


274

BIOINORGANIC

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II

n e a r 250 n m reflects o p t i c a l a c t i v i t y of the o x i n d o l e p r o d u c t of the o x i dation reaction.

Fluorescence

is also m a r k e d l y affected;

residues o x i d i z e d a c c o u n t for 4 8 % of the t o t a l

fluorescence

the first t w o of the e n z y m e

w h i c h f u r t h e r i n d i c a t e s that t h e most r e a c t i v e t r y p t o p h a n residues h a v e u n i q u e properties

(41).

W h a t is most i n t e r e s t i n g a b o u t this m o d i f i c a t i o n is w h a t i t suggests a b o u t the m o l e c u l a r i n t e r a c t i o n s w i t h i n the c o p p e r a c t i v e site.

Prior

e x p e r i m e n t s e s t a b l i s h e d that w i t h the n a t i v e e n z y m e , galactose i n the a b s e n c e of

oxygen

m a r k e d l y reduces

its c o p p e r o p t i c a l a c t i v i t y , b u t

o x y g e n i n the absence of galactose has n o significant effect (40).

The

fact that galactose also m a r k e d l y reduces c o p p e r o p t i c a l a c t i v i t y i n the Downloaded by UNIV LAVAL on April 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/ba-1977-0162.ch015

N B S - i n a c t i v a t e d e n z y m e suggests that the i n a c t i v a t i o n i n v o l v e s a n effect o n catalysis s p e c i f i c a l l y r a t h e r t h a n b i n d i n g . T h e same influence c a n b e drawn from

fluorescence

data

(41).

W h a t is p a r t i c u l a r l y i n t e r e s t i n g

here is that w h i l e b i n d i n g of c o m m o n exogenous l i g a n d s i n v a r i a b l y leads to a u n i f o r m r e d u c t i o n i n e a c h of the o p t i c a l a c t i v i t y transitions, t h e i n a c t i v a t i o n b y selective t r y p t o p h a n o x i d a t i o n is associated w i t h a d e crease at 314 n m , b u t a n increase near 600 n m . W h i l e several alternate inferences are possible for the increase n e a r 600 n m , one p o s s i b i l i t y is that the c h a n g e i n c h e m i s t r y of the c o p p e r site as e x e m p l i f i e d b y a b o l i t i o n of catalysis i n this case m a y reflect a c o n v e r s i o n of the n o r m a l p s e u d o - s q u a r e

planar geometry

to a n e n v i r o n m e n t

c h a r a c t e r i z e d b y a decrease i n a x i a l u n p a i r e d e l e c t r o n d e n s i t y (see III).

T h i s m i g h t b e b r o u g h t a b o u t b y b o n d i n g changes a n d / o r

Table stereo

c h e m i c a l effects, b o t h of w h i c h c a n effect the a x i a l e l e c t r o n d e n s i t y . I n t e r e s t i n g l y , the c u p r i c i o n i n this m o d i f i e d p r o t e i n is r a p i d l y r e d u c e d b y c y a n i d e . A t a 10:1 m o l a r r a t i o , C N " effects c o m p l e t e r e d u c t i o n Table III. Spin Hamiltonian Parameters for Liganded and Non-Liganded Native and Modified Galactose Oxidase" Enzyme Form/Ligand (Ratio) Native C N " (1:1) N -(100:1) N B S oxidized C N " reduces C u ( I I ) Iodoacetamide alkylated" CN(1:1) 6

3

0

Ass

gzz

A χχ

176.5 155.8 166.8 166.0

2.273 2.226 2.262 2.267

28.8 41.6 27.2 38.0

2.048 2.035 2.049 2.055

30.1 45.2 27.9 43.0

2.058 2.048 2.040 2.065

177.8 160.1

2.268 2.234

30.5 38.8

2.035 2.041

30.6 43.0

2.064 2.051

" S p e c t r a were o b t a i n e d o n a V a r i a n E-9 X - b a n d s p e c t r o m e t e r at 110°K with a 100-KH. m o d u l a t i o n a m p l i t u d e of 2 G a u s s a n d a m i c r o w a v e power of 30 m w at 9.5 GHz w i t h p r o t o n G a u s s m e t e r a n d f r e q u e n c y c o u n t e r for spectral m a r k i n g .

{22). (42).

b c d

U4).


15.

275


BEREMAN E T A L .

4

5

6

7

8

9


pH Figure 7. Flot of the pH-dependent values for the oxidation rate of β-methyl-O-galactopyranoside at 1.4 raM 0 by galactose oxidase (k ) and the rate of inactivation of galac tose oxidase by I m M iodoacetamide. The smooth curve for the former (O) is calculated using pK values 6.3 and 7.1 with k t —1350 sec . The curve for the latter process (Φ) corre sponds to pK values 6.3 and 7.6 with a pHindependent rate of 2.2 min' . 2

catapp

a

ca

1

a

1

(Table III).

A t o m i c a b s o r p t i o n analysis shows t h a t the c o p p e r is s t i l l

present i n the e n z y m e . T h e s e t w o facts c o u p l e d w i t h the 6 0 0 - n m r e g i o n i n t e n s i t y increase m i g h t b e i n t e r p r e t e d to suggest t h a t a s t e r e o c h e m i c a l d i s t o r t i o n a c c o m p a n i e s t r y p t o p h a n o x i d a t i o n i n galactose oxidase.

For

e x a m p l e , a s a d d l i n g of the p l a n a r e n v i r o n m e n t w o u l d b e e x p e c t e d l o w e r the r e d u c t i o n p o t e n t i a l of the C u ( I I ) - C u ( I )

to

couple.

T h e a c t i v e site role of one other p r o t e i n m o i e t y has b e e n e x a m i n e d i n d e t a i l . I n the first r e p o r t e d w o r k w i t h galactose oxidase, p H - r a t e d a t a w e r e r e p o r t e d that i m p l i c a t e d a n i m i d a z o l e g r o u p (11).

The pH-depend-

ence of b o t h the e n z y m i c r e a c t i o n as w e l l as e n z y m e i n a c t i v a t i o n b y i o d o a c e t a m i d e reflects the essential i o n i z a t i o n of a conjugate a c i d , p K = a

( F i g u r e 7)

(43).

6.3

I n a c t i v a t i o n is c a u s e d b y the specific a l k y l a t i o n of a

s i n g l e h i s t i d i n e at its N - 3 n i t r o g e n (43).

T h e alkaline

pH-dependence

m a y reflect the i o n i z a t i o n of a c o p p e r - b o u n d w a t e r m o l e c u l e ( v i d e s u p r a ) (36, 37, 43, 44).

L i k e the N B S - m o d i f i e d p r o t e i n , the a l k y l a t e d e n z y m e

s t i l l b i n d s sugar substrate, as i n d i c a t e d b y T h u s , catalysis is a g a i n u n i q u e l y affected. m a l l y a n d does not r e d u c e the C u ( I I )

fluorescence

experiments

(43).

M o r e o v e r , C N " also b i n d s n o r (43).

C o r r e l a t e d to this is the

near i d e n t i t y of the s p i n H a m i l t o n i a n parameters of n a t i v e a n d a l k y l a t e d forms

(Table III).

w i t h i o d o a c e t a m i d e (43).

F u r t h e r m o r e , the a p o e n z y m e

does not

react

T h e r e f o r e , the affected i m i d a z o l e is most l i k e l y

n o t a c o p p e r l i g a n d . T h e a p o e n z y m e result does suggest, h o w e v e r , t h a t


276

BIOINORGANIC

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a c r i t i c a l r e l a t i o n s h i p b e t w e e n h i s t i d i n e r e a c t i v i t y a n d the c o p p e r a t o m does exist. M o r e o v e r , t h e N B S - o x i d i z e d p r o t e i n i s n o t a l k y l a t e d , w h i c h establishes a c r i t i c a l l i n k b e t w e e n the r e a c t i v e t r y p t o p h a n a n d h i s t i d i n e


residues (43) w h i c h are p r o b a b l y b o t h w i t h i n the active site locus.

400

500

600 700 Waveiength.nm

800

900

Figure 8. Copper absorbance spectrum of galactose oxidase ( ) and difference absorbance spectrum with the alkylated enzyme as the reference and the unmodified enzyme as the sample ( ). Spectra were recorded in 5-cm cells, O.I M sodium phosphate buffer, pH 7.0. R e s u l t s w i t h galactose oxidase i l l u s t r a t e that i t is e x t r e m e l y

danger-

ous t o r e l y o n a n y one s p e c t r a l m e t h o d t o evaluate a p e r t u r b a t i o n o f a m e t a l system.

E S R has b e e n a sensitive p r o b e f o r i n n e r

sphere l i g a n d s b u t a p p a r e n t l y a r e l a t i v e l y p o o r one conformation recorded

i n galactose oxidase.

T h e difference

coordination

of metal

spectrum

w i t h the a l k y l a t e d e n z y m e as t h e reference a n d t h e

e n z y m e as the s a m p l e

is i n d i s t i n g u i s h a b l e f r o m

the n a t i v e

a b s o r b a n c e s p e c t r u m , i.e., a l k y l a t i o n v i r t u a l l y abolishes

chelate

w h i c h is native

enzyme's

copper

absorb-

a n c e ( F i g u r e 8 ) . B y C D , some s m a l l m a g n e t i c a n d / o r electric transitions are d e t e c t e d w i t h the a l k y l a t e d e n z y m e ( F i g u r e 9 ) .

T h e l a c k o f shifts

i n energies o f the c o p p e r a b s o r b a n c e transitions f u r t h e r argues against a c h a n g e i n l i g a t i o n . T h e decrease i n t r a n s i t i o n p r o b a b i l i t y c a n b e best r a t i o n a l i z e d b y the c o p p e r chelate a s s u m i n g a m o r e

perhaps

centrosym-

m e t r i c g e o m e t r y . I n a n y event, i n a d d i t i o n t o its role as a specific b a s e


15.


τ

-8—I 300

1

1

277


BEREMAN E T A L .

1

1

1

1

1

1

1

I I ι 320 340 360 380 400 420 440 460 480 Wav6iength,nm

1

1

1

r

I I 1 1— 500 520 540 560

Figure 9. Copper CD spectrum of galactose oxidase ( ) and the alkyl ated enzyme (· · -) which were recorded in 5-cm cells in 0.1 M sodium phos phate buffer, pH 7.0 catalyst, the affected h i s t i d i n e also p r o b a b l y influences a c t i v i t y t h r o u g h a c r i t i c a l role i n m a i n t a i n i n g the c o p p e r chelate c o n f o r m a t i o n (43). G a l a c t o s e oxidase c a n i l l u s t r a t e h o w l i g a n d s , g e o m e t r y , a n d a c t i v e site g r o u p s t o g e t h e r p r o v i d e the basis for the s t r u c t u r e - f u n c t i o n p r o p e r ties o f a m e t a l a c t i v e site.

F i g u r e 10 s u m m a r i z e s m u t u a l i n t e r a c t i o n s

AQ t Histidine

L

^

Tryptophan oxidation prevents Alkylation .·

Figure 10. Summary of interdependent interactions between the three groups proposed to be at the active site. Q = ffuorscence quantum yield, AE = difference absorbance, ΑΘ = change in ellipticity (CD). The dis tance estimation is derived from fluorescence energy-transfer methods. f


278


II

w h i c h m a y p e r t a i n b e t w e e n the c o p p e r chelate a n d a c t i v e site groups i n galactose oxidase. A r e p r e s e n t a t i o n of the a c t i v e site w h i c h contains these s t r u c t u r a l elements is seen i n F i g u r e 11. M o d e l a n d s p e c t r a l studies h a v e suggested the i n - p l a n e l i g a n d s . T h a t the c o p p e r influences the t r y p t o p h a n is s h o w n b y difference

absorbance

and

fluorescence

quantum yield.

The N B S -


m o d i f i c a t i o n i n t u r n demonstrates the c r i t i c a l influence of the t r y p t o p h a n

Figure 11. Diagrammatic representation of active site which includes a four-coordinate copper complex, sugar-substrate bound outer-sphere to the Cu(II) atom, imidazole and indole rings, and nonpolar side chains (X). The distance between the Cu(H) and indole is estimated by fluorescence energytransfer methods. o n the c o p p e r chelate.

T h e s p e c t r a l o v e r l a p b e t w e e n the

fluorescence

s p e c t r u m of the t r y p t o p h a n a n d the c o p p e r a b s o r b a n c e at 314 n m a l l o w s one to estimate the distance b e t w e e n these groups b y a F o r s t e r e n e r g y transfer c a l c u l a t i o n ( 4 1 ) .

T h e t r y p t o p h a n is p r o b a b l y n o t closer t h a n

12 A , b u t e v e n at this distance, i t c o u l d c o m e i n contact w i t h a b o u n d s u g a r substrate.

M o s t l i k e l y , the i n d o l e r i n g is one c o m p o n e n t

of

an

a c t i v e site cluster of h y d r o p h o b i c side chains w h i c h is c r i t i c a l to t h e c o n f o r m a t i o n of the entire a c t i v e site. S t r u c t u r e - f u n c t i o n roles h a v e b e e n suggested for u n i q u e t r y p t o p h a n residues i n other c o p p e r p r o t e i n s as w e l l

(44,45,46).

Moreover, the

s i n g l e t r y p t o p h a n t h a t is q u e n c h e d b y i n c l u d i n g the c o p p e r

atom i n

a z u r i n is a p p a r e n t l y not i n contact w i t h t h e i n d o l e r i n g , as e v i d e n c e d b y metal replacement a n d phosphorescence

results

(45,46).


15.

BEREMAN E T A L .

Copper(II)

Site in Galactose

Oxidation of this tryptophan i n galactose alkylation of the histidine residue.

279

Oxidase

oxidase

also

prevents

Alkylation of the histidine residue

in turn markedly affects the fluorescence quantum yield of this tryptophan ( 43 ) and nearly abolishes the absorbance of the copper atom. T h e copper atom itself is also essential to the reactivity of this histidine. Thus, we appear to have a consistent set of highly interdependent components.

Not unexpectedly, the copper site cannot be fully understood

without considering its interactions with non-ligand protein groups. Acknowledgments


The work outlined here is a product of the cooperative efforts of the several graduate and undergraduate students who are members of the Bioinorganic Graduate Research Group. T h e Group is grateful for the support of the National Science Foundation (B404662) and the Graduate School of this University. Time-averaged E S R spectra were obtained with the aid of a Nicolet L a b 80 C A T ( N S F - M P S 7506183). R. D . B. is a recipient of a Camille and Henry Dreyfus Fellowship.

Literature Cited 1. Evans, G. W., Physiol. Rev. (1973) 54, 535. 2. Vänngård, T., "Copper Proteins," in "Biological Applications of Electron Spin Resonance," H. M. Swartz, J., R. Bolton, D. C. Borg, Eds., p. 441, Wiley-Interscienee, New York, 1972. 3. McMillin, D. R., Holwerda, R. Α., Gray, Η. B., Proc. Natl. Acad. Sci. USA (1974) 71, 1339. 4. McMillin, D. R., Rosenberg, R. G., Gray, Η. B., Proc. Natl. Acad. Sci. USA (1974) 71, 4760. 5. Bereman, R. D., Wang, F. T., Najdzionek, J., Braitsch, D. M., J. Am. Chem. Soc. (1976) 98, 7266. 6. Gray, H. B., ADV. CHEM. SER. (1977) 162, 145. 7. Spiro, T. S., Acc. Chem. Res. (1974) 7, 339. 8. "Biological and Biochemical Applications of Electron Spin Resonance," D. J. E. Ingram, Plenum, New York, 1969. 9. Richardson, J. S., Thomas, Κ. Α., Rubin, P. H., Richardson, D. C., Proc. Natl. Acad. Sci. USA (1975) 72, 1349. 10. Solomen, Ε. I., Dooley, D. M., Wang, R. H., Gray, H. B., Cerdonio, M., Mogno, F., Monani, G. L., J. Am. Chem. Soc. (1976) 98, 1029. 11. Cooper, J. Α., Smith, W., Bacila, M., Medina, H., J. Biol. Chem., (1959) 234, 445. 12. Amaral, D., Kelly-Falcoz, F., Horecker, B. L., Methods Enzymol. (1966) 9, 87. 13. Nobles, M. K., Madhosingh, C., Biochem. Biophys. Res. Commun. (1963) 12, 146. 14. Kosman, D. I., Ettinger, M. I., Weiner, R. E., Massaro, E. J., Arch. Biochem. Biophys. (1974) 165, 456. 15. Bauer, S., Blauer, G., Avigad, G., Isr. J. Chem. Proc. (1967) 5, 126. 16. Giblin, F., unpublished data. 17. Hamilton, G. Α., Libby, R. D., Hartzell, G. R., Biochem. Res. Commun. (1973) 53, 715.



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18. Kosman, D., unpublished data. 19. Blumberg, W., Horecker, B. L., Kelly-Falcoz, F., Peisach, J., Biochim. Biophys. Acta (1965) 96, 336. 20. Cleveland, L., Coffman, R. E., Coon, P., Davis, L., Biochemistry (1975) 14, 1108. 21. Giordano, R. S., Bereman, R. D., J. Am. Chem. Soc. (1974) 96, 1019. 22. Giordano, R. S., Bereman, R. D., Kosman, D. J., Ettinger, M. J.,J.Am. Chem. Soc. (1974) 96, 1023. 23. Katovic, V., Taylor, L. T., Busch, D. H., Inorg. Chem. (1971) 10, 458. 24. Bereman, R. D., Shields, G., unpublished data. 25. Kelly-Falcoz, F., Greenberg, H., Horecker, B. L., J. Biol. Chem. (1965) 240, 2966. 26. Peisach, J., Blumberg, W. E., Arch. Biochem. Biophys. (1974) 165, 691. 27. Ettinger, M. J., Biochemistry (1974) 13, 1242. 28. Solomon, E. J., Hare, L. W., Gray, H. B., Proc. Natl. Acad. Sci. USA (1976) 73, 1389. 29. Taylor, J. S., Mushak, P., Coleman, J. E., Proc. Natl. Acad. Sci. USA (1970) 67, 1410. 30. Taylor, J. S., Coleman, J. E., J. Biol. Chem. (1971) 246, 7058. 31. Williams, R. J. P., in "The Biochemistry of Copper," J. Peisach, P. Aisen, W. E. Blumberg, Eds., p. 131, Academic, New York. 32. Neiman, R., Kievelson, D., J. Chem. Phys. (1961) 35, 149. 33. Ibid. (1961) 35, 156. 34. Ibid. (1961) 35, 159. 35. Marwedel, B. J., Kurland, R. J., Kosman, D. J., Ettinger, M. J., Biochem. Biophys. Res. Commun. (1975) 63, 773. 36. Fabry, T. L., Kim, J. P., Titcomb, L. M., IBM Res. Rept. (1969) RW108, No. 11415. 37. Marwedel, B. J., Kurland, R. J., unpublished data. 38. Dyrkacz, G. R., Libby, R. D., Hamilton, G. Α., J. Am. Chem. Soc. (1976) 98, 626. 39. Nickerson, K. W., Phelan, N. F., Bioinorg. Chem. (1974) 4, 79. 40. Ettinger, M. J., Kosman, D.J.,Biochemistry (1974) 13, 1247. 41. Weiner, R. E., Ettinger, M. J., Kosman, D. J., Biochemistry, in press. 42. Kosman, D. J., Ettinger, M. J., Giordano, R. S., Bereman, R. D., Biochem istry, in press. 43. Kwiatkowski, L. D., Siconolfi, L., Ettinger, M. J., Weiner, R. E., Giordano, R. S., Bereman, R. D., Kosman, D. J., Arch. Biochem. Biophys., in press. 44. Morpurgo, L., Finazzi-Agró, Α., Rotilio, G., Mondovi, B., Biochem. Biophys. Acta (1974) 271, 292. 45. Finazzi-Agró, Α., Giovagnoli, C., Arigliano, L., Rotilio, G., Mondovi, B., Eur. J. Biochem. (1973) 34, 20. 46. Finazzi-Agró, Α., Rotilio, G., Avigliano, L., Guerrieri, P., Boffi, V., Mon dovi, B., Biochemistry (1970) 9, 2009. RECEIVEDJuly26, 1976.


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