Electron Transfer Pathways in Blue Copper Proteins

11 Electron Transfer Pathways in Blue Copper Proteins

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 9, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/ba-1977-0162.ch011

ISRAEL PECHT, OLE FARVER, and MICHEL

GOLDBERG

Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel

Several structural and functional aspects of blue copper pro teins have been investigated. The systematic study of the ultraviolet spectroscopic properties of blue single copper proteins (azurins and stellacyanin) brings evidence for sul fur as a ligand of the copper ion. The energy profile of the electron transfer equilibrium between Ps. Aeruginosa azurin and Fe(CN) was obtained by combined analysis of chemical relaxation times and amplitudes along with micro calorimetry and spectrophotometric titrations. The super -oxide(O ) reduces the type 1 Cu(II) in Rhus laccase. A relatively fast, full reoxidation of the partially reduced en zyme takes place in the presence of oxygen. Rhus laccase reacts specifically and with high affinity with H O to form a stable product which man be a functional intermediate. 3-/4-

6

-

2

2

^pwo

c e n t r a l themes a r e e n c o u n t e r e d

2

i n s t u d y i n g t h e m e c h a n i s m of

a c t i o n of r e d o x p r o t e i n s : ( 1 ) T h e d e t a i l e d p a t h w a y s of r e d o x e q u i v a l e n t s t o a n d f r o m trie a c t i v e centers ( 2 ) T h e i n t r a m o l e c u l a r events i n t h e m u l t i c e n t e r oxidases, w h i c h e n a b l e t h e f u n c t i o n a l c o o p e r a t i o n of t h e different a c t i v e sites i n c a r r y i n g o u t r e d u c t i o n o r o x i d a t i o n of specific substrates, n o t a b l y those w h e r e m u l t i p l e e l e c t r o n transfer steps a r e f a v o r e d p a t h w a y s T h e function of the electron-mediating proteins w h i c h contain a single r e d o x a c t i v e site (e.g., r u b r e d o x i n , a z u r i n s , c y a n i n s ) is m a i n l y r e l a t e d t o t h e first aspect. ficity

flavodoxins,

plasto-

S t i l l , the p r o n o u n c e d s p e c i -

encountered i n their function i n biological energy conversion proc-

esses i n d i c a t e s t h a t t h e i r r e d o x center, often a t r a n s i t i o n m e t a l i o n , is embedded

i n an evolutionarily optimized polypeptide

envelope.

179

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

The

180

BIOINORGANIC

CHEMISTRY

d e t a i l e d t h r e e - d i m e n s i o n a l structures of a n i n c r e a s i n g n u m b e r of

II

these

e l e c t r o n m e d i a t o r s , a l o n g w i t h k i n e t i c studies of t h e i r reactions, h a v e l e d to n e w insights i n t o t h e r e d o x p a t h w a y s of the c - t y p e c y t o c h r o m e s some of the n o n - h e m e i r o n proteins ( J ).

and

Unfortunately no direct struc-

t u r a l i n f o r m a t i o n is yet a v a i l a b l e f o r those e l e c t r o n - m e d i a t i n g proteins w h e r e c o p p e r serves as the a c t i v e center. It is r e m a r k a b l e that a l l m e m bers of this g r o u p h a v e t h e i r s i n g l e c o p p e r b o u n d i n a site w h i c h confers


u p o n t h e m the u n i q u e b l u e state.

T h e y are w i d e l y f o u n d i n n a t u r e ,

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

a n d u m e c y a n i n , w h i c h are f o u n d

i n nonphotosynthetic

plant

tissues, to the l a r g e f a m i l y of a z u r i n s f r o m different b a c t e r i a l sources

(2).

T h i s g r o u p of b l u e single c o p p e r proteins is a c o n v e n i e n t system for p r o b i n g s t r u c t u r a l features of the m e t a l - b i n d i n g site. E a r l y

suggestions

that a s u l f u r l i g a n d is i n v o l v e d i n the c o o r d i n a t i o n sphere ( 2 )

received

f u r t h e r s u p p o r t r e c e n t l y f r o m the s p e c t r a l i n v e s t i g a t i o n s of d e r i v a t i v e s i n w h i c h the c o p p e r w a s r e p l a c e d b y C o ( I I ) ions ( 3 )

and from

resonance

Raman and E S C A

(4,5,6).

We

studies o n the n a t i v e proteins

have

a d o p t e d a different a p p r o a c h to this p r o b l e m , m o n i t o r i n g the i n t r i n s i c p r o b e s of these p r o t e i n s , b y e x a m i n i n g the s p e c t r a l properties i n the u v range. C h a r a c t e r i s t i c alterations w e r e f o u n d w h i c h are p r o b a b l y b y the C u ( I ) - S c h r o m o p h o r e

a l o n g w i t h changes f r o m

caused

conformational

differences b e t w e e n the r e d u c e d a n d o x i d i z e d states of the p r o t e i n . T h e e l e c t r o n transfer m e c h a n i s m of a z u r i n , a w e l l k n o w n

example

for this t y p e of p r o t e i n s , has b e e n s y s t e m a t i c a l l y s t u d i e d u s i n g the c h e m i c a l r e l a x a t i o n m e t h o d a n d a w e l l d e f i n e d i n o r g a n i c outer sphere couple.

redox

I n p a r a l l e l , the investigations of the r e a c t i o n w i t h its p r e s u m e d

p h y s i o l o g i c a l p a r t n e r , c y t o c h r o m e c, w e r e p u r s u e d ( 7 ) .

T h e specificity

of the i n t e r a c t i o n b e t w e e n a z u r i n a n d c y t o c h r o m e c P - 5 5 1 is expressed i n h i g h e r specific rates a n d i n the c o n t r o l of the e l e c t r o n transfer e q u i l i b r i u m b y c o n f o r m a t i o n a l transitions of b o t h p r o t e i n s . I n the b l u e oxidases, the t y p e 1 c o p p e r is o n l y one of at least t h r e e a c t i v e centers.

T h e s t r i k i n g feature of this class of p r o t e i n s , a p a r t f r o m

the c o m p l e x i t y of the different c o p p e r b i n d i n g sites p e r se is the i n t r i c a t e r e l a t i o n s h i p a m o n g these sites. E q u i l b r i u m m e a s u r e m e n t s p r o d u c e d e v i d e n c e for the effect of e x t e r n a l l i g a n d b i n d i n g to t y p e 2 C u ( I I ) o n t h e r e d o x potentials of b o t h t y p e 1 a n d t y p e 3 sites ( 8 ) .

F r o m the k i n e t i c

d a t a it b e c a m e clear that rates of r e d u c t i o n a n d o x i d a t i o n of one site are r a t h e r sensitive to the r e d o x states of the other sites ( 9 ) .

T h i s is f u n c -

t i o n a l l y r e l e v a n t , since a l l three r e d o x sites of laccase are s u p p o s e d undergo

r e v e r s i b l e v a l e n c e changes

d u r i n g the c a t a l y t i c c y c l e

of

to the

e n z y m e . W e h a v e e x a m i n e d the r e d u c t i o n a n d o x i d a t i o n of Rhus laccase u n d e r c o n d i t i o n s w h e r e a r a t h e r s m a l l f r a c t i o n of its sites w a s

reduced

u s i n g 0 " r a d i c a l s as r e d u c t a n t i n o x y g e n - s a t u r a t e d solutions. T h e t y p e 1 2


11.

P E C H T

E T

181

Blue Copper Proteins

AL.

C u ( I I ) ions w e r e r e d u c e d a n d f u l l y r e o x i d i z e d w i t h i n 1-2 sec, a l t h o u g h u n d e r these c o n d i t i o n s n o m o r e t h a n a s i n g l e r e d u c t i o n e q u i v a l e n t m a y b e i n those m o l e c u l e s . T h i s is not the case w h e n r e d u c t i o n of these sites take p l a c e a n a e r o b i c a l l y b y , for e x a m p l e , the C 0 " r a d i c a l i o n . 2

Interaction between o x i d i z e d f u n g a l laccase a n d h y d r o g e n peroxide added

i n excess,

occurs

v i a its t y p e 2 C u ( I I )

site.

T h i s b i n d i n g is

m a n i f e s t e d b y b o t h the E S R a n d a b s o r p t i o n s p e c t r a , y e t t h e affinity of


this c o m p l e x is r a t h e r l o w , a n d i t d e c o m p o s e s r a p i d l y ( J O ) . I n contrast w e f o u n d that tree laccase forms a r e l a t i v e l y stable a n d h i g h affinity c o m p l e x w i t h one m o l e of H 0 2

2

w h i c h m a y b e r e d u c e d together w i t h t h e

e n z y m e sites a n d c o u l d b e of m e c h a n i s t i c s i g n i f i c a n c e i n t h e r e d u c t i o n of d i o x y g e n to w a t e r . Experimental Materials. L a c c a s e a n d s t e l l a c y a n i n w e r e p r e p a r e d f r o m the acetone extract of Rhus vernicifera l a c q u e r a c c o r d i n g to the p r o c e d u r e of R e i n h a m m a r (11) a n d w e r e k e p t at — 2 0 ° i n salt-free solutions. A z u r i n f r o m Pseudomonas aeruginosa a n d Alcaligenes faecalis w a s i s o l a t e d a n d p u r i fied b y t h e m e t h o d of A m b l e r (12) a n d w a s s t o r e d at 4 ° i n 0 . 0 5 M s o d i u m acetate buffer ( p H 3.9). T h e p r o t e i n solutions w e r e e x t e n s i v e l y d i a l y z e d against p o t a s s i u m p h o s p h a t e buffer b e f o r e b e i n g u s e d i n the e x p e r i m e n t s a n d , w h e n necessary, w e r e c o n c e n t r a t e d b y v a c u u m d i a l y s i s . T h e p u r i t y of t h e p r o t e i n s w a s d e t e r m i n e d b y c h e c k i n g t h e r e s p e c t i v e a b s o r p t i o n ratios b e t w e e n the b l u e b a n d a n d the 2 8 0 - n m b a n d ; t h e y w e r e a l w a y s i n g o o d a g r e e m e n t w i t h the l i t e r a t u r e v a l u e s . I n a d d i t i o n , t h e q u a l i t y of the laccase p r e p a r a t i o n was assayed b y m e a s u r i n g its e n z y m a t i c a c t i v i t y w i t h N , N - d i m e t h y l - p - p h e n y l e n e d i a m i n e (11). S t e l l a c y a n i n a n d the t w o a z u r i n s w e r e r e d u c e d b y t h e f o l l o w i n g m e t h o d s — b y ascorbate, f o l l o w e d b y a n a e r o b i c d i a l y s i s ; b y h y d r o g e n , u s i n g p l a t i n u m b l a c k as catalyst ( 7 ) ; b y s o d i u m b o r o h y d r i d e ; or b y d i t h i o n i t e . A l l of these m e t h o d s l e d to the same p r o d u c t as c o n c l u d e d f r o m the a b s o r p t i o n s p e c t r a of the n a t i v e , r e d u c e d , a n d r e o x i d i z e d p r o teins. A p o p r o t e i n s w e r e p r e p a r e d b y d i a l y s i s against 0 . 0 5 M N a C N i n 0 . 1 M p h o s p h a t e buffer p H 7.4, f o l l o w e d b y r e p e a t e d d i a l y s i s against t h e buffer. T h e c o n c e n t r a t i o n s of the n a t i v e p r o t e i n s w e r e d e t e r m i n e d u s i n g €6i4 = 5700 M " c m " f o r laccase (13), c 5 = 4080 M " c m " a n d e o = 1

23,200 M

1

cm

1

1

for s t e l l a c y a n i n (13),

1

G 0

e«

2 5

=

5700 M

1

cm'

1

1

(14),

28

and c

2 8 0

= 10,700 M " c m " ( 1 5 ) for Ps. aeruginosa a z u r i n a n d e 25 = 4620 M " c m " for A. faecalis a z u r i n (16). A l l m a t e r i a l s u s e d w e r e of a n a l y t i c a l g r a d e . A l l solutions w e r e p r e p a r e d i n d o u b l y d i s t i l l e d w a t e r . T h e c o n c e n t r a t i o n of h y d r o g e n p e r o x i d e stock s o l u t i o n w a s d e t e r m i n e d i o d o m e t r i c a l l y (17) b e f o r e a n d after e a c h peroxide titration. Methods. M e a s u r e m e n t s of a b s o r p t i o n s p e c t r a a n d 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 w e r e c a r r i e d o u t o n a C a r y 15 s p e c t r o p h o t o m e t e r e q u i p p e d w i t h a t h e r m o s t a t e d c e l l c o m p a r t m e n t . C i r c u l a r d i c h r o i c spect r a w e r e r e c o r d e d o n a C a r y 60 s p e c t r o p o l a r i m e t e r e q u i p p e d w i t h a 6001 C D attachment. Fluorescence spectra were measured on a H i t a c h i 1

1

G

1


1


182

BIOINORGANIC C H E M I S T R Y

Π

P e r k i n E l m e r M P F - 3 spectrofluorometer. X - a n d Q - b a n d measurements of E P R spectra w e r e c a r r i e d o u t at l i q u i d n i t r o g e n a n d l i q u i d h e l i u m temperatures. M i c r o c a l o r i m e t r i c measurements were performed o n a L K B 10700 b a t c h m i c r o c a l o r i m e t e r . T e m p e r a t u r e - j u m p r e l a x a t i o n k i n e t ics w e r e m e a s u r e d u s i n g a d o u b l e b e a m i n s t r u m e n t (18) w i t h a c e l l a d a p t e d f o r a n a e r o b i c w o r k . T h e r e l a x a t i o n signals w e r e f e d i n t o a n H . P . 2 1 0 0 c o m p u t e r a n d a n a l y z e d as d e s c r i b e d i n R e f . 7. T h e p u l s e r a d i o l y s i s e x e p r i m e n t s w e r e c a r r i e d o u t o n t h e 5 - M e V l i n e a r accelerator at t h e H e b r e w U n i v e r s i t y . D e t a i l s o f t h e system h a v e b e e n p u b l i s h e d p r e v i o u s l y (19). SPECTROSCOPIC STUDIES. A l l measurements w e r e m a d e a t 2 5 ° i n 0 . 0 5 M o r 0.1 M p o t a s s i u m p h o s p h a t e buffer, p H 7.0. E a c h s p e c t r u m w a s r e c o r d e d w i t h s e v e r a l samples, w h i c h g e n e r a l l y d i f f e r e d i n c o n c e n t r a t i o n , a n d w a s s c a n n e d t w o or three times. C i r c u l a r d i c h r o i s m is expressed as m o l a r e l l i p t i c i t y [Θ] i n u n i t s of degrees c m · d m o l e " . R E L A X A T I O N KINETICS. T h e details of t h e e x p e r i m e n t a l p r o c e d u r e h a v e b e e n d e s c r i b e d e a r l i e r (14). 0 . 1 M p h o s p h a t e buffer, p H 7.0, c o n t a i n i n g 2 Χ 10" M E D T A was u s e d i n a l l r e l a x a t i o n e x p e r i m e n t s . T h e s e w e r e p e r f o r m e d w i t h solutions of different i n i t i a l reagent c o m p o s i t i o n — either ferrocyanide was a d d e d to o x i d i z e d azurin or ferricyanide to r e d u c e d a z u r i n . T e m p e r a t u r e j u m p s o f 2.9° o r 4.7° w e r e a p p l i e d to t h e r e a c t i o n s o l u t i o n . T h e s u b s e q u e n t t r a n s m i s s i o n changes w e r e m o n i t o r e d at 625 n m ( a b s o r p t i o n of o x i d i z e d a z u r i n ) o r 420 n m ( a b s o r p t i o n of f e r r i c y a n i d e ). E a c h p l o t t e d v a l u e o f the r e l a x a t i o n t i m e o r a m p l i t u d e r e p r e sents t h e average o f at least f o u r measurements. P U L S E RADIOLYSIS. A d e t a i l e d a c c o u n t o f the e x p e r i m e n t a l p r o c e d u r e has b e e n g i v e n elsewhere (19). Solutions o f tree laccase w e r e p r e p a r e d i n t r i p l y d i s t i l l e d w a t e r a n d c o n t a i n e d f e r f - b u t a n o l as scavenger f o r O H r a d i c a l s . T h e s o l u t i o n was s a t u r a t e d w i t h a r g o n o r o x y g e n b y p r o l o n g e d b u b b l i n g ( > 20 m i n ) i n l a r g e glass syringes e q u i p p e d w i t h s t a n d a r d c a p i l l a r y t a p e r joints. B u b b l i n g w a s d o n e p r i o r t o t h e a d d i t i o n o f t h e r e q u i r e d v o l u m e o f c o n c e n t r a t e d p r o t e i n s o l u t i o n , thus m i n i m i z i n g d e naturation b y foaming. 2

1

5

ANAEROBIC O X I D A T I O N - R E D U C T I O N TITRATIONS.

Oxidation-reduction

titrations of Rhus laccase w e r e c a r r i e d out i n a s p e c i a l l y c o n s t r u c t e d o p t i c a l c e l l d e s c r i b e d i n R e f . 20. T h e solutions w e r e f r e e d f r o m o x y g e n b y a l t e r n a t i v e e v a c u a t i o n a n d flushing w i t h w a t e r - s a t u r a t e d a r g o n f r o m w h i c h traces of o x y g e n w e r e r e m o v e d b y p a s s i n g t h e gas t h r o u g h f o u r c o l u m n s o f m e t h y l v i o l o g e n . D u r i n g t h e titrations a s l i g h t excess a r g o n p r e s s u r e w a s m a i n t a i n e d to a v o i d diffusion of o x y g e n i n t o the c e l l . T i t r a n t was a d d e d through a serological cap w i t h a H a m i l t o n micro syringe. T h e t i t r a t e d solutions w e r e s t i r r e d b y a s m a l l m a g n e t i c b a r . E x p e r i m e n t a l values w e r e c o r r e c t e d f o r d i l u t i o n o f p r o t e i n o n a d d i n g t i t r a n t , f o r r e s i d u a l a b s o r b a n c e of t h e f u l l y r e d u c e d c h r o m o p h o r e , a n d w h e n necessary, f o r a b s o r b a n c e o f the o x i d i z e d a n d r e d u c e d forms of the titrants u s e d . Spectroscopy of Blue Single Copper in the Ultraviolet

Proteins

Region

T h e n a t u r e o f the b l u e c o p p e r site m i g h t b e c o n c e i v e d as t h e r e s u l t of a c o m p r o m i s e b e t w e e n the free energy r e q u i r e m e n t s f o r the p r e f e r r e d


11.

183


PECHT E T A L .

c o o r d i n a t i o n s t r u c t u r e of t h e m e t a l i o n i n its t w o r e d o x states a n d t h e o p t i m a l c o n f o r m a t i o n of t h e p o l y p e p t i d e f o r m i n g i t . I t is therefore ex p e c t e d that changes i n t h e state of t h e c o p p e r w o u l d also b e expressed i n some of the properties of a m i n o a c i d residues, n o t a b l y those w h i c h are r e l a t e d to t h e b i n d i n g site. T h u s a n earlier s t u d y f r o m this l a b o r a t o r y has s h o w n a c o r r e l a t i o n b e t w e e n t h e r e d o x state of t y p e 1 c o p p e r a n d the i n t r i n s i c

fluorescence

of Rhus laccase (20).

H e r e w e have compared


the u v s p e c t r a l properties of three b l u e s i n g l e - c o p p e r proteins monas aeruginosa a z u r i n , Alcaligenes

(Pseudo-

faecalis a z u r i n , a n d Rhus s t e l l a c y

a n i n ) i n t h e i r o x i d i z e d , r e d u c e d , a n d copper-free states, u s i n g a b s o r p t i o n , difference a b s o r p t i o n , c i r c u l a r d i c h r o i s m , a n d

fluorescence

measurements.

T h e c o m p a r i s o n b e t w e e n t h e t w o a z u r i n species is of s p e c i a l significance as t h e y a r e h o m o l o g o u s p r o t e i n s , y e t c e r t a i n residues w h i c h are of p a r t i c u l a r interest (e.g., t h e s i n g l e t r y p t o p h a n ) o c c u p y i n t h e a m i n o a c i d sequence

π — ι — ι — ι — ι — ι

260

different positions

(21).

280

ι

300 Wavelength

ι

320

ι

ι

I

340

(nm)

Figure 1. Reduced-minus-oxidized difference absorption spectrum of P s . a e r u g i n o s a azurin. Sample and reference cell contained 6.8 Χ ΙΟ M solutions of reduced and oxi dized protein, respectively. Hydrogen, with platinum black as catalyst, was used as reductant (7). Medium: 0.1 M potassium phospate buffer, pH 7.0 5

F i g u r e 1 shows t h e difference a b s o r p t i o n s p e c t r u m b e t w e e n r e d u c e d a n d o x i d i z e d Ps. aeruginosa

azurin.

A s is w e l l k n o w n , i n the v i s i b l e

r e g i o n t h e t y p i c a l b l u e b a n d disappears u p o n r e d u c t i o n , a n d n o other changes a r e o b s e r v e d .

H o w e v e r , p r o n o u n c e d a n d c o m p l e x changes a r e

seen i n t h e u v r e g i o n . U p o n r e d u c t i o n a decrease is f o u n d a b o v e 330 n m , w h e r e a s b e l o w this w a v e l e n g t h t h e r e d u c e d a z u r i n has t h e h i g h e r e x t i n c -


184

BIOINORGANIC C H E M I S T R Y

Table I.

II

Ultraviolet Extinction of Reduced Copper Proteins Value Measured (Mr cm- )

Protein

1

Ps. aeruginosa azurin A. faecalis azurin Stellacyanin C u — thionein +

a

1

e

red

e

ox

e

red

e

ox

€

red

c

ox

*red

Wavelength (nm) 250

260

270

5050 4250 5150 3650

3450 3050 4300 3050

3200 2950 4250 2650

2350 2250 3500 2200


° Values calculated from spectral data reported by Rupp and Weser (24). tion.

T h i s is consistent w i t h t h e result o f a p r e l i m i n a r y e x p e r i m e n t r e -

p o r t e d b y Y a m a n a k a et a l . ( 2 2 ) . T h e p e a k i n t h e difference s p e c t r u m at 294 n m is c a u s e d b y a s m a l l r e d shift of t h e w e l l r e s o l v e d fine s t r u c t u r e p e a k of t r y p t o p h a n at 292 n m ( 2 2 ) . A m o n o t o n o u s increase i n t h e e x t i n c t i o n of t h e r e d u c e d p r o t e i n r e l a t i v e t o t h e o x i d i z e d takes p l a c e t o w a r d s

240

260

280 300 Wavelength (nm)

320

340

Figure 2. CD spectra of oxidized ( ) and reduced ( ) Ps. aeruginosa azurin. The samples contained 1.2 X 10~ M protein in 0.05M potassium phosphate, pH 7.0. Reduction was achieved with ascorbate, followed by extensive dialysis. Optical pathlength, 10 mm. 4


11.


PECHT E T AL.

185

l o w e r w a v e l e n g t h s , p r o d u c i n g a difference of ~ 5000 M " c m " at 250 n m . 1

1

T h i s increase is m o d u l a t e d b y s l i g h t , b u t d i s t i n c t , v a r i a t i o n s i n the s t r u c t u r e of t h e a r o m a t i c a b s o r p t i o n b a n d s s h o u l d e r at 270 n m .

(275-290nm)

fine

andby a

E s s e n t i a l l y s i m i l a r difference s p e c t r a b e t w e e n r e

d u c e d a n d o x i d i z e d p r o t e i n h a v e also b e e n m e a s u r e d f o r A. faecalis a z u r i n a n d s t e l l a c y a n i n . T h e isosbestic p o i n t s a r e s o m e w h a t s h i f t e d t o t h e b l u e ( 3 2 4 n m f o r t h e f o r m e r , 304 n m f o r the l a t t e r ) , a n d the p e a k a t


294 n m is o n l y a flat s h o u l d e r , w h i c h is s i m p l y a c o n s e q u e n c e o f t h e t r y p t o p h a n t r a n s i t i o n at 292 n m n o t b e i n g r e s o l v e d i n these p r o t e i n s . B u t a m a r k e d increase of the e x t i n c t i o n difference w i t h d e c r e a s i n g w a v e l e n g t h a n d a s h o u l d e r a t 270 n m is i n a c c o r d w i t h t h e p r e v i o u s over, t h e ( c

r e d

findings.

More

— cox) values f o u n d are a l t o g e t h e r q u i t e s i m i l a r ( T a b l e I ) .

T h e s o m e w h a t h i g h e r values for s t e l l a c y a n i n at 260 a n d 270 n m are c a u s e d b y a m u c h m o r e p r o n o u n c e d s h o u l d e r a t 270 n m .

ο Ε ο

•ë CM* E o σ> 3 Ό

0

I

—I

I

260

280

I

300

ι

320

ι

340

I

Wavelength(nm)

Figure 3. CD spectra of oxidized ( ), reduced ( ), and copper-free (- ' -) stellacyanin. Protein solutions: 5 Χ ΙΟ" M in 0.1 M potassium phosphate, pH 7.0. The reduced sample was prepared by catalytic reduction with hydrogen; the oxidized sample by aerobic reoxidation of the same reduced sample. The apoprotein was prepared as described under "Experimental." Optical pathlength, 10 mm. 5

C D spectra of the oxidized a n d reduced f o r m have been measured for a l l t h r e e p r o t e i n s ; the s p e c t r a o f t w o of t h e m are p r e s e n t e d i n F i g u r e s 2 a n d 3. T h e c o m p l e x set o f d i c h r o i c b a n d s i n the r e g i o n o f the a r o m a t i c transitions is i n a l l cases f u l l y c o n s e r v e d u p o n r e d u c t i o n , w i t h o n l y t h e m a g n i t u d e o f t h e e l l i p t i c i t i e s b e i n g affected.

T h e small b u t significant

differences at 310 a n d at 293 n m as w e l l as the l a r g e increase of t h e e l l i p t i c i t y a t 2 8 0 n m f o u n d f o r Ps. aeruginosa

a z u r i n ( F i g u r e 2 ) are e x a c t l y

analogous to changes f o u n d f o r r e d u c e d A. faecalis a z u r i n c o m p a r e d w i t h


186

BIOINORGANIC

CHEMISTRY

II

the o x i d i z e d p r o t e i n . T h e C D s p e c t r a of o x i d i z e d Ps. aeruginosa a z u r i n a n d A. faecalis a z u r i n ( F i g u r e s 2 a n d 4 )

are essentially s i m i l a r .

They

e x h i b i t exactly the same b a n d p a t t e r n , except for some s m a l l shifts a n d differ o n l y i n the e l l i p t i c i t i e s of the v a r i o u s b a n d s . T h e o n l y m a j o r difference i n the C D p r o p e r t i e s of these t w o proteins is the response of the p o s i t i v e b a n d at 260 n m to r e d u c t i o n . W h e r e a s i n A. faecalis a z u r i n this b a n d changes l i t t l e u p o n r e d u c t i o n , a p r o n o u n c e d c h a n g e t o w a r d s n e g a -


t i v e e l l i p t i c i t y occurs i n the case of Ps. aeruginosa a z u r i n ( F i g u r e 2 ).

The

effect of r e d u c t i o n o n the C D s p e c t r u m of s t e l l a c y a n i n ( F i g u r e 3 ) is also l i m i t e d to the a m p l i t u d e s of the different b a n d s , t h e i r p o s i t i o n r e m a i n i n g p r a c t i c a l l y u n a l t e r e d . T h e increase of m o r e t h a n t w o - f o l d i n t h e e l l i p t i c i t y of the b r o a d n e g a t i v e b a n d a r o u n d 265 is p r o b a b l y r e l a t e d to t h e same t r a n s i t i o n w h i c h p r o d u c e s a p r o n o u n c e d s h o u l d e r at 270 n m i n t h e r e d u c e d - m i n u s - o x i d i z e d difference a b s o r p t i o n s p e c t r u m .

260

280 300 Wavelength

320

340

(nm)

Figure 4. CD spectra of native ( ) and copper-free (- · -) A . faecalis azurin. The apoprotein was prepared as described under "Experimental." Solutions: 9 X JO" M in 0.1 M potassium phosphate, pH 7.0. Optical pathlength, 10 mm. 5

T h e s p e c t r a l p r o p e r t i e s of the three proteins i n the u v are s t r o n g l y i n f l u e n c e d b y the redox state of the c o p p e r .

A l t h o u g h this is p r o b a b l y

t r u e for a l l b l u e c o p p e r p r o t e i n s , v e r y l i t t l e a t t e n t i o n has b e e n p a i d to these changes.

T h e o v e r a l l s i m i l a r i t y of t h e r e d o x - r e l a t e d changes

a b s o r p t i o n a n d C D s p e c t r a s t r o n g l y suggests that most of the

in

changes

are c o m m o n to a l l three proteins a n d are therefore r e l a t e d to some c o m m o n s t r u c t u r a l p r o p e r t y , most p r o b a b l y i n v o l v i n g t h e c o p p e r site. results of v a r i o u s s p e c t r o s c o p i c

The

a n d c h e m i c a l studies h a v e b e e n i n t e r -


11.

PECHT E T A L .

187


p r e t e d to i n d i c a t e that b l u e c o p p e r is c o o r d i n a t e d to s u l f u r (3, 4, 5, 6,23). T h e findings of this s t u d y are consistent w i t h s u c h a c o n c e p t . T h e g e n e r a l course

of the r e d u c e d - m i n u s - o x i d i z e d

difference

a b s o r p t i o n s p e c t r a is

s i m i l a r to the a b s o r p t i o n s p e c t r u m of the C u ( I ) - S c h r o m o p h o r e , as seen f r o m e x t i n c t i o n values ( T a b l e I ) d e r i v e d f r o m C u ( I ) - t h i o n e i n s p e c t r a H o w e v e r , the c h e m i c a l f o r m of the s u l f u r l i g a n d i n the b l u e p r o -

(24).

teins is n o t f u l l y e s t a b l i s h e d , b u t it has g e n e r a l l y b e e n p r o p o s e d to b e


the s u l f h y d r y l g r o u p of a cysteine r e s i d u e . Y e t one s h o u l d also c o n s i d e r t h e p o s s i b i l i t y that m e t h i o n i n e m a y serve as a l i g a n d . S o m e s u p p o r t f o r this h y p o t h e s i s comes f r o m a m o d e l s t u d y w h e r e the chelate c o m p l e x of a t h i a e t h e r w i t h C u ( I I ) has b e e n f o u n d to e x h i b i t a n intense b l u e a b s o r p t i o n b a n d , s i m i l a r to that f o u n d i n b l u e c o p p e r proteins (26). ferent a z u r i n species

( f r o m Pseudomonas,

Bordetella,

In nine dif-

and

Alcaligenes

a n d i n eight different p l a s t o c y a n i n species ( b e a n , potato, a n d g r e e n

(21))

algae (27)), m e t h i o n i n e 121 ( i n the p l a s t o c y a n i n n u m b e r i n g M e t 97) is i n v a r i a n t , a n d the s e q u e n c e a r o u n d it contains some f u r t h e r residues w h i c h h a v e c o n s e r v e d t h e i r a r o m a t i c or h y d r o p h o b i c c h a r a c t e r [ T y r 108 (85),

P h e / T y r 110 ( 8 7 ) ,

P h e / T y r 111 ( 8 8 ) ,

L e u / V a l / I l e 125

(101),

L e u / V a l 127 ( 1 0 3 ) ] a p a r t f r o m the i n v a r i a n t G l y 123 ( 9 9 ) a n d the single cysteine r e s i d u e 112 ( 8 9 ) .

T h i s m a y i n d i c a t e the i n v o l v e m e n t of

both

cysteine a n d m e t h i o n i n e i n the c o o r d i n a t i o n sphere of the b l u e c o p p e r , as far as t h e y are a v a i l a b l e . T h e a d d i t i o n a l effects i n the a r o m a t i c r e g i o n of the difference spect r u m ( 2 5 0 - 3 0 0 n m ) are p r o b a b l y c a u s e d b y a r o m a t i c transitions w h i c h are i n f l u e n c e d b y the r e d o x state of the c o p p e r . T h e s h o u l d e r at 270 n m , w h i c h occurs i n a l l three p r o t e i n s , c o u l d result f r o m a n increase i n t y r o s i n e a b s o r p t i o n . I n this context, it is i n t e r e s t i n g to r e c a l l that T y r 108 ( a z u r i n n u m b e r i n g ) , w h i c h is r e l a t i v e l y close to the p r o p o s e d

copper ligands

C y s 112 a n d M e t 121, is c o m p l e t e l y i n v a r i a n t b o t h i n a z u r i n a n d p l a s t o c y a n i n a n d m a y therefore b e a n o b l i g a t o r y constituent of the c o p p e r site. T h e r e d o x - i n d u c e d changes

i n the C D s p e c t r u m of the a r o m a t i c

r e g i o n d o not seem to b e r e l a t e d to a c o p p e r c h r o m o p h o r e b e c a u s e t h e y are not u n i f o r m i n the three p r o t e i n s . I n s t e a d , t h e y p r o b a b l y arise f r o m differences i n the d i r e c t or i n d i r e c t ( c o n f o r m a t i o n a l )

effects of the r e d o x

state of the c o p p e r o n the d i c h r o i s m of v a r i o u s a r o m a t i c t r a n s i t i o n s . T h e c o m p a r a t i v e l y s m a l l changes i n the 2 8 5 - 2 9 5 - n m r a n g e suggest t h a t t h e t r y p t o p h a n transitions are less affected t h a n those of t y r o s i n e a n d p h e n y l a l a n i n e . T h e changes a r o u n d 250 n m m a y i n v o l v e d i s u l f i d e groups w h i c h p r o d u c e C o t t o n effects at this w a v e l e n g t h s . F o r certain blue single-copper that a C u ( I I ) (28),

proteins it w a s p r o p o s e d

or C u ( I I ) - r e l a t e d b a n d is present b e t w e e n

long

ago

300-350 n m

b u t i t has a t t r a c t e d o n l y v e r y l i m i t e d a t t e n t i o n i n spite of the p o t e n -

t i a l i m p l i c a t i o n s for the s t u d y of b l u e c o p p e r - c o n t a i n i n g oxidases. A s u m -


188

BIOINORGANIC

CHEMISTRY

II

m a r y of the s p e c t r a l m e a s u r e m e n t s i n t h e 3 0 O - 3 5 0 - n m r e g i o n is p r e s e n t e d i n F i g u r e 5. R e m o v i n g t h e c o p p e r almost abolishes t h e a b s o r p t i o n i n this region.

T h i s is confirmed b y t h e C D spectra where t h e p r o b l e m of

a c h i e v i n g s p e c t r a l l y v e r y c l e a n solutions o r m a t c h i n g t h e concentrations of h o l o - a n d a p o p r o t e i n s is less c r u c i a l t h a n f o r a b s o r p t i o n o r difference a b s o r p t i o n spectra. T h e e x t i n c t i o n o f t h e n a t i v e proteins i n this r e g i o n is i n t h e r a n g e 2 0 0 - 7 0 0 M " c m " c o m p a r e d w i t h extinctions o f a b o u t 1 X 1

1

1 0 ( a z u r i n ) or 2.3 Χ 1 0 M " c m " ( s t e l l a c y a n i n ) at 280 n m . S u c h e x p e r i 4

4

1

1


m e n t a l p r o b l e m s m a d e i t difficult to o b t a i n r e l i a b l e d a t a o n t h e shape of the C u ( I I ) b a n d b e l o w 305 n m , w h e r e the a r o m a t i c a b s o r p t i o n b e g i n s . ABS: OX/APO

CD: ox/APo

ABS: RED-ox

CD:OX/RED

EMISSION: ox

Azurin

RED - 12%

from

^

Pseudomonas

\

330

'^^^v;—

aeruginosa Azurin

RED:~O%

from

V

324

Alcaligenes faecalis Stelacyanin

RED'.+18%

from

304

Rhus

i

vernicrfera

/^^^^

Figure 5. Optical properties of three blue copper proteins in the range 300-350 nm. The various protein samples were prepared as indicated under "Experimental." Medium: 0.05 or 0.1M potassium phosphate, pH 7.0, 25°. ( ) oxidized protein, ( ) reduced or copper-free protein. Abscissa: The wavelength range extends from 300 to 350 nm. Ordinate: All absorption spectra were drawn to the same scale, as was done for the CD spectra. Fluorescence emission spectra: the scale is different for each protein. All spectra were recorded in the ratio mode with λ = 280 nm. The effect of reduction is indicated as percentage change of the maximum emission intensity of the oxidized protein. β

χ

F r o m measurements where reconstituted azurin was compared w i t h apoa z u r i n , w e estimate that the c o p p e r - d e p e n d e n t

e x t i n c t i o n does n o t e x c e e d

450 M " c m " at 280 n m , b u t i t r e m a i n s to b e d e t e r m i n e d w h e t h e r i t passes 1

1

t h r o u g h a m a x i m u m b e t w e e n this w a v e l e n g t h a n d 320 n m . A l i k e l y c a n d i date f o r the l i g a n d , w h i c h i n c o n j u n c t i o n w i t h C u ( I I ) gives rise t o t h e a b s o r p t i o n i n this r e g i o n , is p e p t i d e n i t r o g e n . V a r i o u s complexes i n v o l v i n g the coordination of a deprotonated peptide nitrogen to C u ( I I )

were

f o u n d t o absorb i n this r e g i o n (15, 2 9 ) . T h e m o d e r a t e l y intense b a n d has b e e n a s c r i b e d t o a l i g a n d - m e t a l charge transfer t r a n s i t i o n . A n i n t e r e s t i n g


11.

189


PECHT E T A L .

m o d e l system e x h i b i t i n g a s i m i l a r b a n d is the C u ( I I ) - p o l y - L - h i s t i d i n e complex

formed

i n n e u t r a l - t o - s l i g h t l y - a c i d solutions

(30).

This

gains

s p e c i a l significance i n v i e w of N M R d a t a of single b l u e c o p p e r p r o t e i n s (31)

s u g g e s t i n g b o t h p e p t i d e a n d i m i d a z o l e n i t r o g e n s as l i g a n d s to t h e

copper. Considering now

the r e d u c e d - m i n u s - o x i d i z e d

different

absorption

spectra, i t b e c o m e s clear t h a t the isosbestic p o i n t shifts to a shorter w a v e -


l e n g t h w h e n g o i n g f r o m the a z u r i n s to s t e l l a c y a n i n , b e c a u s e the a b s o r b a n c e of o x i d i z e d s t e l l a c y a n i n i n this r e g i o n is h i g h e r t h a n that of

the

o x i d i z e d a z u r i n s . U n d e r l y i n g , of course, is the a s s u m p t i o n that the C u ( I ) s p e c t r u m has a s i m i l a r i n t e n s i t y i n a l l three p r o t e i n s , at least i n this r e g i o n . T h e response of t h e

fluorescence

e m i s s i o n i n t e n s i t y to the r e d u c -

t i o n of the c o p p e r c a n b e e x p l a i n e d o n the same basis. Ps. aeruginosa a z u r i n has its e m i s s i o n c e n t e r e d a r o u n d 308 n m .

W h e n the p r o t e i n is

r e d u c e d , the a b s o r b a n c e i n this w a v e l e n g t h r e g i o n increases.

Therefore

a n y q u e n c h i n g effect o r i g i n a t i n g f r o m i n t e r n a l n o n r a d i a t i v e e n e r g y t r a n s fer b e t w e e n the e x c i t e d t r y p t o p h a n r e s i d u e a n d the c o p p e r is e x p e c t e d

to b e c o m e l a r g e r , t h e r e b y

chromophore

d e c r e a s i n g the q u a n t u m y i e l d .

E x a c t l y the o p p o s i t e h a p p e n s w i t h s t e l l a c y a n i n . H e r e the r e d u c e d

pro-

t e i n absorbs less i n the r e g i o n of the e m i s s i o n m a x i m u m , e n h a n c i n g its intensity.

T h e emission spectrum ( A

e x

= 280 n m ) of A. faecalis a z u r i n

h a r d l y changes o n r e d u c t i o n , p r o b a b l y b e c a u s e q u e n c h i n g a n d e n h a n c i n g effects

approximately

compensate.

R e c a l l that the s i n g l e

tryptophan

r e s i d u e of A. faecalis a z u r i n is not h o m o l o g o u s to the s i n g l e t r y p t o p h a n of Ps. aeruginosa a z u r i n (21), different

fluorescence

a fact w h i c h m a y c o n t r i b u t e b o t h to the

spectra of the n a t i v e proteins a n d to the different

response to r e d u c t i o n . R e m o v a l of the c o p p e r i o n o n l y m o d e r a t e l y effects the a r o m a t i c p a r t of the C D s p e c t r u m ( F i g u r e s 3, 4 ) , a s p e c t r a l r e g i o n w h i c h is i n g e n e r a l less affected b y r e m o v i n g the c o p p e r t h a n b y r e d u c i n g i t . T h i s i n d i c a t e s t h a t the s t r u c t u r a l i n t e g r i t y of b l u e s i n g l e - c o p p e r p r o t e i n s d e p e n d s l i t t l e o n the presence of the c o p p e r i o n as f a r as these a r o m a t i c residues are concerned.

A s i m i l a r result has b e e n o b t a i n e d f r o m the c o m p a r i s o n

the t r y p t o p h a n a z u r i n (23).

fluorescence

of

i n n a t i v e a n d c o p p e r - f r e e Ps. fluorescence

T h e c o n c l u s i o n is that the i n t e r a c t i o n b e t w e e n the c o p p e r

i o n a n d the p r o t e i n , as discussed at the outset, is l a r g e l y d o m i n a t e d b y the c o n f o r m a t i o n of the latter. Electron

Transfer

Profile

of Ps. aeruginosa

Azurin

A n effective a p p r o a c h to r e s o l v i n g the e l e c t r o n p a t h w a y to a n d f r o m t h e redox center of a z u r i n is the systematic i n v e s t i g a t i o n of its e q u i l i b r i a a n d k i n e t i c s of i n t e r a c t i o n s w i t h i n o r g a n i c r e d o x c o u p l e s . ferrate ( I I / H I )

Hexacyano-

is a w e l l d e f i n e d r e d o x c o u p l e , k n o w n to react v i a a n


190

BIOINORGANIC

CHEMISTRY

II

outer sphere p a t h . F u r t h e r m o r e , it is r e l a t e d i n its e l e c t r o n i c s t r u c t u r e to Fe(II/III)

h e m e , the r e d o x center of a l l c y t o c h r o m e s , i n c l u d i n g c y t o

c h r o m e c P 5 5 1 , the n a t u r a l p a r t n e r of a z u r i n ( 7 ) . the a z u r i n - h e x a c y a n o f e r r a t e ( I I / I I I ) l a x a t i o n spectroscopy

(32),

T h u s w e have studied

system b y t e m p e r a t u r e - j u m p r e

spectrophotometric

titrations, a n d

micro-

calorimetry. T h e r e l a x a t i o n s p e c t r u m consists of a single r e l a x a t i o n m o d e .

The

r e l a x a t i o n t i m e , τ, was m e a s u r e d u p to h i g h concentrations of p r o t e i n ( 1 Χ ΙΟ" M ) a n d h e x a c y a n o i r o n


3

t i t r a t e d w i t h f e r r i c y a n i d e , τ" (Figure 6b).

( 8 Χ ΙΟ" M ).

W h e n azurin ( I )

2

was

l e v e l e d off w i t h i n c r e a s i n g c o n c e n t r a t i o n

1

T h i s b e h a v i o r reveals t h a t the e l e c t r o n transfer b e t w e e n

azurin and F e ( C N )

6

4 - / 3

~ is not a s i m p l e one-step process as the s i n g l e

relaxation observed might have indicated. T h e limiting dependence

on

f e r r i c y a n i d e c o n c e n t r a t i o n suggested a k i n e t i c s c h e m e i n v o l v i n g the fast r e v e r s i b l e f o r m a t i o n of a n a z u r i n ( I ) - f e r r i c y a n i d e c o m p l e x f o l l o w e d a

slower

electron

transfer step.

azurin (II)-ferrocyanide of m i c r o s c o p i c

The

formation

of

a

by

corresponding

c o m p l e x is to b e e x p e c t e d f r o m the p r i n c i p l e

r e v e r s i b i l i t y , yet the c o n c e n t r a t i o n d e p e n d e n c e

of

the

r e l a x a t i o n t i m e p r o v i d e d n o e v i d e n c e for i t ; w h e n a z u r i n ( I I ) w a s t i t r a t e d w i t h i n c r e a s i n g a m o u n t s of f e r r o c y a n i d e , τ"

showed a monotonous i n

1

crease u p to v e r y h i g h concentrations ( F i g u r e 6 a ) .

Direct evidence

for

the i n v o l v e m e n t of a n a z u r i n ( I I ) - f e r r o c y a n i d e c o m p l e x w a s o b t a i n e d b y a n a l y z i n g the r e l a x a t i o n a m p l i t u d e s . T h e s e constitute a f u r t h e r source of i n f o r m a t i o n a b o u t the m e c h a n i s m of r e a c t i o n , a p a r t f r o m t h e k i n e t i c d a t a d e r i v e d f r o m the analysis of the r e l a x a t i o n times

(33).

A s s u m i n g the f o l l o w i n g S c h e m e A , the " n o r m a l " e n t h a l p y of

the

r e l a x a t i o n m o d e o b s e r v e d , Δ Η , w h i c h c a n b e c a l c u l a t e d f r o m the r e l a x a -

Az(II) +

Fe(II)

^

Az(II) · Fe(II)

JÎ

Az(I) · Fe(III)

Az(I)

+

ks (fast)

Fe(III) (A)

(slow)

(fast)

t i o n a m p l i t u d e s , is l i n e a r l y r e l a t e d to the i n d i v i d u a l e n t h a l p y changes, as d e s c r i b e d b y E q u a t i o n 1, w h e r e

ά

Η

- Κ

χ

>

([Az(II)] +

is the i n d i v i d u a l r e a c t i o n e n -

AHi°

[Fe(II]) + l

A

H

l

°

K

2

tf

2

+

[Az(I)] +

t h a l p y of the i - t h step, a n d K i a n d K ~

[Fe(III)]

2

steps 1 a n d 2, r e s p e c t i v e l y (14).

l

+

AH ° 2

+

ΔΗ ° 3

(1)

are t h e a s s o c i a t i o n constants o f

I n the e x p e r i m e n t s i n v o l v i n g a d d i t i o n of

f e r r o c y a n i d e to o x i d i z e d a z u r i n ( I I ) , t h e c o n c e n t r a t i o n s of a z u r i n ( I ) a n d


191


PECHT E T A L .


11.

Figure 6. Ps. aeruginosa azurin-hexacyanoiron (II/HI) equilibrium-concentration dependence of the reciprocal relaxation time, (a) Azurin (II) reacted with ferrocyanide (24-860-fold excess), 6.5° C. (b) Azurin (I) (2-5 X 10~ M) reacted with ferricyanide, 16.8° C. τ has a minimal value at [Fe] ^ [Az] and increases again at lower [Fe] . Points in the region [Fe]

10kcal/mole)

s h o u l d b e of a d v a n t a g e i n o v e r -

c o m i n g the b a r r i e r s i n the r e d u c t i o n step. P r e l i m i n a r y experiments w h e r e h a l f - r e d u c e d laccase w a s r e a c t e d w i t h d i o x y g e n seem to s u p p o r t this hypothesis.

We

h a v e f o u n d that a r a p i d l y f o r m e d

intense transient

a b s o r p t i o n decays w i t h a h a l f - l i f e of ~ 27 m i n ( a t 2 5 ° ) to a stable spect r u m s t r i k i n g l y s i m i l a r to the l a c c a s e - p e r o x i d e s p e c t r u m , w i t h the same specific e x t i n c t i o n of the extra b a n d at ~ 330 n m . T h e E P R s p e c t r u m at 10° Κ is the same as for n a t i v e or p e r o x i d e - t r e a t e d laccase, a n d at least


204

BIOINORGANIC CHEMISTRY II

five reducing equivalents are needed for the full reduction of this new species. It has recently been reported (45) that reoxidation of fully reduced Rhus laccase with oxygen is characterized by a second-order rate constant of about 5 · 10 M" s" for both type 1 and type 3 copper. We have inves tigated the kinetics of the reaction between reduced Rhus laccase and hydrogen peroxide spectrophotometrically under anaerobic conditions at 10° and 25 °C by following the changes in absorbance at 330 and 615 nm (39). The rate of reoxidation of the type 1 Cu(I) was found to be first order, independent of either concentration of H 0 or the state of reduction of other sites in the enzyme (10°C, A: = 4.6 · lO^s' ; 25°C, k ~ 0.015 s" ). The oxidation of the type 3 site by hydrogen peroxide is significantly faster (τ < 10 s at 25°C). At 10°C we found the rate to befirstorder both in reduced type 3 site and in hydrogen peroxide, with an overall second-order rate constant k = 1.8 Χ 10 M" s". These find ings indicate that the primary step in this reaction is the reoxidation of the type 3 site by H 0 , in parallel with a slower intramolecular oxidation of the type 1. The reoxidation of type 2 copper cannot be monitored spectrophotometrically, but from the overall stoichiometry as well as from the above equilibrium titrations, it is obvious that this site is also involved in the reaction. A more extensive kinetic investigation of the reduction of H 0 is presently being carried out.


6

1

1

2

2

1

1

1/2

3

2

2

1

1

2

2

Concluding Remarks We have used a range of different physical and chemical approaches in the effort to better understand how the different blue copper proteins function. With the relatively simpler, electron-mediating proteins like azurin, the ultraviolet chromophores were shown to be informative in terms of copper-protein interactions. These proteins are also a useful system for detailed examination of the electron transfer pathways to and from their single copper site. The elaborate mechanism by which blue oxidases react with dioxy gen to produce water was tackled by studying the possible role of H 0 . We have observed the formation of a stable and high affinity complex between tree laccase and H 0 . Moreover, thefindingthat the oxidation of the reduced enzyme with H 0 follows a pattern which is different from that operative in the reduction of the oxidized enzyme may have important implications for the mechanism of action of laccase. 2

2

2

2

2

2

Literature Cited 1. Dickerson, R. E., Timkovich, R., in "The Enzymes," Boyer, P. B., Ed., 3rd ed., Vol. XI, Academic, New York, 1975.



11. PECHT ET AL.


205

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206

BIOINORGANIC CHEMISTRY II

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Electron Transfer Pathways in Blue Copper Proteins

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