Coupled Binuclear Copper Active Sites - American Chemical Society

Chapter 7

Coupled Binuclear Copper Active Sites

Downloaded by UNIV OF AUCKLAND on May 3, 2015 | http://pubs.acs.org Publication Date: June 21, 1988 | doi: 10.1021/bk-1988-0372.ch007

Edward I. Solomon Stanford University, Department of Chemistry, Stanford, CA 94305 A coupled binuclear copper active s i t e i s found i n a variety of different metalloproteins involved i n dioxygen reactions. These include hemocyanin (reversible O binding), tyrosinase ( O activation and hydroxylation) and laccase (O reduction to water). Unique excited state spectral features of active s i t e derivatives and binuclear copper model complexes are used to define exogenous ligand bridging between the two coppers at the active s i t e s of hemocyanin and tyrosinase. A chemical and spectroscopic comparison of the coupled s i t e in hemocyanin to the binuclear center (Type 3, T3) in laccase indicates that the s i t e s are s i m i l a r with respect to an endogenous bridge responsi ble for antiferromagnetic coupling but d i f f e r i n that exogenous ligands bind to only one copper at the T3 site. In addition to the T3 coupled binuclear copper s i t e , laccase contains two additional coppers, the Type 1 and Type 2 centers. X-ray absorption edge spectral studies of the 1s->4p t r a n s i t i o n at 8984 eV are used to demonstrate that i n the absence of the T2 copper, the T3 s i t e i s reduced and will not react with O2. The role of the T2 and T3 coppers i n exogenous ligand binding has been probed through low temperature magnetic c i r c u l a r dichroism (MCD) which allows a c o r r e l a t i o n between the excited state and ground state spectral features. Through these low temperature MCD studies i t i s demonstrated that one azide produces charge transfer transitions to both the paramagnetic T2 and the antiferromagnetic T3 center defining a new trinuclear copper cluster active s i t e which appears to be important i n the i r r e v e r s i b l e multi-electron reduction of dioxygen to water. 2

2

2

The Coupled Binuclear Copper Active Site A coupled binuclear active s i t e i s found i n a wide variety of proteins and enzymes which are involved i n different b i o l o g i c a l 0097-6156/88/0372-0116$09.75A) • 1988 American Chemical Society

In Metal Clusters in Proteins; Que, L.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

7.

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117


functions. A l l reactions involve dioxygen and i t has been the major focus of research of this class of metalloproteins to establish correlations between differences i n active s i t e geometric and electronic structure and v a r i a t i o n i n protein function. Proteins containing a coupled binuclear copper active s i t e are summarized i n Table 1.

Table 1.


HEMOCYANIN:

Proteins Containing Coupled Binuclear Copper Active Sites and Their Functions Arthropod

deoxy + 0^

Mollusc

deoxy + 0 2H 0 2

TYROSINASE:

0

2

deoxy + 0 2H 0 2

2

oxy J

2

2

2

oxy

+ 2H 0 2

600 cm (H - -2JS -S ).(7) Since the ground state is spectroscopically inaccessible, the excited state features must be employed to probe the interactions of dioxygen with the coppers in the oxyhemocyanin active s i t e . Figure 2 presents the spectroscopic changes observed by displacement of peroxide from the oxyhemocyanin s i t e , producing the met derivative [Cu(II)Cu(II)].(1) The two intense bands i n the oxyhemocyanin absorptj-on spectrum at 350 nm ( £=* 20,000 M era ) and 600 nm ( £ » 1000 M cm ) are eliminated as is a feature i n the c i r c u l a r dichroism (CD) spectrum at 486 nm ( A e » +2.53 M~ cm" ) . These three features are therefore assigned as 0 *~ —> Cu(II) charge transfer t r a n s i t i o n s . Weak d-d transitions which are sensitive to_Jhe ayerage ligand f i e l d at the coppers remain at ^ 630 nm (e ^250 M cm ) and r e f l e c t an approximately tetragonal coordination geometry. It i s thus important to define the charge transfer spectral features associated with an 0 ~-Cu(II) bond and to use these features to obtain insight into the charge transfer spectrum of oxyhemocyanin. K a r l i n and coworkers have synthesized a binuclear copper(I) model complex which reacts with dioxygen to give the absorption spectrum changes i l l u s t r a t e d i n Figure 3a.(8) Resonance Raman excitation into these features(9) produces a metal ligand stretch at 488 cm~ and intraligand stretch at 803 cm" which shifts to 750 cm with 0 i n d i c a t i n g that dioxygen i s bound as peroxide (Figure 3b). The mode of peroxide coordination was probed through a mixed isotope perturbation of the resonance Raman spectrum combined with normal coordinate analysis. In Figure 3c i s presented the resonance Raman spectrum of the oxygenated complex i n the Cu-0 2

2

2



7.

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119

\ ! 3 00 5

3 4 '•} ft

Sx w i i

Figure 1. Comparison of absorption (top) and EPR (bottom) spectra of normal tetragonal copper(II) and oxyhemocyanin.


METAL CLUSTERS IN PROTEINS

120

oxy

>•

[ Cu( I I )Cu( I I ) ] 0

me t

=

[Cu( I I ) C u ( I I ) ] H 0

2

2


Abs .

Figure 2 . Absorption and c i r c u l a r dichroism spectra of oxyhemocyanin compared with absorption spectrum of methemocyanin•

A b s o rpt I on

400

000

lOO(nm)

Figure 3 a . Absorption spectra of deoxy, oxy, and met forms of O2 binding binuclear copper model complex. Also included i s the resonance Raman p r o f i l e for the 0 - 0 stretch i n the oxy complex.


7. SOLOMON

•


111


464

1 8

360 350 400 450 500 550 Frequency fcnrr!)

750

Q

650 700 750 800 850 Frequency (cm") 1

Figure 3b. Resonance Raman spectra i n and 1 8 , of the oxy complex. The unlabelled peaks are due to the CH2CI2 solvent. 0

16-16-180 :2 0 0: 2

16

2

Normal Coordinate A n a l y s i s C a l c u l a t i o n s terminal

400

450

500

550 cm"

1

700

750

800

850 cm"

1

Figure 3c. Comparison of observed resonance Raman spectra for isotope mixture with predicted spectra for two possible coordina tion geometries.



122

and 0-0 stretch regions using a s t a t i s t i c a l mixture of oxygen isotopes ( 0 : 2 0 0 : 0 ). Also shown is the v i b r a t i o n a l spectrum calculated using experimental lineshapes for both terminal and bridging peroxide. The metal-ligand stretch c l e a r l y allows these possible binding geometries to be distinguished and indicates that peroxide i s bound asymmetrically and thus to only one copper. Therefore, from the resonance Raman enhancement p r o f i l e of the 0-0 stretch (Figure 3a) a terminally bound peroxide-cupric complex e x h i b i t j two charge transfer t r a n s i t i o n s , a band at 503 nm (e = 6300 M c m - ) and a less intense lower energy shoulder at 625 nm ( £ = 1100 M cm ^. These 0 —> Cu(II) charge transfer bands can be assigned using the energy l e v e l diagram sho^n i n Figure 4. The highest energy occupied o r b i t a l of peroxide is a doubly degenerate TT* l e v e l . Upon coordination to Cu this degeneracy i s l i f t e d and results i n a 7r l e v e l which i s oriented along the Cu-0 bond and a 7i"* which is perpendicular to the Cu-0 bond. The 7TQ is more strongly s t a b i l i z e d by the bonding interaction with the copper. Further, the highest energy, half-occupied o r b i t a l of tetragonal Cu(II) i s d 2 2 which has a lobe directed at the peroxide ligand i n the equatorial plane. Since the intensity of charge transfer transitions i s proportional to overlap (S) of the donor and acceptor o r b i t a l s ( I a (SR) ), this analysis predicts a r e l a t i v e l y weak low energy TT* —> Cu(II) t r a n s i t i o n and a more intense IT* —> Cu(II) t r a n s i t i o n . Oxyhemo cyanin, however, exhibits three charge transfer transitions (arrows i n Figure 2). Since the above analysis shows that only two bands are possible for peroxide bound to a single copper, the three band pattern must have another o r i g i n . This analysis must then be extended to consider the effects of interactions with two coppers on the charge transfer transitions of a bridging ligand.(1) Structurally defined monomers and dimers of Cu(II) with azide exist(10) and as this ligand has a V homo which i s very s i m i l a r to the TT valence l e v e l of peroxide, these complexes are suitable models for probing exogenous ligand bridging effects. As shown i n Figure 5, azide bound to a single copper gives r i s e to one r e l a t i v e l y intense charge transfer absorption band. In analogy to peroxide, this t r a n s i t i o n originates from the *n^ l e v e l ; the corresponding Tr^ —> Cu(II) t r a n s i t i o n i s too weak to be observed. In the dimer, the absorption band i s observed to s p l i t into two bands with the one at higher energy having more i n t e n s i t y . A Transition Dipole Vector Coupling (TOVC) Model has been developed to analyze these data.(1,11) In the dimer, the t r a n s i t i o n dipoles of the —> Cu(II) charge transfer transitions to each copper w i l l couple to form symmetric and antisymmetric combinations (Table 2). From group theory, these coupled t r a n s i t i o n dipoles have A^ and symmetry ( i n C« ), respectively. The intensity of each dimer t r a n s i tion i s given By the square of the vector sum or difference of the individual t r a n s i t i o n moments. For the dimer, the intensity r a t i o I /I i s predicted to be ^ 8 indicating that the higher energy intense band i s the A^ component of i r —> Cu(II) transfer. Experimentally, the intensity r a t i o i s somewhat lower which derives from vibronic mixing of A^ intensity into the B^ t r a n s i t i o n . Further, the coulomb interaction between the two t r a n s i t i o n moments can be estimated for the y-1,3 geometry to obtain an approximate b

2

2


2

Q

v

c t

1

n

n

A 1

B 1

n

a



SOLOMON


U

^

*

sjht x * - y d

O

l

d orbitals j £

ol

Figure 4 . Molecular o r b i t a l diagram for terminally bound peroxide-Cu(II) complex. The two possible charge transfer transitions are indicated with relative i n t e n s i t y .



124


30

25

20

15

Energy (cm" x10' ) 1

3

Figure 5• Absorption spectra with Gaussian resolution for terminally bound and bridging azide model complexes and for met-azide hemocyanin*


7. SOLOMON

125


Table 2. Monomer

Transition Dipole Vector Coupling £

2

v

Pimer A

l

B,

calc.


E

E

A ~ B 1

exp.

1

1

350 cm""

3500 cm"

1

value for the energy difference between the and B^ dimer states. The calculated value gives the correct energy ordering with the A^ component at higher energy but i s much smaller than the observed s p l i t t i n g , which indicates that excited state exchange terms contribute s i g n i f i c a n t l y to the s p l i t t i n g (see reference 12). F i n a l l y , addition of azide to methemocyanin, Figure 5 (Bottom), yields a charge transfer spectrum which shows an A p B, pattern very similar to the spectrum of the y - 1 , 3 azide model complexes. Thus azide binds i n an analogous p-1,3 bridging geometry to the binuclear s i t e i n methemocyanin. This approach can now be applied to interpret the charge transfer spectrum of oxyhemocyanin with peroxide bridging i n a y-1,2 fashion.(1) In the dimer, the TT- * and charge transfer transitions of the monomer each s p l i t into two bands. The symmetry types, selection r u l e s , intensity r a t i o s , and coulomb s p l i t t i n g s have been calculated and are given i n Table 3. The 7T * —> Cu(II) t r a n s i t i o n w i l l s p l i t into two components, a lower energy B band which should contain most of the absorption intensity and a higher energy A t r a n s i t i o n having l i t t l e absorption i n t e n s i t y . This higher energy t r a n s i t i o n i s , however, magnetic dipole allowed and is there fore predicted to be r e l a t i v e l y intense i n the CD spectrum. The predicted r e l a t i v e energies and absorption and CD Intensities correlate with the bands at 600 nm and 486 nm (Figure 6), allowing v

V

2

2

Table 3.

Transition Dipole Vector Coupling-Oxyhemocyanin

Monomer

C ^ Dimer t

\

/

E

A~

I

E

A

\ E

1

b

2

/ \ E

A^" B

1

=

0.0 840 cm"

1

-

2.6

=

-5000 cm"

1


126


their assignment as the B and A components, respectively. The TT * —> Cu(II) t r a n s i t i o n should be analogously s p l i t (Table 3) into a low energy A^ component with most of the intensity and a component at higher energy. The A^ band i s associated with the very intense 350 nm band while the B^ component i s obscured by intense protein absorption at higher energy. From the preceding and e a r l i e r a n a l y s i s ( l ) of the unique spec troscopic features of oxyhemocyanin (and oxytyrosinase) a spectroscopically effective model of the active s i t e has been generated which i s i l l u s t r a t e d i n Figure 7. In the s i t e , two tetragonal cupric ions are bridged by both an endogenous ligand (0R~) and the exogenous y-1,2 peroxide. The endogenous bridge i s responsible for the strong antiferromagnetic coupling between the coppers and hence the lack of an EPR s i g n a l , and the bridging peroxide gives r i s e to the intense charge transfer features in the electronic absorption spectrum of oxyhemocyanin. While, unfortunately, no high resolution c r y s t a l l o graphic information is presently available on oxyhemocyanin(13a), the structure of deoxy hemocyanin has recently been determined to 3.2 A resolution(13b). For deoxy, the two Cu(I) are each found to have two short Cu-N bonds (His at ^ 2.0 A) and one long, a x i a l Cu-His bond (at ^2.6 A) consistent with the tetragonal effective Cu(II) symmetry of oxyhemocyanin. These crystallographic results indicate that the endogenous bridge i s not a protein residue but is derived from water, most l i k e l y i n the form of hydroxide.


2

2

Chemical and Spectroscopic Comparison With Laccase Characterization of the Type 2 Depleted Derivative of Laccase. The model for the coupled binuclear copper s i t e i n hemocyanin and tyrosinase (Figure 7) may now be compared to the p a r a l l e l s i t e in laccase which contains a blue copper (denoted Type 1 or T I ) , a normal copper (Type 2, T2), and a coupled binuclear copper (Type 3, T3) center. As shown i n Figures 8a and b, native laccase has c o n t r i butions from both the TI and T2 copper centers in the EPR spectrum (the T3 cupric ions are coupled and hence EPR nondetectable as i n hemocyanin), and an intense absorption band at ^600 nm ( ~ 5 7 0 0 M cm"") associated with the TI center (a thiolate —> Cu(II) CT transition).(14) The only feature i n the native laccase spectra believed to be associated with the T3 center was the absorption band at 330 nm (e ^ 3200 M~ era ) which reduced with two electrons, independent of the EPR signals.(15) I n i t i a l studies have focussed on the simplified Type 2 depleted (T2D) derivative(16) i n which the T2 center has been reversibly removed. From Figure 8 the T2 c o n t r i bution is c l e a r l y eliminated from the EPR spectrum of T2D and the TI contribution to both the EPR and absorption spectrum remains. However, upon removal of the T2 copper the 330 nm absorption is eliminated, r a i s i n g concern with respect to the involvement of the T2 copper i n this spectral feature. When T2D laccase i s reacted with peroxide the 330 nm band reappears (Figure 8a).(17) This observation suggested the p o s s i b i l i t y that the T3 s i t e might be reduced i n the T2D derivative but could be oxidized by H 0 A l t e r n a t i v e l y , other researchers assigned the 330 nm band as an 0 ~ —> Cu(II) charge transfer e

1

2

2

7




SOLOMON

Figure 7. The Spectroscopically Effective Active Site of hemocyanin and tyrosinase•


128



a

400

500

600

700

nm

c

2500

3000

3500 Gauss

Figure 8. (a) Optical absorption, (b) EPR, and (c) X-ray absorption edge spectra for native and T2D laccase and after reaction of T2D with peroxide.



7. SOLOMON


129

t r a n s i t i o n in analogy to oxyhemocyanin and therefore associated this spectral feature with peroxide binding to the T3 s i t e . ( 1 8 ) An absorption feature at 330 nm, however, cannot d i r e c t l y distinguish between these p o s s i b i l i t e s . A l s o , the EPR spectrum i s not diagnostic of the T3 oxidation state since both a reduced s i t e and an antiferromagnetically coupled oxidized binuclear cupric s i t e would be EPR nondetectable. A direct probe of oxidation state i s , however, available through X-ray absorption edge spectroscopy. The T2D form exhibits a peak at 8984 eV which is eliminated by reaction with peroxide (Figure 8c), q u a l i t a t i v e l y indicating that Cu(I) i s being oxidized to Cu(II).(17) Such conclusions must be drawn cautiously however, since covalent cupric complexes can also exhibit X-ray absorption edge intensity at low energy.(19) The oxidation state dependence of the X-ray absorption edge feature was defined and quantitated by a systematic study of a series of twenty Cu(I) and forty Cu(II) model complexes in collaboration with Professor Keith Hodgson.(6) The Cu K edge band energies and shapes depend on oxidation state, l i g a t i o n , and coordination geometry. Figure 9 (Top) presents representative edges for two, three, and four coordinate Cu(I), and tetragonal Cu(II) complexes with a variety of ligand sets. Two or three coordinate Cu(I) complexes always exhibit a peak at energies below 8985 eV. In addition, the three coordinate complexes display a double peak with lower i n t e n s i t y . No Cu(II) complex studied shows a peak below 8986 eV. There is however a low energy t a i l i n the 8984 eV region which i s associated with a peak at higher energy. These spectral differences can be understood through ligand f i e l d theory.(6) For two coordinate Cu(I) with an approximately l i n e a r geometry, the e l e c t r i c dipole allowed Is —> 4p t r a n s i t i o n i s predicted to s p l i t into Is —> 4p and Is —> 4p components. Further, the Is —> 4p t r a n s i t i o n i s expected to'be highest in energy due to repulsive interactions with the a x i a l ligands. Polarized single c r y s t a l X-ray edge spectroscopy(20) confirms the assignment of the lowest energy peak as Is —> 4p . As shown at the bottom of Figure 9, increasing the ligand fiefc! strength by adding a third ligand along the y axis leads to an additional repulsive interaction which raises the energy of the 4p l e v e l r e l a t i v e to the 4p • This accounts for the experiment i l l y observed edge s p l i t t i n g i n the three coordinate Cu(I) complexes. For a tetrahedral geometry, a l l p o r b i t a l s are equally destabilized by the ligand f i e l d and the Is —> 4p transitions are a l l above 8985 eV. For the tetragonal Cu(II) complexes, both the low energy t a i l and i t s associated higher energy peak s h i f t to lower energy as the covalent interaction with the equatorial ligand set increases. Since this band i s z polarized(20) with an energy shift dependent on equatorial ligand ionization energy, this t r a n s i t i o n can be assigned as the Is —> 4p combined with a ligand —> Cu(II) charge transfer shake-up, which Is also supported by f i n a l state calculations.(21) Turning to the X-ray absorption edge spectrum of the T3 s i t e in T2D laccase (Figure 10a), a peak is observed below 8984 eV. This energy i s c h a r a c t e r i s t i c of Cu(I) while the shape of the peak suggests that i t i s due to three coordinate copper. The amount of reduced copper present can then be quantitated from the normalized edge i n t e n s i t i e s of copper model complexes with the appropriate z

x

z




130

8980 8990 eV

8980 8990

A

Cu

I -"Pz

0 Cu-L

£980 8990

8980 8990

Cu

*cu(n)

r

-4p.

x,y,z

-T-"P,

Y

— 4p.

3 d

xV

•44- Ligand

>

CD i_ Cu(II) charge transfer transitions i n the 400-500 nm region (Figure 18a). The low temperature MCD and absorp tion behavior i n this region i s quite complex and consists of two contributions. The spectral changes associated with high a f f i n i t y (HA) binding are completed with the addition of less than one equiva lent of azide ( K ^ >^ 10 M ). Prior addition of H 0 eliminates these features, suggesting that they may be associated with ligand binding to a f r a c t i o n of native laccase molecules i n which the T3 s i t e i s reduced. This interpretation has been confirmed through X-ray absorption edge studies, shown i n Figure 18b, i n which the edge features of ^ O o treated laccase have been subtracted from those of native enzyme.(5) A peak i s present at 8984 eV with an intensity that corresponds to ^22% reduced T3 s i t e s i n the native enzyme. The features associated with low a f f i n i t y azide binding to f u l l y oxidized laccase sites can then be obtained by subtraction of the high a f f i n i t y azide spectral contributions. The low a f f i n i t y absorption and low temperature MCD spectra are shown i n Figure 19a for increasing azide concentrations. Two -> Cu(II) charge tranfer bands are observed i n absorption, one at 500 nm and a more intense band at 400 nm. Both increase with azide concentration; the 400 nm absorption intensity is plotted as a function of [N^ ] i n Figure 19b. In the low temperature MCD spectrum a negative peak is observed at 485 nm which increases i n magnitude with increasing azide concentration. It i s thus clear that the corresponding ^ 500 nm absorption band Is associated with a paramagnetic ground state and must correspond to azide binding to the T2 center. In the region of the intense 400 nm absorption, a positive feature at 385 nm i s observed i n the low temperature MCD spectrum. The intensity of this feature f i r s t increases, then decreases with increasing azide concentration. As shown i n Figure 19b, the intensity of this low temperature MCD feature does not correlate with the 400 nm absorption 2

2


SOLOMON


4.0

Affinity N

3

i O X

w 2.0


High Affinity

0.0 0.30|

LTMCD

0.00

i -0.30

Uw

Affinity Nj

-0.60h

8970

500

400

300

8980

8990

9000

ENERGY (eV) Figure 18. (a) Absorption and low temperature MCD spectra of azide binding to native laccase. (b) X-ray absorption edge data for reaction of native laccase with peroxide (insert presents normalized difference edge).


144


Wavelength, nm 333

400

3.0

2.5

500

1500


1000

0.00

-0.50

2.0

Energy, 10"Xcm" 4

1

uncoupled T3

Protein Equivalents N~ Figure 19. (a) Absorption and low temperature MCD spectra for binding of low a f f i n i t y azide to native laccase. (b) Correla tion of the LT MCD signals at 4 85 and 385 nm to the 400 nm_ absorption and the g - 1.86 EPR signal with increasing [N^ ] .


7. SOLOMON


145


EPR

i 2400

i 2800

1

3200

Gauss Figure 20. Summary of N3~"/F~ binding competition experiments for native laccase. Continued on next page.




146


7. SOLOMON


147


c

Equiv. FFigure

20.

Continued.

\ / NNN"

R HEMOCYANIN AND TYROSINASE

Type 2

y Cu \

2 2

N

3

T

y

p e

3

R NATIVE LACCASE

Figure 21. Comparison of the spectroscopically effective models for azide binding at the binuclear copper active s i t e i n hemocyanin and the t r i n u c l e a r copper cluster s i t e i n laccase.

American Chemical Society Library 1155 16th St., N.W.

In Metal Clusters in Proteins; Que, L.; Washington, D.C. Society: 20036Washington, DC, 1988. ACS Symposium Series; American Chemical


148

i n t e n s i t y , but i n fact p a r a l l e l s the appearance of the g=1.86 EPR signal which i s associated with the small fraction of uncoupled T3 sites (Figure 11, r i g h t ) . Thus, no low temperature MCD intensity is associated with the intense 400 nm absorption band and i t must be associated with azide binding to the antiferromagnetically coupled T3 site. From the low temperature MCD data i t i s thus found that azide binds to both the T2 and T3 centers with similar binding constants. The stoichiometry of this binding has been determined through a series of ligand competition experiments, one of which i s summarized in Figure 20. From Figure 20a, addition of F~ to native laccase containing 9-fold excess N^" (corresponding to the presence of both HA and LA forms) produces a superhyperfine s p l i t t i n g _ ( I - 1/2 for F) of the T2 hyperfine l i n e , indicating that one F i s binding to the T2 center. In the absorption and low temperature MCD spectra (Figure 20b), the low a f f i n i t y N~"~ -> Cu(II) charge transfer transitions associated with the T2 (485 nm MCD) and the T3 (400 nm absorption) centers are eliminated with the addition of increasing concentrations of f l u o r i d e . The quantitative loss of these features p a r a l l e l each other as shown by the s o l i d l i n e i n Figure 20c. F i t t i n g these data to models(29b) for competitive binding of one or two fluorides c l e a r l y demonstrates that only one fluoride binds to the T2 center and i s involved i n displacing azide from both the T2 and T3 centers. Therefore one exogenous azide ligand must bridge between the T2 and T3 centers i n native laccase defining a trinuclear copper cluster active s i t e i n the multicopper oxidases(29). Figure 21 summarizes the differences between exogenous azide binding to the coupled binuclear copper s i t e i n hemocyanin (and tyrosinase) as compared to laccase. For hemocyanin and tyrosinase exogenous ligands bridge the two coppers at the active s i t e while i n laccase azide binds to only one copper of the T3 center and bridges to the T2 center forming a t r i n u c l e a r copper c l u s t e r . It is the focus of current research to define i n d e t a i l the contribution of the y-1,2 peroxy bridge i n reversible 0 binding and a c t i v a t i o n and the T2-T3 t r i n u c l e a r copper cluster with respect to i t s role in the multi-electron reduction of dioxygen to water.


n

2

Acknowledgments I would l i k e to thank my students and postdoctoral associates who have made major contributions to this research: Dr. Richard S. Himmelwright, Dr. Nancy C. Eickman, Dr. Cynthia D. LuBien, Dr. Yeong T. Hwang, Dr. Marjorie E . Winkler, Dr. Thomas J . Thamann, Dr. Arturo G. Porras, Dr. Dean E . Wilcox, Dr. Mark Allendorf, Dr. Darlene Spira-Solomon, Lung-Shan Kau, Dr. T. David Westmoreland, and Dr. James Pate. I also acknowledge contributions of the following collaborators: Prof. K. Lerch, Prof. K . O . Hodgson, Prof. T . G . S p i r o , Prof. K . D . K a r l i n , Prof. C A . Reed, Prof. T . N . S o r r e l l , Dr. W.B. Mims, and Dr. M.S. Crowder. NIH grant #DK31450 i s g r a t e f u l l y acknowledged for supporting this work. Literature Cited 1.

Eickman, N . C . ; Himmelwright, R . S . ; Solomon, E.I. Proc. N a t l . Acad. S c i . USA 1979, 76, 2094.


7. SOLOMON 2.

3.


4.

5. 6.

7.

8. 9. 10.

11.

12. 13.

14.

15. 16. 17.

18.


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(a) Himmelwright, R . S . ; Eickman, N . C . ; LuBien, C.D.; Lerch, K . ; Solomon, E . I . J. Am. Chem. Soc. 1980, 102, 7339. (b) Winkler, M . E . ; Lerch, K . ; Solomon, E.I. J. Am. Chem. Soc. 1981, 103, 7001. (c) Wilcox, D . E . ; Porras, A . G . ; Hwang, Y.T.; Lerch, K . ; Winkler, M . E . ; Solomon, E.I. J. Am. Chem. Soc. 1985, 107, 4015. (a) Solomon, E.I. i n Copper Proteins, ed. T . G . Spiro, Wiley, NY, 1981, pp. 41-108. (b) Solomon, E.I.; Penfield, K.W.; Wilcox, D . E . i n Structure and Bonding, Springer-Verlag, 1983, V o l . 53, pp. 1-57. (c) Solomon, E.I. Pure and Applied Chemistry 1983, 55, 1069. (a) Freedman, T . B . ; Loehr, J.S.; Loehr, T.M. J. Am. Chem. Soc. 1976, 98, 2809. (b) Eickman, N.C.; Solomon, E.I.; Larrabee, J.A.; Spiro, T . G . ; Lerch, K. J. Am. Chem. Soc. 1978, 100, 6529. Brown, J.M.; Powers, L.; Kincaid, B . ; Larrabee, J.A.; Spiro, T . G . J. Am. Chem. Soc. 1980, 102, 4210. (a) Kau, L.S.; Spira, D.J.; Penner-Hahn, J.E.; Hodgson, K . O . ; Solomon, E.I. J. Am. Chem. Soc. 1987, 109, 6433. (b) Kau, L.S.; Solomon, E.I.; Hodgson, K.O. J. Physique 1986, 47, 289 (1986). (a) Solomon, E.I.; Dooley, D . M . ; Wang, R . H . ; Gray, M . ; Cerdonio, M . ; Mogno, F.; Romani, G . L . J. Am. Chem. Soc. 1976, 98, 1029. (b) Dooley, D . ; Scott, R . A . ; Ellinghaus, J.; Solomon, E.I.; Gray, H . B . Proc. Nat. Acad. S c i . USA 1978, 75, 3019. K a r l i n , K . D . ; Cruse, R.W.; Gultneh, Y . ; Hayes, J.C.; Zubieta, J. J. Am. Chem. Soc. 1984, 106, 3372. Pate, J.E.; Cruse, R.W.; K a r l i n , K . D . ; Solomon, E.I. J. Am. Chem. Soc. 1987, 109, 2624. (a) McKee, V . ; Dagdigian, J.V.; Bau, R . ; Reed, C.A. J. Am. Chem. Soc. 1981, 103, 7000. (b) McKee, V . ; Zvagulis, M . ; Dagdigian, J.V.; Patch, M . G . ; Reed, C.A. J. Am. Chem. Soc. 1984, 106, 4765. (c) K a r l i n , K . D . ; Cohen, B.I.; Hayes, J.C.; Farooq, A . ; Zubieta, J. Inorg. Chem. 1987, 26, 147. (a) Pate, J.E.; Thamann, T.J.; Solomon, E.I. Spectrochimica Acta 1986, 42A, 313. (b) Pate, J.; K a r l i n , K . D . ; Reed, C.A.; S o r r e l l , T . ; Solomon, E.I., manuscript i n preparation. Desjardins, S.R.; Wilcox, D . E . ; Musselman, R.L.; Solomon, E.I. Inorg. Chem. 1987, 26, 288. (a) Magnus, K . A . and Love, W.G. J. Mol. Biol. 1977, 116, 171. (b) Volbeda, A. and H o l , W . G . J . , i n Invertebrate Oxygen C a r r i e r s , ed. B. Linzen, 1986, pp. 135-47. (a) Solomon, E.I.; Hare, J . W . ; Gray, H.B. Proc. Nat. Acad. S c i . USA 1976, 73, 1389. (b) Penfield, K.W.; Gay, R . R . ; Himmelwright, R . S . ; Eickman, N . C . ; Norris, V . A . ; Freeman, H . C . ; Solomon, E.I. J. Am. Chem. Soc. 1981, 103, 4382. (c) Penfield, K.W.; Gewirth, A . A . ; Solomon, E.I. J. Am. Chem. Soc. 1985, 107, 4519. Reinhammar, B. Biochim. Biophys. Acta 1972, 275, 245. G r a z i a n i , M . T . ; Morpurgo, L.; R o t i l i o , G . ; Mondovi, B. FEBS L e t t . 1976, 70, 87. LuBien, C.D.; Winkler, M . E . ; Thamann, T.J.; Scott, R . A . ; Co, M . S . ; Hodgson, K . O . ; Solomon, E.I. J. Am. Chem. Soc. 1981, 103, 7014. (a) Farver, O.; Frank, P . ; Pecht, I . Biochem. Biophys. Res.


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20. 21.

22.

23. 24. 25. 26. 27. 28.

29.

30.

M E T A L CLUSTERS IN PROTEINS

Comm. 1982, 108, 273. (b) Frank, P . ; Farver, O.; Pecht, I. J. B i o l . Chem. 1983, 258, 11112. (c) Frank, P . ; Farver, O.; Pecht, I . Inorg. Chim. Acta 1984, 91, 81. Powers, L.; Blumberg, W . E . ; Chance, B . ; Barlow, C . ; Leight, J . S . Jr.; Smith, J.C.; Yonetani, T . ; V i k , S.; Peisach, J. Biochim. Biophys. Acta 1979, 546, 520. Smith, T.A.G., Ph.D. Thesis, Stanford University, 1985. (a) B l a i r , R . A . ; Goddard, W.A. Phys. Rev. B. 1980, 22, 2767. (b) Kosugi, N . ; Yokoyama, T.; Asakuna, K . ; Kuroda, H. Springer Proc. Phys. 1984, 2, 55 (1984). (c) Kosugi, N . ; Yokoyama, T.; Asakuna, K . ; Kuroda, H. Chem. Phys. 1984, 91, 249. (d) Gewirth, A . A . ; Cohen, S . L . ; Schugar, H.J.; Solomon, E.I. Inorg. Chem. 1987, 26, 1133. It should be noted that there has been some confusion i n the l i t e r a t u r e with respect to the apparent oxidation state of the T3 s i t e i n different T2D laccase preps. This derives from the use of i n d i r e c t methods to define oxidation state. We have run x-ray edge spectra of T2D laccase prepared by four of the research groups strongly involved i n this f i e l d , and in all cases the Type 3 s i t e i s found to be f u l l y reduced. Spira D.J.; Solomon, E.I., J. Am. Chem. Soc. 1987, 109, 6421. Wilcox, D . E . ; Westmoreland, T . D . ; Sandusky, P . O . ; Solomon, E.I., unpublished r e s u l t s . Wilcox, D . E . ; Long, J . R . ; Solomon, E.I. J. Am. Chem. Soc. 1984, 106, 2186. Himmelwright, R . S . ; Eickman, N.C.; Solomon, E.I. J. Am. Chem. Soc. 1979, 101, 1576. Westmoreland, T . D . ; Solomon, E.I., to be published. (a) Stephens, P . J . Ann. Rev. Phys. Chem. 1974, 25, 201. (b) Piepho S . B . ; Schatz, P.N. Group Theory i n Spectroscopy, Wiley-Interscience: New York, 1983. (a) Allendorf, M.D.; Spira, D.J.; Solomon, E.I. Proc. Natl. Acad. S c i . USA 1985, 82, 3063 (1985). (b) D . J . Spira, M.D. Allendorf, and E.I. Solomon, J. Am. Chem. Soc. 1986, 108, 5318. The oxidized T1 s i t e i n laccase i s spectroscopically similar to that of plastocyanin and a z u r i n , indicating that exogenous ligands can coordinate only to the T2 and T3 coppers at the native active s i t e . (see Spira, D.J.; Co, M . S . ; Solomon, E.I.; Hodgson, K.O. Biochem. Biophys. Res. Commun. 1983, 112, 746.)

RECEIVED December 18, 1987


Coupled Binuclear Copper Active Sites - American Chemical Society

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