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Journal of Inorganic Biochemistry 75 ( 1999) 63-69

Structure-function correlation of Cu( II) - and Cu (I) -di-Schiff-base complexes during the catalysis of superoxide dismutation

Jijrg Miiller a, Dieter Schiibl b, Ckilia Maichle-M6ssmer b, Joachim Str&le b, Ulrich Weser a,* a Anorganische Biochemie, Physiologisch-Chemisches Institut der Eberhard-Karls-Universitiit Tiibingen, D-72076 Tiibingen, Germany

’ Anorganische Chemie I, Anorganisch-Chemisches Institut der Eberhard-Karls-Universitiir Tiibingen, D-72076 Tiibingen, Germany

Received 24 November 1998; received in revised form 5 March 1999; accepted 15 March 1999

Abstract

Aquo-[N,N’-bis( 2-( 6-methyl-pyridyl)methylene)-1,4-butanediamine] (N,N’,W’,N”‘)-copper( II) perchlorate, [ Cu( II)-( Pu-6-MePy)- (H,O) ] ( ClO,),, a copper( II)-di-Schiff base was synthesized and reduced to the Cu( I) form. The successful preparation was controlled by IR and in the case of the Cu(I1) complex additionally by EPR spectroscopy. [Cu(II)-(Pu-6-MePy) (H,O)] (C1O4)2 has an electronic absorption maximum at 717 nm of E,,, = 108 M-’ cm-‘. The EPR parameters are g, = 2.059 and gt = 2.235 which are very close to those of the respective values of Cu( II) in intact Cu,Zn, superoxide dismutase (SOD). The electronic absorption maximum of the Cu (I) complex is at 474 nm (edT4= 12 123 M-’ cm-‘) and, as expected, no EPR is seen. The crystal structure of [ Cu( II) -( Pu-6-MePy ) ( H20) ] ( C104) 2 shows a monomer while the reduced Cu( I) form reveals a dimer. Both complexes comprise the same SOD mimetic activity of 0.3% compared to that of the cytosolic enzyme. As the reduction of the Cu( II) complex proceeds within lo-40 min it is unlikely that the Cu(1) dimer is formed during the reduction oxidation cycle of superoxide dismutation which occurs at a timescale of milliseconds. The Cu(1) dimer is a promising superoxide dismutase mimicking compound in an aerobic reducing environment as, for example, in cytosol. 0 1999 Elsevier Science Inc. All rights reserved.

Keywords: Schiff base complexes; Catalysis; Superoxide dismutation; Copper

1. Introduction

Cu,Zn, superoxide dismutase (SOD) accelerates the pro- ton-dependent spontaneous dismutation of superoxide into oxygen and hydrogen peroxide at a rate of 2 X 1 O9 M - ’ s - ’ [ 1,2]. The mechanism of how the enzymatic dismutation takes place is still debated. In comparing the crystal structure of Cu,Zn, SOD in both its Cu(1) and Cu( II) states, the copper coordination geometry remains essentially unchan- ged. Only the N( his)-Cu bond distances are elongated in the Cu(1) form, as expected [ 31. In contrast, previous X-ray crystal structure analyses exhibit a cleavage of the Cu(I)- N(his 61) bond [4] and a change from a fourfold coordi- nation of Cu(I1) by his 44, 46, 61 and 118 to a threefold coordinated Cu (I) centre. The mechanism with the cleaved Cu( I)-N( his 61) bond and protonation of his 61 is proposed to be likely for the case of physiologically occurring concen- trations of superoxide and low reaction rates [5,6]. Under superoxide saturated conditions a mechanism with unchan-

* Corresponding author. Fax: + 49-70-71-29-5564; e-mail: uhich.weser @uni-tuebingende

ged coordination geometry between Cu( II) and Cu( I) sup- ported by the work of Banci et al. is discussed [ 3,7]. A superoxide molecule coordinated to Cu( II) is stabilized by hydrogen bonding to arg 141. During the reaction of a Cu( 11)-O; complex with a second superoxide, radical oxy- gen and a transiently Cu(I)-O- species are formed. The latter complex reacts with two protons releasing hydrogen peroxide and the oxidized enzyme. Copper complexes of the biuret or acetate type including amino acid residues, peptides, and salicylate derivatives are known to be useful functional analogues of SOD [8-l 11. It was of utmost importance to design and successfully achieve Cu complexes not only as functional analogues. These genuine mimics should be struc- turally related to the copper coordination environment of CuzZn, SOD. Thus, imidazolate bridged CuZn [ 12-141, macrocyclic [ 151 and tetradentate Cu-di-Schiff-base com- plexes were [2,16-181 synthesized. As Cu(I1) prefers a tetragonal planar and Cu(I) a tetrahedral ligand field the coordination should be maintained during the reduction oxi- dation cycle. Di-Schiff bases with a butylene bridge between the imino functions fulfil this requirement and allow a flexible coordination. While Cu( II) complexes are well characterized

0162-0134/99/$ - see front matter 0 1999 Elsevier Science Inc. All rights reserved. PDSOl62-0134(99)00035-5

64 J. Miller et al. /Journal of Inorganic Biochemistry 75 (1999) 6349

by X-ray crystal structure analyses, little is known regarding the arrangement of the nitrogen atoms around the copper centre in the reduced Cu( I) form.

In order to obtain more information about the Cu(1) coordination the new Cu di-Schiff base aquo-[N,N’- bis( 2- (6-methyl-pyridyl) methylene) - 1 ,cbutanediamine] - (N,N’,N”,N”‘) -copper( II) perchlorate, [ Cu( II) -( Pu-6- MePy)(H,O)](ClO,), (Fig. l), and the Cu(1) form were synthesized and subjected to crystal structure analyses. The SOD mimetic activity of the complexes was estimated and compared with that of the native enzyme. Further character- ization was carried out by elemental analyses, electronic absorption, IR and electron paramagnetic resonance (EPR) measurements.

2. Experimental

2.1. Reagents

All chemicals were of the highest purity available. Cop- per( II) perchlorate hexahydrate was purchased from Aldrich, Steinheim; 1,4-butanediamine and 6-methylpyridine-Zalde- hyde were obtained from Merck, Darmstadt; N-( 2-hydroxy- ethyl) -piperazine-N’-2-ethane sulfonic acid (HEPES), nitroblue tetrazolium chloride (NBT) , bovine superoxide dismutase (SOD), xanthine and xanthine oxidase from butter milk (XOD) were ordered from Serva, Heidelberg.

2.2. Synthesis of complexes

2.2.1. [Cu(II)(Pu-6-MePy)(H20)](C10,), 2.42 g (20 mmol) 6-Methylpyridine-2-aldehyde and 3.7

g ( 10 mmol) copper( II) perchlorate hexahydrate were dis- solved in 60 ml water under vigorous stirring. 0.88 g (10 mmol) 1,4-Butanediamine were added dropwise while mix- ing was maintained. After a reaction time of 30 min the pH was adjusted to 5. The reaction mixture was heated for 1 h to 50°C and allowed to cool to room temperature. By further cooling to 4°C precipitation was completed. The blue powder was recrystallized once each from ethanol/petrolether and from ethanol/water and dried under vacuum in the presence of potassium hydroxide. The yield of blue crystals was 3.74 g (65.0%). Anal. Found: C, 38.22; H, 4.42; N, 9.89; Cu, 10.94. Calc. for C,8H24C12C~N409: C, 37.61; H, 4.21; N, 9.75; cu, 11.05%.

2.2.2. [Cu,(I)(Pu-6-MePy)J(C10,),. MeOH 1.52 g (2.65 mmol) [Cu(II)(Pu-6-MePy)(H,O)]-

( C1O4)2 were dissolved in 100 ml acetonitrile/methanol at a ratio of 6:4. Reduction was accomplished within 15 min fol- lowing the addition of 3.34 g (26.5 mmol) sodium sulfite under constant stirring. The reaction was complete when the colour had changed from blue to yellowish brown. The reac- tion mixture was cooled to 4°C to remove undissolved sodium sulfite and precipitated sodium sulfate. The total volume was

Fig. 1. Chemical formula of the ligand Pu-6-MePy.

reduced to 15 ml and the solution was repeatedly cooled to 4°C to allow crystallization of brown crystals. The crude product was separated from salt residues by repeated recrys- tallization from methanol with 1 ml of water added. The yield of brown crystals was 0.8 g (63.8%). Found: C, 47.66; H, 5.13; N, 12.29; Cu, 13,14. Calc. for C,,H,,Cl,Cu,N,O,: C, 46.94; H, 5.11; N, 11.84; Cu, 13.42%.

2.3. Crystal structure analyses

A single blue crystal of [Cu(II) (Pud-MePy)- (H,O) ] ( ClO4)2 with dimensions 0.3 X 0.25 X 0.5 mm was mounted on an Enraf-Nonius CAD-4 diffractometer. The unit cell was determined. For crystallographic data see Table 1.

X-ray diffraction was measured at 298 K using graphite monochromated Cu Ko radiation by the w/28scan technique. The intensity was monitored for 1 h. After absorption correc- tion with DIFABS [ 191 (absorption correction coefficients: min. 0.8689, max. 1.4282, av. 1.0029) 2906 reflections with an intensity Z > 3 o(Z) of 4849 data were collected in the range 5 < 0 < 65” and were used in structure determination and refinement. The structure was solved using SHELXS [20] by direct methods and subsequent difference Fourier synthe- sis (SDP [ 211) . All atoms (nonhydrogen) were assigned anisotropic thermal parameters. The H atoms (calculated with HYDRO) were considered in the structure factor cal- culations. The final refinement led to 308 refined parameters converged to the unweighted factor R= 0.059 and the

Table 1 Crystallographic data for [ Cu( II) ( Pu-6-MePy ) (H,O) ] (ClO,)*

Chemical formula Molecular weight (g mol ’ ) Crystal system Space group a (A) b (A) c (A) P (“) v (AZ) Z h (Cu Ka) (A) P (g cm-7 cL (cm-‘) F(mO)

C I sHdACuN,O, 572.8 monoclinic P2,ln 15.506( 1) 9.0816(4) 17.710(l) 107.632(3) 2376 4 1.541838 1.601 38.9 1172

.I. Miller et al. /Journal of Inorganic Biochemistry 75 (1999) 6349 6.5

weighted one R, =0.061. The highest peak in the final AF map was 1.6 e A-’ because one of the perchlorate anions was disordered. Atomic scattering factors were taken from the International Tables for X-ray Crystallography [22]. Molecular graphics were from SCHAKAL [ 231.

model 1106). Copper was assayed on a Zeiss M4Q III atomic absorption spectrometer combined with PMQ II.

2.5. Superoxide dismutase assay

A red-brown crystal of [ Cuz( I) (Pu-6-MePy),] - ( C10,)2.MeOH, size 0.3 X 0.2 X 0.1 mm, was mounted on an Enraf-Nonius CAD-4 single-crystal diffractometer using graphite monochromated Cu Kol radiation. The unit cell was determined from the angular settings of 25 reflections with 16.9 < 8< 26.7”. The intensity data of 4350 reflections were measured in the hkl range ( - 1, 0, - 24) to ( 10, 13, 24) between 0 limits: 5.21 < 0< 64.82” by the w/28 scan tech- nique. For crystal data see Table 2. The intensity was con- trolled for 1 h. A semi-empirical absorption correction was performed with psi scan. 3596 unique reflections of which 3019 have an intensity I> 2a( 1) were observed. The struc- ture was solved by direct methods using SHELXL [ 241.

Superoxide dismutase activity was examined indirectly using the nitroblue tetrazolium (NBT) assay [ 93. 0.5 ml of the reaction volume contained 0.24 mM nitroblue tetrazo- lium, 0.1 M HEPES buffer of pH 8.0, 0.4% (w/v) gelatine and copper complexes or SOD in variable concentrations. EDTA if not equilibrated with the copper complex was added directly to the reaction mixture to yield a final concentration of 5 pM. At this concentration the SOD activity of either the employed complex or SOD remained unchanged. Xanthine/ xanthine oxidase served as a convenient superoxide source.

3. Results and discussion

During the final stage of refinement, the positional and anisotropic thermal parameters of the nonhydrogen atoms were refined using the method of the smallest F2 data. The hydrogen atoms were isotropically refined with a common thermal parameter. The final agreement factors were RI = 0.0456, wR2 = 0.1230 with a fit quality for F2 being 1.063 for 3596 reflections observed and 254 refined para- meters. The final difference Fourier map showed no peaks higher than 0.349 e A-’ or deeper than - 0.451 e A-‘. Atomic scattering factors were taken from International Tables for X-ray Crystallography [ 221. Molecular graphics were from SCHAKAL [ 231.

3.1. Synthesis of complexes

2.4. Spectrometric measurements

IR spectra were run on a Beckman Acculab 4 spectropho- tometer employing KBr disks. Electronic absorption spectra were recorded on two different Beckman spectrometers DU 7400 and 25, respectively. Electron paramagnetic resonance was measured on a Bruker ESP 300E instrument at modula- tion amplitude 0.1 mT, modulation frequency 100 X 10’ s - ’ , microwave power 20 mW, microwave frequency 9.47 X lo9 S - ‘, temperature 100 K. The elemental analyses for C, H and N were performed on an elemental analyser (Carlo Erba

The preparation of [ Cu( II) (Pu-6-MePy) (H,O) ] (Clod) 2 from Cu( C104) *, 1,6butanediamine and 6-methylpyridine- 2-aldehyde was successful. The blue crystals of this copper( II) complex were metastable. A colour change into reddish brown and some deterioration of the crystals are seen after three months of storage under normal atmospheric con- ditions. Attributable to this phenomenon it was attempted to fully reduce this complex and to culture crystals of the cop- per(I) compound aiming at single crystals suitable for crystal structure analyses. For maintenance of the copper(I) state [ Cu( II) ( Pu-6-MePy) ( H20) ] ( C1O4)2 was reduced in a solution containing acetonitrile/methanol at a ratio of 6:4. As acetonitrile is known to be a soft ligand with high affinity to copper(I) this solvent was chosen to effectively stabilize the low oxidation state and to avoid release of Cu( I) from the complex which would undesirably disproportionate into Cu( II) and elemental copper.

3.2. Crystal structures

3.2.1. [Cu(Il)(Pu-6-MePy)(H,O)](ClO,), The preparation of the complex from l,Cbutanediamine,

6-methylpyridine-Zaldehyde and copper( II) perchlorate

Table 2 Bond distances (A) of [Cu(II) (Pu-6-MePy) (H,O)] (ClO,)* with e.s.d.s in parentheses

Atom 1 Atom 2 Distance Atom 1 Atom 2 Distance

CL’ O(l) 2.080(4) N(3) C(l) 1.265(S) CU N(l) 2.214(5) N(3) (32) 1.491(9) cu N(2) 2.038(4) N(4) C(5) 1.478(7) cu N(3) 1.982(4) N(4) C(6) 1.270(S) cu N(4) 1.981(5) C(l) C(l1) 1.45( 1)

N(1) C(11) 1.356(7) C(6) W5) 1.455(8)

N(1) C(l5) 1.356(9) C(3) C(4) 1.53(2)

N(2) C(21) 1.341(7) (38) C( 15) 1.499(9)

N(2) ~(25) 1.347(9) C(7) C(21) 1.52(2)

66 .I. Miller et al. /Journal of Inorganic Biochemistry 75 (1999) 63-69

024

C23

v Cl4 C8

Fig. 2. Crystal structure of [Cu( II) (Pu-6-MePy) (H,O) ] (C104)*

This complex (Fig. 3) was successfully prepared by reduc- tion of [ Cu( II) (Pu-6-MePy) ( H20) ] ( C104)2. Red-brown crystals were obtained. Crystal structure analysis was per- formed using a single crystal. Crystallographic data are given in Table 4, and bond distances and angles in Tables 5 and 6.

was successful. Single crystals were cultured by recrystalli- zation from water. A thus-obtained blue square crystal was subjected to crystal structure analysis. The crystallographic data are given in Table 1.

The Cu( II) centre shows a slightly distorted trigonal bipyr- amidal coordination geometry (Fig. 2). The axial ligands are assigned to one pyridine (N2) and one imino (N3) nitrogen each. The equatorial coordination is formed by one pyridine (Nl ) , one imino (N4) nitrogen and .an oxygen from a water molecule.

The bond distances between copper and the imino nitro- gens are shorter as for the pyridine nitrogens (Table 2). The reason for the relatively long Cu-N bond distance of Nl with 2.214 A is the sterical hindrance of the methyl substituents at the pyridines. This sterical hindrance enables the coordina- tion of water as a fifth ligand and is the main factor for the metastability of the crystals. Different observations have been made with the Cu complex [ Cu( II) ( PuPhePy) ] ( ClO,) 2 being unmethylated in the ortho position to the pyridine nitro- gen and which was found to be stable in the Cu( II) state.

Initially a tetrahedrally coordinated monomeric Cu( I) complex was expected. It was surprising to realize that the crystal structure revealed a dimer. At first it was concluded that the reason for this phenomenon could be assigned to the preparation procedure. In order to ascertain the Cu( I) state a mixture of 60% acetonitrile and 40% methanol was used to avoid the release of ‘free’ copper into the solvent by tightly chelating possible dissociated Cu( I). Due to its high affinity to Cu( I) it seemed plausible that acetonitrile could replace one or two nitrogen ligand atoms and that it would be possible for two Cu( I) monomers to associate and create a dimer. The coordinated acetonitrile would be replaced by an imino-pyr- idine system.

A comparison between [ Cu( II) ( Pud-MePy ) (H,O) ] - (Clod) 2 and Cu,( II) Zn, SOD on the basis of their crystal

If this hypothesis were true the preparation using a water/ methanol mixture of 1: 1 as a solvent would be most suitable to obtain the monomeric form. Much to our surprise the opposite was observed. A dimeric Cu( I) complex of the very same structure was noticed. Both Cu( I) atoms in the dimer show the same distorted tetrahedral coordination geometry. Four nitrogen atoms are bound to the metal centre. They originate from one imino and one pyridine nitrogen from each of the two di-Schiff-base ligands. Taking N( 4) as the tip of the tetraeder the N(4)-Cu-N angles are 81.9, 113.4 and

Table 3 Bond angles (“) of [ Cu( II) (Pu-6-MePy) (H,O) ] (CIO,), with e.s.d.s in parentheses

structure is difficult because the mimic shows a fivefold and the enzyme a fourfold coordination. It is likely that the water molecule is exchanged in solution due to the Jahn-Teller effect and that the whole coordination geometry changes into a fourfold coordinated Cu( II) centre. On the other hand, if one considers the water molecule replacing the superoxide anion under superoxide saturated conditions a fivefold coor- dination is expected in the enzyme and the coordination geometry is very similar. The enzyme shows N&-N angles of 130 and 160” and the complex 112 and 170” (Table 3).

3.2.2. [Cu,(I)(Pu-6-MePy)2](C10,),~MeOH

Atom 1 Atom 2 Atom 3 Angle Atom 1 Atom 2 Atom 3 Angle

O(l) O(l) O(l) O(l) N(l) N(l) N(l) N(2) N(2) N(3) N(3) N(4)

CU cu cu cu cu cu cu cu cu cu (32) (35)

N(l) N(2) N(3) N(4) N(2) N(3) N(4) N(3) N(4) N(4) C(3) C(4)

101.0(2) 97.9(2) 85.2(2)

145.0(2) 109.8(2) 78.9(2)

112.3(3) 170.0( 2) 81.3(2) 90.7( 2)

lllS(5) 110.4(5)

C(ll) (221) C(l) C(5) N(l) N(l) N(2) N(2) N(3) N(4) C(2) C(3)

N(l) N(2) N(3) N(4) C(ll) C(15) C(21) C(25) C(l) (36) C(3) C(4)

C(15) CC251 C(2) (36) C(l) ‘78) C(7) C(6) (311) (225) C(4) C(5)

118.7(5) 117.6(5) 119.2(5) 119.2(6) 116.4(6) 119.5(7) 118.9(5) 116.0(6) 118.9(5) 116.6(6) 115.8(6) 115.6(6)

J. Miiller et al. /Journal of Inorganic Biochemistry 75 (1999) 63-69 67

C46 r c1’( \

0 C1’3/’

c44 C6 c5

Fig. 3. Crystal structure of [C&(I) (I%-6-MePy),] (C104)2*MeOH.

Table 4 Crystallographic data for [Cu,( I) (Pu-6-MePy),] (CIO,),*MeOH

Chemical formula Molecular weight (g mall ‘) Crystal system Space group a (A) b (8) c (A) P (“) v (AZ) 2 A (Cu Ko) (A) P (g cm-“) w (mm’) F(000)

WLsWWW, 946.82 monoclinic P2ln 9.0102(5) 11.2475(4) 20.9589( 14) 94.320( 3) 2118.0(2) 2 1.54056 1.485 29.18 980

123.6”. The Cu-N bond distances range between 1.98 and 2.07 A. As the bridge between the imino functions of the ligands includes four carbon atoms and the conformation is eclipsed, a copper-copper interaction is not possible.

3.3. IR, EPR and electronic absorption spectroscopy

The successful synthesis of [ Cu( II) (Pu-6-MePy) - (H,O) ] (Clod)* was confirmed by IR spectroscopy. The

Table 5

C=N valence vibration characteristic for the imine function appears at 1650 cm- ‘. The N-H valence vibrations from 1,4- butanediamine at 3300-3500 cm- ’ and the C=O valence vibrations originated from the aldehyde at 1660-l 7 15 cm- ’ are clearly levelled off. During the reduction of the Cu(I1) complex employing sodium sulfite the C=N bonds remain unaffected. The C=N imino valence vibration band of [ Cu,( I) (Pu-6-MePy) *] (Clod) 2. MeOH appears at a wave number of 1620 cm-‘. The shift of 30 cm-’ between the vibration bands of the Schiff-base functions of the Cu(I1) and the Cu(I) complex is due to a smaller distortion of the five-membered chelate rings in the case of the Cu( I) dimer.

The electronic absorption spectra of Cu( II) complexes and Cu,Zn, SOD show bands in the ultraviolet region, which are due to p-p* and n-p* transitions inside the ligand or protein. A characteristic feature for the ligand field and the strength of the splitting of the d-orbitals are bands which occur in the region of visible light and have their origin in d-d transitions. Cu,Zn, SOD has its absorption maximum at 680 nm with an extinction coefficient of 150 M- ’ cm-’ (Table 7). For [ Cu( II) ( PuPy) ] (ClO,) 2 a well-known SOD mimic the d- d band is 30 nm red-shifted. The reason for this is the weaker ligand field of the relatively electron-poorpyridinerings com-

Bond distances (A) of [Cu,(I) (Pu-6-MePy),] (ClO.,),.MeOH with e.s.d.s in parentheses

Atom 1 Atom 2 Distance Atom 1 Atom 2 Distance

cu N(l) 1.984(3) N(3) C(45) 1.353(4) cu N(2) 2.004( 3) N(4) (211) 1.343(4) cu N(3) 2.037(3) N(4) C( 15) 1.354(4) CU N(4) 2.071(3) (21) C(l5) 1.462(5) N(l) (31) 1.275(4) C(6) C(45) 1.465(4)

N(l) C(2) 1.469(4) (32) C(3) 1.525(5)

N(2) C(6) 1.274(4) C(3) C(4)#1 1.516(4)

N(2) (35) 1.476(4) C(4) C(5) 1.509(5) N(3) C(41) 1.342(4)

68 .J. Miiller et al. /Journal of Inorganic Biochemistry 75 (1999) 63-69

Table 6 Bond angles (“) of [Cu,(I) (Pt-6-MePy),] (ClO,),+MeOH with e.s.d.s in parentheses

Atom 1 Atom 2 Atom 3 Angle Atom 1 Atom 2 Atom 3 Angle

N(1) N(1) N(l) N(2) N(2) N(3) C(1) (72) C(5) C(6) C(41) C(45)

CU CU CU CU CU CU N(1) N(1) N(2) N(2) N(3) N(3)

N(2) N(3) N(4) N(3) N(4) N(4) CU CU cu cu cu cu

128.68( 11) 132.13( 11) 81.93( 11) 81.86( 10)

123.58( 11) 113.36( 11) 113.4(2) 127.0(2) 127.0(2) 113.3(2) 129.8(2) 111.3(2)

cc111 C(l5) N(l) N(2) N(3) N(4) N(l) N(2) N(3) N(4) (32) C(3)#l

N(4) N(4) C(l) (26) C(45) C(15) C(2) C(5) C(4l) (311) C(3)#1 C(4)

cu CU cc 1.5) C(45) C(6) C(1) C(3) C(4) C(46) CC 16) C(4) C(5)

131.3(2) 110.3(2) 119.6(3) 118.7(3) 114.8(3) 114.6(3) 110.6(3) 109.7(3) 116.6(3) 116.5(3) 113.0(3) 113.7(3)

Table 7 Visible electronic absorption of active site models and SOD (all data are given per g atom of Cu)

Copper complex Solvent A,,, E (nm) (M-l cm-‘)

[Cu(II)(Pu-6-MePy)(HzO)]*+ H,O 717 108 [cu(II)(PuPy)lz+ Hz0 710 116 CuI( II)Zn, SOD H@ 680 150 [&(I) (Pu-6-MePy),l’+ Hz0 474 12123

pared to the electron-rich imidazolates in the enzyme. The inductive effect of the two methyl groups in [ Cu( II) (Pu-6- MePy) ( HzO) ] ( ClOJ 2 should result in a blue shift of the absorption maximum. However, the sterical hindrance of the two methyl groups in the 6-position of the pyridine rings leads to a distortion of the coordination geometry overcom- pensating the electronic effect. The ligand field is weaker compared to [Cu(II)(PuPy)](ClO,), (Table 7). The extinction coefficient of [ Cu( II) (Pu-6-MePy) (H,O) ] - ( ClOJ 2 is in the same range as that of CuzZnz SOD.

The absorption of [ Cu2( I) (Pu-6-MePy ) 2] ( ClOJ 2 * MeOH in the visible region is due to a charge transfer between copper ion and ligand. A characteristic for such bands is the loo-fold higher extinction coefficient in com- parison to that of d-d transitions. The spectrum of [ Cuz( I) (Pu-6-MePy),] (CiO,),*MeOH has an absorption maximum at 474 nm with an extinction coefficient of 12 123 M-’ cm-‘.

The coordination of Cu(I1) was examined by EPR spec- troscopy. The data for [ Cu( II) (Pu-6-MePy ) ( H20) ] - (ClOJ 2 are very close to those of Cu,( II) Znz SOD (Table 8). The g,, /A,, ratio is an empirical factor for the tetrahedral distortion of a tetragonal coordinated complex [ 251. A value between 105 and 135 cm is assigned to a square planar envi- ronment of the Cu( II) centre. A rise of up to 250 cm indicates an obvious distortion of the tetragonal ligand field against tetrahedral. SOD with a ratio of 162 cm, therefore, should show a distinct distortion as confirmed by the X-ray crystal structure of Tamer et al. [ 261. Nearly the same g,, /A,, ratio

with 149 cm was estimated for [Cu( II) (Pu-6-MePy)- (H,O) ] ( C104)2. As expected for a transition metal ion with no unpaired electrons, the Cu( I) dimer remained EPR-silent for more than 12 h. It was intriguing to realize that not a single trace of EPR-detectable Cu(I1) was measured in aqueous solutions and in the presence of air. It is concluded that the Cu(1) complex survives oxidation by oxygen quite effectively.

3.4. Superoxide dismutase assay

The SOD mimetic activity of [ Cu( II) (Pu-6-MePy) - (H,O)] (Clod)* and [%(I) (~-6-MePyM (Cl%L* MeOH was examined using the NBT assay [9]. Although methanol is known as a radical scavenger the copper concen- tration for 50% inhibition of the reduction of NBT (named the I&, value) for both the Cu( II) monomer and the Cu( I) dimer is identical within standard deviation.

The complexes exert 0.3% of the activity, which is one order of magnitude less than the best SOD analogues (Table 9). However, it should be emphasized that these complexes are genuine structure function analogues of the intact enzyme. In this context the sequestering of defined oxidation states promised to be highly rewarding in comparing the oxidation reduction reactions of both the di-Schiff-base complexes and intact SOD. As the reduction of [Cu( II) (Pu-bMePy)- (H,O) ] ( ClO,), in the presence of either ascorbateor sodium sulfite is successful within lo-40 min (data not shown) the

Table 8 EPR parameters of [ Cu( II) (Pu-6-MePy) (H,O) ] ( ClO,) 2 and Cu,( II)Zn, SOD

Copper chelate g I gll AlI d‘%l

(mT) x 1o-4 (cm) (cm-‘)

[Cu(II)(Pu-6- 2.059 MePy)l”

Cu*(II)Zn, 2.083 SOD

2.235 14.4 150 149

2.21 I 13.3 140 162

J. Miiller et al. /Journal of Inorganic Biochemistry 75 (1999) 63-49 69

Table 9 Inhibitory effect of copper complexes on the xanthine-XOD mediated reduc- tion of nitroblue tetrazolium

Copper complex IC, cont. of chelated Cu(II) required to yield 50% inhibition (FM)

[Cu(II)(Pu-6-MePy)(H,0)12+ 2.25 [Cu,(I)(Pu-6-MePy),]Z+ 2.06 [ww(~pY)1*+ 0.60 [Cu(II)(PuPhePy)]‘+ 0.27 Cua(II)Zn, SOD 0.0084

formation of the Cu( I) dimer during the dismutation of supe- roxide in a timescale of milliseconds is rather unlikely. The dimer formation is not attributable to a concentration effect. Upon diluting the concentrated crystallization solution no changes in the absorption maximum are seen. During the reduction of the Cu( II) complex the charge transfer band first occurs at 485 nm and is shifted to 474 nm. This would allow the conclusion that the reaction takes place in two steps. The initial first reaction, i.e. the reduction of the monomer, pro- ceeds very fast while in the second step the formation of the dimer is fairly slow. Di-Schiff-base complexes are frequently known to appear in both monomeric and dimeric forms in solution. Thus, the possible existence of a monomer cannot be excluded in the present case. However, it appears that the equilibrium is shifted towards the dimeric form. The results of a separate study on the exact mode of this monomer/dimer equilibrium following the reduction of Cu( II) to Cu( I) will be awaited with great interest.

In the course of the dismutation of superoxide following the reduction to Cu( I) the transition metal centre is imme- diately oxidized to Cu( II). Thus, a possible dimer formation is much too slow in comparison to the subsequent oxidation process. This thermodynamically stable dimeric Cu( I) SOD mimic offers new possibilities to allow the use of SOD mim- ics in aerobic reducing environments like cytosol and to learn more about the Cu( I) state of these most effective and stable class of SOD mimics, the Cu-di-Schiff bases.

4. Supplementary material

Tables of positional and thermal parameters and structure factors are available from the authors on request.

Acknowledgements

This study was aided by DFG (Grant We 401/24-3) and in part by the Fonds der Chemischen Industrie.

References

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