Journal of Molecular Catalysis, 23 (1984) 369 - 375 369

SYNTHETIC POSSIBILITIES AND STRATEGIES IN THE DEVELOPMENT OF ANALOGUES FOR THE DINUCLEAR AND POLYNUCLEAR METAL SITES IN ENZYMES tiD PROTEINS

JAN REEDIJK, PAUL J. M. W. L. BIRKER and JACOBUS VAN RIJN

Department of Chemistry, Gorleaus Laboratories, State University Leiden, P.O. Box 9502, 2300 RA Leiden (The Netherlands)

Summary

Two synthetic approaches in the synthesis of dinuclear (and polynuclear) coordination compounds are described briefly. The first method uses small external ligands (such as OH- and S*-) that act as a bridge to bring the metal ions together. The second method uses dinucleating chelating ligands to hold two metal ions fixed together, with one (or more) bridging donor groups between the metals.

Application of these methods in the synthesis of model coordination compounds for superoxide dismutase (imidazole-bridged copper and zinc systems) and for hemocyanin (copper dimers with imidazole-type ligands) is described. It is found that the most stable dinuclear systems are formed when a dinucleating chelating ligand system is used.

Introduction

A large number of metalloproteins and metalloenzymes are known to contain more than one metal ion per ‘molecule [l]. Very often these metal ions are quite far apart, e.g. in different sub-units (as in hemoglobin) or even in the same sub-unit (as in dopamine P-hydroxylase and in transferrin). In other cases the metal ions are quite close together, having one or more bridg- ing ligands between them (as in ferredoxin) [ 21. In this paper we will only deal with the last category, because only in these cases can a significant mag- netic exchange between the metal ions occur. The dinuclear and polynuclear centers that occur in Nature can be either homopolyatomic or heteropoly- atomic. Homopolyatomic species occur as dimers (e.g. as copper dimers in tyrosinase and hemocyanin [ 31, as iron dimers in hemerythrin [4] and ferre- doxin [5]), trimers (in ferredoxin [6]), tetramers (in ferredoxins [2] and hydrogenases [7]) and even larger units (in ferritin [8] and in metallo- thioneins [ 91). Heteropolyatomic species are known to occur as dimers (such as in the Cu-Zn site of bovine superoxide dismutase [lo] and the Fe-Cu site in cytochrome C oxidase [ 111) and also as larger units (such as in the Fe-

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370

and Mo-containing cofactor of nitrogenase [12]). The role of the dinuclear and polynuclear sites seems to be structural and/or electronic.

Because most of the dinuclear and polynuclear species in nature are involved in electron-transfer reactions, it seems likely that the presence of two or more metal ions has something to do with this electron transfer. Therefore a better understanding of the coupling between (magnetic) ions in proteins and in low-molecular weight analogues is highly relevant. This paper will mainly deal with the synthesis, structure, and spectroscopic and magnetic properties of dinuclear and polynuclear species that more or less resemble the active site in me~lopro~~s and me~loenzymes.

Synthetic approaches

Several authors have recently reviewed synthetic strategies that might result in dimeric or oligonuclear metal species [13 - 161. Basically, two ap proaches have been followed so far:

(a) Use of small ligands that are well known to form bridges between two (or more) ions. Examples are OH- (for Cu dimers) [1’7], S2- (for iron- sulfur clusters) [18] and deprotonated imidazole (for copper dimers) [19]. Under these circumstances dinuclear or polynuclear metal units can be formed only in certain cases, and gener~izations are difficult to make. A few examples of such structures are shown schematic~ly in Fig. l(a), (b) and (c),

(b) Use of dinucleating (or polynucleating) chelating ligands to hold two (or more) metal ions bound to the same ligand [13 - 161. In addition small bridging ligands, such as OH-, may be present (see Fig. 2(a)). These systems may or may not contain a ligand atom bridging two or more metal ions (see Fig. 2(b)). Asymmetric dinucleating chelating ligands can be de- signed to synthesize asymmetric dimers, which may even hold two different metal ions [20] (see Fig. 2(c)). Steric constraints and/or intramolecular interactions (e.g. hydrogen bonding) can be used to generate a certain coor- dination geometry, or to regulate metal-ligand distances [ 153. Most success- ful results have been obtained so far when method (b) was used. However, many clusters have been prepared according to method (a) also.

In the remaining part of this paper we will briefly summarize our recent results in this area, with special attention to models for superoxide dismutase (type 2 copper) and hemocyanin (type 3 copper). Our work on synthesis, characterization and magnetic exchange studies in dinuclear and polynuclear compounds with fluoride ions as a bridge has been reviewed [21, 221 else- where and will not be discussed here because of the fact that the F- ion is unlikely to be a relevant bridge in biological systems.

ModeIs for the metal site in bovine superoxide dismutase

The X-ray crystal st~cture of this enzyme is now well known [lo], although discussions about the biological functions of the enzyme are still

371

(4 i-1=1.2.3 Cl= added ligand

(b)

(cl

0 \O/ L\C”/N-N\C”/L L’ \/ ‘L

I H

M = Cu.Zn

N/;\N-L

f\l I\ L-Fe -x-cu-s

Fig. 1. Schematic structures of systems having externally added bridging ligands. (a) A system with bridging ligands such as Cl- (usually n = 1, 2, 3), OH- (n = 2, 3). (b) A bridg- ing imidazole-type ligand. (c) A system having both OH- and pyrazolate as bridging ligand.

Fig. 2. Schematic structures of dinuclear systems with a dinucleating chelating ligand. (a) With an added ligand bridging as well (such as Cl-). (b) With a built-in benzimidazole bridge. (c) With an asymmetric dinucleating ligand to bind two different metal ions.

on-going [23]. A striking feature in the structure is the imidazolyl group bridging between a copper and a zinc ion. The role of zinc seems to be only structural [24], as it can be replaced by many other metal ions without sig- nificant changes in activity. A schematic picture of a possible mechanism, in which the copper coordination changes from four nitrogens (in the divalent state) to two nitrogens (in the monovalent state), is depicted in Fig. 3. In this picture two of the imidazole nitrogens bind a proton, thereby allowing the 02- ion to be rapidly protonated after electron transfer from Cu(1). It should be stated that the details of the mechanism are still subject of debate and that many variations of the one depicted in Fig. 3 have been published [ 241. The electronic and magnetic effects of a bridging imidazolyl group are easily seen when the zinc is replaced by Cu(I1). In that case the magnetic exchange coupling between the two copper ions is antiferromagnetic (J = 26 cm-‘) [25]. Therefore, most of the model studies have been initially directed to dimeric copper systems as shown in Fig. l(b). Lippard and others [24] have studied a variety of compounds with several end-group ligands,

372

t Nhis

+J? ) \c;/Nh?z[

di\ I’

*his 1 Nhis

rearrangement +o;

+2 Hi

Fig. 3. Schematic drawing of possible intermediate structures in the catalytic cycle of bovine superoxide dismutase. The mechanism is based on the fact that Cu(I1) has four nitrogen donor ligands preferentially in one plane (plus a weakly bonding axial water iigand) and that Cu(1) has only two imidazole-type ligands preferentially.

such as aliphatic and aromatic amines. We have extended this with imidazole groups as the non-bridging ligands, just as in the enzyme [19]. In all cases the magnetic exchange was of the same order of magnitude and the details of the correlations between the structure and the magnetic exchange have been discussed elsewhere [19, 24, 261. It turned out that in solution disso- ciation of the Cu-imidazole--Cu bridge occurs, but stabilization is possible when a dinucleating ligand as depicted in Fig. 2(a) is used [ 241. We have developed a chelating ligand system containing both bridging and end imi- dazole groups, as depicted in Fig. 4. It was hoped that dinuclear species, as shown schematically in Fig. 2(b), would be formed. In case of ZnClz, how- ever, it turned out that the central benzimidazole groups only bind to one metal ion [ 271. On the other hand, when Cu(II) perchlorate is used, the central group indeed bridged two copper ions, as found by preliminary mea- surements of its X-ray structure [ 281. Attempts to prepareand crystallize a mixed metal species with Cu(I1) and Zn(II) have remained unsuccessful so far. Work is continu~g with derivatives of this chelating ligand system which have an asymmetry built into the molecule.

Models for the dinuclear site in hemocyanin

Although the spectroscopic evidence for dinuclear copper species in the so-called type 3 copper proteins is quite strong, no clear evidence exists for the nature of the structure of the active sites. To date, spectroscopic and structural studies on hemocyanins have yielded the most detailed informa- tion. A structure that explains most of the observations (spectroscopic, mag-

373

-

ti \f

Fig. 4. St;ructure af the chelating benzimidazoie Iigand PDT& diethylenetriamine pentakis- (benzimidazol-2-yl-methyl), or 1,1,4,7,7-pentakis(benzimidazo~-Z-yl-methyl)-1,4,7-tria~~- heptane.

@his

I 1 his

~“L._______~u~ + a2 *

ague b”i

LJ$/ / ‘t L//cu ----‘---~\L

Nhis Nhis Nhis Nhis

L=his or other Ligond cuI-----cu* = 3.8-3,5%,

c&P-----cP= 3.7 -3.4 R

Fig. 5. Proposed structure for the dim&ear copper centre in deoxy- and oxy-hemacya- nine, as based on preliminary X-ray results and EXAFS information [3, 291.

cu ---Cu=3.U5A (3

Fig. 6. ORTEP drawing of ~n’~(E~TB)~* based on the published coordinates j30],

Fig. 7. Schematic drawing of the X-ray structure of the oxidation product of CI.&- (NME~TB~(N~s)~. The Cu-Cu separation amounts to 5.171(2) 8.

net&, X-ray) [ 3,293 is depicted in Fig. 5. The indicated histidines are certain, whereas the other ligands are not yet known in detail.

A dinuclear Cu( I) compound that resembles this site somewhat (Cu- l * Cu, 3.05 A) is depicted in Fig. 6. This dimer also reacts with dioxygen in solu- tion 1301, although reversibility is lost after a couple of cycles. Studies with the ~-me~yla~d derivative allowed us to isolate and crystallize the oxida- tion product. The ligand bridge between the two copper ions, however, ap- peared to be nitrate, rather than the dioxygen (Fig. 7). In an attempt to prepare larger dinucleating units to bind Cu(1) and Cu(II), the ligand ~~E~TB (~,~~lU,~O-tetra~(~-methyl benzimid~ol-Z~l-me~yl)-l,lO-~~a~4,7-dioxa-

314

Fig. 8 ORTEP drawing of the Cun2(NMEGTB)Fa2+ cation The Cu.**Cu distances 5.83 (intramolecular) and 4.81 A (intermolecular) (311, respectively.

are

decane) was synthesized. The Cu(I1) product obtained as [Cu,(NMECTB)- F,] ‘+ has a very large Cu-Cu distance as shown in Fig. 8. In fact, the shortest Cum - . Cu distance (4.81 A) occurs between the dinuclear units (not indicated in Fig. 8) [31].

Other workers in this field have been studying related systems, with or without (benz)imidazole groups [32 - 351. This work has recently been re- viewed [ 361 and will not be repeated here.

Final remarks

Work of our group and others during the past few years has made it clear that the synthesis of new dinuclear and polynuclear coordination com- pounds resembling the active site of multimetal proteins is mainly a matter of ligand design and, hence, of organic synthesis. It is to be expected that in the coming years the development of advanced chelating ligand systems will arrive at a stage at which catalytic and other specific functions of the multi- metal proteins can be imitated to a high degree. Applications of such spe- cially designed compounds in chemical synthesis (specific oxidation, chiral synthesis) may then be close to reality.

References

1 C. D. Garner and P. M. Harrison, Chem. Br., 18 (1982) 1. 2 W. Lovenberg (ed.), Iron-Sulfur Proteins, Vol. III, New York, 1977. 3 W P. J. Gaykema, E. J. M. van Schaik, W. G. Schutter and W. G. J. Hol, Chem. Ser.,

21(1983) 19. 4 J. Sanders-Loehr and T. M. Loehr, Adv. Inorg. Biochem., 1 (1979) 235.

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5 R. H. Holm, Chem. Sot. Rev., (1981) 455, and references cited therein. 6 I. Moura, J. J. G. Moura and A. X. Xavier, Inorg. Chim. Acta, 79 (1983) 128. 7 S. P. J. Albracht, E. G. Graft and R. K. 1‘. Hauer, FEBS Left., 140 (1982) 311. 8 R. R. Crichton, J. Mol. Catal., 7 (1980) 267. 9 M. Vasak, Znorg. Chim. Acta, 79 (1983) 15, and references cited therein.

10 J. S. Richardson, K. A. Thomas, H. H. Rubin and D. C. Richardson, Proc. Nut. Acod. Sci. USA, 72 (1975) 1349.

11 L. Powers, B. Chance, Y. Ching and P. Angiolillo, Biophys. J., 34 (1981) 465. 12 V. K. Shah and W. J. Brill, Proc. Nat. Acad. Sci. USA, 78 (1981) 3483. 13 D. E. Fenton, U. Casellato, P. Vigato and M. Vivaldi, Znorg. Chim. Acta, 62 (1982) 57. 14 S. M. Nelson, Inorg. Chim. Aeta, 62 (1982) 39. 15 J. M, Lehn, Pure Appt. Chem., 52 (1980) 2441, and references cited therein. 16 J. A. Ibers and R. M. Holm, Science, 209 (1980) 223, and references cited therein. 17 V. H. Crawford, H. W. Richardson, J. R. Wasson, D. J. Hodgson and W. E. HatfieId,

Inorg. Chim. Acta, 15 (1976) 2107, and references cited therein. 18 R. H. Helm, Act. Chem. Res., 10 (1977) 427. 19 H. M. J. Hendriks, P. J. M. W. L. Birker, G. C. Verschoor and J. Reedijk, J. Chem.

Sot., Dalton Trans., (1982) 623. 20 0. Kahn, Znorg. Chim. Acta, 62 (1982) 3. 21 J. Reedijk, Commun. Inorg. Chem., I(1982) 379. 22 J. Reedijk and R. W. M. ten Hoedt, Reck Trav. Chim. Pays-Bas, 101 (1982) 49. 23 K. G. Strothkamp and S. J. Lippard, Biochemistry, 20 (1981) 7488, and references

cited therein. 24 K. G. Strothkamp and S. J. Lippard, Act. Chem. Res., 10 (1982) 318. 25 J. A. Fee and R. G. Briggs, Biochim. Biophys. Acta, 400 (1975) 439. 26 M. G; B. Drew, S. M. Nelson and J. Reedijk, Znorg. Chim. Acta, 64 (1982) L189. 27 P. J. M. W. L. Birker, A. J. Schierbeek, G. C. Verschoor and J. Reedijk, J. Chem. Sot.,

Chem. Commun., (1981) 1124. 28 A. J. de Kok, G. C. Verschoor and J. Reedijk, in preparation. 29 E. 1. Salomon, K. W. Penfield and D. E. Wilcox, Struct. Bonding (Berlin], 53 (1983) 1. 30 H. M. J. Hendriks, P. J. M. W. L. Birker, J. van Rijn, G. C. Verschoor and J. Reedijk,

J. Am. Chem. Sot., 104 (1982) 3607. 31 J. van Rijn and J. Reedijk, in preparation. 32 V. McKee, J. V. Dagdagian, R. Bau and C. A. Reed, J. Am. Chem. Sot., 103 (1981)

7000. 33 M. Nakamura, M. Mikuriya, H. Okawa and S. Kida, Bull. Chem. Sot. Jpn., 54 (1981)

1825. 34 K. D. Karlin, J. C. Hayes, R. W. Cruse, Y. Gultneh, J. P. Hutchinson and J. Zubieta,

Inorg. Chim. Acta, 79 (1983) 98. 35 T. N. Sorrell, D. L. Jameson, M. R. Malachowki and A. S. Borovnik, Inorg. Chim.

Acta, 79 (1983) 98. 36 K. D. Karlin and J. Zubieta, Copper Coordination Chemistry: Biochemical and Inor-

ganic Perspectives, Adenine Press, Albany, New York, 1983.