Trinuclear Copper Cluster Research Articles

Theoretical modeling of the hydroxylation of methane as mediated by the particulate methane monooxygenase

We present here the results of density functional theory (DFT) calculations directed toward elucidation of the C H bond activation mechanism that might be adopted by the particulate methane monooxygenase (pMMO) in the hydroxylation of methane and related small alkanes. In these calculations, we considered three of the most probable models for the transition metal active site mediating the “oxo-transfer”: (i) the trinuclear copper cluster bis(μ 3-oxo)trinuclear copper(II, II, III) complex 1 , recently proposed by Chan et al. [S.I. Chan, K.H.-C. Chen, S.S.-F. Yu, C.-L. Chen, S.S.-J. Kuo, Biochemistry 43 (2004) 4421–4430.]; (ii) the most frequently used model complex, bis(μ-oxo)Cu(III) 2 complex 2 ; and (iii) the mixed-valence bis(μ-oxo)Cu(II)Cu(III) complex 3 . The results obtained indicate that the methane hydroxylation chemistry mediated by the trinuclear copper cluster bis(μ 3-oxo)trinuclear copper(II, II, III) complex 1 offers the most facile pathway for methane hydroxylation, and this model yields KIE values that are in good agreement with experiment. In this mechanism, the reaction proceeds along a “singlet” potential surface and a “singlet oxene” is directly inserted across a C H bond in a concerted manner. Kinetic isotope effects ( k H/ k D or KIE) associated with the concerted oxene insertion process mediated by complex 1 are calculated to be 5.2 at 300 K when tunneling effects are included. Overall rate constants for the methane hydroxylation by the three models have been calculated as a function of temperature, and the rates are at least 5–6 orders of magnitude more facile when the chemistry is mediated by complex 1 compared to complex 2 or complex 3 .

One- and Two-Dimensional Coordination Polymers Constructed from Bicapped Keggin Mixed Molybdenum−Vanadium Heteropolyoxoanions and Polynuclear Copper(I) Clusters Bridged by Asymmetrical Bipyridine (2,4‘-bipy and 2,3‘-bipy) Ligands

Two novel trimetallic coordination polymers containing bridging bicapped Keggin mixed Mo/V polyoxoanions and asymmetrical aromatic amine bridging ligands 2,3‘-bipyridine (2,3‘-bipy) or 2,4‘-bipyridine (2,4‘-bipy), [CuI6(2,3‘-bipy)6(2,3‘-bipy-2‘-O)2][VIV2MoV5MoVI7O38(PO4)] [2,3‘-bipy-2‘-OH = 3-(pyridin-2-yl)pyridin-2-ol] (1) and [CuI5(2,4‘-bipy)6(OH)][PMoVI8VV3VIVO40(VIVO)2]·1.5H2O (2), have been prepared under hydrothermal conditions and structurally characterized by single-crystal X-ray diffraction. Gillard type hydration for the 2,3‘-bipy ligand was observed during synthesis of 1, which happens on the 2‘-carbon atom rather than on the α-carbon atom of the coordinated nitrogen atom in the 2,3‘-bipy ligand. Complex 1 is constructed from an alternatively arranged bivanadyl capped α-Keggin polymolybdate anion [VIV2MoV5MoVI7O38(PO4)]4- and double 2,3‘-bipy ligand bridged trinuclear copper(I) cluster [CuI3(2,3‘-bipy)3(2,3‘-bipy-2‘-O)]2+, forming a one-dimensional (1D) ribbon structure. The two-dimensional (2D) extended layer structure of 2 is composed of another type of bicapped Keggin mixed molybdenum−vanadium polyoxoanion [PMoVI8VV3VIVO40(VIVO)2]4- and copper(I) coordination polymer cation chain [CuI5(2,4‘-bipy)6(OH)]4+; the latter consists of a 2,4‘-bipy ligand bridged hexanuclear copper(I) cluster [CuI5(2,4‘-bipy)6]5+ linked by double hydroxyl groups.

A Combined Quantum and Molecular Mechanical Study of the O2 Reductive Cleavage in the Catalytic Cycle of Multicopper Oxidases

The four-electron reduction of dioxygen to water in multicopper oxidases takes place in a trinuclear copper cluster, which is linked to a mononuclear blue copper site, where the substrates are oxidized. Recently, several intermediates in the catalytic cycle have been spectroscopically characterized, and two possible structural models have been suggested for both the peroxy and native intermediates. In this study, these spectroscopic results are complemented by hybrid quantum and molecular mechanical (QM/MM) calculations, taking advantage of recently available crystal structures with a full complement of copper ions. Thereby, we obtain optimized molecular structures for all of the experimentally studied intermediates involved in the reductive cleavage of the O(2) molecule and energy profiles for individual reaction steps. This allows identification of the experimentally observed intermediates and further insight into the reaction mechanism that is probably relevant for the whole class of multicopper oxidases. We suggest that the peroxy intermediate contains an O(2)(2-) ion, in which one oxygen atom bridges the type 2 copper ion and one of the type 3 copper ions, whereas the other one coordinates to the other type 3 copper ion. One-electron reduction of this intermediate triggers the cleavage of the O-O bond, which involves the uptake of a proton. The product of this cleavage is the observed native intermediate, which we suggest to contain a O(2-) ion coordinated to all three of the copper ions in the center of the cluster.

The Copper Clusters in the Particulate Methane Monooxygenase (pMMO) from Methylococcus Capsulatus (Bath)

AbstractThe particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) is a multi‐copper protein that hydroxylates methane and other small n‐alkanes to their corresponding alcohols with high regiospecificity and stereoselectivity. The copper ions appear to be arranged into ˜5 trinuclear copper clusters. Two of these clusters are thought to be involved in the dioxygen chemistry and alkane hydroxylation mediated by the enzyme. Accordingly, these catalytic clusters (C‐clusters) are typically oxidized, as the protein is isolated. The remaining copper ions are normally reduced, and it has been suggested that they provide a reservoir of reducing equivalents needed for the turnover of the enzyme. In this study, we have oxidized the protein to different levels of oxidation of the copper ions using dioxygen, hydrogen peroxide, ferricyanide, and dioxygen in the presence of the suicide substrate acetylene, and have characterized the oxidized copper centers using low temperature electron paramagnetic resonance (EPR) and X‐ray absorption spectroscopy. The results are consistent with the grouping of the copper ions into catalytic clusters and electron transfer clusters (E‐clusters). Quantification indicates that there are, indeed, two C‐clusters. A model in which the two C‐clusters are activated by dioxygen starting from their fully reduced states and mediate the hydroxylation chemistry of methane will be presented and discussed.

Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity.

Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft. In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain. This protein is representative of a new family of enzymes--the two-domain laccases. Disruption of the corresponding gene abrogates laccase activity in the growth media. We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it. The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact. We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family. The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment. The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH. SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules.

Structure and magnetism of two new linear trinuclear copper(II) clusters obtained from the tetradentate N 2O 2 ligand bis(2-hydroxybenzyl)-1,3-diaminopropane

Reaction of bis(2-hydroxybenzyl)-1,3-diaminopropane (H 2bhbd) with copper(II) perchlorate and copper(II) chloride in methanol, respectively, leads to linear trinuclear clusters, namely [Cu 3(bhbd) 2(CH 3OH) 2(ClO 4) 2] ( 1) and [Cu 3(bhbd) 2Cl 2](CH 3OH) 4 ( 2). These coordination compounds were characterized by X-ray crystallography, UV–Vis, IR and EPR spectroscopy, and magnetic susceptibility measurements. Both complexes have a linear trinuclear array of copper ions bridged by means of phenolato O atoms and separated by a distance of 2.985(4) Å ( 1) and 2.937(4) Å ( 2). Strong antiferromagnetic interactions between these adjacent Cu II ions govern the magnetochemistry of 1 ( J = −303(1) cm −1) and 2 ( J = −482(3) cm −1) resulting in S = 1/2 ground states fully populated below 150 K. A correlation between the interaction parameter J and the angles within the trinuclear clusters is proposed.

The Structure of Rigidoporus lignosus Laccase Containing a Full Complement of Copper Ions, Reveals an Asymmetrical Arrangement for the T3 Copper Pair

Laccase is a multicopper blue oxidase that couples the four-electron reduction of oxygen with the oxidation of a broad range of organic substrates, including phenols and arylamines. The enzyme is the object of intense biotechnological research, due to its employment in bioremediation of soils and water as well as in other biotechnological applications. We report here the cDNA and protein sequences, the post-translational modifications, the crystallization and X-ray structure determination of a laccase from the white-rot fungus Rigidoporus lignosus. The amino acid residues sequence deduced from cDNA clearly identified a pre-sequence of 21 residues representing the signal for extra-cellular localization. Mass spectrometry analysis performed on the salvage enzyme, confirmed the deduced sequence and precisely mapped two glycosylation sites at Asn337 and Asn435, determining the nature of the bound glycosidic moieties. The crystal structure was determined at 1.7A resolution from perfectly hemihedrally twinned crystals, by molecular replacement technique. While the overall structure closely resembled those reported for other fungal laccases, the analysis of the T2/T3 trinuclear cluster revealed an unprecedented coordination sphere for the T3 copper pair. No bridging oxygen ligand was present between the two T3 copper ions, which were no longer symmetrically coordinated. The observed structure could represent an intermediate along the process of four-electron reduction of oxygen to water taking place at the trinuclear copper cluster.

Toward delineating the structure and function of the particulate methane monooxygenase from methanotrophic bacteria.

The particulate methane monooxygenase (pMMO) is a complex membrane protein complex that has been difficult to isolate and purify for biochemical and biophysical characterization because of its instability in detergents used to solubilize the enzyme. In this perspective, we summarize the progress recently made toward obtaining a purified pMMO-detergent complex and characterizing the enzyme in pMMO-enriched membranes. The purified pMMO is a multi-copper protein, with ca. 15 copper ions sequestered into five trinuclear copper clusters: two for dioxygen chemistry and alkane hydroxylation (catalytic or C-clusters) and three to provide a buffer of reducing equivalents to re-reduce the C-clusters following turnover (electron transfer or E-clusters). The enzyme is functional when all the copper ions are reduced. When the protein is purified under ambient aerobic conditions in the absence of a hydrocarbon substrate, only the C-clusters are oxidized; there is an apparent kinetic barrier for electron transfer from the E-cluster copper ions to the C-clusters under these conditions. Evidence is provided in support of both C-clusters participating in the dioxygen chemistry, but only one C-cluster supporting alkane hydroxylation. Acetylene modification of the latter C-cluster in the hydrophobic pocket of the active site lowers or removes the kinetic barrier for electron transfer from the E-clusters to the C-clusters so that all the copper ions could be fully oxidized by dioxygen. A model for the hydroxylation chemistry when a hydrocarbon substrate is bound to the active site of the hydroxylation C-cluster is presented. Unlike soluble methane monooxygenase (sMMO), pMMO exhibits limited substrate specificity, but the hydroxylation chemistry is highly regioselective and stereoselective. In addition, the hydroxylation occurs with total retention of configuration of the carbon center that is oxidized. These results are consistent with a concerted mechanism involving direct side-on insertion of an active singlet "oxene" from the activated copper cluster across the "C-H" bond in the active site. Finally, in our hands, both the purified pMMO-detergent complex and pMMO-enriched membranes exhibit high NADH-sensitive as well as duroquinol-sensitive specific activity. A possible role for the two reductants in the turnover of the enzyme is proposed.

Models for biological trinuclear copper clusters. Characterization and enantioselective catalytic oxidation of catechols by the copper(II) complexes of a chiral ligand derived from (S)-(-)-1,1'-binaphthyl-2,2'-diamine.

The dinuclear and trinuclear Cu(II) complexes of an octadentate ligand derived from (S)-1,1'-binaphthyl-2,2'-diamine have been prepared and characterized by UV/Vis, CD, EPR and NMR spectroscopy. The ligand contains two tridentate aminobis(benzimidazole) donor arms connected to a central bidentate diaminobinaphthyl linker, which hosts the chiral unit. In the dinuclear Cu complex the ligation occurs essentially within the tridentate arms of the ligand. The two Cu centers are EPR nonequivalent and noninteracting. The EPR data suggests that one of the Cu ions additionally interacts with one of the tertiary aminonaphthyl donors. In the trinuclear complex the two aminonaphthyl donors bind the third Cu ion. The EPR spectrum of this complex shows the signal for a mononuclear Cu(II) center bound to a tridentate arm, while the remaining two Cu(II) centers are coupled through hydroxo groups. The CD spectrum shows that in the free ligand a severe reduction of the dihedral angle between the naphthyl groups from the strain free range occurs. This conformation is stabilized by ring stacking interactions with the benzimidazole groups. On complex formation this interaction is removed because the benzimidazole groups are involved in metal binding. In the dinuclear Cu complex the conformation of the binaphthyl chromophore probably approaches the strain free range, while in the trinuclear Cu complex a marked flattening of the dihedral angle between the two naphthyl rings occurs. Both complexes are active catalysts in the oxidation of L-/D-Dopa derivatives to quinones. High enantioselectivity is observed in the oxidation of L-/D-Dopa methyl ester catalyzed by the dinuclear Cu complex, which exhibits strong preference for the d enantiomer. The enantioselectivity is largely lost for the trinuclear Cu complex.

Sequential reconstitution of copper sites in the multicopper oxidase CueO.

CueO belongs to the family of multicopper oxidases which are characterized by the presence of multiple copper-binding sites with different structural and functional properties. These enzymes share the ability to couple the one-electron oxidation of substrate to reduction of oxygen to water by way of a functional unit composed of a mononuclear type 1 blue copper site, which is the entry site for electrons, and of a trinuclear copper cluster formed by type 2 and binuclear type 3 sites, where oxygen binding and reduction take place. The mechanism of copper incorporation in CueO has been investigated by optical and EPR spectroscopy. The results indicate unambiguously that the process is sequential, with type 1 copper being the first to be reconstituted, followed by type 2 and type 3 sites.

Crystal Structure of a Laccase from the FungusTrametes versicolor at 1.90-Å Resolution Containing a Full Complement of Coppers

Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccases oxidize a broad range of substrates, preferably phenolic compounds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Until now, only one crystal structure of a laccase in an inactive, type-2 copper-depleted form has been reported. We present here the first crystal structure of an active laccase containing a full complement of coppers, the complete polypeptide chain together with seven carbohydrate moieties. Despite the presence of all coppers in the new structure, the folds of the two laccases are quite similar. The coordination of the type-3 coppers, however, is distinctly different. The geometry of the trinuclear copper cluster in the Trametes versicolor laccase is similar to that found in the ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for the copper oxidation and the O(2) reduction. In contrast to most blue copper proteins, the type-1 copper in the T. versicolor laccase has no axial ligand and is only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.

A novel density functional study of the ground state properties of a localized trinuclear copper(II,II,III) mixed-valence system.

Herein we report the analysis of a mixed-valence localized trinuclear copper(II,II,III) cluster by density functional theory. We focused on two peculiar aspects of this system. First we investigated the triplet ground state potential energy surface on a model system. To this end we computed, on the [E multiply sign in circle e] adiabatic potential energy surface, the potential energy profile along the e mode and constructed ab initio the full potential energy surface (the so called Mexican hat), by a fitting procedure. Next, we analyzed the magnetooptical properties of the minimum energy structures. In particular, we applied the single determinant method to compute the full manifold of states arising from the highest occupied molecular orbitals (magnetic orbitals). This procedure yielded results in agreement with previous calculations and with the available experimental data when using a model closer to the X-ray structure or when directly dealing with the complete structure of the system.

Nature of the intermediate formed in the reduction of O(2) to H(2)O at the trinuclear copper cluster active site in native laccase.

The multicopper oxidases contain at least four copper atoms and catalyze the four-electron reduction of O(2) to H(2)O at a trinuclear copper cluster. An intermediate, termed native intermediate, has been trapped by a rapid freeze-quench technique from Rhus vernicifera laccase when the fully reduced form reacts with dioxygen. This intermediate had been described as an oxygen-radical bound to the trinuclear copper cluster with one Cu site reduced. XAS, however, shows that all copper atoms are oxidized in this intermediate. A combination of EXAFS, multifrequency EPR, and VTVH MCD has been used to understand how this fully oxidized trinuclear Cu cluster relates to the fully oxidized resting form of the enzyme. It is determined that in the native intermediate all copper atoms of the cluster are bridged by the product of full O(2) reduction. In contrast, the resting form has one copper atom of the cluster (the T2 Cu) magnetically isolated from the others. The native intermediate decays to the resting oxidized form with a rate that is too slow to be in the catalytic cycle. Thus, the native intermediate appears to be the catalytically relevant fully oxidized form of the enzyme, and its role in catalysis is considered.

Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli.

CueO (YacK), a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli. The crystal structure of CueO has been determined to 1.4-A resolution by using multiple anomalous dispersion phasing and an automated building procedure that yielded a nearly complete model without manual intervention. This is the highest resolution multicopper oxidase structure yet determined and provides a particularly clear view of the four coppers at the catalytic center. The overall structure is similar to those of laccase and ascorbate oxidase, but contains an extra 42-residue insert in domain 3 that includes 14 methionines, nine of which lie in a helix that covers the entrance to the type I (T1, blue) copper site. The trinuclear copper cluster has a conformation not previously seen: the Cu-O-Cu binuclear species is nearly linear (Cu-O-Cu bond angle = 170 degrees) and the third (type II) copper lies only 3.1 A from the bridging oxygen. CueO activity was maximal at pH 6.5 and in the presence of >100 microM Cu(II). Measurements of intermolecular and intramolecular electron transfer with laser flash photolysis in the absence of Cu(II) show that, in addition to the normal reduction of the T1 copper, which occurs with a slow rate (k = 4 x 10(7) M(-1)x (-1)), a second electron transfer process occurs to an unknown site, possibly the trinuclear cluster, with k = 9 x 10(7) M(-1) x (-1), followed by a slow intramolecular electron transfer to T1 copper (k approximately 10 s(-1)). These results suggest the methionine-rich helix blocks access to the T1 site in the absence of excess copper.

Fast reduction of a copper center in laccase by nitric oxide and formation of a peroxide intermediate.

The rapid reduction of one of the copper atoms (type 2) of tree laccase by nitric oxide (NO) has been detected. Addition of NO to native laccase in the presence of oxygen leads to EPR changes consistent with fast reduction and slow reoxidation of this metal center. These events are paralleled by optical changes that are reminiscent of formation and decay of the peroxide intermediate in a fraction of the enzyme population. Formation of this species is only possible if the trinuclear copper cluster (type 2 plus type 3) is fully reduced. This condition can only be met if, as suggested previously, a fraction of the enzyme contains both type 3 coppers already reduced before addition of NO. Our data are consistent with this assumption. We have suggested recently that fast reduction of copper is the mechanism by which NO interacts with the oxidized dinuclear center in cytochrome c oxidase. The present experiments using laccase strongly support this view and suggest this reaction as a general mechanism by which copper proteins interact with NO. In addition, this provides an unexploited way to produce a stable peroxide intermediate in copper oxidases in which the full complement of copper atoms is present. This enables the O-O scission step in the catalytic cycle to be studied by electron addition to the peroxide derivative through the native electron entry site, type 1 copper.

Temperature-induced changes in copper centers and protein conformation of two fungal laccases from Coriolus hirsutus and Coriolus zonatus

The paper reports on two fungal laccases from Coriolus hirsutus and Coriolus zonatus and their type-2 copper-depleted derivatives. Temperature-induced changes of the copper centers were characterized by optical and electron paramagnetic resonance (EPR) spectroscopy, and the overall protein stability by differential scanning microcalorimetry. The intact enzymes showed highly cooperative thermal unfolding transitions at about 90°C. Type-2 copper depletion led to uncoupling of the domains characterized by a different melting pattern which resolved three subtransitions. Melting curves monitored optically at 290, 340 and 610 nm showed additional transitions below thermal unfolding temperature. EPR spectra of the intact laccases showed the disintegration of the trinuclear copper cluster accompanied by loss of one of the copper ions and disappearance of the strong antiferromagnetic coupling in the type-3 site at 70°C and above 70°C. The copper centers of type-2 copper-depleted laccase showed reduced thermotolerance.

Role of iron and copper in particulate methane monooxygenase of Methylosinus trichosporium OB3b

The role of iron and copper in particulate methane monooxygenase (pMMO) of Methylosinus trichosporium OB3b is described, and an overview of the enzyme's properties is presented. The pMMO from M. trichosporium OB3b was solubilized in the detergent n-dodecyl-β-D-maltoside and purified by chromatographic techniques. The enzyme consists of 0.9 iron atoms and 12.8 copper atoms per molecule. The iron site in pMMO may be mononuclear non-heme iron. Copper exists as either copper ion coupled to four nitrogen atoms and/or trinuclear copper cluster wherein copper ions are ferromagnetically coupled.

Homology modeling of the multicopper oxidase Fet3 gives new insights in the mechanism of iron transport in yeast.

Fet3, the multicopper oxidase of yeast, oxidizes extracellular ferrous iron which is then transported into the cell through the permease Ftr1. A three-dimensional model structure of Fet3 has been derived by homology modeling. Fet3 consists of three cupredoxin domains joined by a trinuclear copper cluster which is connected to the blue copper site located in the third domain. Close to this site, which is the primary electron acceptor from the substrate, residues for a potential iron binding site could be identified. The surface disposition of negatively charged residues suggests that Fet3 can translocate Fe(3+) to the permease Ftr1 through a pathway under electrostatic guidance.

Structure/function relationships in ceruloplasmin.

Ceruloplasmin belongs to the family of multinuclear copper oxidases, which also includes ascorbate oxidase and laccase (Solomon et al., 1996). These proteins are trinuclear copper cluster enzymes, as documented by spectroscopic and functional studies in solution for laccase (Aliendorf et al., 1985; Spira-Solomon et al., 1986) and ceruloplasmin (Calabrese et al., 1988, 1989), and recently confirmed by X-ray crystallography for ascorbate oxidase (Messerschmidt et al., 1992) and ceruloplasmin (Zaitseva et al., 1996). Multiple copper sites are present, with a minimal functional unit constituted by a blue, or type 1, copper site, and three copper atoms clustered in a trinuclear arrangement. The blue copper site takes up an electron from a reducing substrate and transfers it, through long-range intramolecular electron transfer mediated by a Cys-His pathway, to the trinuclear cluster (Fig. 1), which provides four electrons to reduce oxygen to water. Among multicopper oxidases, ceruloplasmin is presently unique in that it possesses additional copper sites beside the four ions of the minimal unit. The three dimensional structure of ceruloplasmin solved at 3 A resolution has shown that the enzyme is made up of six α-barrel domains arranged in a ternary simmetry (Zaitseva et al., 1996). Consistent with spectroscopic data (Wever et al., 1973, Musci et al., 1993), three of these domains (domains 2, 4 and 6) bind type 1 copper ions coordinated by nitrogen and sulphur ligands. Type 1 copper ions bound to domain 4 and 6 (hereafter referred to as Cu4 and Cu6) are coordinated by 2 histidines, a cysteine and a methionine. Type 1 copper bound to domain 2 (Cu2) should be preferably reduced (Musci et al., 1995; Solomon et al., 1996) since the axial methionine is replaced by leucine, in a geometry typical of a high redox potential copper site. The three copper ions of the trinuclear cluster lie at the interface between domain 1 and 6 and are coordinated by 8 histidine ligands and an oxygen ligand (OH or H2O) bridging two of them. Labile metal binding sites have also been observed on domains 4 and 6, close to the respective prosthetic blue sites. Domain 2 appears different also in this respect, since it lacks the proper ligands for the corresponding labile site.

Synthetic models for biological trinuclear copper clusters. Trinuclear and binuclear complexes derived from an octadentate tetraamine-tetrabenzimidazole ligand

The new tetraamino-tetrabenzimidazole ligand N,N′{bis[3-(1-methyl-2-benzimidazolyl]amino}piperazine (L) has been synthesised together with a series of trinuclear and binuclear complexes. Two terminal binding sites with tridentate linkages (A sites) and one central binding site with the bidentate piperazine residue (B site) are used by the ligand to bind divalent metal centres in the trinuclear complexes [CuH3L]6+, [CuH2ZnHL]6+, and [CuH2CoHL]6+. In the binuclear complex [CuH2L]4+ each nitrogen donor of the piperazine rine acts as an axial ligand for the two coppers bound to A sites, but these piperazine donors can be easily displaced by protonation to form the [CuH2LH2)]6+ species. The structure of this protonated complex has been determined by X-ray analysis. The crystals of composition [CuH2(LH2)(CH3CN)4][ClO4]6·2H2O·3CH3CN belong to the monoclinic system. space group P21/n with cell parametrs a = 10.661 (3), b = 23.014(3), c = 17.217(4)Å, β = 96.58(2)°, Z = 2. The citation is arranged on a crystallographic symmetry centre, which is located at the middle of the protonated piperazine ring. The protonation at the piperazine N atoms is supported by the total charge of the cation and by the analysis of the difference Fourier map. The copper ions are five-coordinated, with ligation of the two benzimidazole residues and the tertiary N donor in the basal plane of a distorted square pyramid. Two CH3CN molecules, one at the basal, the other at the apical position, complete the coordination polyhedron. The centrosymmetric spacer, between the tertiary N atoms, deviates from the higher C2h symmetry, so that the two approximately planar N(CH2)3 groupings lie in two parallel planes. Complexes containing reduced copper ions, [CuH2L2+ and [CuH2CuIIL]4+, have also been obtained, but these ions do not bind to the piperazine B site which can only be used to coordinate divalent metal ions. The complexes containing Cu(II) centres exhibit EPR signals indicative of mononuclear species with tetragonal symmetry. The different coordination environment of [CuH2L]4+ with respect to [CuH2ZnHL]6+, [CuH2CoHL]6+ or [CuH2(LH2]6+ is reflected by a difference in the magnetic parameters of the complexes. The EPR spectrum of [CuH3L]6+ is very similar to those of [CuH2ZnHL]6+, typical for Cu(II)-A sites, but the integrated intensity accounts for only about 2.2 paramagnetic centres per molecule. It is likely that a dipolar interaction between one of the Cu(II)-A centres and the Cu(II)-B centre produces severe broadening of the corersponding signals and apparent reduction in the overall EPR intensity. Voltammetric data in acetonitrile solution exhibit quasi-reversible electron transfer for the Cu(II)/Cu(II) couples with estimated reduction potentials in the range 0.39–0.56 V versus NHE. The voltammogram of [CuH3L]6+ run at low concentration and sweep rate shows that the three-electron transfer in split into one-electron and two-electron steps. Binding experiments show that the complexes bind azide molecules in the terminal mode to the copper (II) centres with affinity constants decreasing in the series: [CuH3L]6+ > [CuH2L]4+. The complexes derived from L are catalytically active in the air oxidation of di-tert-butylcatechol (DTBC). The oxidations are biphasic, with a fast initial stoichiometric phae corresponding to reduction of a pair of copper(II) centres and oxidation of DTBC to quinone followed by the catalytic reaction. The catalytic reaction follows substrate saturation behaviour, with kinetic constants decreasing in the order: [CuH3L6+ > [CuH2CoHL]6+ > [CuH2ZnHL]6+ ≈ [CuH2L]4+. Anaerobic experiments show that the two-electron oxidation of DTBC involves reduction of one Cu(II)-A site and the Cu(II)-B site for [CuH3L]6+, both the Cu(II)-A sites of CuH2ZnHL]6+ and [CuH2K]4+, but one Cu(II)-A and Co(III) in the case of the mixed [CuH2Co11L]6+ complex, since the Co(II) ion is rapidly oxidised to Co(III) in the conditions in which the catalytic reaction is carried out.