Dicopper Site Research Articles

Possible Peroxo State of the Dicopper Site of Particulate Methane Monooxygenase from Combined Quantum Mechanics and Molecular Mechanics Calculations.

Enzymatic methane hydroxylation is proposed to efficiently occur at the dinuclear copper site of particulate methane monooxygenase (pMMO), which is an integral membrane metalloenzyme in methanotrophic bacteria. The resting state and a possible peroxo state of the dicopper active site of pMMO are discussed by using combined quantum mechanics and molecular mechanics calculations on the basis of reported X-ray crystal structures of the resting state of pMMO by Rosenzweig and co-workers. The dicopper site has a unique structure, in which one copper is coordinated by two histidine imidazoles and another is chelated by a histidine imidazole and primary amine of an N-terminal histidine. The resting state of the dicopper site is assignable to the mixed-valent Cu(I)Cu(II) state from a computed Cu-Cu distance of 2.62 Å from calculations at the B3LYP-D/TZVP level of theory. A μ-η(2):η(2)-peroxo-Cu(II)2 structure similar to those of hemocyanin and tyrosinase is reasonably obtained by using the resting state structure and dioxygen. Computed Cu-Cu and O-O distances are 3.63 and 1.46 Å, respectively, in the open-shell singlet state. Structural features of the dicopper peroxo species of pMMO are compared with those of hemocyanin and tyrosinase and synthetic dicopper model compounds. Optical features of the μ-η(2):η(2)-peroxo-Cu(II)2 state are calculated and analyzed with TD-DFT calculations.

Correlating the structures and activities of the resting oxidized and native intermediate states of a small laccase by paramagnetic NMR

Multicopper oxidases (MCO) are the fastest and most efficient known catalysts of the oxygen-reduction reaction. When all four copper ions are oxidized during catalysis, the native intermediate state (NI) decays in seconds to the resting oxidized state (RO), which returns to the catalytic cycle via reduction, but at a much slower rate than NI. We report the long-lived (months at 4°C) NI state of the small laccase (SLAC) MCO and the subsequent characterization of both its RO and NI states by paramagnetic 1H NMR. We find that the RO state of the trinuclear cluster (TNC) is best described as an isolated Type-3 dicopper site, antiferromagnetically coupled by a hydroxo group with −2J=500cm−1. The NI state is more complicated; we develop a theoretical treatment for the case in which all three copper ions in the TNC are coupled, and find that the results are consistent with three coupling constants of −2J=300, 240, and 160cm−1. These couplings result in a ground doublet state, a low-lying excited doublet state at 121cm−1, and a quartet excited state at 411cm−1, in good agreement with DFT models in which the Type-2 copper has a terminal hydroxo ligand.

Dimetallic functionalities in liposome bilayers

A dicopper(II) complex can be covalently linked to palmitate/palmitoyl-oleoyl-phosphatidylcholine (PA/POPC) liposomes using the following one-pot strategy: preformed [Cu2(bpbp)(PA)](ClO4)2 (bpbp− = 2,6-bis((N,N′-bis(2-picolyl)amino)methyl)-4-tertbutylphenolato) was incorporated into POPC liposomes with a loading of up to 10 mol%. Despite its shape and charge, the decoration of PA/POPC liposomes with {Cu2(bpbp)}3+ did not disrupt the liposome structure; however, the mean liposome diameter increased from about 130 nm (0 mol% dicopper complex) to about 150 nm (10 mol% dicopper complex). Single-crystal X-ray structures furnish ‘snapshots’ of the pH-dependent solution state derivatives of {Cu2(bpbp)}3+, and model the structure of the [Cu2(bpbp)(PA)]2+ head group at the surface of the liposomes. An impressive plasticity in the intramolecular non-bonded Cu….Cu distance for these ions, ranging from 3 to 4 Å, in [Cu2(bpbp)OH]2+, [Cu2(bpbp)(OAc)(H2O)]2+ and [Cu2(bpbp)(H2O)2]3+ allows for their utility as labile reagents in water. Remarkably, the flexible dicopper site is selective for a single carboxylate ligand, so that [Cu2(bpbp)(PA)]2+ is favoured even in the presence of other chemically similar oxoanions, such as CO32 − , HCO3− , NO3− , ClO4− , ReO4− and CF3SO3− .

Chemical Plausibility of Cu(III) with Biological Ligation in pMMO.

The mechanisms of dioxygen activation and methane C-H oxidation in particulate methane monooxygenase (pMMO) are currently unknown. Recent studies support a binuclear copper site as the catalytic center. We report the low-temperature assembly of a high-valent dicopper(III) bis(μ-oxide) complex bearing marked structural fidelity to the proposed active site of pMMO. This unprecedented dioxygen-bonded Cu(III) species with exclusive biological ligation directly informs on the chemical plausibility and thermodynamic stability of the bis(μ-oxide) structure in such dicopper sites and foretells unusual optical signatures of an oxygenation product in pMMO. Though the ultimate pMMO active oxidant is still debated, C-H oxidation of exogenous substrates is observed with the reported Cu(III) complexes. The assembly of a high valent species both narrows the search for relevant pMMO intermediates and provides evidence to substantiate the role of Cu(III) in biological redox processes.

Discrimination of the prochiral hydrogens at the C-2 position of n-alkanes by the methane/ammonia monooxygenase family proteins.

The selectivity of ammonia monooxygenase from Nitrosomonas europaea (AMO-Ne) for the oxidation of C4-C8n-alkanes to the corresponding alcohol isomers was examined to show the ability of AMO-Ne to recognize the n-alkane orientation within the catalytic site. AMO-Ne in whole cells produces 1- and 2-alcohols from C4-C8n-alkanes, and the regioselectivity is dependent on the length of the carbon chain. 2-Alcohols produced from C4-C7n-alkanes were predominantly either the R- or S-enantiomers, while 2-octanol produced from n-octane was racemic. These results indicate that AMO-Ne can discriminate between the prochiral hydrogens at the C-2 position, with the degree of discrimination varying according to the n-alkane. Compared to the particulate methane monooxygenase (pMMO) of Methylococcus capsulatus (Bath) and that of Methylosinus trichosporium OB3b, AMO-Ne showed a distinct ability to discriminate between the orientation of n-butane and n-pentane in the catalytic site.

A Ferromagnetically Coupled (S=1) Peroxodicopper(II) Complex

AbstractCopper enzymes play important roles in the binding and activation of dioxygen in biological systems. Key copper/dioxygen intermediates have been identified and studied in synthetic analogues of the metalloprotein active sites, including the μ‐η2:η2‐peroxodicopper(II) motif relevant to type III dicopper proteins. Herein, we report the synthesis and characterization of a bioinspired dicopper system that forms a stable μ‐η1:η1‐peroxo complex whose Cu‐O‐O‐Cu torsion is constrained to around 90° by ligand design. This results in sizeable ferromagnetic coupling between the copper(II) ions, which is detected by magnetic measurements and HF‐EPR spectroscopy. The new dicopper peroxo system is the first with a triplet ground state, and it represents a snapshot of the initial stages of O2 binding at type III dicopper sites.

A Ferromagnetically Coupled (S=1) Peroxodicopper(II) Complex

Copper enzymes play important roles in the binding and activation of dioxygen in biological systems. Key copper/dioxygen intermediates have been identified and studied in synthetic analogues of the metalloprotein active sites, including the μ-η(2):η(2)-peroxodicopper(II) motif relevant to type III dicopper proteins. Herein, we report the synthesis and characterization of a bioinspired dicopper system that forms a stable μ-η(1):η(1)-peroxo complex whose Cu-O-O-Cu torsion is constrained to around 90° by ligand design. This results in sizeable ferromagnetic coupling between the copper(II) ions, which is detected by magnetic measurements and HF-EPR spectroscopy. The new dicopper peroxo system is the first with a triplet ground state, and it represents a snapshot of the initial stages of O2 binding at type III dicopper sites.

Identification of the valence and coordination environment of the particulate methane monooxygenase copper centers by advanced EPR characterization.

Particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol in methanotrophic bacteria. As a copper-containing enzyme, pMMO has been investigated extensively by electron paramagnetic resonance (EPR) spectroscopy, but the presence of multiple copper centers has precluded correlation of EPR signals with the crystallographically identified monocopper and dicopper centers. A soluble recombinant fragment of the pmoB subunit of pMMO, spmoB, like pMMO itself, contains two distinct copper centers and exhibits methane oxidation activity. The spmoB protein, spmoB variants designed to disrupt one or the other or both copper centers, as well as native pMMO have been investigated by EPR, ENDOR, and ESEEM spectroscopies in combination with metal content analysis. The data are remarkably similar for spmoB and pMMO, validating the use of spmoB as a model system. The results indicate that one EPR-active Cu(II) ion is present per pMMO and that it is associated with the active-site dicopper center in the form of a valence localized Cu(I)Cu(II) pair; the Cu(II), however, is scrambled between the two locations within the dicopper site. The monocopper site observed in the crystal structures of pMMO can be assigned as Cu(I). 14N ENDOR and ESEEM data are most consistent with one of these dicopper-site signals involving coordination of the Cu(II) ion by residues His137 and His139, the other with Cu(II) coordinated by His33 and the N-terminal amino group. 1H ENDOR measurements indicate there is no aqua (HxO) ligand bound to the Cu(II), either terminally or as a bridge to Cu(I).

Mutagenesis of the hydrocarbon monooxygenase indicates a metal centre in subunit-C, and not subunit-B, is essential for copper-containing membrane monooxygenase activity

The hydrocarbon monooxygenase (HMO) of Mycobacterium NBB4 is a member of the copper-containing membrane monooxygenase (CuMMO) superfamily, which also contains particulate methane monooxygenases (pMMOs) and ammonia monooxygenases (AMOs). CuMMOs have broad applications due to their ability to catalyse the oxidation of difficult substrates of environmental and industrial relevance. Most of our understanding of CuMMO biochemistry is based on pMMOs and AMOs as models. All three available structures are from pMMOs. These share two metal sites: a dicopper centre coordinated by histidine residues in subunit-B and a 'variable-metal' site coordinated by carboxylate and histidine residues from subunit-C. The exact nature and role of these sites is strongly debated. Significant barriers to progress have been the physiologically specialized nature of methanotrophs and autotrophic ammonia-oxidizers, lack of a recombinant expression system for either enzyme and difficulty in purification of active protein. In this study we use the newly developed HMO model system to perform site-directed mutagenesis on the predicted metal-binding residues in the HmoB and HmoC of NBB4 HMO. All mutations of predicted HmoC metal centre ligands abolished enzyme activity. Mutation of a predicted copper-binding residue of HmoB (B-H155V) reduced activity by 81 %. Mutation of a site that shows conservation within physiologically defined subgroups of CuMMOs was shown to reduce relative HMO activity towards larger alkanes. The study demonstrates that the modelled dicopper site of subunit-B is not sufficient for HMO activity and that a metal centre predicted to be coordinated by residues in subunit-C is essential for activity.

Access to a Cu(II)-O-Cu(II) motif: spectroscopic properties, solution structure, and reactivity.

We report a complex with a rare Cu(II)-O-Cu(II) structural motif that is stable at room temperature, which allows its in-depth characterization by a variety of spectroscopic methods. Interest in such compounds is fueled by the recent discovery that a Cu(II)-O-Cu(II) species on the surface of Cu-ZSM-5 is capable of oxidizing methane to methanol, and this in turn ties into mechanistic discussions on the methane oxidation at the dicopper site within the particulate methane monooxygenase. For the synthesis of our Cu2O complex we have developed a novel, neutral ligand system, FurNeu, exhibiting two N-(N',N'-dimethylaminoethyl)(2-pyridylmethyl)amino binding pockets connected by a dibenzofuran spacer. The reaction of FurNeu with CuCl yielded [FurNeu](Cu2(μ-Cl))(CuCl2), 1, demonstrating the geometric potential of the ligand to stabilize Cu-X-Cu moieties. A Cu(I) precursor with weakly coordinating anions was chosen in the next step, namely [Cu(NCCH3)4]OTf, which led to the formation of [FurNeu](Cu(NCCH3))2(OTf)2, 3. Treatment of 3 with O2 or PhIO led to identical green solutions, whose UV-vis spectra were markedly different from the one displayed by [FurNeu](Cu)2(OTf)4, 4, prepared independently from FurNeu and Cu(OTf)2. Further investigations including PhIO consumption experiments, NMR and UV-vis spectroscopy, HR-ESI mass spectrometry, and protonation studies led to the identification of the green product as [FurNeu](Cu2(μ-O))(OTf)2, 5. DOSY NMR spectroscopy confirmed its monomeric character. Over longer periods of time 5 decomposes to give [Cu(picoloyl)2], formed through an oxidative N-dealkylation reaction followed by further oxidation of the ligand. Due to its slow decomposition reaction, all attempts to crystallize 5 failed. However, its structure in solution could be determined by EXAFS analysis in combination with DFT calculations, which revealed a Cu-O-Cu angle that amounts to 105.17°. Moreover, TDDFT calculations helped to rationalize the UV-vis absorptions of 5. The reactivity of complex 5 with 2,4-di-tert-butylphenol, DTBP, was also investigated; the initially formed biphenol product, TBBP, was found to further react in the presence of excessive O2 to yield 2,4,7,9-tetra-tert-butyloxepino[2,3-b]benzofuran, TBOBF, via an intermediate diphenoquinone. It turned out that 5, or its precursor 3, can even be employed as a catalyst for the oxidation of DTBP to TBBP or for the oxidation of TBBP to TBOBF.

Role of Tyrosine Residue in Methane Activation at the Dicopper Site of Particulate Methane Monooxygenase: A Density Functional Theory Study

Methane hydroxylation at the dinuclear copper site of particulate methane monooxygenase (pMMO) is studied by using density functional theory calculations. The electronic, structural, and reactivity properties of a possible dinuclear copper species (μ-oxo)(μ-hydroxo)Cu(II)Cu(III) are discussed with respect to the C-H bond activation of methane. We propose that the tyrosine residue in the second coordination sphere of the dicopper site donates an H atom to the μ-η(2):η(2)-peroxoCu(II)Cu(II) species and the resultant (μ-oxo)(μ-hydroxo)Cu(II)Cu(III) species can hydroxylate methane. This species for methane hydroxylation is more favorable in reactivity than the bis(μ-oxo)Cu(III)Cu(III) species. The H-atom transfer or proton-coupled electron transfer from the tyrosine residue can reasonably induce the O-O bond dissociation of the μ-η(2):η(2)-peroxoCu(II)Cu(II) species to form the reactive (μ-oxo)(μ-hydroxo)Cu(II)Cu(III) species, which is expected to be an active species for the conversion of methane to methanol at the dicopper site of pMMO. The rate-determining step for the methane hydroxylation is the C-H cleavage, which is in good agreement with experimental KIE values reported so far.

Electrophilic Arene Hydroxylation and Phenol OH Oxidations Performed by an Unsymmetric μ‐η1:η1‐O2‐Peroxo Dicopper(II) Complex

Reactions of the unsymmetric dicopper(II) peroxide complex [Cu(II)(2)(μ-η(1):η(1)-O(2))(m-XYL(N3N4))](2+) (1 O(2), where m-XYL is a heptadentate N-based ligand), with phenolates and phenols are described. Complex 1 O(2) reacts with p-X-PhONa (X = MeO, Cl, H, or Me) at -90 °C performing tyrosinase-like ortho-hydroxylation of the aromatic ring to afford the corresponding catechol products. Mechanistic studies demonstrate that reactions occur through initial reversible formation of metastable association complexes [Cu(II)(2)(μ-η(1):η(1)-O(2))(p-X-PhO)(m-XYL(N3N4))](+) (1 O(2)⋅X-PhO) that then undergo ortho-hydroxylation of the aromatic ring by the peroxide moiety. Complex 1 O(2) also reacts with 4-X-substituted phenols p-X-PhOH (X = MeO, Me, F, H, or Cl) and with 2,4-di-tert-butylphenol at -90 °C causing rapid decay of 1 O(2) and affording biphenol coupling products, which is indicative that reactions occur through formation of phenoxyl radicals that then undergo radical C-C coupling. Spectroscopic UV/Vis monitoring and kinetic analysis show that reactions take place through reversible formation of ground-state association complexes [Cu(II)(2)(μ-η(1):η(1)-O(2))(X-PhOH)(m-XYL(N3N4))](2+) (1 O(2)⋅X-PhOH) that then evolve through an irreversible rate-determining step. Mechanistic studies indicate that 1 O(2) reacts with phenols through initial phenol binding to the Cu(2)O(2) core, followed by a proton-coupled electron transfer (PCET) at the rate-determining step. Results disclosed in this work provide experimental evidence that the unsymmetric 1 O(2) complex can mediate electrophilic arene hydroxylation and PCET reactions commonly associated with electrophilic Cu(2)O(2) cores, and strongly suggest that the ability to form substrate⋅Cu(2)O(2) association complexes may provide paths to overcome the inherent reactivity of the O(2)-binding mode. This work provides experimental evidence that the presence of a H(+) completely determines the fate of the association complex [Cu(II)(2)(μ-η(1):η(1)-O(2))(X-PhO(H))(m-XYL(N3N4))](n+) between a PCET and an arene hydroxylation reaction, and may provide clues to help understand enzymatic reactions at dicopper sites.

Phosphatase reactivity of a dicopper(ii) complex of a patellamide derivative – possible biological functions of cyclic pseudopeptides

A possible biological function of cyclic pseudo-octapeptides is presented. The dinuclear copper(II) complex of a synthetic analogue ([Cu(2)(H(2)Pat(1))(μ-OH)(OH(2))(2)]) of the naturally occurring ascidiacyclamide is known to have a hydroxo-bridged dicopper(II) site which is able to catalytically transform CO(2) into CO(3)(2-). This complex is shown here to function as a phosphatase mimic, suggesting that the so far unknown biological function of these macrocycles within the ascidians may involve phosphoester hydrolysis.

Theoretical studies of the magnetic couplings and the chemical indices of the biomimetic models of oxyhemocyanin and oxytyrosinase

Magnetic interactions and the nature of the chemical bonds of the biomimetic Cu 2(μ–η 2:η 2–O 2) complexes of oxyhemocyanin and oxytyrosinase have been investigated with hybrid density functional theory. The Cu 2(μ–η 2:η 2–O 2) species has drawn attention in type III copper proteins because this structure is suggested as an important motif in biological systems. Many synthetic modeling approaches have been performed and greatly developed our understanding of the character of the Cu 2(μ–η 2:η 2–O 2) species. Natural orbital analysis clearly shows that the superexchange interaction of the d xy orbitals of the Cu ions through the π ∗ orbitals of μ–η 2:η 2 peroxide is responsible for the antiferromagnetic couplings of these Cu 2O 2 system and that the distortion of the Cu 2O 2 core from a planar structure to a butterfly structure and elongation of the Cu–O bonds cause the reduction of orbital interactions between the d xy ± d xy orbitals of the dicopper site and the σ ∗ and π ∗ orbitals of peroxide, weakening the magnetic coupling between the Cu sites via μ–η 2:η 2-peroxide.

Diphenoxo-Bridged Copper(II) Complexes of Reduced Schiff Base Ligands as Functional Models for Catechol Oxidase

The design and development of synthetic analogues for the active dicopper(ii) sites of catechol oxidase, with the help of binucleating ligands in particular, is an attractive strategy to generate relevant information on structure–function relationships. Dicopper(ii) complexes of different yet closely related series of reduced Schiff base ligands (N-(2-hydroxybenzyl)-amino acids; N-(2-hydroxybenzyl)-amino amides, N-(2-hydroxybenzyl)-aminomethane or ethanesulfonic acids, and N-(2-hydroxy-5-substituted-benzyl)-cyclopentane or hexanecarboxylic acids) derived from various substituted salicylaldehydes and natural or unnatural amino acids or amides explored as functional models for the Type 3 copper enzyme catecholase oxidase are reviewed in the present paper. The catalytic activity of different series of dicopper(ii) complexes to oxidize the model substrate 3,5-di-tert-butylcatechol to the corresponding 3,5-di-tert-butylquinone is discussed with respect to the various ligand properties such as the length and chelating ability of the amino acid side-arm of the ligands, conformation of the ligand, nature of the donor groups on the amino acid backbone and role of different para-substituents. This article provides a short review summarizing the trend observed in the catecholase activity of different series of dicopper(ii) complexes investigated in our laboratory.

Quantum Chemical Approach to the Mechanism for the Biological Conversion of Tyrosine to Dopaquinone

Tyrosinase catalyzes the biological conversion of tyrosine to dopaquinone with dioxygen at the dinuclear copper active site under physiological conditions. On the basis of the recent X-ray crystal structural analysis of tyrosinase (J. Biol. Chem. 2006, 281, 8981), a possible mechanism for the catalytic cycle of tyrosinase is proposed by using quantum mechanical/molecular mechanical calculations, which can reasonably take effects of surrounding amino-acid residues, hydrogen bonding, and protein environment into account. The (mu-eta2:eta2-peroxo)dicopper(II) species plays a role in a series of elementary processes mediated by the dicopper species of tyrosinase. A stable phenoxyl radical is involved in the reaction pathway. The catalysis has five steps of proton transfer from the phenolic O-H bond to the dioxygen moiety, O-O bond dissociation of the hydroperoxo species, C-O bond formation at an ortho position of the benzene ring, proton abstraction and migration mediated by His54, and quinone formation. The energy profile of the calculated reaction pathway is reasonable in energy as biological reactions that occur under physiological conditions. Detailed analyses of the energy profile demonstrate that the O-O bond dissociation is the rate-determining step. The activation energy for the O-O bond dissociation at the dicopper site is computed to be 14.9 kcal/mol, which is in good agreement with a measured kinetic constant. As proposed recently, the His54 residue, which is flexible because it is located in a loop structure in the protein, would play a role as a general base in the proton abstraction and migration in the final stages of the reaction to produce dopaquinone.

Conversion of Methane to Methanol at the Mononuclear and Dinuclear Copper Sites of Particulate Methane Monooxygenase (pMMO): A DFT and QM/MM Study

Methane hydroxylation at the mononuclear and dinuclear copper sites of pMMO is discussed using quantum mechanical and QM/MM calculations. Possible mechanisms are proposed with respect to the formation of reactive copper-oxo and how they activate methane. Dioxygen is incorporated into the Cu(I) species to give a Cu(II)-superoxo species, followed by an H-atom transfer from a tyrosine residue near the monocopper active site. A resultant Cu(II)-hydroperoxo species is next transformed into a Cu(III)-oxo species and a water molecule by the abstraction of an H-atom from another tyrosine residue. This process is accessible in energy under physiological conditions. Dioxygen is also incorporated into the dicopper site to form a (mu-eta(2):eta(2)-peroxo)dicopper species, which is then transformed into a bis(mu-oxo)dicopper species. The formation of this species is more favorable in energy than that of the monocopper-oxo species. The reactivity of the Cu(III)-oxo species is sufficient for the conversion of methane to methanol if it is formed in the protein environment. Since the sigma orbital localized in the Cu-O bond region is singly occupied in the triplet state, this orbital plays a role in the homolytic cleavage of a C-H bond of methane. The reactivity of the bis(mu-oxo)dicopper species is also sufficient for the conversion of methane to methanol. The mixed-valent bis(mu-oxo)Cu(II)Cu(III) species is reactive to methane because the amplitude of the sigma singly occupied MO localized on the bridging oxo moieties plays an essential role in C-H activation.

Catalytic Transesterification of Dialkyl Phosphates by a Bioinspired Dicopper(II) Macrocyclic Complex

For a number of phosphoryltransfer enzymes, including the exonuclease subunit of DNA polymerase I, a mechanism involving two-metal ions and double Lewis-acid activation of the substrate, combined with leaving group stabilization, has been proposed. Inspired by the active site structure of this enzyme, we have designed as a synthetic phosphoryl transfer catalyst the dicopper(II) macrocyclic complex LCu(2). Crystal structures of complexes [(L)Cu(2)(mu-NO(3))(NO(3))](NO(3))(2) (1), [(L)Cu(2)(mu-CO(3))(CH(3)OH)](BF(4))(2) (2), and [(L)Cu(2)(mu-O(2)P(OCH(3))(2))(NO(3))](NO(3))(2) (3) illustrate various possibilities for the interaction of oxoanions with the dicopper(II) site. 1 efficiently promotes the transesterification of dimethyl phosphate (DMP) in CD(3)OD, k(cat) = 2 x 10(-)(4) s(-)(1) at 55 degrees C. 1 is the only available catalyst for the smooth transesterification of highly inert simple dialkyl phosphates. From photometric titrations and the pH dependence of reactivity, we conclude that a complex [(L)Cu(2)(DMP)(OCH(3))](2+) is the reactive species. Steric bulk at the -OR substituents of phosphodiester substrates O(2)P(OR)(2)(-) drastically reduces the reactivity of 1. This is explained with -OR leaving group stabilization by Cu coordination, an interaction which is sensitive to steric crowding at the alpha-C-atom of substituent R. A proposed reaction mechanism related to that of the exonuclease unit of DNA polymerase I is supported by DFT calculations on reaction intermediates. The complex [(L)Cu(3)(mu(3)-OH)(mu-CH(3)O)(2)(CH(3)CN)(2)](ClO(4))(3) (4) incorporates a [Cu(OH)(OCH(3))(2)(CH(3)CN)(2)](-) complex anion, which might be considered as an analogue of the [PO(2)(OCH(3))(2)(OCD(3))](2)(-) transition state (or intermediate) of DMP transesterification catalyzed by LCu(2).

Mimics of copper proteins: structural and functional aspects.

The importance of copper as an essential element can be estimated by the wide range of copper proteins and enzymes playing different roles in biological systems. In the last decades many bioinorganic studies were developed on mimetic complexes of copper-dependent proteins, in order to verify the interrelations between structural and functional properties of active copper centers. Among the most studied copper ion ligand, diimine compounds have deserved special attention due their flexibility, facility of preparation, and ability to stabilize both oxidation states of this metal. In our laboratory, we have been investigating some Schiff base copper complexes as mimics of different proteins, with emphasis on functional aspects, trying to elucidate mechanisms of reaction, based on proposed intermediary species, in addition to molecular shapes. Particularly, mimics of the copper-zinc superoxide dismutase, and of monooxigenases and oxidases exhibiting dicopper sites are discussed in this work.

Metallobiosites and their synthetic analogues—a belief in synergism1997–1998 Tilden Lecture

Just as it is possible to use coordination compounds to try and gain insight into the nature of metallobiosites so it is also possible to use the knowledge acquired through crystallography concerning the structure of a metallobiosite to try to develop new chemistry. The aim of this lecture was to illustrate this philosophy by referring to the interplay that led to the confirmation of the dicopper site in haemocyanin and by speculating on the use of knowledge acquired concerning the dinuclear site in urease to facilitate simulation of the functioning of the site.