Binding of water to "types I and II" Cu2+ in proteins

Water proton spin-lattice relaxation times have been measured at 30MHz between 280 – 333 K in aqueous solutions of proteins containing Type I Cu²⁺ ions (azurin and umecyanin) and Type II Cu²⁺ ions (benzylamine oxidase and superoxide dismutase). These measurements show that Type II Cu²⁺ is accessible to exchangeable water molecules but Type I is not. This behaviour is consistent with the EPR and optical properties of these ions and their likely biochemical functions.     

The Analysis of Cu(II)/Zn(II) Cyclopeptide System as Potential Cu,ZnSOD Mimic Center

In this paper are presented the features of copper (II) and zinc (II) heteronuclear complexes of the cyclic peptide—c(HKHGPG)₂. The coordination properties of ligand were studied by potentiometric, UV–Vis and CD spectroscopic methods. These experiments were carried out in aqueous solutions at 298 K depending on pH. It turned out that in a physiological pH dominates Cu(II)/Zn(II) complex ([CuZnL]⁴⁺) which could mimic the active center of superoxide dismutase (Cu,ZnSOD). In next step we performed in vitro research on Cu,ZnSOD activity for [CuZnL]⁴⁺ complex existing in 7.4 pH by the method of reduction of nitroblue tetrazolium (NBT). Also mono- and di-nuclear copper (II) complexes of this ligand were examined. The ability of inhibition free radical reaction were compared for all complexes. The results of these studies show that Cu(II) mono-, di-nuclear and Cu(II)/Zn(II) complexes becoming to new promising synthetic superoxide dismutase mimetics, and should be considered for further biological assays.     

Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.     

Superoxide dismutase–mimicking activities of dinuclear Cu(II) complexes with ligands containing a tetrathioether-tetraamino moiety

The superoxide scavenging activities of copper(II) complexes with the ligands, 6,6′-methylene-bis(5′-amino-3′,4′-benzo-2′-thiapentyl)-1,11-diamino-2,3:9,10-dibenzo-4,8-dithiaundecane (H₄L), and 6,6′-bis(5′-amino-3′,4′-benzo-2′-thiapentyl)-1,11-diamino-2,3:9,10-dibenzo-4,8-dithiaundecane (H₄L′), were investigated by xanthine–xanthine oxidase (X/XO) assays using nitroblue tetrazolium (NBT) as indicator molecule, and the results were compared with respect to the particular type of anion (ClO·4, Cl^·, NO·3) on the apical site of the copper(II) complexes. All of the complexes inhibited the reduction of NBT by superoxide radicals, with the [Cu₂(L′)](ClO₄)₂ complex exhibiting the highest scavenging activity against superoxide radicals among the complexes examined. The catalytic efficiency of the complexes for dismutation of superoxide radicals depends on the particular anion liganded to Cu(II) ion in the complexes, and the order of potency was observed to be ClO₄ > Cl > NO·3 in phosphate buffer at pH 7.40. The Cu(II)-H₄L′ complexes had the lowest IC₅₀ and catalytic rate constant values indicating that the distorted geometry of the Cu(II)-H₄L′ complexes influence their catalytic activities for dismutation of superoxide radicals more efficiently. The difference in the activities of the complexes toward superoxide radicals can also be attributed to the nature of the anions on the apical site of the copper(II) complexes and the superoxide dismutase-like activity. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 53–59, 1998     

Spectroscopic and equilibrium dialysis studies of poly(α-L-glutamic acid)–Cu(II) complexes in the pH range 4–7

The formation of complex between the Cu²⁺ ion and poly(α-L -glutamic acid) [poly(Glu)] in 150 mM NaCl solutions was studied by uv–visible absorption and equilibrium dialysis methods at the mixing ratios of Glu residues to Cu²⁺, R, of 32, 16, and 8 and in the pH range 4–7. The results showed that more than 90% of Cu²⁺ ions bind to the poly(Glu) at pH > 4.9, but the bound Cu(II) begins to dissociate with a decrease in pH. The absorption spectra of bound Cu(II) varied with pH and R in a complicated manner. Three different component spectra were disclosed from the analysis of the pH dependence of the bound spectra. We concluded that poly(Glu)–Cu(II) complexes fall into three classes in the pH range 4–7, with the proportions of these complexes varying with both pH and R. The three complexes predominate either in the helix or extended-coil region, in the helix–coil transition region, or in the helix-aggregate region. The stability constant and binding mode of each Cu(II)–Glu complex were estimated from the dialysis data. With these results, the possible structure of each complex is discussed.     

Superoxide dismutase-like activities of copper(II) complexes tested in serum

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.     

Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

 The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ^• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ^• form of amine oxidases, and thus preventing the reaction of O₂ with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQ_red redox state, which is in equilibrium with Cu(I)-TPQ^•. Received: 12 December 1996 / Accepted: 20 March 1997     

Catalytic activity studies of some copper(II)-histidine-containing dipeptide complexes on aqueous superoxide ion dismutation

The reactivity of histidine-containing copper(II)-dipeptide complexes toward O₂^? produced by the pulse radiolysis technique was investigated. Whereas the His-X-Cu^2? complexes (X = Phe, Ala, Val, Tyr) display a relatively high superoxide dismutase (SOD) activity, that of the Ala-His-Cu²⁺ complex is very low and Val-His-Cu²⁺ as well as Phe-His-Cu²⁺ are ineffective. This difference in behavior can be explained by the fact that the Cu ion is not chelated in the same way in the different complexes, as shown by stability constant determination and other spectral studies. This allows one to conclude that in His-X-Cu²⁺, the dipeptide coordinates to the metal ion by only two nitrogens, whereas in X-His-Cu²⁺ it behaves as a tridentate ligand with three nitrogen atoms bound to the metal.     

Amine oxidase-like activity of polyphenols. Mechanism and properties.

Polyphenols in several oxidation systems gained amine oxidase-like activity, probably due to the formation of the corresponding quinones. In the presence of Cu(II), o- and p-phenolic compounds exhibited amine oxidase-like activity, whereas only the o-phenolic compounds showed the activity in the presence of 1,1-diphenyl-2-picrylhydrazyl radical. The activity was determined by measuring the conversion of benzylamine to benzaldehyde by HPLC. Moreover, gallic acid, chlorogenic acid, and caffeic acid, which are plant polyphenols, converted the lysine residue of bovine serum albumin to alpha-amino-adipic semialdehyde residue, indicating lysyl oxidase-like activity. We also characterized the activity of pyrocatechol, hydroquinone, and pyrogallol in the presence of Cu(II). The oxidative deamination was accelerated at a higher pH, and required O2 and transition metal ions. Furthermore, EDTA markedly inhibited the reaction but not beta-aminopropionitrile, which is a specific inhibitor of lysyl oxidase. Catalase significantly inhibited the oxidation, implying the participation of hydroxyl radical in the reaction, but superoxide dismutase stimulated the oxidation, probably due to its radical formation activity. We discussed the mechanism of the oxidative deamination by polyphenols and the possible significance of the activity for biological systems.     

Magnetic, spectroscopic, structural and biological properties of mixed-ligand complexes of copper(II) with N,N,N,N″,N″-pentamethyldiethylenetriamine and polypyridine ligands

Two new mixed ligand complexes of copper(II) with N,N,N^′,N″,N″-pentamethyldiethylenetriamine and polypyridine ligands have been prepared and characterized by means of spectroscopic, magnetic and single-crystal X-ray diffraction methods. These two complexes are isomorph and isostructure in which the coordination polyhedron about the copper(II) ion is distorted square pyramidal. [Cu(PMDT)(bipy)]²⁺ and [Cu(PMDT)(phen)]²⁺ show an absorption wavelength maximum at 625 and 678 nm, respectively, assigned to the d-d transition. Antibacterial, antifungal and superoxide dismutase activities of these complexes have also been measured. It was observed that [Cu(PMDT)(bipy)](ClO₄)₂ was more effective against P. Pyocyanea and Klebsiella sp. than S. aureus. Similarly, Fusarium sp. was highly susceptible against [Cu(PMDT)(bipy)](ClO₄)₂ but less susceptible against [Cu(PMDT)(phen)](ClO₄)₂.     

Singlet oxygen and superoxide dismutase (cuprein).

Singlet oxygen of elongated lifetime was generated photochemically in ²H₂O-solutions in the presence of molecular oxygen and methylene blue. The oxidation of 1,3-diphenyliso-benzofuran by singlet oxygen was both significantly and specifically inhibited by superoxide dismutase. No such inhibition was observed using the apoprotein, CuSO₄, Cu(tyr)₂, Cu(his)₂, Cu(lys)₂, Cu-serum albumin, hemocyanin, ceruloplasmin, diamine oxidase, horseradish peroxidase, catalase and hemoglobin. Cyanide and azide added in approximately equimolar concentrations proved to be potent inhibitors of this biochemical action of cuprein. The extraordinary stability of superoxide dismutase before and after singlet oxygen treatment was demonstrated by the identical parameters obtained from circular dichroism, electron paramagnetic resonance and X-ray photoelectron spectroscopy (see also ref. 1). There was also no detectable change in the electron absorption spectra compared to the untreated copper protein.     

Two macrocyclic pentaaza compounds containing pyridine evaluated as novel chelating agents in copper(II) and nickel(II) overload

Two pentaaza macrocycles containing pyridine in the backbone, namely 3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene ([15]pyN₅), and 3,6,10,13,19-pentaazabicyclo[13.3.1]nonadeca-1(19),15,17-triene ([16]pyN₅), were synthesized in good yields. The acid-base behaviour of these compounds was studied by potentiometry at 298.2 K in aqueous solution and ionic strength 0.10 M in KNO₃. The protonation sequence of [15]pyN₅ was investigated by ¹H NMR titration that also allowed the determination of protonation constants in D₂O. Binding studies of the two ligands with Ca²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Pb²⁺ metal ions were performed under the same experimental conditions. The results showed that all the complexes formed with the 15-membered ligand, particularly those of Cu²⁺ and especially Ni²⁺, are thermodynamically more stable than with the larger macrocycle. Cyclic voltammetric data showed that the copper(II) complexes of the two macrocycles exhibited analogous behaviour, with a single quasi-reversible one-electron transfer reduction process assigned to the Cu(II)/Cu(I) couple. The UV-visible-near IR spectroscopic and magnetic moment data of the nickel(II) complexes in solution indicated a tetragonal distorted coordination geometry for the metal centre. X-band EPR spectra of the copper(II) complexes are consistent with distorted square pyramidal geometries. The crystal structure of [Cu([15]pyN₅)]²⁺ determined by X-ray diffraction showed the copper(II) centre coordinated to all five macrocyclic nitrogen donors in a distorted square pyramidal environment.     

Synthesis and structure of copper(II) complexes of two new poly(2-pyridylalkyl)diamine ligands

The new pentapyridyldiamine ligand, L¹, which incorporates two bis(2-pyridylethyl)amine donor domains held together by a 2,6-dimethylenepyridine linker, is readily prepared. In the presence of metal salts, L¹ is unstable due to facile elimination of vinyl pyridine. Complexes of L¹ are therefore difficult to isolate. Nonetheless, a novel copper dimer [Cu₂(L¹)(μ-OH)(CH₃CN)](ClO₄)₃ has been isolated in small quantities along with the interesting monomer [Cu(L²)](ClO₄)₂, in which L² is the tetrapyridyldiamine ligand derived from the decomposition of L¹ by loss of one pyridylethyl `arm'. The crystal structures of the two complexes are reported: the [Cu<sub>2</sub>(L<sup>1</sup>)(μ-OH)(CH<sub>3</sub>CN)]<sup>2+</sup> cation exhibits a μ-hydroxo-bridged dicopper(II) core and a coordinated acetonitrile molecule, akin to a putative intermediate in nitrile hydrolysis, and the chiral [Cu(L<sup>2</sup>)]<sup>2+</sup> cation is revealed to have a five-coordinate copper(II) centre that is stabilised by an intramolecular hydrogen-bond between the 2° amine group and a pendant pyridylethyl `arm'.     

Characteristics of non-specific permeability and H+-ATPase inhibition induced in the plasma membrane of Nitella flexilis by excessive Cu2+

Effects of Cu²⁺ on a non-specific conductance and H⁺-ATPase activity in the plasma membrane of the freshwater alga Nitella flexilis L. Agardh was studied using a conventional microelectrode voltage-clamp technique. We show that a Cu²⁺-induced increase in the non-specific conductance is related to the formation of pores in the plasma membrane. Pore formation is the result of unidentified chemical reactions, since the Q₁₀ for the rate of increase of conductance over time was about 3. Various oxidants and antioxidants (10 mmol/l H₂O₂, 10 mmol/l ascorbate, 100 μg/ml superoxide dismutase, and 100 μg/ml catalase) did not alter Cu²⁺-induced changes in the plasma membrane conductance, suggesting that the effect of Cu²⁺ was unrelated to peroxidation of plasma-membrane lipids. In contrast, organic and inorganic Ca²⁺-channel antagonists (nifedipine, Zn²⁺, Cd²⁺, Fe²⁺, Ni²⁺) inhibited the Cu²⁺-induced non-specific conductance increase. This suggests that changes in Ca²⁺ influx underlie this effect of Cu²⁺. Decreasing the pH or the ionic strength of external solutions also inhibited the Cu²⁺-induced plasma-membrane conductance increase. Copper was also found to inhibit plasma-membrane H⁺-ATPase activity with half-maximal inhibition occurring at about 5–20 μmol/l and full inhibition at about 100–300 μmol/l. The Hill coefficient of Cu²⁺ inhibition of the H⁺-ATPase was close to two. Received: 8 December 1999 / Accepted: 16 August 2000     

Chiral Discrimination of Ofloxacin Enantiomers Using DNA Double Helix Regulated by Metal Ions

DNA‐based chiral selectors are constructed to discriminate ofloxacin enantiomers through metal‐ion anchoring on a special DNA double helix that contains successive GC pairs. The effects of metal ions involving Mg²⁺, Ni²⁺, Cu²⁺, Ag⁺, and Pt²⁺ were studied on the regulation of DNA chiral discrimination towards ofloxacin enantiomers. It is shown that DNA‐Cu(II) complexes exhibit the highest enantioselectivities at the [Cu²⁺]/base ratio of 0.1. The enantiomeric excess can reach 59% in R‐enantiomer after being adsorbed by the RET‐Cu(II) complex. Stereoselective recognition of ofloxacin enantiomers on the double helix is tunable via external stimulus, providing a programmable desorption process to regenerate DNA. This DNA‐based chiral selector exhibits excellent reusability without apparent loss of enantioselectivity after three cycles of adsorption and desorption. Chirality 26:249–254, 2014. © 2014 Wiley Periodicals, Inc.     

A pH-dependent superoxide dismutase activity for zinc-free bovine erythrocuprein. Reexamination of the role of zinc in the holoprotein

The zinc-free derivative of bovine erythrocuprein, Cu₂E₂BE, was prepared and its superoxide dismutase activity was measured and compared with that of the holoprotein, Cu₂Zn₂BE. The dismutase activity of these proteins was measured by quantitating their inhibition of the superoxide-mediated autooxidation of 6-hydroxydopamine, dihydroxyfumaric acid, pyrogallol, and epinephrine. It was found that the superoxide dismutase activity of the zinc-free protein is pH dependent, ranging between 82 ± 5% (relative to Cu₂Zn₂BE) at pH 5.8, and 25 ± 10% at pH 10.2. The overlapping range of assays and buffers verified that these measurements are independent of the method of assay, buffer, and ionic strength (in the range of μ = 0.10 to 0.20). The variation in activity with pH is probably due, at least in part, to the migration of Cu(II) at high pH as described previously [J.S. Valentine, M.W. Pantoliano, P.J. McDonnell, A.R. Burger, and S.J. Lippard, Proc. Natl. Acad. Sci. USA 76, 4245 (1979)], since Cu(II) bound at the zinc binding site has been shown to have little or no dismutase activity. The observation of high activity (82%) for the zinc-free protein at pH 5.8, where Cu(II) is predominantly in the native Cu binding site, and less susceptible to removal by ethylenediaminetetraacetic acid, demonstrates that the presence of Zn(II) in Cu₂Zn₂BE does not greatly enhance the inherent dismutase activity of Cu(II) in the holoprotein.     

Copper(II) PMDTA and copper(II) TMEDA complexes: precursors for the synthesis of dinuclear dicationic copper(II) complexes

Copper(II) cations coordinated with PMDTA (pentamethyldiethylenetriamine) and TMEDA (tetramethylethylenediamine) possess a high synthetic potential. The synthesis of these cations was carried out by metathesis reactions with silver salts. The cationic copper(II) complexes, [Cu(PMDTA)(Me₂CO)Cl]⁺, [Cu(PMDTA)(H₂O)Cl]⁺, [Cu(PMDTA)(DMF)]⁺, [Cu(PMDTA)Cl]⁺, [Cu(PMDTA)OAc]⁺, [Cu(PMDTA)(MeCN)₂]²⁺, [Cu₂(TMEDA)₂Cl₃]⁺ and [Cu(TMEDA)(MeCN)₃]²⁺ were synthesised as PF₆ salts, crystallised and characterised by single-crystal X-ray diffraction.     

Purification and Characterization of Cu,Zn Superoxide Dismutase from Ark Shell Scapharca broughtonii

A superoxide dismutase has been purified to apparent homogeneity from the muscular tissue of the ark shell, Scapharca broughtonii, by ammonium sulfate fractionation, and consecutive column chromatographies using DEAE-Sephadex and Sephadex G-100. This enzyme has a molecular weight of 71,700 and is composed of two identical subunits of M _r 35,800, which are joined by noncovalent interactions. The purified enzyme was stable over the range of pH 5.0-10.0 at 4°C for 24 h and at temperatures below 45°C. Cyanide at 0.1 and 1 mM inhibited the activity of the superoxide dismutase 56 and 100%, but 5 mM azide caused 8% inhibition. The optical spectrum of this enzyme had a maximum at 265 nm, and the amino acid composition of the enzyme was similar to that of the other Cu, Zn superoxide dismutases except for the contents of threonine, serine, proline, and leucine. Atomic absorption spectroscopy showed that this enzyme has approximately 2 atoms of Cu²⁺ and Zn²⁺ per mole of enzyme. These results indicate that the purified enzyme from ark shell, Scapharca broughtonii, is a Cu, Zn superoxide dismutase.     

Cyanide as a copper and quinone-directed inhibitor of amine oxidases from pea seedlings (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and<Emphasis Type="Italic"> Arthrobacter globiformis</Emphasis>: evidence for both copper coordination and cyanohydrin derivatization of the quinone cofactor

The interactions of cyanide with two copper-containing amine oxidases (CuAOs) from pea seedlings (PSAO) and the soil bacterium Arthrobacter globiformis (AGAO) have been investigated by spectroscopic and kinetic techniques. Previously, we rationalized the effects of azide and cyanide for several CuAOs in terms of copper coordination by these exogenous ligands and their effects on the internal redox equilibrium TPQ_amr-Cu(II)TPQ_sq-Cu(I). The mechanism of cyanide inhibition was proposed to occur through complexation to Cu(I), thereby directly competing with O₂ for reoxidation of TPQ. Although cyanide readily and reversibly reacts with quinones, no direct spectroscopic evidence for cyanohydrin derivatization of TPQ has been previously documented for CuAOs. This work describes the first direct spectroscopic evidence, using both model and enzyme systems, for cyanohydrin derivatization of TPQ. K_d values for Cu(II)-CN^– and Cu(I)-CN^–, as well as the K_i for cyanide inhibition versus substrate amine, are reported for PSAO and AGAO. In spite of cyanohydrin derivatization of the TPQ cofactor in these enzymes, the uncompetitive inhibition of amine oxidation is determined to arise almost exclusively through CN^– complexation of Cu(I).Abbreviations AGAO Arthrobacter globiformis amine oxidase - APAO Arthrobacter P1 amine oxidase - APT attached proton test - BPAO bovine plasma amine oxidase - CuAO quinone-copper containing amine oxidase - LTQ lysyl tyrosylquinone - MAO monoamine oxidase - PKAO porcine kidney amine oxidase - PPAO porcine plasma amine oxidase - PSAO pea seedling amine oxidase - TPQ 2,4,5-trihydroxyphenylalaninequinone - TPQ_amr TPQ aminoresorcinol - TPQ_imq TPQ iminoquinone - TPQ_ox TPQ oxidized - TPQ_sq TPQ semiquinone - WT wild-typeE.M. Shepard and G.A. Juda contributed equally to this workThis revised version was published online in February 2004: Hansenula polymorpha was not italicised at the end of the Introduction, Equation 3 appeared twice, and the resolution of Scheme 3 was insufficient.An erratum to this article can be found at     

Phenoxyl-copper(II) complexes: models for the active site of galactose oxidase

 The reaction of the macrocycles 1,4,7-tris (3,5-di-tert-butyl-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L¹H₃, or 1,4,7-tris(3-tert-butyl-5-methoxy-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L²H₃, with Cu(ClO₄)₂·6H₂O in methanol (in the presence of Et₃N) affords the green complexes [Cu^II(L¹H)] (1), [Cu^II(L²H)]·CH₃OH (2) and (in the presence of HClO₄) [Cu^II(L¹H₂)](ClO₄) (3) and [Cu^II(L²H₂)] (ClO₄) (4). The Cu^II ions in these complexes are five-coordinate (square-base pyramidal), and each contains a dangling, uncoordinated pendent arm (phenol). Complexes 1 and 2 contain two equatorially coordinated phenolato ligands, whereas in 3 and 4 one of these is protonated, affording a coordinated phenol. Electrochemically, these complexes can be oxidized by one electron, generating the phenoxyl-copper(II) species [Cu^II(L¹H)]^+ ·, [Cu(L²H)]^+ ·, [Cu^II(L¹H₂)]^2+ ·, and [Cu^II(L²H₂)]^2+ ·, all of which are EPR-silent. These species are excellent models for the active form of the enzyme galactose oxidase (GO). Their spectroscopic features (UV-VIS, resonance Raman) are very similar to those reported for GO and unambiguously show that the complexes are phenoxyl-copper(II) rather than phenolato-copper(III) species. Received: 10 February 1997 / Accepted: 7 April 1997